A SURVEY OF ECTOPARASITES OF SMALL MAMMALS ON MOUNT SERAPI, KUBAR NATIONAL PARK, SARAWAK

Hanim Syuhada Mahyudin

Bachelor of Science with HonoursQ1. (Animal Resource Science and Management)757

20078239 2007

HANIM SYUHADA MAHYUDIN

This project is submitted in partial fulfillment of the requirement for the Degree of Bachelor of Science with Honours

(Animal Resource Science and Management Programme)

t'usat Khidmat Maklumat Akaden UNlVERsm MALAYSIA SARAWII

Q4300 KOla SamarabaD

A SURVEY OF ECTOPARASIl'ES OF SMALL MAMMALS ON MOUNT SERAPI, KUBAH NATIONAL PARK,

SARAWAK

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARA W AK

2007

I

DECLARATION

No portion of the work referred to in this dissertation has been submitted in support of an application for another degree of qualification of this or any other university or institution of higher learning.

Hanim Syuhada Binti Mahyudin

Programme of Animal Resource Science and Management Faculty ofRe ource Science and Technology

niversiti Malaysia Sarawak

ACKNOWLEDGEMENTS

Fir t of all, I would like to express my appreciation and gratitude to Prof. Dr. Fatimah Bt. Haji

Abang who has given much advice, support and guidance throughout this study and critized this

paper in draft. I also wish to sincerely thank to my co-supervisor, Assoc. Prof. Dr. Andrew Alek

Tuen, the Head of Department of Zoology, Assoc. Prof. Dr. Mohd Tajuddin B. Abdullah and my

lecturer, Mr. Azlan layasilan B. Gulam Azad for guiding me while doing the field work as well as

for making the field study at Kubah National Park possible.

Th i appreciation is also forwarded to Sarawak Forestry Corporation for allowing me to conduct

th i tudy at Kubah National Park (NPW.907.4.2. (I)-55). Not forget to the wardens of Kubah

National Park, Mr. Mohyiddin and Miss Suziani as well as their staff for concerning on our safety

while taying in the national park.

Special appreciation is also forwarded to Mr. Wahap B. Marni, Mr. Mohamad lalani B. Mortada,

Mr. Besar B. Ketol, Mr. Huzal Irwan B. Husin and Miss Ratnawati Bt. Hazali for their guidance and

assistance during field sampling and support in completing this report.

Last but not least, many thanks to my fellow friends and coursemates notably Siti Hasmah Bt. Taha

and Mohd. Ridwan B. Tahir for their co-operation and helping hands during sampling in the field as

well as teaching me the right way of handling small wild mammals.

TABLE OF CONTENTS

Acknowledgements

Table of Contents

List of Tables

List of Figures

List of Appendices

Abstract

1.0 Introduction

2.0 Literature Review

2.1 Ectoparasitic Insects

2.2 Ectoparasitic Ticks and Mites

3.0 Materials and Methods

3.1 Study Site

3.2 Field Sampling

3.2.1 Ectoparasite Collection and Preservation

3.3 ~Iide Preparation

3.3.1 Blood Clearing

3.3.2 Neutralization

3.3.3 Spreading and Hardening

3.3.4 Alcohol Dissolving

3.3.5 Mounting

3.3.6 Labeling

ii

I'usat Khidmat Maktumat Akaaemtll UNIVERSITI MALAYSIA SARAWA'J

QA':\()() KOla Samaral'lan

Pages

1\

IV

V

VII

VIII

5

5

8

10

10

II

12

13

13

14

14

14

14

15

,.....

3.3.7 Identification 15

4.0 Results and Discussion 16

4.1 Composition of Ectoparasites of Small Mammals on 16

Mount Serapi

4.2 Ectoparasitic Insects 18

4.2.1 Streblidae (bat flies) 18

4.2.2 Nycteribiidae (spider-like bat flies) 20

4.2.3 Pygiopsyllidae (fleas) 24

4.3 Ectoparasitic Ticks and Mites 26

4.3.1 Ticks 26

4.3.2 Mesostigmatid Mites 29

5.0 Conclusions and Recommendation 33

6.0 References 34

Appendices

III

I

,.. ,..

Table I

Table 2

Table 3

LIST OF TABLES

Checking time for small mammals based on cage trap,

mist net and harp trap in Kubah National Park.

