Hong Kong Baptist University

MASTER'S THESIS

Biological control of golden apple snails (Pomacea canaliculata) infreshwater wetland using black carp (Mylopharyngodon piceus)Ip, Ka Lok Kelvin

Date of Award:2013

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Biological Control of Golden Apple Snails (Pomacea canaliculata)

in Freshwater Wetland

using Black Carp (Mylopharyngodon piceus)

IP Ka Lok Kelvin

M. Phil. Thesis

Department of Biology

Hong Kong Baptist University

October 2013

Biological Control of Golden Apple Snails (Pomacea canaliculata)

in Freshwater Wetland

Using Black Carp (Mylopharyngodon piceus)

IP Ka Lok Kelvin

A thesis submitted in partial fulfilment of the requirements

for the degree of

Master of Philosophy

Principal Supervisor: Dr. QIU Jianwen

Hong Kong Baptist University

October 2013

i

Declaration

I hereby declare that this thesis represents my own work which has been done

after registration for the degree of MPhil at Hong Kong Baptist University, and

has not been previously included in a thesis, dissertation submitted to this or

other institution for a degree, diploma or other qualification.

Signature

October, 2013

ii

Abstract

The apple snail Pomacea canaliculata Lamarck is a native of South America but

has invaded Hong Kong since early 1980s. Its feeding has resulted in a

tremendous loss in semi-aquatic agriculture, especially rice (Oryza sativa L.) and

other aquatic crops such as taro (Colocasia esculenta L.) and water spinach

(Ipomoea aquatica Forssk). While spreading to freshwater wetlands, its feeding

threatens macrophyte diversity. Owing to its voracious appetite, this invasive

snail has also become a competitor of lowland indigenous mollusks. On

ecosystem level, over-grazing by high density of apple snails could also induce

excessive release of nutrients from macrophytes to water bodies, thus promoting

phytoplankton growth and primary production.

Measures to control invasive apple snails fall into three categories:

mechanical / cultural, chemical, and biological. Among them, biological control

methods are appealing because they are usually considered relatively less labor-

intensive and more cost-effective. However, both the control efficacy and

potential non-target effects should be carefully evaluated before adopting a

species in biological control. Although various fish species have been proposed

as biological control agents for apple snails, their effectiveness and non-target

effects on wetland flora and fauna are largely unknown.

This study investigated the feasibility of black carp (Mylopharyngodon

piceus Richardson) as bio-control agent for apple snails in both laboratory and

field experiments.

iii

The laboratory experiment compared the feeding of black carp, common

carp (Cyprinus carpio L.) and white-spotted catfish (Clarias fuscus Lacepède) on

apple snails. These three species are indigenous and widely aquacultured in

southern China. The three species of fish of comparable body length were each

offered apple snails of various sizes ad libitum in aquaria. Black carp (fork length:

165 mm; maximum gap width: 16 mm) was the most effective predator, with a

predatory rate of 70.5 apple snails in 48 hours. Common carp and white-spotted

catfish of similar fork lengths consumed only 58.6 and 15.7 apple snails on

average within the same experimental period. Apple snails preyed upon by black

carp and common carp were juveniles, with their respective shell length ranged

from 3 - 16mm and 3 - 17mm, while that for white-spotted catfish ranged from 3

- 21mm.

An 8-week mesocosm experiment was conducted in a constructed wetland

during the dry season of 2011 to determine whether black carp (fork length: 170 -

185 mm) is as effective as common carp (fork length: 170 - 195 mm) as a bio-

control agent for apple snails, but causes less herbivory to macrophytes and

predation to non-Pomacea snails. Both species of carp preyed effectively on P.

canaliculata, removing almost all apple snail individuals (~ 200 per enclosure)

that were small enough to fit into their mouths. The effects of the two fish

species on macrophytes were different. Black carp reduced herbivory on

macrophytes through reducing apple snail density. However, common carp

reduced apple snail density but did not result in a lower level of herbivory

because it also grazed on macrophytes. Non-target mollusk density was reduced

by both fish species.

iv

A one-year whole-pond experiment was also conducted in June 2012 to

June 2013 to investigate the applicability of black carp as a biological control

agent of apple snails in constructed freshwater wetlands. Three separate

constructed freshwater wetlands were used as replicates of the experiment. Each

wetland was divided into a control side without black carp and a treatment side

with black carp. Four individuals of black carp (fork length 260 - 310 mm) were

released to the side of wetland assigned as treatment. Prior to starting the

experiment and every three months, density of apple snails and other macro-

invertebrates, apple snail egg clutch size and abundance, water quality

parameters (total nitrogen, ammonia nitrogen, total phosphorus and reactive

phosphorus) were recorded. Black carp was highly tolerant to the low dissolved

oxygen in the shallow stagnant waters. It was an effective predator of juvenile

apple snails (<5 – 25mm), but it did not result in significant reduction of adult

apple snails (shell length >25mm) nor affected their reproduction. In addition,

black carp preyed on non-apple snail macro-invertebrates, especially mollusks.

In conclusion, our study has shown that juvenile black carp (minimum total

length: 300mm) is a suitable bio-control agent of apple snails in shallow water

wetlands as it is tolerant of stagnant poor water quality and is an effective

predator of apple snails. A major decline of 89.2% in average overall density of

apple snail has been recorded in the treatment plots of the three experimental

sites after one year. Juvenile snails would be eradicated before they get to mature

minimum size (male SL: 25.2 ± 3.3mm; female: 29.8 ± 3.6mm) for reproduction.

Given the longevity of black carp, a low stocking density (80-89 individuals ha-1

)

is sufficient to control apple snail populations. However, black carp reduces the

v

abundance and diversity of non-target macro-invertebrates. Therefore the

benefits of the biological control must be weighed against the potential

undesirable effects on wetland diversity before adopting in the pest management.

To maximize the control efficacy, mechanical methods to eradicate adult snails,

for instance hand-picking in the shallow water, should be implemented with

biological control effort in an integrated apple snail management program.

vi

Acknowledgements

I would like to take this opportunity to thank my principal supervisor Dr.

Jianwen Qiu for his insightful guidance, generous sharing of knowledge,

continuous support and patience throughout my course of study. I would like to

thank whole-heartedly for his encouragement and support for me in pursuing this

degree with such an inspiring topic that could provide practical management

recommendations for wetlands in future. Special thanks to my co-supervisor Dr.

Yan Liang who has provided vital comments for my study and manuscripts as

well.

I must thank the Mass Transit Railway Corporation Ltd. for providing

financial support for this research. I would like to deliver my thanks to Dr. Kam

Foon Chan, Mr. Peter Choi, Ms. Catherine Leung, Ms. Ida Yu and Mr. John

Allcock who has provided technical comments and all necessary on-site support

for field experiments in Kam Tin compensation wetlands.

I would also like to thank Ms. Yingxuan Li for her kind assistance in

handling the technical and financial problems for us throughout the study. I am

thankful to my dear labmates, Dr. Jin Sun, Ms. Yanan Sun, Mr. Paul Fong, Mr.

James Xie, Ms. Huawei Wu who have provided support in field work,

encouragements, comfort and laughter whenever we chat. I am also grateful to

receive useful comments from two previous students of my principal supervisor:

Mr. King Lun Allan Kwok, Mr. Pak Ki Wong who conducted their in-depth

studies and contributed to understanding in ecology of apple snails in Hong Kong.

Special thanks to Mr. Mak who provided us with precious black carp specimens;

Mr. Aaron Au and Dr. Dickey Lau for assistance in contacting local fish farmers;

vii

Mr. Tim Chung, Ms. Phoebe Lam, Mr. Lam Ngo Lun Alan and Ms. Priscilla Li

for their assistance to my tedious field work in summer.

I would like to extend my thanks to my parents. I am forever indebted to

their love, care, encouragement and understanding in my current research and

previous 25 years. Their support for my freedom in exploring personal academic

interest and career pathway has always been the most precious gift of my lifetime.

I would also like to thank Ms. Flora Leung for her love and encouragement for

me in the last four years, whilst I hope that would last till ever.

Last but not least, thanks to all indigenous fauna and flora of Hong Kong.

You have strived through the time where even few trees could ever survive after

wartime. With your contribution, we are proud of being one of the wealthiest

cities in the world with such huge biodiversity. Though we have deprived your

rights to thrive within several decades by over-exploitation, habitat degradation,

and introduction of invasive species, I hope our efforts and enthusiasm would

finally bridge the knowledge gaps in biodiversity conservation and ecological

consequences brought forth by rapid development.

viii

Table of Contents

Declaration .......................................................................................................... i

Abstract.............................................................................................................. ii

Acknowledgements ........................................................................................... vi

Table of Contents ............................................................................................ viii

List of Tables ..................................................................................................... x

List of Figures ................................................................................................... xi

Chapter 1 Introduction........................................................................................ 1

1.1 Natural distribution range and pathway of introduction of apple snails .. 1

1.2 Distribution and identity of apple snails in Hong Kong .......................... 2

1.3 Impact of apple snails as invasive species to Chinese wetlands .............. 4

1.4 Current control methods of apple snails in Hong Kong .......................... 8

1.5 Biological control and indigenous predators of apple snails in Hong

Kong ......................................................................................................... 10

1.6 Research objectives and general organization of thesis ........................ 13

Chapter 2 Predatory potential of indigenous fish as bio-control agent of apple

snails ................................................................................................................ 19

2.1 Background ......................................................................................... 19

2.2 Material and method ............................................................................ 22

2.3 Results ................................................................................................ 24

2.4 Discussion ........................................................................................... 25

Chapter 3 Biological control of invasive apple snails in a freshwater wetland by

two carp species: subtle differences matter ....................................................... 32

3.1 Background ......................................................................................... 32

3.2 Materials and methods ......................................................................... 35

3.3 Results ................................................................................................ 39

3.4 Discussion ........................................................................................... 42

Chapter 4 Wetland scale application of biological control of apple snails using

black carp: efficacies and subtle effect to ecosystem......................................... 51

4.1 Background ......................................................................................... 51

4.2 Study site ............................................................................................ 54

4.3 Method ................................................................................................ 55

ix

4.4 Results ................................................................................................ 61

4.5 Discussion ........................................................................................... 67

5.1 Summary of information on local invasion history and possible

indigenous predators of apple snails .................................................... 94

5.2 Predatory potential of different local fish species on apple snails ......... 96

5.3 Mesocosm studies on functional practicability of fish: testing efficacies

and subtle effects in a nutshell ............................................................. 97

5.4 Wetland scale application of biological control of apple snails using

black carp: efficacies and subtle effect to ecosystem............................ 98

5.5 Implications on integrated pest management ....................................... 99

Appendices .................................................................................................... 100

References ..................................................................................................... 104

Curriculum Vitae............................................................................................ 128

x

List of Tables

Page

Table 1.1 List of possible indigenous predators of aquatic mollusks

in Hong Kong wetlands

15

Table 2.1Results of one-way ANOVA and post-hoc Tukey test on

total abundance of consumed apple snails after the treatment 28

Table 4.1 Fish survival in three experimental sites in June 2012-

September 2013

74

Table 4.2 Results of two-way ANOVA testing the difference in

estimated hatchlings between treatments and among sampling time

75

Table 4.3 Effect of black carp on density of different size range of

apple snail and their reproductivity

76

Table 4.4 Hatchability of collected egg clutches in laboratory 77

Table 4.5 Effect of black carp to non-target gastropods

78

Table 4.6 Effect of black carp on abundance of non-target benthic

invertebrates except gastropods. Data are total abundance of each

family of invertebrates in 1.5m2 quadrat area

79

Table 4.7 Effect of black carp on abundance of non-target benthic

invertebrates except gastropods. Data are total abundance of each

family of invertebrates in 1.5m2 quadrat area

80

Table 4.8 Results of two-way ANOVA testing the difference in

nutrient parameters in individual sites between treatment and among

sampling period. Nutrient parameters abbreviations: NH3 =

ammoniacal nitrogen; TN = total nitrogen; P = phosphates; TP =

total phosphorus

81

Table 4.9 The black carp experiment. Effect of black carp and apple

snails on water quality

Times 1-5 represented June 2012, September 2012, December 2012,

March 2013 and June 2013 respectively. Data are means with SE in

parentheses

82

Table 4.10 Effect of fish and apple snails on plant abundance. Data

are mean of eight quadrats with S.D. in parentheses. Time T1 and

T2 represented June 2012 and June 2013 respectively

84

xi

List of Figures

Page

Fig. 2.1 Size-dependent predation of three fish species on apple

snails in aquaria. Each graph for each species showed total

abundance of snails survived in six aquaria before (t=0hrs) and after

(t = 48hrs) the experiment

29

Fig. 2.2 Mean percentage of survival for golden apple snails in six

aquaria, after individual treatments with three species of fish

30

Fig. 2.3 Comparison of mean amount of snails consumed per

experimental tank among three treatments. Letters above the bars

are results of one-way ANOVA and the Tukey test comparing the

difference in mean amount of golden apple snails consumed per

tank among treatments. Different letters and asterisk indicate

significant difference at p < 0.05

31

Fig. 3.1 Size frequency distribution of Pomacea canaliculata at the

end of the 8-week experiment. Abbreviations: AS = treatment

containing apple snails; AS+CC = treatment containing apple snails

and common carp; AS+BC = treatment containing apple snails and

black carp

47

Fig. 3.2 Abundance of non-Pomacea mollusks at the end of the 8-

week experiment. Each bar shows the mean number of mollusks per

replicate for each treatment

48

Fig. 3.3. Size-frequency distribution of the two most abundant non-

target mollusks – Sinotaia quadrata and Melanoides tuberculata.

Each graph shows the pooled data from five replicate enclosures for

each treatment

49

Fig. 3.4. Biomass of three species of macrophytes at the end of the

8-week experiment. Each bar represents the mean ± S.D. of all

available (3-5) replicate enclosures. Letters above the bars are

results of the Tukey tests comparing the difference in biomass

among treatments

50

Fig. 4.1 Mean densities of apple snails in each plot from June 2012

to June 2013

85

Fig. 4.2 Number of egg clutches encountered during walkover

survey from June 2012 – June 2013 86

Fig. 4.3 Abundance of apple snail hatchlings (individuals m-2

) from

June 2012 to March 2013

87

xii

Fig. 4.4 Size-frequency distribution of Pomacea canaliculata in

experimental and control plots from June 2012 to March 2013

88

Fig. 4.5 Densities of three major species of non-target mollusks

(individuals m-2

) in June 2012 – March 2013

89

Fig. 4.6 Comparison of physical water quality parameters within

June 2012 to June 2013. Mean values of each data were shown with

standard deviations as error bars

90

Fig. 4.7 Comparison of chemical water quality parameters within

June 2012 to June 2013. Mean values of each data were shown with

standard deviations as error bars

91

Fig. 4.8 2D nMDS plots showing impact of black carp and apple

snail on vegetation structure from initial (June 2012 – June 2013),

using three sites as replicates

92

1

Chapter 1 Introduction

1.1 Natural distribution range and pathway of introduction of apple snails

Pomacea (Ampullariidae) is a large genus of apple snails with more than 100

described species (Cowie and Thiengo, 2003). They are indigenous to freshwater

marshes in South America. At least three species (i.e. Pomacea canaliculata

Lamarck, P. maculata Perry and P. scalaris D’ Orbigny) have been introduced

into Asia since the 1980s (Anderson, 1993; Jhang, 1985; Joshi, 2007; Yusa and

Wada, 1999; Joshi et al., 2001; Chen et al., 2004; Cowie et al., 2006; Hayes et al.,

2008; Hayes et al., 2012; Lv et al., 2013). P. canaliculata was the first species

introduced to Asia, through importation of egg clutches to establish snail

aquaculture in Taiwan in 1980. This species was soon introduced to Japan, China

and several countries in Southeast Asia including the Philippines, Vietnam,

Cambodia and Thailand (Hamada and Matsumoto, 1985; Cha, 1989; Mochida,

1991; Joshi and Sebastian, 2006). In addition to P. canaliculata, P. maculata and

P. scalaris were also introduced into Asia (Cowie et al. 2006; Hayes et al. 2008;

2012). In China, P. canaliculata and P. maculata exhibited a wide and mosaic

distribution in southern China (Lv et al., 2009; Lv et al., 2013). In Taiwan, P.

canaliculata is widely distributed, but the distribution of P. scalaris is restricted

to southern part (Chen, 2011; Lee and Wu, 1996; Wu et al., 2011). Due to their

rapid growth and high protein content, apple snails were initially widely

promoted as human food and protein supplement in animal feed. However, apple

snail aquaculture farms soon stopped operation due to a number of reasons: lack

of tenderness of apple snail meat, high processing cost, and infestation by

eosinophilic meningitis causing rat lungworm Angiostrongylus cantonensis

2

(Mochida, 1991; Yu et al., 2001; Lv et al., 2008). Abandoned snails soon

established large populations in various freshwater habitats through active

crawling and passive transportation by water flow in drainage systems (Mochida,

1991; Yusa and Wada, 1999; Cowie, 2002). In this chapter we provide a review

of the ecology (distribution, secondary production, and impact to wetland flora

and fauna and wetland function) and management of apple snails in the

agricultural and non-agricultural areas of Hong Kong.

1.2 Distribution and identity of apple snails in Hong Kong

Apple snails were first reported in northern New Territories as a result of a

territory-wide survey conducted in 1988 by Yipp et al. (1991), who estimated

that these snails were introduced to agricultural area with many vegetable

gardens and abandoned rice paddies in 1980 to 1983. This estimation was

consistent with the results of an earlier territory-wide survey of mollusks

conducted in 1980-1981 by Yipp (1983), who did not find apple snails. There is

no consensus about the source and invasion pathway for apple snails in Hong

Kong. Because the first report of apple snails in the agricultural area of the New

Territories of Hong Kong was very close in timing of the first report of apple

snails in Guangdong (Cai, 1990), and the agricultural areas of Hong Kong and

Shenzhen (southern Guangdong) are connected by a network of streams and

drainage channels, it was difficult to determine apple snails were first introduced

to which of these two areas.

