the conservation of horseshoe crabs in hong...

Copyright Warning

Use of this thesisdissertationproject is for the purpose of private study or scholarly research only Users must comply with the Copyright Ordinance Anyone who consults this thesisdissertationproject is understood to recognise that its copyright rests with its author and that no part of it may be reproduced without the authorrsquos prior written consent

CITY UNIVERSITY OF HONG KONG 香港城市大學

The Conservation of Horseshoe Crabs in Hong Kong

香港馬蹄蟹的保育研究

Submitted to Department of Biology and Chemistry

生物及化學系 in Partial Fulfillment of the Requirements

for the Degree of Master of Philosophy 哲學碩士學位

by

LI Hiu Yan 李曉恩

September 2008 二零零八年九月

i

Declaration

The research described in this M Phil thesis was conducted under the supervision

of Dr SG Cheung at the Department of Biology and Chemistry City University of

Hong Kong It was an independent work of the author unless otherwise stated and has

not been included in any other thesis or dissertation submitted to this or other institution

for a degree diploma or any other qualifications Attention is drawn to the fact that

anyone without the authorrsquos prior consent strictly may not copy reproduce transform

or publish any data derived from the authorrsquos own work in this project

LI Hiu Yan

September 2008

ii

Abstract of thesis entitled



Submitted by

LI Hiu Yan

for the Degree of Master of Philosophy at the City University of Hong Kong

Horseshoe crabs in Hong Kong are facing a rapid population decline and local

extinction risk This study focused on some aspects of the conservation of horseshoe

crabs in Hong Kong including 1) most up-to-date information of the present status and

distribution of horseshoe crabs 2) morphological measurements and genetic analysis of

two horseshoe crab species Tachypleus tridentatus and Carcinoscorpius rotundicauda

and geographical variations in population genetics of T tridentatus 3) the degree of

human exploitation of horseshoe crabs and 4) trials on artificial insemination and

breeding of horseshoe crabs and investigations on the optimal environmental conditions

for enhancing the survival and hatching success of horseshoe crab eggs

Juvenile horseshoe crabs were sampled on the soft shores in Hong Kong using a

quantitative random quadrat sampling along shore transects at 17 survey sites in Hong

Kong A total of 15 juvenile Tachypleus tridentatus were found at Tsim Bei Tsui Pak

Nai two locations at Ha Pak Nai in northwestern New Territories and San Tau and Yi

O on Lantau Island In comparison with a similar territory-wide study in 2002 by

Morton and Lee (2003) a significant decline in the mean population density was

iii

recorded with the decrease of 96 90 and 80 at Pak Nai and two locations in Ha Pak

Nai respectively No Carcinoscorpius rotundicauda was found in the transect study at

the 17 sites Additional walk-through observations on the shores revealed the presence

of T tridentatus at other sites including Tsim Bei Tsui in the northwestern New

Territories and Shui Hau Wan Tai Ho Wan Sham Wat Yi O and Tung Chung on

Lantau Island The walk-through study also revealed the presence of C rotundicauda at

Tsim Bei Tsui Sheung Pak Nai Pak Nai two locations at Ha Pak Nai in northwestern

New Territories Luk Keng and Lai Chi Wo in the northeastern New Territories and Tai

Ho Wan Yi O and Tung Chung on Lantau Island

Further intensive distribution surveys were conducted at four key horseshoe crab

nursery grounds namely Pak Nai Ha Pak Nai Shui Hau Wan and San Tau at monthly

intervals from March to August 2005 using both the transect study and walk-through

observations All the sites supported T tridentatus populations with a maximum

abundance of 85 individuals at Shui Hau Wan in April Few (1-2 individuals) C

rotundicauda were found at Pak Nai and Ha Pak Nai while a higher abundance (total

64 individuals) was recorded at San Tau on Lantau Island in the 6-month survey

However its overall population was much lower as compared with T tridentatus

Among the sites San Tau and Shui Hau Wan were the key nursery grounds for C

rotundicauda and T tridentatus respectively The active period for horseshoe crabs

lasted from May to July with more individuals being found as compared with the

inactive period in March April and August A wide range of age groups of horseshoe

crabs were recorded with a maximum prosomal width ranging from 101 to 961 mm

and 109 to 433 mm for T tridentatus and C rotundicauda respectively A significant

spatial variation in the abundance of T tridentatus on the shore was obtained with larger

individuals on the lower shores As individuals of T tridentatus lt120 mm (maximum

iv

prosomal width) were recorded in the present survey these sites were certainly

important nursery grounds for juveniles and newly hatched individuals

Both morphological measurements and genetic analysis were used for the

differentiation of the two commonly occurring horseshoe crab species in Hong Kong

For morphological measurements T tridentatus and C rotundicauda were significantly

different in various ratios of body parts Only 5 out of the 9 parameters were proven

important in differentiating the two species they are the ratios of prosomal length to

maximum prosomal width maximum prosomal width to distance between two

compound eyes carapace length to telson length first opisthosomal spine length to

maximum prosomal width and sixth opisthosomal spine length to maximum prosomal

width For phylogenetic comparisons the inter-specific variations of both 18S and 28S

rDNA sequence were very small and was lt1 and 157 respectively Thus these

two rDNA regions may not be powerful enough for the differentiation of T tridentatus

and C rotundicauda The 18S and 28S rDNA of ldquoabnormalrdquo juvenile T tridentatus

individuals with only 1 immovable spine on the dorsal surface of the opisthosoma above

the insertion of a post-anal spine and ldquonormalrdquo individuals with 3 immovable spines

were also sequenced Both juvenile groups were genetically closer to C rotundicauda

but morphologically closer to adult T tridentatus There was no significant spatial

variation in the genetic pattern of juvenile T tridentatus at five nursery grounds Pak

Nai Ha Pak Nai San Tau Shui Hau Wan and Yi O based on 18-28S intergenic spacer

sequence (ITS) rDNA suggesting that these horseshoe crab populations may come from

a common population

The degree of human exploitation of horseshoe crabs in Hong Kong was estimated

through interviewing 34 seafood restaurants 150 fish sellers and fish handlers A total

v

of 1023 horseshoe crabs were caught in 2004-05 with 72 from mainland Chinese

waters Of these 33 were released back to the sea after being caught and only 690

individuals were retained on board The majority (62) of them were sold and used for

the Chinese traditional set-free rituals while the remaining were kept and sold in fish

stalls and seafood restaurants An average sale of 17 horseshoe crabs per month was

estimated throughout the 13-month study with 45 of them being obtained from Hong

Kong waters The sale of horseshoe crabs was high by comparing with other popular

commercial marine species in view of the low population density long maturity period

and low breeding rate of horseshoe crabs Hence human exploitation is possibly one of

the major impacts affecting the long-term survival of this animal in Hong Kong

In view of the low natural breeding and hatching success and low juvenile

survival artificial insemination and breeding may be reasonable options to enhance the

horseshoe population in the natural environment Trials of artificial breeding were

conducted in the summer of 2004 Several thousand trilobite larvae hatched with some

of them further molted into juveniles of second to fifth instars Mortality rate was high

after hatching and varied over time After almost two years of laboratory rearing only

some 20 individuals of the first batch of trilobites survived to become juveniles To

elucidate the optimum conditions for the survival and hatching of eggs a two factorial

experiment was conducted with combinations of four salinities (15permil 20permil 25permil and

30permil) and three temperatures (20degC 25degC and 32degC) being studied Horseshoe crab

eggs survived at a wide range of temperatures (20ndash32degC) and salinities (20ndash30permil)

However no hatching was observed at 15permil Although the survivorship at low

temperatures and low salinities were relatively high the developmental and hatching

rates were reduced The highest hatching rate was obtained at 32degC and 30permil which

should be recommended for future artificial breeding practices Trials on non-invasive

vi

artificial breeding methods using electrical stimulation for the collection of unfertilized

eggs and sperms were also performed Several successful trials were undertaken

however the number of eggs obtained was small Further studies are required to

ascertain the applicability and repeatability of this method

The present study provides comprehensive baseline information on the horseshoe

crabs in Hong Kong that may form a basis for the implementation of conservation

measures in the future

vii

Significance of the Study

Horseshoe crabs have been facing global decline in the past decades Species

specific conservation and management strategy of horseshoe crab are urgently required

The present study provides comprehensive baseline information on the horseshoe crabs

in Hong Kong including

1) Status evaluation of horseshoe crabs in Hong Kong

The distributions and population densities of the juveniles of the two horseshoe

crab species at various nursery grounds in Hong Kong was updated by both extensive

and intensive 6-month population studies The distribution of adult horseshoe crabs was

also investigated by interviewing the local seafood markets and fishermen These

distribution studies provided scientific evidence on the sharp decline in local horseshoe

crab populations from 2002 to 2005

2) Threat determination of horseshoe crabs in Hong Kong

Human exploitation including harvest and sale of horseshoe crabs (purpose for

set-free rituals display and sale for dishes) in Hong Kong was evaluated in the present

study The market survey showed a potential risk of human exploitation on the local

horseshoe crab populations

3) Population enhancement of horseshoe crabs in Hong Kong

Various artificial breeding and rearing practices for T tridentatus have been

explored in the present study It provided important basic knowledge for further

artificial breeding and restocking of horseshoe crabs in Hong Kong

viii

4) Taxonomic and population genetic study of horseshoe crabs in Hong Kong

Differentiating the juvenile forms of the two commonly occurred horseshoe crab

species T tridentatus and C rotundicauda in Hong Kong using both morphological

and genetic approaches and population genetic of T tridentatus in various nursery

grounds have been performed in this study It provided basic knowledge of the

speciation and spatial genetic variation of juvenile horseshoe crabs in local nursery

grounds

Based on these baseline data on local horseshoe crabs species specific

management tools can be designed and undertaken to minimize the threats conserve the

species and foster sustainability or recovery of it by a dynamic and responsive process

Hence this study provided vital information for future conservation and management

planning of horseshoe crabs in Hong Kong

x

Acknowledgements

I would like to thank my supervisor Dr SG Cheung for his invaluable instruction

and constructive criticism throughout my postgraduate study Moreover his kindness

and guidance were helpful to my research process At the same time I would like to

thank Dr Paul Shin for his comments and suggestions that are crucial and necessary to

the success of my research and Dr Richard Kong for his advices on the phylogenetic

and population genetic studies of local horseshoe crabs The work described in this

thesis was fully funded by a grant from the Environment and Conservation Fund (ECF

Project 122003) and Woo Wheelock Green Fund (WWGF)

Besides I would like to thank Dr Kevin Gao for advice on statistical analysis I

express my sincere appreciation to my student helpers Stella Kwok Grace Tsui and

Scottie Cheung for their assistance in field and laboratory works The assistance and

support from my lab-mates Nelson Lam Harry Chai Joseph Lo Leo Tai Melody Mak

Cheung Kwok Leung Carmen Yip Rita Chan Chen Yan Fish Ho and Candy Yang are

appreciated Help from department laboratory technicians Helen Ng Raymond Chan

Eric Shum and Amy So is also important and valuable Study management and schedule

by the staff of School of Graduate Study are greatly appreciated

Special thanks are expressed to Prof CH Ke from the College of Oceanography

and Environmental Science of Xiamen University for his guidance and professional

advice on the method of artificial insemination of horseshoe crabs

I express my sincere thanks to my family members Dad Mum and my young

sister Samantha They provide me spiritual support to explore in the field of scientific

research in the past four years Special thanks are given to my dear friends Sze-man Li

xi

Mavis Li Stella Wai Gigi Ho Liza Yeung Idy Mo Sandy Kwong and Hannah Tai

who devote love to share happiness and sadness with me during this 4-year research

period They always give me a great encouragement and support particularly in my

hardest time dealing with the project work Also thanks are expressed to the Christian

fellows in CCC Kam Kong Memorial Church especially members in my cell group

and members in ldquoChildren Sunday Schoolrdquo service who always pray for me and give

me endless support

Last but not least I deeply thank my Heavenly Father Thanks for His amazing

creation works As Bible says ldquoFor the invisible things of him from the creation of the

world are clearly seen being understood by the things that are made even his eternal

power and Godhead so that they are without excuserdquo (Romans 120) Furthermore God

gives me intelligence strength and peace during my hardest time dealing with the

project works Without Him I can never have such an achievement

xii

Table of Contents

Declaration i

Abstract ii

Significance of the Study vii

Thesis Acceptance ix

Acknowledgements x

Table of Contents xii

List of Tables xvii

List of Figures xxii

Chapter 1 General Introduction 1

11 History of Horseshoe Crabs 1

12 Biology of Horseshoe Crabs 1

13 Population Study on Horseshoe Crabs 4

14 Importance of Horseshoe Crabs 6

15 Decline in Horseshoe Crab Populations 9

16 Risks Facing by Horseshoe Crabs 10

17 Conservation of Horseshoe Crabs 11

18 Artificial Breeding of Horseshoe Crabs 12

19 Genetic Study of Horseshoe Crabs

110 Objectives

13

15

111 Organization of the Thesis 16

Chapter 2 Survey of Horseshoe Crabs in Hong Kong 17

21 Introduction 17

22 General Distribution and Abundance of Juvenile Horseshoe Crabs

on Soft Shores in Hong Kong

18

221 Materials and Methods 18

2211 Site Characteristics 18

2212 Sample Collection 23

2213 Statistical Analysis 26

222 Results 26

2221 Distribution and Abundance of Horseshoe Crabs 26

2222 Environmental Parameters of the Study Sites 41

xiii

2223 Size of the Horseshoe Crabs on the Shores 50

23 Temporal Variations in the Population Density of Horseshoe Crabs

at Four Nursery Grounds

57





232 Results 59

2321 Temporal Variations in the Population Density of

Horseshoe Crabs

59

2322 Environmental Parameters of the Shores 68


24 Discussion 102

241 Updated Distribution of Juvenile Horseshoe Crabs in Hong

Kong

102

2411 T tridentatus 104

2412 C rotundicauda 106

242 Local Distribution Patterns of the Two Horseshoe Crab

Species

107

243 Status of Horseshoe Crabs in Hong Kong 110



244 Temporal Variations in the Distribution of Horseshoe Crabs 112

245 Size Distributions of Horseshoe Crabs 113

Chapter 3 To Differentiate Tachypleus tridentatus from Carcinoscorpius

rotundicauda using Morphological and Genetic Studies and to

Study Genetic Relationships among Horseshoe Crabs from

Various Nursery Grounds in Hong Kong

116

31 Introduction 116

32 Morphological Study of the Two Horseshoe Crab Species 119


3211 Measurement of Various Body Parts 119


xiv

322 Results 123

3221 Qualitative Comparisons 123

3222 Quantitative Comparisons 125

33 Genetic Differentiation of Horseshoe Crab Species 133



3212 DNA Extraction PCR Amplification and Sequencing 135


332 Results 136

3221 18S rDNA Gene Comparison 136


34 Genetic Relationships among Horseshoe Crabs from Various

Nursery Grounds in Hong Kong

146





342 Results 148

35 Discussion 153

351 Morphological and Genetic Differentiation in Horseshoe

Crabs

153

352 Genetic Relationships among Horseshoe Crabs from

Various Nursery Grounds

155

Chapter 4 Assessment of Human Exploitation of Horseshoe Crabs in Hong

Kong

159

41 Introduction 159



422 Data Collection 159

43 Results 159

431 Catch of Horseshoe Crabs 161

432 Sale of Horseshoe Crabs 166

4321 Set-free Rituals 166

xv

4322 Display of Horseshoe Crabs 168

4323 Serving Horseshoe Crabs for Dishes 170

43 Discussion 173

441 Population of Horseshoe Crabs in Open Waters 173

442 Human Exploitation of Horseshoe Crabs 175

443 Potential Risks to Horseshoe Crabs 179

Chapter 5 Trials on Artificial Breeding of Horseshoe Crabs 181

51 Introduction 181



522 Results 185

53 Effects of Temperature and Salinity on Egg Development 190


5311 Fertilization of Eggs 190

5312 Incubation under Different Combinations of

Temperature and Salinity

190


532 Results 192

5321 Survival Rate of Horseshoe Crab Eggs 192

5322 Hatching Rate of Horseshoe Crab Eggs 197

54 Preliminary Study on Alternative Artificial Breeding Method 199


5411 Check for the Maturity of Horseshoe Crab Eggs 199

5412 Electrical Stimulation 199

542 Results 201

55 Discussion 203

551 Trials on Artificial Insemination 203

552 Incubation of Eggs in the Laboratory 205

553 Survival of Trilobites and Juveniles 206

Chapter 6 General Discussion 208

61 Recommendations for Further Study

210

xvi

611 Walk-through Method and Mark-recapture Method for

Distribution Study

210

612 Study on Adult Horseshoe Crabs 212

613 In-depth Studies on Phylogenetic and Geographical Genetic

Variations in Juvenile Horseshoe Crabs

213

614 Risk Assessment of Horseshoe Crabs 215

615 More Studies on Carcinoscorpius rotundicauda 216

616 Non-destructive Artificial Breeding Technique 217

617 Further Study on the Requirements for Juvenile Rearing 218

62 Proposed Conservation Measures 219

621 Re-introduction Programme 220

622 Ban on Fishing and Possession of Horseshoe Crabs in Hong

Kong

221

623 Designation of Protected Areas 222

624 Regular Monitoring Study on Horseshoe Crabs Distribution 223

625 Public Education 223

Chapter 7 Conclusion 225

Chapter 8 References 227

Appendix 1 Questionnaire for Assessing Human Exploitation of Horseshoe

Crabs in Hong Kong

250

xvii

List of Tables

Table 21 Information on the 17 sampling sites in New Territories and on

Lantau Island and Lamma Island in Hong Kong

21

Table 22 Total number and density of T tridentatus and various

environmental parameters of the 17 sampling sites in New

Territories and on Lantau Island and Lamma Island in Hong

Kong in the summer distribution study

28




Kong in the winter distribution study

30

Table 24 Total number of T tridentatus at various tidal levels in New


Kong

32

Table 25 Results of the Kruskal-Wallis test for the differences in the

population density of juvenile T tridentatus at the 17 sites in the

summer and winter

36

Table 26 Abundance and population density (individual hour-1 person-1) of

juvenile T tridentatus obtained by the walk-through survey at

the 17 study sites in the summer and winter

38


juvenile C rotundicauda at the 17 study sites in the summer and

winter

39

Table 28 Results of the 3-way ANOVA test for the differences in

temperature at the 17 sites in the summer and winter (tidal level

times site times season)

43

xviii

Table 29 Results of the 3-way ANOVA test for the differences in salinity

at the 17 sites in the summer and winter (tidal level times site times

season)

46

Table 210 Results of the 3-way ANOVA test for differences in the DO level


season)

49

Table 211 3-way ANOVA (tidal level site and season) results on the

prosomal width of juvenile T tridentatus recorded by the

walk-through survey

52


prosomal width of juvenile C rotundicauda recorded by the

walkndashthrough survey

55

Table 213 Results of the Kruskal-Wallis test for differences in the

population density of T tridentatus at the 4 sites from March to

August 2005

63

Table 214 Abundance (total number of individuals) and population density

(individual hour-1 person-1) of juvenile T tridentatus and C

rotundicauda recorded by the walk-through survey at the four

sites from March to August 2005

65

Table 215 Results of the 3-way ANOVA test for differences in temperature

at the four sites from March to August 2005 (tidal level times site times

time)

70

Table 216 Results of the 3-way ANOVA test for differences in salinity at

the four sites from March to August 2005 (tidal level times site times

time)

74

xix

Table 217 Results of the 3-way ANOVA test for differences in DO level at


time)

77

Table 218 Prosomal width (PW) of T tridentatus found at the 4 sites

79

Table 219 Results of the Kruskal-Wallis test for the differences in prosomal

width of T tridentatus at the four sites from March to August

2005

84

Table 220 Average prosomal width (plusmn SD) of individual cohorts of T

tridentatus at the four sites as identified by FiSAT II

91

Table 221 Measurements of prosomal width (PW) of C rotundicauda

found at the four sites

94


width of C rotundicauda at the four sites from March to August

2005

98

Table 223 Records of juvenile horseshoe crabs obtained from previous

surveys by Chiu and Morton (1999a) Morton and Lee (2003)

and the AFCD monitoring survey (AFCD field data)

103

Table 31 The means (plusmnSD) and ranges of various morphological ratios in

juvenile T tridentatus and C rotundicauda

127

Table 32 Results of the test of equality of discriminant analysis for

differences in various morphological ratios between T

tridentatus and C rotundicauda

128

Table 33 Comparisons of the morphology between T tridentatus and C

rotundicauda

129

xx

Table 34 Significant morphological ratios and their discriminant function

coefficients of T tridentatus and C rotundicauda derived from

the stepwise multiple discriminant analysis

131

Table 35 Grouping of horseshoe crab specimens used in the study of 18S

and 28S rDNA

134

Table 36 Percentage of pair-wise difference in 18S rDNA gene of

horseshoe crab individuals and number of

transitionstransversions

138

Table 37 Percentage of pair-wise difference in 18 rDNA gene within and

among each horseshoe crab species (mean plusmn SD)

139




143


among each horseshoe crab species (mean plusmn SD) Bracketed

values indicate the range of the data

144

Table 310 Grouping of horseshoe crab specimens used in the study of

18-28S intergenic spacer sequence (ITS) rDNA

147

Table 311 Percentage of pair-wise difference in the 18-28S ITS rDNA gene

of horseshoe crab individuals and number of transitions

transversions

150


for T tridentatus within each study site and among different

study sites (mean plusmn SD)

151

xxi

Table 41 Historical records of adult horseshoe crabs in open waters in

Hong Kong with reference to Chiu and Morton (1999a)

174

Table 51 Different combinations of temperature and salinity were used in

culturing the eggs of T tridentatus

191

Table 52 Results of the two-way ANOVA test followed by the Tukey

multiple comparison test for differences in the survival rate of

the horseshoe crab eggs at 3 temperatures and 4 salinities

193

Table 53 Results of the multiple comparisons of the effect of temperature

at individual salinities and the effect of salinity at individual

temperatures on the survival rate of horseshoe crab eggs

196

Table 54 Number of juvenile horseshoe crabs hatched under different

combinations of temperature and salinity for 90 days

198

xxii

List of Figures

Figure 11 Three major body parts of horseshoe crab (Tachypleus

tridentatus)

3

Figure 21 The 17 distribution study sites in New Territories on Lantau

Island and on Lamma Island

20

Figure 22 Sampling design for the distribution survey of juvenile

horseshoe crabs in Hong Kong

25

Figure 23 Mean density (+SE) of juvenile T tridentatus recorded by the

random quadrat sampling

34

Figure 24 Mean density (+SE) of juvenile T tridentatus found at the four

tidal levels recorded by the random quadrat sampling

35

Figure 25 Population distribution of the horseshoe crabs at the 17 sites by

the walk-through survey

40

Figure 26 Temporal variations in temperature (plusmn SD) at the 17 sites in the

summer and winter

42

Figure 27 Temporal variations in salinity of the interstitial waters (plusmn SD) at

the 17 sites in the summer and winter

45

Figure 28 Temporal variations in the DO level (plusmn SD) of the interstitial

waters at the 17 sites in the summer and winter

48

Figure 29 Mean (+SE) prosomal width of juvenile T tridentatus recorded

by the walk-through survey at the 17 sites in the summer and

winter

51

Figure 210 Mean (+SE) prosomal length of juvenile T tridentatus found at

the four tidal levels

53

xxiii

Figure 211 Mean (+SE) prosomal width of juvenile C rotundicauda

recorded by the walk-through survey at the 17 sites in the

summer and winter

54

Figure 212 Mean (+SE) prosomal length of juvenile C rotundicauda found

at the four tidal levels recorded by the walk-through survey

56

Figure 213 Mean density of juvenile T tridentatus at the four sites recorded

by the random sampling method from March to August 2005

61

Figure 214 Spatial distribution (tidal levels) (+SE) of juvenile T tridentatus

at the four sites recorded by the random sampling method from

March to August 2005

62


at the four sites recorded by the walk-through survey from


66

Figure 216 Spatial distribution (tidal levels) (+SE) of juvenile C

rotundicauda at the four sites recorded by the walk-through

survey from March to August 2005

67

Figure 217 Temporal variations in average temperature (plusmn SD) at the four


69

Figure 218 Temporal variations in the average interstitial water salinity (plusmn

SD) at the four sites from March to August 2005

73

Figure 219 Temporal variations in the average DO level (plusmn SD) of the

interstitial water at the four sites from March to August 2005

76

Figure 220 Spatial variations (among sites and tidal levels) of the prosomal

width (+SE) of T tridentatus from March to August 2005

81

xxiv

Figure 221 Temporal variations of the prosomal width (+SE) of T

tridentatus from March to August 2005

83

Figure 222 Size-frequency distributions of T tridentatus at Site 1 (Pak Nai)

Site 2 (Ha Pak Lai) Site 3 (Shui Hau Wan) and Site 4 (San

Tau)

87

Figure 223 Spatial variations (both among sites and tidal levels) of the

prosomal width (+SE) of C rotundicauda from March to August

2005

96

Figure 224 Temporal variations of the prosomal width (+SE) of C

rotundicauda from March to August 2005

97

Figure 225 Size-frequency distributions of C rotundicauda at Site 1 (Pak

Nai) Site 2 (Ha Pak Lai) and Site 4 (San Tau)

100

Figure 31 Photos of juvenile horseshoe crabs found on the shores

121

Figure 32 Various body parts of a horseshoe crab were measured to the

nearest 01mm

122

Figure 33 The dorsal view of a) Tachypleus tridentatus b)

Carcinoscorpius rotundicauda

124

Figure 34 Plot of the stepwise multiple discriminant analysis based on

various morphological ratios in T tridentatus (Tt) and C

rotundicauda (Cr)

132

Figure 35 Neighbor-joining tree for the horseshoe crab 18S rDNA gene

sequences

140


sequences

145

xxv

Figure 37 Neighbor-joining tree for the horseshoe crabs 18-28S ITS rDNA

gene sequences

152

Figure 41 Number of Tachypleus tridentatus being caught in Hong Kong

and China waters from September 2004 to September 2005

163


waters with proportions for sale and set-free from September

2004 to September 2005

164

Figure 43 Number of Tachypleus tridentatus being caught in China waters

with proportions for sale and set-free from September 2004 to

September 2005

165

Figure 44 The sale of horseshoe crabs for the release and non-release

activities from September 2004 to September 2005

167

Figure 45 Number of T tridentatus displayed in fish stalls and seafood

restaurants from September 2004 to September 2005

169

Figure 46 The advertisements of horseshoe crab dishes were shown in a)

Cheung Chau seafood restaurant b) Causeway Bay seafood

restaurant

171

Figure 47 Estimated sale of horseshoe crabs for local consumption from

September 2004 to September 2005 Individuals being caught

from both Hong Kong and China waters are shown

173

Figure 51 The direct extraction of horseshoe crab eggs a) body view of a

female horseshoe crab b) extraction of eggs from one side of the

ovary of a female horseshoe crab

183

Figure 52 Fertilized eggs were incubated in a water table with aeration and

temperature control

184

xxvi

Figure 53 Various developmental stages of horseshoe crab eggs

188

Figure 54 Different instars of the horseshoe crab a) second instar b) third

instar c) fourth instar

189

Figure 55 The survival rate (plusmn SE) of the horseshoe crab eggs under

different temperatures at the salinity of a) 15permil b) 20permil c)

25permil d) 30permil

194

Figure 56 The survival rate (plusmn SE) of horseshoe crab eggs under different

salinities at the temperature of a) 20degC b) 25degC c) 32degC

195

Figure 57 Photos showing a) the position of the gonopores on the ventral

side of the genital operculum b) electrical shock applied 1 cm

beneath the gonopores of a female T tridentatus

200

Figure 58 Photos showing a) a wide view b) a close up of the horseshoe

crab eggs released from the gonopores after electrical

stimulation

202

1

Chapter 1 General Introduction

11 History of Horseshoe Crabs

Horseshoe crabs also called Horsefoot King Crab or Sauce-pan are well known

to scientists as they have already lived on earth for the past 420 million years It is

believed that they belong to a close relative of the trilobite (an ancient marine organism)

and were evolved far before the dinosaurs and flowering plants with fossil records

dated back to the Palaeozoic Devonian period the Age of Visible Life Horseshoe crabs

belong to Phylum Arthropoda which consists of major Classes including Insecta (eg

ants beetles) Arachnida (eg spiders scorpions mites and ticks) Crustacea (eg

lobsters crabs and shrimps) and Class Merostomata At present there are 4 species of

horseshoe crabs occurring in limited areas of the world including Limulus polyphemus

the only American horseshoe crab species and three Indo-Pacific species Tachypleus

tridentatus (Leach 1819) Tachypleus gigas (Muumlller 1785) and Carcinoscorpius

rotundicauda (Latreille 1802)

12 Biology of Horseshoe Crabs

Horseshoe crabs have a simple body structure with no remarkable morphological

changes in the past millions of years The body is divided into three parts prosoma

opisthosoma and telson (Fig 11) The prosoma is the front semicircular part of the

body combining the head and thorax The opisthosoma attaching to the prosoma with a

2

hinge protects the five pairs of book gills and two genital pores underneath whereas

the telson is the tail part used for flipping the body over from upside down

Two large compound eyes are located on the dorsal side of the prosoma with other

light receptors scattered all over the body Six pairs of leg-like appendages are on the

ventral side of the prosoma and mainly used for moving as well as gathering and eating

food The first pair of appendages ie chelicerae is smaller in size and end with a

pincer for detecting clams and worms on the sea bottom When the chelicerae find a

prey one of the claws picks it up and pushes it toward the mouthpart near the base of

the walking legs The next pair of appendages is the pedipalps which are the first

ambulatory legs In the adult male the tarsus (first section of the appendage) of the

pedipalp is modified as a grasping appendage allowing the male to clasp the female

during spawning The second third and fourth pair of appendages are called ldquowalking

legsrdquo which are mainly for moving on the seabed and also aid in pushing food into the

mouth during feeding The last pair of appendages is the ldquoswimming legsrdquo which have

leaf-like flaps used for pushing body forward during swimming The small pincers on

the last appendage pair are also used for cleaning the gills on the abdomen

3

pr

op

te

Figure 11 Three major body parts of horseshoe crab (Tachypleus tridentatus) (pr

prosoma op opisthosoma te telson)

4

In the breeding season from late March to August the adult male horseshoe crab

clasps the female by the pincer-like pedipalps in open waters The mating pair then

crawls onto the soft shore during spring tides The female makes nests at a depth of 15

to 20 cm in the sand in the upper shore and releases eggs in the sand while the male

fertilizes the eggs by releasing the sperms After spawning the mating pair leaves the

shore and the waves wash the sand over the nest A range of 15000 to 64000 eggs will

be released per spawning season per female (Ehlinger and Tankersley 2003) After

fertilization the first instar will hatch in several weeks The juvenile horseshoe crabs

will spend their first ten years on the shore but when they become sexually mature they

will migrate to deep waters for the rest of their life During the first year the juveniles

molt several times with approximately 30 increase in body length for each molting

After three to four years juvenile horseshoe crabs molt only once a year usually in mid

summer while the increments of growth generally decrease during the life cycle

(Cierpich et al 2004) It is believed that the life span of horseshoe crab can be up to 20

to 24 years

13 Population Study on Horseshoe Crabs

As mentioned before there are four extant horseshoe crab species all over the

world The American L polyphemus is distributed along the eastern coast of North

America from Yucatan Peninsula (19degN) to Northern Maine (44degN) (Mikkelsen 1988)

5

with 90 of the populations aggregate along the mid-Atlantic coast of the Delaware

Bay (Shuster 2001) In Indo-Pacific waters three horseshoe crab species can be found

They are T tridentatus T gigas and C rotundicauda T tridentatus the ldquoJapaneserdquo

horseshoe crab is the only species found in Japanese waters and distributed along the

western coast of Japan the coast of China and south of the Philippines and Malaysia T

gigas the ldquoChineserdquo horseshoe crabs is distributed throughout South East Asia while

C rotundicauda the ldquoIndonesianrdquo horseshoe crab is distributed further to the west of

Indonesia Malaysia the Philippines Thailand and India (Chatterji 1994)

To study the life cycle and populations of this living fossil various baseline studies

have been undertaken for both the American and Indo-Pacific horseshoe crab species

For L polyphemus numerous studies on the population dynamics in the coastal areas

were carried out such as in Pleasant Bay Cape Cod (Carmichael et al 2003) New

Jersey (Botton and Haskin 1984 Botton et al 2003a) Jamaica Bay (Hanna 2001) and

Delaware Bay (Michels 1996 Smith et al 2002 Botton et al 2003a) It provides

comprehensive information on the population structure age-size relationship natural

mortality rate and environmental criteria for spawning and nursery grounds Population

studies on the three Indo-Pacific horseshoe crab species have also been investigated in

various countries including Philippines (Almendral and Schoppe 2005) Taiwan (Chen

and Yeh 2005) Singapore (Hong 2004) and Hong Kong (Morton and Lee 2003)

6

In Hong Kong horseshoe crabs including Tachypleus tridentatus T gigas and

Carcinoscorpius rotundicauda were first recorded in 1950rsquos However studies on

Hong Kong horseshoe crabs only started in 1997 by Professor Brian Morton Studies

including local population distribution study in open waters and various soft shores and

habitat characteristics study were conducted (Chiu and Morton 1999a) In Year 2002

Professor Morton further studied the spatial and temporal distribution of juvenile

horseshoe crabs and sediment physiography of the shores (Morton and Lee 2003)

Apart from the population distribution study behaviour growth and allometry study of

juvenile and adult horseshoe crabs in Hong Kong have also been undertaken (Chiu and

Morton 1999a Morton and Lee 2003)

14 Importance of Horseshoe Crabs

Horseshoe crabs have important values in the intertidal food web scientific and

medical studies and economics Horseshoe crabs especially L polyphemus are very

important to migratory birds including various protected species which stop along the

Delaware Bay in spring to fuel up for the flight north to Arctic nesting grounds (Carl

and Shuster 1982 Tanacredi 2001) Numerous studies have shown that a reduction in

the number of shorebirds such as Red Knot throughout the Delaware Bay is tied to a

decline in horseshoe crabs (Clark et al 1993 Botton et al 1994 Baker et al 2004

Haramis et al 2007 Sweka et al 2007) Meanwhile shorebirds are not the only

7

organisms depending on horseshoe crabs and their eggs as vital food resource

Horseshoe crab eggs and larvae are also food for invertebrates such as crabs

gastropods and fish including American eel killifish and silver perch (Carl and Shuster

1982 Chatterji 1994 Berkson and Shutter 1999) Juvenile and adult horseshoe crabs

are common prey for the shark and threatened sea turtles such as loggerhead (Carl and

Shuster 1982 Mikkelsen 1988 Berkson and Shuster 1999)

Horseshoe crabs have important values in the scientific and medical fields As

horseshoe crabs have changed very little in both morphology and functions in the

evolutionary history they serve as a very good model in the studies of aquatic

merostome fauna The simple compound eyes of horseshoe crabs are one of the most

popular research topics as the visual system of horseshoe crabs is an important model

system for the studies of basic visual processes and the role of efferent feedback and

circadian rhythms in vision (Behrens and Wulff 1965 Barlow et al 2001) The brain

architecture and anatomy of the central nervous system of L polyphemus have been

reported upon in recent years (Harzsch et al 2005 Hudson et al 2005) In addition as

juvenile horseshoe crabs spend their first ten years on the soft shore the levels of

contaminants in their bodies also serve as good bioindicators of the pollution levels of

shore sediments (Burger et al 2002)

8

L polyphemus is exploited as bait in the American eel and conch fisheries

(Berkson and Shutter 1999 Botton et al 2003b) In 1996 at least 2 million individuals

were collected for this purpose throughout the Atlantic Coast In addition a

multi-million dollars medical industry centres around the horseshoe crab blood Limulus

Amoebocyte Lysate (LAL) a chemical extracted from the horseshoe crab blood cells is

used for the detection of bacterial endotoxins in pharmaceutical products By reacting

with bacterial toxins in the sample solution LAL would clot and turn the solution into

milky colour (Loveland et al 1996 Mikkelsen 1988) The LAL test has been used to

assess food spoilage (such as fish milk and beef) air and water quality and determine

the ability of new drugs to neutralize the toxic effects of endotoxin

Besides LAL a number of reagents and medically useful compounds have been

discovered in the blood of horseshoe crabs For example a new test for fungal

infections (G-Test) is in use in Japan and an endotoxin-neutralizing protein extracted

from the horseshoe crab blood has a potential to be an antibiotic and used in an

alternative endotoxin assay A number of other proteins have also shown anti-viral and

anti-cancer activities Since the mid-1950s medical researchers have known that

chitin-coated suture material reduces healing time by 35-50 The chitin from

horseshoe crabs has been used in the manufacturing of chitin-coated filament for

suturing and chitin-coated wound dressing for burn victims (Hall 1992)

9

15 Decline in Horseshoe Crab Populations

In the past few decades horseshoe crabs have been facing the problem of

population decline all over the world According to spawning censuses trawl surveys

and anecdotal data the abundance of L polyphemus has dropped sharply by an order of

magnitude on the East coast of the USA including Delaware Bay (Michels 1996

Widener and Barlow 1999 Swan et al 1996 Ruth et al 2003 Rutecki et al 2004)

Annual horseshoe crab spawning data revealed that from 1991 to 1994 a decrease from

1225000 to 535000 individuals was observed in Delaware Bay (University of

Delaware 1996) The three Indo-Pacific species in Taiwan Japan Thailand Malaysia

and China have also decreased in numbers in recent years (Botton 2001 Itow 1998

Chen et al 2004) Earle (1991) even claimed that if no comprehensive conservation

measures are implemented all horseshoe crab species would not be able to survive this

century

All the 3 Indo-Pacific species in Hong Kong are facing the same problem

(Mikkelsen 1988 Chiu and Morton 1999a 2003a) Their populations have dropped

sharply in recent years with T gigas being disappeared 20 years ago (Chiu and Morton

1999b) while T tridentatus and C rotundicauda disappeared in Tolo Harbour 5 years

ago (Chiu and Morton 1999a 2003a)

10

16 Risks Facing by Horseshoe Crabs

Although the survival rate of juvenile horseshoe crabs particularly in the first year

is extremely low in the wild adult horseshoe crabs do not have major predators and thus

have a low natural mortality Instead human activities generally account for the greatest

mortality (Berkson and Shutter 1999 Rutecki et al 2004) The major threats are

human exploitation and habitat loss and degradation

As mentioned in Section 14 horseshoe crabs are of importance in various fields

hence heavily exploited L polyphemus is currently harvested for biomedical scientific

and fishing purposes (Berkson and Shutter 1999 Rutecki et al 2004) For the Asian

horseshoe crabs they are exploited for medicinal uses but a majority of them are

harvested as food South-east Asian residents especially the Fukien and Hokklo of

South China consider horseshoe crabs as a delicacy and highly nutritious food (Chiu

and Morton 2003a) In Hong Kong horseshoe crabs are sale in local seafood

restaurants such as Sai Kung Some cook books and magazines even teach people how

to cook these animals and advertise the high nutritional value and good taste of their

meat and the soup prepared from them (Cheung 1995) Therefore it is suspected that

human exploitation may be one of the major factors contributing to the decline of these

animals in Hong Kong (Chiu and Morton 1999a 2003a)

11

The habitat loss and deterioration of nursery grounds are also potential factors

causing the population decline of horseshoe crabs in Hong Kong In the past 15 years

various infrastructural and coastal developments have been carried out such as the

Western Corridor in northwestern New Territories and the New Airport on Lantau

Island while various big infrastructural developments are also in the planning stage

such as Logistic Park and Macau bridge on northern Lantau These developments are

adjacent to various horseshoe crab nursery grounds in northwestern New Territories

such as Sheung Pak Nai and Pak Nai and on Lantau Island including Tung Chung San

Tau and Tai O (Morton and Lee 2003) These developments have highly modified the

coastal configuration and environments and caused marine and coastal water pollution

which may affect the quality and condition of horseshoe crab nursery grounds in Hong

Kong

17 Conservation of Horseshoe Crabs

In view of the population decline conservation measures should be implemented

to preserve the horseshoe crab populations all over the world Hambler (2004) defined

conservation as the protection of wildlife from irreversible harm and the act or process

of conserving to either sustainably use preserve or restore the wildlife or natural

resources in which sustainable use refers to using resources in ways that do not deplete

them Preservation is protecting resources ecosystems and structures for present and

12

future generations while restoration is returning resources ecosystems and structures

to their original (or near-original) condition (National Geographic Society 2000)

To design a comprehensive conservation plan for local horseshoe crabs the degree

of human exploitation has to be investigated Besides the population distribution has to

be updated and key nursery grounds identified The information gathered is essential for

setting up legislation and protection areas to preserve this animal In view of the low

natural breeding and hatching success and low juvenile survival (Botton et al 2003a

Carmichael et al 2003) artificial insemination and incubation is one of the reasonable

options to restore and enhance the population of horseshoe crabs in the natural

environment in Hong Kong

18 Artificial Breeding of Horseshoe Crabs

The methods of artificial insemination and incubation are well established for L

polyphemus Numerous studies on the methodology of artificial insemination including

the collection of sperms and eggs from male and female horseshoe crabs respectively

(Brown and Clapper 1981 Smith and Berkson 2005) and on the optimal

environmental conditions (salinity and temperature) for in vitro fertilization and

incubation of the embryos larvae and juveniles have been conducted (Brown and

Clapper 1981 Laughlin 1983 Sekiguchi et al 1988) Nevertheless the understanding

13

of the interplay among environmental factors on the survival and hatching success of

the juveniles is essential but poorly known for Indo-Pacific species (Hong et al 2002

Li et al 1999 Wang et al 2001a) Therefore trials on artificial insemination and

incubation for Indo-Pacific horseshoe crabs are essential for the restoration of wild

populations in Hong Kong


Genetic analysis is commonly used nowadays in environmental and ecological

studies Phylogenetic analysis provides valuable information on the clarification of

evolutionary relationships among species with similar morphologies while population

genetics is commonly used to determine the geographical differences in different forms

and groups of a species in order to enhance the understanding of population distribution

and geographical range of the species Various genetic studies on horseshoe crabs have

been undertaken especially for L polyphemus Several sub-populations of L

polyphemus were determined by using different genetic analyses such as mitochondrial

DNA (Saunders et al 1986) Random Amplification of Polymorphic DNA (RAPD)

footprint and mitochondrial COI (Pierce et al 2000) and microsatellite DNA markers

(King and Eackles 2004) However few genetic studies have been carried out on the

Indo-Pacific species (Kato et al 2005 2006 Yang et al 2007) Population genetic

study is necessary in designing a good conservation and management plan for horseshoe

14

crabs through enhancing the understanding of the spawning pattern of adult and

migration of adult and juvenile

The three Indo-Pacific horseshoe crab species have similar morphologies

therefore it is difficult to distinguish them by external appearance especially in the

juvenile stage (Chiu and Morton 2003b) DNA footprint provides a more accurate way

for distinguishing these species Phylogenetic analysis of these species has been studied

However they constitute a phylogenetically unresolvable trichotomy in which T gigas

and T tridentatus are grouped together on the basis of morphological traits (Fisher

1984) whereas C rotundicauda and T tridentatus appear to be more closely related on

the basis of amino acid sequence divergence of a fibrinopeptide-like protein (Shishikura

et al 1982) and coagulogen (Srimal et al 1985) and through inter-specific

hybridization studies (Sekiguchi and Sugita 1980) In the study of Miyazaki et al

(1987) the similarity index between T gigas and C rotundicauda was the greatest for

the two- dimensional electrophoresis of cardiac muscles however for skeletal muscles

the greatest value was obtained between T tridentatus and T gigas Phylogenetic

analyses based on two partial mitochondrial genes 16S ribosomal RNA (rRNA) and

cytochrome oxidase subunit I (COI) also yielded conflicting topologies (Avise et al

1994)