12

Ectoparasitic infestation rates on small mammals in

Kubah National Park, Sarawak (27 Aug-I Sept 2006

and I Dec-6 Dec 2006).

17

The number of ectoparasite fauna found on small

mammals in Kubah National Park, Sarawak (27 Aug­

1 Sept 2006 and I Dec-6 Dec 2006).

27

iv

..... I

LIST OF FIGURES

~igure 1 Locality map of Mount Serapi, Kubah National Park 10

(Anon, 2006a).

-=-igure 2 Labelling of the slide. 15

~igure 3a Female individual of Megastrebla spp. 20

Figure 3b Thoracic segment of Megastrebla spp. 20

~igure 4a Dorsal view of head and wings of Raymondia spp. 20

~igure 4b Closer view of head of Raymondia spp. 20

!Figure 5a Female individual of Stre I. 20

f igure 5b Thoracic segment of Stre I. 20

!Figure 6a Dorsal view of Basilia spp. 22

tf' igure 6b Closer view of head of Basilia spp. 22

~igure 7a Dorsal view of Eucampsipoda sundaicum. 23

Figure 7b Closer view of head of E. sundaicum. 23

Wigure 8a Dorsal view ofNyct I. 23

f igure 8b C;loser view of head ofNyct I. 23

f igure 9a Dorsal view ofNyct 2. 23

Figure 9b Closer view of head ofNyct 2. 23

Figure lOa Dorsal view ofNyct 3. 24

Figure lOb Clo er view of head ofNyct 3. 24

Figure Ita Dor al view ofNyct 4. 24

~f" re lIb Clo er view of head ofNyct 4. 24

v

l

12a

12b

13a

13b

14

15a

Dorsal view of Nyct 5. 24

Closer view of head ofNyct 5. 24

Male individual of L. vomerus. 26

Female individual of L.vomerus. 26

Dorsal view of Haemaphysalis spp. 29

Dorsal view of Der 1. 31

Dorsal view of Lae 1. 31

Dorsal view of spinturnicid mite. 31

VI

I

r

LIST OF APPENDICES

~pendix I Preparation of potassium hydrochloride J0% (KOH).

~pendix II Preparation of hydrochloric acid 10% (Hel).

vii

A Survey of Ectoparasites of Small Mammals on Mount Serapi, Kubah National Park, Sarawak

Hanim Syuhada Binti Mahyudin

Animal Resource Science and Management Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

survey on ectoparasites of small mammals was carried out on Mount Serapi, Kubah National Park from August to tember 2006. A total of 401 individuals comprising 37 species of small mammals were captured. Of these, 20.7% of small mammmais (83 individuals) were found to be infested by a total of 418 individuals comprising 18

rphospecies of ectoparasites, 54% (226 individuals) of which were insects and 46% were ticks and mites (192 ividuals). The mo t highly infested small mammal was the Penthelor lucasi of Chiroptera with 44.1 % infestation by nycteribiid flies. Among the species of ectoparasites collected, ticks from the genus Dermacenlur could pose ntial health risks to visitors of Kubah National Park because ticks of this genus had been reported as vectors of ~an virus in Malay ia. All families of ectoparasites except the Spinturnicidae (Mesostigmata) and Pygiopsyllidae

iphonaptera) were collected at both elevations (119 m a.s.1. and 787 m a.s.I.). Spinturnicidae and Pygiopsyllidae were Iy collected from the upper elevation. Host-ectoparasite association is further discussed.

eywords: ectoparasites, small mammals, Kubah National Park.

TRAK

u /cajian mengenai ekJoparasil daripada mamalia kecillelah dijalankan di Gunllng Serapi, Taman Negara Kubah i Ogos hingga &ptember 2006. Sejwnlah 401 individu yang lerdiri daripada 37 species mamalia kecil lelah

ong/cap. 20.7% (83 individu) daripada mamalia kedl tersebul dipenuhi oleh sejllmlah 418 individll yang lerdiri ipoda 18 morfospecies ekJoparasit, 54% (226 individll) adalah ekloparasit serangga dan 46% adalah sengkenil dan