Within the subsequent two decades of their invasion, apple snails spread to

most lowland wetlands in the New Territories including streams, ponds,

freshwater marshes, abandoned rice paddies, vegetable gardens and drainage

3

channels in the New Territories. The periodic flooding during the summer rainy

season must have helped apple snail dispersion in the lowland wetlands, which

are connected through streams and drainage channels. A territory-wide survey of

apple snails conducted in 2006 (Kwong et al., 2008) revealed the wide-

distribution of apple snails in the New Territories (Fig. 1). The sites inhabited by

apple snails typically had high levels of phosphate and alkalinity, but apple snails

were also occasionally found in streams where nutrient and alkalinity levels were

low. It was also found that geographical isolation and a lack of agricultural

activities may have halted the spread of apple snails to other areas with apparent

suitable habitats in Hong Kong. Specifically, although the water quality

parameters of sites, i.e. abandoned rice paddies, on Lantau Island were

apparently suitable for apple snails, they had not been colonized due to a lack of

physical connection with the New Territories. However, the building of bridges

connecting the New Territories and Lantau Island in 1997 has greatly enhanced

the traffic, and there is concern of introducing apple snails to Lantau Island

through replanting abandoned rice paddies with agricultural crops, which may

carry apple snail eggs, as a result of the development of ecotourism. There was

some confusion regarding the identity of apple snails introduced into Hong Kong

(Kwong et al., 2008). Cha (1989) and Yipp et al. (1991) identified two species of

apple snails - Ampullaria levior and A. gigas. Although both species were found

in northern New Territories, they showed some differences in their distribution

(Cha, 1989). Yipp et al. (1991) concluded that A. gigas preferred habitats with

bottom sediment of coarser grain sizes while A. levior was a dominant grazer in

finer ones. Lam (1994) identified the apple snails collected from a stream in the

4

New Territories as P. levior, whereas Dudgeon and Corlett (2004) considered P.

lineata to be the species of apple snails that are widely spread in Hong Kong. To

determine the species identity of apple snails in Hong Kong, selective snails

collected from different locations (Kwong et al., 2008) showing the greatest

morphological variations with respect to size, shell color and shell shape were

sent to Robert Cowie and Kenneth Hayes of University of Hawaii, who

successfully amplified a segment of the cytochrome oxidase I gene for 12

individuals. An analysis of these mitochondrial DNA sequences showed that all

of these individuals were P. canaliculata (Kenneth Hayes, pers. comm.),

therefore P. canaliculata is likely the only species of Pomacea currently present

in Hong Kong. Based on shell morphology, this species shall be A. levior in Cha

(1989) and Yipp et al. (1991), P. levior in Lam (1994) and P. lineata in Dudgeon

and Corlett (2004). A. gigas in Cha (1989) and Yipp et al. (1991) was likely P.

maculata, which can reach a larger maximum body size than P. canaliculata

(Hayes et al. 2012). P. maculata has successfully established in some areas of

southern China (Lv et al., 2013), but may have become locally extinct in Hong

Kong.

1.3 Impact of apple snails as invasive species to Chinese wetlands

Natural existence of apple snails in agricultural wetlands of South America has

been reported to be harmless (Cazzaniga, 2006). Yet, it has been widely regarded

as pests to freshwater agricultural plants in many tropical and sub-tropical

countries, especially to rice (Oryza sativa) and other aquatic crops such as taro

(Colocasia esculenta) and water spinach (Ipomoea aquatica ) (Anderson, 1993;

Joshi, 2007; Naylor, 1996; Yusa and Wada, 1999a; Yusa and Wada, 1999b). In

5

Taiwan, an annual loss of US $3.89 million has been estimated, with 100,000 ha

of agricultural lands devastated by apple snails yearly (Huang, 2005).

Approximately 660,000 ha and 167,000 ha of rice paddies has been infested with

apple snails in Guangdong and Guangxi province in 2006 respectively, and apple

snail population is expanding further north at rate of 8-10 km per year (Yin et al.,

2006).

In fact, the impact of apple snails to agricultural lands is not restricted to

taros and rice. Grazing by apple snails also significantly reduce the yield of

various aquatic crops in shallow wet agricultural lands, and crops that are planted

immediately adjacent to paddies for instance watercress (Nasturtium officinale)

(Hue et al., 1997) and Manchurian wild rice (Zizania latifolia) (Dong et al.,

2012). Since these wet agricultural lands remain key habitats of farmland birds in

Asia (Amano et al., 2008), infestation of apple snails as an invasive species could

further reduce incentives for conserving these habitats (Pascual and Perrings,

2007) due to their potential impacts on wetland flora and fauna. Apple snails can

disrupt the natural growth cycle of phytoplankton and macrophyte in shallow

wetlands, such as ponds, lakes and floodplains. In natural and artificial wetlands,

macrophytes are a key component in habitat complexity. In general, they provide

surface for the growth of cyanobacteria and periphyton, refuge for animals

against predation, nutrient sink (phosphorus and nitrogen) and reduce re-

suspension of sediments. In shallow wetlands, there are few invertebrate grazers

of macrophytes in southern China including Hong Kong (Dudgeon 1999c). There

is however plenty of shredders that slowly consume allochthonous leaf litter. Yet,

periphyton attachs and grows on the surface of macrophytes constitutes a large

6

portion in the diet of scrapers, while detritus from decomposition of dead

macrophytes comprise a large portion in diet of collector-gatherers (which

include most of the snails) (Dudgeon and Yipp 1983). Previous studies have

reported that exotic apple snails could consume macrophytes and promote the

growth of phytoplankton, thus shifting wetland ecosystems towards a state of low

macrophyte diversity and high nutrient concentration (Carlsson et al., 2004a;

Carlsson and Lacoursière, 2005; Fang et al., 2010).

Apple snails may have out-competed local freshwater invertebrates including

mollusks by predation. Studies have shown that apple snails could approach its

prey in a much quicker rate than the other snails (Kwong et al., 2009) and graze

heavily on fresh aquatic plants with low phenolic content and dry matter content

(Fang et al., 2010; Qiu and Kwong, 2009; Wong et al., 2010). In addition, apple

snails are generalists in feeding and could utilize a much broader spectrum of diet

than other indigenous snails (Kwong et al. 2010). As a highly productive

consumer, its secondary production estimate is approximately 10-20 folds more

than that of Melanoides tuberculata, the most productive exotic snail in the

lowland lentic habitats of Hong Kong (Kwong et al., 2010). Cha (1989) reported

that apple snails could influence the directional movement of indigenous

mollusks, especially Sinotaia quadrata. But it is still unclear whether the feeding

efficiency of S. quadrata would be affected by apple snails in the field. Apple

snails could feed on juveniles and eggs of local freshwater snails as a part of its

diet in supplement to nutrients obtained from macrophytes (Kwong et al., 2009).

Apple snails could complete three generations under favorable conditions

before cold spells come in winter (Kwong et al., 2010; Yin et al., 2006). With the

7

ability to bury in substratum, the adults could hibernate and survive through

winter and become active in the next wet season. Adult apple snail that survive

through winter could reproduce when conditions become favorable, therefore

once apple snails invade an area, they often become established and their impact

can persist for many years.

Although apple snails are notorious invasive species in Asia, there were

advocates on the application of apple snails as bio-control agents, but it was later

concluded that the side-effects of apple snails on non-target flora and fauna are

difficult to remediate. The release of apple snails and their close relatives has

been reported to be beneficiary in some countries, where they were used to

control exotic weeds of weeds (Perera and Walls, 1996; Porte et al. 2006). But

they were also intended to be introduced to countries with severely infestation of

water-borne schistosomiasis disease (Pointier and David, 2004; Hofkin et al.

1991; Frandsen 1987). Parasitic trematodes in the genus Schistosoma

(Platyhelminthes; Trematoda; Digenea; Schistosomatidae) are responsible for

this disease after infesting human through contact or ingestion of contaminated

water (Collins III et al. 2013). Mollusks like Oncomelania spp. and

Biomphalaria spp. are intermediate host of human-related trematodes to

complete its lifecycle (Huang et al., 2010; Tang, 1985). There are advocates on

the use of apple snails and their close relatives as biological control agents for

trematode-hosting mollusks. Marisa cornuarietis, a close relative of apple snails,

was the most widely used snail in controlling Biomphalaria spp. (Haridi and

Jobin, 1985; Nguma et al., 1982; Pointier and Jourdane, 2000; Pointier et al.

1994; Pointier et al. 1991). P. canaliculata has also been used in field

8

experiments in Thailand to control trematode-hosting mollusks (Kruatrachue and

Upatham, 1993). Although these studies may have achieved certain extent of

success in controlling the target snails, the ecological impacts on non-target

macrophytes and invertebrates are difficult to estimate (Cowie, 2001). In addition,

P. canaliculata is the intermediate host of the rat lungworm (Angiostrongylus

cantonensis), which was responsible for a mass infection of over 100 citizens of

Guangxi province (Hao et al., 2007) and 8 citizens of Fujian province (Chen et

al., 2003).

1.4 Current control methods of apple snails in Hong Kong

Since their first invasion in the 1980s, apple snails have become a major pest of

rice in Asia and taro in Hawaii (Cowie, 2002). Although Hong Kong is one of

the most densely populated cities with more than seven million people living on

small area of 1,108 km2, there is still approximately 51 km

2 agricultural land.

While existing rice paddies (approximately 10% of agricultural land) are mainly

for demonstration of tradition rice cultivation in Yuen Long, small-scale farms

still provide 8% of vegetables for local consumption. Several species of semi-

aquatic vegetable, especially Ipomoea aquatica (water spinach), Nasturtium

officinale (watercress), Nelumbo nucifera (Indian lotus) and Sagittaria

sagittifolia (Chinese arrowhead) are commonly cultivated species. Apple snails

are widely distributed in these semi-aquatic vegetable gardens and drainage

channels connecting them. In response to the infestation of apple snails in

vegetable gardens, local farmers have adopted cultural and chemical control

methods. The cultural method involved simple hand picking of apple snail eggs

and large individuals throughout the year, especially the summer when apple

9

snails reach their peak of reproduction and growth (HKBWS, 2006). The

chemical methods involved application of lime and / or tea seed cake (residue of

seeds in the Camellia family, after oil extraction, which contains saponin)

between two crops to control apple snails (Lau, S.P., pers. comm.).

Due to the increasing development pressure, many wetlands in the New

Territories have been lost to construction for residential buildings and associated

roads and highways. As required by the Environmental Impact Assessment

Ordinance (Cap 499), compensation wetlands of a similar or larger size of those

destroyed were created with various macrophytes used to build up the floral

community (Lau et al., 2004). An example of such wetland loss is the large-scale

(220 ha) residential development in Tin Shui Wai for a population of 340,000

(Cha, 2004). As part of the compensation, Hong Kong Wetland Park (60 ha) was

created with channels of different depths connecting many ponds separated by

weirs for flow and water depth control, and macrophytes were planted to filter

intake water and to create habitats of different floral diversity. The invasion of

apple snails to these constructed wetlands may have greatly reduced floral

biodiversity, and their value as habitats of animals, such as birds. Current

wetland management activities in Mai Po Nature Reserve (managed by World

Wide Fund, Hong Kong) and Hong Kong Wetland Park (managed by Agriculture,

Fisheries and Conservation Department and Development Bureau, HKSARG)

and West Rail wetlands in Kam Tin (managed by Mass Transit Railway

Corporation Ltd., Hong Kong) to control apple snail populations in constructed

wetlands mainly involve hand-picking, which can be an effective method, but is

10

labor-intensive and requires repeated efforts (Cowie, 2002; Hung T.T.H., pers.

comm.; Yau V.P.T., pers. comm.).

1.5 Biological control and indigenous predators of apple snails in Hong

Kong

A summary of all potential predators of golden apple snails in Hong Kong

wetlands based upon local and international literatures have been compiled as

table 1.1. Among all resident species of Hong Kong lowland wetlands, there are

only twenty indigenous species have recorded diet on gastropods. It should be

noted that most of these predators are omnivores and apple snails might only

comprised a portion of their diet. Yet, lack of any local studies provided practical

evidences in demonstrating importance of particular species of fauna or

integrated ecosystem in controlling apple snail population.

Biological control of snail species susceptible to contamination has been

practiced in Southeast Asia, including China, Taiwan, Japan, Vietnam, Thailand,

Malaysia, Lao P.D.R., Sri Lanka, Israel and Bangladesh (Mukherjee et al. 1991).

Ducks are well known predators of pest snails in paddy fields (Cagauan et al.,

1999; Gallebu et al., 1992). Before 1970s, fish farmers in the whole North-

western New Territories, including the Deep Bay Area, once widely practiced

duck farming (Tuen Mun and Yuen Long District Planning Office, 2002; Carey

et al. 2001). Previous literature has cited about 58% and 8% of all fish farms

raised ducks and gooses respectively (Kausar, 1983; Sin and Cheng, 1976). Yet,

it has not been practiced anymore locally. In addition, wild Anatidae are only

winter passage migrants of Hong Kong through East Asia-Australian Flyway

(WWF, 2012) instead of resident species. Other than ducks, fish has been

11

suggested and implemented as agent for integrated pest control in paddyfields

(Fernando, 1993; FAO, 1998; Halwart, 1998; Lardans and Dissous, 1998; Teo,

2006; Ichinose et al., 2002; Yusa et al., 2006). Yet, few studies have considered

their potential subtle effects to wetland flora and fauna. Recent mesocosm

experiment by Wong et al. (2009) studied the effectiveness and non-target effects

of common carp (Cyprinus carpio L.) as a biological control agent against P.

canaliculata in a 2-month enclosure experiment. They examined the impact of

common carp on nine species of gastropods including the apple snail, and three

species of macrophytes in a constructed wetland in Hong Kong Wetland Park.

Their results showed that common carp completely eliminated apple snail

juveniles that are small enough to fit into their months. Although they could not

prey on large apple snails due to the use of small fish as constrained by the size

of the enclosures, its ability to grow in local ponds showed common carp is an

effective bio-control agent of apple snails. However, common carp also caused a

significantly reduction of plant biomass, as well as of the densities of most non-

Pomacea gastropods, indicating common carp’s strong non-target effects on

local wetland flora and fauna. To continue finding a cost-effective mean to

control invasive apple snails, this research focused on investigating the predatory

potential and practical control efficacy of black carp Mylopharyngodon piceus

Richardson. on apple snails. Black carp has been suggested in recent years as

remediation in particular to reduce pest mollusks (Ben-Ami and Heller, 2001;

Collins, 1996; Hung et al. 2013; Leventer, 1979; Mochida, 1991; Shelton et

al.,1995; Thomforde, 1999; Venable et al.,2001) and are traditionally reared in

farm ponds (Wu et al., 1964) , fish ponds (Hickling, 1971), rice paddies (Wang,

12

2010) and reservoirs (Leventer and Teltsch, 1990), while being food fish to

generate profit in complement with that from crops yield (Lin, 1955; Liu, 1955).

13

1.6 Research objectives and general organization of thesis

The present thesis consists of five chapters. Chapter 1 is a general introduction

and Chapter 5 is a summary of research findings. Chapter 2 to 4 describes the

laboratory (Chapter 2) and field experiments (Chapter 3-4) conducted to

evaluating the effectiveness and non-target effects of fish as predators of apple

snails with fish as predators. Details of three studies are as follows:

1. Predatory potential of indigenous fish as bio-control agent of apple snails

(Chapter 2) – This chapter mentioned an aquarium study (Chapter 2). It laid

the foundation for the field studies described in Chapters 3 and 4. Three

indigenous fish species were evaluated regarding their predatory potential on

apple snails. The results were useful for selecting a candidate for the field

experiments.

2. Biological control of invasive apple snails in a freshwater wetland by two

species of carp: subtle differences matter (Chapter 3) – this study aimed to

explore and compare the performance of two fish species that fed voraciously

on apple snails in laboratory experiments. In addition, I would like to

understand their possible side-effects on water quality, aquatic plants and

various epi- and benthic fauna in freshwater constructed wetland habitat. Two

species of fish, common carp C. carpio and black carp M. piceus were

selected due to their promising performance in the laboratory experiments

(Chapter 2). The field experiment lasted two months. The results were useful

for selecting a fish for the following whole wetland experiment.

3. Wetland scale application of biological control of apple snails using black

carp: efficacies and subtle effect to ecosystem (Chapter 4) – Black carp,

which was found to be a more desirable predator of apple snails from

previous field trial experiment (Chapter 3), was released in semi-natural

constructed wetlands and was tested upon its practical efficacy in reducing

apple snail abundance. Previous studies only used black carp in small scaled

trials and only its direct impact on the target mollusks were tested (Ben-Ami

and Heller, 2001; Collins, 1996; Mochida, 1991; Shelton et al., 1995;

14

Thomforde, 1999; Venable et al., 2001). They were conducted in semi-

natural habitats with low biodiversity especially shallow rice paddies, without

evaluating the effects on non-target benthic macroinvertebrates and water

quality, while only large scale study in Israel (Leventer 1979) failed to

provide many details of his experimental design and results. In our study,

side effects on water quality and co-existing macrobenthos were also

evaluated (Chapter 4). Macrophyte abundance and diversity were also

analyzed to test on treatment effect.

15

Table 1.1 List of possible indigenous predators of aquatic mollusks in Hong Kong wetlands. Growth stage of apple snails: juvenile

(3-25mm); adult (>25mm) based upon a study by Estoy et al. (2002).

Species / group Family Snail growth stage Major diet

(if noted in references) References and notes

Class Aves

Ducks, geese and

swans Anatidae Adult

Aquatic invertebrates, especially snails, young

vegetation, seeds

Cagauan et al., 1999;

Gallebu et al., 1992

Greater Painted Snipes

Rostratula

benghalensis

Rostratulidae Juvenile; Adult

Insects (e.g. crickets and

grasshoppers) (Urban et al. 1986), snails, earthworms,

crustaceans and seeds

Kirwan, 1996

Grey-headed Lapwings

Vanellus cinereus Charadriidae Juvenile

Worms, insects,

crustaceans, mollusks Kirwan, 1996

Pintail / Swinhoe’s

Snipes

Gallinago spp.

Scolopacidae Juvenile

Earthworms, adult and larval insects (e.g. glow-

worms, beetles, ants and

grasshoppers), terrestrial mollusks and seeds

Johnsgard, 1981; Piersma et al., 1996

Blue Whistling Thrush

Myophonus caeruleus Turdidae Juvenile; adult

Snails, earthworm, various

types of insects, frogs,

lizards and skinks, juvenile birds and small rats

Ali and Ripley, 1998;

Collar, 2005; Yoong,

2012

Starlings and mynas Sturnidae Adult Mainly insects Craig and Feare, 2009

16

Species / group Family Snail growth stage Major diet

(if noted in references) References and notes

Magpie and crows Corvidae Adult

Invertebrates, nestlings,

small mammals, mollusks, berries, fruits, seeds, and

carrion

Debus et al., 2009

Class Mammalia

Bandicoot Rat

Bandicota indica Muridae Juvenile? Insects, seeds, crops

Shek, 2006; Sridhara and Srihari, 1983

Eurasian otter

Lutra lutra Mustelidae Adult

Fish, crab, aquatic insects,

reptiles, amphibians, birds,

small mammals, and other crustaceans; snails as

minor supplement

de Silva, 1996; de Silva, 1997; Ruiz-Olmo and

Palazon, 1997; Shek,

2006

Small Indian

Mongoose

Herpestes javanicus

Mustelidae Adult

Rats, birds, reptiles, frogs,

crabs, insects; gastropod shell fragments in food

debris

Lekagul and Mcneely, 1988; Shek, 2006

Crab-eating Mongoose

Herpestes urva Mustelidae Adult

Mammals, reptiles, insects,

fish, frogs, mollusks, other crustaceans

Shek, 2006; Van Rompaey, 2001; Wang

and Fuller, 2001; Wang

and Fuller, 2003

Class Anura

Hong Kong Newt

Paramesotriton

hongkongensis

Salamandridae Juvenile? Records of predation on

Brotia hainanensis only Fu, 2010; Kirwan, 1996

17

Species / group Family Snail growth stage Major diet

(if noted in references) References and notes

Class Reptilia

White-spotted slug snake

(Pareas

margaritophorus)

Colubridae Juvenile

Gastropods, feed readily on

terrestrial species when

held in captive conditions

Karsen et al., 1998

Reeve’s terrapin

Chinemys reevesii Geoemydidae Juvenile; Adult

Fish, frogs, carrions,

carcass, minor supplement

with mollusks; pond weed Hydrilla spp.