15

In addition Morton and Lee (2003) discovered that some juvenile T tridentatus in

nursery grounds contained only 1 proanal spine in contrast to 3 in most of the

conspecifics It is suspected that these ldquoabnormalrdquo individuals may be caused by

mutation or are hybrids of T gigas and T tridentatus In-depth phylogenetic analysis of

the Indo-Pacific species should provide valuable information on the identification of

juvenile horseshoe crabs and evolutionary relationships among these species

110 Objectives

This thesis is divided into three main parts They include

1 ecological assessment of horseshoe crabs in Hong Kong which aims

a to provide most up-to-date information of the present status and distribution of

horseshoe crabs in Hong Kong and

b to assess the degree of human exploitation of horseshoe crabs in Hong Kong

2 conservation and population enhancement of horseshoe crabs in Hong Kong through

a trials on artificial insemination and breeding and

b determination of the optimal environmental conditions for enhancing the

survival and hatching success of horseshoe crab eggs

3 genetic study on horseshoe crab populations by examining

a the genetic differentiation between the two closely related species T

tridentatus and T gigas and

16

b geographical variations in population genetics of T tridentatus

111 Organization of the Thesis

This thesis comprises seven chapters Chapter 1 is a general review of the

background information and research objectives of the present study Chapter 2

investigates the distribution and abundance of horseshoe crabs in various nursery

grounds in Hong Kong The morphology and taxonomy of juvenile horseshoe crabs

recorded in Hong Kong are described in Chapter 3 with population genetic studies

among various nursery grounds in Hong Kong Chapter 4 investigates the degree of

human exploitation of horseshoe crabs in Hong Kong Chapter 5 presents the results of

trial studies on artificial insemination and laboratory rearing of the horseshoe crab

species Tachypleus tridentatus A general discussion and conclusion of the findings and

implications of the present study are provided in Chapter 6

17

Chapter 2 Survey of Horseshoe Crabs in Hong Kong

21 Introduction

Horseshoe crabs in Hong Kong have been facing the problem of population decline

in the past few decades (Mikkelsen 1988 Chiu and Morton 1999a 2003a) Tachypleus

gigas disappeared in Hong Kong 20 years ago (Chiu and Morton 1999b) while T

tridentatus and Carcinoscorpius rotundicauda could not be found in Tolo Harbour 5

years ago (Chiu and Morton 1999a 2003a) Hence a species-specific conservation plan

for local horseshoe crabs is urgently needed before it is too late

In the planning of a species-specific conservation strategy adequate knowledge of

the species including its ecology and population biology and the direct and indirect

threats to the species are essential However like many endangered species

information on horseshoe crabs in Hong Kong is very limited Therefore a

comprehensive survey of horseshoe crabs in Hong Kong is an initial and critical step

towards formulating the conservation plan

All the three Indo-Pacific species ie T tridentatus T gigas and C rotundicauda

were once recorded in Hong Kong (Mikkelsen 1988 Chiu and Morton 1999a 2003a)

Although T tridentatus and T gigas frequently appeared in early local ecological

books (Hill et al 1978 Hill and Phillipps 1981 Mikkelsen 1988) and were regarded

as the key species on clean sand-flats in Hong Kong (Morton amp Morton 1983) very

few scientific investigations and population studies on them have been undertaken

The first qualitative survey on local horseshoe crabs was conducted by Chiu and

Morton (1999b) from March 1995 to June 1998 to determine the occurrence of

18horseshoe crabs including adult and juvenile in territorial waters and local beaches in

Hong Kong In this survey nursery habitats for juvenile horseshoe crabs were

identified including mud flats of Deep Bay and various sandy beaches on Lantau

Island In 2002 another survey on juvenile horseshoe crabs was conducted in eight

mudflats along the Deep Bay including Northern Lau Fau Shan Sha Kong Temple

Sheung Pak Nai two locations at Long Chuk Hang (Pak Nai) two locations at Ha Pak

Nai and Nim Wan (Morton and Lee 2003) This comprehensive survey has provided

baseline information on the distribution and population density of the juvenile

horseshoe crabs and further confirmed the soft shores along Deep Bay as important

nursery grounds

The present study included a comprehensive survey which provided most

up-to-date information on the distribution and population density of juvenile horseshoe

crabs along Deep Bay in Northeastern New Territories as well as on Lantau Island and

Lamma Island In addition a six-month study was conducted at four important nursery

grounds to investigate the population dynamics of the juveniles and environmental

factors affecting their abundances

22 General Distribution and Abundance of Juvenile Horseshoe Crabs on Soft

Shores in Hong Kong

221 Materials and Methods

2211 Site Characteristics

A total of 17 Hong Kong soft shores were surveyed in this study (Fig 21)

including Tsim Bei Tsui Sheung Pak Nai Pak Nai and two locations at Ha Pak Nai in

northwestern New Territories and San Tau Shui Hau Wan Pui O Wan Tai Ho Wan

Sham Wat Yi O Tung Chung and Hau Hok Wan on Lantau Island where horseshoe

19crabs once were reported in abundance (Chiu and Morton 1999b) Survey sites in

northeastern waters and on Lamma Island included Luk Keng Lai Chi Wo Pak Kok

Wan and Sok Kwu Wan (Fig 21)

All the shores have sediments intermediate between mud and sand However five

sites in northwestern New Territories are located in eastern Deep Bay Region and

adjacent to the Pearl River Estuary with a large influx of freshwater (30826 billion msup3

yr-1) and sediment (3389 x 107 tons yr-1) (Morton and Lee 2003 Morton and Wu

1975) The sediments at these sites are relatively muddier The hydrography in this

region is also very different from that in the northeastern waters which is influenced by

oceanic currents (Morton and Morton 1983) The characteristics of each site are

described in Table 21

20

Figure 21 The 17 distribution study sites in New Territories on Lantau Island and on

Lamma Island

21

Table 21 Information on the 17 sampling sites in New Territories and on Lantau Island and Lamma Island in Hong Kong

Site Shore width (m)

Distance of 07m water line from the shore (m)

Total area of the site (msup2)

Average mud depth (cm)

Vegetation Human activities on the mudflat

Buildings on neighbouring land

Northwestern New Territories

Tsim Bei Tsui 500 250 125000 25-60 Fringed by mangrove trees Fishermens homes

Sheung Pak Nai 510 325 - 40-70 Fringed by mangrove trees Occasional oyster farming A food store and fishermens

homes

Pak Nai (Long Chuk Hang) 490 190 93100

0-25 (muddier at the northern side)

Mangrove trees (and a ship wreck) Intensive oyster farming A Thai temple and two fishing

sites

Ha Pak Nai (1) 170 105 17850 ~45 None None A Tin Hau Temple

Ha Pak Nai (2) 600 190 114000 0-45 (muddier at the northern side) Mangrove trees Occasional oyster farming

Ha Pak Nai village a Chinese temple and a garbage disposal site

Lantau Island

Pui O 935 58 54230 0-5 None None A restaurant and public swimming beach facilities

Shui Hau Wan 221 356 78676 0-5 Mangrove trees and grass

Occasional fishing at the times of spring low tides

Shui Hau village and a football court

San Tau 352 102 35904 0-30 Fringed by mangrove trees None San Tau village and a food

store Tai Ho Wan 223 77 17171 30-70 Mangrove trees None Tai Ho village

Sham Wat 210 100 21000 30-50 Mangrove trees Occasional fishing at the times of spring low tides

Fishermens homes and a food store

Yi O 460 854 39284 0-50 (muddier at the southern side)

Fringed by mangrove trees None None

22

Table 21 (Continued)







Tung Chung 275 100 27500 0-10 Mangrove trees None A football court and recreation camp site

Hau Hok Wan 155 50 7750 0-5 None None None

Northeastern New Territories

Luk Keng 430 213 91590 40-70 Fringed by mangrove trees None A Chinese temple

Lai Chi Wo 300 71 21300 0-50 (muddier at the northern side)

Fringed by mangrove trees None A village

Pak Kok Wan 93 40 3720 0-5 Mangrove trees None None

Lamma Island

Sok Kwu Wan 170 66 11220 0-5 None None A Tin Hau temple and seafood restaurants

232212 Sample Collection

The 13 sites in northwestern New Territories and on Lantau Island were surveyed

once in summer from August to early November 2004 and in winter from late

December 2004 to February 2005 For Luk Keng Pak Kok Wan and Sok Kwu Wan the

summer survey was conducted from August to October 2005 while that at Lai Chi Wo

was undertaken in June 2006 The winter distribution studies at the four sites were

conducted in January 2006

The survey method was based on Morton and Lee (2003) with modifications On

each shore 4 horizontal transects were set equally apart from 07 m to 16 m above

chart datum (CD) The length of each transect was similar to the width of the shore and

along each transect 5 quadrats (8 m times 8 m) were randomly selected for counting of

horseshoe crab juveniles (Fig 22) The total survey area on a shore was 1280 m2 The

number of individuals of each horseshoe crab species found on the sediment surface

within the quadrat was counted with the carapace length (prosomal width) measured

using vernier calipers For survey locations near freshwater streams the sampling was

further stratified by studying additional 20 random quadrats (05 m times 05 m) within the

stream area As horseshoe crabs may bury in the sediment and could not be seen on the

surface such sampling bias was assessed by using 20 random quadrats (2 m2 each) and

each quadrat was examined for horseshoe crabs by digging the sediment up to 5 cm

depth in the distribution study in northeastern New Territories and on Lamma Island in

2005-06

Apart from the random sampling method the population density of horseshoe

crabs was also obtained by walk-through sampling method On each shore two

researchers walked side-by-side along the entire 4 horizontal transects within a fixed

24time (3 hours ie time between low and high tides) All the individuals of horseshoe

crabs found were counted and measured in prosomal width The data were standardized

by calculating the population density per unit searching effort ie number of horseshoe

crabs hour-1 person-1

Environmental parameters including temperature salinity and dissolved oxygen of

the interstitial waters in each quadrat were monitored using a glass thermometer a

hand-held refractometer (Model ATAGO SMill-E) and an oxygen electrode (YSI

Model 58) respectively

25

Figure 22 Sampling design for the distribution survey of juvenile horseshoe crabs in

Hong Kong

262213 Statistical Analysis

As the population density data did not follow the normal distribution the effects of

site tidal level and season on the population density of horseshoe crabs were compared

using the non-parametric Kruskal-Wallis (KW) Test A Bonferroni adjustment was used

to correct for Type I error and the significance for each KW test was evaluated against

α = 005 divided by the number of comparisons being made Thus the significance level

for each KW test was p = 0017 For the hydrological parameters and the spatial and

temporal variations in the size of horseshoe crabs the 3-way ANOVA was used to

address the interacting effects among the sampling site tidal level and season When

significant differences among treatments were observed Tukey multiple comparison

tests were performed to determine the differences between site and tidal level with a

significance level of p = 005 All the statistical analyses were undertaken using the

software SPSS 110

222 Results

2221 Distribution and Abundance of Horseshoe Crabs

Table 21 summarizes the characteristics of the 17 survey shores Shores within

Deep Bay (Tsim Bei Tsui Sheung Pak Nai Pak Nai and Ha Pak Nai) Luk Keng in

northeastern New Territories and Tai Ho Wan and Sham Wat on Lantau Island have

deeper mud and are mostly fringed by mangrove plants with villages nearby Oyster

farming was found practicing on some of these shores

Juveniles of T tridentatus were only found on six shores in summer and one shore

in winter with a total number of 15 individuals (13 in summer and 2 in winter) in the

quadrats of the transect survey (Tables 22 and 23) and no C rotundicauda was found

Figure 23 shows the mean density of juvenile T tridentatus in the summer and winter

27surveys In the summer Tsim Bei Tsui and one location at Ha Pak Nai had the highest

mean density of 031 individual 100 m-2 and 023 individual 100 m-2 respectively

followed by another location at Ha Pak Nai (016 individual 100 m-2) and San Tau (016

individual 100 m-2) Pak Nai and Yi O had the lowest mean density of 008 individual

100 m-2 among the six shores In the winter horseshoe crabs were only found at one site

at Ha Pak Nai with an average density of 016 individual 100 m-2 However no

significant difference in density was found among shores (KW test H = 29811 p =

0019) (Table 25) For spatial variations within sites the density on the upper shore (16

m above CD) was higher than that on the lower shore (07 ndash 13 m above CD) in both

summer and winter (Table 24 and Fig 24) the differences however were not

statistically significant (KW test H = 1625 p = 0654) (Table 25) From the limited

data obtained from the random surveys juvenile horseshoe crabs tended to distribute

patchily at the study sites There was no significant difference (KW test H = 4554 p =

0033) in the distribution of horseshoe crabs between the two sampling seasons The

mean density was 0056 individual 100 m-2 and 0009 individual 100 m-2 in summer and

winter respectively (Table 24) Horseshoe crabs were neither found in stream areas nor

by the digging method in the distribution study in the northeastern New Territories and

on Lamma Island

28

Table 22 Total number and density of T tridentatus and various environmental parameters of the 17 sampling sites in New Territories and on Lantau

Island and Lamma Island in Hong Kong in the summer distribution study Survey months are in the brackets

Summer

No of individuals

Density by random sampling (individual 100 m-2) Temperature (degC) Salinity (permil) Dissolved oxygen (mg l-1)

Northwestern New Territories Tsim Bei Tsui (Sep 04) 4 031 2873 2580 504 Sheung Nak Nai (Sep 04) 0 000 2950 2450 - Pak Nai (Long Chuk Hang) (Sep 04) 1 008 - 2757 -

Ha Pak Nai (1) (Sep 04) 2 016 2908 2438 438 Ha Pak Nai (2) (Sep 04) 3 023 - 2482 - Lantau Island Pui O (Oct 04) 0 000 2965 3150 763 Shui Hau Wan (Oct 04) 0 000 3337 2159 456 San Tau (Oct 04) 2 016 2901 3088 798 Tai Ho Wan (Oct 04) 0 000 2640 2689 532 Sham Wat (Nov 04) 0 000 2839 3155 607 Yi O (Nov 04) 1 008 2631 2600 831 Tung Chung (Nov 04) 0 000 2863 3100 - Hau Hok Wan (Nov 04) 0 000 2740 2767 686 - = no measurement due to the absence of interstitial waters in sediment

29

Table 22 (continued)

No of individuals


Northeastern New Territories Luk Keng (Oct 04) 0 000 3303 1753 630 Lai Chi Wo (Jun 05) 0 000 3145 1911 523 Pak Kok Wan (Aug 04) 0 000 2920 2000 475 Lamma Island Sok Kwu Wan (Sep 04) 0 0000 3272 1544 691 - = no measurement due to the absence of interstitial waters in sediment

30


Island and Lamma Island in Hong Kong in the winter distribution study Survey months are in brackets

Winter

No of individuals


Northwestern New Territories Tsim Bei Tsui (Dec 04) 0 000 1836 3145 - Sheung Nak Nai (Dec 04) 0 000 1794 2800 511 Pak Nai (Long Chuk Hang) (Dec 04) 0 000 1010 4000 1012

Ha Pak Nai (1) (Dec 04) 2 016 2067 3365 846 Ha Pak Nai (2) (Dec 04) 0 000 1010 3700 1011 Lantau Island Pui O (Jan 05) 0 000 2027 2524 770 Shui Hau Wan (Jan 05) 0 000 1305 3583 794 San Tau (Jan 05) 0 000 1280 4325 895 Tai Ho Wan (Jan 05) 0 000 1372 3933 687 Sham Wat (Feb 05) 0 000 1552 3718 837 Yi O (Feb 05) 0 000 1760 1000 769 Tung Chung (Feb 05) 0 000 - - - Hau Hok Wan (Feb 05) 0 000 2270 3900 809 - = no measurement due to the absence of interstitial waters in sediment

31


No of individuals


Northeastern New Territories Luk Keng (Jan 06) 0 000 1830 3057 734 Lai Chi Wo (Jan 06) 0 000 1120 2257 778 Pak Kok Wan (Jan 06) 0 000 1130 2775 726 Lamma Island Sok Kwu Wan (Jan 06) 0 000 2185 2313 729 - = no measurement due to the absence of interstitial waters in sediment

32

Table 24 Total number of T tridentatus at various tidal levels in New Territories and on Lantau Island and Lamma Island in Hong Kong

Summer Winter

1st Transect 2nd Transect 3rd Transect 4th Transect 1st Transect 2nd Transect 3rd Transect 4th Transect

16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD

Northwestern New Territories Tsim Bei Tsui 4 0 0 0 0 0 0 0 Sheung Nak Nai 0 0 0 0 0 0 0 0

Pak Nai (Long Chuk Hang) 0 0 1 0 0 0 0 0

Ha Pak Nai (1) 0 2 0 0 0 1 1 0 Ha Pak Nai (2) 1 0 2 0 0 0 0 0 Lantau Island Pui O 0 0 0 0 0 0 0 0 Shui Hau Wan 0 0 0 0 0 0 0 0 San Tau 2 0 0 0 0 0 0 0 Tai Ho Wan 0 0 0 0 0 0 0 0 Sham Wat 0 0 0 0 0 0 0 0 Yi O 0 0 0 1 0 0 0 0 Tung Chung 0 0 0 0 0 0 0 0 Hau Hok Wan 0 0 0 0 0 0 0 0

33


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD

Northeastern New Territories Luk Keng 0 0 0 0 0 0 0 0 Lai Chi Wo 0 0 0 0 0 0 0 0 Pak Kok Wan 0 0 0 0 0 0 0 0 Lamma Island Sok Kwu Wan 0 0 0 0 0 0 0 0

34

000

010

020

030

040

050

060

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)

Summer Winter

Figure 23 Mean density (+SE) of juvenile T tridentatus recorded by the random

quadrat sampling

35

000

005

010

015

020

025

1st Transect16m above CD

2nd Transect13m above CD

3rd Transect10m above CD

4th Transect07m above CD

Tidal level

Popo

ulat

ion

Den

sity

(ind

100

msup2)

Summer Winter

Figure 24 Mean density (+SE) of juvenile T tridentatus found at the four tidal levels

recorded by the random quadrat sampling

36Table 25 Results of the Kruskal-Wallis test for the differences in the population

density of juvenile T tridentatus at the 17 sites in the summer and winter The

Bonferroni adjustment was used (p lt 0017)

Kruskal Wallis Test on Population Density Chi-Square df p-value Between Site 29811 16 0019 Between Season 4554 1 0033 Between Tidal Level 1625 3 0654

37For the walk-through survey among these 17 shores juvenile T tridentatus were

found on nine shores in summer and two shores in winter including Tsim Bei Tsui Pak

Nai and two locations at Ha Pak Nai in Deep Bay Shui Hau Wan San Tau Sham Wat

Yi O and Tung Chung on Lantau Island However no T tridentatus was found in

northeastern New Territories and on Lamma Island (Tables 26) C rotundicauda was

found on 10 shores in summer and one shore in winter including Tsim Bei Tsui

Sheung Pak Nai Pak Nai and two locations at Ha Pak Nai in Deep Bay Tai Ho Wan

Yi O and Tung Chung on Lantau Island Luk Keng and Lai Chi Wo in northeastern

New Territories (Table 27) Figure 25 shows the distribution of juvenile T tridentatus

and C rotundicauda recorded by the walk-through survey

More individuals of T tridentatus were collected by the walk-through survey than

the random quadrat sampling both in summer (43 vs 13) and winter (7 vs 2) (Table 26)

This indicated that the walk-through survey is a better sampling method for horseshoe

crabs occurring at low densities Similar to the random sampling method higher

densities of horseshoe crabs were found at Tsim Bei Tsui Ha Pak Nai (2) San Tau and

Pak Nai However no individual was found at Shui Hau Wan by the random sampling

but eight individuals were found by the walk-through survey in the summer study

(Table 26 and Fig 25)

No individual of C rotundicauda was found using the random quadrat sampling

but 30 and 4 individuals were found in the summer and winter respectively using the

walk-through survey (Table 27) with highest densities being found at Tsim Bei Tsui

and Pak Nai Only C rotundicauda was found in northeastern New Territories

including Luk Keng and Lai Chi Wo (Table 27 and Fig 25)

38Table 26 Abundance and population density (individual hour-1 person-1) of juvenile T

tridentatus obtained by the walk-through survey at the 17 study sites in the summer and

winter

Summer Winter

Total no of

T tridentatusDensity (individual

hour-1 person-1) Total no of

T tridentatus

Density (individual hour-1

person-1)

Northwestern New Territories Tsim Bei Tsui 4 067 0 000 Sheung Pak Nai 0 000 0 000 Pak Nai 10 167 0 000 Ha Pak Nai (1) 1 017 4 067 Ha Pak Nai (2) 10 167 0 000 Lantau Island Pui O 0 000 0 000 Shui Hau Wan 8 133 0 000 San Tau 10 167 0 000 Tai Ho Wan 0 000 0 000 Sham Wat 2 033 0 000 Yi O 2 033 3 050 Tung Chung 1 017 0 000 Hau Hok Wan 0 000 0 000 Northeastern New Territories Luk Keng 0 000 0 000 Lai Chi Wo 0 000 0 000 Pak Kok Wan 0 000 0 000 Lamma Island Sok Kwu Wan 0 000 0 000

39Table 27 Abundance and population density (individual hour-1 person-1) of juvenile C

rotundicauda at the 17 study sites in the summer and winter

Summer Winter

Total no of

C rotundicauda

Density (individual

hour-1 person-1)

Total no of C rotundicauda

Density (individual

hour-1 person-1)


40

Figure 25 Population distribution of the horseshoe crabs at the 17 sites by the

walk-through survey

412222 Environmental Parameters of the Study Sites

Temperature

Temporal and spatial variations in temperature at the 17 study sites in two seasons

are illustrated in Figure 26 A mean sediment temperature of 294 plusmn 22 degC in the

summer and 170 plusmn 36 degC in the winter was obtained for all the sites (Fig 26)

Temperature varied significantly among site (F 16 156 = 83392 p lt 0001) tidal level (F

3 156 = 8558 p lt 0001) and season (F 1 156 = 5187483 p lt 0001) (Table 28) All

interactions among site tidal level and season also had significant effects on

temperature variations

42

0

5

10

15

20

25

30

35

40

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Tem

pera

ture

(degC

)

Summer Winter

Figure 26 Temporal variations in temperature (plusmn SD) at the 17 sites in the summer and

winter

43Table 28 Results of the 3-way ANOVA test for the differences in temperature at the

17 sites in the summer and winter (tidal level times site times season) (p lt 005) The Tukey

multiple comparison test was used to compare the differences between tidal levels

3-way ANOVA on Temperature

F df p-value Observed power

Between Tidal Level 8558 3 lt 0001 0993 Between Site 83392 16 lt 0001 1000 Between Season 5187483 1 lt 0001 1000 Interaction Between Tidal Level and Site 4093 29 lt 0001 1000

Interaction Between Tidal Level and Season 17651 3 lt 0001 1000

Interaction Between Site and Season 91428 10 lt 0001 1000

Interaction Between Tidal Level Site and Season 4576 12 lt 0001 1000

Within Error 156 Tukey Test for Tidal Level Comparison Mean Difference p-value Tidal Level 1 vs Tidal Level 2 08921 lt 0001 Tidal Level 1 vs Tidal Level 3 03517 0046 Tidal Level 1 vs Tidal Level 4 07598 lt 0001 Tidal Level 2 vs Tidal Level 3 -05405 0001 Tidal Level 2 vs Tidal Level 4 -01323 0781 Tidal Level 3 vs Tidal Level 4 04082 0013 Significant at p lt 005

44Salinity of Interstitial Water

Salinity varied significantly with site (F 16 203 = 18854 p lt 0001) tidal level (F 3

203 = 5728 p = 0001) and season (F 1 203 = 11747 p lt 0001) as tested by 3-way

ANOVA (Table 29) The interactions among site tidal level and season also

significantly affected the salinity except the interaction between tidal level and season

(F 3 203 = 1186 p = 0316) and the interaction among tidal level site and season (F 20

203 = 1233 p = 0230) An average salinity of 251 plusmn 72permil was obtained in the summer

and 325 plusmn 66permil in the winter among the sites (Fig 27) Sites on the northern Lantau

Island including San Tau Hau Hok Wan and Tai Ho Wan were more saline than the

other sites The lowest salinity was obtained at Yi O on the southern Lantau Island

45

00

50

100

150

200

250

300

350

400

450

500

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Salin

ity o

f Int

erst

itial

Wat

ers

(permil)

Summer Winter

Figure 27 Temporal variations in salinity of the interstitial waters (plusmn SD) at the 17

sites in the summer and winter

46Table 29 Results of the 3-way ANOVA test for the differences in salinity at the 17

sites in the summer and winter (tidal level times site times season) (p lt 005) The differences

between tidal levels were compared using the Tukey test

3-way ANOVA on Salinity


Between Tidal Level 5728 3 0001 0946 Between Site 18854 16 lt0001 1000 Between Season 117470 1 lt0001 1000 Interaction Between Tidal Level and Site 2769 40 lt0001 1000

Interaction Between Tidal Level and Season 1186 3 0316 0316

Interaction Between Site and Season 3115 14 lt0001 0997

Interaction Between Tidal Level Site and Season 1233 20 0230 0844

Within Error 203 Tukey Test for Tidal Level Comparison Mean Difference p-value Tidal Level 1 vs Tidal Level 2 -24905 0002 Tidal Level 1 vs Tidal Level 3 -17998 0047 Tidal Level 1 vs Tidal Level 4 -25450 0002 Tidal Level 2 vs Tidal Level 3 06907 0752 Tidal Level 2 vs Tidal Level 4 00054 1000 Tidal Level 3 vs Tidal Level 4 -07452 0703 Significant at p lt 005

47Interstitial Water Dissolved Oxygen Level

The dissolved oxygen (DO) level in the sediment varied significantly with site (F 15

170 = 9241 p lt 0001) and season (F 1 170 = 69180 p lt 0001) but not tidal level (F 3 170

= 0471 p = 0703) (Table 210) The interaction between site and season was also

significant (F 12 170 = 6672 p lt 0001) An average DO value of 60 plusmn 17 mg l-1 and 77

plusmn 14 mg l-1 was obtained in the summer and winter among the sites respectively (Fig

28) Pak Nai and two locations at Ha Pak Nai were better aerated than Tsim Bei Tsui

and Sheung Pak Nai in northwestern New Territories while all the sites on Lantau

Island and in northeastern New Territories had an average DO concentration of 77 plusmn

08 mg l-1 and 64 plusmn 04 mg l-1 respectively (Fig 28)

48

0

2

4

6

8

10

12

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Dis

solv

ed O

xyge

n C

oncc

entr

atio

n (m

gl) Summer Winter

Figure 28 Temporal variations in the DO level (plusmn SD) of the interstitial waters at the

17 sites in the summer and winter

49Table 210 Results of the 3-way ANOVA test for differences in the DO level at the 17

sites in the summer and winter (tidal level times site times season) (p lt 005)

3-way ANOVA on DO Level


Between Tidal Level 0471 3 0703 0144 Between Site 9241 15 lt0001 1000 Between Season 69180 1 lt0001 1000 Interaction Between Tidal Level and Site 1301 37 0134 0969



Interaction Between Tidal Level Site and Season

0649 15 0831 0414

Within Error 170 Significant at p lt 005

502223 Size of the Horseshoe Crabs on the Shores

Figure 29 shows the mean carapace length (prosomal width) of juvenile T

tridentatus obtained from the walk-through survey The mean carapace length varied

from 76 to 960 mm According to the size-age relationship established by Sekiguchi

(1988a) these corresponded to an age of four to eight years old Horseshoe crabs at

Tsim Bei Tsui Shui Hau Wan and Yi O had the highest average prosomal length of ~56

mm whereas those at Tung Chung had the lowest average prosomal length (266 mm)

(Fig 29) The size of juvenile T tridentatus did not vary significantly with site (F 8 35 =

1721 p = 0128) season (F 1 35 = 0265 p = 061) tidal level (F 3 35 = 1048 p = 0383)

and their interactions (p gt 005) (Table 211) The size of T tridentatus at different tidal

levels is shown in Figure 210 The largest T tridentatus was collected at 13 m above

CD and the smallest at 07 m above CD the differences among tidal levels however

were not statistically significant (Table 211)

Figure 211 shows the prosomal width of C rotundicauda at different sites and

ranged from 259 mm at Ha Pak Nai (1) to 895 mm at Lai Chi Wo The size of

horseshoe crabs did not vary significantly with tidal level (F 3 18 = 292 p = 0062)

season (F 1 18 = 14307 p = 0932) and their interaction (F 1 18 = 0119 p = 0734) but

varied significantly with site (F 9 18 = 14307 p lt 0001) (Table 212) The largest

horseshoe crabs were collected from 10 m above CD and the smallest at 07 m above

CD (Fig 212) the differences among tidal levels however were not statistically

significant (Table 212)

51

00

10

20

30

40

50

60

70

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter

Figure 29 Mean (+SE) prosomal width of juvenile T tridentatus recorded by the

walk-through survey at the 17 sites in the summer and winter

52Table 211 3-way ANOVA (tidal level site and season) results on the prosomal width

of juvenile T tridentatus recorded by the walk-through survey (p lt 005)

3-way ANOVA on Prosomal Width

Factor F df p-value Observed power

Between Tidal Level 1048 3 0383 0259 Between Site 1721 8 0128 0646 Between Season 0265 1 0610 0079 Interaction Between Tidal Level and Site 0616 5 0689 0199

Interaction Between Site and Season 0031 1 0861 0053


53

00

10

20

30

40

50

60





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)

Figure 210 Mean (+SE) prosomal length of juvenile T tridentatus found at the four

tidal levels (Number of individuals shown in the brackets)

(12)

(2)

(26)

(14)

54

00102030405060708090

100

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter

Figure 211 Mean (+SE) prosomal width of juvenile C rotundicauda recorded by the



of juvenile C rotundicauda recorded by the walkndashthrough survey (p lt 005)





56

00

20

40

60

80

100

120





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)

Figure 212 Mean (+SE) prosomal length of juvenile C rotundicauda found at the four

tidal levels recorded by the walk-through survey (Number of individuals shown in the

brackets)

(11)

(3)

(2)

(18)

5723 Temporal Variations in the Population Density of Horseshoe Crabs at Four

Nursery Grounds



Pak Nai and Ha Pak Nai in northwestern New Territories and San Tau and Shui

Hau Wan on Lantau Island were selected for the monthly distribution survey (Fig 25)

These sites were key nursery grounds for juvenile horseshoe crabs and had the highest

abundance among the 17 study sites The detailed descriptions of the four sites are

shown in Table 21



monthly in the summer from March to August 2005 The sampling strategy for the

study was similar to that described in Section 22 On each shore 4 horizontal transects

were set equally apart from 07 m to 16 m above CD with five quadrats (8 m times 8 m)

being randomly selected at each tidal level (Fig 22) The total survey area on a shore

was 1280 m2 The number of individuals of each horseshoe crab species found on the

sediment surface in the quadrat was counted The prosomal width of all the horseshoe

crabs found along the transects was measured using vernier calipers The population

density of horseshoe crabs was also obtained by the walk-through survey On each

shore two researchers walked side-by-side along the entire 4 horizontal transects within

a fixed time (3 hours ie time between low and high tides) All the horseshoe crabs

found were counted and measured in prosomal width The data were normalized by

calculating the population density per unit effort ie number of individual hour-1

person-1 However owing to the bad weather with heavy showers in June and the

58logistics problems of field arrangements the distribution study at Shui Hau Wan could

not be conducted

The temperature salinity and DO of the interstitial waters in each quadrat were

monitored using a glass thermometer a hand-held refractometer (Model ATAGO

SMill-E) and an oxygen electrode (YSI Model 58) respectively


As the population density and size distribution of horseshoe crabs did not follow

the normal distribution differences among the study sites were examined using the

non-parametric Kruskal-Wallis (KW) test to assess the differences among site tidal

level and sampling month A Bonferroni adjustment was used to correct for Type I

error and the significance level for each KW test was evaluated against α = 005

divided by the number of comparisons being made Thus the significance level for each

KW test was set at 0017 If a significant difference was obtained among sites or tidal

levels a series of non-parametric 2-way Mann-Whitney (U) multiple comparisons tests

were computed A Bonferroni adjustment was used and the significance level for each

U test was evaluated against α = 005 divided by the number of comparisons being

made Thus the significance level for each U test was 00083 A series of nonparametric

paired Wilcoxon (Z) tests was also used if there was a significant effect of time A

Bonferroni adjustment was used and the significance level for each Z test was

evaluated against α = 005 divided by the number of comparisons being made The

significance level for each Z test was 00033

Interactions among site tidal level and season on hydrological parameters

(temperature salinity and DO of the interstitial waters) were examined by the 3-way

59ANOVA with p = 005 All the statistical analyses were undertaken using SPSS 110

For the analysis of population structure of juvenile horseshoe crabs length-frequency

data were grouped into size classes of 10 mm intervals and the percentage frequency of

the whole sample contributed by each size class was plotted as a length-frequency

histogram In order to follow the growth of separate cohorts over the sampling period

individual cohorts (size classes) were separated using the modal progression analysis of

Fish Stock Assessment Tool II (FiSAT II httpwwwfaoorgfistatistfisoftfisat)

FiSAT II applies the maximum likelihood concept to separate the normally distributed

components of size-frequency samples allowing accurate demarcation of the

component cohorts from the composite polymodal population size frequency

distribution For each cohort identified mean lengths with standard deviations group

sizes (in numbers) and separation index were presented in the results of FiSAT II

analysis

232 Results


Spatial Distributions among Sites and Tidal Levels

Among these shores 31 juvenile horseshoe crabs were found using the random

quadrat sampling with 25 individuals belonging to T tridentatus and six to C

rotundicauda Figure 213 shows the mean density of juvenile T tridentatus recorded at

each site from March to August 2005 Pak Nai had the highest average density of T

tridentatus (014 individual 100 m-2) followed by Ha Pak Nai (013 individual 100 m-2)

and Shui Hau Wan (0047 individual 100 m-2) San Tau had the lowest mean density of

0013 individual 100 m-2 However the difference in mean density among sites was

statistically insignificant (KW test H = 8639 p = 0034) (Table 213) There was a

significant difference (KW test H = 1119 p = 0011) in the distribution of horseshoe

60crabs at different tidal levels with more individuals of T tridentatus on the upper shore

(an average of 012 and 016 individual 100 m-2 at 16 and 13 m above CD

respectively) than on the lower shore (an average of 0054 individual 100 m-2 at 10 m

above CD and none at 07 m above CD) (Fig 214 Table 213)

C rotundicauda was recorded only at Ha Pak Nai and San Tau by the random

sampling method with most of them (five out of six individuals) being found at 16 m

above CD at San Tau whereas the only individual found at Ha Pak Nai was located at

07 m above CD In view of the limited number of individuals being recorded no

statistical analysis of the spatial pattern was undertaken

Temporal Distributions

Significant temporal variations in the abundance of T tridentatus was found (KW

test H = 19683 p = 0001) with highest density being recorded in May and June (021

individual 100 m-2) Less than 006 individual 100 m-2 of T tridentatus was found in

July August and April and no horseshoe crab was found in March by the random

sampling method (Fig 213) Temporal variations in the abundance however were

statistically insignificant owing to large variance of the data (Table 213)

C rotundicauda was recorded only from April to June with three individuals being

found in April two in June and one in May No statistical analysis was performed

because of the small number of individuals

61

000

020

040

060

080

100

120

140

Mar Apr May Jun Jul AugMonth (2005)

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)

Pak NaiHa Pak NaiShui Hau WanSan Tau

Figure 213 Mean density of juvenile T tridentatus at the four sites recorded by the

random sampling method from March to August 2005

62

000

010

020

030

040

050

060

070





Tidal Level

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)


Figure 214 Spatial distribution (tidal levels) (+SE) of juvenile T tridentatus at the

four sites recorded by the random sampling method from March to August 2005

63Table 213 Results of the Kruskal-Wallis test for differences in the population density

of T tridentatus at the 4 sites from March to August 2005 The Bonferroni adjustment

was used (p lt 0017) Pairwise comparisons of time effect were tested by Wilcoxon (Z)

test with Bonferroni adjustment (p lt 00033) Mann-Whitney (U) test was used to test

for the differences among tidal levels with Bonferroni adjustment (p lt 00083)

Kruskal Wallis Test on Population Density Chi-Square df p-value Between Site 8639 3 0034 Between Time 19683 5 0001 Between Tidal Level 1119 3 0011 Wilcoxon Test for Time Comparison Z p-value March vs April -1000 0317 March vs May -2264 0024 March vs June -2828 0005 March vs July -1414 0157 March vs August -1732 0083 April vs May -1930 0054 April vs June -2333 0020 April vs July -0577 0564 April vs August -1000 0317 May vs June -0034 0973 May vs July -1611 0107 May vs August -1310 0190 June vs July -1897 0058 June vs August -1890 0059 July vs August -0447 0655 Mann-Whitney Test for Tidal Levels Comparison U p-value Tidal level 1 vs tidal level 2 65595 0817 Tidal level 1 vs tidal level 3 62720 0080 Tidal level 1 vs tidal level 4 60950 0002 Tidal level 2 vs tidal level 3 63270 0126 Tidal level 2 vs tidal level 4 61525 0004 Tidal level 3 vs tidal level 4 64400 0082 Significant at p lt 0017 for the Kruskal Wallis test p lt 00033 for the

Wilcoxon (Z) test or p lt 00083 for Mann-Whitney test

64Population density of T tridentatus and C rotundicauda obtained by the

walk-through survey is shown in Table 214 A total of 879 individuals of T tridentatus

and 67 individuals of C rotundicauda were obtained from March to August 2005 The

highest mean density was obtained in April at Shui Hau Wan (2833 individual hour-1

person-1) for T tridentatus and in August at San Tau (11 individual hour-1 person-1) for

C rotundicauda Population densities of T tridentatus and C rotundicauda at different

tidal levels are shown in Figures 215 and 216 respectively For both T tridentatus and

C rotundicauda most of the individuals were collected at the uppermost part of the

shore ie 16 m above CD

65

Table 214 Abundance (total number of individuals) and population density (individual hour-1 person-1) of juvenile T tridentatus and C rotundicauda

recorded by the walk-through survey at the four sites from March to August 2005

Pak Nai Ha Pak Nai Shui Hau Wan San Tau Tachypleus tridentatus Month Abundance Density Abundance Density Abundance Density Abundance Density

March 4 133 2 067 0 000 0 000 April 28 933 33 1100 85 2833 1 033 May 37 1233 13 433 33 1100 8 267 June 16 533 46 1533 - - 1 033 July 8 267 32 1067 63 2100 3 100 August 15 500 38 1267 3 100 3 100 Carcinoscorpius rotundicauda Month Abundance Density Abundance Density Abundance Density Abundance Density

March 0 000 0 000 0 000 2 067 April 0 000 1 033 0 000 6 200 May 0 000 0 000 0 000 3 100 June 0 000 1 033 0 000 14 467 July 1 033 0 000 0 000 6 200 August 0 000 0 000 0 000 33 1100

66

0

2

4

6

8

10

12

14

16

18





Tidal level (upper = 1)

Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)



four sites recorded by the walk-through survey from March to August 2005

67

0

05

1

15

2

25

3

35

4

45






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)


Figure 216 Spatial distribution (tidal levels) (+SE) of juvenile C rotundicauda at the


682322 Environmental Parameters of the Shores

Temperature

Temperature varied significantly with site (F 3 83 = 131755 p lt 0001) tidal level

(F 3 83 = 32173 p lt 0001) and time (F 5 83 = 85718 p lt 0001) Interactions among

site tidal level and time were also significant (Table 215) Temperature increased

significantly from March with an average temperature of 206 plusmn 43 degC to a maximum

in July (352 plusmn 25 degC) and then decreased in August (331 plusmn 19degC) (Fig 217 Table

215)

69

0

5

10

15

20

25

30

35

40

Mar Apr May Jun Jul Aug

Month (2005)

Tem

pera

ture

(degC

)


Figure 217 Temporal variations in average temperature (plusmn SD) at the four sites from


70Table 215 Results of the 3-way ANOVA test for differences in temperature at the four

sites from March to August 2005 (tidal level times site times time) (plt005) The differences

among tidal level site and time were tested by the Tukey multiple comparison test

(plt005)



Between Tidal Level 32173 3 lt0001 1000 Between Site 131755 3 lt0001 1000 Between Time 85718 5 lt0001 1000 Interaction Between Tidal Level and Site 12249 9 lt0001 1000

Interaction Between Tidal Level and Time 2622 15 0003 0984

Interaction Between Site and Time 45765 13 lt0001 1000

Interaction Between Tidal Level Site and Time 4108 9 lt0001 0994

Within Error 83 Tukey Test for Tidal Level Comparison Mean difference p-value Tidal level 1 vs tidal level 2 03806 0928 Tidal level 1 vs tidal level 3 50751 lt0001 Tidal level 1 vs tidal level 4 21006 0011 Tidal level 2 vs tidal level 3 46945 lt0001 Tidal level 2 vs tidal level 4 17200 0079 Tidal level 3 vs tidal level 4 -29745 0001 Tukey Test for Site Comparison Mean difference p-value Pak Nai vs Ha Pak Nai 25539 lt0001 Pak Nai vs Shui Hau Wan 156950 lt0001 Pak Nai vs San Tau 183936 lt0001 Ha Pak Nai vs Shui Hau Wan 131411 lt0001 Ha Pak Nai vs San Tau 158396 lt0001 Shui Hau Wan vs San Tau 26986 0022

Significant at p lt 005 for ANOVA and Tukey Test

71Table 215 (Continued)

Tukey Test for Time Comparison Mean difference p-value March vs April 12957 0819 March vs S May 59879 lt0001 March vs June -47886 lt0001 March vs July -146545 lt0001 March vs August -124483 lt0001 April vs May 46922 lt0001 April vs June -60842 lt0001 April vs July -159502 lt0001 April vs August -137439 lt0001 May vs June -107765 lt0001 May vs July -206424 lt0001 May vs August -184362 lt0001 June vs July -98660 lt0001 June vs August -76597 lt0001 July vs August 22063 0228 Significant at p lt 005 for ANOVA and Tukey tests


There were significant differences in salinity among sites (F 5 90 = 7314 p lt 0001)

and times (F 3 90 = 49409 p lt 0001) but not tidal levels (F 3 90 = 081 p = 0492) (Fig

218 Table 216) The interaction between site and time was also significant (F 14 90 =

8171 p lt 0001) Salinity decreased significantly from March to July with an average

salinity of lt 4permil in June and July (Fig 218 Table 216) and was mainly due to the

rainy weather in the mid summer as the freshwater diluted the saline interstitial water on

the shores Salinity increased up to 157 plusmn 72permil in August

73

-5

0

5

10

15

20

25

30

35

40


Month (2005)

Salin

ity o

f Int

erst

itial

Wat

ers

(permil) Pak Nai

Ha Pak Nai

Shui Hau Wan

San Tau

Figure 218 Temporal variations in the average interstitial water salinity (plusmn SD) at the

four sites from March to August 2005

74Table 216 Results of the 3-way ANOVA test for differences in salinity at the four sites

from March to August 2005 (tidal level times site times time) (p lt 005) The Tukey test was

used to compare the differences among site and time (p lt 005)

3-way ANOVA on Salinity F df p-value Observed power

Between Tidal Level 0810 3 0492 0219 Between Site 7314 3 lt0001 0981 Between Time 49409 5 lt0001 1000 Interaction Between Tidal Level and Site 0797 9 0620 0371



Interaction Between Tidal Level Site and Time 0616 12 0824 0329

Within Error 90 Tukey Test for Site Comparison Mean difference p-value Pak Nai vs Ha Pak Nai 34444 lt0001 Pak Nai vs Shui Hau Wan 50000 lt0001 Pak Nai vs San Tau 37971 0001 Ha Pak Nai vs Shui Hau Wan 15556 0525 Ha Pak Nai vs San Tau 03527 0984 Shui Hau Wan vs San Tau -12029 0790 Tukey Test for Time Comparison Mean difference p-value March vs April 93391 lt0001 March vs May 213576 lt0001 March vs June 233647 lt0001 March vs July 248609 lt0001 March vs August 114621 lt0001 April vs May 120184 lt0001 April vs June 140256 lt0001 April vs July 155217 lt0001 April vs August 21229 0382 May vs June 20071 0296 May vs July 35033 0017 May vs August -98955 lt0001 June vs July 14962 0716 June vs August -119026 lt0001 July vs August -133988 lt0001 Significant at p lt 005 for ANOVA and Tukey Test