ma (192 individu). Mamalia kecil yang paling ban yak dijangkili adalah Penlhelorlucasi daripada order Chimplera gan /cadar infestasi 44.1% oleh Nycleribiidae (Diplera). Anlara spesies-spesies ekloparasit yang dijumpai, sengkenil i genus Dermacentor mungkin mempunyai kepenlingan kesihatan kepada para pengunjllng di Taman Negara Kubah una spesies sengkenit daripada genus ini lelah dilapur/can sebagai veklor kepada virus Lanjan di Malaysia. 'Semlla famili ektopara il kecuali Spinlurnicidae (Mesosligmala) dan Pygiopsyl/idae (Siphonap/era) dijllmpai di dua-dua ketinggian (1 19 m a.s./. and 787 m a.s.l.). Spinturnicidae dan Pygiopsy/lidae hanya dijumpai pada ;nggian a/as sahaja. Hllbungan anlara pemmah dan ekJoparasil dibincangkan dengan lebih lanjul di dalm /cajian

kunci: ekJoparasit, mamalia kecil, Taman Negara Kubah

Vltt

Introduction

Ectoparasitic arthropod can be defined as those families or higher taxa of arthropods whose

members spend much of their lives closely associated with the dermecos (the habitat created

by the skin and its outgrowths in mammlas and birds) (Marshall , 1976). They posses

obvious adaptations to this habitat and derive their food from the host. They are also

considered as animals that live at the expense of other animals (hosts) that they do not kill

(the host is being parasitized) because that relationship is one sided (Gullan and Cranston,

1994). Even though the host is harmed, they are not immediately killed by the ectoparasites

(Elzinga, 2000).

Ectoparasite are host specific which require a specific species or genus of host to complete

their life cycle (specificity for particular taxa) (Gannon and Willig, 1995). They are most

likely to be entirely dependent on their host and among the members of this group, some

remain on the host throughout their life cycle, some live on the host only during particular

stage of life cycle or otherwise free-living (Romoser and Stoffolano, 1994). Still, there are

some ectoparasites that remain in the host's roost only and feed during periods of host

inactivity ,(Gannon and Willig, 1995), some only occuring on a single hosts species whereas

others have a wide range of suitable hosts (Marshall , 1987). They tend to be specialized

because living intimately with a host requires special adaptations that suit it only to one or a

narrow range of hosts (Price, 1984).

Of the 26 orders of Insecta, only eight orders are known as ectoparasitic insects; Mallophaga

(43%), Anoplura (8%), Siphonaptera (34%), Diptera (11%), Hemiptera (2%), Coleoptera

II

,...

(I %), and less than I % of Dermaptera and Lepidoptera (Marshall, 1981; Cheng, 1986).

According to Marshall (1987), there are 6000 known species of ectoparasitic insects with

60% of them associated with mammals and the rest associated with birds. These

ectoparasitic insects spend much of their adult lives in close association with the habitat

created by the skin and its outgrowth of mammals and birds (vertebrates), or with the host's

nest and roost (Marshall, 1987). According to Gullan and Cranston (1994), about 25% of

insect species are parasitic in feeding habit in some life-history stages. They live and feed on

external surfaces and provide no net benefit to their host (Gannon and Willig, 1995). There

are two types of ectoparasites based on their feeding behavior; the one that derive all or part

oftheir sustenance from the host externally (Romoser and Stoffolano, 1994) by invading the

tissue or feeding through the outer of the host by ingesting cells or blood seepage with their

chewing mouthparts and the one that penetrate and take blood directly from blood vessels

(solenophages) or lacerate blood vessels and feed from the resulting blood pool, namely

telmophage (Elzinga, 2000). The relationship between ectoparasitic insects and their hosts

are complex because of their strictly limited number of natural hosts. Their feeding and

reproduction are not really successful if ther are placed upon other hosts experimentally

(Marshal.! , 1987).

Acarina is the most heterogeneous of the eleven or so living subclass of the Arachnida

(Macfarlane, 1986). This subclass comprised of seven orders; Notostigmata, Holothyrida,

Ixodida, Mesostigmata, Prostigmata, Astigmata and Oribatida (Evans, 1992). Of these,

Ixodida and Mesostigmata are known to be parasites (either ecto- or endo-) of vertebrates

(reptiles, birds, mammals and amphibians) as well as few invertebrates (Woolley, 1988) and

2

are known as the parasitic acarines. According to Evans (1992), four main categories of

feeding habits in the Acarina can be classified as zoophagy (camivory), phytophagy

(herbivory), omnivory and saprophagy (detritivory). Arlian and Vyszenski-Moher (1987)

stated that parasitic acarines are no different from other arachnid parasites as they exhibit a

wide range of interactions with their hosts in which feeding is ultimately accomplished.