Karsen et al., 1998;

Yoshie and Yusa, 2008

Chinese soft-shelled turtle

Pelodiscus sinensis Trionychidae Juvenile; Adult

Fish, frogs, insect larvae,

carrions, carcass, mollusks

Karsen et al., 1998;

Zheng et al., 2005

Class Osteichthyes

Nile Tilapia

Oreochromis niloticus Cichlidae Juvenile

Suggested controlling

agent for golden apple

snails; feed on planktons, benthic algae, macrophytes

and soft-bodied benthic

invertebrates, while apple

snails would only be its supplement food item

Halwart et al., 1998;

Lee et al., 2004

White-spotted Walking Catfish

Clarias fuscus

Clariidae Juvenile; Adult

Small fishes, worms,

crustaceans and insects as major diet; gastropods as

supplement or alternatives

Cui and Zhao, 2012; Lee et al., 2004

18

Species / group Family Snail growth stage Major diet

(if noted in references) References and notes

Class Crustacea

Hainan Swamp Shrimp

Macrobrachium

hainanense

Palaemonidae Juvenile

Small crustaceans,

Chironomidae, Tricoptera

as major diet; Brotia

hainanensis at around 3 –

9% of diet

Mantel and Dudgeon,

2004a; Mantel and

Dudgeon, 2004b

Somanniathelphusa

sinensis Parathelphusidae Juvenile; Adult

Prefer Biomphalaria

straminea, Physa acuta

and Radix plicatulus

Dudgeon and Cheung,

1990

Japanese mitten crab

Eriocheir japonicas Varunidae Juvenile; Adult

Feed readily upon golden

apple snails ad libitum

Yusa et al., 2006

Class Insecta / Odonata

Lesser Emperors

Anax parthenope Aeshnidae Juvenile

Various pond snails;

feed readily upon golden

apple snails ad libitum

Turner and Chislock,

2007; Yusa et al., 2006

Wandering Glider

Pantala flavescens Libelluidae Juvenile

Various pond snails;

feed readily upon golden

apple snails ad libitum

Turner and Chislock,

2007; Yusa et al., 2006

Class Insecta / Coleoptera

Aquatica leii Lampyridae Juvenile Various pond snails Li et al., 2011

Aquatica ficta Lampyridae Juvenile Various pond snails Li et al., 2011

Pteroptyx maipo Lampyridae Juvenile Various pond snails Ballantyne et al., 2011

Diving beetle

Cybister sp.

Dytiscidae Juvenile Feed readily upon golden

apple snails ad libitum

Yusa et al., 2006

19

Chapter 2 Predatory potential of indigenous fish as bio-control

agent of apple snails

2.1 Background

There has been an increasing incidence of exotic species becoming invasive

when it is removed from its natural enemies (predators or parasites) in its original

niche and upon introduction to a new favorable habitat (Forrest et al. 2012;

Higgins & Vander Zanden 2010; Hermoso et al., 2011; Mack et al., 2000). High

impact invaders established in new habitats and caused more chronic negative

impact to native biota than native species (in terms of diversity loss, change in

disturbance regime and retarding ecosystem succession) (Light and Marchetti,

2007; Simberloff et al., 2012; Paolucci et al., 2013). These species also degraded

ecosystem services of our native ecosystem (Pimentel et al., 2001; Pejchar and

Mooney, 2009) and eventually homogenize aquatic biodiversity (Lodge 1993;

Rahel 2002). Eradication of invasives was therefore crucial before they continue

to spread to suitable habitats and alter the ecosystem (Myers et al. 2000)

Apple snails (Pomacea canaliculata Lamarck.) have first infested Hong Kong

in 1970s and are well-known major pests of rice in Asia and taro in Hawaii (Cha

1989; Cowie, 2002). They have established sustainable population and

distributed widely in these semi-aquatic vegetable gardens, inter-connecting

drainage channels and some compensation wetlands (Kwong et al. 2008). In

response to the infestation of apple snails in vegetable gardens, local farmers

have adopted cultural and chemical control methods. The cultural method

involved detachment of apple snail egg clutches and hand-picking of large

individuals throughout the year, especially in summer when apple snails reach

20

their peak of reproduction and growth (HKBWS, 2006; Hung T.T.H., pers. comm.;

Yau V.P.T., pers. comm). The chemical methods involved application of lime or

tea seed cake (residue of seeds in the Camellia family, after oil extraction, which

contains saponin) between two crops to apple snails (Lau, S.P., pers. comm.).

However, these methods are usually labor-intensive. In addition, chemical

control could not be applied to wetlands with deeper water levels (>1.0m),

especially during wet season as rain-induced spate usually flood those areas and

provide favorable breeding grounds for apple snails.

Classical biological control mechanism that involves introducing predators

existing in the natural area of origin might not be a good solution for controlling

apple snails. These South America originated predators are namely snail kites

(Rostrhamus sociabilis), glossy ibis (Plegadis falcinellus), redear sunfish

(Lepomis microlophus), exotic fire ants (Solenopsis geminata) and introduced

duck varieties (Gallebu et al., 1992; Teo, 2001). Yet, none of these predators are

native in Hong Kong. In addition, there is no consent on an efficient agent that

could minimize their invasion by predation or competition temporally in global

scale.

Natural biological control would be a better option of management, in

which diversity and abundance of native species are manipulated to reduce the

invasion impact to minimal. It is yet difficult to locate a predator or competitor

specific for apple snails. Previous experiment has revealed secondary production

by apple snails outweighed all lowland wetlands fauna in Hong Kong (Kwong et

al., 2010a). In addition, though apple snails have invaded for more than a decade

in Hong Kong (Yipp et al., 1991), there are few observations on predators that

21

feed upon them, while these records focus on only a handful of omnivorous

wetland-dependent residents (Chapter 1, table 1).

Using single fish species as direct controlling agent or in complement with

ducks, is most widely studied bio-control mechanism against apple snail invasion

in Southeast Asia. It originated from studies on a number of cichlid species

(Slootweg et al., 1993; Slootweg et al., 1994), Nile tilapia (Oreochromis niloticus

Linnaeus.) and common carp (Cyprinus carpio L.) (Cagauan et al., 2000;

Halwart, 1994a). Biological control using common carp has then been widely

explored in the laboratory (Caguan 2002; Halwart et al., 1998; Teo, 2006; Yusa

et al. 2006), and rice fields in Taiwan (Mochida, 1991), Vietnam (FAO, 1998),

Japan (Ichinose et al., 2002) and Malaysia (Teo, 2006). In China, black carp

Mylopharyngodon piceus Richardson. was long considered as a potent

molluscivore in fish ponds (Hickling, 1971; Lin, 1955; Liu, 1955). Similar

researches in fish ponds have subsequently continued in Taiwan (Mochida, 1991),

Israel (Ben-Ami and Heller, 2001; Leventer, 1979) and Russia (Denisov, 1982).

Other than using black carp, various species of catfish with wide gape has also

been tested in different countries regarding their effectiveness in controlling

apple snails. These include broadhead catfish Clarias macrocephalus Günther.

(Cagauan and Joshi, 2002; Carlsson and BrÖnmark, 2006), African catfish C.

gariepinus Burchell. (Slootweg et al., 1993) and Philippine catfish C. batrachus

L. (Liao et al., 2004). Though African catfish might prey upon apple snails

(Coates 1984), it is classified as an invasive species in Asia (ISSG 2013). The

only resident species in Genus Clarias in Hong Kong is white-spotted catfish

(Clarias fuscus Lacépède.).

22

The current objective of the present study was to identify two species as

predators of apple snails in wetlands. As fish would be a preferable candidate at

the present stage, we compared the predatory potential of three indigenous fish

species on apple snails by an aquaria study. This study included estimation of the

maximum amount of ingestion and maximum size of snail consumed by the

tested fish species under optimum conditions. By comparing the figures, we

located two most suitable ones with high predatory potential on apple snails for

conducting field trial experiment to further explore their possible side-effects

upon release.

2.2 Material and method

Fish and apple snails used in aquaria experiment

We have used three species of fish for this experiment. They are black carp,

common carp and white-spotted walking catfish. Juvenile black carp (fork length

170-185 mm), common carp (fork length 170-195 mm) and white-spotted catfish

(fork length 150-180mm) were purchased from a commercial fish farm and an

aquarium shop, respectively. The gape width of each fish specimen was

measured by a digital caliper. They are acclimated for at least 2 weeks under

experimental conditions (25oC and in separate tanks for each specimen with 8.0L

dechlorinated water and water circulation system) before the experiment

commenced. All specimens selected for experiment are ensured to feed

simultaneously upon provision of juvenile apple snails reared in laboratory (8-

15mm), with daily supplement by commercial fish feed.

Juvenile (8-25mm) and adult (>25mm, minimum reproductive size due to

previous literature (Estebenet and Cazzaniga, 1992; Martin, 1986) apple snails

23

with variety of sizes were caught from drainage channels of a vegetable garden in

Long Valley (Yin Kong Tsuen, Sheung Shui), They were then transported to

laboratory and were allowed to acclimatize in three separate aquaria with 8.0L of

dechlorinated water and water circulation system for at least 7 days before use.

Aeration was provided in each aquarium and the snails were fed daily with

romaine lettuce (Lactuca sativa L.).

Aquaria set-up

There were six aquaria (size: 67cm*51cm*33.5cm) with 30.0L dechlorinated

water and water circulation system for each species of fish. A day prior to the

experiment, all fish specimens and snails were supplied with no food to allow

natural starvation. Similar size spectrum of approximately 100-140 apple snails

was first introduced into each experiment aquarium. Shell length of each snail

introduced into the tank has been measured by digital caliper to the nearest mm.

For each fish specimens, total length, fork length and gape width were measured.

Upon start of the experiment, only a single fish specimen was allowed to enter

each experimental tank. The experimental period then lasted for 48 hours.

After the experiment, fish specimens were first removed from the tanks.

Surviving snails were collected and measured individually for their shell length.

Dead snails were observed for signs of predation or natural death if exist with

entire shell.

An extra aquarium has been prepared for control treatment, where

approximately same quantity and size spectrum of apple snails was allowed to

24

stay in an aquarium without later introduction of fish. This would serve as a

comparison of snail’s survival with experimental treatments.

Data analysis

Total abundances of snails consumed in six replicates are compared among fish

species and control using one-way ANOVA. In order to investigate the effect of

size-dependent predation, amount of consumption within two size ranges of

apple snails (Juvenile: 3-25mm; adult: >25mm) is compared among three species

of fish and control with one-way ANOVA.

Relationship between fish total length and amount of apple snails

consumed was tested using linear regression analysis.

2.3 Results

Aquaria studies on predatory potential of three fish species on apple snails

All of the apple snails in control treatment survived throughout the experimental

period. Among six replicates of each treatment, mean survival rate of apple snails

with presence of catfish was 86.1%, while that with presence of common carp

and black carp was 45.8% and 39.0% respectively (Fig. 2.1). Abundance of apple

snails after 48hours reduced significantly in all fish treatments, with compared to

that before treatments. Significant difference was found between total amount of

consumed apple snail among treatments (F3,20 = 118.50, p < 0.01). Results of

Tukey test comparing among treatments revealed significant difference in control

treatment and all treatments with fish. Among all with fish treatments, abundance

of apple snails consumed was not significantly different within treatments with

two species of carp, but that of treatment with catfish is significantly lower. On

average, estimated daily predatory rate of black carp was 35.25 individuals (SE =

25

6.28) while that of common carp was 29.33 individuals (SE = 3.78). Maximum

predatory rate for black carp and common carp was 45.0 and 31.5 individuals per

day respectively.

Predation of apple snails by two species of carp was size-dependent, with

juvenile snails being more susceptible. Snails within shell length (SL) of 3-8mm

were totally eradicated in both carp treatments (Fig. 2.2). Significant difference

was found in amount of juvenile snails (SL = 3 - 25mm) consumed in all

treatments (F3,20= 118.835, p <0.01*). Tukey test results indicated that both carp

species specimens consumed significantly larger amount of apple snails than that

by catfishes, though no significant differences were observed between results of

two species of carp (Fig. 2.3). In treatment tanks with catfish, only snails with

shell length below 6mm was consumed most significantly. This accounted for

50% of the mean total snail consumption while the rest scattered within juvenile

sized snails. For all fish species, no significant differences were drawn among

abundance of adult snails provided (SL >25mm) (F3,20= 1.0, p = 0.413).

Maximum size of ingestion by common carp was 18mm, which was higher

than 16mm in tanks with black carp. Yet, a point to note was that the maximum

size of ingestion by catfish was 26mm. This far exceeded that of the other two

carp species, though catfish consumed much less in quantity of snails.

2.4 Discussion

Fish in family Cyprinids do not have jaw teeth. Benthic dwellers, like black carp

and common carp share the same characteristics of being pharyngeal crushers,

having pharyngeal teeth, horny and callous pad specialized for preying upon

26

mollusks (Liu et al., 1990; Sibbing, 1988). In this study, black carp and common

carp demonstrated high predatory potential against apple snails. But common

carp possess protruding mouthparts allowing them to prey upon mollusks of

higher shell length, by sucking the soft tissues of mollusks with intact shells

(Sibbing et al. 1986). Given that omnivorous common carp has been

demonstrated as good example of biological control against apple snails (FAO

1998), molluscivorous black carp with similar predatory potential might also be

another option in controlling mollusk population with lower trophic interactions

with aquatic vegetation and other invertebrates. Further field manipulative

experiment would thus be needed for more in-depth study.

White-spotted walking catfish C. fuscus, an omnivorous native predator that

equipped with large broad flat head and huge mouthpart, has demonstrated weak

predatory effect to apple snails in this study. Indeed gastropods might only

compose a small portion in their natural diet. Local literatures noted small fish,

crustaceans, insects, planktons and rotten meat and plants as diet items of this

species (Man and Hodgkiss 1981; Lee et al. 2004). Though there were some

literatures supporting catfish including African catfish C. gariepinus (Coates

1984; Phonekhampheng et al. 2009) and Philippine catfish C. batrachus (Liao

2005) as suitable bio-control agents for snails, catfish are indeed opportunistic

predators having ontogenic diet shift upon different habitats. Moving targets,

including small fishes, worms, crustaceans and insects might be their optimal

prey items instead of gastropods (Cagauan and Joshi, 2002; Cui and Zhao, 2012).

To conclude, the present study has clearly demonstrated high potential of

common carp and black carp as predators of the golden apple snails, and that

27

white-spotted catfish has comparatively limited ability in preying upon the snails.

Both carp species are gape-limited predators and snails were first engulfed and

crushed before being swallowed. Indigenous White-spotted catfish have a wider

gape but have low diet preference towards exotic apple snails.

28

Table 2.1 Statistical results for one-way ANOVA test and post-hoc Tukey test on total abundance of consumed apple snails after the treatment.

Treatment abbreviation: 1 – control; 2 – with black carp; 3 – with common carp; 4 – with white-spotted catfish. Data with statistical difference at

p < 0.05 were shown with asterisk.

Factor df F p

Corrected Model 3 118.500 .000

Intercept 1 546.189 .000

Treatment 3 118.500 .000*

Error 20

Total 24

Tukey test

Treatment N

Subset

1 2 3 4

1.00 6 .0000

4.00 6 15.6667

3.00 6 58.6667

2.00 6 70.5000

p 1.000 1.000 .061

29

Fig. 2.1 Size-dependent predation of three fish species on apple snails in aquaria. Each graph for each species showed total abundance of snails

survived in six aquaria before (t=0hrs) and after (t = 48hrs) the experiment. Treatment abbreviations: BC – black carp; CC– common carp; WC-

white-spotted catfish.

Before experiment

Treatment: WCn = 677

0 5 10 15 20 25 30

After experiment

Treatment: WCn = 583

Shell Length (mm)

0 5 10 15 20 25 30

After experiment

Treatment: CCn = 297

Before experiment

Treatment: CCn = 649

Num

ber

of

indiv

iduals

0

10

20

30

40

50

60

0 5 10 15 20 25 30

0

10

20

30

40

50

60 After experiment

Treatment: BCn = 270

Before experiment

Treatment: BCn = 693

30

Fig. 2.2 Mean percentage of survival for golden apple snails in six aquaria, after

individual treatments with three species of fish. Treatment abbreviation as Fig.1.

0

20

40

60

80

100

BC

2D Graph 2

Pe

rce

nta

ge

su

rviv

ed

(%

)

0

20

40

60

80

100

CC

Shell length (mm)

0 5 10 15 20 25

0

20

40

60

80

100

WC

31

Fig. 2.3 Comparison of mean amount of snails consumed per experimental tank among three treatments. Treatment abbreviations: BC – Black

carp; CC – Common carp; WC: White-spotted walking catfish. Letters above the bars are results of Tukey test comparing the difference in mean

amount of golden apple snails consumed per tank among treatments. Individual analysis has been conducted for number of juvenile and adult

size class of apple snails consumed after the treatment. Different letter indicate significant difference at p < 0.05.

Treatment

BC CC WC

Mean

con

sum

ed

ap

ple

sna

il (indiv

idu

als

pe

r ta

nk)

0

20

40

60

80 Juvenile (3-25mm)

Adult (>25mm)

a

a

b

a a a

32

Chapter 3 Biological control of invasive apple snails in a

freshwater wetland by two carp species: subtle differences matter

3.1 Background

With the rapid development in travel and trade in the last century, there has been

a dramatic increase in the introduction of non-indigenous species worldwide

(Sala et al., 2000). While some introduced species can have economic benefits,

they can also become invasive, causing significant changes to the structure and

function of the invaded ecosystems through predation, competition, habitat

alteration and transmission of pathogens (Woodward and Hildrew, 2001;

Dudgeon et al., 2006; Gozlan et al., 2010; Paterson et al., 2011).

Biological control has been widely used as a tool to manage invasive

species, but there has been a long-standing debate regarding its benefits, risks

and costs (Cowie, 2001; Messing and Wright, 2006; Hulme, 2009; Strayer, 2010;

De Clercq et al., 2011). Critics of biological control argue that the risks are

difficult to predict due to the complexity of species interactions, and negative

impacts on non-target organisms and ecosystem functioning are often not

detected until it is too late (Howarth, 1991; Simberloff and Stiling, 1996).

Proponents of biological control emphasize that controlling or eradicating

invasive species benefits the economy, public health and ecosystems (Van

Driesche et al., 2010), and that precautionary procedures are sufficient to protect

non-target organisms (DeLoach, 1991). Very often, biological control is

perceived as more environmentally benign than chemical control, which is

usually expensive, non-selective and requires repeated application. This is

especially the case in the management of the apple snail, Pomacea canaliculata

33

Lamarck, an indigenous species of the freshwater wetlands of South America

that has invaded several Asian countries and Pacific islands including Hawaii

(Hayes et al., 2008). This species is a large freshwater gastropods and has a

voracious appetite for vegetation (Boland et al., 2008; Tamburi and Martín, 2009;

Qiu et al., 2011), causing tremendous losses to rice (Halwart, 1994b; Naylor,

1996) and taro agriculture (Cowie, 1995), as well as structural and functional

changes to non-agricultural wetlands (Carlsson et al., 2004a; Carlsson and

Lacoursière, 2005). Due to its huge economic and ecosystem impacts, this

species is listed among the 100 most notorious invasive species worldwide

(Lowe et al., 2000), and has become a prime target for control (Cowie, 2002;

Joshi and Sebastian, 2006).