75Interstitial Water DO Level

DO level in the sediments varied significantly with site (F 3 74 = 17559 p lt 0001)

and time (F 5 74 = 6203 p lt 0001) but not tidal level (F 3 74 = 0579 p = 0631) (Fig

219 Table 217) Interactions among site tidal level and time were also significant (p lt

005)

76

0

2

4

6

8

10

12

14


Dis

solv

ed O

xyge

n C

once

ntra

tion

(mg

l) Pak NaiHa Pak NaiShui Hau WanSan Tau

Figure 219 Temporal variations in the average DO level (plusmn SD) of the interstitial

water at the four sites from March to August 2005

77Table 217 Results of the 3-way ANOVA test for differences in DO level at the four

sites from March to August 2005 (tidal level times site times time) (p lt 005) The Tukey test

was used to compare the differences among site and time (p lt 005)

3-way ANOVA on Dissolved Oxygen F df p-value Observed power





Within Error 74 Tukey Test for Site Comparison Mean difference p-value Pak Nai vs Ha Pak Nai 07057 0154 Pak Nai vs Shui Hau Wan 31492 lt0001 Pak Nai vs San Tau 44281 lt0001 Ha Pak Nai vs Shui Hau Wan 24435 lt0001 Ha Pak Nai vs San Tau 37224 lt0001 Shui Hau Wan vs San Tau 12789 0076 Tukey Test for Time Comparison Mean difference p-value March vs April 10744 0594 March vs May 11390 0358 March vs June -11389 0354 March vs July -18670 0102 March vs August -08437 0692 April vs May 00646 1000 April vs June -22133 0001 April vs July -29414 lt0001 April vs August -19181 0006 May vs June -22779 lt0001 May vs July -30060 lt0001 May vs August -19827 lt0001 June vs S July -07281 0797 June vs August 02953 0976 July vs August 10233 0492 Significant at p lt 005 for ANOVA and Tukey Test


The prosomal width of all the horseshoe crabs found using the quadrat sampling

and walk-through methods were measured In the six month distribution study 539

horseshoe crabs were found by the walk-through method as compared with only 31

being recorded by the quadrat sampling Among all the horseshoe crabs found 472

were T tridentatus and 67 belonged to C rotundicauda In view of the low occurrence

of horseshoe crabs on the shores the present study indicated that the walk-through

survey is a better method in retrieving horseshoe crabs and therefore should be

recommended in future studies as a standard monitoring protocol to assess the status of

the juveniles

Size Distribution of T tridentatus

- Spatial Variations in Size among Sites

Table 218 shows the mean prosomal width of the juvenile T tridentatus which

ranged from 101 to 961 mm According to the size-age relationship established by

Sekiguchi (1988a) these corresponded to an age of one to ten years old The

Kruskal-Wallis test showed significant differences in the prosomal width among sites

(Table 219) with T tridentatus at San Tau being significantly larger than those at other

sites In which the average size of horseshoe crabs at San Tau was 567 mm

Individuals at Pak Nai and Ha Pak Nai were similar in size (~ 40 mm) but larger than

those at Shui Hau Wan (313 plusmn 13 mm) (Table 219)

79Table 218 Prosomal width (PW) of T tridentatus found at the 4 sites

Total number of T tridentatus Average PW (mm) Min PW (mm) Max PW (mm)

Northwestern New Territories Pak Nai 108 384 148 961 Ha Pak Nai 164 385 101 688 Lantau Island Shui Hau Wan 184 288 153 623 San Tau 16 567 326 824

80- Spatial Variations in Size at Different Tidal Levels

There was a significant difference in the prosomal width of T tridentatus among

tidal levels (KW test H = 13974 p = 0003) (Fig 220 Table 219) Pairwise

comparisons showed that the average size of individuals was significantly different

between 16 m above CD and 10 m above CD as tested by the Mann-Whitney tests

(p = 0007) (Table 219)

81

00051015202530354045505560657075





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)


Figure 220 Spatial variations (among sites and tidal levels) of the prosomal width

(+SE) of T tridentatus from March to August 2005

82- Temporal Variations in Size

Significant difference in the size of horseshoe crabs was found among sampling

times (KW test H = 27374 p lt 0001) (Table 219) with the size increasing gradually

from 298 plusmn 42 mm in March to 489 plusmn 32 mm in August except for a decrease in July

(338 plusmn 22 mm) (Fig 220) Pairwise comparisons showed that significant differences

were obtained between June and July and May and July (Fig 221 Table 219)

83

00051015202530354045505560657075


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)


Figure 221 Temporal variations of the prosomal width (+SE) of T tridentatus from


84Table 219 Results of the Kruskal-Wallis test for the differences in prosomal width of

T tridentatus at the four sites from March to August 2005 with Bonferroni adjustment

(p lt 0017) The effect of time was tested by the Wilcoxon (Z) test with Bonferroni

adjustment (p lt 00033) The site and tidal effects were tested by the Mann-Whitney

(U) test with Bonferroni adjustment (p lt 00083)

Kruskal Wallis Test on Size Chi-Square df p-value Between Site 70579 3 0000 Between Time 27374 5 0000 Between Tidal Level 13974 3 0003 Wilcoxon Test for Time

Comparison Z p-value March vs April -1153 0249 March vs May -0314 0753 March vs June -0105 0917 March vs July -1153 0249 March vs August -2201 0028 April vs May -0087 0931 April vs June -1075 0282 April vs July -2885 0004 April vs August -1363 0173 May vs June -1514 0130 May vs July -3214 0001 May vs August -1782 0075 June vs July -3091 0002 June vs August -1363 0173 July vs August -0314 0753 Mann-Whitney Test for Site Comparison U p-value Pak Nai vs Ha Pak Nai 54335 0377 Pak Nai vs Shui Hau Wan 54405 0000 Pak Nai vs San Tau 2785 0000 Ha Pak Nai vs Shui Hau Wan 68395 0000 Ha Pak Nai vs San Tau 3945 0000 Shui Hau Wan vs San Tau 1595 0000 Significant at p lt 0017 for the Kruskal Wallis test p lt 00033 for the

Wilcoxon test and p lt 00083 for the Mann-Whitney test


Mann-Whitney Test for Tidal Level Comparison U p-value Tidal level 1 vs tidal level 2 97065 0078 Tidal level 1 vs tidal level 3 31660 0007 Tidal level 1 vs tidal level 4 20340 0011 Tidal level 2 vs tidal level 3 10320 0273 Tidal level 2 vs tidal level 4 6385 0141 Tidal level 3 vs tidal level 4 2940 0557

Significant at p lt 0017 for the Kruskal Wallis test p lt 00033 for the Wilcoxon

test and p lt 00083 for the Mann-Whitney test

86Size-frequency Distribution Histograms of T tridentatus

Figure 222 shows the size-frequency distribution of T tridentatus at the four sites

Newly recruited juveniles started to occur on the shore in May at Pak Nai and Ha Pak

Nai in April at Shui Hau Wan and in June at San Tau The cohorts were identified

using the software FiSAT II with the average length of each cohort shown in Table 220

Older cohorts were only found in some periods of time eg cohort 5 at Pak Nai and Ha

Pak Nai cohort 4 at Pak Nai and Shui Hau Wan and cohort 3 at San Tau This may be

due to small sample sizes being collected from these sites or the older cohorts have

migrated to the sea when they become sexually mature Based on the more

comprehensive data set for cohort 3 at Pak Nai and Ha Pak Nai the growth in prosomal

width for T tridentatus at Pak Nai during summer was estimated at 84 mm (March to

April) 7 mm (April to May) 184 mm (May ndash June) and 32 mm (June ndash July) and that

at Ha Pak Nai during the same period of time was 26 mm 71 mm 211 mm and 47

mm respectively

87

88

89

90

Figure 222 Size-frequency distributions of T tridentatus at Site 1 (Pak Nai) Site 2 (Ha Pak Lai) Site 3 (Shui Hau Wan) and Site 4 (San Tau) (10^1 = 101)

91

Table 220 Average prosomal width (plusmn SD) of individual cohorts of T tridentatus at

the four sites as identified by FiSAT II

Site Pak Nai

Cohort Number Average Prosomal Width (mm) (plusmnSD)

No of Individuals

March 3 300 plusmn 82 4 April 3 384 plusmn 76 21

4 496 plusmn 72 6 5 695 plusmn 74 1

May 2 221 plusmn 35 16 3 454 plusmn 76 15 4 702 plusmn 108 5

June 1 175 plusmn 05 2 2 336 plusmn 57 12 3 638 plusmn 103 3

July 1 275 plusmn 47 6 3 670 plusmn 237 2

Site Ha Pak Nai

Cohort Number Average Prosomal Width (mm) (plusmnSD) No of Individuals

March 3 180 1 4 410 1

April 3 206 plusmn 38 5 4 468 plusmn 72 21 5 675 plusmn 07 6

May 3 277 plusmn 90 10 4 537 plusmn 19 3


July 1 238 plusmn 51 14 2 354 plusmn 38 10 3 535 plusmn 67 8

92Site Shui Hau Wan





August 2 427 plusmn 49 3

Site San Tau


April 3 220 1 May 3 418 plusmn 99 8 June 1 100 1 July 1 160 1

2 280 2 August 1 297 plusmn 31 3

93Size Distribution of C rotundicauda

- Spatial Variations in Size Among Sites

Table 221 shows the mean prosomal width of the juvenile C rotundicauda which

varied from 109 to 433 mm Using the size data obtained by Chiu and Morton (1999a)

individuals collected in the present study corresponded to an age of 3 to 9-10 instars

Only one individual of C rotundicauda was found at Pak Nai with a prosomal width of

310 mm and none was found at Shui Hau Wan C rotundicauda at San Tau were

bigger than those at Ha Pak Nai with an average size of 301 and 181 mm

respectively the difference however was statistically insignificant as analyzed by the

KW test (H = 4112 p = 0128) (Table 222)

94Table 221 Measurements of prosomal width (PW) of C rotundicauda found at the

four sites

Total number of C rotundicauda Average PW (mm) Min PW (mm) Max PW (mm)

Northwestern New Territories Pak Nai 1 310 - - Ha Pak Nai 2 181 161 201 Lantau Island Shui Hau Wan 0 - - - San Tau 64 301 109 433

95- Variations in Size at Different Tidal Levels

C rotundicauda was recorded at all tidal levels (Fig 223) the size however was

not significantly different (KW test H = 2397 p = 0494) (Table 222) The average

prosomal width was 222 - 340 mm

- Temporal Variations in Size

The size of C rotundicauda varied significantly with time (KW test H = 35836 p

lt 0001) (Table 222) with the average prosomal width increasing gradually from 135

mm in March to 351 mm in August (Fig 224) Pairwise comparisons showed that a

significant difference was found between June and August (Table 222)

96

00

05

10

15

20

25

30

35

40

45

50





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)


Figure 223 Spatial variations (both among sites and tidal levels) of the prosomal width

(+SE) of C rotundicauda from March to August 2005

97

00

05

10

15

20

25

30

35

40


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)


Figure 224 Temporal variations of the prosomal width (+SE) of C rotundicauda from



C rotundicauda at the four sites from March to August 2005 with the Bonferroni

adjustment (p lt 0017) The effect of time was tested by the Wilcoxon (Z) test with the

Bonferroni adjustment (p lt 00033)

Kruskal Wallis Test on Prosomal Width Chi-Square df p-value Between sites 4112 2 0128 Between time 35836 5 0000 Between tidal levels 2397 3 0494 Wilcoxon Test for Time Comparison Z p-value March vs April -1342 0180 March vs May -0447 0655 March vs June -1342 0180 March vs July -1342 0180 March vs August -1342 0180 April vs May 0000 1000 April vs June -1014 0310 April vs July -2366 0018 April vs August -2366 0018 May vs June 0000 1000 May vs July -0535 0593 May vs August -1069 0285 June vs July -1690 0091 June vs August -3039 0002 July vs August -1859 0063 Significant at p lt 0017 for the Kruskal Wallis test or p lt 00033 for the

Wilcoxon test

99Size-frequency Distribution Histograms of C rotundicauda

The size-frequency distribution of C rotundicauda at the three study sites is shown

in Figure 225 A single cohort of C rotundicauda was found in July at Pak Nai with a

mean prosomal width of 31 mm At Ha Pak Nai a single cohort was found in April in

which the prosomal width increased from 16 mm to 20 mm in June Two cohorts were

identified each month from April to August for the population at San Tau As there was

a large overlap in the size of different cohorts growth of individual cohorts could not be

determined with certainty

100

101

Figure 225 Size-frequency distributions of C rotundicauda at Site 1 (Pak Nai) Site

2 (Ha Pak Lai) and Site 4 (San Tau)

10224 Discussion

241 Updated Distribution of Juvenile Horseshoe Crabs in Hong Kong

In this distribution study both western and eastern shores of Hong Kong were

investigated Local distribution survey of horseshoe crabs was conducted twice in the

past once in the period from 1995 to 1998 and another in 2002 In the first distribution

survey (Chiu and Morton 1999a) juvenile horseshoe crabs were recorded from the

mudflat at Shui Hau Wan and San Tau on Lantau Island Pak Nai Lung Kwu Sheung

Tan and Nim Wan in northwestern New Territories (Table 223) They were also found

at Sheung Pak Nai Pak Nai (Long Chuk Hang) and two locations at Ha Pak Nai in the

distribution survey in 2002 (Morton and Lee 2003) Most of the juvenile horseshoe

crabs collected belonged to T tridentatus while C rotundicauda was only observed

sporadically at Ha Pak Nai and San Tau in the first survey and Sheung Pak Nai and Pak

Nai in the second one

In addition Agriculture Fisheries and Conservation Department (AFCD) have

been performing monitoring surveys of local juvenile horseshoe crabs since 2000

(Morton and Lee 2003) Horseshoe crabs were searched by walking around the site for

2-3 hours during ebbing tides ie about 1-2 hours prior to the predicted time of low tide

(Everitt 2004) In the period of 2000 to 2004 juvenile horseshoe crabs were found at

Sheung Pak Nai Pak Nai and Ha Pak Nai in the Deep Bay region at Shui Hau Wan

San Tau Sham Wat Tai Ho Wan Yi O Tung Chung and Hau Hok Wan on Lantau

Island and at Pak Kok Wan and Kai Kuk Shue Ha in northeastern New Territories

(Table 223)

103Table 223 Records of juvenile horseshoe crabs obtained from the present survey and

previous surveys by Chiu and Morton (1999a) Morton and Lee (2003) and the AFCD

monitoring survey (AFCD field data)

Location Year Number of individuals

Species Source of information

Northwestern New Territories Mai Po 1988-1993 1 Chiu and Morton 1999a 2005 1 C rotundicauda Unpublished data Tsim Bei Tsui 2004-2005 4 T tridentatus Present extensive survey 7 C rotundicauda Sheung Pak Nai Sept 1997 ~5 T tridentatus Chiu and Morton 1999a 2002 1 C rotundicauda Morton and Lee 2003 2000ndash2004 11 (average) T tridentatus AFCD field data 2004-2005 1 C rotundicauda Present extensive surveyPak Nai 2002 24

(2 ind 100m-sup2)T tridentatus Morton and Lee 2003

2002 3 C rotundicauda Morton and Lee 2003 2000-2004 16 (average) T tridentatus amp

C rotundicaudaAFCD field data

2004-2005 10 T tridentatus Present extensive survey 7 C rotundicauda 2005 18 (average) T tridentatus Present 6-month survey 02 (average) C rotundicauda Ha Pak Nai Sept 1997 ~22 T tridentatus Chiu and Morton 1999a Jun 1998 ~20 T tridentatus Chiu and Morton 1999a ~3 C rotundicauda Chiu and Morton 1999a 2002 18

(15ind 100m-sup2)T tridentatus Morton and Lee 2003

2002 12 (1 ind 100m-sup2)

T tridentatus Morton and Lee 2003

2000-2004 103 (average) T tridentatus amp C rotundicauda

AFCD field data

Ha Pak Nai (1) 2004-2005 5 T tridentatus Present extensive survey 2 C rotundicauda Ha Pak Nai (2) 2004-2005 10 T tridentatus Present extensive survey 3 C rotundicauda 2005 274 (average) T tridentatus Present 6-month survey 03 (average) C rotundicauda Nim Wan May 1995 8 Chiu and Morton 1999aLung Kwu Sheung Tan

Mar 1998 1 Chiu and Morton 1999a




Lantau Island Shui Hau Wan 1977 2 T tridentatus Chiu and Morton 1999a Aug 1997 ~40 T tridentatus Chiu and Morton 1999a 2000-2004 86 (average) T tridentatus AFCD field data

2004-2005 8 T tridentatus Present extensive survey 2005 368 (average) T tridentatus Present 6-month survey San Tau May 1995 ~13 Chiu and Morton 1999a Oct 1997 ~15 T tridentatus Chiu and Morton 1999a Jun 1998 1 C rotundicauda Chiu and Morton 1999a 2000-2004 72 (average) T tridentatus amp


2004-2005 10 T tridentatus Present extensive survey 2005 27 (average) T tridentatus Present 6-month survey

107 (average) C rotundicauda Tai Ho Wan 2000-2004 10 (average) C rotundicauda AFCD field data

2004-2005 2 C rotundicauda Present extensive surveySham Wat 2000-2004 138 (average) T tridentatus amp


2004-2005 2 T tridentatus Present extensive surveyYi O 2000-2004 9 (average) T tridentatus amp


2004-2005 5 T tridentatus Present extensive survey 1 C rotundicauda Tung Chung 2000-2004 11 (average) T tridentatus amp


2004-2005 1 T tridentatus Present extensive survey 1 C rotundicauda Hau Hok Wan 2000-2004 5 (average) T tridentatus amp


Northeastern New Territories Kei Lai Ha ~1975 1 T tridentatus Chiu and Morton 1999aStarfish Bay ~1988 1 Chiu and Morton 1999aPak Kok Wan 2000-2004 14 (average) C rotundicauda AFCD field data Kai Kuk Shue Ha

2000-2004 55 (average) C rotundicauda AFCD field data

Luk Keng 2004-2005 5 C rotundicauda Present extensive surveyLai Chi Wo 2004-2005 5 C rotundicauda Present extensive survey Accidental catch in the mudflat at Mai Po during sediment collection by the core

sampling in the summer in 2005

1052411 T tridentatus

Along the northwestern coast of New Territories from Lau Fau Shan to Ha Pak Nai

T tridentatus was only recorded at Pak Nai and two locations at Ha Pak Nai by the

random quadrat sampling in 2002 (Table 223) (Morton and Lee 2003) While it could

still be found at these three sites in the present study by using a similar sampling

method Apart from Pak Nai and Ha Pak Nai four T tridentatus were also found at

Tsim Bei Shui in the present survey by the walk-through survey It indicated that T

tridentatus still has a wide distribution range in the Deep Bay from Tsim Bei Shui to Ha

Pak Nai However no individual of T tridentatus was recorded at Sheung Pak Nai in

the present study whereas several individuals were reported in 1997 by Chiu and

Morton (1999a) and an average of 11 juveniles were observed in the AFCD survey in

2000-2004 Such a big difference in the distribution records may be due to the different

sampling methods being used in the AFCD survey and the present one In the AFCD

survey the whole site was actively searched for the record of horseshoe crabs whereas

only four tidal levels within the survey site were searched in the present survey

Furthermore only two surveys (summer and winter) were conducted in the present

study further surveys are hence recommended to be conducted to confirm the reocrd of

T tridentatus at Sheung Pak Nai

In addition this study has also confirmed the distribution of T tridentatus on

Lantau Island Scientific and systematic study on the distribution of horseshoe crabs on

Lantau Island has not been conducted previously In recent years only preliminary

distribution data were obtained from the interviews with fishermen and old villagers

(Chiu and Morton 1999a) and the AFCD monitoring study undertaken from 2000-2004

In which Shui Hau Wan San Tau Sham Wat Yi O Tung Chung and Hau Hok Wan

were identified as nursery grounds for T tridentatus In the present study juvenile T

106tridentatus were recorded at San Tau and Yi O by the random quadrat sampling and

Shui Hau Wan Sham Wat and Tung Chung by the walk-through survey Whilst no

horseshoe crab was found at Hau Hok Wan However as only two surveys (summer

and winter) were conducted in the present study further surveys are recommended to

confirm the presence of T tridentatus at Hau Hok Wan

2412 C rotundicauda

Only very few individuals of C rotundicauda were recorded in the past surveys

with 2 individuals being reported upon by Chiu and Morton (1999a) 4 in the 8-month

survey by Morton and Lee (2003) and one in Mai Po during sediment sampling in 2005

(unpublished data) They were mainly recorded in the Deep Bay region including

Sheung Pak Nai Pak Nai and Ha Pak Nai and at San Tau on Lantau Island A more

extensive distribution of C rotundicauda was reported in the AFCD survey with C

rotundicauda being found at Pak Nai and Ha Pak Nai as well as San Tau Tai Ho Wan

Sham Wat Yi O Tung Chung and Hau Hok Wan on Lantau Island

In the present survey no C rotundicauda was recorded using the similar random

quadrat sampling as in the study of Morton and Lee (2003) However using the

walk-through method C rotundicauda was recorded from those identified nursery

grounds in the Deep Bay and on Lantau Island except for Sham Wat where only T

tridentatus was recorded Such differences in the distribution records may be due to the

different sampling methods being used in the AFCD survey and the present one as

mentioned before

In the eastern waters the AFCD survey indicated the presence of C rotundicauda

at Pak Kok Wan and Kai Kuk Shue Ha our study has shown that their distribution has

107extended to Luk Keng and Lai Chi Wo Similar to T tridentatus C rotundicauda has a

wide distribution in Hong Kong including the Deep Bay northeastern New Territories

and Lantau Island (Fig 25)

242 Local Distribution Patterns of the Two Horseshoe Crab Species

Based on the distribution data obtained in this study horseshoe crabs were not

evenly distributed throughout Hong Kong Both T tridentatus and C rotundicauda

were more abundant in the western waters on mudflats or sandy shores However only

C rotundicauda was found in the eastern waters including Luk Keng Lai Chi Wo Pak

Kok Wan and Kai Kuk Shue Ha Such distribution pattern may be due to interspecific

differences in habitat preference (Sekiguchi and Nakamura 1979 Mikkelsen 1988)

with salinity and substratum granulometry being two possible environmental factors

responsible for the absence of T tridentatus in the eastern waters

Hong Kong waters are characterized by the interaction between oceanic and

estuarine waters (HKSAR 2006) During summer Hong Kong is influenced by the

warm and oceanic Hainan Current from the southwest and the sediment-laden

freshwater from the Pearl River in the northwest which eventually divide Hong Kong

waters into three distinct zones In the west where the freshwater influence is the

greatest the environment is estuarine and the water is brackish In the east the water is

mainly oceanic with relatively minor dilution from rainfall and runoff from streams

The limits of the central transitional zone vary depending upon the relative influence of

the Pearl River and marine currents During the winter dry season the Kuroshio oceanic

current brings warm water of high salinity from the Pacific through the Luzon Strait

The freshwater discharge of the Pearl River is much lower than in the summer and

108salinity is more uniform across Hong Kong The coastal Taiwan current also brings cold

water from the northeast down the South China coast affecting inshore waters

Both T tridentatus and C rotundicauda can tolerate a wide range of salinities

(Stormer 1952 Shuster 1957) and embryos of both species could be developed within

the range of 20-35permil without a delay in the development (Sekiguchi 1988a) In view of

this embryos and juveniles of both two species should be able to develop at the sites in

western New Territories with oceanic waters In addition comparing with the other

three horseshoe crab species C rotundicauda prefer to breed in fresh or brackish water

(Sekiguchi 1988b) Salinity therefore seems not to be the determining factor for the

distribution of the two horseshoe crab species in Hong Kong waters

Sediment particle size may be a factor responsible for the absence of T tridentatus

in the eastern waters Among the 13 nursery grounds being studied Luk Keng and Lai

Chi Wo in the eastern waters are muddy whereas most of the sites in the western waters

are sandy or sandy to muddy The maximum depth of mud at Luk Keng and Lai Chi Wo

was 70 and 50 cm respectively (Table 21) while most of the shores in western Hong

Kong have a mud depth varied from 5 to 50 cm The studies of Sekiguchi and

Nakamura (1979) Sekiguchi (1988b) and Chatterji (1994) indicated the preference

towards sandy shores in T tridentatus Liao et al (2002) also showed that more C

rotundicauda could be recorded in the waters with muddy substratum where only few

adult and juvenile T tridentatus were found This is further supported by the fact that T

tridentatus was absent in muddy shores in western Hong Kong including Sheung Pak

Nai and Tai Ho Wan where the sediments are silt-clay with a maximum mud depth of

60-70 cm

109Pollution is another factor which may affect the distribution of horseshoe crabs in

certain areas such as the Tolo Harbour Horseshoe crabs were recorded at Kei Lai Ha

and Starfish Bay in the Tolo Harbour twenty years ago (Cheung et al 1975) however

they became disappear at these sites in the past 10 years (Chiu and Morton 1999a

2003a) This might be due to the water pollution problem caused by industrial and

agricultural wastes and urban development in the harbour where it was once regarded as

a ldquopristinerdquo site Owing to anthropogenic activities both the water and sediment

qualities of the Tolo Harbour were deteriorating The increase in the concentration of

various metals in the sediment including Cu Zn Pb and Ni suggested human-derived

inputs (Owen and Sandhu 2000) The problem was aggravated by the bottle-necked

topography of the harbour which resulted in poor flushing and accumulation of

sediments (clay organic carbon) suitable for metal adsorption (Owen and Sandhu

2000) Meanwhile eutrophication caused by agricultural and domestic wastes increased

organic loading in the water and resulted in occasional hypoxia and red tide during

summer which eventually reduced the biodiversity of the marine ecosystem in the Tolo

Harbour (Horikoshi and Thompson 1980) For example the bivalve Paphia undulata

was found to decline in the late 1970s (Horikoshi and Thompson 1980) Subsequent

studies on the benthic ecosystem (Shin 1985 Taylor 1992 Taylor and Shin 1990) also

showed continuous reduction in the bivalve species diversity and an increase in

opportunistic scavengers (Morton et al 1996)

The reclamation for urban development in the harbour is another serious problem

For instance the Victoria Harbour originally was about 7000 hectares in size in which

around 2500 hectares had already been reclaimed by 1990 while a further 661 hectares

had been reclaimed in 1990-1997 An extra of 636 hectares may be reclaimed in the

future (Friends of the Harbour 2003) Such a large scale of reclamation results in the

110deterioration of marine environment and highly reduces the natural coastal shores All

these pollution impacts and the reclamation in the harbour have severely destroyed the

natural breeding and nursery grounds of the horseshoe crabs (Chiu and Morton 1999a)

243 Status of Horseshoe Crabs in Hong Kong

2431 T tridentatus

Using a sampling strategy similar to the random quadrat sampling in our study T

tridentatus was found at Pak Nai (Long Chuk Hang) and two locations at Ha Pak Nai

along the Deep Bay area in 2002 with an average density of 197 155 and 114

individual 100 m-2 respectively (Morton and Lee 2003) In the present study the

density at these sites decreased sharply in the summer of 2004 to 0078 016 and 023

individual 100 m-2 respectively The decreases in the population density at Pak Nai and

two locations at Ha Pak Nai were 96 90 and 80 respectively In the six-month survey

similar results were obtained with an average density of 014 individual 100 m-2 and

013 individual 100 m-2 at Pak Nai and Ha Pak Nai respectively

Apart from the Deep Bay area no quantitative data on the population density of

horseshoe crabs were available from the previous scientific researches With only the

simple overview on the distribution and population data from Chiu and Mortonrsquos study

and the AFCD monitoring study it is impossible to have any valid conclusion on the

population changes of T tridentatus in recent years at these sites Nevertheless no

individual was obtained by the random sampling method in the six-month survey at

each sampling site in northeastern New Territories and on Lantau Island therefore it is

reasonable to conclude that T Tachypleus is now being restricted mostly to the Deep

Bay and rare in other parts of Hong Kong

1112431 C rotundicauda

Using a sampling strategy similar to the random quadrat sampling in our study

only 4 individuals of C rotundicauda were found at Sheung Pak Nai and Pak Nai (Long

Chuk Hang) in 2002 (Morton and Lee 2003) However in the present study no C

rotundicauda was found in all the 17 sampling sites by the random sampling method

This implies a decline in the population density of C rotundicauda in the Deep Bay

area although they could still be observed at Tsim Bei Tsui Sheung Pak Nai Pak Nai

and two locationas at Ha Pak Nai in northwestern New Territories at San Tau Tai Ho

Wan Yi O and Tung Chung on Lantau Island at Luk Keng and Pai Chi Wo in

northeastern New Territories by a more intensive walk-through survey (Tables 27 and

214)

Similar to T tridentatus the lack of past data on the population density of C

rotundicauda at other sites did not allow me to make any conclusion on the population

changes of this species However the present study has indicated an unexpected wide

distribution of C rotundicauda on Lamma Island and eastern Hong Kong Although the

population size obtained in these areas was much smaller than that in the Deep Bay area

more C rotundicauda were recorded at San Tau on Lantau Island than in the Deep Bay

area

In comparing with the AFCD field data obtained between 2000 and 2004 fewer

individuals of both species of horseshoe crabs were obtained in the present survey

particularly at Tai Ho Wan Sham Wat Yi O Tung Chung and Hau Hok Wan (Tables

26 27 and 223) The sharp decline in T tridentatus and C rotundicauda populations

in recent years therefore is apparent

112T tridentatus and C rotundicauda are also distributed in mainland China and other

Asian countries including Japan Taiwan Malaysia and the Philippines In the

Philippines recent population studies on juvenile T tridentatus were conducted on two

soft shores in Palawan in 2001 and 2002 (Kaiser 2002 Almendral and Schoppe 2005)

with an average density of 200 individual 100 m-2 and 147 individual 100 m-2

respectively In Singapore a density of 205 individual 100 m-2 was obtained for

juvenile C rotundicauda (Hong 2004) As the density found in Hong Kong was lt 10

of those obtained in other southeast Asian countries the local population is definitely

facing a great threat and there is a pressing need to develop a specific conservation plan

for the long term survival of these species in Hong Kong

Identifying key nursery grounds for the horseshoe crabs provides essential

information for specific conservation plans for example reducing pollution levels and

setting up protected areas in the nursery grounds In the 6-month survey Shui Hau Wan

and San Tau on Lantau Island were found to be the most important nursery grounds for

T tridentatus and C rotundicauda respectively Continuous monitoring of the

populations of horseshoe crabs at these sites is essential

244 Temporal Variations in the Distribution of Horseshoe Crabs

Significant seasonal variations in the population density of horseshoe crabs were

obtained between summer and winter and may be caused by seasonal differences in

their behaviour Affected by the low water and sediment temperatures horseshoe crabs

become inactive bury into the sediment or even undergo diapause in winter

(Kawahara 1982 Morton and Lee 2003 Chiu and Morton 2004) However they

become active as temperature increases This helps explain why more individuals were

found in the summer in this study Our laboratory experiment on the temperature effect

113on the activity of T tridentatus also showed that they became inactive at 9degC as

compared with 19degC 29degC and 39degC (unpublished data)

Similar temporal variations in the population density of horseshoe crabs were

observed in the 6-month (March to August) survey of the four important nursery

grounds A lower frequency of occurrence of T tridentatus was recorded in April and

the abundance peaked in the mid summer (May to August) Similar results were

obtained by Morton and Lee (2003) The temporal variations in the occurrence of

juvenile T tridentatus appeared to follow the seasonal variations in temperature In

general temperature increased gradually from early summer (March) to late summer

(August) with an average temperature of 206degC in March a maximum of 352degC in

July and a slight drop in August (331degC) A similar occurrence pattern was observed

for C rotundicauda

245 Size Distributions of Horseshoe Crabs

The age of T tridentatus recorded was between one and ten years old A similar

study was conducted in Hong Kong in 1999 and 2002 (Chiu and Morton 1999a

Morton and Lee 2003) in which individuals varied between 115 mm and 900 mm in

the prosomal width ie two to ten years old along the coast of northwestern New

Territories while a range of 20 mm to 58 mm for T tridentatus was found at Shui Hau

Wan The size ranges obtained in these studies were narrower than those obtained in the

present study which ranged from 101 to 961 mm in the Deep Bay area and 153 mm to

762 mm at Shui Hau Wan Individuals of lt 120 mm in the prosomal width were also

recorded in the present survey with an individual of 101 mm being registered at Ha

Pak Nai and two individuals of 55 and 10 mm at San Tau The results implied that all

114these sites ie Pak Nai Ha Pak Nai Shui Hau Wan and San Tau are important nursery

grounds for juvenile or even newly hatched T tridentatus

Using the cohort analysis a maximum of five cohorts have been identified for T

tridentatus from the study sites with new recruits occurred from April to June

However owing to the small number of individuals being collected the growth could

only be followed throughout the 6-month survey for a single cohort at Pak Nai and Ha

Pak Nai The growth in the prosomal width ranged from 26 mm to 211 mm per month

or 009 mm to 07 mm per day The results agreed with those obtained from a laboratory

study of which the growth of T tridentatus was 01 mm per day (Lee and Morton

2005)

For C rotundicauda individuals between three instars and nine to ten instars with

the prosomal width between 109 and 433 mm were recorded at the four sites in the

6-month survey Individuals between 292 mm and 620 mm ie seven to eleven

instars were obtained along the coast of northwestern New Territories by Morton and

Lee (2003) Two cohorts have been identified for C rotundicauda at San Tau from

April to August However as there was a large overlap in the size of different cohorts

growth of individual cohorts could not be determined with certainty

Significant spatial variations in the size of T tridentatus with tidal levels was

identified The size of animals on the lower shores (07 ndash 10 m above CD) was

significantly larger with an average prosomal width of 40-50 mm followed by 388

mm and 356 mm at 13 m and 16 m above CD respectively It is generally believed

that horseshoe crab eggs are laid on the upper shore After hatching the juveniles will

inhabit in the intertidal areas and move further offshore with increasing age (Sekiguchi

1151988b Shuster 1979) Similar behaviour has been reported upon for the American

species L polyphemus However no significant spatial variation in the size of C

rotundicauda was observed

116

Chapter 3 To Differentiate Tachypleus tridentatus from

Carcinoscorpius rotundicauda using Morphological and Genetic

Studies and to Study Genetic Relationships among Horseshoe Crabs

from Various Nursery Grounds in Hong Kong

31 Introduction

The taxonomic data are vital and a major concern in conservation especially with

regard to status designation and protection policies (Ganders et al 2003 Valdecasas

and Camacho 2003) In cases where the taxonomic status of threatened species has not

been sufficiently clarified there is a risk that potentially valid species could become

extinct before conservation action is initiated (Gjershaug 2006)

There are four horseshoe crab species in the world Detailed morphological

descriptions of all the species have been given in previous literature (Mikkelsen 1988

Sekiguchi 1988c Yamasaki et al 1988) whereas Chiu and Morton (1999a 2003b)

described the various body part dimensions in adult T tridentatus and C rotundicauda

in Hong Kong Among the four species T tridentatus is the largest one whereas C

rotundicauda the smallest One of the most obvious morphological differences is the

telson in which the cross-section of the telson of C rotundicauda is subtriangular with

round edges and a triangular hollow while that of the other three species are triangular

with a hollow of the same shape (Sekiguchi 1988c) The ventral side of the telson is

convex in L polyphemus and C rotundicauda but is concave in the two Tachypleus

species (Sekiguchi 1988c) T gigas bears a row of small thin spinnerets only on the

top edge of the telson whereas T tridentatus has a row of sharply pointed small

spinnerets on every edge (Sekiguchi 1988c)

117

In contrast to the distinctive morphological features in their adult forms the

juveniles of these species are much more difficult to be distinguished This section

focuses on the morphological differences between the juveniles of T tridentatus and C

rotundicauda the two common horseshoe crab species in Hong Kong based on the

measurement of various body parts

In addition to morphological measurements genetic analysis is a reliable

speciation method for taxonomic purposes In this study 18S and 28S ribosomal DNA

(rDNA) were used for the phylogenetic analysis of horseshoe crab species Eukaryotic

rDNA genes are parts of repeat units that are arranged in tandem arrays located at the

chromosomal sites known as nucleolar organizing regions (NORs) They regulate

amplification and transcription initiation of the DNA sequences and play a major role

in the informational processing machinery (Brocchieri 2000) Each repeat unit consists

of transcribed and nontranscribed regions in which 18S and 28S are the small and large

subunits of the transcribed rDNA regions With a low rate of polymorphism the rDNA

transcription unit (including 18S and 28S) is specific for each species and hence 18S

and 28S rDNA are useful for interspecific comparisons and commonly used for the

phylogenetic analysis for speciation

The small ribosomal DNA sequence 18S rDNA has been used in molecular

phylogenetic analyses of various organisms including Annelida (Rota et al 2001

Struck et al 2002 Struck and Purschke 2005) Heteroptera (Li et al 2005) and Insecta

(Belshaw and Quicke 2002) The nuclear 28S rDNA coding the second expansion

segment of the nuclear ribosome subunit was also used for speciation analysis in

Platyhelminthes (Mollaret et al 2000) Pulmonata (Mejia and Zuniga 2007) and

Insecta (Reiko et al 2006) Besides 18S and 28S rDNA were also used for

118

differentiation of horseshoe crabs at the species level with high precision (Xia 2000)

In this study genomic sequences of 18S and 28S rDNA of juvenile T tridentatus

and C rotundicauda in Hong Kong were determined and compared Morton and Lee

(2003) discovered that some juvenile T tridentatus at some study sites contained only

one immovable spine on the dorsal surface of the opisthosoma above the insertion of a

post-anal spine in contrast to three in most of the conspecifics (Fig 31a-b) They

suspected that these ldquoabnormalrdquo individuals may be caused by mutation or are hybrids

of T gigas and T tridentatus To solve this taxonomic problem 18S and 28S rDNA of

the ldquoabnormalrdquo horseshoe crabs were also sequenced and results were compared with

those obtained from the two known horseshoe crab species

Apart from the taxonomic issue the understanding of the dispersal of individuals

between nursery grounds and their geographical borders will be critical to management

programs (Bowen et al 1993 Paetkau et al 1995) particularly for migratory species

With the knowledge of the geographical movement and migration effective

trans-boundary management and broad cross-country coordination and planning could

be effectively developed (Mansourian and Dick 2006) An understanding of the

movement range also initiates a strategic management plan for the endangered species

such as polar bear in order to reduce any potential impacts and risks to the species

(Paetkau et al 1995)

However migration studies of species at risk are usually restricted by logistic

problems including limited access to populations small sample sizes and restrictions

prohibiting manipulative experimentation (Steinberg and Jordan 1998) Thus even the

most basic geographical population and migration data can be difficult to acquire In

contrast recent technological advances in molecular population genetics have greatly

119

reduced the cost and simultaneously increase the ease of field genetic studies Also

employing these non-intrusive sampling methods mean that we can obtain genetic data

on highly endangered species without sacrificing a single individual (Steinberg and

Jordan 1998) Genetic variations in juveniles at various nursery grounds in Hong Kong

were examined in this study to identify possible gene flow among populations This

would help determine the geographical range of reproductive adult in Hong Kong and

clarify if individuals would spawn on more than one beach as postulated by Morton and

Lee (2003) Compared to other genomic sequences such as mitochondrial COI

(cytochrome oxidase subunit I) and other protein-coding genes 18-28S intergenic

spacer sequence (ITS) rDNA has been successfully used for molecular differentiation

andor identification of closely related species of pathogenic plant fungi (Wang et al

2003) and Asian mitten crabs (Tang et al 2003) Therefore 18-28S intergenic spacer

sequence (ITS) rDNA of T tridentatus from five sites were examined and compared

32 Morphological Study of the Two Horseshoe Crab Species


3211 Measurement of Various Body Parts

A total of 131 juvenile horseshoe crabs were collected from Hong Kong soft shores

from March to June 2005 with 65 individuals of T tridentatus from Pak Nai and Ha

Pak Nai and 66 individuals of C rotundicauda from San Tau Various body parts of the

horseshoe crabs were measured using digital vernier calipers to a tenth of a millimeter

(Fig 32) The ratios of the body parts of the two species were compared


As ratio data were not normally distributed they were transformed logarithmically

Differences in body part ratios between the two species were compared using the

120

stepwise discriminant analysis (Tabachnick and Fidell 1996) Tests of equality of group

means were assessed using the F-statistic with significance of α = 005 and the

important discriminating parameters for inter-specific differences were determined All

the statistical analyses were undertaken using the software SPSS 110

121

a

b

Figure 31 Photos of juvenile horseshoe crabs found on the shores a) normal form

with three immovable spines on the dorsal surface of the opisthosoma above the

insertion of the post-anal spine b) with only one immovable spine on the surface of the

opisthosoma (indicated with an arrow)

122

Figure 32 Various body parts of a horseshoe crab were measured to the nearest 01mm

(a carapace length b prosomal length c prosomal width d distance between the two

compound eyes e telson width f telson length g first opisthosomal length h sixth

opisthosomal length)

123

322 Results

The prosomal width of T tridentatus ranged between 229 and 772 mm with an

average of 476 mm Referring to the size-age relationship established by Sekiguchi et

al (1988) the individuals were presumed to be three to nine years old For C

rotundicauda the prosomal width ranged between 109 and 433 mm with an average

of 296 mm Although the data from Sekiguchi et al (1988) on the growth stages of C

rotundicauda were incomplete due to the short rearing periods they believed that the

stepwise growth of C rotundicauda progressed at almost the same rate as that of T

tridentatus Referring to Sekiguchi et al (1988) individuals collected in the present

study were classified as the 3rd to 10th instar or two to seven years old

3221 Qualitative Comparisons

Figure 33 shows the dorsal view of the juvenile T tridentatus and C rotundicauda

The cross-section of the telson of T tridentatus is triangular in shape while that of C

rotundicauda is spherical The telson surface is spiny for T tridentatus but smooth for

C rotundicauda Furthermore the opisthosomal spine of T tridentatus is darker in

colour while that of C rotundicauda is lighter and whitish yellow in colour

a b

Figure 33 The dorsal view of a) Tachypleus tridentatus b) Carcinoscorpius rotundicauda

124

125

3222 Quantitative Comparisons

Prosoma and Opisthosoma

The carapace length (prosomal length + opisthosomal length) of C rotundicauda

was shorter than that of T tridentatus (Fig 32) Both species had an average ratio of

carapace length to prosomal width (ac) lt 1 indicating that the carapace was ellipse in

shape with longer carapace width than length (Table 31) A significantly lower ac ratio

was obtained for C rotundicauda (F 1 129 = 4411 p = 0038) (Table 32) The ratio of

prosomal length to prosomal width (bc) was also lower for C rotundicauda (F 1 129 =

6014 p = 0016) Both species also showed significantly longer prosomal length than

opisthosomal length Although both species had the carapace length to prosomal length

ratio (ab) lt 2 a significantly lower ratio was obtained for T tridentatus (F 1 129 = 5087

p = 0026) (Tables 31 and 32) As C rotundicauda was more elliptical the ratio of

prosomal width to the distance between two compound eyes (cd) was also significantly

higher than that for T tridentatus (F 1 129 = 49519 p lt 0001) (Table 32)

Telson

Both species showed lt 1 in the ratio of carapace length to telson length (af) (Table

31) indicating that both species had the telson being longer than the total carapace

length and no difference between the two species was found (F 1 129 = 0161 p = 0689)

However a higher ratio of telson width to telson length (ef) was obtained for C

rotundicauda (F 1 129 = 9782 p = 0002) (Table 32) This implied that a wider telson in

proportion to telson length was obtained for C rotundicauda

Opisthosomal Spine Length

There are six pairs of opisthosomal spines in horseshoe crabs The opisthosomal

spines of T tridentatus were longer than those of C rotundicauda (Fig 33) For both