Studies and information on ectoparasites of small mammals pertaining to West Malaysia is

abundant (Leong and Marshall, 1968). However, not much published data were available for

Sarawak as well as Sabah. Therefore, this study is crucial to know the faunistic composition

of ectoparasites in Kubah National Park as this area is known as a recreation and tourism

spot. Certain species of ectoparasites are globally known because of their publ ic health

importance particularly the ticks and mites.

In this study, the ectoparasite fauna of small mammals on Mount Serapi, Kubah National

Park were examined. Three orders of mammals were chosen, which are Chiroptera (bats),

Rodentia (rats and squirrels) and Scadentia (treeshrews) to represent the small wild

mammals. They were chosen because any assemblage of mammal species whose individual

live weights do not exceed 5 kg when adult is considered as small mammals (Hayward and

Phillipson, 1979).

The primary objective of this research was to study the host associations of ectoparasites

from small mammals on Mount Serapi, Kubah National Park. The other two objectives were

to determine whether each species of ectoparasite is associated with a group of hosts that are

3

closely related or have similar bionomics (host-specificity) and to examine whether the

presence of ectoparasites in Kubah National Park have any potential health risks to the

visitors.

4

,...

Literatu re Review

Ectoparasites that commonly infest small wild mammals are included mostly in the Ixodida

and Mesostigrnata of the Acari subclass as well as a few orders of Insecta (e.g. Dermaptera,

Hemiptera, Diptera, Siphonaptera, etc) (Allen, 1939; Marshall, 1981; Nava et ai., 2003;

Nieri-Bastos et al., 2004). Rodents are considered as one of the most important hosts due to

the fact that, taxonomically this group includes the largest number of species (Nieri-Bastos

el al., 2004). Finally yet importantly, Chiroptera or bats are also considered as preferential

hosts and is second in the volume after rodents.

Ectoparasitic Insects

Anoplura with fewer than 500 species are ectoparasites of mammals mainly rodents

(Marshall, 1981; Roberts and Janovy, 2000). According to Marshall (1981), generally they

are highly ho t-specific but they may be transferred to unusual hosts in a mixed-species

flock at communal roosting or nesting sites or burrows, through the activities of helpers

during hybridization, or from prey to predator.

Hemip~era is one of the largest insect orders but only about 100 species are ectoparasitic

upon mammals (Roberts and Janovy, 2000). All of these ectoparasitic bugs belong to the

family Cimicidae and Polyctenidae (Marshall, 1981). Heteroptera, the suborder of

Hemiptera are known to be able to feed upon vertebrate blood as well as a few Reduviidae.

Fifty-nine out of eighty-nine species of Cimicidae are associated with bats, particularly

Vespertilionidae and Molossidae whereas 32 species of Polyctenidae are all exclusively

ectoparasitic upon microchiropteran bats. Dermaptera consists of very few parasitic species

5

because most of the members are hemimetabolous or free-living earwigs, feeding upon a

wide variety of animal and plant material (Marshall , 1977; Roberts & Janovy, 2000). Of

1000 described species, only 16 species of two families ; Arixeniidae and Hemimeridae are

known to be ectoparasitic (Marshall, 1981). The former are known only from the roosts of

the Molossidae bats in the Oriental Region whereas the latter are found only in association

with murine rodents in Ethiopian Region. According to Marshall (1981), ectoparasitic

dermapterans are all narrowly host-specific because all of the five species of Arixeniidae

were monoxenous (only one host species), being associated only with the hairless molossids

bat. Besides that their principal diet is some product of the glandular skin of that bat.

Marshall (1977) in a study of Dermaptera ofNiah Caves, Sarawak found that Arixenia esau

is primarily associated with Cheiromeles torquatus only.

Five families of Diptera; Carnidae, Mystacinobiidae, Hippoboscidae, Nycteribiidae and

Streblidae are ectoparasitic (Marshall, 1981). Of these, only Nycteribiidae and Streblidae are

all exclusively ectoparasitic upon bats. The former are all wingless with a curious spidery

appearance in the flattened body, minute head which is folds back into a groove on the upper

side of ~he thorax and wide-spreading legs whereas the latter are winged, though in some

species the wings are small and remnants with head is not bent backwards upon the thorax