Cultural, mechanical, chemical and biological methods have been used to

control populations of invasive apple snails (Litsinger and Estano, 1993; Cowie,

2002; Joshi and Sebastian, 2006). Cultural methods, such as changing from direct

seeding to transplanting older seedlings, and mechanical methods such as placing

grills at water inlets and hand-picking the snails, are effective but labour

intensive. Chemical methods, which involve the application of pesticides, are

expensive when used in large areas, and pose risks to human and ecosystem

health. Biological control using predators, especially common carp, has been

widely explored in the laboratory (Halwart et al., 1998; Teo, 2006), and rice

fields in Taiwan (Mochida, 1991), Vietnam (FAO, 1998), Japan (Ichinose et al.,

2002) and Malaysia (Teo, 2006). These experiments and field applications have

focused on effectiveness of control, without considering other potential

ecosystem effects. This focus is understandable as the studies were conducted in

34

rice paddies where biodiversity was low and the risk to non-target species might

be minor.

However, since P. canaliculata has also invaded non-agricultural wetlands

(Carlsson et al., 2004a; Kwong et al., 2008; Fang et al., 2010), the risks that

biological control agents may pose to non-target flora and fauna should be

carefully evaluated before releasing them to the field. Fish as predators of apple

snails in wetlands have been studied using enclosures, but the results were

unsatisfactory: the fish did not prey on apple snails but escaped from the

enclosures (Carlsson and Brönmark, 2006); the predation effect was not strong

enough to reduce herbivory by apple snails (Carlsson et al., 2004b); or the fish

were effective in controlling apple snails, but reduced macrophyte biomass as

much as did the apple snails and fed on macroinvertebrates in the sediment

(Wong et al., 2009). The present study was therefore conducted to compare the

performance of black carp (Mylopharyngodon piceus Richardson) and common

carp (Cyprinus carpio L.) as biological control agents of P. canaliculata in non-

agricultural wetlands. Black carp feeds on snails (Venable et al., 2000; Ben-Ami

and Heller, 2001). Five million juveniles of this species were released to control

P. canaliculata in rice planting areas in Taiwan, but its effectiveness was not

reported (Mochida, 1991). In this study, we compared not only the effectiveness

of the two biological control agents, but also their potential side effects on

wetland flora and fauna.

35

3.2 Materials and methods

Field site

The experimental site (22°26'40.69" N, 114°3'18.99" E) is in Kam Tin,

northwestern Hong Kong. The climate of Hong Kong is subtropical with a wet

season (April–September) and a dry season (October–March) (Dudgeon and

Corlett, 2004). The study lasted eight weeks from early October to early

December 2011, during which mean air temperatures declined from 23.7 °C in

the first week to 19.1°C in the last week. Total precipitation during the study

period was 301.3 mm, with 210.7 mm in the first 4 weeks and 86.1 mm in the

last 4 weeks. The experiment was conducted in a freshwater earthen wetland

created in 2002 as a compensation for the loss of the original wetland due to the

construction of a railway. The wetland was roughly oval, with a total area of 2.36

hectare. The bottom sloped gently from the edge to the centre, the deepest part

with over 2.0 m of water. The water is slightly alkaline (pH = 7.1), mildly

oxygenated (5.4 mg l-1

) and quite clear (turbidity = 2.4 NTU). The edge of the

wetland was vegetated with a variety of macrophytes, e.g. water dragon

(Ludwigia x taiwanensis Peng), water spinach (Ipomoea aquatica Forssk.),

bahiagrass (Paspalum sp.), knotgrass (Polygonum barbatum L.), common reed

(Phragmites australis Trin.). and bulrush (Typha angustifolia L.). From around

1.0 m to the centre, the wetland was unvegetated. The bottom was sandy clay,

with a community of macrobenthos dominated by the detritivorus snails Sinotaia

quadrata Benson (Viviparidae) and Melanoides tuberculata Müller (Thiaridae).

Pomacea canaliculata was present in the wetland at a low density (0.4 individual

m-2

, surveyed prior to the field experiment).

36

Experimental setup

The experiment was conducted in 18 1 x 1 x 1 m enclosures. There were five

replicates of three experimental treatments: with apple snails and black carp –

AS+BC; with apple snails and common carp – AS+CC; with apple snails but

without fish – AS; and three replicates of the control treatment without fish or

apple snails – C (originally five replicates but two enclosures were damaged by a

typhoon). The enclosures were placed in five haphazardly chosen blocks in the

shallow water (~ 0.7 m) near the shore (Supplementary material 1, Photographs

of the site showing the experimental enclosures), with each block containing one

enclosure of each of the four treatments (with the exception of two blocks in

which the original control enclosures had been damaged). Enclosures were made

of four woven plastic side-walls and a sewn nylon net bottom with a pore size of

200 m. An external frame, made of 4.3 cm PVC pipes and joints, was used to

support each enclosure. The lower part of the frame had four 15 cm legs, one at

each corner. To enhance the stability, the legs were pushed into the bottom

sediment, and a brick was tied to each lower corner of the enclosure. Surface

sediment (~ the top 3 cm of the substrate) was collected from the wetland for

introduction into the enclosures. The sediment was sieved through a 1.6 mm

mesh and washed into each enclosure to a depth of 3 cm. The residues retained

were then checked to remove apple snails by hand-picking. Other mollusks were

introduced into each enclosure at natural density (i.e. mollusks in 1 x 1 m surface

sediment). On the following three days, the enclosure walls and surface sediment

were swept to collect any apple snails that had been missed previously.

Afterwards, each enclosure was stocked with natural abundance of three species

37

of macrophytes common in local wetlands to test for the grazing effects of apple

snails and fish. Two of them were emergent (water dragon Ludwigia x

taiwanensis Peng, water spinach Ipomoea aquatica Forssk), whereas one was

floating (water lettuce Pistia striatiotes L.). Each plant was trimmed to

standardize the weight. The initial wet weight of the emergent species was 100

1 g, whereas that of the floating species was 50 1 g.

Black carp (fork length 170-185 mm) and common carp (fork length 170-

195 mm) were purchased from a commercial fish farm and an aquarium shop,

respectively. Both species are native to southern China, and are traditional

aquaculture species. The fish were allowed to acclimatize in enclosures in the

field for at least one week. One fish was introduced into each of the designated

enclosures. Enclosures were checked twice per week to ensure fish survival.

Replacement of fish was done immediately after dead specimen was observed.

Apple snails of different sizes were introduced into the enclosures. The

juveniles (shell length (SL): 8-15 mm) and adults (SL: 30-40 mm) were collected

from drainage channels of a vegetable garden in Yin Kong Tsuen, Sheung Shui

(22°30'22.08" N, 114°6'38.88" E), 15 km from the experimental wetland. They

were allowed to acclimatize in the field in an empty enclosure for at least 3 days

before use. Four adults (2 males and 2 females) and 20 juveniles were introduced

into each of the experimental enclosure in treatments AS, AS+BC and AS+CC

along with ten neonates (SL: 3 mm), obtained by allowing field collected egg

clutches to hatch in the laboratory. An additional 200 neonates were introduced

38

into each of these enclosures in the fifth week of the experiment to simulate the

hatching of one clutch of apple snail eggs.

Enclosure maintenance, data collection and analysis

During the experiment, the enclosures were covered with a nylon net (1 mm

mesh) to exclude birds and prevent the apple snails escaping. The enclosures

were inspected daily at the start of the experiment and roughly weekly after one

month to examine their integrity, the presence of grazing marks on the

macrophyte leaves and the health of the fish. Dead fish would float on the water

surface. Dead fish were removed and replaced with fish with the same species

and size ( 1 cm fork length of the dead one). Apple snail egg clutches laid on

the enclosure walls were recorded, removed from site and brought back to

laboratory. The number of eggs in each egg clutch was then counted after

separating the eggs in 1 M sodium hydroxide solution (Joshi et al., 2002)

At the end of the 8-week experiment, macrophytes were collected from

each enclosure, blotted dry with a paper towel, and each species weighed to the

nearest 0.1 gram. Macrophytes from each enclosure were weighted separately in

the field and then placed in separate Ziploc bags. They were brought back to the

laboratory, stored temporarily in a cold room at 4C, and later examined to

collect juvenile apple snails hidden among the roots. After removing the

macrophytes, the enclosures were detached from the supporting frames and

hauled ashore. Fish were collected and their length and gape width measured.

The enclosures were then transported to the laboratory where they were stored at

4C, and within 3 days the contents were washed into a sieve with a pore size of

39

500 m, in order to retrieve mollusks. The contents retained on the sieve were

stained with 0.01% Rose Bengal to facilitate sorting and preserved in 70%

ethanol. The preserved samples were then sorted to recover mollusks, which

were then identified to the lowest possible taxonomic level counted and the shell

length and width measured to the nearest 0.01 cm using digital calipers.

Differences among the four treatments were compared using one-way

randomized block ANOVA followed by the Tukey test. Parameters compared

included the number of survivors for each of the two size classes of apple snails

(juveniles and adults), the number of egg clutches deposited and the total number

of eggs, the biomass of each of the three species of macrophytes, total

macrophyte biomass, and the abundance of non-Pomacea mollusks. The t-test

was used to compare difference in predation of the two most abundant non-

Pomacea mollusks by the two species of carp.

3.3 Results

Fish survival and effect on apple snail abundance

All black carp in the enclosures survived through the 8-week experiment.

Common carp died in three of the five replicates, and in total six individuals died.

The mortality occurred during the first month and thereafter the replacement

individuals survived well.

Of the 234 apple snails introduced into each of the enclosures, the mean

survival rate was as high as 93% in the control, but only 5% in the common carp

treatment and 2% in the black carp treatment. Apple snail mortality was size-

dependent, with smaller individuals suffering the most severe mortality (Fig. 3.1).

40

In the AS+CC treatment, the survivors were restricted to individuals ≥ 0.8 cm

shell width (SW). In the AS+BC treatment, except for 6 individuals of 0.3 cm

SW, all survivors were ≥ 2.0 cm SW. Statistical analysis showed that the

abundances of juvenile (F2,12 = 165.619, p < 0.001) snails in the treatments with

carp were significantly reduced when compared with the control. However, the

abundance of adult apple snails that survived through the experiment did not

differ significantly among the treatments (F2,12 = 0.105, p = 0.901). The Tukey

tests showed that the abundance of the two size classes of apple snails did not

differ significantly between the two fish treatments, even for juvenile snails for

which the mean value in the AS+BC treatment (1.2 individuals) was much lower

than the mean value in the AS+CC treatment (8.6 individuals) due to the great

variation among replicates (range: 0–17 individuals; S.D.: 7.02). The number of

apple snail egg clutches and the total number of eggs laid during the experiment

varied substantially among replicates in each treatment (AS: 1–3 clutches,146–

393 eggs/enclosure; AS+CC: 0–3 clutches, 0–396 eggs/enclosure; AS+BC: 0–2

clutches, 0–316 eggs/enclosure) but there was no significant difference in either

reproductive parameter among the three treatments (number of clutches: F2,12 =

0.20, p = 0.821; number of eggs: F2,12 = 0.426, p = 0.663).

Predation on non-Pomacea mollusks by carp

Seven species of non-Pomacea mollusks were recovered from the enclosures,

two bivalve species (Corbicula fluminea Müller., Anodonta woodiana Lea.), four

operculate snail species (Sinotaia quadrata, Melanoides tuberculata, Bithynia sp.,

Segmentina succinea Gredl.) and one pulmonate snail species (Physella acuta

Draparnaud.). M. tuberculata ranked first and S. quadrata ranked second in

41

abundance, representing 44% and 33% of the overall abundance, respectively

(Fig. 3.2). The overall abundance of these non-Pomacea mollusks differed

significantly among the four treatments (one-way ANOVA, F3,14 = 8.628, p =

0.002). The Tukey tests showed that the four treatments were clustered into two

subsets (C, AS) and (AS+CC, AS+BC).

Comparison of the number of the two most abundant non-Pomacea

mollusks across the treatments showed that black carp exerted a stronger

predatory effect than common carp, and in both treatments M. tuberculata was

more susceptible than S. quadrata to fish predation (Fig. 3.2). Comparing with

the treatment with apple snails, the abundance of S. quadrata was reduced by

74.3% in enclosures with black carp, while only by 31.8% in enclosures with

common carp. In contrast, the abundance of M. tuberculata reduced by 95.7%

and 67.5% in enclosures with black carp and common carp, respectively. The t-

test detected significantly fewer S. quadrata (t = 3.373, p = 0.012) and M.

tuberculata (t = 2.657, p = 0.033) in the black carp treatment than in the common

carp treatment. To determine whether there was size-dependent predatory effect

on the two most abundant snails, abundance data from all replicates from each

treatment were pooled to show the size frequency distribution (Fig. 3.3). For S.

quadrata, the small individuals (< 1 cm SW) were clearly eaten by both fish

species. For M. tuberculata, there appeared to be no size refugium from

predation due to its elongate shell with a small maximum width, but the mortality

of this snail in the black carp treatment was much higher than in the common

carp treatment.

42

Treatment effects on macrophytes

The three species of macrophytes had different patterns of growth and loss to

herbivory (Appendix: Plate 1). In the control, the biomass of water spinach (I.

aquatica) and water dragon (L. x taiwanensis) increased by 0.5- and 3-fold

respectively during the experiment (from the initial 100 g per replicate), but that

of water lettuce (P. stratiotes) declined to only 41% of its initial value (50g) (Fig.

3.4). Of these three species, the greatest reduction due to herbivory by apple

snails or apple snails plus carp occurred in L. x taiwanensis, the species that had

the highest increase in biomass in the control. There was a great increase in the

total macrophyte biomass in the control, from 250 g in the beginning to 542.6 g

at the end (Fig. 3.4). Macrophyte biomass in treatments with apple snails or apple

snails plus carp was remarkably lower than the control: only 30% in the AS

treatment, 32.5% in the AS+CC treatment and 56% in the AS+BC treatment. The

total plant biomass was significantly different among treatments (F3,16 = 38.746,

p < 0.001). The Tukey tests showed that the difference between the AS and

AS+CC treatments was not significant (p = 0.988), but plant biomass in the

AS+BC treatment was significantly higher than that of the AS and AS+CC

treatments (p < 0.05).

3.4 Discussion

Our study shows that the invasive apple snail P. canaliculata can exert a strong

and selective grazing pressure on wetland macrophytes. This is consistent with

the results from previous field enclosure studies (Carlsson et al., 2004a; Wong et

al., 2009). Laboratory experiments show that P. canaliculata can consume many

species of macrophtytes with clear feeding selectivity (Estebenet, 1995; Lach et

43

al., 2000; Boland et al., 2008; Tamburi and Martín, 2011), with its feeding rate

being determined by the nutrient (nitrogen and phosphorus) and defense

chemical (phenolics) contents of the macrophyte leaves (Qiu and Kwong, 2009;

Wong et al., 2010; Qiu et al., 2011). Such strong and selective grazing may alter

floral composition, and thus highlights the need to control P. canaliculata

population in non-agricultural freshwater wetlands.

Although most attempts to control apple snail populations by biological

methods have been undertaken in agricultural areas due to the obvious need to

protect crops (Liao et al., 2005; Teo, 2006), with the realization that apple snails

can reduce macrophtyte biomass and alter floral structure in non-agricultural

wetlands (Carlsson et al., 2004a), biological control of apple snails in these more

natural ecosystems has been explored in two mesocosm studies conducted in the

field (Carlsson et al., 2004b; Carlsson and Brönmark, 2006). In one of the studies,

Carlsson et al. (2004b) used climbing perch Anabas testudineus (Bloch) as a

predator of P. canaliculata, and examined the amelioration of the snail’s

herbivory due to the biological control. Their results were encouraging as the

perch showed some level of control by feeding on the neonate apple snails with

2-3 mm SL. Predation on these small apple snails, however, did not reduce the

overall herbivory on macrophytes when compared with the no-fish treatment.

Furthermore, potential predation on non-target benthic invertebrates was not

examined in these studies.

The present study not only determined the effectiveness of common carp

and black carp as potential predators of apple snails, but also examined the side-

effects on wetland benthic invertebrates and macrophytes. In a previous study

44

using similar experimental design that included common carp as the only

predator (Wong et al., 2009), we found that common carp removed almost all the

small and medium-sized P. canaliculata from the enclosures, being much more

effective than the climbing perch and catfish (Carlsson et al., 2004b; Carlsson

and Brönmark, 2006). However, common carp also greatly reduced the biomass

of macrophytes by herbivory, as well as the abundance of non-Pomacea snails by

predation. The present study, conducted in a different wetland with somewhat

different floral and faunal composition in the enclosures, confirmed common

carp’s desirable predatory effect on apple snails and undesirable side effects on

macrophytes and non-target invertebrates.

Using black carp as another potential biological control agent for apple

snails in this study has allowed us to conduct a side-by-side comparison with

common carp. Both species are effective in removing the apple snails, but

perhaps black carp is a stronger predator of the snails as it fed on more apple

snails than common carp. Although common carp had a wider gape size than

black carp (mean ± S.D.: 17.1± 0.6 mm vs. 15.9 ± 0.5mm), black carp still

consumed a wider size spectrum and higher quantity of apple snails than

common carp. Both fish were unable to feed on large-sized apple snails, but their

long life span of up to 13 years (Nico et al., 2005), their ability to consume larger

apple snails as the carp grow, and the high natural mortality of large-sized apple

snails (Kwong et al., 2010) should make both species of carp effective biological

control agents in Asian freshwater wetlands. Compared with common carp,

however, black carp appeared to be more beneficial as it preys on invertebrates

only, whereas common carp feed on both invertebrates and macrophytes.

45

Black carp was used to control snails (Melanoides tuberculata and Physella

acuta) in an outdoor concrete pond in Isreal (Ben-Ami and Heller, 2001), as well

as Planorbella trivolvis in a commercial catfish culture pond in the USA

(Venable et al., 2000). However, in a field application involving the use of both

carp species to control P. canaliculata in the paddy fields of Vietnam, black carp

had a lower survivorship than common carp (FAO, 1998). This is probably

because black carp has adapted to live in lakes and middle or lower stretches of

rivers (Pan et al., 1991). It is more sensitive to the overheating commonly

associated with the shallow water of paddies during summer in Southeast Asia.

Thus the water level was raised to 40 cm when black carp were used to control P.

canaliculata in a water bamboo field in Taiwan (Liao et al., 2005). In the present

study, overheating was probably not a problem as the part of the wetland where

we conducted the experiment was around 0.7 m deep, and there was no mortality

of black carp. However, common carp introduced into three of the five

enclosures died. Since the mortality occurred during the first month of study,

longer acclimation in the field might have improved the survival of common carp.

Although we replaced the fish as soon as possible, in one of the enclosures where

the replacement fish also died, substantial number of non-Pomacea snails

survived to the end of the experiment (498 individuals). Nevertheless, in the

other four replicates, common carp clearly exerted strong control of the non-

Pomacea mollusks, with only 160 ± 45 individuals (mean ± S.D.) surviving to

the end of the experiment.