126

the ratios of first opisthosomal spine length to prosomal width (gc) and sixth

opisthosomal spine length to prosomal width (hc) T tridentatus had significantly

higher values than C rotundicauda (F 1 129 = 201207 p lt 0001 F 1 129 = 15686 p lt

0001) (Table 32) Although the first spine was longer than the sixth one (gh ratio in

Table 31) no significant difference was found between the two horseshoe crab species

(F 1 129 = 0608 p = 0437)

The morphological differences between T tridentatus and C rotundicauda are

summarized in Table 33

127

Table 31 The means (plusmnSD) and ranges (bracketed values) of various morphological

ratios in juvenile T tridentatus and C rotundicauda For details of abbreviations from a

to h refer to Figure 32

Morphological Ratio

T tridentatus

C rotundicauda

ab

180plusmn015 (146-230)

186plusmn023 (145-245)

bc

053plusmn006 (034-069)

050plusmn008 (034-068)

ac

095plusmn005 (079-112)

093plusmn006 (080-107)

cd

178plusmn011 (157-218)

191plusmn010 (157-213)

ef

008plusmn001 (006-012)

009plusmn001 (006-012)

af

099plusmn013 (077-159)

099plusmn016 (072-151)

gh

135plusmn032 (072-238)

134plusmn050 (020-260)

gc

008plusmn001 (005-012)

004plusmn001 (000-006)

hc

006plusmn001 (003-009)

003plusmn001 (001-005)

128

Table 32 Results of the test of equality of discriminant analysis for differences in

various morphological ratios between T tridentatus and C rotundicauda with the

significance level of p lt 005 For details of abbreviations from a to h refer to Figure

32

Morphological Ratio Wilksrsquo Lambda F df1 df2 p-value ab 0962 5087 1 129 0026 bc 0955 6014 1 129 0016 ac 0967 4411 1 129 0038 cd 0723 49519 1 129 0000 ef 0930 9782 1 129 0002 af 0999 0161 1 129 0689 gh 0995 0608 1 129 0437 gc 0391 201207 1 129 0000 hc 0345 15686 1 129 0000

Significant at p lt 005

129

Table 33 Comparisons of the morphology between T tridentatus and C rotundicauda

T tridentatus C rotundicauda Carapace shape elliptical (carapace width gt carapace length) less elliptical more elliptical longer prosomal length shorter prosomal length

Distance between two compound eyes

longer

shorter

Telson telson length gt total carapace length narrower telson

wider telson

Opisthosmal spine Longer Shorter 1st spine length gt 6th spine length

130

Results of the stepwise discriminant analysis are shown in Table 34 with

significant differences being found between the two species (F 5 125 = 80646 p lt 0001)

Only five out of the nine morphological parameters were proven important in

discriminating the two species they are the ratios of prosomal length to prosomal width

(bc) prosomal width to distance between the two compound eyes (cd) carapace length

to telson length (af) first opisthosomal spine length to prosomal width (gc) and sixth

opisthosomal spine length to prosomal width (hc) Figure 34 depicts the plot of the two

species according to the discriminant function with the centroid values of 1796 and

-1769 for T tridentatus and C rotundicauda respectively As there was only one

discriminant function (ie number of discriminant functions = n-1 where n is the

number of groups in this case n = 2 for T tridentatus and C rotundicauda) all the

specimens of T tridentatus and C rotundicauda were plotted along this discriminant

function as shown on the x-axis of the graph (Fig 34)

131

Table 34 Significant morphological ratios and their discriminant function coefficients

of T tridentatus and C rotundicauda derived from the stepwise multiple discriminant

analysis For details of abbreviations from a to h refer to Figure 32

Morphological ratio Factor structure coefficients bc -0226 cd -0289 af 0451 gc 0652 hc 0606

132

Tt

Cr

-5 -4 -3 -2 -1 0 1 2 3 4

Driminant Function Score

T tridentatus C rotundicauda

Figure 34 Plot of the stepwise multiple discriminant analysis based on various

morphological ratios in T tridentatus (Tt) and C rotundicauda (Cr) Centroid values

of these two species were shown in black symbols

133

33 Genetic Differentiation of Horseshoe Crab Species



Since no T gigas was found in this study only T tridentatus and C rotundicauda

were examined Adult T tridentatus were bought from a seafood restaurant at Cheung

Chau while juvenile C rotundicauda were collected at Luk Keng No genetic analysis

was conducted for adult C rotundicauda as they could not be found in the field study as

well as in seafood restaurants Four individuals of each species were used for this DNA

analysis Eight juveniles of T tridentatus three with 3 spines and five with 1 spine on

the dorsal surface of the opisthosoma were also collected at Pak Nai to determine their

DNA fingerprint (Table 35) For the species genetic differentiation study the small and

large ribosomal DNA sequences and the conserved regions in the flanking 18 and 28S

rDNA genes were examined

134

Table 35 Grouping of horseshoe crab specimens used in the study of 18S and 28S

rDNA

Species No of individuals

analyzed Population Origin Adult T tridentatus (3 spines ) 4 TtHK Hong Kong Juvenile C rotundicauda 4 CrHK Hong Kong Juvenile T tridentatus (1 spine) 5 UnSHK Hong Kong Juvenile T tridentatus (3 spines) 3 UnTHK Hong Kong

Note The population abbreviations correspond to their species name and origins

Immovable spine(s) on the dorsal surface of the opisthosoma above the insertion of a post-anal spine

135

3312 DNA Extraction PCR Amplification and Sequencing

Twenty-five mg of tissue of each horseshoe crab individual was used for the DNA

extraction using the DNeasy Tissue Kit One pair of primer was designed for the

amplification of the 18S DNA

forward primer 5rsquo-CCTGGTTGATCCTGCCAGTAG-3rsquo

reverse primer 5rsquo-CGCAGGTTCACCTACGGTAACCTT-3rsquo

Another pair of primer was designed for the amplification of 28S DNA



Double-stranded DNA amplification was performed in 50 μl reaction volumes

containing 05 μl of Taq polymerase 5 μl of 10times Reaction Buffer 1 μl dNTPs 1 μl of

each primer and approximately 200 ng of the template DNA Thermal cycling

amplification conditions were as follows initial denaturation at 95 degC for 2 min

followed by 35 cycles of strand denaturation at 95 degC for 30 s annealing at 60 degC for 30

s and primer extension at 68 degC for 2 min and a final 10 min elongation time at 68 degC

The size of the PCR product was checked against a 50-bp DNA ladder

(MBI-Fermentas) in agarose gel run in 1times TAE buffer and stained with ethidium

bromide The DNA product was then sequenced by direct sequencing in order to

examine the genetic differences among species


Nucleotide sequences of two large ribosome genes (18S and 28S rDNA) were used

in this study For both ribosome genes a brine shrimp Artemia salinia (strain AsGB1

accession number X01723) and a tick Ixodes cookie (strain IxGB1 accession number

L76351) were used as outgroups 18S and 28S rDNA sequences were aligned using

136

Clustal X (Thompson et al 1997) and pair-wise genetic distances and transitions and

transversions were calculated using the MEGA 21 program (Kumar et al 2001)

Phylogenetic analysis was performed on genetic distance matrices using the

Neighbor-Joining software in the MEGA 21 package Some related 18S and 28S rDNA

sequences from GenBank were included in the analysis with 1 individual of C

rotundicauda (strain CrGB1 accession number U91491) and 4 individuals of Limulus

polyphemus (strains LpGB1 LpGB2 LpGB3 LpGB4 accession numbers U91490

L81949 AF062947 X90467) being used as outgroups

332 Results

3321 18S rDNA Gene Comparison

Table 36 shows the 18S rDNA gene comparison including the percentage of

pair-wise difference and the sequence differences (transitions transversions) among all

the tested individuals The mean difference of each species is summarized in Table 37

Intra-specific differences were small in which the pair-wise difference was 014 for T

tridentatus 017 for C rotundicauda and 042 for L polyphemus For inter-specific

sequences the differences were larger than the intra-specific sequences in which the

percentage of pair-wise difference was 032 between T tridentatus and C

rotundicauda 126 between T tridentatus and the American species L polyphemus

and 099 between C rotundicauda and L polyphemus The two juvenile T tridentatus

groups collected from the shores had similar intra-specific differences averaging 011

and 009 The inter-specific differences within these two groups were also small

(010) The inter-specific differences between T tridentatus C rotundicauda and L

polyphemus were also similar and ranged from 013 to 097 In view of the

extremely small inter-specific variations between the juveniles of T tridentatus with 1

immovable spine and those with 3 immovable spines and similar percentage

137

differences for the three horseshoe crab species these two juvenile T tridentatus groups

should belong to the same species according to the 18S rDNA analysis with only

011plusmn013 intra-specific variation For inter-specific variations the two juvenile T

tridentatus groups had a closer genetic relationship with the indo-Pacific horseshoe crab

species than the American species L polyphemus which has a smaller difference with C

rotundicauda than T tridentatus The number of transitions transversions between

these groups and C rotundicauda ranged between 0ndash30-1 while that between juvenile

T tridentatus groups and adult T tridentatus ranged between 2-30-3 (Table 36) For

the two outgroups Ixodes cookei and Artemia salina significantly larger differences for

the two horseshoe crab species and the two juvenile T tridentatus groups were observed

and ranged between 994-1050 and 1317-1373 respectively (Table 37)

In the Neighbor-joining tree (Fig 35) L polyphemus was separated as an

independent clade adult T tridentatus was also separated as an independent clade with

one individual of C rotundicauda of which the data were obtained from the GenBank

while other individuals of C rotundicauda and the two juvenile T tridentatus groups

were clustered under the same clade Ixodes cookei formed an independent clade from

the horseshoe crab species and Artemia salina formed a single clade

Table 36 Percentage of pair-wise difference in 18S rDNA gene of horseshoe crab individuals (below diagonal) and number of

transitionstransversions (above diagonal) (CrGB = C rotundicauda from GenBank LpGB = Limulus polyphemus from GenBank IcGB = Ixodes

cookei from GenBank AsGB = Artemia salina from GenBank For other species codes refer to Table 35 number represents individual)

Tt Tt Tt Tt Cr Cr Cr Cr Cr Lp Lp Lp Lp UnS UnS UnS UnS UnS UnT UnT UnT Ic As

HK1 HK2 HK3 HK4 HK1 HK2 HK3 HK4 GB1 GB1 GB2 GB3 GB4 HK1 HK2 HK3 HK4 HK5 HK1 HK2 HK3 GB1 GB1

TtHK1 00 00 02 20 20 30 20 00 25 25 45 56 20 20 20 31 20 30 20 20 4627 5539 TtHK2 000 00 02 20 20 30 20 00 25 25 45 56 20 20 20 31 20 30 20 20 4627 5539 TtHK3 000 000 02 20 20 30 20 00 25 25 45 56 20 20 20 31 20 30 20 20 4627 5539 TtHK4 028 028 028 22 22 32 22 02 27 27 47 58 22 22 22 33 22 32 22 22 4629 5541 CrHK1 028 028 028 056 00 10 00 20 05 05 25 36 00 00 00 11 00 10 00 00 4427 5739 CrHK2 028 028 028 057 000 10 00 20 05 05 25 36 00 00 00 11 00 10 00 00 4427 5739 CrHK3 042 042 042 070 014 014 10 30 15 15 35 46 10 10 10 21 10 20 10 10 4527 5839 CrHK4 028 028 028 056 000 000 014 20 05 05 25 36 00 00 00 11 00 10 00 00 4427 5739 CrGB1 000 000 000 028 028 028 042 028 25 25 45 56 20 20 20 31 20 30 20 20 4627 5539 LpGB1 098 098 098 126 070 070 084 070 098 00 20 31 05 05 05 16 05 15 05 05 4228 5736 LpGB2 098 098 098 126 070 070 084 070 098 000 20 31 05 05 05 16 05 15 05 05 4228 5736 LpGB3 126 126 126 154 098 098 112 098 126 028 028 51 25 25 25 36 25 35 25 25 4128 5736 LpGB4 154 154 154 182 126 126 140 126 154 056 056 084 36 36 36 47 36 46 36 36 4529 5837 UnSHK1 028 028 028 056 000 000 014 000 028 070 070 098 126 00 00 11 00 10 00 00 4427 5739 UnSHK2 028 028 028 056 000 000 014 000 028 070 070 098 126 000 00 11 00 10 00 00 4427 5739 UnSHK3 028 028 028 056 000 000 014 000 028 070 070 098 126 000 000 11 00 10 00 00 4427 5739 UnSHK4 056 056 056 084 028 028 042 028 056 098 098 126 154 028 028 028 11 21 11 11 4528 5840 UnSHK5 028 028 028 056 000 000 014 000 028 070 070 098 126 000 000 000 028 10 00 00 4427 5739 UnTHK1 042 042 042 007 014 014 028 014 042 084 084 112 140 014 014 014 042 014 10 10 4527 5839 UnTHK2 028 028 028 056 000 000 014 000 028 070 070 098 126 000 000 000 028 000 014 00 4427 5739 UnTHK3 028 028 028 056 000 000 014 000 028 070 070 098 126 000 000 000 028 000 014 000 4427 5739 IcGB1 1022 1022 1022 1050 994 994 1008 994 1022 980 980 966 1036 994 994 994 1022 994 1008 994 994 7048 AsGB1 1317 1317 1317 1345 1345 1345 1359 1345 1317 1303 1303 1303 1331 1345 1345 1345 1373 1345 1359 1345 1345 1653

138

139

Table 37 Percentage of pair-wise difference in 18 rDNA gene within and among each horseshoe crab species (mean plusmn SD) Bracketed values indicate

the range of the data

T tridentatus Juvenile C rotundicauda

L polyphemus Juvenile T tridentatus with 1 immovable spine

Juvenile T tridentatus with

3 immovable spines Adult T tridentatus

014plusmn015 (000-028)

Juvenile C Rotundicauda 032plusmn019 (000-070)

017plusmn014 (000-042)

L polyphemus

126plusmn027 (098-182)

099plusmn026 (070-154)

042plusmn029 (000-084)

Juvenile T tridentatus with 1 immovable spine

041plusmn017 (028-084)

014plusmn016 (000-056)

097plusmn026 (070-154)

011plusmn014 (000-028)

Juvenile T tridentatus with 3 immovable spines

040plusmn014 (028-070)

013plusmn013 (000-042)

096plusmn025 (070-140)

010plusmn013 (000-042)

009plusmn008 (000-014)

Ixodes cookei

1029plusmn014 (1022-1050)

1002plusmn013 (994-1022)

991plusmn031 (966-1036)

1000plusmn013 (994-1022)

999plusmn008 (994-1008)

Artemia salina

1324plusmn014 (1317-1345)

1342plusmn015 (1317-1359)

1310plusmn014 (1303-1331)

1351plusmn013 (1345-1373)

1350plusmn008 (1345-1359)

Note From GenBank

140

UnSHK1

UnSHK3

CrHK2

UnTHK2

UnSHK5

UnSHK2

UnTHK3

CrHK4

CrHK1

CrHK3

UnTHK1

UnSHK4

TtHK4

TtHK2

TtHK3

TtHK1

CrGB1

LpGB4

LpGB3

LpGB2

LpGB1

IcGB1

AsGB1

91

30

78

25

35

58

70

43

80

69

100

002 Figure 35 Neighbor-joining tree for the horseshoe crab 18S rDNA gene sequences

Sequences from GenBank

CrGB1 Carcinoscorpius rotundicauda

LpGB1 LpGB2 LpGB3 LpGB4 Limulus polyphemus

IcGB1 Ixodes cookei

AsGB1 Artemia salina

For other species codes refer to Table 35 Number close to the major branches is the

bootstrap value which is a measure of the confidence of the separation of branches of

the Neighbor-joining tree with a maximum value of 100

141


Table 38 shows the comparisons of the 28S rDNA gene including the percentages

of pair-wise difference and the sequences difference (transitions transversions) among

all the tested individuals The mean differences for each species are summarized in

Table 39 Intra-specific differences were small in which the pair-wise difference was

000 for both the adult T tridentatus and the two juvenile T tridentatus groups

009 for L polyphemus and 065 for C rotundicauda In contrast inter-specific

sequence differences were large in which the percentage of pair-wise difference

between L polyphemus and other horseshoe crab species between T tridentatus and C

rotundicauda and between the two juvenile T tridentatus groups were all 882 This

was in agreement with the 18S rDNA results that the American species L polyphemus

showed the largest genetic difference among the three horseshoe crab species For the

two juvenile T tridentatus groups there was no inter-specific difference detected in 28S

rDNA thus these two groups should belong to the same species The pair-wise

difference between these two groups and T tridentatus was 196 and was 039 for C

rotundicauda The number of transitions transversions between the juvenile T

tridentatus groups and adult T tridentatus was 20 while that between the juvenile T

tridentatus groups and C rotundicauda was 0-20-2 (Table 38) This was in agreement

with the 18S rDNA results that the juvenile T tridentatus groups were genetically

closer to C rotundicauda despite they shared more morphological similarities with

adult T tridentatus However owing to the absence of 28S rDNA data from T gigas in

the GenBank and the relatively low inter-specific variations among Indo-Pacific

horseshoe crab species the speciation of juvenile T tridentatus groups especially those

with 1 immovable spine requires further genetic sequence studies For the two

outgroups Ixodes cookei and Artemia salina significantly larger differences with the

two horseshoe crab species and the two groups of juvenile T tridentatus were observed


142

In the Neighbor-joining tree (Fig 36) the American species L polyphemus was

separated as an independent clade adult T tridentatus from Hong Kong was also

separated as an independent clade with one individual of C rotundicauda from the

GenBank while other individuals of C rotundicauda and the two juvenile T tridentatus

groups were clustered under the same clade Ixodes cookei formed an independent clade

from the horseshoe crab species and Artemia salina formed a single clade







143

144



T tridentatus C rotundicauda L polyphemus Juvenile T tridentatus with1 immovable spine


T tridentatus

000plusmn000

C rotundicauda

157plusmn080 (000-196)

065plusmn097 (000-196)

L polyphemus

882plusmn000

882plusmn000

009plusmn015 (000-041)


196plusmn000

039plusmn080 (000-196)

882plusmn000

000plusmn000


196plusmn000

039plusmn081 (000-196)

882plusmn000

000plusmn000

000plusmn000

Ixodes cookei

2843plusmn000

2843plusmn000

2549plusmn000

2843plusmn000

2843plusmn000

Artemia salina

5000plusmn000

4922plusmn044 (4902-5000)

5000plusmn000

4902plusmn000

4902plusmn000

Note From GenBank

145

CrHK3

UnSHK3

CrHK1

CrHK2

UnSHK4

UnSHK5

UnSHK2

UnSHK1

UnTHK3

CrHK4

UnTHK2

UnTHK1

TtHK3

TtHK2

CrGB1

TtHK1

TtHK4

LpGB1

LpGB2

LpGB3

LpGB4

IcGB1

AsGB1

100

87

87

98

100


Sequences from the GenBank



IcGB1 Ixodes cookei





146

34 Genetic Relationships among Horseshoe Crabs from Various Nursery Grounds

in Hong Kong

Different coding regions of the rDNA repeats usually show distinct evolutionary

rates As a result rDNA can provide information about almost any systematic level

(Hillis and Dixon 1991) In the transcribed region spacers are found on the sides of the

transcribed sequences and described as internal transcribed spacers (ITS) For example

18-28S ITS rDNA is the ITS between the 18S and 28S sequences Comparing to 18S

and 28S rDNAs the 18-28S ITS rDNA is fast evolving even amongst genetically closely

related organisms and thus has been successfully used for molecular differentiation


2003) and Asian mitten crabs (Tang et al 2003) Based on this information the 18-28S

ITS rDNA was examined in this study to identify possible gene flow among juvenile

horseshoe crabs at various nursery grounds



A total of five soft shores were visited including Pak Nai and Ha Pak Nai in the

northwestern New Territories and San Tau Shui Hau Wan and Yi O on northern

eastern and southern Lantau Island respectively (Fig 21) Three individuals of T

tridentatus were collected from each site One C rotundicauda was also collected at

Luk Keng as an outgroup (Table 310)The animals were kept alive in the laboratory or

in 95 ethanol for storage

147

Table 310 Grouping of horseshoe crab specimens used in the study of 18-28S

intergenic spacer sequence (ITS) rDNA

Species Population Origin Tachypleus tridentatus TtPN Pak Nai TtHPN Ha Pak Nai TtST San Tau TtSH Shui Hau Wan TtYO Yi O Carcinoscorpius rotundicauda CrHK Hong Kong (Luk Keng)

Note The population abbreviations correspond with their species name and origin

148


Twenty-five mg of tissue from each horseshoe crab was used for the DNA

extraction using DNeasy Tissue Kit One pair of primer was designed for the

amplification of the 18-28S intergenic spacer sequence (ITS) rDNA with forward

primer 5rsquo-AAGGTTACCGTAGGTGAACCTGCG-3rsquo and reverse primer

5rsquo-GGGAATCCCGTATTGGTTTCTTTT-3rsquo

For PCR amplification and sequencing the methodology and materials followed

the procedure of the study of genetic relationships among horseshoe crabs from various

locations in Hong Kong Experimental details can be referred to Section 3312


18-28S ITS rDNA sequences were aligned using Clustal X (Thompson et al 1997)

and pair-wise genetic distances transitions and transversions were calculated using the

MEGA 21 program (Kumar et al 2001) Phylogenetic analysis was performed on

genetic distance matrices using the Neighbor-Joining software in the MEGA 21

package Related ITS rDNA sequences from an individual of Hong Kong C

rotundicauda was used as an outgroup

342 Results

Table 311 shows the comparisons of the 18-28S ITS rDNA gene and the mean

differences of each species are summarized in Table 312 Intra-site differences were

extremely small in which the pair-wise difference within the sites including Pak Nai

Ha Pak Nai San Tau Shui Hau Wan and Yi O all averaged 005The geographical

genetic differences among T tridentatus were also very small in which the percentage

of pair-wise differences among Pak Nai Ha Pak Nai San Tau and Shui Hau Wan were

149

004 while between Yi O and the other four sties were 005 The inter-site

differences were even smaller than those within sites (Table 312) Table 311 shows

that the number of transitions transversions among all the T tridentatus individuals

collected at the five sites was extremely small and ranged between 0 and 0-1 For the

outgroup C rotundicauda significantly larger differences with T tridentatus collected

at the five sites were observed and ranged between 734 and 743

The phylogenetic Neighbor-joining tree (Fig 37) showed that T tridentatus from

all the five sites were mixed together under two clades whereas C rotundicauda formed

a single clade

Table 311 Percentage of pair-wise difference in the 18-28S ITS rDNA gene of horseshoe crab individuals (below diagonal) and number of transitions

transversions (above diagonal) (refer to Table 310 for species codes number represents individual)

Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Cr

PN1 PN2 PN3 HPN1 HPN2 HPN3 ST1 ST2 ST3 SH1 SH2 SH3 YO1 YO2 YO3 HK1

TtPN1 01 00 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtPN2 008 01 01 01 00 00 01 01 01 01 00 00 00 00 5235

TtPN3 000 008 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN1 000 008 000 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN2 000 008 000 000 01 01 00 00 00 00 01 01 01 01 5236

TtHPN3 008 000 008 008 008 00 01 01 01 01 00 00 00 00 5235

TtST1 008 000 008 008 008 000 01 01 01 01 00 00 00 00 5235

TtST2 000 008 000 000 000 008 008 00 00 00 01 01 01 01 5236

TtST3 000 008 000 000 000 008 008 000 00 00 01 01 01 01 5236

TtSH1 000 008 000 000 000 008 008 000 000 00 01 01 01 01 5236

TtSH2 000 008 000 000 000 008 008 000 000 000 01 01 01 01 5236

TtSH3 008 000 008 008 008 000 000 008 008 008 008 00 00 00 5235

TtYO1 008 000 008 008 008 000 000 008 008 008 008 000 00 00 5235

TtYO2 008 000 008 008 008 000 000 008 008 008 008 000 000 00 5235

TtYO3 008 000 008 008 008 000 000 008 008 008 008 000 000 000 5235

CrHK1 743 734 743 743 743 734 734 743 743 743 743 734 734 734 734

150

151

Table 312 Percentage of pair-wise difference in the 18-28S ITS rDNA gene for T tridentatus within each study site and among different study sites

(mean plusmn SD) Bracketed values indicate the range of the data

Pak Nai Ha Pak Nai San Tau Shui Hau Wan Yi O Pak Nai

005plusmn005 (000-008)

Ha Pak Nai

004plusmn004 (000-008)

005plusmn005 (000-008)

San Tau

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Shui Hau Wan

004plusmn004 (000-008)

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Yi O

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

000plusmn000


740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

734plusmn000

152

TtPN1

TtPN3

TtST2

TtHPN2

TtST3

TtHPN1

TtSH1

TtSH2

TtPN2

TtHPN3

TtSH3

TtYO1

TtYO3

TtST1

TtYO2

CrHK1

66

66

001

Figure 37 Neighbor-joining tree for the horseshoe crabs 18-28S ITS rDNA gene

sequences For species codes refer to Table 310 Number close to the major branches is

the bootstrap value which is a measure of confidence of the separation of branches of


153

35 Discussion

351 Morphological and Genetic Differentiation in Horseshoe Crabs

Based on the results obtained locally the adult form of T tridentatus and C

rotundicauda can be easily distinguished by their body size as T tridentatus is the

biggest among the four horseshoe crab species while C rotundicauda is the smallest

For the juveniles the body size of T tridentatus is still bigger than that of C

rotundicauda at the same age with the prosomal width at the age of seven being 550

cm and 433 cm for T tridentatus and C rotundicauda respectively However as the

body size of the juvenile varies with age it is not a good parameter for speciation of

juvenile horseshoe crabs

Among various ratios of body parts significant variations between the two species

were found in ab bc ac cd ef gc and hc (Fig 32) with bc cd af gc and hc

being useful in distinguishing the species as indicated by the discriminant analysis Chiu

and Morton (1999a) showed that the ratio of prosomal width to distance of the two

compound eyes (cd) being significantly different between the two species for both

juveniles and adult with a shorter eye width for C rotundicauda which is associated

with better vision competence (Chiu and Morton 1999a) Although no significant

differences in ab bc and ac were obtained significant differences were found for the

ratios af and gh between mature individuals of these species Nevertheless qualitative

differences including the cross-section of the telson and the presence of spines on the

telson are still reliable and fast identification methods for these two species in field

studies

In the phylogenetic comparison speciation is determined by the distance

measurement in phylogenetic analysis Distance measurement is determined by the

154

percentage of nucleotides that differ between two genomic sequences in which the

lower the percentage of pairwise difference the closer is the genomic relationship

between two individuals The species cutoff level of the distance measurement however

varies from genus to genus Sometimes the species that comprise a particular genus are

very closely related (ie low genetic distance) as with the enterics but in other genera

the species are distantly related (ie high genetic distance) For amphioxus the

inter-specific sequence differences in 18S rDNA were large in which the percentage of

pairwise differences among B malayanum B belcheri and B japonicum ranged from

1900 to 26 with the number of transitionstransversions ranged 60-7349-68 (Chen

et al 2007) However in the present study although the inter-specific variations of

both 18S and 28S rDNA sequence were larger than the intra-specific variations the

percentage of pair-wise differences was only lt 1 and 157 respectively with small

differences in terms of the number of transitionstransversions Thus these two rDNA

regions are not very powerful for the speciation of T tridentatus and C rotundicauda

as low genetic distances were observed among the two horseshoe crab species even

though they are in different genera

From previous literature the three Indo-Pacific horseshoe crab species constituted

a phylogenetically irresolvable trichotomy (Miyazaki et al 1987 Avise et al 1994)

Xia (2000) showed that T gigas and C rotundicauda were genetically closer and they

were grouped together as a monophyletic taxon based on the 16S rDNA and DNA (COI)

genomic sequence In the present study the juvenile T tridentatus including both the

normal (group with three immovable spines) and abnormal forms (group with one

immovable spine) are morphologically closer to the adult T tridentatus but genetically

closer to C rotundicauda as shown by 18S rDNA However the absence of 18S rDNA

data from T gigas in the GenBank precludes a direct comparison with data obtained

155

from the present study Besides with the relatively low inter-specific variations in 18S

and 28S rDNA sequence among the Indo-Pacific horseshoe crab species the speciation

of the juvenile T tridentatus groups especially the abnormal form (with one immovable

spine) requires further investigations on their genetic sequences Other molecular

studies such as the use of mitochondrial DNA (mtDNA) are suggested to be used in the

survey of the overall genetic differences for closely related species Because of the

energy-producing reactions mtDNA carries out mitochondria mutate their DNA at a

rapid rate and are less able to ldquoproofreadrdquo these errors than genes in nuclear DNA

Hence mtDNA has a high mutation rate and thus is a perfect tool for spotting genetic

differences between individuals of closely related species and within a species


Apart from the genetic and morphological variations among the four horseshoe

crabs species geographical variations in the morphology and genetic sequence within

species are receiving much wider attention in recent years Morphological variations

sush as body size in North American species were documented by Riska (1981) and

Sekiguchi (1988c) whereas Sekiguchi et al (1976 1978) studied variations in the

morphological features such as body size colour of the prosoma marginal spine length

etc in T gigas and C rotundicauda collected from different localities Previous studies

have also indicated a difference in the body size of T tridentatus with the populations

from Xiamen of South China the Philippines the coast near Manado in Celebes and

Padang and Sibolga in Sumatra being similar but smaller than those from Kota Kinabalu

(Sekiguchi 1988c) The Japanese population was smaller than those of Southeast Asia

and the smallest individuals were found on the Chou Shan Islands off Ningpo South

China (Sekiguchi 1988c) In addition variations in the shape of the marginal spines of

156

T tridentatus were also recorded on Iki Island (Japan) and in Bomeo (Indonesia) and

Sumatra (Indonesia) (Kato et al 2005)

Morphology of a species is highly influenced by the environment which causes

individualsrsquo variations For example the horseshoe crab in Sundarban West Bengal

was once considered as a subspecies of C rotundicauda due to its unique

morphological differences in body size marginal spines and body colouration (Itow et

al 2004) However genetic analysis has shown that the different forms of C

rotundicauda in Bengal are only individual variations (Kato et al 2006) Hence to

resolve these mysteries of speciation the geographical variation in DNA sequences of

horseshoe crabs has been investigated recently For the American species L polyphemus

populations in the Delaware Bay and Chesapeake Bay were identified as genetically

distinct by using both Random Amplification of Polymorphic DNA (RAPD) and DNA

(COI) genomic sequence (Pierce et al 2000) while regional genetic variations in

Atlantic coast and Gulf of Mexico were identified using the mitochondrial DNA (COI)

genomic sequence and microsatellite DNA markers (King and Eackles 2004)

Geographical variations in genetic sequence were also observed for the Indo-Pacific

species For C rotundicauda seven substitutions of the 16S rRNA locus in the

mitochondrial DNA were confirmed at three localities in a small region including the

Gulf of Bengal in Sittwe City Myanmar the Gulf of Martaban in Mawlamyine City

Myanmar and the Gulf of Siam in Bangkok City Thailand (Kato et al 2006) while six

substitutions were found for L polyphemus between the Atlantic and Gulf of Mexico

(Avise 1994) However only two substitutions of the 16S rRNA locus were confirmed

for T tridentatus on Iki Island in Japan and in Borneo and Sumatra in Indonesia (Kato

et al 2005)

157

In the present study the inter-specific variation of the sequence of 18-28S rDNA

between T tridentatus and the outgroup C rotundicauda was larger than the genomic

18S and 28S rDNA sequence with the percentage of pair-wise difference ranged from

734 to 740 As the number of differences in both transversion and transition among

T tridentatus individuals collected at various sites was very small it implied that there

was no significant spatial variation in the genetic pattern of juvenile T tridentatus in

Hong Kong In contrast significant differences in the sequence of mitochondrial DNA

of T tridentatus were obtained at three localities in Taiwan including Kinmen

Tiexianwei and Dongwei (Yang et al 2007) It is mainly due to the differences in the

spatial distance among the study sites in Hong Kong and Taiwan with Taiwan (35801

square km) being 35 times larger than Hong Kong (1104 square km) Besides mtDNA

is a more sensitive genetic measurement for variations within species than rDNA

Other more sensitive molecular studies such as the use of amplified fragment

length polymorphism (AFLP) are suggested for studying the geographical genetic

variations AFLP is a highly sensitive method for detecting polymorphism in DNA

There are many advantages of AFLP when compared to other marker technologies

including randomly amplified polymorphic DNA (RAPD) restriction fragment length

polymorphism (RFLP) and microsatellites AFLP not only has higher reproducibility

resolution and sensitivity at the whole genome level but the capability to amplify

between 50 and 100 fragments at one time In addition no prior sequence information is

needed for amplification (Vos et al 1995 Meudth amp Clarke 2007) As a result AFLP

has become widely used for the identification of genetic variation in strains or closely

related species of plants fungi animals and bacteria and population genetic studies

Hence further investigations in the spatial variations of the genetic makeup of

horseshoe crabs in local habitats by AFLP are suggested Meanwhile the geographical

158

population genetic study is also suggested to compare horseshoe crabs from different

Asian countries such as Japan Indonesia Malaysia Singapore mainland China and

Hong Kong

Among the three genomic markers 18S rDNA 28S rDNA and 18-28S rDNA ITS

sequence 18-28S rDNA ITS sequence can distinguish T tridentatus from C

rotundicauda better than the genomic 18S and 28S rDNA sequence The 18-28S rDNA

ITS sequence not only provides information on speciation but also some indications of

population differences Therefore it is recommended to use 18-28S rDNA ITS

sequence as a genomic marker for further studies of population genetics in horseshoe

crabs

The horseshoe crabs normally lay their eggs on the upper shore of soft shores

Although the larvae can swim they spend most of the time under the sediment for

avoidance of predators The chance they will be carried by water currents to another site

during high tide therefore is low Hence owing to the limited gene flow among

habitats there may be genetic variations among populations of horseshoe crabs on

different shores Similar observations have been found for L polyphemus in Delaware

and Chesapeake Bay (Pierce et al 2000) and a 17-year tagging experiment also

confirmed that the horseshoe crabs would return to their release beach during the

spawning season (Swan 2005) Therefore it is important to protect the key nursery

grounds for juvenile horseshoe crabs in Hong Kong so as to preserve the genetic

variations among local horseshoe crab populations

159

Chapter 4 Assessment of Human Exploitation of Horseshoe Crabs in

Hong Kong

41 Introduction

Effective conservation involves the identification of the causes of environmental

change and the implementation of practices to manage those changes (Caughley and

Gunn 1996) Hence characterizing and managing the potential threats that harmfully

affect the survival abundance distribution or potential for evolutionary development of

a species are indispensable in a conservation strategy (Falk 1990 Bradstock et al

1995) Most of the human activities fall into five major categories including

overexploitation habitat destruction the introduction of non-native species the spread

of diseases and pollution (Wilson 1992 Burgman et al 2007) In most of the

assessments of species of conservation importance habitat destruction by urban

development and disease are usually the most serious threats around the world but

overexploitation is also serious (Wilcove et al 1998)

Adult horseshoe crabs do not have many predators in the natural environment and

thus have a low mortality due to predation Human activities generally account for the

greatest mortality of the horseshoe crabs and result in a rapid decline in horseshoe crab

populations (Berkson and Shuster 1999 Rutecki et al 2004) Among the five major

categories of human activity overexploitation is one of the key factors for the

population decline For Limulus polyphemus it is currently harvested for biomedical

scientific and fishing bait purposes (Loveland et al 1996 Rutecki et al 2004) In

contrast although Asian horseshoe crabs are also used for medicinal uses a majority of

them are harvested as a source of food Southeast Asian residents especially the Fukien

and Hokklo of South China consider horseshoe crabs as a delicacy and highly

160

nutritious food (Cheung et al 1975 Chiu and Morton 2003a) In Hong Kong

horseshoe crabs could also be seen for sale in local seafood restaurants Some cook

books and magazines even taught people how to cook horseshoe crabs and promoted

horseshoe crab eating by emphasizing its taste and nutritious value (Cheung 1995

Zhao 2006) Therefore it is suspected that human exploitation may be one of the major

factors contributing to the decline of horseshoe crabs in Hong Kong (Chiu and Morton

1999a 2003a) In this section market surveys on the harvest and sale of these animals

were investigated in order to evaluate the impact of local human exploitation on the




A total of 11 popular sites for sale of seafood including Po Toi O Sai Kung Lei

Yue Mun Aberdeen Ap Lei Chau Lau Fau Shan Sam Shing Street Tai O Lamma

Island Cheung Chau and Ping Chau 34 seafood restaurants and 150 fish sellers and

fish handlers were surveyed Fishermen in Cheung Chau and at the Aberdeen fish

wholesale market were also interviewed

422 Data Collection

The seafood restaurants and fish sellers were interviewed monthly for information

on the sale of adult horseshoe crabs for 13 months from September 2004 to September

2005 using a standard questionnaire (Appendix 41) Meanwhile interviews of fish

sellers at the Aberdeen fish wholesale market were also carried out monthly for 13

months to record the number of horseshoe crabs being caught in local waters and

elsewhere during the survey period

161

43 Results

431 Catch of Horseshoe Crabs

Through interviewing the fish sellers in fish stalls seafood restaurants and the

wholesale market in Aberdeen a total of 1023 individuals of horseshoe crabs (mostly T

tridentatus as its size is relatively larger than C rotundicauda) were caught from

September 2004 to September 2005 (13-month period) (Fig 41) Most of the horseshoe

crabs were caught by shrimp trawlers occasionally by netting and cage fishing

methods A maximum of 258 individuals was obtained in September 2004 while only 4

individuals were caught in both May and June 2005 For seasonal variations higher

catches (~50 individuals month-1) were obtained from September to December 2004

than in other months (~10 individuals month-1) The total catch in these four months

constituted 79 of the annual catch from 2004 to 2005 By contrast an average of only

11 individuals month-1 was caught from March to August 2005 and none was obtained

in Chinese waters in the summer (from April to August) Similar phenomenon of high

catch of horseshoe crabs in the winter was obtained from the interviews with the

fishermen The highest catch in September was possibly caused by the resumption of

fishing after the seasonal fishing moratorium from June to July in South China Sea

Referring to the results of the interviews the size of horseshoe crabs caught ranged

from 15 cm to 40 cm (maximum prosomal width) with an average weight of 21 kg

(from 075 to 625 kg) Among 1023 horseshoe crabs caught two-third (68) of them

were obtained from South China Sea the remaining (332 individuals or 32) were

caught in Hong Kong waters (Fig 41) From the results of the present survey the sites

where horseshoe crabs were caught in Hong Kong waters included the eastern waters

(Port Shelter Sai Kung Tseung Kwan O Lei Yue Mun and Kwo Chau Wan) western

waters (Pak Nai Lung Kwu Tan and Tuen Mun) southern waters (Sandy Bay

162

Aberdeen Shek Pai Wan Tai Shue Wan Tai Tam Cheung Chau Tung Wan Lamma

Island Shek Kwu Chau and Po Toi) and near Lantau Island (Shek Pik Cheung Sha and

Chok Ko Wan (Tai Siu A Chau)) Among these locations more horseshoe crabs were

obtained near Sai Kung Kwo Chau Wan and Cheung Chau however none were caught

in the waters of Ping Chau in the past ten years

Owing to the low market value some of the horseshoe crabs caught in the net (103

individuals or 31 of total catch in Hong Kong waters) were released immediately back

to the sea with the highest number of the release being obtained in September 2004 (52

individuals or ~50 of the annual release) In other months from October 2004 to

September 2005 only an average of 43 individuals month-1 was released After

subtracting the number of horseshoe crabs being set free the sale of local horseshoe

crabs to seafood markets and restaurants was 229 individuals or an average of 17

individuals month-1

For the horseshoe crabs caught in China waters most of them were caught by

Hong Kong fishing vessels while a small number was transported to Hong Kong by

trucks Similarly one-third of them were released back to the sea after catch with the

remaining two-thirds (or 461 individuals) being transported and sold to Hong Kong (Fig

43) Similar to the horseshoe crabs being caught in Hong Kong higher catches in China

waters were obtained in the winter with all the set-free activities being carried out in

September and October 2004 Considering all the catches from Hong Kong and China

waters a total of 690 horseshoe crabs were sold in a year in which 67 (or 461

individuals) of them were caught in China waters

163

0

50

100

150

200

250

300

Sept Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sept

04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Hong Kong Waters China Waters

Figure 41 Number of Tachypleus tridentatus being caught in Hong Kong and China

waters from September 2004 to September 2005

164

0

20

40

60

80

100

120

140


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free

Figure 42 Number of Tachypleus tridentatus being caught in Hong Kong waters with

proportions for sale and set-free from September 2004 to September 2005

165

0

20

40

60

80

100

120

140

160


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free

Figure 43 Number of Tachypleus tridentatus being caught in China waters with


166

432 Sale of Horseshoe Crabs

A total of 690 horseshoe crabs were sold from September 2004 to September 2005

They were sold to seafood restaurants or local people from fish stalls in Sai Kung Lei

Yue Mun Sam Shing Street Ap Lei Chau Tai O and Cheung Chau or to the Aberdeen

wholesale market which were then transported to local seafood restaurants However

no horseshoe crabs were observed in the seafood restaurants and markets in Po Toi O

Lau Fau Shan Lamma Island and Ping Chau According to the interviews with the

fishermen and old villagers at these sites horseshoe crabs had not been sold since five

years ago Horseshoe crabs sold served three major purposes namely set-free ritual

display and food

4321 Set-free Rituals

Setting animals free is a traditional ritual in Buddhism Many people think that

releasing animals from captivity will bring people good luck or fortune or atone for

their sins especially in Buddhist and Chinese festivals (KFBG 2006) Birds tortoises

and fish are common animals for such set-free activities The horseshoe crab is also one

of the common set-free animals due to its relatively long life span (up to 20 years old)

large body size and historical background as a living fossil

In the present survey about 62 (or 425 individuals) of the sale of horseshoe crabs

were purposed for the set-free activity with the highest sales in November and

December (170 individuals month-1) (Fig 44) Only 265 individuals were actually

retained and sold to fish stalls and seafood restaurants

167

0

20

40

60

80

100

120

140

160

180

Sept Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sept04 05

Month

Sale

of H

orse

shoe

Cra

bs

Release Non-release

Figure 44 The sale of horseshoe crabs for the release and non-release activities from

September 2004 to September 2005

168

4322 Display of Horseshoe Crabs

Horseshoe crabs can be commonly found in aquaria of large seafood restaurants

and fish stalls in Hong Kong including Sai Kung Lei Yue Mun Sam Shing Street and

Cheung Chau throughout the year However not all the horseshoe crabs kept in seafood

restaurants would be served for dishes In large seafood restaurants and fish stalls

marine animals such as seahorses and rays are displayed in the aquaria as attractions to

customers Owing to their special appearance horseshoe crabs are also kept for display

and photo-taking by tourists During the survey period an average of 30 horseshoe

crabs were found in seafood restaurants and fish stalls (Fig 45) All of them belonged

to T tridentatus with an average prosomal width of 25 cm They were usually kept for

several months The number in captivity did not vary significantly over the study period

although fewer horseshoe crabs were observed in the summer Those horseshoe crabs

which had been kept in captivity for a long time may also die if no proper care and food

were offered

169

0

10

20

30

40

50

60


Time

Num

ber o

f Hor

sesh

oe C

rabs

Figure 45 Number of T tridentatus displayed in fish stalls and seafood restaurants

from September 2004 to September 2005

170

4323 Serving Horseshoe Crabs for Dishes

In this market survey only 3 restaurants served horseshoe crabs for dishes with an

average sale of 17 individuals month-1 or 224 individuals throughout the year They

were located at Sai Kung Lamma Island and Cheung Chau In addition a restaurant in

Causeway Bay also served horseshoe crabs with an average of sale of 17 individuals

month-1 (Fig 46)

Only 45 of the horseshoe crabs being sold were caught in Hong Kong waters

while the remaining individuals were transported from mainland China (Fig 47) More

horseshoe crabs were served as dishes in the first half of the survey (September 2004 to