In conclusion, our study has clearly shown that both black carp and

common carp are effective predators of the invasive apple snail P. canaliculata

46

in non-agricultural wetlands. However, the two fish species exhibited subtle

differences in their effects on non-target organisms. Black carp prey on non-

target mollusks, whereas common carp feed on both non-target mollusks and

macrophytes. These subtle differences should be considered before adopting the

fish as management tools for invasive apple snails. For instance, if wetland

macrophyte diversity is a major management concern, black carp would be a

better choice than common carp. If wetland invertebrate biodiversity is a major

concern, mechanical and cultural control measures should be included in long

term management plan, and perhaps neither species of carp should be used to

control apple snails. Our empirical study, driven by the need to control an

invasive species, has provided solid evidence to support the call for considering

the complex predator-prey interactions when evaluating the potential of a

predator as a bio-control agent (Cowie, 2002).

47

Fig. 3.1 Size frequency distribution of Pomacea canaliculata at the end of the 8-

week experiment. Abbreviations: AS = treatment containing apple snails;

AS+CC = treatment containing apple snails and common carp; AS+BC =

treatment containing apple snails and black carp.

0

50

100

150

200

250

300

350

Treatment: ASn = 1039

Nu

mb

er

of

su

rviv

ing

in

div

idu

als

0

50

100

150

200

250

300

350

Treatment: AS + CCn = 61

Shell width (mm)

0 5 10 15 20 25 30

0

50

100

150

200

250

300

350

Treatment: AS + BCn = 23

48

Fig. 3.2 Abundance of non-Pomacea mollusks at the end of the 8-week

experiment. Each bar shows the mean number of mollusks per replicate

for each treatment. (There were only three control replicates as opposed

to five of each experimental treatment). Abbreviations: C = Control with

no fish or apple snails; AS: treatment with apple snails only; AS+CC =

treatment with apple snails and common carp; AS+BC: treatment with

apple snails and black carp. Letters above the bars are results of the

Tukey tests comparing the difference in biomass among treatments.

C AS AS+CC AS+BC

0

100

200

300

400

500

Sinotaia quadrata

Melanoides tuberculata

Bithynia sp

Segmentina succinea

Corbicula fluminea

Anodonta woodiana

Physella acuta

Treatment

Nu

mb

er

of

ind

ivid

uals

a a

b

b

49

Fig. 3.3 Size-frequency distribution of the two most abundant non-target mollusks – Sinotaia quadrata and Melanoides tuberculata. Each graph

shows the pooled data from five replicate enclosures for each treatment. Treatment abbreviations are identical to those in Fig. 3.2.

M. tuberculata

0 5 10 15 20

0

50

100

150

200

250

300M. tuberculata

0 5 10 15 200 5 10 15 20

S. quadrata

0

50

100

150

200

250

300

n = 1049

n = 414

Shell width (mm)

Treatment AS Treatment AS+CC Treatment AS+BC

n = 1286

Nu

mb

er

of

ind

ivid

uals

n = 50

S. quadratan = 585 n = 184

S. quadrata

M. tuberculata

50

Fig. 3.4 Biomass of three species of macrophytes at the end of the 8-week

experiment. Each bar represents the mean ± S.D. of all available (3-5)

replicate enclosures. Letters above the bars are results of the Tukey tests

comparing the difference in biomass among treatments. Treatment

abbreviations are identical to those in Fig. 3.2.

TreatmentC AS AS+CC AS+BC

0

100

200

300

400

500

600

700

C AS AS+CC AS+BC

Total plant biomassLudwigia x taiwanensis

Ipomoea aquatica Pistia stratiotes

a

bb

c

a

bab

ab

a

b

a

a

ab b b

Bio

mass (

gra

ms in w

et w

eig

ht)

0

100

200

300

400

500

600

700 a

bb

c

a

b b

b

Total plant biomass Ludwigia x taiwanensis

51

Chapter 4 Wetland scale application of biological control of apple

snails using black carp: efficacy and subtle effect to ecosystem

4.1 Background

Invasive mollusks have altered many aquatic ecosystems, either by replacing

indigenous species through predation or competition, coexisting with indigenous

species though facilitation, or colonizing modified habitats where indigenous

species have already been eradicated by abrupt environmental changes (Cowie,

1998; Ricciardi et al., 1998). However, whether an introduced species could

eventually establish to become invasive is dependent on both the characteristics

of the recipient ecosystem such as diversity, resource abundance, frequency and

scale of disturbances (Davis and Pelsor, 2001) and also propagule pressure

(Simberloff, 2009) and life-history traits of the introduced species such as dietary

flexibility and tolerance to adverse environmental conditions (Kolar and Lodge,

2001). In Asia, three species of apple snails (Pomacea canaliculata Lamarck, P.

maculata Perry and P. scalaris D’Orbigny) have been introduced from South

America (Anderson, 1993; Jhang, 1985; Joshi, 2007; Yusa and Wada, 1999;

Joshi et al., 2001; Chen et al. 2004; Cowie et al., 2006; Hayes et al. 2008; Hayes

et al., 2012; Lv et al., 2013). Among them, two (P. canaliculata and P. maculata)

have become highly successful agricultural pests (Joshi, 2005; Joshi, 2007), but

their impact has currently proven to extend beyond agricultural context once

established and spread. As unique grazers with enlarged jaws with radula and a

digestive system with amylase for macrophyte digestion, these apple snails have

invaded natural wetlands by reversing succession pressure, promoting growth of

phytoplankton by releasing nutrients stored in living aquatic plants (Carlsson et

52

al., 2004) and drive shallow lakes towards turbid conditions (Jones and Sayer

2003; Carlsson and Lacoursière, 2005). In addition, wetland plant and

phytoplankton diversity can be altered by the feeding selectivity of P.

canaliculata that exhibit clear selection over macrophytes with high nitrogen

content, low dry matter content and chemical defense (Qiu and Kwong, 2009;

Wong et al., 2010). As most indigenous snails are mainly detritivores (Dudgeon

and Yipp, 1985), apple snails have successfully established its own niche in

consumption of underutilized wetland resource - riparian macrophytes in Hong

Kong and South-east China.

Fish, especially carp, is natural predators that regulate macroinvertebrate

density and diversity. Use of fish as bio-control agents for trematode and

schistosomiasis-hosting snails was common practice in human-modified habitats,

like fishponds and paddyfields (FAO, 1998; Lardans and Dissous, 1998). In this

aspect, common carp (Cyprinus carpio L.), Nile tilapia (Oreochromis niloticus L.)

and catfishes (Clarias spp.) have been most widely studied and implemented in

Asia (Ali, 1992; Fernando, 1993; Halwart, 1998; Liao et al. 2005; Teo, 2006).

Yet, they are omnivores that feed upon macroinvertebrates and aquatic plants

(Batzer et al., 2000), which in turn might alter original ecosystem functioning.

Chinese carp species are indigenous to riverine and floodplain habitats and have

high degree of niche segregation including specialized diet regime (Dudgeon,

1999b). Black carp (Mylopharyngodon piceus Richardson) are one of the most

abundant residents of the Great rivers (Nanjing Institute of Geography, Academia

Sinica, 1981; Liu et al., 1986; Qiu et al., 2002). They are traditionally reared in

farm ponds (Wu et al., 1964), fish ponds (Hickling, 1971) and rice paddies

53

(Wang, 2010). Other than being food fish to generate profit in complement with

that from crops yield (Lin, 1955; Liu, 1955), Black carp is also widely used as

biocontrol agents of pest mollusks.

Previous field experiment using black carp as controlling agent, however,

only addressed the applicability of this fish to slow down the invasion of snails

bearing disease-causing agents in semi-natural wetlands and within a short term

(less than a year). Experiments were conducted mainly in deeper waters, such as

nursery ponds (Hung et al., 2013), fish ponds (Venable et al. 2000) and reservoirs

(Ben-Ami and Heller, 2001). Only one experiment was conducted with apple

snails in Taiwan in shallow water paddy fields (Liao et al .2004). A large-scaled

project to control pest snails was implemented in Israeli reservoirs using black

carp, but the effects of black carp on non-target macroinvertebrates and water

quality were not evaluated (Leventer, 1979). Above all, no study has been

conducted to evaluate the application of black carp as a bio-control agent in

shallow water non-agricultural wetlands where non-target effects on macrophytes

and indigenous invertebrates might be of management concern. Nor did they

consider the effects of season on apple snail reproduction and development, and

consequently on the effectiveness of the biological control.

This field study was conducted in a constructed freshwater wetland being

infested by apple snails. Specifically, the objectives for experiment were: first, to

determine the effectiveness of black carp on apple snails in natural settings;

second, to determine the direct predatory effect of black carp on non-target

macroinvertebrates and indirect effect of predation of apple snails on macrophyte

diversity and biomass, as well as macroinvertebrates and water quality.

54

4.2 Study site

The study was conducted in three constructed freshwater wetlands that are nearly

permanent lentic ponds. The study lasted one year encompassing the wet season

(June - September 2012 & April - June2013) and dry season (October 2012 -

March 2013). Mean monthly air temperature declined from a maximum of

30.5°C in June 2012 to a minimum of 15.9 °C in December 2012. Total

precipitation during the study period was 2595.5 mm (till June 2013), with

1855.2 mm in the wet season and 307.1 mm in the dry season. These constructed

wetlands are all freshwater earthen ponds created in 2002 as a compensation for

the loss of the original wetlands due to the construction of the West Rail. Mean

water depth ranged from 1.1 – 2.2 m in the wet season; and 0.6 – 1.3 m in the dry

season. Site 1 and site 2 were roughly rectangular, with a total area of 2525

m2and 1000 m

2 respectively.

A partition was set up with woven nylon clothes supported by a steel frame

in the middle of each of these two sites, creating one experimental plot and one

control plot. The partitions prevented snails from migrating and minimized water

exchange between the control and treatment plots. Site 3 was comprised of two

separated but immediately adjacent ponds; one with an area of 450 m2 served as

control and the other with an area of 625 m2 as treatment. In general, the water in

sites 1 and 2 was mildly acidic (6.4 ± 0.44), mildly oxygenated (4.8 ± 2.3 mg/L)

and quite turbid (20.3 ± 11.9 NTU). Site 3 was quite hypo-oxygenated (2.39 ±

2.44 mg/L) at the initial stage. In subsequent months during dry season 2012,

mechanical water pumping was conducted to maintain stable water levels within

all plots above 0.5 m. Dissolved oxygen levels in Site 3 had thus been raised and

maintained at approximately 3 mg/L until the experiment ended in June 2013.

55

Several emergent macrophytes dominated the sites. These included

Panicum dichotomiflorum Michaux, Paspalum distichum Linnaeus, Polygonum

barbatum Linnaeus, P. lapthifolium L. and Ipomoea aquatica Forsskål.

Apple snail P. canaliculata inhabited the three ponds at initial mean

densities of 24.67 – 39.3 individuals per m2, and were similar between each of

the control and treatment pairs. Indigenous snails co-existing with apple snails

within our study site were all common residents at lowland lentic wetlands of

Hong Kong. These included Sinotaia quadrata Benson, Melanoides tuberculata

Müller and Bithynia sp. in sites 1 and 2. For site 3, only exotic snail Segmentina

succinea co-existed with apple snails. In April and May 2012 before experiment

commenced, various attempts of stocking S. quadrata and M. tuberculata were

made in Site 3 as these two species are very common in local lentic water. It was

thus important to include these two species to test the non-target effects of carp

predation. However, since subsequent field surveys did not show that these two

species successfully colonized Site 3, no further reintroduction was conducted.

4.3 Method

Black carp specimen preparation and collection

Thirty individuals of black carp were obtained from a local fish farm in Sheung

Shui, New Territories. They were kept in an indoor aquarium and fed with

commercial fish feed for at least one month before the experiment. One day prior

to the experiment, no food was given to all specimens. 12 specimens were

randomly selected and transported to site. Four black carp were released into

each experimental plot (treatment), whereas no fish were introduced into the

control. The average total length and wet weight for black carp was 294 mm

56

(S.D.: 17.40mm) and 231.3g (S.D.: 42.13g), respectively. Experiment started

immediately once black carp were released to each experimental plot. Fish

specimens were not fed during the experimental period.

At the end of the experiment, fish were extracted by drying the whole site and

subsequent extensive netting. They were euthanized and freezed as soon as they

were returned to laboratory. Respective total length, weight and gape width were

measured for each retrieved fish specimen. Within the subsequent three days

after retrieval, fish extracted from each of the three sites were dissected to

remove their stomach. Each stomach was then preserved in 10% formalin. Prey

composition was determined by dissecting the foregut of each stomach, where

there was least active amount of digestion and allowed easier identification. Gut

fullness was estimated visually by a dissecting microscope. Indirect volumetric

analysis was deployed in estimating the percentage of different food items in

comparing with the total volume of all stomach content (Hyslop 1980).

Seasonal field surveys

Field surveys were conducted from June 2012 to June 2013 to test the

effectiveness of black carp as a bio-control agent for apple snails, as well as their

non-target effects on macrophytes and benthos. In these surveys, macrobenthic

community analysis and water quality measurement were conducted every three

months, while macrophyte composition was determined only in the beginning

and at the end of the study, due to low water levels in the dry season which

prevented apple snails from feeding on the macrophytes.

Macrobenthic survey

57

The benthic surveys were conducted at fixed points marked with bamboo sticks.

In each plot, six 0.5 m2 were sampled by enclosing the area with a throw trap and

intensively collecting the macrobenthos on the sediment surface, in the sediment

as well as on the macrophytes using a hand-net with a mesh size of 500μm. A

total of 36 benthic samples were collected from the six sites (i.e. three control

and three treatment sites) during each sampling. The samples stored in numbered

Ziploc bags and were transported to Hong Kong Baptist University, and kept in a

cold room at 4ºC before further processing.

Sieving was conducted with a metallic sieve (pore size 500μm) within three

days after sampling to remove fine sediment. Macrobenthos were extracted from

the residuals retained on the sieve. They were identified into the lowest

taxonomic level and counted. One sample for each identified taxon was

preserved in 70% ethanol for detailed identification. For any species of identified

mollusk, its amount within each sample was enumerated, with shell length of

each individual measured. Same procedures applied to any species of identified

invertebrates, except that their sizes were not measured.

Determination of water quality parameters

Water quality parameters were either measured in-situ or in the laboratory. In-

situ measurements were conducted for temperature, pH, salinity, dissolved

oxygen, conductivity and turbidity. Temperature, pH, salinity, dissolved oxygen,

conductivity and turbidity were measured using a TPS 90-FLMV meter, whereas

turbidity was measured using a Thermo Scientific Orion AQ4500 meter. A total

of three measurements were taken for each parameter in each site. Two 1-L water

samples were also collected from each treatment and control site for laboratory

58

measurement of nutrient parameters (ammonia nitrogen, total nitrogen, reactive

phosphate and total phosphorus). A total of 12 water samples were collected

from three sites during each sampling. They were kept in a cool box with ice and

immediately transported to the laboratory for analysis using standard methods

(Eaton and Franson, 2005): total nitrogen (TN) using a flow injection analyzer

(Method 4500 N B); ammoniacal nitrogen (NH3) using a flow injection analyzer

(Method 4500-NH3 H); total phosphorus (TP) and total phosphate (P) using the

ascorbic acid method (Method 4500-P B & E; Method 4500-P E respectively).

Abundance of apple snail hatchlings

The number of neonates that might enter the pond was estimated by determining

the number of eggs available in the ponds and estimating their hatchability. The

number of eggs available was determined by counting the actual number of egg

clutches observed (a walkover survey throughout the whole pond bund during

each sampling time). Once an egg clutch was detected, we measured the

maximum length and width to the nearest 0.1 mm using digital calipers. This was

repeated until all egg clutches in each site were measured accordingly. The

number of eggs in each egg clutch was estimated by a regression relationship

between maximum area (maximum length* maximum width) and the actual

number of eggs in each egg clutch from previous literature as follows (Ichinose

et al., 2002).

Y = 0.1 * X1.24

(r2 = 0.75, n=272),

where X is the product of the maximum length and width of the egg masses and

Y is the number of eggs.

59

Hatchability of a given egg mass was estimated by hatching field-

collected egg clutches in the laboratory. In each month of the experiment, 25 - 30

egg masses were collected from wetlands within 200 m from our experimental

ponds. Five egg clutches were placed on a steel rack on top of a plastic container

(total number of containers = 5 - 6) with de-chlorinated water, and allowed to

hatch for 21 days at a rooftop. At the end of the experiment, the hatched neonates

and non-hatched eggs were counted. Monthly mean hatchability data were then

obtained. The estimated hatchling number from all individual egg clutches was

then estimated by the following equation:

N = Σ Hhatch * Y

where Hhatch is the monthly mean hatchability and Y is the number of eggs

estimated from egg clutch.

Macrophytes

Macrophyte surveys were conducted to understand the indirect effects of black

carp on macrophytes, or whether controlling apple snails would enhance

macrophyte biomass and diversity. Since macrophytes were present only on the

edges of the ponds during the wet season when the water levels are sufficiently

high, surveys were conducted in the beginning and at the end of the experiment

only. Eight 1 m x 1 m permanent quadrats were set up in each plot of each site

with the corners marked with bamboo sticks. A total of 48 1m2 quadrats were

established along in the six plots. In each quadrat, vegetative cover (%) was

estimated by visual observation, and the species composition was recorded.

Data analysis

60

Hatchling numbers were compared among sampling seasons and treatments

using a two-way Analysis of Variance (ANOVA). Change in apple snail

abundance over time from the same set of replicates was compared using

repeated measures ANOVA.

Abundance of juvenile apple snails was compared between the control and

treatment among the three sites using repeated measures ANOVA to determine

the effects of black carp. Abundances of three major non-target mollusks –

Sinotaia quadrata, Melanoides tuberculata and Bithynia sp. were compared

among sampling period and treatments by repeated measures ANOVA.

Physical and chemical water quality parameters were compared in each

individual sites using two-way ANOVA to determine the significance of

treatment and period effect.

Vegetation coverage data were square-root transformed. Non-metric

multidimensional scaling ordination (nMDS) was used to ordinate the Bray-

Curtis similarities computed from transformed vegetation data according to

treatment and period (data taken at the start versus that at the end of experiment).

One-way Analysis of Similarities (ANOSIM) was used to determine the

significance of treatment effect in each site. To determine whether particular

species was susceptible to treatment effects, individual one-way ANOVA test

were done on abundance in the first four dominant aquatic plant species of

aquatic plant observed before and after the treatment.

61

4.4 Results

Fish growth and survival

All fish were captured using landing nets, after full water extraction of all three

sites by September 2013 (Table 4.1). Mean monthly growth rate for length and

weight was 3.27 cm (S.D.: 0.20 cm) and 279.24g (S.D. = 20.26 g). While

average total length increment of more than one fold was recorded, fish specimen

weights increased in average for 3350.85g (S.D.: 243.15g) within one year.

Average survival rate was 66.67% (8 out of 12 individuals survived throughout

treatment). All four specimens were retrieved in site 2, while only two specimens

were retrieved from site 1 and 3 respectively. Death of two black carp was

detected in site 1 during September 2012. Even after replenishment of two new

juveniles in September 2012, we failed to retrieve them by the end of the

experimental period. For site 3, one specimen died in October 2012 and another

two specimens in April 2013 was found to be dead. Release of another two

specimens was done by mid-April 2013. Yet, none of newly released specimens

survived by September 2013.

Eggs clutches in the field

Eggs were laid most abundantly in June 2012 and then declined continuously to

minimum in March 2013, thereafter egg abundance rose again till June 2013

(Table 4.3). In treatment site 1 and 2, amount of egg clutches was reduced in

higher proportions than control sites during each sampling period until June 2013.

By the end of the experiment, a reduction of 55.8% of egg clutch abundance was

recorded in treatment site 1 by comparing the data of June 2012 and 2013. In

control site 1, amount of egg clutches in control site increased by three folds

62

within the same period. Same degree of reduction was not observed in treatment

site 2 by June 2013. Egg clutches abundance sharply rose for 2 folds during

March to June 2013 and reached similar amount to that in control site. Yet in

March 2013, percentage of egg clutches abundance in treatment site still

decreased by 15.38% since the trough reached during December 2012, compared

to an increase of more than one fold in control within the same period. No

comparable differences due to treatment were observed in data of site 3 (Fig. 4.2).

Hatchability of egg clutches in laboratory

The number of successful hatched eggs per clutch ranged from 90 to 355 (n =

25 – 30 egg clutches per sampling period). Mean hatchability ranged from 40%

in December 2012 to 83% in March 2013 (Table 4.4).

Apple snail and hatchling densities

Mean snail densities ranged from 1.33 – 92.0 individuals per m2 throughout the

experimental period (Fig. 4.1). There was a major decline of 96.6% (site 1),

94.1% (site 2) and 77.0% (site 3) in overall apple snail density in sites with black

carp introduced within the first three months (Jun 2012 – Sept 2012), while it

remained fairly stable till the breeding season in June 2013. Snail densities in all

three sites were significantly different both between treatments (F1,16 = 5.590, p <

0.05*) and among sampling time (F3,16 = 3.456, p < 0.05*).

Total estimated abundance of hatchlings ranged from 612.6 to 23973.0

individuals per month, and exhibited a clear seasonal pattern, with high values in

wet season (June – September 2012; April – June 2013) and low values in the dry

season (October – March 2013) (Fig. 4.3). A gradual decline in hatchlings was

recorded in both the control and treatment sites from June to December 2012, but

63

the number of hatchlings rose since March 2013. A comparison using three sites

as replicates showed significant differences between treatments (F1,16 = 4.450, p

< 0.05*) but not among sampling times (F3,16 = 2.011, p = 0.132) nor interaction

between the two factors (F3,16 = 0.0475, p = 0.766) (Table 4.2).

From the data of snail densities and hatchling abundance, we could

summarize that there was a clear seasonal differences in density and reproduction.

During June 2012, apple snail density reached a peak due to the high recruitment

from egg clutches. In September to December 2012, snail densities declined

which reduced the amount of egg clutches. Snail densities rose again in March

2013, thus causing the egg clutches abundance to grow again until it peaked in

June 2013.

For the juvenile apple snails (SL: <5 – 25mm), there were significant

differences in snail density recorded between treatments (F1,7 = 4.633, p < 0.05*)

but not among sampling times (F3,7 = 1.765, p = 0.194). For the adults,

significant differences in densities were detected only among sampling times

(F1,7 = 4.759, p = 0.01*) but not among treatments (F1,7 = 1.681, p = 0.213).

These results suggested size-dependent predation of juvenile snails by black carp

in experimental plot, while adults population was not affected (Fig. 4.4).

Fish gut content analysis

Among all eight specimens retrieved, mean gut fullness was 36.25% (S.D. =

30.67%). No distinguishable features in bolus could be identified to species level

even under dissection microscope.

Effects to non-target mollusks and other invertebrates

64

Mean densities of S. quadrata, M. tuberculata and Bithynia sp. ranged from 0.7 -

250, 0.0 - 126 and 0 - 110 individuals per m2, respectively (Fig. 4.5 & Table 4.6).

While no statistical differences in all parameters were detected in Bithynia sp.

densities, significant differences were detected in S. quadrata densities between

treatments (F1,7 = 6.229, p < 0.05*) and among sampling times (F3,7 = 4.477, p <

0.05). Densities of M. tuberculata differed between treatments (F1,7 = 9.236, p =

0.01*) and among sampling times (F3,7 = 5.301, p = 0.01*). In experimental site

1, average densities of S. quadrata and M. tuberculata reduced by 98.8% and

100% by June 2013 when compared to data in one year before, while that in

control site 1 decreased by 42.13% and increased by 33.33% respectively.

Similar magnitude of reduction was detected in experimental site 2. Average

densities for S. quadrata and M. tuberculata declined by 94.34% and 87.88%

respectively, while only a minor reduction of 0.01% in densities of S. quadrata

and 41.27% for that of M. tuberculata were recorded in control site 2.

No significant reductions due to treatment were found in abundance of

other non-target invertebrates. This was probably because of a high seasonal

variation in amount of benthic invertebrates, especially insects in ponds (Table

4.6).

Fish influence on water quality

There were significant seasonal differences among all physical water quality

parameters in all three sites (Fig. 4.6 & Table 4.9). Three parameters showed

additional significant difference between treatments (Table 4.7). These include

dissolved oxygen (site 2 and 3), pH (site 1, 2 and 3) and turbidity (site 2 and 3). It

was noted that turbidity levels shown significant differences between treatments

65

in both site 2 and 3. In site 2, treatment pond was 4.74% more turbid than control

pond in June 2012. By March and June 2013, treatment pond was 19.78% and

31.58% clearer than control pond respectively. Yet, in site 3, it was found that

treatment pond is 364.28% and 84.31% more turbid than control during

September and December 2012. Turbidity levels in treatment pond turned

53.60% and 17.85% clearer than control pond in two subsequent sampling

periods (March and June 2013).

In all three sites, nutrient level fluctuated throughout the experimental

period (Fig. 4.7). In general, total phosphorus (TP) and total nitrogen (TN) levels

reached maximum in June 2012, then dropped to minimum in December, while

they rose again thereafter. Among three sites, site 3 showed the highest level in

total phosphorus (≥ 0.3 mgL-1

), no matter in experiment or control treatments.

Ranges for four nutrient parameters were 0.0 – 1.45 mgL-1

(NH3); 0.8 – 2.25

mgL-1

(TN); 0.0 – 0.24 mgL-1

(PO4-) and; 0.01 – 0.45 mgL

-1 (TP) (Table 4.9).

While seasonal effect was highly significant in all parameters among sites, only

data of P and TP in site 2 and 3 showed significant differences among treatments

(Table 4.8). In site 2, significantly lower phosphate concentrations (F1,4 = 5.000,

p < 0.05*) and total phosphorus concentration (F1,4 = 8.333, p = 0.01*) were

recorded in treatment pond, while interactions with period effect was both

significant. In site 3, significant differences were also found in two phosphorus

parameters. Significantly lower concentrations of phosphates (F1,4 = 96.729, p <

0.01) and total phosphorus (F1,4 = 34.416, p < 0.01) was recorded by June 2013,

while positive interactions with period effect were recorded as well (Table 4.8).

Vegetation abundance

66

Vegetation diversities were quite consistent across three sites. From mean

abundance of each plant species among three sites, dominant vegetation before

the experiment was mostly emergent plants comprised of Paspalum conjugatum

(30.71%), Paspalum distichum (12.50%), Ipomoea aquatica (12.29%), Panicum

dichotomiflorum (11.73%). P. dichotomiflorum (25.19%), Paspalum distichum

(17.18%), Ipomoea aquatica (13.14%, especially at site 3), and Paspalum

conjugatum (12.43%) became the most dominant plant species after the treatment

(Table 4.10). Ordination of plant abundance communities before and after the

treatment yet gave poor representation in three nMDS plots (stress = 0.1) (Fig.

4.8). No clear change in overall community structure was found after fish

implementation in all three experimental plots. Results from one-way ANOSIM

indicated that there was unclear segregation of results due to treatment (site 1:

Global R = 0.088, p = 3.8%; site 2: Global R = 0.119, p = 2.1%; site 3: Global R

= 0.126, p = 1.5%).

Though no significant changes in vegetation community structure were

recorded, particular plant species grew vigorously in treatment with fish. Yet,

these changes were restricted in one site, while not observed in all three

replicates. By comparing plant abundances from same eight quadrats in June

2012 and June 2013, average abundances of P. dichotomiflorum increased

dramatically by 100% in experimental site 1, while that in control reduced by

63%; in experimental site 3, mean abundance of I. aquatica increased by 400%

in treatment with black carp and decreased by 99.1% in control treatment.

Significant statistical differences in abundance of P, dichotomiflorum (F1,14 =

23.33, p < 0.01*) in site 1 and I. aquatica (F1,14 = 4.876, p < 0.05*) in site 3 were

67

shown individually after treatment with black carp for one year, while no

significant differences were observed by comparing them on June 2012 (P.

dichotomiflorum: F1,14 = 0.066, p = 0.801; I. aquatica: F1,14 = 7.177, p = 0.059).

Comparison on maximum height of these two emergent aquatic plant species

showed same pattern of results. Mean maximum height of P. dichotomiflorum in

quadrats of experimental site 1 reached 1.3 m (S.D. = 0.063, n = 8), while that in

control has only reached 0.91 m (S.D. = 0.13, n = 2). In site 3, maximum height

of I. aquatica has reached 0.82 m on average (S.D. = 0.21, n = 5) in experimental

site and 0.50 m (S.D. = 0.11, n = 4) in control site.

4.5 Discussion

In natural shallow water bodies, presence of predatory fish, especially

molluscivorous specialists, impose top-down control on mollusk populations in

aquatic ecosystems (Brönmark, 1988; Brönmark and Hansson, 2005; Martin et

al., 1992). In Hong Kong, due to the absence of large water bodies and artificial

channelization of natural lowland streams, there are insufficient predators to

control apple snail populations (refer to Chapter 1, table 1.1). In addition, escape

of edible exotic fish fingerlings from fish ponds and purposeful release facilitate

invasion of exotic predatory and omnivorous fish, like omnivorous Nile tilapia

(Oreochromis niloticus Linnaeus) and piscivorous snakehead murrels (Channa

striata Bloch), in disturbed rivers and channels (Dudgeon, 1996; Hodgkiss &

Man, 1977; Lee et al., 2004). However, among all existing predatory fish in local

freshwater habitats, none are specialized molluscivores (Lee et al., 2004). Re-

introduction of an indigenous mollusk specialist might thus be a viable solution

in controlling apple snails, especially in lowland wetlands with impoverished

biodiversity.

68

Black carp (Mylopharyngodon piceus) is a highly tolerant species that

migrates annually from riverine habitats to lakes and floodplains to complete

reproductive cycle. This species has longevity of 13 – 15 years, making it a good

candidate for biological control (Nico et al., 2005). Due to synchronization with

annual flooding events, it has broad environmental tolerances. For example, it

can survive in a wide range of water temperature from 4 to 30oC (Chu and Chen,

1989; Gangstad, 1980; Li and Fang, 1990). In our study, even under the poor

conditions of site 3, 2 of the 4 individuals released survived throughout the whole

study period. The water depth at this site was as low as 0.4 m during the dry

season, while temperature reached 31.1oC and dissolved oxygen level was

0.41mgL-1

in the wet season. At the start of the experiment, dissolved oxygen

was even as low as 0.16 mgL-1

but no fish specimens were observed to die within

the first week after release even being reared in stagnant water.

Our study is the first pond-scale experiment that has examined the

effectiveness of black carp as a biological control agent of apple snails. The one-

year experimental period was essential to determine how seasonal changes in

apple snail life-history characteristics could modulate the predatory effect of

black carp. Within the first three months of experiment (June – September 2012),

apple snail density had already diminished by an average of 89.25%, while that

ranged from 77.0% (site 3) to 96.6% (site 1). The maximum reduction in snail

density of 98.3% was achieved in December 2012 in site 1, where no more

neonates were recorded until the final survey conducted in June 2013. It was also

in site 1 where the abundance of juvenile snails (< 25 mm) declined most quickly

and remained low until the end of experiment. In March 2013, this size range of

69

snails was also depleted by black carp in site 3 after low recruitment rate in dry

season, though the level rose again by June 2013. This re-elevation in snail

densities might be related to the availability of favorable habitat structure for

apple snails. During wet season in June 2013, the site was flooded by rainwater

and generated additional shallow habitats (<0.2m). These habitats were known to

increase apple snails’ fitness (Karunaratne et al., 2006), but low water depth

increased difficulties for black carp in reaching their prey. This could be proved

from body depths of all retrieved black carp, in which it ranged from 0.13 -

0.14m. In site 3, large number of adult snails, which were too big to fit into the

mouth of the fish released into the pond, sustained a huge population of over

20000 individuals of hatchlings in June 2012 and 2013. Though carp could

significantly reduce the abundance of juvenile snails, eradication of the whole

population could only be achieved after the adult snails died out after completing

their lifespan. Nevertheless, since the adults suffered great over-winter mortality,

even measures that can control only the abundance of juveniles should be

effective for the whole population control by limiting new recruits from reaching

adulthood. Gut content analysis of black carp specimens were insufficient in

reflecting the diet of black carp. Even in the foregut where food items were least

digested, no distinguishable features among all prey present within the habitat

could be found. This is probably because pharyngeal mastication (Sibbling, 1982)

and repeated re-suspension of food materials with mucus in pharynx (Sibbling,

1991) has highly increased the feeding efficiency in all cyprinid fishes.

By controlling the population of herbivorous apple snails, black carp might

have indirect effects to macrophytes and nutrients. Grazing by apple snails could

70

release nitrogen and phosphorus originally stored in macrophytes (Carlsson et al.,

2004a), although the sediment might re-absorb some of the nutrients. Previous

studies conducted in wetlands with greater macrophyte diversity showed that the

existence of juvenile apple snails with high abundance might become more

competitive than adult ones by having higher feeding efficiency (Carlsson and

Brönmark, 2006; Boland et al. 2008). It is therefore possible that significant

reduction of juvenile apple snails could eventually minimize nutrient flux after

the adults died, thus preventing shift of primary production from macrophytes to

phytoplanktons. Results from our present study showed significantly different

phosphates and total phosphorus concentrations in two out of three treatment

sites than control sites since treatment started. However, comparing between

results of June 2012 and June 2013, we could only observe a significant

reduction in phosphorus concentrations in site 3, but not among the rest two

treatment sites. Insignificant results in only one but not all treatment sites might

be due to continued addition of contaminated water source from an adjacent

channel. As there was only one reliable water source, constant water supply was

provided to only site 1 and 2 to maintain sufficient water levels during November

2012 – February 2013 from this channel. As a high population of tilapia and

apple snails was sustained inside this channel, water inside this channel might

had a steadily high concentrations of nitrogen and phosphorus. It would therefore

highly possible that fluctuation in dissolved nutrient concentrations in our

treatment sites was caused by this process. Our results from present study were

therefore inconclusive in explaining reduction in population of a voracious

consumer in aquatic plants could reduce excessive nutrient influx to water bodies.

71

Although black carp is known to be mollusk-eating, most studies conducted

using this fish species as subject however provided insufficient data in evaluating

the fish performance as a bio-control agent. In one of publication at Fuxian Lake

at Yunnan, China, bivalves namely Margarya spp. and Corbicula fluminea have

been cited as food items of black carp (Yang and Chen, 1995), though no

experiment was cited as reference. A number of experimental studies have shown

that black carp is an effective predator of molluscs that are hosts of human

parasites, such as Planorbella spp. (Ledford and Kelly, 2006), Physa acuta and

Melanoides tuberculata (Ben-Ami and Heller, 2001) Ram-horn snails Heliosoma

sp.and Physa sp. (Collins, 1996; Thomforde, 2000). Though some of these

experiments were conducted in field, there were no data evaluating densities of

co-existing benthic organisms. The only field based experiment that considered

subtle effects in controlling exotic snails on existing indigenous mollusks was

conducted in Israeli reservoirs by Leventer (1979). From the results, it has been

reported that visibly all target and non-target specimens of snails including

Lymnea auricularia, Bulinus truncates, M. tuberculata, Melanopsis praemorsa

and M. costata plus one bivalve Corbicula fluminea were eliminated. Yet, only

their abundance data estimated after the experiment were used to justify the

success of deploying fish as effective bio-control agent, while seasonal predatory

effect was not considered. Results from our experiment indicated that black carp

would prey upon golden apple snails and at least three species of non-target

snails including Sinotaia quadrata, M. tuberculata and Bithynia sp., though

significant reduction was not recorded in Bithynia sp. due to variation in densities

among plots. Other benthic invertebrates including various species of dragonflies

and damselflies also existed within the sites. But no significant changes could be

72

concluded from densities of any co-exiting soft-bodied invertebrates other than

mollusks between two treatments.

Only some species of aquatic plants but not all showed increased growth

performance by reduction in apple snail densities. In this study, though black

carp succeeded in controlling abundance of juvenile apple snails, only two

emergent or floating macrophytes P. dichotomiflorum and I. aquatica were

shown to have benefited. Reduced abundance of apple snails as voracious grazers

had promoted their heights and densities in sites with fish treatment. Wong et al.

(2010) has suggested I. aquatica to be preferably consumed by apple snails

according to their relatively high nutrient content and low dry matter content.

Recent mesocosm experiment (Ip et al. submitted) had also shown increased

abundance and biomass of I. aquatica when black carp were in coexistence with

apple snails. For P. dichotomiflorum, its habitat is restricted to shallow water and

marshy places. It is a hydrophious herbaceous plant with emergent and floating

culms (Lin, 2009; Xia et al., 2011). No current data was available considering its

nutrient data and physical constituents. Yet their differences in growth

performance might be related to their natural growth strategies. Since our present

study did not devise sufficient replicates to compare growth performances among

plant species and between treatments, we could not reach conclusive summary on

reduction of apple snail abundance could lead to more favorable growth

performance of co-existing aquatic plants.

In conclusion, black carp is a highly tolerant species that can survive in

shallow stagnant waters. This species could be used as a biological control agent

of apple snails due to its high effectiveness as a predator in shallow freshwater

73

wetlands, which are common habitat in southern China. Given the longevity of

black carp, a low stocking density (80 - 89 individuals ha-1

; stocking density for

site 2 and 3 in this experiment) will be sufficient to control apple snail

populations. Additionally, black carp culture in wetlands can provide commercial

incentives as it is an edible fish. Yet implementation of black carp to control

apple snails should only be carried out in habitats with low biodiversity of

macro-invertebrates, especially mollusks. Juvenile (total length: at least

approximately 300mm) specimens are most preferable option for release as they

could reach a broader range of habitats including shallow riparian area where

snails aggregate, while still beholding their high adaptability to adverse

environmental conditions in shallow wetlands.

74

Table 4.1 Fish survival in three experimental sites in June 2012-September 2013. Time abbreviation is as follows: Jun-12 refers to June 2012;

Sep-13 refers to September 2013; Absent refers to missing fish specimen during retrieval in August to September 2013.