February 2005) with an average of 34 individuals month-1 In the second half of the

survey from March to September 2005 horseshoe crabs were still found in seafood

restaurants and fish stalls in Sai Kung Lei Yue Mun Sam Shing Street and Cheung

Chau regularly however fewer horseshoe crabs were served as dishes especially for

Sai Kung with only 23 individuals There was a significant temporal variation in the

consumption of horseshoe crabs

In general female horseshoe crabs are preferable than males owing to their larger

body size People cook the horseshoe crab eggs by steaming or frying them with

chicken eggs Serving as soup or in stew is another common cooking method for

horseshoe crabs One seafood restaurant owner even claimed that it have a vital

nutritious value from the Chinese medical point of view Furthermore legs are also

served as dishes by frying just like common decapod crustaceans In the past it was

even salted and cooked with dried fish peanuts beans sugar or vinegar

171

a

b

Figure 46 The advertisements of horseshoe crab dishes were shown in a) Cheung

Chau seafood restaurant b) Causeway Bay seafood restaurant

172

0

10

20

30

40

50

60

70


Month

Sale

of H

orse

shoe

Cra

bs

HK Waters China Waters

Figure 47 Estimated sale of horseshoe crabs for local consumption from September

2004 to September 2005 Individuals being caught from both Hong Kong and China

waters are shown

173

44 Discussion

441 Population of Horseshoe Crabs in Open Waters

Horseshoe crabs spend their first ten years at soft shores and move into open

waters in the adult stage In Chapter 2 the extensive and intensive (6-month study)

surveys on various soft shores have provided the most up-to-date distribution and

population status of juvenile horseshoe crabs in Hong Kong This section provides an

updated distribution of adult horseshoe crabs in Hong Kong through the market survey

and interviews

Horseshoe crabs are previously named as ldquorock crabrdquo or ldquotankrdquo in the old villages

They were commonly found on beaches and in deeper waters and easily caught by

shrimp trawlers stern trawlers and netting According to the interviews conducted with

various local fishermen adult horseshoe crabs were previoiusly found in a wide range

of locations from the western estuarine waters such as Tuen Mun Pak Nai and Lung

Kwu Tan to the eastern oceanic waters including Port Shelter Sai Kung Tseung Kwan

O Lei Yue Mun and Kwo Chau Wan In addition horseshoe crabs also occurred in the

southern waters near Hong Kong Island and outlying islands including Cheung Chau

Tung Wan Lamma Island Shek Kwu Chau Po Toi and around Lantau Island

Furthermore the local distribution of adult horseshoe crabs was studied by visiting

various sandy and muddy shores fishing villages markets and restaurants in the period

of 1995 to 1998 (Chiu and Morton 1999a) By interviewing local fishermen coastal

villagers and middle- and old-aged people local distribution of adult horseshoe crabs

prior to 1995 was also recorded (Chiu and Morton 1999a) The past distribution of

horseshoe crabs is summarized in Table 41

174

Table 41 Historical records of adult horseshoe crabs in open waters in Hong Kong

with reference to Chiu and Morton (1999a)

Location Year Number recorded

Species

Western waters

Deep Bay 1988 - T tridentatus and T gigas 1997 2

Lau Fau Shan 1997 1

Lung Kwu Sheung Tan 1994 1 T tridentatus Tuen Mun 1995 1 T tridentatus Eastern Waters

Ping Chau Mirs Bay 1988 - T tridentatus and T gigas 1994 -

Tai Po Tolo Harbour 1988 - T tridentatus and T gigas

Silverstrand beach Sai Kung 1997 2

Port Shelter Sai Kung 1997 2 Southern Waters

Lobster Bay Cape drsquo Aguilar 1991 2 T tridentatus

Waglan Island 1993 2 T tridentatus

Tai Tam Bay 1994 1 T tridentatus East Lamma Channel 1994 1 T tridentatus 1997 4 T tridentatus West Lamma Channel 1997 2 T tridentatus Sok Ko Wan Lamma Island 1995 1 T tridentatus Sha Chau 1994 1 1995 1 C rotundicauda South of Shek Kwu Chau 1995 2 T tridentatus Silver Mine Bay Lantau Island 1995 1 T tridentatus Tung Chung Wan Lantau Island 1997 32 T tridentatus

1997 18 T tridentatus Northwest of Lantau Island (Tai O Yi O Sam Wat Wan Sha Lo Wan) 1997 63 C rotundicauda

175

When compared with previous records the distribution pattern of adult horseshoe

crabs remained the same in the past ten years The present survey indicated that

horseshoe crabs were still found in the eastern waters western waters southern waters

outlying islands and Lantau Island According to Chiu and Morton (1999a) adult

horseshoe crabs could also be observed in the mudflats in Mai Po Tsim Bei Tsui and

Lung Kwu Sheung Tan in northwestern New Territories Starfish bay Sharp Island and

Pak Sha Wan in northeastern New Territories Kong Sin Wan and Kau Pei Chau on

Hong Kong Island Tung O Wan on Lamma Island and Pui O and Yi O on Lantau

Island before 1998 with mating pairs being occasionally encountered in the mudflats

However none of the mating pairs or adult horseshoe crabs was recorded in the 17 local

sandy or muddy shores in the present distribution study including Tsim Bei Tsui Pui O

and Yi O (Chapter 2)

According to the old fishermen being interviewed the population of horseshoe

crabs has dropped sharply in Hong Kong waters in recent years They were rarely found

in the past 5 years comparing with 15 to 20 years ago when about 15 horseshoe crabs

day -1 were caught as by-catch The catch has dropped to lt 10 individuals year-1 fishing

vessel-1 and in the present study a total of only 332 horseshoe crabs were reported to be

caught in Hong Kong waters in 13 months

442 Human Exploitation of Horseshoe Crabs

Human exploitation refers to the use of natural resources by man for various

purposes including consumption medical use and research etc For the American

species Limulus polyphemus as early as 1850s millions were harvested annually from

the breeding beaches of Delaware Bay and grounded up as fertilizer and livestock food

They also became preferred bait for fisheries of eels and whelks since 1980s (Rudloe

176

1982) In addition horseshoe crabs have also been harvested to support the production

of Limulus amebocyte lysate from their blood by the pharmaceutical industry (Cohen

1979 Pearson and Weary 1980) About 3000-5000 individuals year-1 of L

polyphemus were used in the neurophysiological and other basic research (Rudloe

1982)

For the Indo-Pacific species they have also been served for medical purposes since

early times Indians used them to reduce pains by tying them on the arms (Chatterji

1994) The tail tips were used for healing arthritis or other joint pains and the carapace

boiled with mustard oil was used for treating rheumatic pain in West Bengal and Orissa

India (Chatterji 1994) In addition the nearly pure form of chitin extracted from the

horseshoe crabrsquos carapace has been explored as possible dressing for burns that

promotes faster healing (Chiu and Morton 1999a) In China as early as in Song

Dynasty the Chinese medical book ldquo嘉佑本草rdquo recorded that the meat of horseshoe

crabs can be used for curing sore the eggs for eye disease the liver for cold and cough

and the carapace for fever (Wan 2005)

Apart from medical uses horseshoe crabs have been served as dishes for a long

history Eggs of T gigas and C rotundicauda have been reported to be eaten in

Thailand (Rudloe 1982 Kungsuwan et al 1987) People in Singapore Malaysia and

Borneo also regard the eggs of T gigas as a delicacy (Chatterji 1994) In China

horseshoe crabs have been regarded as valuable food since Tong Dynasty (Wang 1986)

In Hong Kong many local villagers and fishermen think that T tridentatus has a high

nutritional value

177

In the present survey horseshoe crabs were kept and sold in local seafood markets

and restaurants in Sai Kung Lei Yue Mun Sam Shing Street Causeway Bay Ap Lei

Chau Aberdeen Tai O and Cheung Chau In the interview survey by Chiu and Morton

(1999a) adult horseshoe crabs were also kept in local markets in similar locations

including Tsuen Wan Yuen Long Peng Chau Sai Kung Shau Kei Wan Cheung Chau

Lamma Island and Mui Wo Lantau Island from 1995 to 1998 Surprisingly although

horseshoe crabs are commonly seen in seafood restaurants the present survey indicated

that only one-third of them were served as food Another 62 was sold for set-free

rituals and 5 for display

Although setting wildlife free is a traditional ritual for gaining luck recent

researches have shown that this activity may cause mass death of wildlife or damages to

ecosystems For wild animals such as birds as they are kept in crowded cages some of

them may die before being set free Some animals are released into unsuitable habitats

and die in a short period of time after being released More importantly animals may be

released to a new environment and become invasive species which may cause damages

to the ecosystem by transmitting new diseases or reducing the number of the native

species due to their aggressive behaviour and competition (Yan 2007) Owing to the

adverse impact of set-free rituals China and some regions in Taiwan have restricted the

set-free rituals by introducing a permit system Without permissions from the

government the maximum penalty of the set-free activity is RM$10000 in China (Lau

2007) In view of the disadvantages of the set-free rituals some of the Buddhism

associations have recommended environmental protection activities such as tree

planting activity or donating money to charity instead of having the set-free rituals

(Lam 2006) For horseshoe crabs as their distribution is not restricted to certain

localities of Hong Kong waters their release should not cause any problems to the

178

environment where they are released However if the horseshoe crabs are not properly

handled and maintained in captivity before being released the set-free activity will have

a potential risk to their health and survival

Although eating horseshoe crabs has a long history in China including Hong Kong

they are not popular seafood with high commercial value Many of them are only caught

as by-catch not as target organisms by shrimp trawlers People who consume the

horseshoe crabs are mainly curious for the taste of this special ldquomonsterrdquo The results of

the present survey have confirmed this observation as only 224 individuals of horseshoe

crabs served as dishes in 2004-2005 Another reason for the low consumption rate is

due to the risk of food poisoning by eating horseshoe crabs According to Liao and Li

(2001) the major cause of the poisoning is tetrodotoxin from C rotundicauda although

some cases were due to allergic reactions from eating T tridentatus Similar result were

also obtained in the study of Ngy et al (2007) in which tetrodotoxin was the main toxin

found in C rotundicauda where no paralytic shellfish toxins were detected Although it

is believed that T tridentatus is not poisonous it is difficult for the public to distinguish

between the juveniles of T tridentatus and C rotundicauda so eating horseshoe crabs

may impose a potential risk of food poisoning Besides it is claimed that by removing

the intestine of horseshoe crabs carefully horseshoe crabs should be safe for

consumption (Cheung 1995) However as few people know how to cook horseshoe

crabs properly nowadays they hence become rarely served as dishes

Furthermore the present survey has indicated seasonal variations of the

consumption of horseshoe crabs with a higher rate being obtained in the winter from

November to December It may be due to the traditional Chinese medical view that

different types of food are good to health in different seasons For example snake soup

179

and Chinese mitten crab (Eriocheir sinensis H Milne-Edwards) are only served as

dishes in late autumn and winter as they are believed to help keep warm and preserve

energy by providing an unique ldquoinner warmthrdquo especially in the cold days It is also

believed that these foods have their own properties of ldquoheatingrdquo for helping the balance

of the bodyrsquos natural forces strengthening the ankles curing malaria and preventing all

sorts of diseases that come from exposure to cold winds (Specter 1984 Hong Kong

Tourism Board 2008 Wikipedia 2008) Similarly horseshoe crabs are also regarded as

good at nourishing both vital energy and blood from the Chinese medical view

therefore they are favored in winter for ldquoinner warmthrdquo supply

443 Potential Risks to Horseshoe Crabs

Based on the market survey the sale of horseshoe crabs was relatively low in

comparing with other popular commercial marine species in Hong Kong However

considering the long maturity period and low breeding rate of horseshoe crabs human

exploitation still contributes a potential impact and even puts further pressure on the

scattered populations of this animal in Hong Kong Although most of the horseshoe

crabs kept in the seafood restaurants were mainly for display without properly care and

food supply they may eventually die in captivity This also reduces the number of

potential breeding horseshoe crabs in the wild and further decreases the chance of

successful pair matching and breeding The traditional set-free rituals also create

potential threats to the survival of those ldquoreleasingrdquo horseshoe crabs as they may have

been injured or mishandled Chiu and Morton (1999a) suggested that horseshoe crabs

should become a banned fishery target and be protected under the Wild Animals

Protection Ordinance (Cap 170) Educating the public not to select this species for

set-free activities or for consumption will be helpful in protecting the existing

populations in local waters Since horseshoe crabs are not popular seafood in Hong

180

Kong banning their sale should not cause any serious effects on the livelihood of

fishermen restaurant owners and citizens

181

Chapter 5 Trials on Artificial Breeding of Horseshoe Crabs

51 Introduction

Artificial breeding has become one of the major tools used in conservation to

enhance the population of endangered species in recent years Artificial breeding

programmes of some endangered species have been well developed and are in practice

nowadays One of the successful cases is the Giant Panda (Ailuropoda melanoleuca) in

China Up to now a total of 161 individuals of Giant Panda were bred in captivity

Thanks to the advances in artificial fertilization technologies the birth rate of artificially

bred Giant Panda has increased 90 (Cao 2004) Together with the increase in wild

populations there is a 40 increase in panda population in the past 20 years

In view of extrinsic factors including habitat destruction and degradation of

spawning andor nursery grounds due to urbanization human exploitation and pollution

and intrinsic factors such as slow juvenile growth and late maturity horseshoe crabs are

facing serious threats which may lead to extinction of the species Apart from

conserving local horseshoe crabs through conserving natural habitats and reducing

human exploitation artificial insemination and breeding in the laboratory may be a

viable option in mass production of the juveniles which could be released to the wild to

enhance the population To make this a success the understanding of the interplay

among environmental factors on the survival and hatching success of the juvenile

horseshoe crabs is essential but at present such information is poorly known for

Indo-Pacific species (Li et al 1999 Wang et al 2001a) In contrast the methods of

artificial insemination are well established for Limulus polyphemus (Brown and

Clapper 1981 Smith and Berkson 2005) to aim at providing eggs trilobites and

juveniles for laboratory experiments (Laughlin 1983) This section reports the results of

several trials of artificial breeding in T tridentatus including the effects of salinity and

182

temperature on the survival and hatching rates of fertilized eggs Owing to the limited

number of adults available in the market and the rapid decline of horseshoe crabs in the

wild a preliminary trial on a non-destructive artificial breeding method was also

conducted



Three pairs of adult horseshoe crabs were purchased in July 2004 They were

dissected and the sperms and eggs were extracted separately The procedures of

collection of eggs and sperms were adopted from Sekiguchi (1988a) and Hong et al

(2002) Eggs were obtained from females by direct extraction from the ovaries by

cutting open the carapace of the ventral side of the prosoma (Fig 51) The eggs were

washed several times with filtered seawater to remove the blood and body fluid Sperms

were extracted by cutting open the ventral side of the prosoma and the base of the

appendages of male horseshoe crabs to obtain a mixture of body fluid and sperm

solution The eggs were mixed with the sperm solution for 15 minutes with a ratio of

sperms to filtered seawater being 1 20 Filtered seawater was then used to remove

excess sperms Fragments of body tissue sticking on the eggs were removed and the

eggs were transferred to a water table for further incubation (Fig 52) The fertilized

eggs were incubated in filtered seawater at 28degC with oxygen supply and addition of

antibiotics including penicillin and streptomycin which are common antibiotics for

larval culture (Strathmann 1987)

183

b

a

Figure 51 The direct extraction of horseshoe crab eggs a) body view of a female

horseshoe crab b) extraction of eggs from one side of the ovary of a female horseshoe

crab

184


temperature control

185

522 Results

Over 10000 eggs were extracted from three female horseshoe crabs and fertilized

in vitro The diameter of fertilized eggs ranged from 2-3 mm and increased during

development The development of the eggs is divided into eight stages 1) just fertilized

2) period of cleavage 3) period of gastrula 4) period of tissue differentiation (ie the

first embryonic molting) 5) period of tissue differentiation (ie the second embryonic

molting) 6) period of tissue differentiation (ie the third embryonic molting) 7) period

of fast growth (ie the fourth embryonic molting) and 8) hatching (Fig 53) (Brown

and Clapper 1981 Wang et al 2001b Hong et al 2002) These stages of development

were also observed in the present study

Both fertilized and unfertilized eggs had indentations on the surface and were

sticky in nature The fertilized eggs however became spherical and the indentations

disappeared after 1 day (Fig 53a) The eggs then underwent cell division at Stage 2

(period of cleavage) Segmentation of the yolk inside the egg membrane was observed

Different segmentations could be observed among the eggs ie some with deep grooves

and some with the appearance of blocks of blastomeres on the surface (Fig 53b)

Owing to rapid cell division blastomeres became smaller in size and more uniform in

shape At Stage 3 (the period of gastrula) the irregularly-shaped indentation was

observed (Fig 53c) The indentation represented the area of the formation of germ disc

At Stage 4 (period of tissue differentiation) the yellowish outermost embryonic shell

was broken with a transparent inner embryonic shell observed The first embryonic

molting was undergoing at this stage The external organ was formed while the internal

tissue was undergoing differentiation The initial shape of the larva could be observed

The prosoma of the larva was developed however lifted together connecting with a

pyramid shape as the initial formation of the opisthosma and tail (Fig 53d) The second

186

embryonic molting was undergoing at Stage 5 (period of tissue differentiation) The egg

became larger (about 55 mm) The prosoma of the larva was opened horizontally with

two compound eyes formed The 6 pairs of appendages could also be observed with

slight movement (Fig 53e) The third embryonic molting occurred at Stage 6 of the

period of tissue differentiation Meanwhile the diameter of the egg increased to about 6

mm The opisthosomal carapace was formed The appendages of the larva became

longer and the larva moved freely within the extra-embryonic shell (Fig 53f) The

larval movement caused rolling around of the shell in the container The larva increased

in size rapidly at Stage 7 (period of fast growth) and underwent the fourth embryonic

molting The opisthosomal appendages were greatly flattened after molting and the

larva reached the size of the extra-embryonic shell (Fig 53g) Hence at Stage 7 the

larva failed to move around within the shell At Stage 8 the tribolite larva continued to

flex and straighten the opisthosoma in order to break the shell The hatched larva swam

freely and frequently in an upside down position (Fig 53h)

The first instar looked similar to the adult however the prosomal width was only 7

mm and light yellow in colour After 60 days the first batch of first instar underwent

first molting and the prosomal width increased to 9 mm The telson of the larva was

formed in the second instar with the body colour of light brown The larvae were reared

in a fish tank with distilled seawater and fed with newly hatched Artemia (brine shrimp)

larvae

During the 2-year experimental period from 2004-2006 some horseshoe crabs had

molted four times and became the fifth instars The prosomal width of the third and

fourth instar was about 12 mm and 15 mm respectively The body colour became

darker with a longer telson comparing with the second instar They were reared in a

187

fish tank with filtered seawater and 5 cm sediment obtained from the nursery ground at

Pak Nai A mixture of newly hatched Artemia and adult Artemia were fed to the third

and fourth instars Figure 54 shows the second to fourth instars After two years of the

experiment about 15 of the eggs developed into larvae (around 4000 individuals) but

only some 20 individuals survived for two years It was mainly due to the massive

mortality of the second instars from February to July 2005 Some of the surviving

juveniles (now over 3 years old) are now in display at the Ocean Park Hong Kong

188

a b

d c

f e

g h

Figure 53 Various developmental stages of horseshoe crab eggs a) fertilized egg b) period of cleavage c) period of gastrula d) period of tissue differentiation (first embryonic molting) e) period of tissue differentiation (second embryonic molting) f) period of tissue differentiation (third embryonic molting) g) period of fast growth (fourth embryonic molting) h) hatching Scale bar in 10-4 m

189

Figure 54 Different instars of the horseshoe crab a) second instar b) third instar c) fourth instar Scale bar in mm

190

53 Effects of Temperature and Salinity on Egg Development


5311 Fertilization of Eggs

Two pairs of horseshoe crabs were purchased from a seafood restaurant in Cheung

Chau in July 2005 They were dissected in the laboratory and the sperms and eggs were

extracted separately on 11th July 2005 using the method mentioned in Section 521 The

eggs were then removed and transferred to petri dishes (9 cm diameter x 15 cm high)

for further incubation Antibiotics including penicillin and streptomycin were added

5312 Incubation under Different Combinations of Temperature and Salinity

To investigate the effect of temperature and salinity on the survival rate of the

eggs a combination of 3 different temperatures (20 25 and 32degC) and 4 salinities (15

20 25 and 32permil) were tested Therefore 12 treatments were examined (Table 51)

Horseshoe crab eggs were reared in thermostatically controlled incubators until

hatching Filtered seawater was changed every other day


Percent hatchingsurvival data were arcsine square-root transformed to conform to

normality (Zar 1996) Differences in the survival rate of the eggs under different

combinations of temperature and salinity were compared using two-way ANOVA with

a significance level of α = 005 All the statistical analyses were undertaken using

software SPSS 110 If a significant difference was obtained among the treatments the

multiple comparison Tukey test was used with a significance level of α = 005 For

significant interactions between temperature and salinity a series of one-way ANOVA

was used to test the effect of each factor separately

191

Table 51 Different combinations of temperature and salinity were used in culturing the

eggs of T tridentatus

Treatment Temperature degC Salinity permil No of eggs per dish 1 20 15 50 2 20 20 50 3 20 25 50 4 20 30 50 5 25 15 50 6 25 20 50 7 25 25 50 8 25 30 50 9 32 15 50 10 32 20 50 11 32 25 50 12 32 30 50

192

532 Results

5321 Survival Rate of Horseshoe Crab Eggs

After 90 days about 63 of the eggs survived after artificial breeding with the

survival rate being significantly affected by both temperature (F 2 181 = 76110 p lt

0001) and salinity (F 3 181 = 12082 p lt 0001) (Table 52) The survival rates of the

eggs at 20 and 25degC were significantly higher than that at 32degC For the effect of

salinity the survival rate of the eggs decreased gradually as salinity increased with a

significantly higher survival rate being obtained at 15permil and 20permil

Figure 55(a-d) shows the effect of temperature on the survivorship of the eggs

under 4 different salinities At all salinities there was a significant variation in the

survivorship among the three tested temperatures with higher survival rates at 20 and

25degC (Table 53) Figure 56(a-c) shows the effect of salinity on the survivorship of the

eggs under the three different temperatures At 20degC there was no significant difference

in the survivorship of the eggs among the three salinities (F 3 58 = 2072 p = 0114)

(Table 53) At 25degC although significant differences (F 3 66 = 3114 p = 0032) in

survivorship were found among different salinities the differences were marginal and

could not be detected by the Tukey multiple comparison test The effect of salinity

however was significant (F 3 59 = 10094 plt0001) at 32degC with the results obtained at

15permil and 20permil being higher than those obtained at 25permil and 30permil

193

Table 52 Results of the two-way ANOVA test followed by the Tukey multiple

comparison test for differences in the survival rate of the horseshoe crab eggs at 3

temperatures and 4 salinities (p lt 005)

Two-way ANOVA on Survival Rate F df p-value Between Temperature 76110 2 lt0001 Between Salinity 12082 3 lt0001 Interaction between Temperature and Salinity 3806 6 0001

Within error 181 Tukey Test for Temperature Comparison Mean Difference p-value 20degC vs 25degC 00334 0712 20degC vs 32degC 05035 lt0001 25degC vs 32degC 04701 lt0001 Tukey Test for Salinity Comparison Mean Difference p-value 15permil vs 20permil 01520 0012 15permil vs 25permil 02975 lt0001 15permil vs 30permil 02790 lt0001 20permil vs 25permil 01455 0016 20permil vs 30permil 01269 0047 25permil vs 30permil -00185 0982 Significant at p lt 005

Figure 55 The survival rate (plusmn SE) of the horseshoe crab eggs under different temperatures at the salinity of a) 15permil b) 20permil c) 25permil d) 30permil

Same letter denoted no difference between treatments

0102030405060708090

100

20 25 32Temperature (degC)

Surv

ivor

ship

()

(a) 15 permil (b) 20 permil

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(c) 25 permil

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(d) 30 permil

194

195

Figure 56 The survival rate (plusmn SE) of horseshoe crab eggs under different salinities at the temperature of a) 20degC b) 25degC c) 32degC Same letter

denoted no difference between treatments

(b) 25

70

80

90

)

0

10

20

30

40

50

60

15 20 25 30Salinity (permil)

Surv

ivor

ship

(

(a) 20

8090

100

)

010203040506070


Surv

ivor

ship

(

(c) 32

0

10

20

30

40

50

60

70

80


Surv

ivor

ship

()

196 Table 53 Results of the multiple comparisons of the effect of temperature at individual

salinities and the effect of salinity at individual temperatures on the survival rate of

horseshoe crab eggs (p lt 005)

One-way ANOVA on Survival Rate I Temperature Comparisons Salinity F p-value Tukey test 15permil 9142 lt0001 20 degC = 25 degC gt 32 degC 20permil 6468 0003 25 degC = 20 degC gt 32 degC 25permil 34009 lt0001 20 degC = 25 degC gt 32 degC 30permil 55936 lt0001 20 degC = 25 degC gt 32 degC II Salinity Comparisons Temperature F p-value Tukey test 20degC 2072 0114 15 permil = 20 permil = 25 permil = 30 permil 25degC 3114 0032 15 permil = 20 permil = 25 permil = 30 permil 32degC 10094 lt0001 15 permil = 20 permil gt 25 permil = 30 permil


197 5322 Hatching Rate of Horseshoe Crab Eggs

After culturing the eggs at different combinations of temperature and salinity in an

incubator for 90 days 14 tribolite larvae hatched (Table 54) with half of them being

found at the highest temperature and salinity (32degC and 30 permil respectively) Hatching

was successful at all the three tested temperatures but no hatching was observed at a

salinity of 15permil Therefore salinity seemed to be a more important determining factor

than temperature in the hatching of the eggs

198 Table 54 Number of juvenile horseshoe crabs hatched under different combinations of

temperature and salinity for 90 days

Treatment Temperature degC Salinity permil No of juveniles hatched

1 20 15 0 2 20 20 2 3 20 25 0 4 20 30 1 5 25 15 0 6 25 20 0 7 25 25 1 8 25 30 0 9 32 15 0 10 32 20 2 11 32 25 1 12 32 30 7

199 54 Preliminary Study on Alternative Artificial Breeding Method


5411 Check for the Maturity of Horseshoe Crab Eggs

To ensure the presence of mature eggs inside the female horseshoe crab a small cut

was made on the ventral side of the prosomal carapace near the ovary to check the

presence and size of the eggs inside Mature and healthy horseshoe crab eggs are

yellowish white in colour with a size gt 25 mm

5412 Electrical Stimulation

An electrical stimulus (3 ndash 7 V 1 mA ac) was applied in several short intervals

(1s each) for about 1 minute approximately 1 cm beneath the two gonopores which are

beneath the genital operculum (Fig 57a-b)

200

a

b

Figure 57 Photos showing a) the position of the gonopores on the ventral side of the

genital operculum (indicated by the red arrow) b) electrical shock applied 1 cm beneath

the gonopores (indicated by the red arrow) of a female T tridentatus

201

542 Results

For female T tridentatus (carapace width of 320 cm) three trials were performed

in September and October 2005 In the first trial following the previous electrical

stimulation study on the horseshoe crab species L polyphemus (Brown and Clapper

1981) only 3 ndash 4 V was applied but no egg was released

In the second trial a larger range of voltage (3 ndash 7 V) was examined No release of

horseshoe crab eggs was observed with the range of 3 - 65 V However when the

voltage was increased to 7 V with the aid of a plastic pipette to widen the openings of

the gonopores eggs were released and could be collected from both gonopores (Fig

58a-b) The same voltage was applied for three times with about 100-150 eggs being

collected from each gonopore each time More eggs were released when the gonophores

were slightly pressed by hand However this electric stimulation method failed in the

third trial in early October 2005 In which no egg was released from the gonopores

although the same voltage (ie 7 V) was used

Some preliminary studies were also undertaken on several male T tridentatus

(carapace width 200 - 283 cm) from July to August 2006 As the male horseshoe crab

is much smaller in size smaller voltages (5-6 V) were used Several drops of milky

fluid were collected from the gonopores after electrical stimulation The fluid was

examined under a compound microscope and a high density of sperm was observed

However this method was not applicable for male horseshoe crabs smaller than 240 cm

Thus for both female and male T tridentatus further studies on the applicability and

repeatability of this electrical stimulation method are necessary

202

a

b

Figure 58 Photos showing a) a wide view b) a close up of the horseshoe crab eggs

released from the gonopores after electrical stimulation

203 55 Discussion

Owing to the small wild population of horseshoe crabs in Hong Kong artificial

breeding in the laboratory may be a viable option to enhance the horseshoe crab

population in the natural environment The intent of the present study is to breed and

raise juveniles to an age of 2-3 years in the laboratory prior to releasing them back to

the existing nursery grounds To make this successful apart from the artificial

insemination well-developed laboratory conditions for incubation of eggs and tribolites

and rearing of juveniles are also essential Hence the interplay between salinity and

temperature on the hatching success of larvae and the survival of juveniles was

investigated

551 Trials on Artificial Insemination

Artificial insemination of horseshoe crabs was started in 1970rsquos (Brown and

Clapper 1981) and now the procedures for collecting and fertilizing gametes of Limulus

polyphemus in vitro are well developed This makes L polyphemus highly suitable for

the investigation of developmental processes in horseshoe crabs Chinese scientists

started developing artificial breeding techniques for Indo-Pacific horseshoe crab species

particularly T tridentatus in late 1980rsquos For L polyphemus electrical stimulation was

commonly used for the collection of sperm and eggs (Brown and Clapper 1981) while

dissecting the mating pairs and extracting gametes directly from them (direct extraction

method) were commonly used for the study of T tridentatus in China (Liao and Hong

1997 Wang et al 2001a Wang et al 2001b Hong et al 2002)

In the present study the direct extraction method was applied in the trials of

artificial breeding in 2004 The potential of using non-destructive electrical stimulation

method has also been explored According to Brown and Clapper (1981) an electrical

204 stimulus of 3-4 V applied below and slightly lateral to the gonopores of L polyphemus

could induce spawning successfully The same voltage however failed in T tridentatus

and a higher voltage (7 V) was required This is probably due to the difference in body

size as L polyphemus is much smaller than T tridentatus L polyphemus is the second

smallest horseshoe crab species with an average prosomal width of 18 and 21 cm for

male and female respectively while T tridentatus is the largest species with an average

prosomal width of 25 and 30 cm respectively (Sekiguchi 1988c) Nevertheless the

voltage applied in the present study was much lower than that reported upon by Dong

and Ng (1985) the only Chinese literature applying the method of electrical stimulation

with a voltage of 10 V However great care must be taken as an excessive stimulation

would cause lesions and results in the risk of losing reproductive ability of the

individuals Owing to the limited description of the voltage and current applied and the

lack of quantitative results of the study of Dong and Ng (1985) future studies are

required with regard to the voltage and current used as well as the frequency of

stimulation

Apart from the direct extraction and electrical stimulation methods two other

artificial breeding methods have been used by Taiwan scientists The first method

involved collecting fertilized eggs in the field and incubated them in the laboratory

(Chen et al 2004) The fertilized eggs in the horseshoe crab spawning ground could be

identified by observing the spawning foam resulted from air bubbles emerging when

adults dug into the sand during spawning (Chen et al 2004) However this method is

difficult to be applied in Hong Kong as it is very rare to have mating pairs being spotted

in the spawning grounds in recent years (see Section 2) Another method is to induce

the horseshoe crabs to spawn in the laboratory In the breeding season mating pairs

were transferred from a tank without sediment to a tank containing sediment as

205 substratum during flood tide In the study of Chen et al (2004) the mating pairs had

spawn naturally with over 10000 eggs by this laboratory stimulating experiment The

potential of this method should be further explored

552 Incubation of Eggs in the Laboratory

The present study has indicated that although the survivorship of the eggs was

relatively high at low temperatures and salinities the hatching rate was reduced with no

hatching at 15permil This was probably due to the low developmental rate of the eggs On

the other hand the highest hatching rate was obtained at 32degC and 30 permil indicating that

high temperature and high salinity appeared to be the optimal environmental conditions

for artificial breeding practices Similar results were obtained in other studies In a

preliminary study of the artificial breeding of T tridentatus by Liao and Hong (1997)

the incubation period was highly related to temperature with a shorter hatching period

around 30 degC In another study by Chen et al (2004) the hatching rate of T tridentatus

between 25 and 34 degC was conducted The median hatching time decreased as

temperature increased with the values being 46 days at 34 degC 48 days at 31 degC 528

days at 28 degC and 706 days at 25 degC Therefore the optimal temperature range for

hatching of T tridentatus is around 31 to 34 degC

Salinity was another important environmental factor affecting the developmental

and hatching rate of horseshoe crabs In the present survey a higher hatching rate was

obtained at 30 permil as compared with 15 20 and 25 permil while T tridentatus failed to

hatch at 15 permil However a wider salinity range for hatching was obtained by Li et al

(1999) in which the optimum salinity range for hatching was between 16 and 33 permil

206

The effects of temperature and salinity on the survival and hatching rates for the

American species L polyphemus were conducted recently The embryos of L

polyphemus tolerated a wide range of temperature-salinity combinations (Jegla and

Costlow 1982) with higher survival and developmental rates at around 30 degC and 33

permil Laughlin (1983) showed that the lowest survival of the embryos occurred at low

temperatures and salinities (20 degC 10 permil) while the survival was high at temperatures

ranged from 20 to 35 degC and salinities from 10 to 35 permil Similar results were obtained

by Jegla and Costlow (1982) with a shorter duration for hatching (ie faster

developmental rate) at 30-35 degC than 20-25 degC and 25-30 degC However a delay in the

embryonic development was obtained at salinities above 40permil (Jegla and Costlow 1982

Ehlinger and Tankersley 2004) and temperatures at 35 degC or above (Ehlinger and

Tankersley 2004) In conclusion embryos of both T tridentatus and L polyphemus can

tolerate a wide range of temperature-salinity combinations but L polyphemus is more

sensitive to low temperatures (Laughlin 1983) while T tridentatus is more sensitive to

low salinities

553 Survival of Trilobites and Juveniles

In the present study the survival of the juveniles was low after rearing in the

laboratory for 2 years despite addition of antibiotics There could be a combination of

factors responsible for such high mortality including the quality of food (Artemia)

sediment layer for burying andor requirements of natural light or other essential

elements for molting

The optimum temperature range for the survival of juvenile horseshoe crabs in the

laboratory varied with species with 28-34 degC being obtained for T tridentatus and

25-35 degC for L polyphemus (Sekiguchi et al 1988 Sugita 1988 Chen et al 2004 Lee

207

and Morton 2005 Botton et al 2006) For salinity the optimal value was 40 permil for T

gigas and 20-30 permil for L polyphemus (Jegla and Costlow 1982 Chatterji and Mathew

2004 Ehlinger and Tankersley 2004) The growth rate and time for molting of

juveniles also depend on the presence of sediment The study of Chen et al (2006)

showed that the juvenile T tridentatus spent most of the time burrowed into sediments

juveniles living in tanks with sediments also grew better and faster than those in tanks

without sediments In my study as 5-cm sediment layer was only used for rearing the

third and fourth instars whether the absence of sediment reduced the survival of the

first two instars deserves further investigations

Food quality may also contribute to a low survival rate in the juveniles In the

natural environment juvenile horseshoe crabs preferentially select insect larvae

(Chatterji et al 1992 Zhou and Morton 2004) while those rearing in the laboratory in

my study were fed with Artemia Although it is common to feed the marine suspension

feeding invertebrate larvae with Artemia (Strathmann 1987) the diet may not provide

adequate nutrients for the growth and molting of the horseshoe crab juveniles In fact

the inclusion of algae zooplankton and organic detritus was suggested by Liao and

Hong (1997) while a mixture of squid prawn and fish was used by Lee and Morton

(2005) In a feeding experiment of the juvenile T tridentatus (Gao et al 2003)

cuttlefish was found to be the most preferred food comparing with fish prawn oyster

and sipunculid worm The size of the food also affects the food consumption of the

juveniles with the highest consumption being obtained for small cuttlefish (03-05 cm)

More detail studies of the effects of various environmental factors and diet on the

growth of juvenile horseshoe crabs are necessary in order to find out the best conditions

for rearing this animal in the laboratory

208

Chapter 6 General Discussion

Horseshoe crabs have been facing the problem of population decline in the past

few decades The abundance of L polyphemus has dropped sharply by an order of

magnitude on the east coast of the USA including Delaware Bay (Michels 1996

Widener and Barlow 1999 Swan et al 1996 Shuster 2001 Rutecki et al 2004) The

three Indo-Pacific species in Taiwan Japan Thailand Malaysia and China are also

decreasing in numbers in recent years (Itow 1998 Botton 2001 Chen et al 2004) All

the 3 Indo-Pacific species in Hong Kong are facing the same problem (Mikkelsen

1988 Chiu and Morton 1999a 2003a) Their populations have dropped sharply in

recent years with T gigas being disappeared 20 years ago (Chiu and Morton 1999b)

while T tridentatus and C rotundicauda disappeared in Tolo Harbour 5 years ago

(Chiu and Morton 1999a 2003a) The present study also demonstrated that the

abundance of T tridentatus and C rotundicauda at Pak Nai and Ha Pak Nai has

declined substantially from 2002 to 2005 Between May and December 2002 Lee and

Morton (2003) collected 165 individuals of T tridentatus and 4 individuals of C

rotundicauda in the Deep Bay area by the random sampling method In the present

study in 2004-2005 however only 15 individuals of T tridentatus were found in the

extensive distribution study at the 17 study sites The decrease in T tridentatus

population density at Pak Nai and two locations in Ha Pak Nai were 96 90 and 80

respectively No C rotundicauda was found by the random sampling method Therefore

immediate action to conserve local horseshoe crabs is required before they become

extinct

Species conservation is a popular and familiar conservation approach in recent

years (New 2000) A comprehensive species conservation plan includes a sequence of

status evaluation threat determination and its operation Based on the information

209

gathered for the target species species specific management tools can be designed and

undertaken to minimize the threats conserve the species and foster sustainability or

recovery of it by a dynamic and responsive process (New 2000) The present

investigation included studies on the population dynamics taxonomy and population

genetics evaluation of threats and potential conservation tools Firstly Section 3 aimed

at differentiating the juvenile forms of the two commonly occurred horseshoe crab


and genetic approaches It provided the basic knowledge of the speciation of juvenile

horseshoe crabs in the field essential for the evaluation of the status of each of the local

horseshoe crab species In Section 2 the status of the juveniles of the two horseshoe

crab species at various nursery grounds in Hong Kong was updated including their

distributions and population densities A more intensive 6-month population study was

also undertaken at four key nursery shores In Section 4 the distribution of adult

horseshoe crabs in Hong Kong was investigated by interviewing the local seafood

markets and fishermen These distribution studies have provided scientific evidence on

the sharp decline in local horseshoe crab populations from 2002 to 2005 and updated

information on the ecology of local horseshoe crabs which is essential for the planning

of a conservation strategy In response to the rapid decline in the populations

identification and evaluation of potential threats should be conducted because an

effective conservation strategy relies on how these potential threats can be minimized

(New 2000) In the present study human exploitation of horseshoe crabs in Hong Kong

was evaluated and recommendations were made in Section 4 Finally Section 5

explored various artificial breeding and rearing practices for T tridentatus as potential

tools to restock horseshoe crabs in Hong Kong

210


611 Walk-through Method and Mark-recapture Method for Distribution Study

The quadrat sampling was commonly used in recent population studies of juvenile

horseshoe crabs (Carmichael et al 2003 Chen et al 2004) In the study of Carmichael

et al (2003) four randomly placed quadrats (2 m x 2 m) were sampled and juveniles

were hand-picked at three sediment depths (5 mm 10 mm and 50 mm) Chen et al

(2004) set a 125 m long transect line perpendicular to the shoreline with fiver plots (5

m x 5 m) being placed at 25 m intervals However given that the distribution of local

juvenile horseshoe crabs is patchy with high temporal and spatial variations a detailed

walkndashthrough survey along transects parallel to the shore may be a better sampling

method to assess the population status of the juvenile horseshoe crabs than the random

quadrat method The results of the present distribution study have shown an advantage

of the walk-through method and the data gathered will serve as the baseline for future

comparison purposes

Apart from the walk-through survey the mark-recapture method is another

possible survey method for population estimation of juvenile horseshoe crabs in nursery

grounds In the Philippines the population of juvenile T tridentatus was assessed using

the mark-recapture method with the individuals being marked by cutting the tips of the

opisthosomal spines (Almendral and Schoppe 2005) It allowed the calculation of the

total number of individuals (abundance) based on the ratio of known (marked) to

unknown (new) individuals (Krebs 1989) The usefulness of this method in studying

the population distribution of the juvenile horseshoe crabs in Hong Kong deserves

further investigations

211

Furthermore the present study has confirmed the further decline of juvenile

horseshoe crabs on local shores in Hong Kong as compared to a similar study in 2002

In particular there were less shores within the Deep Bay area in which C rotundicauda

was recorded To further ascertain if such a declining trend continues it is suggested

that monitoring should be conducted every year or every two years particularly at those

key nursery shores including Pak Nai Ha Pak Nai in Deep Bay and Shui Hau Wan and

San Tau on Lantau Island

Apart from the four key nursery shores monitoring of other existing nursery shores

identified in this study should also be conducted to update the local distribution of the

juvenile horseshoe crabs In addition further distribution surveys on sandy and muddy

beaches where horseshoe crabs were reported upon by Chiu and Morton (1999a) should

also be conducted particularly in northeastern New Territories and Tolo Harbour These

included Mai Po and Lung Kwu Sheung Tan in northwestern New Territories Kei Lai

Ha and Starfish Bay in northeastern New Territories Sites where adult had been found

in the past including Sharp Island and Pak Sha Wan in northeastern New Territories

and Kau Pei Chau in Cape drsquo Aguilar Hong Kong Island should also be studied To

confirm the presence of juvenile horseshoe crabs at these sites distribution studies are

recommended to be performed twice in summer particularly in June and July when the

highest abundance of horseshoe crabs was recorded in the present study If horseshoe

crabs are recorded regular distribution studies should be conducted annually to update

the population status of the juvenile horseshoe crabs at each nursery ground

612 Study on Adult Horseshoe Crabs

Apart from further studies on the juvenile horseshoe crabs at the nursery grounds

it is also worthwhile to investigate the population of adult horseshoe crabs in Hong

212

Kong waters especially where these adult horseshoe crabs are thriving At present no

such data are available in Hong Kong One idea is to launch a tagging study of the adult

horseshoe crabs based on the capture and re-capture method (Rudloe 1980 Tanacredi

2001 James-Pirri et al 2005 Swan 2005) Apart from the population estimation

tagging of adult helps understand the breeding behaviour and identify the spawning

grounds of horseshoe crabs (Rudloe 1980) It helps evaluate the frequency of beach

visits for spawning the fecundity of the females and the length of a femalersquos

reproductive life (Tanacredi 2001) It also provides evidence on site specificity for

horseshoe crabs (Rudloe 1980 James-Pirri et al 2005) All these information related

to the breeding movement and behaviour and location of the spawning grounds is

essential for the development of a strategic conservation plan The tagging experiment

also aids at defining and confirming the range of discrete spawning populations (Swan

2005)

To investigate where the adult horseshoe crabs forage and live a tracking study of

these animals using ultrasonic devices can be initiated Radio and acoustic telemetry

have been used for studying short term (less than one month) movement of horseshoe

crabs in the spawning season (Kurz and James-Pirri 2002 Brousseau et al 2004

Moore and Perrin 2007) Sonic transmitters were usually fastened to the top of the

prosoma using marine epoxy or monofilament harness (Kurz and James-Pirri 2002

Moore and Perrin 2007) Each sonic transmitter emits a unique signal which is

detectable up to 1000 m while a hydrophone and a handheld geographic positioning

system (GPS) unit can be used to locate and record the geographic coordinates of the

signals from the tagged horseshoe crabs (Kurz and James-Pirri 2002 Moore and Perrin

2007) By using the telemetry equipment home range and movement pattern of adult

horseshoe crabs underneath the water could be determined Comparing with the

213

traditional tagging capture-recapture method a significantly higher probability of

recapture was obtained by using the radio and acoustic telemetry (Brousseau et al