Location Fish specimen number

Site 1 S1 S2 S3 S4

Time Jun-12 Sep-13 Jun-12 Sep-13 Jun-12 Sep-13 Jun-12 Sep-13

Fork Length (cm) 31 69.3 26 Absent

31 67.5 28 Absent

Weight (g) 242.4 3730 143 255.9 3230 200.6

Location Fish specimen number

Site 2 S1 S2 S3 S4

Time Jun-12 Sep-13 Jun-12 Sep-13 Jun-12 Sep-13 Jun-12 Sep-13

Fork Length (cm) 31 70.5 31 70 27.5 71 29 65

Weight (g) 260.6 3720 289 3691.2 186.5 3715 215.1 3165

Location Fish specimen number

Site 3 S1 S2 S3 S4

Time Jun-12 Sep-13 Jun-12 Sep-13 Jun-12 Sep-13 Jun-12 Sep-13

Fork Length (cm) 29.5 70 31.5 72 29 Absent

28.3 Absent

Weight (g) 242.5 3730 287.4 3805 238 214.5

75

Table 4.2 Results of 2-way ANOVA testing the difference in estimated

hatchlings between treatments and among sampling time.

Factor df F P

Treatment 1 4.450 0.048*

Sampling time 4 2.011 0.132

Treatment * Sampling time 4 0.457 0.766

Error 20

Data with statistical differences (P < 0.05) are shown with asterisk.

76

Table 4.3 Effect of black carp on density of different size range of apple snail

and their reproductivity.

Treatment site Control site

Factors Time 1 2 3 1 2 3

Apple

snail density

(indiv

.m-2

)

T1 39.33 56.67 24.67 36.00 32.00 12.00

T2 1.33 3.33 5.67 17.33 18.00 18.00

T3 0.67 3.33 6.50 15.67 16.33 18.00

T4 0.67 2.33 7.50 25.00 28.67 92.00

T5 2.00 12.67 17.33 26.67 23.33 47.33 Juvenile apple

snail

density (indiv.

m-2

)

T1 26.67 52.00 10.33 13.33 18.67 6.67

T2 0.00 0.33 0.50 16.00 17.67 15.00

T3 0.00 0.33 0.50 5.67 15.67 16.67

T4 0.00 2.33 0.00 20.50 20.67 76.00

T5 0 10.67 3.33 6.67 9.33 21.33

Adult snail

density

(indiv.

m-2

)

T1 12.67 4.67 14.33 22.67 13.33 5.33

T2 1.33 3.00 5.17 1.33 1.33 3.00

T3 0.67 0.00 6.00 10.00 0.67 1.33

T4 0.67 0.00 7.50 4.50 8.00 16.00

T5 2.00 2.00 14.00 20.00 14.00 26.00

Number

of egg

clutches on site

(indiv.)

T1 181 37 111 40 27 96

T2 104 37 180 21 50 44

T3 35 13 23 15 17 40

T4 5 11 39 7 38 11

T5 80 36 103 164 33 17

Estimat-

ed hatchling

(indiv.)

T1 11170.04 6119.71 23973.08 791.72 1525.77 11141.32

T2 7328.93 2356.83 13141.99 1292.67 2649.05 3964.06

T3 1779.51 560.64 1933.39 612.61 738.09 1342.11

T4 1250.45 1888.04 13731.93 1584.60 4553.04 2153.56

T5 17860.58 2035.61 16364.69 46431.81 2060.85 1178.76

77

Table 4.4 Hatchability of collected egg clutches in laboratory

Month Number of egg

clutches collected Hatched amount Unhatched amount

Total number of

eggs

Hatching percentage

(%)

June 2012 30 3235 710 3945 82.00

September 2012 25 1825 1201 3026 60.31

December 2012 25 770 1158 1928 39.93

March 2013 25 1684 341 2025 83.16

June 2013 30 2337 1579 3916 59.68

78

Table 4.5 Effect of black carp to non-target gastropods.

Treatment site Control site

Density

(indiv.m-1

)

Time 1 2 3 1 2 3

Sinotaia

quadrata

T1 222.67 106.00 0.00 250.00 159.33 0.00

T2 88.67 125.33 0.00 130.00 260.00 0.00

T3 22.00 119.33 0.00 44.00 170.67 0.00

T4 6.67 72.67 0.00 162.67 246.67 0.00

T5 2.67 6.00 0.00 144.67 157.33 0.00

Melanoides

tuberculata

T1 6.00 22.00 0.00 8.00 42.00 0.00

T2 0.00 38.67 0.00 77.33 126.00 0.00

T3 0.00 3.33 0.00 0.67 3.33 0.00

T4 3.33 0.00 0.00 77.33 19.33 0.00

T5 0.00 2.67 0.00 10.67 24.67 0.00

Bithynia

sp.

T1 14.67 22.67 0.00 110.00 23.33 0.00

T2 0.00 0.67 0.00 12.67 11.33 0.00

T3 0.00 2.67 0.00 0.67 4.00 0.00

T4 0.00 14.67 0.00 19.33 19.33 0.00

T5 0.00 6.00 0.00 31.33 20.67 0.00

79

Table 4.6 Effect of black carp on abundance of non-target benthic invertebrates

except gastropods. Data are total abundance of each family of invertebrates in

1.5m2 quadrat area.

Treatment site Control site

Family Time 1 2 3 1 2 3

Odonata

T1 4 1 2 5 0 6

T2 4 1 4 5 0 8

T3 0 3 19 0 2 17

T4 16 1 8 0 2 1

T5 0 2 5 2 1 5

Corixidae

T1 28 1 5 79 0 5

T2 38 1 5 69 0 5

T3 0 13 3 2 0 0

T4 365 0 57 62 0 6

T5 0 0 0 0 0 0

Notonectidae

T1 0 4 29 3 1 7

T2 0 1 35 4 1 7

T3 0 0 5 0 0 2

T4 0 1 51 4 1 31

T5 0 0 23 0 0 0

Naucoridae

T1 0 0 5 0 0 12

T2 0 0 0 0 0 12

T3 0 0 0 0 0 0

T4 0 0 0 0 0 0

T5 0 0 3 0 0 1

Pleidae

T1 0 0 15 0 0 2

T2 0 0 15 0 0 3

T3 2 0 41 1 0 140

T4 0 0 23 0 0 73

T5 0 0 0 0 0 0

Palaemonidae

T1 0 5 0 0 2 0

T2 0 12 0 0 2 0

T3 0 1 0 0 2 0

T4 3 1 0 0 2 0

T5 0 1 0 0 0 0

Anura larvae

T1 0 0 4 0 0 0

T2 0 0 5 0 0 1

T3 0 0 0 0 0 0

T4 6 195 1 1 117 19

T5 0 0 22 0 0 4

80

Table 4.7 Results of two-way ANOVA testing the difference in physical water quality parameters in individual sites between treatment and

among sampling period.

Site 1 Site 2 Site3

Factor df F P F P F P

Temperature

Treatment 1 6.000 0.024* 7.579 0.012* 2.042 0.168

Period 4 12045.063 <0.001* 12046.711 <0.001* 3388.370 <0.001*

Treatment * period 4 17.771 <0.001* 35.079 <0.001* 2.901 0.048

Dissolved oxygen

Treatment 1 69.467 <0.001* 1.272 0.273 49.573 <0.001*

Period 4 267.037 <0.001* 219.401 <0.001* 73.582 <0.001*

Treatment * Period 10 21.621 <0.001* 9.679 <0.001* 165.349 <0.001*

pH

Treatment 1 23.237 <0.001* 909.091 <0.001* 50.241 <0.001*

Period 4 60.563 <0.001* 2108.055 <0.001* 74.675 <0.001*

Treatment * Period 10 .751 0.569 182.182 <0.001* 28.189 <0.001*

Turbidity

Treatment 1 .340 0.567 112.372 <0.001* 219.290 <0.001*

Period 4 312.566 <0.001* 964.059 <0.001* 210.976 <0.001*

Treatment * Period 10 13.693 <0.001* 49.413 <0.001* 178.921 <0.001*

Conductivity

Treatment 1 2.046 0.172 101.250 0.071 .260 0.617

Period 4 15.850 <0.001* 34.602 <0.001* 5.922 0.006*

Treatment * Period 10 .146 0.931 .212 0.842 .140 0.929

81

Table 4.8 Results of two-way ANOVA testing the difference in nutrient parameters in individual sites between treatment and among sampling

period. Nutrient parameters abbreviations: NH3 = ammoniacal nitrogen; TN = total nitrogen; P = phosphates; TP = total phosphorus.

Site 1 Site 2 Site3

Factor df F P F P F P

NH3

Treatment 1 .319 0.585 0.418 0.532 10.370 0.009*

Period 4 566.807 <0.001* 8.767 0.003* 69.962 <0.001*

Treatment * period 4 2.657 0.096 0.359 0.832 2.793 0.086

TN

Treatment 1 .900 0.365 0.138 0.718 3.716 0.083

Period 4 23.378 <0.001* 11.888 0.001* 26.244 <0.001*

Treatment * Period 10 3.020 0.071 2.372 0.122 2.880 0.080

P

Treatment 1 2.006 0.187 5.000 0.049* 96.729 <0.001*

Period 4 26.856 <0.001* 1167.700 <0.001* 214.377 <0.001*

Treatment * Period 10 8.256 0.003 9.500 0.002* 35.141 <0.001*

TP

Treatment 1 1.210 0.297 8.333 0.016* 34.416 <0.001*

Period 4 57.035 <0.001* 326.583 <0.001* 131.451 <0.001*

Treatment * Period 10 .416 0.794 4.583 0.023* 7.523 0.005*

Error 10

82

Table 4.9 The black carp experiment. Effect of black carp and apple snails on water quality.

Times 1-5 represented June 2012, September 2012, December 2012, March 2013 and June 2013

respectively. Data are means with SE in parentheses. Parameters abbreviations: NH3: ammonia;

TN: total nitrogen; P: phosphates; TP: total phosphorus; Temp: temperature; DO: dissolved

oxygen; Cond: conductivity; Turb: turbidity.

Treatment Site Control site

Para-

meters

Time 1 2 3 1 2 3

Chemical parameters

NH3

(mgL-1)

T1 0.035(0.013) 0.195 (0.01) 0 (0) 0.021 (0.01) 0.115 (0.01) 0.005 (0.01)

T2 1.45 (0.07) 1.15 (0.07) 0.745 (0.04) 1.3 (0.14) 1.35 (0.07) 0.585 (0.12)

T3 0 (0) 0.27 (0.38) 0.71 (0.11) 0 (0) 0.65 (0.92) 0.43 (0.14)

T4 0.025 (0.01) 0.22 (0.03) 0.215 (0.02) 0.06 (0) 0.215 (0.02) 0.205 (0.01)

T5 0 (0) 0.11 (0.16) 0.11 (0.03) 0.065 (0.02) 0.08 (0.09) 0.044 (0.02)

TN

(mgL-1)

T1 1.95 (0.49) 2.1 (0.28) 1.95 (0.01) 1.9 (0) 1.5 (0) 1.8 (0.42)

T2 2.2 (0) 2.1 (0.14) 1.7 (0) 2.25 (0.07) 2.2 (0.14) 1.65 (0.07)

T3 1.035 (0.23) 0.835 (0.37) 2.15 (0.07) 0.795 (0.26) 1.75 (0.92) 1.4 (0.42)

T4 1.55 (0.01) 2 (0.28) 1.45 (0.01) 1.5 (0.28) 1.9 (0.14) 1.5 (0)

T5 0.7555 (0.21) 0.635 (0.22) 0.595 (0.09) 1.52 (0.01) 0.615 (0.11) 0.645 (0.02)

P

(mgL-1)

T1 0.055 (0.01) 0.035 (0.01) 0.135 (0.01) 0.055 (0.01) 0.025 (0.01) 0.04 (0)

T2 0.04 (0.01) 0.025 (0.01) 0.105 (0.01) 0.055 (0.01) 0.03 (0) 0.125 (0.01)

T3 0.02 (0) 0 (0) 0.38 (0.04) 0.01 (0) 0.015 (0) 0.24 (0)

T4 0.05(0) 0.03 (0) 0.19 (0) 0.02 (0) 0.02 (0) 0.06 (0)

T5 0.043 (0) 0.2 (0) 0.0215 (0) 0.049 (0) 0.225 (0) 0.0565 (0)

TP

(mgL-1)

T1 0.18 (0.01) 0.11 (0) 0.35 (0.03) 0.19 (0) 0.1 (0.01) 0.26 (0.04)

T2 0.13 (0.06) 0.095 (0.01) 0.315 (0.04) 0.11 (0.01) 0.1 (0) 0.305 (0.04)

T3 0.02 (0) 0 (0) 0.345 (0.01) 0.01 (0) 0.015 (0.01) 0.235 (0.01)

T4 0.04 (0) 0.015 (0.01) 0.16 (0) 0.02 (0) 0.02 (0) 0.045 (0.01)

T5 0.045 (0.01) 0.15 (0.03) 0.018 (0.01) 0.038 (0) 0.195 (0.01) 0.0415 (0.01)

Physical parameters

Temp

(oC)

T1 27.07 (0.23) 23.53 (0.06) 30.03 (0.40) 26.77 (0.06) 23.50 (0) 29.90 (0.10)

T2 27.60 (0) 24.80 (0) 28.53 (0.06) 27.53 (0.06) 25.13 (0.06) 28.57 (0.12)

T3 18.50 (0) 16.97 (0.21) 20.23 (0.32) 18.57 (0.06) 18.70 (0) 20.37 (0.12)

T4 22.10 (0) 21.63 (0.06) 22.87 (0.12) 22.27 (0.06) 21.00 (0.10) 22.80 (0)

T5 27.07 (0.12) 25.60 (0) 28.50 (0) 27.60 (0) 25.80 (0) 29.00 (0)

DO

(mgL-1)

T1 5.9 (0.26) 5.72 (0.41) 4.60 (0.48) 5.92 (0.19) 6.33 (0.15) 0.18 (0.02)

T2 5.91 (0.09) 6.51 (0.06) 1.19 (0.30) 6.62 (0.04) 6.38 (0.21) 1.51 (0.09)

T3 5.97 (0.20)

6.446667 (0.01) 2.43 (0.08) 6.57 (0.12) 6.58 (0.11) 4.57 (0.22)

T4 5.9 (0.26) 5.72 (0.41) 3.97 (0.23) 5.92 (0.19) 6.33 (0.15)

2.056667 (0.10)

T5 1.64 (0.30) 3.61 (0.22) 2.87 (0.03) 3.77 (0.39) 2.85 (0.12) 3.59 (0.38)

pH

T1 6.78 (0.03 6.56 (0.05) 5.93 (0.03) 6.66 (0.07) 6.50 (0.03) 6.03 (0.15)

T2 6.68 (0.22) 6.72 (0.01) 7.58 (0.22) 6.43 (0.02) 7.29 (0.01) 6.22 (0.22)

T3 6 (0.22)

6.716667 (0.01) 7.58 (0.22) 6 (0.02) 7.29 (0.01) 6.22 (0.22)

T4 6.24 (0.22) 6.72 (0.01) 6.12 (0.29) 6.33 (0.02) 7.29 (0.01) 6.09 (0.17)

T5 5.76 (0.26) 5.71 (0.06) 5.25 (0.03) 5.34 (0.39) 5.7 (0.12) 5.49 (0.38)

83

Treatment Site Control site

Parame

ters

Time 1 2 3 1 2 3

Physical parameters (continued)

Cond

(µS/cm)

T1 107.33 (1.53) 122.67 (9.87) 127.00 (6.24) 119.00 (4.58) 116.00 (5.29) 166.67 (6.11) T2 34.00 (21.00) 72.67 (6.66) 147.67 (5.69) 60.67 (2.08) 169.67 (23.07) 80.00 (18.00) T3 n/a n/a n/a n/a n/a n/a

T4 109.00 (2.65) 113.00 (2.65) 127.00 (6.24) 119.00 (4.58) 117.33 (4.04) 166.67 (6.11) T5 140.00 (62.48) 143.67

(11.59) 165.00 (18.08)

151.00 (26.85)

154.00 (2.65) 138.67 (33.83)

Turb

(NTU)

T1 35.43 (7.15) 5.97 (1.03) 24.87 (0.70) 23.87 (1.50) 5.70 (0.25) 25.66 (1.89) T2 43.83 (4.01) 13.07 (0.38) 52.87 (1.42) 55.27 (1.31) 13.03 (0.49) 11.39 (0.88) T3 2.01 (0.19) 1.50 (0.04) 54.80 (0.44) 1.79 (0.06) 1.87 (0.26) 29.73 (5.60) T4 4.98 (1.50) 3.89 (0.18) 15.63 (0.45) 7.19 (0.14) 6.87 (0.60) 7.25 (0.15) T5 12.67 (0.32) 13.07 (0.15) 18.10 (0.46) 13.70 (0.10) 19.10 (0.46) 22.03 (0.40)

Salinity

(ppm)

T1 0 0 0 0 0 0 T2 0 0 0 0 0 0 T3 0 0 0 0 0 0 T4 0 0 0 0 0 0 T5 0 0 0 0 0 0

84

Table 4.10 Effect of fish and apple snails on plant abundance. Data are mean of eight quadrats with S.D. in parentheses. Time T1 and T2

represented June 2012 and June 2013 respectively.

Treatment Site Control site

Species Time 1 2 3 1 2 3

Panicum dichotomiflorum T1 15.00 (31.51) 0 (0) 0.63 (1.77) 11.88 (13.87) 0 (0) 1.25 (3.54) T2 31.25 (15.06) 0 (0) 19.38 (22.43) 4.38 (4.57) 5.00 (8.018) 6.88 (19.45)

Polygonum barbatum T1 0 (0) 4.50 (4.84) 0 (0) 0.63 (1.77) 2.13 (5.25) 7.5 (10.35) T2 3.50 (4.41) 11.00 (17.18) 0.75 (1.39) 2.50 (3.63) 1.63 (3.54) 2.75 (3.45)

Paspalum conjugatum T1 4.00 (4.24) 18.75 (32.81) 9.00 (8.07) 5.88 (2.27) 18.88 (17.73) 18.75 (13.82) T2 4.38 (8.63) 20.63 (23.37) 3.38 (6.95) 0 (0) 2.13 (4.02) 2.5 (4.63)

Panicum repens T1 0.625 (1.77) 0 (0) 0 (0) 1.50 (2.27) 0 (0) 0 (0) T2 0 (0) 0 (0) 0 (0) 5.63 (15.91) 0 (0) 0 (0)

Paspalum distichum T1 21.25 (21.67) 0.25 (0.71) 0 (0) 3.50 (5.55) 2.50 (4.63) 3.13 (3.72) T2 5.63 (6.23) 0 (0) 9.38 (15.22) 6.25 (11.88) 5.00 (14.14) 22.14 (29.13)

Cynodon dactylon T1 0.88 (1.81) 0 (0) 7.50 (13.88) 0 (0) 0 (0) 0 (0) T2 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

Polygonum lapathifolium T1 0.63 (1.77) 14.38 (27.96) 0 (0) 0 (0) 0 (0) 0 (0) T2 0.63 (1.77) 28.75 (40.16) 0 (0) 0 (0) 0 (0) 0 (0)

Polygonmum glabrum T1 1.00 (1.51) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) T2 0 (0) 0 (0) 0.63 (1.77) 0 (0) 0 (0) 0 (0)

Ipomoea aquatica T1 0.13 (0.35) 0 (0) 3.50 (5.15) 0 (0) 0 (0) 26.50 (31.25) T2 0.25 (0.46) 0 (0) 34.38 (43.71) 0 (0) 0 (0) 0.25 (0.71)

Brachiara mutica T1 0 (0) 0 (0) 7.50 (21.21) 0 (0) 0 (0) 0 (0) T2 0.25 (0.71) 0 (0) 0 (0) 0 (0) 0 (0) 1.25 (3.54)

Cyclosorus interruptus T1 0 (0) 0.63 (1.77) 0 (0) 0 (0) 10.00 (21.3809) 0 (0) T2 0 (0) 6.25 (17.68) 0 (0) 0 (0) 11.88 (18.50) 0 (0)

Aster subulatus T1 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) T2 0 (0) 0.50 (0.93) 0 (0) 0 (0) 0 (0) 0 (0)

Alternanthera philoxeroides T1 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1.50 (3.51) T2 0 (0) 1.25 (3.54) 0 (0) 0 (0) 0 (0) 0.25 (0.71)

Commenlina communis T1 0 (0) 0 (0) 3.13 (8.83) 0 (0) 0 (0) 0 (0) T2 0 (0) 2.50 (7.07) 0 (0) 0 (0) 0 (0) 0.50 (1.41)

Kyllinga aromatica T1 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 5.63 (15.91) T2 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.75 (1.75)

Echinochloa crusgalli T1 0 (0) 0 (0) 1.25 (3.53) 0 (0) 0 (0) 9.38 (11.78) T2 0 (0) 0 (0) 1.88 (5.30) 0 (0) 0 (0) 0 (0)

85

Fig. 4.1 Mean densities of apple snails in each plot from June 2012 to June 2013.