2004) While a trial experiment on the movement of adult horseshoe crabs by using an

ultra-sound emitter has been conducting by Dr Paul Shin and Dr S G Cheung from

the City University of Hong Kong since 2007 (Lai 2007)

Scuba diving and underwater videography can also provide more information on

the behaviour and ecology of the adult horseshoe crabs Behaviour of adult horseshoe

crabs out of the water was previously studied particularly for the spawning behaviour

(Penn and Brockmann 1995 Brockmann 1996 Brockmann et al 2000 Schwab and

Brockmann 2007) Other kinds of underwater behaviour including feeding burrowing

resting and swimming however are rarely documented In a study by Kurtzke (2001)

scuba diving with the aid of underwater videography was used to investigate the

underwater behaviour of the horseshoe crabs in Jamaica Bay including feeding on

mussel beds swimming in the intertidal zone and burying in the sand

613 In-depth Studies on Phylogenetic and Geographical Genetic Variations in Juvenile

Horseshoe Crabs

This was the first attempt to investigate the phylogenetic relationship among

different populations of horseshoe crabs in Hong Kong waters The present data showed

that the inter-specific variations of both 18S and 28S rDNA sequence were larger than

the intra-specific variations with the percentage of pair-wise difference being lt 1 and

157 respectively Thus these two rDNA regions are not very powerful for the

speciation of T tridentatus and C rotundicauda This also applied to the differentiation

between juveniles of T tridentatus with one or three spines Other genetic markers such

as the 18-28S ITS rDNA which not only provides information on speciation but also

214

some indications of population genetics can be adopted for further studies of the

population genetics in horseshoe crabs For the differentiation of closely related species

the mitochondrial DNA (mtDNA) can be used to indicate the overall genetic

differences Because of the energy-producing reactions mtDNA carries out

mitochondria mutate their DNA at a rapid rate and are less able to ldquoproofreadrdquo these

errors than genes in nuclear DNA Hence mtDNA has a high mutation rate and serves

as a perfect tool for spotting genetic differences between individuals of closely related

species and within a species In fact the mt AT-rich region DNA sequences had been

used for the determination of the subdivision of T tridentatus at three localities

Kinmen Tiexianwei and Dongwei in Taiwan (Yang et al 2007)

To examine the geographical variations in genetic diversity of the same species of

horseshoe crabs among different regions in Asia-Pacific Region a geographical genetic

study is suggested Individuals of horseshoe crabs can be collected from different

countries such as Japan Indonesia Malaysia mainland China and Hong Kong and the

genetic diversity among different populations can be examined and compared using the

method of amplified fragment length polymorphism (AFLP) and DNA (COI) genomic

sequence (Pierce et al 2000) AFLP is a highly sensitive method for detecting

polymorhism in DNA There are many advantages of AFLP over other marker

technologies including randomly amplified polymorphic DNA (RAPD) restriction

fragment length polymorphism (RFLP) and microsatellites AFLP not only has higher

reproducibility resolution and sensitivity at the whole genome level compared to other

techniques but the capability to amplify between 50 and 100 fragments at one time In

addition no prior sequence information is needed for amplification (Vos et al 1995

Meudt and Clarke 2007) As a result AFLP has become widely used for the

identification of genetic variations in strains or closely related species of plants fungi

215

animals and bacteria and population genetic studies DNA (COI) genomic sequence

had been used to survey the intraspecific differences of the populations of L

polyphemus in Delaware Bay and Chesapeake Bay (Pierce et al 2000) Hence further

investigations in the spatial variations of the genetic make up of horseshoe crabs in local

habitats by AFLP and DNA (COI) genomic sequence are suggested

614 Risk Assessment of Horseshoe Crabs

Concerns about the possible impact of heavy metal pollution upon horseshoe crabs

have been raised (Chiu and Morton 1999a Morton and Lee 2003) Chiu and Morton

(1999a) showed that the concentrations of cadmium and lead in sediments at Ha Pak

Nai were comparable to those in polluted bays in Tolo and Victoria Harbours Although

previous studies have shown that horseshoe crab embryos and larvae are highly tolerant

to heavy metals (Botton et al 1998) and organic contaminants such as TBT (Botton et

al 1999) heavy metals or organic pollutants would be accumulated in the body and

even sequestrated from the female during egg formation (Kannan et al 1995 Burger

1997) A high toxicity of heavy metals including cadmium lead manganese and

selenium and tetrodotoxin was detected in the eggs of L polyphemus and C

rotundicauda in the wild (Burger 1997 Burger et al 2002 Ngy et al 2007) while a

high toxic level of copper (2070 μg g-1 wet weight) and zinc (2890 μg g-1 wet weight)

was detected in the eggs of T tridentatus (unpublished data) High heavy metal levels

may increase the mortality rate and abnormality of the embryos Some previous studies

showed that heavy metals had sublethal effects on the horseshoe crab embryos larvae

and juveniles including defective embryos inhibition of limb regeneration delay of

molting and abnormal eyes (Itow et al 1998a 1998b) Whether water pollution in

Hong Kong causes sublethal effects or even mortality in horseshoe crabs is unknown

and deserves further investigations

216

Therefore an ecological risk assessment (ERA) of various pollutants in particular

heavy metals to local horseshoe crab species is recommended ERA has been performed

for various organisms in Hong Kong including marine neogastropods Thais clavigera

and Thais luteostoma (Leung et al 2006) Indo-pacific humpback dolphin and Finless

porpoise (Hung et al 2004 2006) and Little egret and Black-crowned night heron

(Connell et la 2002 2003) For horseshoe crab species the probabilistic ERA would

be based on tissue burden of heavy metals Relationships between body burden of heavy

metals and sublethal effects such as defective embryos and inhibition of limb

regeneration could be established The ERA results would provide an essential

ldquobaselinerdquo for a long-term monitoring programme in Hong Kong and provide a

methodological framework for ERA of heavy metals in Southeast Asia An ERA of

heavy metals to horseshoe crabs in Hong Kong therefore is recommended to be done

with urgency

615 More Studies on Carcinoscorpius rotundicauda

C rotundicauda is found in coastal areas from the South China Sea to the Indian

subcontinent However information on C rotundicauda is scarce worldwide including

habitat characteristics and the ecology of the juveniles (Rao and Rao 1972 Sekiguchi

1988b Chatterji and Parulekar 1992) Some of the previous studies have suggested that

C rotundicauda go upstream during the rising tide from the river in the breeding season

(Rao and Rao 1972) In the Gulf of Siam egg clusters of C rotundicauda were also

recorded on muddy banks of rivers flowing through the mangroves (Chatterji and

Parulekar 1992) Sekiguchi (1988b) showed that the breeding environment for C

rotundicauda was quite different from that for the other three species with a preference

towards fresh or brackish waters (Sekiguchi 1988b) However in the present survey no

C rotundicauda was recorded in the additional 20 random quadrats (05 m times 05 m)

217

within the stream area at each sampling site Therefore more studies of the habitat

requirements of C rotundicauda should be conducted Nevertheless in this study

nursery grounds for this species were identified in Deep Bay area northeastern New

Territories and even on Lantau Island Detailed investigation on the hydrology and

sediment characteristics of these areas can provide more information on the habitat

preference of this species In addition although the living environments of T

tridentatus and C rotundicauda seem to be different (Sekiguchi et al 1977 Sekiguchi

1988b) they co-occurred on some of the shores A more in-depth study of their

microhabitat preferences should be conducted

616 Non-destructive Artificial Breeding Technique

Electrical stimulation has been regularly used in L polyphemus for artificial

breeding for over 20 years (Brown and Clapper 1981) It was mainly used in

experimental studies of the horseshoe crab eggs and larva such as developmental study

(Jegia and Costlow 1982 Laughlin 1983 Ehlinger and Tankersley 2004) and

toxicology test (Botton et al 1999) However it failed to apply to the Indo-Pacific

horseshoe crab species including T tridentatus (Sekiguchi 1988a) In the present study

an electric stimulus of 7 V and 1 mA ac was successful to stimulate the release of eggs

and sperm whereas 10 V was used in the study by Dong and Ng (1985) However the

electrical stimulation practice was not repeatable in this study Therefore a more

in-depth study on this method is needed especially with regard to the frequency of the

electrical stimulation and the most suitable sizes of adult horseshoe crabs to be used

Besides to avoid any harm caused by high voltages to the adults voltages ranged from

7 to 10 V are recommended for further investigations on this method In addition

applying a slight pressure anterior to the genital operculum is another possible

non-destructive method for egg collection (French 1979) However as only hundreds

218

of eggs could be collected by these two non-destructive gamete collection methods they

are more suitable for embryonic and larval study than massive population enhancement

practices

Apart from the direct extraction and electrical stimulation induction of spawning

in the laboratory is another possible alternative which is non-destructive with more

gametes collected (Chen et al 2004) By simulating the flood tide and substratum of

the natural spawning ground of the horseshoe crabs adult can be induced to spawn

during flood tide in the laboratory (Chen et al 2004) In a study by Chen et al (2004)

sand grain size ranged from 04 to 18 mm corresponding to medium to very coarse

sand were collected from the natural spawning ground as bedding and the horseshoe

crab mating pairs were induced to spawn during spring tides with a maximum of 10000

eggs being laid in a 5-hour period As this method can collect more gametes a

feasibility study of this method for local horseshoe crabs should be conducted

617 Further Study on the Requirements for Juvenile Rearing

Artificial insemination and breeding in the laboratory is a viable option to enhance

the horseshoe population in the natural environment By maintaining the artificially

fertilized eggs in the optimal environmental conditions (eg appropriate temperature

and salinity) the survival rate of the hatched trilobites can be enhanced

Laboratory-reared juveniles can be released to the field after they have grown to certain

size (may be ge 2 years old) to reduce predation risk Although this study has

successfully fertilized the eggs in vitro and larvae could be reared at least up to the

fourth instar in laboratory conditions mass mortality of the juveniles becomes an

obstacle in the mass production of the juveniles for the release purpose As the reasons

for the mass mortality are unknown further studies should be conducted to enhance the

219

survivorship with regard to the rearing conditions including formulation of the best diet

improvement of water quality and optimization of environmental conditions

62 Proposed Conservation Measures

The present study showed an obvious decline in the juvenile horseshoe crabs from

2002 to 2005 Comparing with the results obtained by Morton and Lee (2003) the

population has dropped by 80-96 (Section 2) Further decline in the wild population is

expected if no immediate conservation measures are taken considering the urbanization

and rapid development of infrastructure projects in the vicinity of these shores in Hong

Kong and the Pearl River Delta Hence a species specific conservation strategy should

be planned and implemented before this living fossil disappears in Hong Kong

A conservation strategy should contain eight elements including species

concerned habitats problems conservation actions monitoring strategy review

interagency coordination and public involvement (ODFW 2006) For the former two

elements they aim at gathering information on the distribution and abundance of the

species and describing the locations and relative conditions of key habitats essential to

the conservation of the species (ODFW 2006) These two elements have been achieved

by Chiu and Morton (1999a) Morton and Lee (2003) and the present study (Section 2

and 3) by providing comprehensive baseline information on the local horseshoe crabs

including morphology taxonomy growth behaviour ecology and their key habitats

The present study has also identified the ldquoproblemsrdquo which may adversely affect the

species or their habitats and identified information required to improve the situation

(ODFW 2006) For example Section 4 identified and evaluated the impact of human

exploitation on the horseshoe crab populations through market surveys on the sale of

220

horseshoe crabs in Hong Kong while Section 2 identified possible impacts of habitat

reclamation and pollution on local horseshoe crabs

This section suggests necessary conservation actions for the horseshoe crabs and

their associated habitats with monitoring measures for evaluating and reviewing the

effectiveness of the proposed conservation actions To formulate a successful and

comprehensive conservation strategy co-operation with international regional and state

agencies and engagement of the public are also suggested (Walls et al 2002 ODFW

2006)

621 Re-introduction Programme

Introduction of captive breeding for animal conservation has increased markedly in

the last decade To compensate for the loss through natural mortality and increase the

wild population a re-introduction programme for juvenile horseshoe crabs may be a

feasible conservation approach with a great potential in Hong Kong China and Taiwan

started the re-introduction program for horseshoe crabs in the past few years In Taiwan

more than 10000 individuals of second-instar juveniles produced artificially in the

laboratory were released in the protected area in Kinmen in 2002 (Chen and Yeh 2005)

while about 40000 larvae were released to the wild in Xiamen China in 2004 Some

people may query the effectiveness and feasibility of the re-introduction programme as

the juveniles may be carried away from the shore by waves or eaten by fish or other

organisms However Chen et al (2006) showed that the re-introduction of juvenile

horseshoe crabs to sandymuddy shores is feasible as the sediment layer on the shores

would protect the juveniles from predators and waves

221

In Hong Kong studies are being carried out to improve the rearing technique for

juvenile horseshoe crabs Dr Paul Shin and Dr S G Cheung of the City University of

Hong Kong have been collaborating with the Ocean Park Hong Kong since October

2006 Being a world class aquarium the Ocean Park has provided expert advice and

technical help on laboratory rearing technique and skills and hopefully the survival of

the juveniles can be improved (Lai 2007)

622 Ban on Fishing and Possession of Horseshoe Crabs in Hong Kong

Apart from enhancing the wild population by re-introduction Hong Kong

government should also protect the local horseshoe crabs through legislation As the

threat of horseshoe crabs associated with human consumption is still high legislation

should be drawn up to ban the fishery and sale of the horseshoe crabs in Hong Kong In

Taiwan a ban on the catch of horseshoe crabs was made in the protected area in

Kinmen from 2000 to 2004 to avoid any human exploitation of the horseshoe crab

populations in the first four years after the establishment of the protected area (Chen and

Yeh 2005) Regional governments in China including Ningbo and Xiamen have drawn

up legislation to ban the catch of horseshoe crabs in the regional waters (Cheung and

Fan 2006) while USA has restricted the catch of horseshoe crabs by a fishing permit

system (Eagle 2001)

In addition public education should be introduced to increase the public awareness

on the life history and fragile status of this animal so as to reduce the fishing pressure

More importantly efforts should be made to place Asia-Pacific horseshoe crabs on the

endangered species list of CITES through international consensus so that the imports

and exports of these animals can be better managed to further reduce human

consumption and fishing pressure especially in this part of the world

222

623 Designation of Protected Areas

Apart from human exploitation the decline in horseshoe crab populations may also

be due to human activities like water pollution and degradation and loss of habitats

Among the potential threats scientists generally agree that habitat destruction is

currently the primary lethal agent for most of the endangered species (Wilcove et al

1998) In Hong Kong several developmental projects adjacent to the horseshoe crab

key nursery grounds have been planned and undergone environmental impact

assessments in the past few months One of them involved the construction of a golf

course and some estates adjacent to Pak Nai one of the key nursery grounds for the two

local horseshoe crab species (Fulland International Limited 2007) Another project is

the proposed Tuen Mun - Chek Lap Kok Link which may cause adverse impacts on the

water quality of the nursery grounds of the horseshoe crabs including Tai Ho Tung

Chung and San Tau (Highways Department 2007) If these environmental threats are

not lessened it is meaningless to breed and release the juveniles into the wild as they

cannot survive in a deteriorating environment (HKDCS 2007)

Protected areas for horseshoe crabs have been set up in various countries including

Taiwan China and Japan Early in 1928 T tridentatus has been protected with an

establishment of the largest horseshoe crab protected area in the Oe-hama Beach in

Japan (Botton 2001) In Taiwan a size of 800 ha protected area was established in

Kinmen in 1999 (Chen and Yeh 2005) while several horseshoe crab special reserves

have been set up in Pingtan Fujian China (Wang et al 2002) In Hong Kong

important nursery shores such as Shui Hau Wan and San Tau should be designated as

Sites for Special Scientific Interest (SSSIs) andor Special Conservation Area to prevent

future developments at these shores Although the designation of horseshoe crab

protected areas was proposed as early as 1999 by Professor Morton (Chiu and Morton

223

1999a) no action has been taken so far Immediate action should be taken before it is

too late

624 Regular Monitoring Study on Horseshoe Crabs Distribution

In Taiwan apart from releasing the laboratory-reared juveniles into the wild their

populations in the protected area have been monitored monthly after the setting up of

the protected area (Chen et al 2004) As the populations of horseshoe crabs are

declining rapidly in Hong Kong regular monitoring is essential to provide most

up-to-date information of the population status which may also help evaluate the

effectiveness of the conservation measures if any especially for the four key nursery

grounds including Pak Nai Ha Pak Nai Shui Hau Wan and San Tau For a long-term

monitoring programme a standard protocol should be established Given that the

distribution of juvenile horseshoe crabs is patchy with high temporal and spatial

variations this study confirmed that the more efficient and effective walkndashthrough

survey is a better sampling method to assess the population status of the juvenile

horseshoe crabs than the random quadrat method Data obtained from the present study

can serve as baseline for future comparison purposes Apart from the walk-through

survey the mark-recapture method is another possible survey method for population

estimation of juvenile horseshoe crabs in nursery grounds (Almendral and Schoppe

2005)

625 Public Education

According to the experience of the conservation of T tridentatus in Kinmen

Taiwan the success of a conservation strategy relies on the consensus and participation

of the public at the community level (Chen and Yeh 2005) Hence a series of public

education programs and publications for horseshoe crabs have been designed and

224

developed in Taiwan including symposiums workshops television programs and

published materials such as pamphlets and books (Chen et al 2004) Similar kinds of

public participation program could also be implemented in Hong Kong With the

increasing popularity of the internet information on various aspects of the conservation

of the horseshoe crabs for example the most up-to-date information of the population

status habitat characteristics of the nursery grounds general biology and ecology of the

horseshoe crabs etc can be uploaded onto the web for public access Public display of

juvenile horseshoe crabs reared from artificial breeding can be an effective tool in

promoting pubic awareness

In the past few years the general public is becoming more familiar with this animal

through news articles TV programs and live animals displayed in the Hong Kong

Wetland Park and the Ocean Park Hong Kong Researches on various aspects of the

conservation of horseshoe crabs have been carried out in universities with funding

support from the government and private organizations Nevertheless a comprehensive

conservation strategy is critical to the success of the conservation of this living fossil

Although this animal has survived all the major catastrophic events which caused

extinction of most of the species in the past 4 billion years its survival in the future

depends on our determinations and concerted efforts

225

Chapter 7 Conclusion



crabs in Hong Kong The ecological assessment of horseshoe crabs was conducted by

providing updated status and distribution of juvenile horseshoe crabs and assessing the

degree of human exploitation of horseshoe crabs in Hong Kong While the

conservation and population enhancement were also focused including trials on

artificial insemination and breeding and determination of the optimal environmental

conditions for the survival and hatching success of horseshoe crab eggs In addition

genetic studies were performed including genetic differentiation between Tachypleus

tridentatus and Carcinoscorpius rotundicauda and population genetics of Tachypleus

tridentatus

The present study provided an update of the status of horseshoe crabs in Hong

Kong Horseshoe crabs could still be recorded in a wide range of soft shores in Deep

Bay region northeastern New Territories and on Lantau Island However a rapid

decline (with 80-90 decrease) in horseshoe crab population in soft shores was

observed The degree of human exploitation of horseshoe crabs in Hong Kong was

estimated through interviewing 34 seafood restaurants 150 fish sellers and fish

handlers Refer to the results the sale of horseshoe crabs was relatively low comparing

with other commercial important marine organisms however combing the human

exploitation in term of sale for dishes set-free ritual and display it caused potential

risks to the scattered population of adult horseshoe crabs in local waters In view of the

low natural breeding and hatching success and low juvenile survival artificial

insemination and breeding may be reasonable options to enhance the horseshoe

population in the natural environment Trials of artificial breeding by direct dissection

226

and electrical simulation were conducted While the effects of salinity and temperature

on the hatching of horseshoe crabs were also investigated Lastly taxonomy and

population genetic study were also conducted


crabs in Hong Kong that is essential for setting up the conservation and management

plans for horseshoe crabs in Hong Kong

227

Chapter 8 References

Almendral MA Schoppe S 2005 Population structure of Tachypleus tridentatus

(Chelicerata Merostomata) at a nursery beach in Puerto Princesa City Palawan

Philippines Journal of Natural History 29(25) 2319-2329

Avise JC Nelson WS Sugita H 1994 A speciational history of lsquoliving fossilsrsquo

Molecular evolutionary patterns in horseshoe crabs Evolution 48 1986-2001

Baker AJ Gonzalez PM Piersma T Niles LJ de Lima I do Nascimento S

Atkinson PW Clark NA Minton CDT Peck MK Aarts G 2004 Rapid

population decline in red knots fitness consequences of decreased refueling rates

and late arrival in Delaware Bay Proceedings of the Royal Society of London

Section B 27 875-882

Barlow RB Hitt JM Dodge FA 2001 Limulus vision in the marine environment

Biological Bulletin 200 169-176

Behrens ME Wulff VJ 1965 Light-initiated responses of retinula and eccentric

cells in the Limulus lateral eye The Journal of General Physiology 48(6)

1081-1093

Belshaw R Quicke DLJ 2002 Robustness of ancestral character state estimates

evolution of life history strategy in ichneumonoid parasitoids Systematic Biology

51(3) 450-477

Berkson JM Shuster CNJr 1999 The horseshoe crab The battle over a true

multiple-use resource Fisheries 24(11) 6-10

Botton ML 2001 The conservation of horseshoe crabs What can we learn from the

Japanese experience In Tanacrei JT (Ed) Limulus in the Limelight ndash A Species

350 Million Years in the Marking and in Peril Kluwer AcademicPlenum

Publishers New York pp 41-51

228

Botton ML Haskin HH 1984 Distribution and feeding of the horseshoe crab

Limulus polyphemus on the continental shelf New Jersey Fishery Bulletin USA

82 383-389

Botton ML Hodge M Gonzalez TI 1999 High tolerance to Tributyltin in embryos

and larvae of the horseshoe crab Limulus polyphemus Estuaries 21(2) 340-346

Botton ML Johnson K Helleby L 1998 Effects of copper and zinc on embryo and

larvae of the horseshoe crab Limulus polyphemus Archives of Environmental

Contamination and Toxicology 35 25-32

Botton ML Loveland RE Jacobsen TR 1994 Site selection by migratory

shorebirds in Delaware Bay and its relationship to beach characteristics and

abundance of horseshoe crab (Limulus polyphemus) eggs Auk 111 605-616

Botton ML Loveland RE Tiwari A 2003a Distribution abundance and

survivorship of young-of-the-year in a commercially exploited population of

horseshoe crabs Limulus polyphemus Marine Ecology Progress Series 265

175-184

Botton ML Pogorzelska M Smoral L Shehata A Hamilton MG 2006 Thermal

biology of horseshoe crab embryos and larvae A role for heat shock proteins

Journal of Experimental Marine Biology and Ecology 336 65-73

Botton ML Shuster CN Keinath J 2003b Horseshoe crabs in a food web Who

eats whom In Shuster CN Barlow RB Brockmann HJ (Eds) The American

Horseshoe Crab Cambridge (MA) Harvard University Press pp 33-153

Bowen B Avise JC Richardson JI Meylan AB Margaritoulis D

Hopkins-Murphy SR 1993 Population structure of loggerhead turtles (Caretta

caretta) in northwestern Atlantic Ocean and Mediterranean Sea Conservation

Biology 7 834-844

229

Bradstock RA Auld TD Keith DA Kingsford RT Lunney D Sivertsen D

1995 Conserving biodiversity Threats and solutions Surrey Beatty Chipping

Norton

Brocchieri L 2000 Phylogenetic inferences from molecular sequences Review and

critique Theoretical Population Biology 59 27-40

Brockmann HJ 1996 Satellite male groups in horseshoe crabs Limulus polyphemus

Ethology 102(1) 1-21

Brockmann HJ Nguyen C Potts W 2000 Paternity in horseshoe crabs when

spawning in multiple-male groups Animal Behaviour 60 837-849

Brousseau LJ Sclafani M Smith DR Carter DB 2004 Acoustic tracking and

radio tracking of horseshoe crabs to assess spawning behavior and subtidal

habitat-use in Delaware Bay North American Journal of Fisheries Management

241376-1384

Brown GG Clapper DL 1981 Procedures for maintaining adults collecting

gametes and culturing embryos and juveniles of the horseshoe crab Limulus

polyphemus In Committee on Marine Investebrates (Ed) Laboratory Animal

Management Marine Invertebrates National Academy Press Washington DC

pp 268-290

Burger J 1997 Heavy metals in the eggs and muscle of horseshoe crabs (Limulus

polyphemus) from Delaware Bay Environmental Monitoring and Assessment 46

279-287

Burger J Dixon C Shukla T Tsipoura N Gochfeld M 2002 Metal levels in

horseshoe crabs (Limulus polyphemus) from Maine to Florida Environmental

Research 90 227-236

230

Burgman MA Keith D Hopper SD Widyatmoko D Drill C 2007 Threat

syndromes and conservation of the Australian flora Biological Conservation

134(1) 73-82

Cao D 2004 Chinarsquos panda population up 40 China Daily

lthttpwwwchinadailycomcnenglishdoc2004-0610content_338386htmgt

Accessed on 14022008

Carl N Shuster Jr 1982 A pictorial review of the natural history and ecology of the

horseshoe crab Limulus polyphemus with reference to other Limulidae In

Bonaventura J Bonaventura C Tesh S (Eds) Physology and Biology of

Horseshoe Crabs ndash Studies on Normal and Environmentally Stressed Animals

Alan R Liss Inc New York pp 1-52

Carmichael RH Rutecki D Valiela I 2003 Abundance and population structure of

the Atlantic horseshoe crab Limulus polyphemus in Pleasant Bay Cape Cod

Marine Ecology Progress Series 246 225-239

Caughley GC Gunn A 1996 Conservation Biology in Theory and Practice

Blackwell Science Cambridge Massachusetts

Chatterji A 1994 The Horseshoe Crab ndash A Living Fossil A Project Swarajya

Publication Orissa India

Chatterji A Mathew R 2004 Effect of salinity on larval growth of horseshoe crab

Tachypleus gigas (Muller) Current Science (Bangalore) 87(2) 248-250

Chatterji A Mishra JK Parulekar AH 1992 Feeding behaviour and food selection

in the horseshoe crab Tachypleus gigas (Muller) Hydrobiologia 246 41-48

Chatterji A Parulekar AH 1992 Fecundity of the Indian horseshoe crab

Carcinoscorpius rotundicauda (Latreille) Tropical Ecology 33 97-102

231

Chen CP Yeh HY 2005 Records of the conservation of horseshoe crab at Kinmen

In Wong QG (Ed) Proceeding of International Academic Symposium on

History Culture and Ecology of Kinmen pp 51-82

Chen CP Yeh HY Lin PF 2004 Conservation of the horseshoe crab at Kinmen

Taiwan strategies and practices Biodiversity and Conservation 13 1889-1904

Chen P Huang XM Liu RZ Lu XN Weng ZC Xu HX Hong SG 2006

Study on adaptability to different environment for juvenile of horseshoe crab

Tachypleus tridentatus with artificial incubation Xiamen Daxue Xuebao (Ziran

Kexue Ban) 45(3) 404-408

Chen Y Shin PKS Cheung SG Kong RYC 2007 Morphological and

molecular comparisons of amphioxus populations in the China Seas Marine

Biology 153 189-198

Cheung JJ Fan CH 2006 Conserving horseshoe crabs in Xiangshan Port Fishery

Guide to be Rich 178-9

Cheung M 1995 The Complete Book of Seafood Hong Kong Food Press Hong

Kong pp 222 (In Chinese)

Cheung YM Poon WF Hwang JC 1975 The disappearing horseshoe crab Its

contributions to physiology In Morton B (Ed) Special Symposium on Marine

Sciences The Pacific Science Association Special Symposium on Marine Sciences

Hong Kong 1973 Hong Kong The Government Printer pp 128

Chiu HMC Morton B 1999a The biology distribution and status of horseshoe

crabs Tachypleus tridentatus and Carcinoscorpius rotundicauda (Arthropoda

Chelicerata) in Hong Kong Recommendations for conservation and management

Final Report The Swire Institute of Marine Science The University of Hong

Kong

232

Chiu HMC Morton B 1999b The distribution of horseshoe crabs (Tachypleus

tridentatus and Carcinoscorpius rotundicauda) in Hong Kong Asian Marine

Biology 16 185-196

Chiu HMC Morton B 2003a The status of horseshoe crabs in Hong Kong In

perspectives on marine environmental change in Hong Kong and Southern China

In Morton B (Ed) Proceedings of the Hong Kong Workshops Reunion

Conference on Perspectives on Marine environmental Change in Hong Kong and

Southern China (1977-2001) Hong Kong Hong Kong University Press pp

743-758

Chiu HMC Morton B 2003b The morphological differentiation of two horseshoe

crab species Tachypleus tridentatus and Carcinoscorpius rotundicauda

(Xiphosura) in Hong Kong with a regional Aisan comparison Journal of Natural

History 37(19) 2369-2382

Chiu HMC Morton B 2004 The behaviour of juvenile horseshoe crabs Tachypleus

tridentatus (Xiphosura) on a nursery beach at Shui Hau Wan Hong Kong

Hydrobiologia 523 29-35

Cierpich SB Grady SP Valiela I 2004 Life history analysis of the juvenile

horseshoe crab in Pleasant Bay Cape Cod Biological Bulletin 207 175

Clark KE Niles LJ Burger J 1993 Abundance and distribution of migrant

shorebirds in Delaware Bay Condor 95 694-705

Cohen E 1979 Biomedical Applications of the Horseshoe Crab (Limulidae) Progress

in Clinical and Biological Research Alan R Liss New York

Connell DW Fung CN Minh TB Tanabe S Lam PKS Wong BSF Lam

MHW Wong LC Wu RSS Richardson BJ 2003 Risk to breeding success

of fish-eating Ardeids due to persistent organic contaminants in Hong Kong

evidence from organochlorine compounds in eggs Water Research 37 459-467

233

Connell DW Wong BSF Lam PKS Poon KF Lam MHW Wu RSS

Richardson BJ Yen YF 2002 Risk to breeding success of Ardeids by

contaminants in Hong Kong Evidence from trace metals in feathers

Ecotoxicology 11 49-59

Dong X Ng ZQ 1985 The electric stimulation of Tachypleus tridentatus Leach

Fujian Fisheries 3 24-25 (In Chinese)

Eagle J 2001 Issues and approaches in the regulation of the horseshoe crab fishery In

Tanacredi JT (Ed) Limulus in the Limelight ndash A Species 350 Million Years in the

Making and in Peril Kluwer AcademicPlenum Publishers New York pp 85-92

Earle SA 1991 Sharks squids and horseshoe crabs - The significance of marine

biodiversity BioScience 41 506-509

Ehlinger GS Tankersley RA 2003 Larval hatching in the horseshoe crab Limulus

polyphemus facilitation by environmental cues Journal of Experimental Marine

Biology and Ecology 292 199-212

Ehlinger GS Tankersley RA 2004 Survival and development of horseshoe crab

(Limulus polyphemus) embryos and larvae in hypersaline conditions Biological

Bulletin 206 87-94

Everitt S 2004 Per Comm

Falk DA 1990 Endangered forest resources in the US Integrated strategies for

conservation of rare species and genetic diversity Forest Ecology and

Management 35 91-107

Fisher D C 1984 The xiphosura archetypes of bradytely In Eldredge N Stanley

SM (Eds) Living Fossils New York Springer-Verlag pp 196ndash213

French KA 1979 Laboratory culture of embryonic and juvenile Limulus In Cohen

E Bang FB Levin J Marchalonis JJ Pistole TG Predergast RA Shuster

CNJr Watson SW (Eds) Progress in clinical and biological research Volume

234

29 Biomedical applications of the horseshoe crab (Limulidae) Alan R Liss Inc

New York pp 61ndash71

Friends of the Harbour 2003 Harbour Primer

lthttpwwwfriendsoftheharbourorgindexphpgt Accessed on 31032008

Fulland International Limited 2007 Planning Statement of Rezoning Request from

ldquoAGRrdquo to ldquoOU (Rural Use)rdquo at Various Lots amp Adjoining Government Land in

DD 133 amp 135 Pak Nai NT

Ganders FR Klinkenberg B Klinkenberg R 2003 Taxonomy in conservation

biology the enigmatic Vancouver Island beggarticks Davidsonia 14 63-70

Gao F Liao Y Ye F 2003 Feed study of the Tachypleus tridentatus juvenile

Marine Science Bulletin 22(4) 92-96 (In Chinese)

Gjershaug JO 2006 Taxonomy and conservation status of hawk-eagles (genus

Nisaetus) in South-East Asia Doctoral Thesis from Norwegian University of

Science and Technology

Hall W R Jr 1992 The Horseshoe CrabmdashA Reminder of Delawares Past University

of Delaware Sea Grant Marine Advisory Service Newark Delaware

Hambler C 2004 Conservation ndash Studies in Biology Cambridge University Press UK

pp 1-11

Hanna DH 2001 An estimate of population sizes of two horseshoe crab (Limulus

polyphemus) sites in Jamaica Bay In Tanacredi JT (Ed) Limulus in the

Limelight A Species 350 Million Years in the Making and in Peril Kluwer

AcademicPlenum Publishers New York pp 147-153

Haramis GM Link WA Osenton PC Carter DB Weber RG Clark NA

Teece MA Mizrahi DS 2007 Stable isotope and pen feeding trial studies

confirm the value of horseshoe crab Limulus polyphemus eggs to spring migrant

shorebirds in Delaware Bay Journal of Avian Biology 38(3) 367-376

235

Harzsch S Wildt M Battelle B Waloszek D 2005 Immunohistochemical

localization of neurotransmitters in the nervous System of larval Limulus

polyphemus (Chelicerata Xiphosura) Evidence for a conserved protocerebral

architecture in Euarthropoda Arthropod Structure and Development 34(3)

327-342

Highways Department 2007 Project profile of Tuen Mun-Chek Lap Kok Link (ESB

1752007)

Hill DS Phillipps K 1981 A Colour Guide to Hong Kong Animals ndash a Naturalistrsquos

Book on Local Widlife The Hong Kong Government Hong Kong

Hill DS Gott B Morton B Hodgkiss J 1978 Hong Kong Ecological Habitats

Flora and Fauna Department of Zoology The University of Hong Kong Hong

Kong

Hillis DM Dixon MT 1991 Ribosomal DNA Molecular evolution and

phylogenetic inference Quarterly Review of Biology 66 411-453

Hong RF 2004 Population and Distribution of Horseshoe Crab Carcinoscorpius

rotundicauda at the Kranji Nature Trail estuaries Western Johor Straits

Singapore The National University of Singapore Department of Biological

Sciences Project report pp 49

Hong S Li Q Chen M Huang D 2002 Studies on the embryo development of

horseshoe crabs Tachypleus tridentatus Journal of Xiamen University Natural

Science 41 239-246 (In Chinese)

Hong Kong Dolphin Conservation Society (HKDCS) 2007 Captivity

lthttpwwwhkdcsorgDolinfoinfo_Capfor_enhtmgt Accessed on 24022008

Hong Kong Special Administrative Region (HKSAR) 2006 Hydrography and

oceanography In Year Book of Hong Kong 2006 Hong Kong Government Press

236

Hong Kong Tourism Board 2008 Feel fabulous the Chinese way ndash The Chinese art of

healthy eating

lthttpwwwdiscoverhongkongcomkormeetingsfeelme_feel_chinjhtmlgt


Horikoshi M Thompson GB 1980 Distribution of subtidal molluscs collected by

trawling in Tolo Harbour and Tolo Channel Hong Kong with special reference to

habitat segregation in two venerid bivalves In Morton B (Ed) Proceedings of

the First International Workshop on the Malacofauna of Hong Kong and Southern

China Hong Kong University Press Hong Kong pp 140-162

Hudson TA McAdory B Townsel JG 2005 Localization of semaphorin 2A in the

central nervous system of Limulus polyphemus by in situ hybridization Integrative

and Comparative Biology 45(6) 1147

Hung CLH So MK Connell DW Fung CN Lam MHW Nicholson S

Richardson BJ Lam PKS 2004 A preliminary risk assessment of trace

elements accumulated in fish to the Indo-Pacific Humpback dolphin (Sousa

chinensis) in the northwestern waters of Hong Kong Chemosphere 56 643-651

Hung CLH Xu Y Lam JCW Jefferson TA Hung SK Yeung LWY Lam

MHW OrsquoToole DK Lam PKS 2006 An assessment of the risks associated

with polychlorinated biphenyls found in the stomach contents of stranded

Indo-Pacific Humpback Dolphins (Sousa chinensis) and Finless Porpoises

(Neophocaena phocaenoides) from Hong Kong waters Chemosphere 63 845-852

Itow T 1998 The pollution of coastal waters and malformations of horseshoe crab

embryos Bulletin of the Faculty of Education Shizuoka University Natural

Science Series 48 15-33

237

Itow T Igarashi T Botton ML Loveland RE 1998a Heavy metals inhibit limb

regeneration in horseshoe crab larvae Archives of Environmental Contamination

and Toxicology 35 457-463

Itow T Loveland RE Botton ML 1998b Developmental abnormalities in

horseshoe crab embryos caused by exposure to heavy metals Archives of

Environmental Contamination and Toxicology 35 33-40

Itow T Mishra KR Ahmed AT 2004 Horseshoe crabs (King crabs) in the Bay of

Bengal South Asia Bulletin of the Faculty of Education Shizuoka University

Natural Sciences Series 54 13-30

JamesndashPirri MJ Tuxbury K Marino S Koch S 2005 Spawning densities egg

densities size structure and movement patterns of spawning horseshoe crabs

Limulus polyphemus within four coastal embayments on Cape Cod

Massachusetts Estuaries 28(2) 296ndash313

Jegia TC Costlow JD 1982 Temperature and salinity effects on developmental and

early posthatch stages of Limulus In Bonaventura J Bonaventura C Tesh S

(Eds) Physiology and Biology of Horseshoe Crabs ndash Studies on Normal and

Environmentally Stressed Animals Alan R Liss Inc New York

Kadoorie Farm and Botanic Garden (KFBG) 2006 Good fortune Or misfortune ndash a

leaflet highlighting problems of captive animal releases

Kaiser D 2002 Zur kologie und Morphologie von Tachypleus tridentatus (Leach

1819) (ChelicerataMerostomata) im Gezeitenwatt der Ostkuuml ste von Palawan

(Philippinen) [graduate thesis] Rostock (Germany) University of Rostock pp

132 (In Germen)

Kannan K Tanabe T Tatsukawa R 1995 Phenyltin residues in horseshoe crabs

Tachypleus tridentatus from Japanese coastal waters Chemosphere 30 925-932

238

Kato H Hara M Etoh H 2005 Geographical variations in mitochondrial DNA

sequence and morphological features of the horseshoe crab Tachypleus tridentatus

Biogeography 7 55-59


sequence and morphological features of the horseshoe crab II Carcinoscorpius

rotundicauda Biogeography 8 35-39

Kawahara D 1982 Investigations on ecology of horseshoe crab larvae Aquabiology 4

380-382 (In Japanese)

King TL Eackles MS 2004 Microsatellite DNA markers for the study of horseshoe

crab (Limulus polyphemus) population structure Molecular Ecology Notes 4(3)

394-396

Krebs CJ 1989 Ecological Methodology New York Harper Collins 654 pp

Kumar S Tamura K Jakobsen IB Nei M 2001 MEGA2 Molecular Evolutionary

Genetics Analysis software Bioinformatics 17 1244-1245

Kungsuwan A Nagashima Y Noguchi T Shida Y Suvapeepan S

Suwansakorakal P Hashimoto K 1987 Tetrodotoxin in the horseshoe crab

Carcinoscorpius rotundicauda inhabiting Thailand Bulletin of the Japanese

Society of Scientific Fisheries 53 261-266

Kurtzke C 2001 Horseshoe crab surveys using underwater videography In Tanacredi

JT (Ed) Limulus in the Limelight ndash A Species 350 Million Years in the Making

and in Peril Kluwer AcademicPlenum Publishers New York pp 119-129

Kurz W James-Pirri MJ 2002 The impact of biomedical bleeding on horseshoe

crab Limulus polyphemus movement patterns on Cape Cod Massachusetts

Marine and Freshwater Behavior and Physiology 35261ndash268

Lai K 2007 Saving endangered marine life preserving the ecology City Today 16

10-17

239

Lam QX 2006 Set-free activity and environmental protection Fjdhcom

lthttpwwwfjdhcomArticleHTMLArticle_20060518223437htmlgt Accessed

on 28042008 (in Chinese)

Lau T 2007 Setting free of tortoises into the Yangtze River Sohucom

lthttpnewssohucom20070510n249929042shtmlgt Accessed on 12022008

(In Chinese)

Laughlin R 1983 The effects of temperature and salinity on larval growth of the

horseshoe crab Limulus polyphemus Biological Bulletin 164 93-103

Lee CN Morton B 2005 Experimentally derived estimates of growth by juvenile

Tachypleus tridentatus and Carcinoscorpius rotundicauda (Xiphosura) from

nursery beaches in Hong Kong Journal of Experimental Marine Biology and

Ecology 318 39-49

Leung KMY Kwong RPY Ng WC Horiguchi T Qiu JW Yang R Song

M Jiang G Zheung GJ Lam PKS 2006 Ecological risk assessments of

endocrine disrupting organotin compounds using marine neogastropods in Hong

Kong Chemosphere 65 922-938

Li F Liao Y Dong X 1999 The influence of salinity upon embryogeny of

Tachypleus tridentatus Journal of Zhanjiang Ocean University 19 4-8 (In

Chinese)

Li HM Deng RQ Wang JW Chen ZY Jia FL Wang XZ 2005 A

preliminary phylogeny of the Pentatomomorpha (Hemiptera Heteroptera) based

on nuclear 18S rDNA and mitochondrial DNA sequences Molecular

Phylogenetics and Evolution 37 313-326

Liao Y Hong S 1997 Artificical insemination and artificial incubation of Tachypleus

tridentatus Journal of Zhanjiang Ocean University 17(2) 13-16 (In Chinese)

240

Liao Y Li XM 2001 Poisoning from eating horseshoe crab and its prevention and

treatment Journal of Hygiene Research 2 24 (In Chinese)

Liao Y Li XM Hong SG 2002 Morphology of sallow horseshoe crab (Tachypleus

Tridentatus) Acta Zoologica Sinica 48(1) 93-99 (In Chinese)