0

10

20

30

40

50

60 Treatment site 1

Control site 1M

ean d

ensity o

f g

old

en a

pple

snails

(P

om

acea c

analic

ula

ta)

in p

lots

(in

div

idual per

mete

r square

)

0

10

20

30

40

50

60 Treatment site 2

Control site 2

Sampling month

0

10

20

30

40

50

60

70

80

90

100

110

120

130Treatment site 3

Control site 3

Jun 2012 Sept 2012 Dec 2012 Mar 2013 Jun 2013

86

Fig. 4.2 Number of egg clutches encountered during walkover survey from June

2012 – June 2013.

0

25

50

75

100

125

150

175

200 Treatment site 1

Control site 1

Nu

mb

er

of

eg

g c

lutc

he

s e

nco

un

tere

d a

nd

me

asu

red

(in

div

.)

0

25

50

75

100

125

150

175

200 Treatment site 2

Control site 2

Sampling month

Jun-12 Sep-12 Dec-12 Mar-13 Jun-13

0

25

50

75

100

125

150

175Treatment site 3

Control site 3

87

Fig. 4.3 Abundance of apple snail hatchlings (individuals m-2

) from June 2012 to

June 2013.

0

10

20

30

40

50

Treatment site 1

Control site 1

Estim

ate

d n

um

ber

of

apple

sna

il h

atc

hlin

gs (

per

mete

r squ

are

)

0

10

20

30

40

50

Treatment site 2

Control site 2

Sampling month

Jun-12 Sep-12 Dec-12 Mar-13 Jun-13

0

10

20

30

40

50

60Treatment site 3

Control site 3

88

Fig. 4.4 Size-frequency distribution of Pomacea canaliculata in experimental and control plots from June 2012 to June 2013. Each bar

of the graph represented sum of total number of apple snail in 1.5m2.

<5

5-1

0

11

-15

16

-20

21

-25

26

-30

31

-35

36

-40

41

-45

46

-50

51

-55

56

-60

Fre

qu

en

cy o

f o

ccu

ren

ce

(in

div

idu

als

)

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

0

5

10

15

20

25

30

35

<5

5-1

0

11-1

5

16-2

0

21-2

5

26-3

0

31-3

5

36-4

0

41-4

5

46-5

0

51-5

5

56-6

0

0

5

10

15

20

25

30

35

Snail shell length (mm)

<5

5-1

0

11

-15

16

-20

21

-25

26

-30

31

-35

36

-40

41

-45

46

-50

51

-55

56

-60

<5

5-1

0

11-1

5

16-2

0

21-2

5

26-3

0

31-3

5

36-4

0

41-4

5

46-5

0

51-5

5

56-6

0

<5

5-1

0

11-1

5

16-2

0

21-2

5

26-3

0

31-3

5

36-4

0

41-4

5

46-5

0

51-5

5

56-6

0

0

5

10

15

20

25

30

35

40

45

50

0

5

10

15

20

25

30

35

40

45

50

0

5

10

15

20

25

30

35

40

45

50

0

5

10

15

20

25

30

35

40

45

50

<5

5-1

0

11-1

5

16-2

0

21-2

5

26-3

0

31-3

5

36-4

0

41-4

5

46-5

0

51-5

5

56-6

0

0

5

10

15

20

25

30

35

40

45

50

Treatment site 1 Control site 1 Treatment site 2 Control site 2 Treatment site 3 Control site 3

Jun-12 (59)

Sept-12 (2)

Dec-12 (1)

Mar-13 (1)

Jun-13 (3)

Jun-12 (54)

Sep-12 (24)

Dec-12 (23)

Mar-13 (38)

Jun-13 (10)

Jun-12 (87)

Sep-12 (5)

Dec-12 (6)

Mar-13 (11)Mar-13 (8)

Jun-13 (19)

Jun-12 (41)

Sep-12 (15)

Dec-12 (28)

Mar-13 (43)

Jun-13 (35)

Jun-12 (37)

Sep-12 (8)

Dec-12(40)

Mar-13(11)

Jun-13 (26)

Jun-12 (18)

Sep-12 (21)

Dec-12 (28)

Mar-13 (138)

Jun-13(71)

89

Fig. 4.5 Densities of three major species of non-target mollusks (individuals m-2

)

in June 2012 – June 2013.

M

ea

n a

bu

nd

an

ce

of M

ela

no

ide

s tub

erc

ula

ta

(in

div

idu

al pe

r m

ete

r sq

ua

re)

Me

an

ab

un

da

nce

of B

ithyn

ia s

p.

(in

div

idu

al pe

r m

ete

r sq

ua

re)

0

25

50

75

100

125

150 Treatment site1

Control site 1

Treatment site 2

Control site 2

Sampling month

Jun 2012 Sept 2012 Dec 2012 Mar 2013 Jun 2013

Me

an

ab

un

da

nce

of S

ino

taia

qu

ad

rata

(in

div

idu

al p

er

me

ter

sq

ua

re)

0

50

100

150

200

250

300 Treatment site 1

Control site 1

Treatment site 2

Control site 2

0

50

100

150

200

250

300 Treatment site 1

Control site 1

Treatment site 2

Control site 2

90

Fig. 4.6 Comparison of physical water quality parameters within June 2012 to

June 2013. Mean values of each data were shown with standard deviations as

error bars.

Jun 2012 Sept 2012 Dec 2012 Mar 2013 Jun 2013 Jun 2012 Sept 2012 Dec 2012 Mar 2013 Jun 2013 Jun 2012 Sept 2012 Dec 2012 Mar 2013 Jun 2013

Site 1 Site 2 Site 3

Tem

pera

ture

(degre

e c

els

ius)

0

10

20

30

Turb

idity (

NT

U)

0

10

20

30

40

50

60

Dis

solv

ed o

xygen (

mg p

er

L)

0

1

2

3

4

5

6

7

pH

0

1

2

3

4

5

6

7

8

Sampling period

91

Fig. 4.7 Comparison of chemical water quality parameters within June 2012 to

June 2013. Mean values of each data were shown with standard deviations as

error bars.

Site 1 Site 2 Site 3

0.0

0.3

0.6

0.9

1.2

1.5

0.0

0.1

0.2

0.3

0.4

0.0

0.5

1.0

1.5

2.0

2.5

Nutr

ient concentr

ations (

mg/L

)

0.0

0.1

0.2

0.3

0.4

Sampling month

Jun 2012 Sept 2012 Dec 2012 Mar 2013 Jun 2013 Jun 2012 Sept 2012 Dec 2012 Mar 2013 Jun 2013 Jun 2012 Sept 2012 Dec 2012 Mar 2013 Jun 2013

Ammonia nitrogen (NH3)

Total nitrogen (TN)

Phosphates (P)

Total phosphorus

(TP)

92

Fig. 4.8 2D nMDS plots showing impact of black carp and apple snail on

vegetation structure from initial (June 2012 – June 2013), using three sites as

replicates. Treatment abbreviation: T – Treatment sites; C – Control sites; whilst

for the number 1 and 2 followed corresponds to June 2012 and June 2013

respectively.

a) Site 1

b) Site 2

5

93

c) Site 3

94

Chapter 5 General summary and discussion

5.1 Summary of information on local invasion history and possible

indigenous predators of apple snails

Studies since the 1980s have showed that two species of apple snails (P.

canaliculata Lamarck and P. maculata) may have invaded Hong Kong wetlands

(Hayes et al. 2008; Hayes et al. 2012; Kwong et al. 2008; Lv et al.2013; Mochida

1991). They have first been reported to present in water channels, in which are

often organically enriched in Hong Kong. The high tolerance to physical stress

might have allowed these apple snails to adapt and breed in channels, as

numerous egg clutches are seen on various solid surfaces and on vegetation

during wet season (Ip pers. obs.). This inevitably adds propagule pressure in

facilitating its subsequent spread in the northern New Territories through the

interconnected water channels (Simberloff 2009). During a systematic survey

conducted in 2008 (Kwong et al. 2008), apple snails were found to be widespread

in New Territories and cause economic loss by devouring semi-aquatic

vegetables in farmlands. In non-agricultural wetlands, the presence of apple

snails resulted in dramatic changes to the lowland agricultural and non-

agricultural freshwater ecosystems. Apple snails graze native and exotic aquatic

macrophytes (Qiu and Kwong 2009; Wong et al. 2009; Wong et al. 2010).

Grazing could trigger excessive influx of nutrients from macrophytes into water,

and shifting succession pressure to favor growth of phytoplanktons (Carlsson et

al. 2004a; Fang et al. 2010). In addition, apple snail grazes selectively on more

nutritious aquatic plants with lower dry matter content (Wong et al. 2010).

Among them, some native macrophytes are preferred over exotic ones from

South America, the home region for apple snails. This finding supports the

95

notion that apple snails are not a safe bio-control agent without invasion risk

(Cowie, 2001). Apple snails are unique invasive species that have devoured

living aquatic plants, directly compete with other grazing macroinvertebrates

through more efficient feeding, resulting in a much higher secondary production

estimate than any other invertebrates recorded within the territory (Dudgeon

1999; Kwong et al. 2009). In short, apple snails possess multiple traits of a

superior competitor and a high impact invader (van der Valk 2012).

Current management of apple snails in Hong Kong is effective but labor

intensive and time-consuming. Control of apple snails in the agricultural area

relies on application of lime and natural molluscides with saponin. In non-

agricultural wetlands, managers mainly rely on hand-picking of adult snails and

detachment of their egg masses, but bio-control of gastropods using fish has

never been explored in local wetlands. A literature review and communication

with local wetland managers suggested there are no effective natural predators in

local waters. Ducks have been demonstrated to be effective predators of apple

snails in Malaysia (Teo, 2001), Philippines (Cagauan et al., 2000), Japan (Yusa et

al., 2006) and China (Liang et al., 2013). But duck farming industry (Carey et al.

2001; Kausar, 1983; Sin and Cheng, 1976; Tuen Mun and Yuen Long District

Planning Office, 2002) had stopped in two decades ago, and since 2006 it has

become illegal for individual residents to keep rearing ducks due to the issue of

public health as to Avian Flu infections (Department of Justice, HKSAR, 2013).

Various species of fish, especially carp, are known to be effective predators

of apple snails (Carlsson et al., 2004b; Halwart et al., 1998; Ichinose et al., 2002;

Liao et al., 2004; Teo, 2006; Wong et al., 2009; Yusa et al., 2006). However, no

96

study has been conducted to evaluate the application of black carp as a bio-

control agent in shallow water non-agricultural wetlands where non-target effects

on macrophytes and indigenous invertebrates might be of management concern.

Nor have previous studies considered the effects of season on apple snail

reproduction and development, and consequently on the effectiveness of the

biological control. We have conducted laboratory test and field implementation

trials to select indigenous fish as biological control agents against apple snails

and to detect possible side-effects of these fish on non-target wetland flora and

fauna.

5.2 Predatory potential of different local fish species on apple snails

An indoor experiment has been conducted in October 2011 in Hong Kong

Baptist University to screen out effective predators of apple snails. Three

indigenous freshwater fish species were selected due to their promising results

based on literature review. The experiment was conducted in separate aquaria

using one individual of each species of fish. Six aquaria of each fish species were

inspected after 48 hours. It aimed to compare the predatory potential of black

carp (Mylopharyngodon piceus), common carp (Cyprinus carpio) and white-

spotted catfish (Clarias fuscus). Both black and common carp are highly

effective predators of apple snails. Results from our aquarium experiment

suggested the two species of carp are more effective predators on apple snails

than catfish. With 100-140 individuals of apple snails offered ad libitum, an

average of 54.2% and 61.0% were predated by common carp and black carp

respectively. Both species are gape limited predators and snails were engulfed

and crushed by their pharyngeal teeth before being swallowed. Catfish crushed a

97

significantly lower amount of apple snails than both carp species, in which

86.1% on average survived.

5.3 Mesocosm studies on functional practicability of fish: testing efficacies

and subtle effects in a nutshell

Molluscivorous fish, especially carp, have been adopted as biological control

agents of golden apple snail, but previous studies have mainly focused on their

effectiveness, with little attention paid to their undesirable side effects on the

non-target plants and animals. To further explore fish as a bio-control agent of

apple snails, a mesocosom study was conducted using black carp

(Mylopharyngodon piceus). The aim of the study was to determine whether black

carp is as effective as common carp as a bio-control agent for apple snails, and

whether it will graze on macrophytes and prey on non-Pomacea snails. The

experimental setup was very similar with that in Wong et al. (2009), except using

two fish species, common carp and black carp in this study. The experimental

duration was also 2 months. Both species of carp preyed effectively on P.

canaliculata, removing almost all individuals that were small enough to fit into

their mouths. Of the 234 apple snails introduced into each of the enclosures, the

mean survival rate was as high as 93% in the control, but only 5% in the common

carp treatment and 2% in the black carp treatment. Apple snail mortality was

size-dependent, with smaller individuals (≤2.0 cm SW) suffering the most severe

mortality.

The effects of the two fish species on macrophytes were different. There

was a great increase in the total macrophyte biomass in the control, from 250 g in

the beginning to 542.6 g at the end. Macrophyte biomass in treatments with apple

98

snails or apple snails plus carp was remarkably lower than the control: only 30%

in the AS treatment, 32.5% in the AS+CC treatment and 56% in the AS+BC

treatment. This experiment proved the ability of black carp to reduce herbivory

on macrophytes through minimizing apple snail density. However, common carp

reduced apple snail density but did not result in a lower level of herbivory

because it also grazed on macrophytes. Non-target mollusk density was reduced

by both fish species.

5.4 Wetland scale application of biological control of apple snails using black

carp: efficacies and subtle effect to ecosystem

In order to demonstrate the practical efficacies of black carp against apple snails

and its subtle effects to organisms and ecosystem, a large-scale field experiment

was conducted during June 2012- June 2013 in three pieces of separated

freshwater wetland as replicates.

We found that black carp is a highly tolerant species that can survive in

shallow stagnant waters. This species can be used as a biological control agent of

apple snails as a predator of juvenile apple snails (SL: <5 – 25mm) in shallow

freshwater wetlands that are common in southern China. A major decline of

89.2% in apple snail overall densities have been recorded on average in three

experimental sites with treatment after one year. Juvenile snails would be

eradicated before they get to mature minimum size (male SL: 25.2 ± 3.3mm;

female: 29.8 ± 3.6mm (Estoy et al. 2002)) for reproduction. Given the longevity

of black carp, a low stocking density (80 - 89 individuals ha-1

) will be sufficient

to control apple snail populations, as adults die out. Additionally, black carp

culture in wetlands can provide commercial incentives as it is an edible fish. Yet

99

implementation of black carp to control apple snails should only be carried out in

habitats with low biodiversity of macro-invertebrates, especially mollusks.

Juvenile specimens (total length: at least approximately 300mm) are most

preferable option for release as they could reach a wider range of habitats

including shallow riparian area where snails aggregate, while still beholding their

high adaptability to adverse environmental conditions in shallow wetlands. Our

results also indicated that application of black carp to control apple snail

populations reduced phosphorus nutrients influx thus preventing aquatic

macrophyte community from shifting towards plankton-dominating state.

5.5 Implications on integrated pest management

A call for integrated pest management has prevailed in recent years (Peshin and

Dhawan 2009). Our study has implications on methods to eradicate pest snails in

shallow water wetlands, with much lower input of labor and management-

associated cost than hand-picking. Using fish as a single controlling agent is a

viable method in permanent and seasonal wetlands infested with apple snails,

where pre-treatment options like natural molluscicides application could not be

applied. To maximize treatment efficacies, it should be supplemented with

mechanical methods, including regular removal of adult snails and detachment of

egg clutches. Supplement methods above should be practiced in the riparian

region of wetland with very shallow water levels and dense vegetation that could

not allow fish access. In seasonality aspect, hand-picking should be conducted

most frequently during wet season (April – September annually) where plenty of

shallow pools generate as the ponds are in spate with rainwater.

100

Appendices

Plate 1Site photos and general growth condition of three species of macrophytes

at the end of 8-week mesocosm experiment. A: Blocks of enclosures at

experimental site; B: Enclosures with meshed nylon cover; C: Control enclosure;

D: Enclosure with AS treatment; E: Enclosure with AS+BC treatment; F:

Enclosure with AS+CC treatment.

101

Plate 2 Photos showing the three sites used as replicates in large-scaled field

implementation experiment in Kamtin, New Territories.

Control plot 1 Experimental plot 1

Control plot 2 Experimental plot 2

Control plot 3 Experimental plot 3

102

Plate 3 Photos showing a list of common benthic macro-invertebrates found in

samples.

Sinotaia quadrata

Melanoides tuberculata

Class Aeshnidae

Darner dragonfly

Orthetrum sabina

Green Skimmer

Ischnura senegalensis

Common Bluetail

9mm

17mm 16mm

Bithynia sp.

103

Pantala flavescens

Wandering Glider

Family Pleidae

Pygmy backswimmer

Family Naucoridae

Creeping waterbug

Family Notonectidae

Backswimmer

Family Palemonidae

Shrimp

Family Corixidae

Water Boatmen

104

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Curriculum Vitae

Academic qualifications of the thesis author, Mr.IP Ka Lok Kelvin:

Received the degree of Bachelor of Science in Environmental

Life Science (Honors, second class division 2) from the

University of Hong Kong, August 2009.

Research experiences:

2013/April: Attended the 18th

International Conference on

Aquatic Invasive Species (ICAIS), organized by Invasive

Species Centre in Canada – Oral presentation.

2008-2009: BSc final year project at University of Hong Kong

on “Fruit and Seed Resources for birds in Long Valley, Hong

Kong” with Dr. Hau Chi Hang Billy.

2008/August: Summer Research Assistant, Division of

Ecology and Biodiversity, University of Hong Kong.

2008/June-July: Voluntary work in assisting ground survey

with Forest Wardens in Yinggeling Nature Reserve Hainan,

supervised by Dr. Chan Pui Lok, Bosco.

October2013