Loveland RE Botton ML Shuster JrCN 1996 Life history of the American

horseshoe crab (Limulus polyphemus L) in Delaware Bay and its importance as a

commercial resource In Farrell J Martin C (Eds) Proceedings of the

Horseshoe Crab Forum Status of the Resource University of Delaware Sea Grant

College Program Lewes Delaware USA pp 15-22

Mansourian S Dick G 2006 WWF Global Species Programme WWF Global

Species Programme

Mejia O Zuniga G 2007 Phylogeny of the three brown banded land snail genus

Humboldtiana (Pulmonata Humboldtianidae) Molecular Phylogenetics and

Evolution 45(2) 587-595

Meudt HM Clarke AC 2007 Almost forgotten or latest practice AFLP

applications analyses and advances Trends in Plant Science 12(3) 106-117

Michels SF 1996 Summary of trends in horseshoe crab abundance in Delaware In

Farrell J Martin C (Eds) Proceedings of the Horseshoe Crab Forum Status of

the Resource University of Delaware Sea Grant College Program Lewes DE pp

26ndash29

Mikkelsen T 1988 The Secret in the Blue Blood Science Press Beijing China pp

140

Miyazaki J Sekiguchi K Hirabayashi T 1987 Application of an improved method

of two-dimensional electrophoresis to the systematic study of horseshoe crabs


241

Mollaret I Jamieson BGM Justine JL 2000 Phylogeny of the Monopisthocotylea

and Polyopisthocotylea (Platyhelminthes) inferred from 28S rDNA sequences

International Journal for Parasitology 30 171-185

Moore S Perrin S 2007 Seasonal movement and resource-use patterns of resident

horseshoe crab (Limulus polyphemus) populations in a maine USA estuary

Estuaries and Coasts 30(6) 1016-1026

Morton B Lee CNW 2003 The biology and ecology of juvenile horseshoe crabs

along the northwestern coastline of the New Territories Hong Kong Prospects

and recommendations for conservation Final Report to China Light and Power

Company Limited The Swire Institute of Marine Science The University of Hong

Kong Hong Kong 91 pp

Morton B Morton J 1983 The Sea Shore Ecology of Hong Kong Hong Kong

University Press Hong Kong 350 pp

Morton B Williams GA Lee SY 1996 The benthic marine ecology of Hong

Kong a dwindling heritage In Civil Engineering Office (Ed) Coastal

Infrastructure in Hong Kong A review Hong Kong Government Press pp

233ndash267

Morton B Wu SS 1975 The hydrology of the coastal waters of Hong Kong

Environmental Research 10 319-347

National Geographic Society 2000 Conservation Triangle

lthttpwwwnationalgeographiccomgawconservationhtmlgt Accessed on

02052008

New TR 2000 Conservation Biology ndash An Introduction for Southern Australia

Oxford Unviersity Press Australia

242

Ngy L Yu CF Takatani T Arakawa O 2007 Toxicity assessment for the

horseshoe crab Carcinoscorpius rotundicauda collected from Cambodia Toxicon

49(6) 843-847

Oregon Department of Fish and Wildlife (ODFW) 2006 Section A A strategy for

action smmary and first steps In Orgon Conservation Strategy pp 1-34

Owen RB Sandhu N 2000 Heavy metal accumulation and anthropogenic impacts

on Tolo Harbour Hong Kong Marine Pollution Bulletin 40(2) 174-180

Paetkau D Calvert W Stirling I Strobeck C 1995 Microsatellite analysis of

population structure in Canadian Polar Bears Molecular Ecology 4 347-354

Pearson FC Weary M 1980 The Limulus amebocyte lysate test for endotoxin

Biological Science 30 461-464

Penn D Brockmann HJ 1995 Age-biased stranding and righting in male horseshoe

crabs Limulus polyphemus Animal behaviour 49(6) 1531-1539

Pierce JC Tan G Gaffney PM 2000 Delaware Bay and Chesapeake Bay

populations of the horseshoe crab Limulus polyphemus are genetically distinct

Estuaries 23(5) 690-698

Rao KVR Rao KVS 1972 Studies on Indian king crab (Arachnid Xiphosura)

Proceedings of Indian National Science Academy 38(B3-4) 206-211

Reiko SO Yuichi O Hitoo O 2006 Phylogenetic relationships of click beetles

(Coleoptera Elateridae) inferred from 28S ribosomal DNA Insights into the

evolution of bioluminescence in Elateridae Molecular Phylogenetics and

Evolution 42(2) 410-421

Riska B 1981 Morphological variation in the horseshoe crab Limulus polyphemus

Evolution 35 647-658

Rota E Martin P Ersersquous C 2001 Soil-dwelling polychaetes enigmatic as ever

Some hints on their phylogenetic relationship as suggested by maximum

243

parsimony analysis of 18S rRNA gene sequences Contributions to Zoology 70

127ndash138

Rudloe A 1982 Manrsquos influence as an environmental threat to Limulus In

Bonaventura J Bonaventura C Tesh S (Eds) Physiology and Biology of

Horseshoe Crabs Studies on Normal and Environmentally Stressed Animals Alan

R Liss Inc New York pp 297-300

Rudloe A Hernkind AE 1980 The breeding behavior and patterns of movement of

horseshoe crab Limulus polyphemus in the vicinity of breeding beaches in

Apalachee Bay Florida Estuaries 3 177-183

Rutecki D Carmichael RH Valiela I 2004 Magnitude of harvest of Atlantic

horseshoe crabs Limulus polyphemus in Pleasant Bay Massachusetts Estuaries

27(2) 179-187

Saunders NC Kessler LG Avise JC 1986 Genetic variation and geographic

differentiation in mitochondrial DNA of the horseshoe crab Limulus polyphemus

Genetics 112 613-627

Schwab RL Brockmann HJ 2007 The role of visual and chemical cues in the

mating decisions of satellite male horseshoe crabs Limulus polyphemus Animal

Behaviour 74 837-846

Sekiguchi K 1988a Normal development In Biology of Horseshoe Crabs Science

House Co Ltd Tokyo Japan pp 133-224

Sekiguchi K 1988b Ecology In Biology of Horseshoe Crabs Science House Co

Ltd Tokyo Japan pp 50-68

Sekiguchi K 1988c Morphology In Biology of Horseshoe Crabs Science House Co


244

Sekiguchi K Nakamura K Sen TK Sugita H 1976 Morphological variation and

distribution of a horseshoe crab Tachypleus gigas from the Bay of Bengal and the

Gulf of Siam Proceedings of the Japanese Society of Systematic 12 13-20

Sekiguchi K Nakamura K Seshimo H 1978 Morphological variation of a

horseshoe crab Carcinoscorpius rotundicauda from the bay of Bengal and gulf of

Siam Proceedings of the Japanese Society of Systematic 15 24-30

Sekiguchi K Nakamura T 1979 Ecology of the extant horseshoe crabs In Cohen E

(Ed) Biomedical Applications of the Horseshoe Crab (Limulidae) Alan R Liss

Inc New York USA pp 37-45

Sekiguchi K Nishiwaki S Makioka T Srithunya S Machjajib S Nakamura K

Yamasaki T 1977 A study on the egg-laying habits of the horseshoe crabs

Tachypleus tridentatus and Carcinoscorpius rotundicauda in Chonburi area of

Thailand Proceedings of the Japanese Society of Systematic Zoology 13 39-45

Sekiguchi K Seshimo H Sugita H 1988 Post-embryonic development of the

horseshoe crab Biological Bulletin 174 337-345

Sekiguchi K Sugita H 1980 Systematics and hybridization in the four living species

of horseshoe crabs Evolution 34 712-718

Shin PKS 1985 A trawl survey of the subtidal Mollusca of Tolo Harbour and Mirs

Bay Hong Kong In Morton B Dudgeon D (Eds) Proceedings of the Second

International Workshop on the Malacofauna of Hong Kong and Southern China

Hong Kong University Press Hong Kong pp 439ndash447

Shishikura S Nakamura FS Takahashi S Sekiguchi K 1982 Horseshoe crab

phylogeny based on amino acid sequences of the fibrino-peptide-like peptide C

Journal of Experimental Zoology 223 89-91

Shuster CNJr 1957 Xiphosura (with special reference to Limulus polyphemus)

Geological Society of America 67 1171-1173

245

Shuster CNJr 1979 Distribution of the American horseshoe crab Limulus

polyphemus (L) In Cohen E (Ed) Biomedical Applications of the Horseshoe

Crab (Limulidae) Alan R Liss Inc New York USA pp 3-26

Shuster CNJr 2001 Two perspectives Horseshoe crabs during 420 million years

worldwide and the past 150 years in Delaware Bay In Tanacredic JT (Ed)

Limulus in the Limelight A Species 350 Million Years in the Making and in Peril

Kluwer AcademicPlenum Publishers New York USA pp 17-40

Smith DR Pooler PS Swan BL Michels S Hall WR Himchak P Millard

M 2002 Spatial and temporal distribution of horseshoe crab (Limulus

polyphemus) spawning in Delaware Bay Implications for monitoring Estuaries

25(1) 115-125

Smith SA Berkson JM 2005 Laboratory culture and maintenance of the horseshoe

crab (Limulus polyphemus) Laboratory Animals 34(7) 27-34

Specter ME 1984 Snake soup can be a charmer The New York Times ndash Travel

lthttpquerynytimescomgstfullpagehtmlsec=travelampres=9C05E6D6163BF93

AA15752C0A962948260gt Accessed on 28042008

Srimal S Miyata T Kawabata S Miyata T Iwanaga S 1985 The complete amino

acid sequence of coagulogen isolated from southeast Asian horseshoe crab

Carcinoscorpius rotundicauda Journal of Biochemistry 98305-318

Steinberg EK Jordan CE 1998 Using molecular genetics to learn about the ecology

of threatened species the allure and the illusion of measuring genetic structure in

natural populations In Fiedler PL Kareiva PM (Eds) Conservation Biology

Chapman amp Hall New York

Stormer L 1952 Phylogeny and taxonomy of fossil horseshoe crabs Journal of

Paleontology 26630-639

246

Strathmann MF 1987 Reproduction and Development of Marine Invertebrates on the

Northern Pacific Coast University of Washington Press Seattle USA 670 pp

Struck T Westheide W Purschke G 2002 The phylogentic position of the

Aeolosomatidae and Parergodrilidae two enigmatic oligochaete-like taxa of the

ldquoPolychaetardquo based on molecular data from 18S rDNA sequences Journal of

Zoological Systematic and Evolutionary Research 40 1-10

Struck_TH Purschke G 2005 The sister group relationship of Aeolosomatidae and

Potamodrilidae (Annelida lsquolsquoPolychaetarsquorsquo) mdash a molecular phylogenetic approach

based on 18S rDNA and cytochrome oxidase I Zoologischer Anzeiger 243

281-293

Sugita H 1988 Environmental adaptation of embryos In Sekiguchi K (Ed) Biology

of Horseshoe Crabs Science House Tokyo pp 195-224

Swan BL 2005 Migration of adult horseshoe crabs Limulus polyphemus in the

Middle Atlantic Bight A 17-year tagging study Estuaries 28(1) 28-40

Swan BL Hall WR Shuster CN 1996 Annual Survey of Horseshoe Crab

Spawning Activity along the Shores of Delaware Bay 1990ndash1995 Summary In



35-39

Sweka JA Smith DR Millard MJ 2007 An age-structured population model for

horseshoe crabs in the Delaware Bay area to assess harvest and egg availability for

shorebirds Estuaries and Coasts 30(2) 277-286

Tabachnick BG Fidell LS 1996 Using Multivariate Statistics HaperCollins

College Publishers New York NJ USA

Tanacredi JT 2001 Horseshoe crabs imperiled The fate of a species 350 million

years in the making In Tanacredi JT (ed) Limulus in the Limelight A Species

247

350 Million Years in the Making and in Peril Kluwer AcademicPlenum


Tang BP Zhou KY Song DX 2003 Molecular systematics of the Asian mitten

crabs genus Eriocheir (Crustacea Brachyura) Molecular Phylogenetics and

Evolution 29(2) 309-316

Taylor JD 1992 Long term changes in the gastropod fauna of Tolo Channel and Mirs

Bay a record of change from 1976ndash1986 In Morton B (Ed) Proceedings of the

Second International Marine Biological the Marine Flora and Fauna of Hong

Kong and Southern China Hong Kong University Press Hong Kong pp

557ndash573

Taylor JD Shin PKS 1990 Trawl surveys of sublittoral gastropods in Tolo Channel

and Mirs Bay a record of change from 1976ndash1986 In Morton B (Ed) The

Marine Flora and Fauna of Hong Kong and Southern China (II) Hong Kong

University Press Hong Kong pp 1203-1220

Thompson JD Gibson T Plewniak F Jeanmougin F Higgins DG 1997 The

Clustal X windows interface Flexible strategies tools Nucleic Acids Research 24

4876-4882

University of Delaware Hugh R Sharp Campus February 23 1996 Proceedings of the

Horseshoe Crab Forum Status of the Resource 60 pp

Valdecasas AG Camacho AI 2003 Conservation to the rescue of taxonomy

Biodiversity and Conservation 12 1113-1117

Vos P Hogers R Bleeker M Reijans M van de Lee T Hornes M Frijters A

Pot J Peleman J Kuiper M Zabeau M 1995 AFLP - A new technique for

DNA-fingerprinting Nucleic Acids Research 23 4407-4414

248

Walls EA Berkson J Smith SA 2002 The horseshoe crabs Limulus polyphemus

200 million years of existence 100 years of study Reviews in Fisheries Science

10(1) 39-73

Wan CQ 2005 Valuable aquaculture species ndash horseshoe crab Fishery Guide to be

Rich 23 5 (In Chinese)

Wang CS Li ZZ Typas MA 2003 Nuclear large subunit rDNA group I intron

distribution in a population in a population of Beauveria bassiana strains

Phylogenetic implications Mycological Research 107 1189-1200

Wang DX Su YQ Wang J Liang JR 2001a Influence of environmental factors

on development of embryo and larvae in Tachypleus tridentatus Journal of

Fishery Sciences of China 8(3) 10-14 (In Chinese)

Wang J Wang DX Su YQ Liang JR 2001b Embryonic development of

Tachypleus tridentatus Chinese Journal of Zoology 36(4) 9-14 (In Chinese)

Wang Q Lam NF You H Lai XX 2002 Fujian Pingtan horseshoe crab special

reserve highly demanded Fujian Environment 19(6) 14-15 (In Chinese)

Wang SS 1986 Horseshoe crab sauce in southeast coasts Peng Ren Shi Hua Xin hua

shu dian Beijing fa xing suo fa xing pp 27 (in Chinese)

Widener JW Barlow RB 1999 Decline of a horseshoe crab population on Cape

Cod Biological Bulletin (Woods Hole) 197 300-302

Wikipedia 2008 Chinese mitten crab

lthttpenwikipediaorgwikiChinese_mitten_crabgt Accessed on 28042008

Wilcove DS Rothstein D Dubow J Phillips A Losos E 1998 Quantifying

threats to imperiled species in the United States BioScience 48(8) 607-615

Wilson EO 1992 The Diversity of Life Harvard University Press Massachusetts

Xia X 2000 Phylogenetic relationship among horseshoe crab species Effect of

substitution models on phylogenetic analyses Systematic Biology 49(1) 87-100

249

Yamasaki T Makioka T Saito J 1988 Morphology In Sekiguchi K (Ed) Biology

of Horseshoe Crabs Science House Co Ltd Tokyo Japan pp 69-132

Yan RD 2007 Problem of invasive species and set-free activities Society for Wildlife

and Nature SWAN

lthttpe-infoorgtwissuebiotechissue-biotech00111501htmgt Accessed on

12022008 (In Chinese)

Yang MC Chen CA Hsieh HL Chen CP 2007 Population subdivision of the

tri-spine horseshoe crab Tachypleus tridentatus in Taiwan strait Zoological

Science 24 219-224

Zar JH 1996 Biostatistical Analysis 3rd Edition Prentice Hall International Inc NJ

USA

Zhao YC 2006 Four dishes of horseshoe crab Cooking Knowledge 9 7 (In Chinese)

Zhou H Morton B 2004 The diets of juvenile horseshoe crabs Tachypleus

tridentatus and Carcinoscorpius rotundicauda (Xiphosura) from nursery beaches

proposed for conservation in Hong Kong Journal of Natural History 38

1915-1925

250

Appendix 41 Questionnaire for assessing human exploitation of horseshoe crabs in Hong

Kong

Date Time

Site RestaurantFish Stall

Ind Species

(T tridentatus C rotundicauda)Sex Prosomal Length Prosomal Width

Questions

1) Catching place

_South China Sea Hong Kong _________________________________________

2) Catching method

___________________________________________________________________

3) Catch of horseshoe crab (individuals month)

___________________________________________________________________

4) Sale of horseshoe crab (individuals month)

___________________________________________________________________

5) Size range

___________________________________________________________________

6) Market price range(uncooked amp with cook fee)

___________________________________________________________________

7) Cooking methods

___________________________________________________________________

Remarks

______________________________________________________________________

front pagepdf

Whole thesis_final

front pagepdf

Whole thesis_final

acceptance form

front page

Whole thesis

Thesis (TOC)

chapter_1_Introduction_final

chapter_2_Distribution_final

chapter_3_Genetic_final

chapter_4_Market_survey_final

chapter_5_Artificial_breeding_final


chapter_6_General_discussion _final

chapter_7_Conclusion _final

chapter_8_Reference

appendix

CITY UNIVERSITY OF HONG KONG 香港城市大學



Submitted to Department of Biology and Chemistry

生物及化學系 in Partial Fulfillment of the Requirements

for the Degree of Master of Philosophy 哲學碩士學位

by

LI Hiu Yan 李曉恩

September 2008 二零零八年九月

i

Declaration








LI Hiu Yan

September 2008

ii




Submitted by

LI Hiu Yan

















iii


























iv






















a common population



v


























vi








vii






















viii







grounds






x

Acknowledgements























xi







me endless support







xii

Table of Contents

Declaration i

Abstract ii



Acknowledgements x


List of Tables xvii












110 Objectives

13

15



21 Introduction 17



18





222 Results 26



xiii




57





232 Results 59


Horseshoe Crabs

59



24 Discussion 102


Kong

102




Species

107










116

31 Introduction 116





xiv

322 Results 123








332 Results 136





146





342 Results 148

35 Discussion 153


Crabs

153



155


Kong

159

41 Introduction 159




43 Results 159




xv



43 Discussion 173





51 Introduction 181



522 Results 185






190


532 Results 192







542 Results 201

55 Discussion 203






210

xvi


Distribution Study

210




213








Kong

221







Crabs in Hong Kong

250

xvii

List of Tables



21





28





30



Kong

32



summer and winter

36




38



winter

39




43

xviii



season)

46



season)

49



walk-through survey

52




55



August 2005

63





65



time)

70



time)

74

xix



time)

77


79



2005

84



91



94



2005

98




103



127




128


rotundicauda

129

xx




131


and 28S rDNA

134




138



139




143




144



147



transversions

150




151

xxi



174



191




193




196



198

xxii

List of Figures


tridentatus)

3



20



25



34



35



40


summer and winter

42



45



48



winter

51



53

xxiii



summer and winter

54



56



61




62




66




67



69



73



76



81

xxiv



83



Tau)

87



2005

96



97



100


121


nearest 01mm

122



124



rotundicauda (Cr)

132


sequences

140


sequences

145

xxv


gene sequences

152



163




164



September 2005

165



167



169



restaurant

171




173




183


temperature control

184

xxvi


188



189




194



195




200



stimulation

202

1




















2

















3

pr

op

te



4















to 24 years





5




















6




















7



















8




















9













century






10



















11












Kong








12



















13




















14


















1994)

15







110 Objectives













16













17


21 Introduction






























nursery grounds








Shores in Hong Kong


















20


Lamma Island

21











homes




sites




Lantau Island











22















Lamma Island






















2005-06












25


Hong Kong













software SPSS 110

222 Results




























on Lamma Island

28



Summer

No of individuals




29


No of individuals



30



Winter

No of individuals




31


No of individuals



32


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD




33


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD


34

000

010

020

030

040

050

060

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)

Summer Winter


quadrat sampling

35

000

005

010

015

020

025





Tidal level

Popo

ulat

ion

Den

sity

(ind

100

msup2)

Summer Winter
































winter

Summer Winter

Total no of



T tridentatus


person-1)




Summer Winter

Total no of

C rotundicauda

Density (individual

hour-1 person-1)


Density (individual

hour-1 person-1)


40


walk-through survey


Temperature








42

0

5

10

15

20

25

30

35

40

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Tem

pera

ture

(degC

)

Summer Winter


winter





















45

00

50

100

150

200

250

300

350

400

450

500

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Salin

ity o

f Int

erst

itial

Wat

ers

(permil)

Summer Winter























48

0

2

4

6

8

10

12

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Dis

solv

ed O

xyge

n C

oncc

entr

atio

n (m

gl) Summer Winter











0649 15 0831 0414























51

00

10

20

30

40

50

60

70

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter










53

00

10

20

30

40

50

60





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



(12)

(2)

(26)

(14)

54

00102030405060708090

100

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter









56

00

20

40

60

80

100

120





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



brackets)

(11)

(3)

(2)

(18)


Nursery Grounds







shown in Table 21

















not be conducted


































analysis

232 Results































61

000

020

040

060

080

100

120

140


Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




62

000

010

020

030

040

050

060

070





Tidal Level

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




















65






66

0

2

4

6

8

10

12

14

16

18






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)




67

0

05

1

15

2

25

3

35

4

45






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)





Temperature






215)

69

0

5

10

15

20

25

30

35

40


Month (2005)

Tem

pera

ture

(degC

)







(plt005)



















73

-5

0

5

10

15

20

25

30

35

40


Month (2005)

Salin

ity o

f Int

erst

itial

Wat

ers

(permil) Pak Nai

Ha Pak Nai

Shui Hau Wan

San Tau
















005)

76

0

2

4

6

8

10

12

14


Dis

solv

ed O

xyge

n C

once

ntra

tion

(mg






















the juveniles



















(p = 0007) (Table 219)

81

00051015202530354045505560657075





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)










83

00051015202530354045505560657075


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)





























mm respectively

87

88

89

90


91



Site Pak Nai


No of Individuals


4 496 plusmn 72 6 5 695 plusmn 74 1




Site Ha Pak Nai


March 3 180 1 4 410 1





92Site Shui Hau Wan






Site San Tau



2 280 2 August 1 297 plusmn 31 3










KW test (H = 4112 p = 0128) (Table 222)


four sites












96

00

05

10

15

20

25

30

35

40

45

50





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)




97

00

05

10

15

20

25

30

35

40


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)









Wilcoxon test









100

101



10224 Discussion





















(Table 223)












2002 12 (1 ind 100m-sup2)



AFCD field data





















































2412 C rotundicauda















mentioned before
















































60-70 cm






























2431 T tridentatus





























214)







area









































for C rotundicauda





















2005)

















116





31 Introduction



















117

























118

























119

























120





121

a

b





122





123

322 Results
















a b


124

125















Telson










126






(F 1 129 = 0608 p = 0437)



127




Morphological Ratio

T tridentatus

C rotundicauda

ab

180plusmn015 (146-230)

186plusmn023 (145-245)

bc

053plusmn006 (034-069)

050plusmn008 (034-068)

ac

095plusmn005 (079-112)

093plusmn006 (080-107)

cd

178plusmn011 (157-218)

191plusmn010 (157-213)

ef

008plusmn001 (006-012)

009plusmn001 (006-012)

af

099plusmn013 (077-159)

099plusmn016 (072-151)

gh

135plusmn032 (072-238)

134plusmn050 (020-260)

gc

008plusmn001 (005-012)

004plusmn001 (000-006)

hc

006plusmn001 (003-009)

003plusmn001 (001-005)

128




32



129




longer

shorter


wider telson


130














131





132

Tt

Cr

-5 -4 -3 -2 -1 0 1 2 3 4






133














134


rDNA





135

























136









332 Results

















137
























138

139







014plusmn015 (000-028)


017plusmn014 (000-042)

L polyphemus

126plusmn027 (098-182)

099plusmn026 (070-154)

042plusmn029 (000-084)


041plusmn017 (028-084)

014plusmn016 (000-056)

097plusmn026 (070-154)

011plusmn014 (000-028)


040plusmn014 (028-070)

013plusmn013 (000-042)

096plusmn025 (070-140)

010plusmn013 (000-042)

009plusmn008 (000-014)

Ixodes cookei

1029plusmn014 (1022-1050)

1002plusmn013 (994-1022)

991plusmn031 (966-1036)

1000plusmn013 (994-1022)

999plusmn008 (994-1008)

Artemia salina

1324plusmn014 (1317-1345)

1342plusmn015 (1317-1359)

1310plusmn014 (1303-1331)

1351plusmn013 (1345-1373)

1350plusmn008 (1345-1359)

Note From GenBank

140

UnSHK1

UnSHK3

CrHK2

UnTHK2

UnSHK5

UnSHK2

UnTHK3

CrHK4

CrHK1

CrHK3

UnTHK1

UnSHK4

TtHK4

TtHK2

TtHK3

TtHK1

CrGB1

LpGB4

LpGB3

LpGB2

LpGB1

IcGB1

AsGB1

91

30

78

25

35

58

70

43

80

69

100





IcGB1 Ixodes cookei





141




























142













143

144





T tridentatus

000plusmn000

C rotundicauda

157plusmn080 (000-196)

065plusmn097 (000-196)

L polyphemus

882plusmn000

882plusmn000

009plusmn015 (000-041)


196plusmn000

039plusmn080 (000-196)

882plusmn000

000plusmn000


196plusmn000

039plusmn081 (000-196)

882plusmn000

000plusmn000

000plusmn000

Ixodes cookei

2843plusmn000

2843plusmn000

2549plusmn000

2843plusmn000

2843plusmn000

Artemia salina

5000plusmn000

4922plusmn044 (4902-5000)

5000plusmn000

4902plusmn000

4902plusmn000

Note From GenBank

145

CrHK3

UnSHK3

CrHK1

CrHK2

UnSHK4

UnSHK5

UnSHK2

UnSHK1

UnTHK3

CrHK4

UnTHK2

UnTHK1

TtHK3

TtHK2

CrGB1

TtHK1

TtHK4

LpGB1

LpGB2

LpGB3

LpGB4

IcGB1

AsGB1

100

87

87

98

100





IcGB1 Ixodes cookei





146


in Hong Kong




















147





148

















342 Results







149









a single clade





TtPN1 01 00 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtPN2 008 01 01 01 00 00 01 01 01 01 00 00 00 00 5235

TtPN3 000 008 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN1 000 008 000 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN2 000 008 000 000 01 01 00 00 00 00 01 01 01 01 5236

TtHPN3 008 000 008 008 008 00 01 01 01 01 00 00 00 00 5235

TtST1 008 000 008 008 008 000 01 01 01 01 00 00 00 00 5235

TtST2 000 008 000 000 000 008 008 00 00 00 01 01 01 01 5236

TtST3 000 008 000 000 000 008 008 000 00 00 01 01 01 01 5236

TtSH1 000 008 000 000 000 008 008 000 000 00 01 01 01 01 5236

TtSH2 000 008 000 000 000 008 008 000 000 000 01 01 01 01 5236

TtSH3 008 000 008 008 008 000 000 008 008 008 008 00 00 00 5235

TtYO1 008 000 008 008 008 000 000 008 008 008 008 000 00 00 5235

TtYO2 008 000 008 008 008 000 000 008 008 008 008 000 000 00 5235

TtYO3 008 000 008 008 008 000 000 008 008 008 008 000 000 000 5235

CrHK1 743 734 743 743 743 734 734 743 743 743 743 734 734 734 734

150

151




005plusmn005 (000-008)

Ha Pak Nai

004plusmn004 (000-008)

005plusmn005 (000-008)

San Tau

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Shui Hau Wan

004plusmn004 (000-008)

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Yi O

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

000plusmn000


740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

734plusmn000

152

TtPN1

TtPN3

TtST2

TtHPN2

TtST3

TtHPN1

TtSH1

TtSH2

TtPN2

TtHPN3

TtSH3

TtYO1

TtYO3

TtST1

TtYO2

CrHK1

66

66

001





153

35 Discussion





















studies



154


























155

























156
























et al 2005)

157


























158



Hong Kong







crabs












159


Hong Kong

41 Introduction






















160

















422 Data Collection







161

43 Results

























162













individuals month-1










163

0

50

100

150

200

250

300


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs




164

0

20

40

60

80

100

120

140


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



165

0

20

40

60

80

100

120

140

160


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



166










display and food












167

0

20

40

60

80

100

120

140

160

180


Month

Sale

of H

orse

shoe

Cra

bs

Release Non-release



168














were offered

169

0

10

20

30

40

50

60


Time

Num

ber o

f Hor

sesh

oe C

rabs



170






month-1 (Fig 46)

















171

a

b



172

0

10

20

30

40

50

60

70


Month

Sale

of H

orse

shoe

Cra

bs




waters are shown

173

44 Discussion







and interviews
















174




Species

Western waters


Lau Fau Shan 1997 1










175

























176





1982)

















nutritional value

177


























178

























179


























180



181


51 Introduction
























182




conducted


















183

b

a



crab

184


temperature control

185

522 Results

























186




















larvae





187








188

a b

d c

f e

g h


189


190
























191




192

532 Results



















193








0102030405060708090

100


Surv

ivor

ship

()


0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(c) 25 permil

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(d) 30 permil

194

195



(b) 25

70

80

90

)

0

10

20

30

40

50

60


Surv

ivor

ship

(

(a) 20

8090

100

)

010203040506070


Surv

ivor

ship

(

(c) 32

0

10

20

30

40

50

60

70

80


Surv

ivor

ship

()
















1 20 15 0 2 20 20 2 3 20 25 0 4 20 30 1 5 25 15 0 6 25 20 0 7 25 25 1 8 25 30 0 9 32 15 0 10 32 20 2 11 32 25 1 12 32 30 7












200

a

b




201

542 Results






















202

a

b



203 55 Discussion









investigated





























stimulation

































206















low salinities










207

























208






















extinct




209

























210














comparison purposes










211

























212













2005)













213
















Horseshoe Crabs









214


























215


























216













with urgency













217


























218



practices






















219
























220








2006)















221


















system (Eagle 2001)







222


























223


too late

















2005)






224



















225












tridentatus














226







227
















1081-1093



51(3) 450-477







228



82 383-389












175-184










Biology 7 834-844

229



Norton










241376-1384





pp 268-290



279-287



Research 90 227-236

230



134(1) 73-82






















231









Kexue Ban) 45(3) 404-408



Biology 153 189-198













Kong

232



Biology 16 185-196






743-758




History 37(19) 2369-2382














233

















Bulletin 206 87-94










234


















pp 1-11









235





327-342


1752007)





Kong














236


healthy eating























237























132 (In Germen)



238











394-396















10-17

239






(In Chinese)






Ecology 318 39-49







Chinese)







240











Species Programme



Evolution 45(2) 587-595






26ndash29


140




241

















233ndash267





02052008



242



49(6) 843-847













Estuaries 23(5) 690-698






Evolution 42(2) 410-421





243


127ndash138










27(2) 179-187













244



























245











25(1) 115-125















246










281-293









35-39








247





Evolution 29(2) 309-316





557ndash573







4876-4882








248



10(1) 39-73
























249




and Nature SWAN





Science 24 219-224


USA





1915-1925

250


Kong

Date Time


Ind Species


Questions

1) Catching place


2) Catching method

___________________________________________________________________


___________________________________________________________________


___________________________________________________________________

5) Size range

___________________________________________________________________


___________________________________________________________________

7) Cooking methods

___________________________________________________________________

Remarks

______________________________________________________________________

front pagepdf

Whole thesis_final

front pagepdf

Whole thesis_final

acceptance form

front page

Whole thesis

Thesis (TOC)









chapter_8_Reference

appendix

i

Declaration








LI Hiu Yan

September 2008

ii




Submitted by

LI Hiu Yan

















iii


























iv






















a common population



v


























vi








vii






















viii







grounds






x

Acknowledgements























xi







me endless support







xii

Table of Contents

Declaration i

Abstract ii



Acknowledgements x


List of Tables xvii












110 Objectives

13

15



21 Introduction 17



18





222 Results 26



xiii




57





232 Results 59


Horseshoe Crabs

59



24 Discussion 102


Kong

102




Species

107










116

31 Introduction 116





xiv

322 Results 123








332 Results 136





146





342 Results 148

35 Discussion 153


Crabs

153



155


Kong

159

41 Introduction 159




43 Results 159




xv



43 Discussion 173





51 Introduction 181



522 Results 185






190


532 Results 192







542 Results 201

55 Discussion 203






210

xvi


Distribution Study

210




213








Kong

221







Crabs in Hong Kong

250

xvii

List of Tables



21





28





30



Kong

32



summer and winter

36




38



winter

39




43

xviii



season)

46



season)

49



walk-through survey

52




55



August 2005

63





65



time)

70



time)

74

xix



time)

77


79



2005

84



91



94



2005

98




103



127




128


rotundicauda

129

xx




131


and 28S rDNA

134




138



139




143




144



147



transversions

150




151

xxi



174



191




193




196



198

xxii

List of Figures


tridentatus)

3



20



25



34



35



40


summer and winter

42



45



48



winter

51



53

xxiii



summer and winter

54



56



61




62




66




67



69



73



76



81

xxiv



83



Tau)

87



2005

96



97



100


121


nearest 01mm

122



124



rotundicauda (Cr)

132


sequences

140


sequences

145

xxv


gene sequences

152



163




164



September 2005

165



167



169



restaurant

171




173




183


temperature control

184

xxvi


188



189




194



195




200



stimulation

202

1




















2

















3

pr

op

te



4















to 24 years





5




















6




















7



















8




















9













century






10



















11












Kong








12



















13




















14


















1994)

15







110 Objectives













16













17


21 Introduction






























nursery grounds








Shores in Hong Kong


















20


Lamma Island

21











homes




sites




Lantau Island











22















Lamma Island






















2005-06












25


Hong Kong













software SPSS 110

222 Results




























on Lamma Island

28



Summer

No of individuals




29


No of individuals



30



Winter

No of individuals




31


No of individuals



32


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD




33


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD


34

000

010

020

030

040

050

060

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)

Summer Winter


quadrat sampling

35

000

005

010

015

020

025





Tidal level

Popo

ulat

ion

Den

sity

(ind

100

msup2)

Summer Winter
































winter

Summer Winter

Total no of



T tridentatus


person-1)




Summer Winter

Total no of

C rotundicauda

Density (individual

hour-1 person-1)


Density (individual

hour-1 person-1)


40


walk-through survey


Temperature








42

0

5

10

15

20

25

30

35

40

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Tem

pera

ture

(degC

)

Summer Winter


winter





















45

00

50

100

150

200

250

300

350

400

450

500

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Salin

ity o

f Int

erst

itial

Wat

ers

(permil)

Summer Winter























48

0

2

4

6

8

10

12

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Dis

solv

ed O

xyge

n C

oncc

entr

atio

n (m

gl) Summer Winter











0649 15 0831 0414























51

00

10

20

30

40

50

60

70

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter










53

00

10

20

30

40

50

60





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



(12)

(2)

(26)

(14)

54

00102030405060708090

100

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter









56

00

20

40

60

80

100

120





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



brackets)

(11)

(3)

(2)

(18)


Nursery Grounds







shown in Table 21

















not be conducted


































analysis

232 Results































61

000

020

040

060

080

100

120

140


Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




62

000

010

020

030

040

050

060

070





Tidal Level

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




















65






66

0

2

4

6

8

10

12

14

16

18






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)




67

0

05

1

15

2

25

3

35

4

45






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)





Temperature






215)

69

0

5

10

15

20

25

30

35

40


Month (2005)

Tem

pera

ture

(degC

)







(plt005)



















73

-5

0

5

10

15

20

25

30

35

40


Month (2005)

Salin

ity o

f Int

erst

itial

Wat

ers

(permil) Pak Nai

Ha Pak Nai

Shui Hau Wan

San Tau
















005)

76

0

2

4

6

8

10

12

14


Dis

solv

ed O

xyge

n C

once

ntra

tion

(mg






















the juveniles



















(p = 0007) (Table 219)

81

00051015202530354045505560657075





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)










83

00051015202530354045505560657075


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)





























mm respectively

87

88

89

90


91



Site Pak Nai


No of Individuals


4 496 plusmn 72 6 5 695 plusmn 74 1




Site Ha Pak Nai


March 3 180 1 4 410 1





92Site Shui Hau Wan






Site San Tau



2 280 2 August 1 297 plusmn 31 3










KW test (H = 4112 p = 0128) (Table 222)


four sites












96

00

05

10

15

20

25

30

35

40

45

50





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)




97

00

05

10

15

20

25

30

35

40


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)









Wilcoxon test









100

101



10224 Discussion





















(Table 223)












2002 12 (1 ind 100m-sup2)



AFCD field data





















































2412 C rotundicauda















mentioned before
















































60-70 cm






























2431 T tridentatus





























214)







area









































for C rotundicauda





















2005)

















116





31 Introduction



















117

























118

























119

























120





121

a

b





122





123

322 Results
















a b


124

125















Telson










126






(F 1 129 = 0608 p = 0437)



127




Morphological Ratio

T tridentatus

C rotundicauda

ab

180plusmn015 (146-230)

186plusmn023 (145-245)

bc

053plusmn006 (034-069)

050plusmn008 (034-068)

ac

095plusmn005 (079-112)

093plusmn006 (080-107)

cd

178plusmn011 (157-218)

191plusmn010 (157-213)

ef

008plusmn001 (006-012)

009plusmn001 (006-012)

af

099plusmn013 (077-159)

099plusmn016 (072-151)

gh

135plusmn032 (072-238)

134plusmn050 (020-260)

gc

008plusmn001 (005-012)

004plusmn001 (000-006)

hc

006plusmn001 (003-009)

003plusmn001 (001-005)

128




32



129




longer

shorter


wider telson


130














131





132

Tt

Cr

-5 -4 -3 -2 -1 0 1 2 3 4






133














134


rDNA





135

























136









332 Results

















137
























138

139







014plusmn015 (000-028)


017plusmn014 (000-042)

L polyphemus

126plusmn027 (098-182)

099plusmn026 (070-154)

042plusmn029 (000-084)


041plusmn017 (028-084)

014plusmn016 (000-056)

097plusmn026 (070-154)

011plusmn014 (000-028)


040plusmn014 (028-070)

013plusmn013 (000-042)

096plusmn025 (070-140)

010plusmn013 (000-042)

009plusmn008 (000-014)

Ixodes cookei

1029plusmn014 (1022-1050)

1002plusmn013 (994-1022)

991plusmn031 (966-1036)

1000plusmn013 (994-1022)

999plusmn008 (994-1008)

Artemia salina

1324plusmn014 (1317-1345)

1342plusmn015 (1317-1359)

1310plusmn014 (1303-1331)

1351plusmn013 (1345-1373)

1350plusmn008 (1345-1359)

Note From GenBank

140

UnSHK1

UnSHK3

CrHK2

UnTHK2

UnSHK5

UnSHK2

UnTHK3

CrHK4

CrHK1

CrHK3

UnTHK1

UnSHK4

TtHK4

TtHK2

TtHK3

TtHK1

CrGB1

LpGB4

LpGB3

LpGB2

LpGB1

IcGB1

AsGB1

91

30

78

25

35

58

70

43

80

69

100





IcGB1 Ixodes cookei





141




























142













143

144





T tridentatus

000plusmn000

C rotundicauda

157plusmn080 (000-196)

065plusmn097 (000-196)

L polyphemus

882plusmn000

882plusmn000

009plusmn015 (000-041)


196plusmn000

039plusmn080 (000-196)

882plusmn000

000plusmn000


196plusmn000

039plusmn081 (000-196)

882plusmn000

000plusmn000

000plusmn000

Ixodes cookei

2843plusmn000

2843plusmn000

2549plusmn000

2843plusmn000

2843plusmn000

Artemia salina

5000plusmn000

4922plusmn044 (4902-5000)

5000plusmn000

4902plusmn000

4902plusmn000

Note From GenBank

145

CrHK3

UnSHK3

CrHK1

CrHK2

UnSHK4

UnSHK5

UnSHK2

UnSHK1

UnTHK3

CrHK4

UnTHK2

UnTHK1

TtHK3

TtHK2

CrGB1

TtHK1

TtHK4

LpGB1

LpGB2

LpGB3

LpGB4

IcGB1

AsGB1

100

87

87

98

100





IcGB1 Ixodes cookei





146


in Hong Kong




















147





148

















342 Results







149









a single clade





TtPN1 01 00 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtPN2 008 01 01 01 00 00 01 01 01 01 00 00 00 00 5235

TtPN3 000 008 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN1 000 008 000 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN2 000 008 000 000 01 01 00 00 00 00 01 01 01 01 5236

TtHPN3 008 000 008 008 008 00 01 01 01 01 00 00 00 00 5235

TtST1 008 000 008 008 008 000 01 01 01 01 00 00 00 00 5235

TtST2 000 008 000 000 000 008 008 00 00 00 01 01 01 01 5236

TtST3 000 008 000 000 000 008 008 000 00 00 01 01 01 01 5236

TtSH1 000 008 000 000 000 008 008 000 000 00 01 01 01 01 5236

TtSH2 000 008 000 000 000 008 008 000 000 000 01 01 01 01 5236

TtSH3 008 000 008 008 008 000 000 008 008 008 008 00 00 00 5235

TtYO1 008 000 008 008 008 000 000 008 008 008 008 000 00 00 5235

TtYO2 008 000 008 008 008 000 000 008 008 008 008 000 000 00 5235

TtYO3 008 000 008 008 008 000 000 008 008 008 008 000 000 000 5235

CrHK1 743 734 743 743 743 734 734 743 743 743 743 734 734 734 734

150

151




005plusmn005 (000-008)

Ha Pak Nai

004plusmn004 (000-008)

005plusmn005 (000-008)

San Tau

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Shui Hau Wan

004plusmn004 (000-008)

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Yi O

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

000plusmn000


740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

734plusmn000

152

TtPN1

TtPN3

TtST2

TtHPN2

TtST3

TtHPN1

TtSH1

TtSH2

TtPN2

TtHPN3

TtSH3

TtYO1

TtYO3

TtST1

TtYO2

CrHK1

66

66

001





153

35 Discussion





















studies



154


























155

























156
























et al 2005)

157


























158



Hong Kong







crabs












159


Hong Kong

41 Introduction






















160

















422 Data Collection







161

43 Results

























162













individuals month-1










163

0

50

100

150

200

250

300


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs




164

0

20

40

60

80

100

120

140


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



165

0

20

40

60

80

100

120

140

160


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



166










display and food












167

0

20

40

60

80

100

120

140

160

180


Month

Sale

of H

orse

shoe

Cra

bs

Release Non-release



168














were offered

169

0

10

20

30

40

50

60


Time

Num

ber o

f Hor

sesh

oe C

rabs



170






month-1 (Fig 46)

















171

a

b



172

0

10

20

30

40

50

60

70


Month

Sale

of H

orse

shoe

Cra

bs




waters are shown

173

44 Discussion







and interviews
















174




Species

Western waters


Lau Fau Shan 1997 1










175

























176





1982)

















nutritional value

177


























178

























179


























180



181


51 Introduction
























182




conducted


















183

b

a



crab

184


temperature control

185

522 Results

























186




















larvae





187








188

a b

d c

f e

g h


189


190
























191




192

532 Results



















193








0102030405060708090

100


Surv

ivor

ship

()


0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(c) 25 permil

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(d) 30 permil

194

195



(b) 25

70

80

90

)

0

10

20

30

40

50

60


Surv

ivor

ship

(

(a) 20

8090

100

)

010203040506070


Surv

ivor

ship

(

(c) 32

0

10

20

30

40

50

60

70

80


Surv

ivor

ship

()
















1 20 15 0 2 20 20 2 3 20 25 0 4 20 30 1 5 25 15 0 6 25 20 0 7 25 25 1 8 25 30 0 9 32 15 0 10 32 20 2 11 32 25 1 12 32 30 7












200

a

b




201

542 Results






















202

a

b



203 55 Discussion









investigated





























stimulation

































206















low salinities










207

























208






















extinct




209

























210














comparison purposes










211

























212













2005)













213
















Horseshoe Crabs









214


























215


























216













with urgency













217


























218



practices






















219
























220








2006)















221


















system (Eagle 2001)







222


























223


too late

















2005)






224



















225












tridentatus














226







227
















1081-1093



51(3) 450-477







228



82 383-389












175-184










Biology 7 834-844

229



Norton










241376-1384





pp 268-290



279-287



Research 90 227-236

230



134(1) 73-82






















231









Kexue Ban) 45(3) 404-408



Biology 153 189-198













Kong

232



Biology 16 185-196






743-758




History 37(19) 2369-2382














233

















Bulletin 206 87-94










234


















pp 1-11









235





327-342


1752007)





Kong














236


healthy eating























237























132 (In Germen)



238











394-396















10-17

239






(In Chinese)






Ecology 318 39-49







Chinese)







240











Species Programme



Evolution 45(2) 587-595






26ndash29


140




241

















233ndash267





02052008



242



49(6) 843-847













Estuaries 23(5) 690-698






Evolution 42(2) 410-421





243


127ndash138










27(2) 179-187













244



























245











25(1) 115-125















246










281-293









35-39








247





Evolution 29(2) 309-316





557ndash573







4876-4882








248



10(1) 39-73
























249




and Nature SWAN





Science 24 219-224


USA





1915-1925

250


Kong

Date Time


Ind Species


Questions

1) Catching place


2) Catching method

___________________________________________________________________


___________________________________________________________________


___________________________________________________________________

5) Size range

___________________________________________________________________


___________________________________________________________________

7) Cooking methods

___________________________________________________________________

Remarks

______________________________________________________________________

front pagepdf

Whole thesis_final

front pagepdf

Whole thesis_final

acceptance form

front page

Whole thesis

Thesis (TOC)









chapter_8_Reference

appendix

ii




Submitted by

LI Hiu Yan

















iii


























iv






















a common population



v


























vi








vii






















viii







grounds






x

Acknowledgements























xi







me endless support







xii

Table of Contents

Declaration i

Abstract ii



Acknowledgements x


List of Tables xvii












110 Objectives

13

15



21 Introduction 17



18





222 Results 26



xiii




57





232 Results 59


Horseshoe Crabs

59



24 Discussion 102


Kong

102




Species

107










116

31 Introduction 116





xiv

322 Results 123








332 Results 136





146





342 Results 148

35 Discussion 153


Crabs

153



155


Kong

159

41 Introduction 159




43 Results 159




xv



43 Discussion 173





51 Introduction 181



522 Results 185






190


532 Results 192







542 Results 201

55 Discussion 203






210

xvi


Distribution Study

210




213








Kong

221







Crabs in Hong Kong

250

xvii

List of Tables



21





28





30



Kong

32



summer and winter

36




38



winter

39




43

xviii



season)

46



season)

49



walk-through survey

52




55



August 2005

63





65



time)

70



time)

74

xix



time)

77


79



2005

84



91



94



2005

98




103



127




128


rotundicauda

129

xx




131


and 28S rDNA

134




138



139




143




144



147



transversions

150




151

xxi



174



191




193




196



198

xxii

List of Figures


tridentatus)

3



20



25



34



35



40


summer and winter

42



45



48



winter

51



53

xxiii



summer and winter

54



56



61




62




66




67



69



73



76



81

xxiv



83



Tau)

87



2005

96



97



100


121


nearest 01mm

122



124



rotundicauda (Cr)

132


sequences

140


sequences

145

xxv


gene sequences

152



163




164



September 2005

165



167



169



restaurant

171




173




183


temperature control

184

xxvi


188



189




194



195




200



stimulation

202

1




















2

















3

pr

op

te



4















to 24 years





5




















6




















7



















8




















9













century






10



















11












Kong








12



















13




















14


















1994)

15







110 Objectives













16













17


21 Introduction






























nursery grounds








Shores in Hong Kong


















20


Lamma Island

21











homes




sites




Lantau Island











22















Lamma Island






















2005-06












25


Hong Kong













software SPSS 110

222 Results




























on Lamma Island

28



Summer

No of individuals




29


No of individuals



30



Winter

No of individuals




31


No of individuals



32


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD




33


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD


34

000

010

020

030

040

050

060

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)

Summer Winter


quadrat sampling

35

000

005

010

015

020

025





Tidal level

Popo

ulat

ion

Den

sity

(ind

100

msup2)

Summer Winter
































winter

Summer Winter

Total no of



T tridentatus


person-1)




Summer Winter

Total no of

C rotundicauda

Density (individual

hour-1 person-1)


Density (individual

hour-1 person-1)


40


walk-through survey


Temperature








42

0

5

10

15

20

25

30

35

40

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Tem

pera

ture

(degC

)

Summer Winter


winter





















45

00

50

100

150

200

250

300

350

400

450

500

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Salin

ity o

f Int

erst

itial

Wat

ers

(permil)

Summer Winter























48

0

2

4

6

8

10

12

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Dis

solv

ed O

xyge

n C

oncc

entr

atio

n (m

gl) Summer Winter











0649 15 0831 0414























51

00

10

20

30

40

50

60

70

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter










53

00

10

20

30

40

50

60





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



(12)

(2)

(26)

(14)

54

00102030405060708090

100

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter









56

00

20

40

60

80

100

120





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



brackets)

(11)

(3)

(2)

(18)


Nursery Grounds







shown in Table 21

















not be conducted


































analysis

232 Results































61

000

020

040

060

080

100

120

140


Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




62

000

010

020

030

040

050

060

070





Tidal Level

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




















65






66

0

2

4

6

8

10

12

14

16

18






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)




67

0

05

1

15

2

25

3

35

4

45






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)





Temperature






215)

69

0

5

10

15

20

25

30

35

40


Month (2005)

Tem

pera

ture

(degC

)







(plt005)



















73

-5

0

5

10

15

20

25

30

35

40


Month (2005)

Salin

ity o

f Int

erst

itial

Wat

ers

(permil) Pak Nai

Ha Pak Nai

Shui Hau Wan

San Tau
















005)

76

0

2

4

6

8

10

12

14


Dis

solv

ed O

xyge

n C

once

ntra

tion

(mg






















the juveniles



















(p = 0007) (Table 219)

81

00051015202530354045505560657075





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)










83

00051015202530354045505560657075


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)





























mm respectively

87

88

89

90


91



Site Pak Nai


No of Individuals


4 496 plusmn 72 6 5 695 plusmn 74 1




Site Ha Pak Nai


March 3 180 1 4 410 1





92Site Shui Hau Wan






Site San Tau



2 280 2 August 1 297 plusmn 31 3










KW test (H = 4112 p = 0128) (Table 222)


four sites












96

00

05

10

15

20

25

30

35

40

45

50





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)




97

00

05

10

15

20

25

30

35

40


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)









Wilcoxon test









100

101



10224 Discussion





















(Table 223)












2002 12 (1 ind 100m-sup2)



AFCD field data





















































2412 C rotundicauda















mentioned before
















































60-70 cm






























2431 T tridentatus





























214)







area









































for C rotundicauda





















2005)

















116





31 Introduction



















117

























118

























119

























120





121

a

b





122





123

322 Results
















a b


124

125















Telson










126






(F 1 129 = 0608 p = 0437)



127




Morphological Ratio

T tridentatus

C rotundicauda

ab

180plusmn015 (146-230)

186plusmn023 (145-245)

bc

053plusmn006 (034-069)

050plusmn008 (034-068)

ac

095plusmn005 (079-112)

093plusmn006 (080-107)

cd

178plusmn011 (157-218)

191plusmn010 (157-213)

ef

008plusmn001 (006-012)

009plusmn001 (006-012)

af

099plusmn013 (077-159)

099plusmn016 (072-151)

gh

135plusmn032 (072-238)

134plusmn050 (020-260)

gc

008plusmn001 (005-012)

004plusmn001 (000-006)

hc

006plusmn001 (003-009)

003plusmn001 (001-005)

128




32



129




longer

shorter


wider telson


130














131





132

Tt

Cr

-5 -4 -3 -2 -1 0 1 2 3 4






133














134


rDNA





135

























136









332 Results

















137
























138

139







014plusmn015 (000-028)


017plusmn014 (000-042)

L polyphemus

126plusmn027 (098-182)

099plusmn026 (070-154)

042plusmn029 (000-084)


041plusmn017 (028-084)

014plusmn016 (000-056)

097plusmn026 (070-154)

011plusmn014 (000-028)


040plusmn014 (028-070)

013plusmn013 (000-042)

096plusmn025 (070-140)

010plusmn013 (000-042)

009plusmn008 (000-014)

Ixodes cookei

1029plusmn014 (1022-1050)

1002plusmn013 (994-1022)

991plusmn031 (966-1036)

1000plusmn013 (994-1022)

999plusmn008 (994-1008)

Artemia salina

1324plusmn014 (1317-1345)

1342plusmn015 (1317-1359)

1310plusmn014 (1303-1331)

1351plusmn013 (1345-1373)

1350plusmn008 (1345-1359)

Note From GenBank

140

UnSHK1

UnSHK3

CrHK2

UnTHK2

UnSHK5

UnSHK2

UnTHK3

CrHK4

CrHK1

CrHK3

UnTHK1

UnSHK4

TtHK4

TtHK2

TtHK3

TtHK1

CrGB1

LpGB4

LpGB3

LpGB2

LpGB1

IcGB1

AsGB1

91

30

78

25

35

58

70

43

80

69

100





IcGB1 Ixodes cookei





141




























142













143

144





T tridentatus

000plusmn000

C rotundicauda

157plusmn080 (000-196)

065plusmn097 (000-196)

L polyphemus

882plusmn000

882plusmn000

009plusmn015 (000-041)


196plusmn000

039plusmn080 (000-196)

882plusmn000

000plusmn000


196plusmn000

039plusmn081 (000-196)

882plusmn000

000plusmn000

000plusmn000

Ixodes cookei

2843plusmn000

2843plusmn000

2549plusmn000

2843plusmn000

2843plusmn000

Artemia salina

5000plusmn000

4922plusmn044 (4902-5000)

5000plusmn000

4902plusmn000

4902plusmn000

Note From GenBank

145

CrHK3

UnSHK3

CrHK1

CrHK2

UnSHK4

UnSHK5

UnSHK2

UnSHK1

UnTHK3

CrHK4

UnTHK2

UnTHK1

TtHK3

TtHK2

CrGB1

TtHK1

TtHK4

LpGB1

LpGB2

LpGB3

LpGB4

IcGB1

AsGB1

100

87

87

98

100





IcGB1 Ixodes cookei





146


in Hong Kong




















147





148

















342 Results







149









a single clade





TtPN1 01 00 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtPN2 008 01 01 01 00 00 01 01 01 01 00 00 00 00 5235

TtPN3 000 008 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN1 000 008 000 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN2 000 008 000 000 01 01 00 00 00 00 01 01 01 01 5236

TtHPN3 008 000 008 008 008 00 01 01 01 01 00 00 00 00 5235

TtST1 008 000 008 008 008 000 01 01 01 01 00 00 00 00 5235

TtST2 000 008 000 000 000 008 008 00 00 00 01 01 01 01 5236

TtST3 000 008 000 000 000 008 008 000 00 00 01 01 01 01 5236

TtSH1 000 008 000 000 000 008 008 000 000 00 01 01 01 01 5236

TtSH2 000 008 000 000 000 008 008 000 000 000 01 01 01 01 5236

TtSH3 008 000 008 008 008 000 000 008 008 008 008 00 00 00 5235

TtYO1 008 000 008 008 008 000 000 008 008 008 008 000 00 00 5235

TtYO2 008 000 008 008 008 000 000 008 008 008 008 000 000 00 5235

TtYO3 008 000 008 008 008 000 000 008 008 008 008 000 000 000 5235

CrHK1 743 734 743 743 743 734 734 743 743 743 743 734 734 734 734

150

151




005plusmn005 (000-008)

Ha Pak Nai

004plusmn004 (000-008)

005plusmn005 (000-008)

San Tau

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Shui Hau Wan

004plusmn004 (000-008)

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Yi O

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

000plusmn000


740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

734plusmn000

152

TtPN1

TtPN3

TtST2

TtHPN2

TtST3

TtHPN1

TtSH1

TtSH2

TtPN2

TtHPN3

TtSH3

TtYO1

TtYO3

TtST1

TtYO2

CrHK1

66

66

001





153

35 Discussion





















studies



154


























155

























156
























et al 2005)

157


























158



Hong Kong







crabs












159


Hong Kong

41 Introduction






















160

















422 Data Collection







161

43 Results

























162













individuals month-1










163

0

50

100

150

200

250

300


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs




164

0

20

40

60

80

100

120

140


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



165

0

20

40

60

80

100

120

140

160


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



166










display and food












167

0

20

40

60

80

100

120

140

160

180


Month

Sale

of H

orse

shoe

Cra

bs

Release Non-release



168














were offered

169

0

10

20

30

40

50

60


Time

Num

ber o

f Hor

sesh

oe C

rabs



170






month-1 (Fig 46)

















171

a

b



172

0

10

20

30

40

50

60

70


Month

Sale

of H

orse

shoe

Cra

bs




waters are shown

173

44 Discussion







and interviews
















174




Species

Western waters


Lau Fau Shan 1997 1










175

























176





1982)

















nutritional value

177


























178

























179


























180



181


51 Introduction
























182




conducted


















183

b

a



crab

184


temperature control

185

522 Results

























186




















larvae





187








188

a b

d c

f e

g h


189


190
























191




192

532 Results



















193








0102030405060708090

100


Surv

ivor

ship

()


0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(c) 25 permil

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(d) 30 permil

194

195



(b) 25

70

80

90

)

0

10

20

30

40

50

60


Surv

ivor

ship

(

(a) 20

8090

100

)

010203040506070


Surv

ivor

ship

(

(c) 32

0

10

20

30

40

50

60

70

80


Surv

ivor

ship

()
















1 20 15 0 2 20 20 2 3 20 25 0 4 20 30 1 5 25 15 0 6 25 20 0 7 25 25 1 8 25 30 0 9 32 15 0 10 32 20 2 11 32 25 1 12 32 30 7












200

a

b




201

542 Results






















202

a

b



203 55 Discussion









investigated





























stimulation

































206















low salinities










207

























208






















extinct




209

























210














comparison purposes










211

























212













2005)













213
















Horseshoe Crabs









214


























215


























216













with urgency













217


























218



practices






















219
























220








2006)















221


















system (Eagle 2001)







222


























223


too late

















2005)






224



















225












tridentatus














226







227
















1081-1093



51(3) 450-477







228



82 383-389












175-184










Biology 7 834-844

229



Norton










241376-1384





pp 268-290



279-287



Research 90 227-236

230



134(1) 73-82






















231









Kexue Ban) 45(3) 404-408



Biology 153 189-198













Kong

232



Biology 16 185-196






743-758




History 37(19) 2369-2382














233

















Bulletin 206 87-94










234


















pp 1-11









235





327-342


1752007)





Kong














236


healthy eating























237























132 (In Germen)



238











394-396















10-17

239






(In Chinese)






Ecology 318 39-49







Chinese)







240











Species Programme



Evolution 45(2) 587-595






26ndash29


140




241

















233ndash267





02052008



242



49(6) 843-847













Estuaries 23(5) 690-698






Evolution 42(2) 410-421





243


127ndash138










27(2) 179-187













244



























245











25(1) 115-125















246










281-293









35-39








247





Evolution 29(2) 309-316





557ndash573







4876-4882








248



10(1) 39-73
























249




and Nature SWAN





Science 24 219-224


USA





1915-1925

250


Kong

Date Time


Ind Species


Questions

1) Catching place


2) Catching method

___________________________________________________________________


___________________________________________________________________


___________________________________________________________________

5) Size range

___________________________________________________________________


___________________________________________________________________

7) Cooking methods

___________________________________________________________________

Remarks

______________________________________________________________________

front pagepdf

Whole thesis_final

front pagepdf

Whole thesis_final

acceptance form

front page

Whole thesis

Thesis (TOC)









chapter_8_Reference

appendix

iii


























iv






















a common population



v


























vi








vii






















viii







grounds






x

Acknowledgements























xi







me endless support







xii

Table of Contents

Declaration i

Abstract ii



Acknowledgements x


List of Tables xvii












110 Objectives

13

15



21 Introduction 17



18





222 Results 26



xiii




57





232 Results 59


Horseshoe Crabs

59



24 Discussion 102


Kong

102




Species

107










116

31 Introduction 116





xiv

322 Results 123








332 Results 136





146





342 Results 148

35 Discussion 153


Crabs

153



155


Kong

159

41 Introduction 159




43 Results 159




xv



43 Discussion 173





51 Introduction 181



522 Results 185






190


532 Results 192







542 Results 201

55 Discussion 203






210

xvi


Distribution Study

210




213








Kong

221







Crabs in Hong Kong

250

xvii

List of Tables



21





28





30



Kong

32



summer and winter

36




38



winter

39




43

xviii



season)

46



season)

49



walk-through survey

52




55



August 2005

63





65



time)

70



time)

74

xix



time)

77


79



2005

84



91



94



2005

98




103



127




128


rotundicauda

129

xx




131


and 28S rDNA

134




138



139




143




144



147



transversions

150




151

xxi



174



191




193




196



198

xxii

List of Figures


tridentatus)

3



20



25



34



35



40


summer and winter

42



45



48



winter

51



53

xxiii



summer and winter

54



56



61




62




66




67



69



73



76



81

xxiv



83



Tau)

87



2005

96



97



100


121


nearest 01mm

122



124



rotundicauda (Cr)

132


sequences

140


sequences

145

xxv


gene sequences

152



163




164



September 2005

165



167



169



restaurant

171




173




183


temperature control

184

xxvi


188



189




194



195




200



stimulation

202

1




















2

















3

pr

op

te



4















to 24 years





5




















6




















7



















8




















9













century






10



















11












Kong








12



















13




















14


















1994)

15







110 Objectives













16













17


21 Introduction






























nursery grounds








Shores in Hong Kong


















20


Lamma Island

21











homes




sites




Lantau Island











22















Lamma Island






















2005-06












25


Hong Kong













software SPSS 110

222 Results




























on Lamma Island

28



Summer

No of individuals




29


No of individuals



30



Winter

No of individuals




31


No of individuals



32


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD




33


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD


34

000

010

020

030

040

050

060

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)

Summer Winter


quadrat sampling

35

000

005

010

015

020

025





Tidal level

Popo

ulat

ion

Den

sity

(ind

100

msup2)

Summer Winter
































winter

Summer Winter

Total no of



T tridentatus


person-1)




Summer Winter

Total no of

C rotundicauda

Density (individual

hour-1 person-1)


Density (individual

hour-1 person-1)


40


walk-through survey


Temperature








42

0

5

10

15

20

25

30

35

40

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Tem

pera

ture

(degC

)

Summer Winter


winter





















45

00

50

100

150

200

250

300

350

400

450

500

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Salin

ity o

f Int

erst

itial

Wat

ers

(permil)

Summer Winter























48

0

2

4

6

8

10

12

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Dis

solv

ed O

xyge

n C

oncc

entr

atio

n (m

gl) Summer Winter











0649 15 0831 0414























51

00

10

20

30

40

50

60

70

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter










53

00

10

20

30

40

50

60





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



(12)

(2)

(26)

(14)

54

00102030405060708090

100

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter









56

00

20

40

60

80

100

120





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



brackets)

(11)

(3)

(2)

(18)


Nursery Grounds







shown in Table 21

















not be conducted


































analysis

232 Results































61

000

020

040

060

080

100

120

140


Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




62

000

010

020

030

040

050

060

070





Tidal Level

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




















65






66

0

2

4

6

8

10

12

14

16

18






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)




67

0

05

1

15

2

25

3

35

4

45






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)





Temperature






215)

69

0

5

10

15

20

25

30

35

40


Month (2005)

Tem

pera

ture

(degC

)







(plt005)



















73

-5

0

5

10

15

20

25

30

35

40


Month (2005)

Salin

ity o

f Int

erst

itial

Wat

ers

(permil) Pak Nai

Ha Pak Nai

Shui Hau Wan

San Tau
















005)

76

0

2

4

6

8

10

12

14


Dis

solv

ed O

xyge

n C

once

ntra

tion

(mg






















the juveniles



















(p = 0007) (Table 219)

81

00051015202530354045505560657075





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)










83

00051015202530354045505560657075


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)





























mm respectively

87

88

89

90


91



Site Pak Nai


No of Individuals


4 496 plusmn 72 6 5 695 plusmn 74 1




Site Ha Pak Nai


March 3 180 1 4 410 1





92Site Shui Hau Wan






Site San Tau



2 280 2 August 1 297 plusmn 31 3










KW test (H = 4112 p = 0128) (Table 222)


four sites












96

00

05

10

15

20

25

30

35

40

45

50





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)




97

00

05

10

15

20

25

30

35

40


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)









Wilcoxon test









100

101



10224 Discussion





















(Table 223)












2002 12 (1 ind 100m-sup2)



AFCD field data





















































2412 C rotundicauda















mentioned before
















































60-70 cm






























2431 T tridentatus





























214)







area









































for C rotundicauda





















2005)

















116





31 Introduction



















117

























118

























119

























120





121

a

b





122





123

322 Results
















a b


124

125















Telson










126






(F 1 129 = 0608 p = 0437)



127




Morphological Ratio

T tridentatus

C rotundicauda

ab

180plusmn015 (146-230)

186plusmn023 (145-245)

bc

053plusmn006 (034-069)

050plusmn008 (034-068)

ac

095plusmn005 (079-112)

093plusmn006 (080-107)

cd

178plusmn011 (157-218)

191plusmn010 (157-213)

ef

008plusmn001 (006-012)

009plusmn001 (006-012)

af

099plusmn013 (077-159)

099plusmn016 (072-151)

gh

135plusmn032 (072-238)

134plusmn050 (020-260)

gc

008plusmn001 (005-012)

004plusmn001 (000-006)

hc

006plusmn001 (003-009)

003plusmn001 (001-005)

128




32



129




longer

shorter


wider telson


130














131





132

Tt

Cr

-5 -4 -3 -2 -1 0 1 2 3 4






133














134


rDNA





135

























136









332 Results

















137
























138

139







014plusmn015 (000-028)


017plusmn014 (000-042)

L polyphemus

126plusmn027 (098-182)

099plusmn026 (070-154)

042plusmn029 (000-084)


041plusmn017 (028-084)

014plusmn016 (000-056)

097plusmn026 (070-154)

011plusmn014 (000-028)


040plusmn014 (028-070)

013plusmn013 (000-042)

096plusmn025 (070-140)

010plusmn013 (000-042)

009plusmn008 (000-014)

Ixodes cookei

1029plusmn014 (1022-1050)

1002plusmn013 (994-1022)

991plusmn031 (966-1036)

1000plusmn013 (994-1022)

999plusmn008 (994-1008)

Artemia salina

1324plusmn014 (1317-1345)

1342plusmn015 (1317-1359)

1310plusmn014 (1303-1331)

1351plusmn013 (1345-1373)

1350plusmn008 (1345-1359)

Note From GenBank

140

UnSHK1

UnSHK3

CrHK2

UnTHK2

UnSHK5

UnSHK2

UnTHK3

CrHK4

CrHK1

CrHK3

UnTHK1

UnSHK4

TtHK4

TtHK2

TtHK3

TtHK1

CrGB1

LpGB4

LpGB3

LpGB2

LpGB1

IcGB1

AsGB1

91

30

78

25

35

58

70

43

80

69

100





IcGB1 Ixodes cookei





141




























142













143

144





T tridentatus

000plusmn000

C rotundicauda

157plusmn080 (000-196)

065plusmn097 (000-196)

L polyphemus

882plusmn000

882plusmn000

009plusmn015 (000-041)


196plusmn000

039plusmn080 (000-196)

882plusmn000

000plusmn000


196plusmn000

039plusmn081 (000-196)

882plusmn000

000plusmn000

000plusmn000

Ixodes cookei

2843plusmn000

2843plusmn000

2549plusmn000

2843plusmn000

2843plusmn000

Artemia salina

5000plusmn000

4922plusmn044 (4902-5000)

5000plusmn000

4902plusmn000

4902plusmn000

Note From GenBank

145

CrHK3

UnSHK3

CrHK1

CrHK2

UnSHK4

UnSHK5

UnSHK2

UnSHK1

UnTHK3

CrHK4

UnTHK2

UnTHK1

TtHK3

TtHK2

CrGB1

TtHK1

TtHK4

LpGB1

LpGB2

LpGB3

LpGB4

IcGB1

AsGB1

100

87

87

98

100





IcGB1 Ixodes cookei





146


in Hong Kong




















147





148

















342 Results







149









a single clade





TtPN1 01 00 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtPN2 008 01 01 01 00 00 01 01 01 01 00 00 00 00 5235

TtPN3 000 008 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN1 000 008 000 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN2 000 008 000 000 01 01 00 00 00 00 01 01 01 01 5236

TtHPN3 008 000 008 008 008 00 01 01 01 01 00 00 00 00 5235

TtST1 008 000 008 008 008 000 01 01 01 01 00 00 00 00 5235

TtST2 000 008 000 000 000 008 008 00 00 00 01 01 01 01 5236

TtST3 000 008 000 000 000 008 008 000 00 00 01 01 01 01 5236

TtSH1 000 008 000 000 000 008 008 000 000 00 01 01 01 01 5236

TtSH2 000 008 000 000 000 008 008 000 000 000 01 01 01 01 5236

TtSH3 008 000 008 008 008 000 000 008 008 008 008 00 00 00 5235

TtYO1 008 000 008 008 008 000 000 008 008 008 008 000 00 00 5235

TtYO2 008 000 008 008 008 000 000 008 008 008 008 000 000 00 5235

TtYO3 008 000 008 008 008 000 000 008 008 008 008 000 000 000 5235

CrHK1 743 734 743 743 743 734 734 743 743 743 743 734 734 734 734

150

151




005plusmn005 (000-008)

Ha Pak Nai

004plusmn004 (000-008)

005plusmn005 (000-008)

San Tau

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Shui Hau Wan

004plusmn004 (000-008)

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Yi O

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

000plusmn000


740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

734plusmn000

152

TtPN1

TtPN3

TtST2

TtHPN2

TtST3

TtHPN1

TtSH1

TtSH2

TtPN2

TtHPN3

TtSH3

TtYO1

TtYO3

TtST1

TtYO2

CrHK1

66

66

001





153

35 Discussion





















studies



154


























155

























156
























et al 2005)

157


























158



Hong Kong







crabs












159


Hong Kong

41 Introduction






















160

















422 Data Collection







161

43 Results

























162













individuals month-1










163

0

50

100

150

200

250

300


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs




164

0

20

40

60

80

100

120

140


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



165

0

20

40

60

80

100

120

140

160


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



166










display and food












167

0

20

40

60

80

100

120

140

160

180


Month

Sale

of H

orse

shoe

Cra

bs

Release Non-release



168














were offered

169

0

10

20

30

40

50

60


Time

Num

ber o

f Hor

sesh

oe C

rabs



170






month-1 (Fig 46)

















171

a

b



172

0

10

20

30

40

50

60

70


Month

Sale

of H

orse

shoe

Cra

bs




waters are shown

173

44 Discussion







and interviews
















174




Species

Western waters


Lau Fau Shan 1997 1










175

























176





1982)

















nutritional value

177


























178

























179


























180



181


51 Introduction
























182




conducted


















183

b

a



crab

184


temperature control

185

522 Results

























186




















larvae





187








188

a b

d c

f e

g h


189


190
























191




192

532 Results



















193








0102030405060708090

100


Surv

ivor

ship

()


0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(c) 25 permil

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(d) 30 permil

194

195



(b) 25

70

80

90

)

0

10

20

30

40

50

60


Surv

ivor

ship

(

(a) 20

8090

100

)

010203040506070


Surv

ivor

ship

(

(c) 32

0

10

20

30

40

50

60

70

80


Surv

ivor

ship

()
















1 20 15 0 2 20 20 2 3 20 25 0 4 20 30 1 5 25 15 0 6 25 20 0 7 25 25 1 8 25 30 0 9 32 15 0 10 32 20 2 11 32 25 1 12 32 30 7












200

a

b




201

542 Results






















202

a

b



203 55 Discussion









investigated





























stimulation

































206















low salinities










207

























208






















extinct




209

























210














comparison purposes










211

























212













2005)













213
















Horseshoe Crabs









214


























215


























216













with urgency













217


























218



practices






















219
























220








2006)















221


















system (Eagle 2001)







222


























223


too late

















2005)






224



















225












tridentatus














226







227
















1081-1093



51(3) 450-477







228



82 383-389












175-184










Biology 7 834-844

229



Norton










241376-1384





pp 268-290



279-287



Research 90 227-236

230



134(1) 73-82






















231









Kexue Ban) 45(3) 404-408



Biology 153 189-198













Kong

232



Biology 16 185-196






743-758




History 37(19) 2369-2382














233

















Bulletin 206 87-94










234


















pp 1-11









235





327-342


1752007)





Kong














236


healthy eating























237























132 (In Germen)



238











394-396















10-17

239






(In Chinese)






Ecology 318 39-49







Chinese)







240











Species Programme



Evolution 45(2) 587-595






26ndash29


140




241

















233ndash267





02052008



242



49(6) 843-847













Estuaries 23(5) 690-698






Evolution 42(2) 410-421





243


127ndash138










27(2) 179-187













244



























245











25(1) 115-125















246










281-293









35-39








247





Evolution 29(2) 309-316





557ndash573







4876-4882








248



10(1) 39-73
























249




and Nature SWAN





Science 24 219-224


USA





1915-1925

250


Kong

Date Time


Ind Species


Questions

1) Catching place


2) Catching method

___________________________________________________________________


___________________________________________________________________


___________________________________________________________________

5) Size range

___________________________________________________________________


___________________________________________________________________

7) Cooking methods

___________________________________________________________________

Remarks

______________________________________________________________________

front pagepdf

Whole thesis_final

front pagepdf

Whole thesis_final

acceptance form

front page

Whole thesis

Thesis (TOC)









chapter_8_Reference

appendix

iv






















a common population



v


























vi








vii






















viii







grounds






x

Acknowledgements























xi







me endless support







xii

Table of Contents

Declaration i

Abstract ii



Acknowledgements x


List of Tables xvii












110 Objectives

13

15



21 Introduction 17



18





222 Results 26



xiii




57





232 Results 59


Horseshoe Crabs

59



24 Discussion 102


Kong

102




Species

107










116

31 Introduction 116





xiv

322 Results 123








332 Results 136





146





342 Results 148

35 Discussion 153


Crabs

153



155


Kong

159

41 Introduction 159




43 Results 159




xv



43 Discussion 173





51 Introduction 181



522 Results 185






190


532 Results 192







542 Results 201

55 Discussion 203






210

xvi


Distribution Study

210




213








Kong

221







Crabs in Hong Kong

250

xvii

List of Tables



21





28





30



Kong

32



summer and winter

36




38



winter

39




43

xviii



season)

46



season)

49



walk-through survey

52




55



August 2005

63





65



time)

70



time)

74

xix



time)

77


79



2005

84



91



94



2005

98




103



127




128


rotundicauda

129

xx




131


and 28S rDNA

134




138



139




143




144



147



transversions

150




151

xxi



174



191




193




196



198

xxii

List of Figures


tridentatus)

3



20



25



34



35



40


summer and winter

42



45



48



winter

51



53

xxiii



summer and winter

54



56



61




62




66




67



69



73



76



81

xxiv



83



Tau)

87



2005

96



97



100


121


nearest 01mm

122



124



rotundicauda (Cr)

132


sequences

140


sequences

145

xxv


gene sequences

152



163




164



September 2005

165



167



169



restaurant

171




173




183


temperature control

184

xxvi


188



189




194



195




200



stimulation

202

1




















2

















3

pr

op

te



4















to 24 years





5




















6




















7



















8




















9













century






10



















11












Kong








12



















13




















14


















1994)

15







110 Objectives













16













17


21 Introduction






























nursery grounds








Shores in Hong Kong


















20


Lamma Island

21











homes




sites




Lantau Island











22















Lamma Island






















2005-06












25


Hong Kong













software SPSS 110

222 Results




























on Lamma Island

28



Summer

No of individuals




29


No of individuals



30



Winter

No of individuals




31


No of individuals



32


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD




33


Summer Winter


16 m above CD

13 m above CD

10 m above CD

07m above CD

16 m above CD

13 m above CD

10 m above CD

07m above CD


34

000

010

020

030

040

050

060

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)

Summer Winter


quadrat sampling

35

000

005

010

015

020

025





Tidal level

Popo

ulat

ion

Den

sity

(ind

100

msup2)

Summer Winter
































winter

Summer Winter

Total no of



T tridentatus


person-1)




Summer Winter

Total no of

C rotundicauda

Density (individual

hour-1 person-1)


Density (individual

hour-1 person-1)


40


walk-through survey


Temperature








42

0

5

10

15

20

25

30

35

40

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Tem

pera

ture

(degC

)

Summer Winter


winter





















45

00

50

100

150

200

250

300

350

400

450

500

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Salin

ity o

f Int

erst

itial

Wat

ers

(permil)

Summer Winter























48

0

2

4

6

8

10

12

Tsim

Bei

Tsu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(F)

Ha

Pak

Nai

(G)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Dis

solv

ed O

xyge

n C

oncc

entr

atio

n (m

gl) Summer Winter











0649 15 0831 0414























51

00

10

20

30

40

50

60

70

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter










53

00

10

20

30

40

50

60





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



(12)

(2)

(26)

(14)

54

00102030405060708090

100

Tsim

Bei

Shu

i

Sheu

ng P

ak N

ai

Pak

Nai

Ha

Pak

Nai

(1)

Ha

Pak

Nai

(2)

Pui O

Shui

Hau

Wan

San

Tau

Tai H

o W

an

Sham

Wat

Yi O

Tung

Chu

ng

Hau

Hok

Wan

Luk

Keng

Lai C

hi W

o

Pak

Kok

Wan

Sok

Kwu

Wan

Site

Max

imum

Pro

som

al W

idth

(cm

)

Summer Winter









56

00

20

40

60

80

100

120





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)



brackets)

(11)

(3)

(2)

(18)


Nursery Grounds







shown in Table 21

















not be conducted


































analysis

232 Results































61

000

020

040

060

080

100

120

140


Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




62

000

010

020

030

040

050

060

070





Tidal Level

Popu

latio

n D

ensi

ty (i

nd1

00 m

sup2)




















65






66

0

2

4

6

8

10

12

14

16

18






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)




67

0

05

1

15

2

25

3

35

4

45






Popu

latio

n D

ensi

ty (i

ndh

ourp

erso

n)





Temperature






215)

69

0

5

10

15

20

25

30

35

40


Month (2005)

Tem

pera

ture

(degC

)







(plt005)



















73

-5

0

5

10

15

20

25

30

35

40


Month (2005)

Salin

ity o

f Int

erst

itial

Wat

ers

(permil) Pak Nai

Ha Pak Nai

Shui Hau Wan

San Tau
















005)

76

0

2

4

6

8

10

12

14


Dis

solv

ed O

xyge

n C

once

ntra

tion

(mg






















the juveniles



















(p = 0007) (Table 219)

81

00051015202530354045505560657075





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)










83

00051015202530354045505560657075


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)





























mm respectively

87

88

89

90


91



Site Pak Nai


No of Individuals


4 496 plusmn 72 6 5 695 plusmn 74 1




Site Ha Pak Nai


March 3 180 1 4 410 1





92Site Shui Hau Wan






Site San Tau



2 280 2 August 1 297 plusmn 31 3










KW test (H = 4112 p = 0128) (Table 222)


four sites












96

00

05

10

15

20

25

30

35

40

45

50





Tidal Level

Max

imum

Pro

som

al W

idth

(cm

)




97

00

05

10

15

20

25

30

35

40


Month (2005)

Max

imum

Pro

som

al W

idth

(cm

)









Wilcoxon test









100

101



10224 Discussion





















(Table 223)












2002 12 (1 ind 100m-sup2)



AFCD field data





















































2412 C rotundicauda















mentioned before
















































60-70 cm






























2431 T tridentatus





























214)







area









































for C rotundicauda





















2005)

















116





31 Introduction



















117

























118

























119

























120





121

a

b





122





123

322 Results
















a b


124

125















Telson










126






(F 1 129 = 0608 p = 0437)



127




Morphological Ratio

T tridentatus

C rotundicauda

ab

180plusmn015 (146-230)

186plusmn023 (145-245)

bc

053plusmn006 (034-069)

050plusmn008 (034-068)

ac

095plusmn005 (079-112)

093plusmn006 (080-107)

cd

178plusmn011 (157-218)

191plusmn010 (157-213)

ef

008plusmn001 (006-012)

009plusmn001 (006-012)

af

099plusmn013 (077-159)

099plusmn016 (072-151)

gh

135plusmn032 (072-238)

134plusmn050 (020-260)

gc

008plusmn001 (005-012)

004plusmn001 (000-006)

hc

006plusmn001 (003-009)

003plusmn001 (001-005)

128




32



129




longer

shorter


wider telson


130














131





132

Tt

Cr

-5 -4 -3 -2 -1 0 1 2 3 4






133














134


rDNA





135

























136









332 Results

















137
























138

139







014plusmn015 (000-028)


017plusmn014 (000-042)

L polyphemus

126plusmn027 (098-182)

099plusmn026 (070-154)

042plusmn029 (000-084)


041plusmn017 (028-084)

014plusmn016 (000-056)

097plusmn026 (070-154)

011plusmn014 (000-028)


040plusmn014 (028-070)

013plusmn013 (000-042)

096plusmn025 (070-140)

010plusmn013 (000-042)

009plusmn008 (000-014)

Ixodes cookei

1029plusmn014 (1022-1050)

1002plusmn013 (994-1022)

991plusmn031 (966-1036)

1000plusmn013 (994-1022)

999plusmn008 (994-1008)

Artemia salina

1324plusmn014 (1317-1345)

1342plusmn015 (1317-1359)

1310plusmn014 (1303-1331)

1351plusmn013 (1345-1373)

1350plusmn008 (1345-1359)

Note From GenBank

140

UnSHK1

UnSHK3

CrHK2

UnTHK2

UnSHK5

UnSHK2

UnTHK3

CrHK4

CrHK1

CrHK3

UnTHK1

UnSHK4

TtHK4

TtHK2

TtHK3

TtHK1

CrGB1

LpGB4

LpGB3

LpGB2

LpGB1

IcGB1

AsGB1

91

30

78

25

35

58

70

43

80

69

100





IcGB1 Ixodes cookei





141




























142













143

144





T tridentatus

000plusmn000

C rotundicauda

157plusmn080 (000-196)

065plusmn097 (000-196)

L polyphemus

882plusmn000

882plusmn000

009plusmn015 (000-041)


196plusmn000

039plusmn080 (000-196)

882plusmn000

000plusmn000


196plusmn000

039plusmn081 (000-196)

882plusmn000

000plusmn000

000plusmn000

Ixodes cookei

2843plusmn000

2843plusmn000

2549plusmn000

2843plusmn000

2843plusmn000

Artemia salina

5000plusmn000

4922plusmn044 (4902-5000)

5000plusmn000

4902plusmn000

4902plusmn000

Note From GenBank

145

CrHK3

UnSHK3

CrHK1

CrHK2

UnSHK4

UnSHK5

UnSHK2

UnSHK1

UnTHK3

CrHK4

UnTHK2

UnTHK1

TtHK3

TtHK2

CrGB1

TtHK1

TtHK4

LpGB1

LpGB2

LpGB3

LpGB4

IcGB1

AsGB1

100

87

87

98

100





IcGB1 Ixodes cookei





146


in Hong Kong




















147





148

















342 Results







149









a single clade





TtPN1 01 00 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtPN2 008 01 01 01 00 00 01 01 01 01 00 00 00 00 5235

TtPN3 000 008 00 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN1 000 008 000 00 01 01 00 00 00 00 01 01 01 01 5236

TtHPN2 000 008 000 000 01 01 00 00 00 00 01 01 01 01 5236

TtHPN3 008 000 008 008 008 00 01 01 01 01 00 00 00 00 5235

TtST1 008 000 008 008 008 000 01 01 01 01 00 00 00 00 5235

TtST2 000 008 000 000 000 008 008 00 00 00 01 01 01 01 5236

TtST3 000 008 000 000 000 008 008 000 00 00 01 01 01 01 5236

TtSH1 000 008 000 000 000 008 008 000 000 00 01 01 01 01 5236

TtSH2 000 008 000 000 000 008 008 000 000 000 01 01 01 01 5236

TtSH3 008 000 008 008 008 000 000 008 008 008 008 00 00 00 5235

TtYO1 008 000 008 008 008 000 000 008 008 008 008 000 00 00 5235

TtYO2 008 000 008 008 008 000 000 008 008 008 008 000 000 00 5235

TtYO3 008 000 008 008 008 000 000 008 008 008 008 000 000 000 5235

CrHK1 743 734 743 743 743 734 734 743 743 743 743 734 734 734 734

150

151




005plusmn005 (000-008)

Ha Pak Nai

004plusmn004 (000-008)

005plusmn005 (000-008)

San Tau

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Shui Hau Wan

004plusmn004 (000-008)

004plusmn004 (000-008)

004plusmn004 (000-008)

005plusmn005 (000-008)

Yi O

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

005plusmn004 (000-008)

000plusmn000


740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

740plusmn005 (734-743)

734plusmn000

152

TtPN1

TtPN3

TtST2

TtHPN2

TtST3

TtHPN1

TtSH1

TtSH2

TtPN2

TtHPN3

TtSH3

TtYO1

TtYO3

TtST1

TtYO2

CrHK1

66

66

001





153

35 Discussion





















studies



154


























155

























156
























et al 2005)

157


























158



Hong Kong







crabs












159


Hong Kong

41 Introduction






















160

















422 Data Collection







161

43 Results

























162













individuals month-1










163

0

50

100

150

200

250

300


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs




164

0

20

40

60

80

100

120

140


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



165

0

20

40

60

80

100

120

140

160


04 05 Month

Cat

ch o

f Hor

sesh

oe C

rabs

Sale Set-free



166










display and food












167

0

20

40

60

80

100

120

140

160

180


Month

Sale

of H

orse

shoe

Cra

bs

Release Non-release



168














were offered

169

0

10

20

30

40

50

60


Time

Num

ber o

f Hor

sesh

oe C

rabs



170






month-1 (Fig 46)

















171

a

b



172

0

10

20

30

40

50

60

70


Month

Sale

of H

orse

shoe

Cra

bs




waters are shown

173

44 Discussion







and interviews
















174




Species

Western waters


Lau Fau Shan 1997 1










175

























176





1982)

















nutritional value

177


























178

























179


























180



181


51 Introduction
























182




conducted


















183

b

a



crab

184


temperature control

185

522 Results

























186




















larvae





187








188

a b

d c

f e

g h


189


190
























191




192

532 Results



















193








0102030405060708090

100


Surv

ivor

ship

()


0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(c) 25 permil

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

0

10

20

30

40

50

60

70

80

90


Surv

ivor

ship

()

(d) 30 permil

194

195



(b) 25

70

80

90

)

0

10

20

30

40

50

60


Surv

ivor

ship

(

(a) 20

8090

100

)

010203040506070


Surv

ivor

ship

(

(c) 32

0

10

20

30

40

50

60

70

80


Surv

ivor

ship

()
















1 20 15 0 2 20 20 2 3 20 25 0 4 20 30 1 5 25 15 0 6 25 20 0 7 25 25 1 8 25 30 0 9 32 15 0 10 32 20 2 11 32 25 1 12 32 30 7












200

a

b




201

542 Results






















202

a

b



203 55 Discussion









investigated





























stimulation

































206















low salinities










207

























208






















extinct




209

























210














comparison purposes










211

























212













2005)













213
















Horseshoe Crabs









214


























215


























216













with urgency













217


























218



practices






















219
























220








2006)















221


















system (Eagle 2001)







222


























223


too late

















2005)






224



















225












tridentatus














226







227
















1081-1093



51(3) 450-477







228



82 383-389












175-184










Biology 7 834-844

229



Norton










241376-1384





pp 268-290



279-287



Research 90 227-236

230



134(1) 73-82






















231









Kexue Ban) 45(3) 404-408



Biology 153 189-198













Kong

232



Biology 16 185-196






743-758




History 37(19) 2369-2382














233

















Bulletin 206 87-94










234


















pp 1-11









235





327-342


1752007)





Kong














236


healthy eating























237























132 (In Germen)



238











394-396















10-17

239






(In Chinese)






Ecology 318 39-49







Chinese)







240











Species Programme



Evolution 45(2) 587-595






26ndash29


140




241

















233ndash267





02052008



242



49(6) 843-847













Estuaries 23(5) 690-698






Evolution 42(2) 410-421





243


127ndash138










27(2) 179-187













244



























245











25(1) 115-125















246










281-293









35-39








247





Evolution 29(2) 309-316





557ndash573







4876-4882








248



10(1) 39-73
























249




and Nature SWAN





Science 24 219-224


USA





1915-1925

250


Kong

Date Time


Ind Species


Questions

1) Catching place


2) Catching method

___________________________________________________________________


___________________________________________________________________


___________________________________________________________________

5) Size range

___________________________________________________________________


___________________________________________________________________

7) Cooking methods

___________________________________________________________________

Remarks

______________________________________________________________________

front pagepdf

Whole thesis_final

front pagepdf

Whole thesis_final

acceptance form

front page

Whole thesis

Thesis (TOC)









chapter_8_Reference

appendix

the conservation of horseshoe crabs in hong...

Documents