(Allen, 1939). They are grouped together with Hippoboscidae in ' Pupipara' due to their

method of reproduction (Marshall, 1981). There are 256 species in 12 genera and three

subfamilies of nycteribiids and they appear to be host specific (Marshall, 1981). The same

species of nycteribiids is usually not found on both fruit- and insect-eating bats. The

treblidae contains 221 species in 31 genera and five subfamilies and like nycteribiids, they

6

are also host specific (Marshall, 1981). These ectoparasites spend their entire lives either on

the body or in the roosts of their hosts and survive in a comparatively warm, moist and

equable environment because of a large tropical distribution of bats (Marshall, 1981). Lim

(1973), in his study on parasitic infestation of bats in Gunung Brinchang at Pahang captured

a total of 18 species of chiropterans represented by 741 indivliduals in five families;

Pteropodidae, Emballonuridae, Megadermatidae, Rhinolophidae, Hipposideridae and

Verpertilionidae. Of these, only bats of the family Pteropodidae, Rhinolophidae,

Hipposideridae and Vespertilionidae were infested with both streblids and nycteribiids,

whereas bats of the family Emballonuridae and Megadermatidae were infested with streb'lids

only.

Siphonaptera or fleas are largely parasites of rodents and about 74% of known forms had

been recorded from this order of mammals (Marshall, 1981). Some bats seem on the whole

rather free of them eventhough no less than 19 genera of fleas are listed from bats (Allen,

1939). According to Bittercourt and Rocha (2002), some members of fleas living on rodent

hosts show a preference for particular sites on the host body, and that some ectoparasite

speci~s may overlap, largely on sites of difficult access to the host which increases the

chance of their occurrences there. Other than that, even though they have preferred hosts,

they also can transfer from one of their hosts to another or to a host of a different species

meaning that they are not very host specific (Roberts and Janovy, 2000). They are capable of

detecting their hosts from a distance not exceeding a few centimeters and attracted to hosts

primarily by their sense of smell, which is sufficiently acute to enable them to distinguish

between different species of vertebrates (Cheng, 1986). The distribution of fleas is limited

7

by the distribution of their main hosts (Kotti et ai., 2001). Malul (1987), in his taxonomic

study on fleas of Tambunan, Sabah found a total of 94 specimens of fleas comprising four

families; Pulicidae, Pygiosyllidae, Ceratophyllidae and Leptosyllidae. Most of these fleas

were not found in these area host specific except for Gryphopsylla mjoebergi which was

collected only from Tupaia montana.

Ectoparasitic Ticks and Mites

The Acari which comprises of mites and ticks forms one of the largest and most biologically

diverse groups of the Arachnida. They are worldwide in distribution and can be found in

habitats where moisture is more abundant (Woolley, 1988).

Ticks (Ixodida) consist of three families; Argasidae, Ixodidae and Nuttalliellidae (Cheng,

1986). A large number of them have developed intimate associations with other animals

from commensalism to parasitism and are obligate external parasites of a wide range of

vertebrate hosts including man and domesticated animals (Evans, 1992). Ticks are vectors

of a wide range of disease organisms than any other group of arthropods which trasmit the

pathogenic organisms directly (during attachment and feeding) or indirectly (by contact with

tick coxal gland fluid and faeces) (Macfarlane, 1986; Evans, 1992). All ticks undergo four

basic stages in their life cycles; egg, larva, nymph and adult (Roberts and Janovy, 2000).

They take only one meal of blood per instar (larva, nymph and adult) either on the same

host, on two different hosts (larva and nymph remaining attached to the same host, adult on

a different host) or on a maximum of three hosts during their I ife cycle (Evans, 1992). Some

8

ticks are rather host specific, but most are opportunists that will feed on a variety of hosts

(Roberts and Janovy, 2000).

The mesostigmatid mites include the free-living as well as symbiotic mites. Of these, about

250 species are para itk on vertebrates and invertebrates and are of considerable economic

importance (Cheng, 1986; Woolley, 1988). Their association with vertebrates has notably

diverse host relationships, but nearly all are in one superfamily, the Dermanyssoidea

(Radovsky, 1994). Some of them remain on the host throughout their life cycle or spend part

of their life cycle in the nest, roost, or other dwelling. Even so, they still can be found on the

host (Radovsky, 1994). Most of the Dermanyssoidea show little or no preference for the

type of prey and feeding on any small animals that they are able to overcome and locate

their prey by random contact or by chemical cues produced by the prey itself (Evans, 1992).

Like those of the ticks, the four stages in the life history of the mites include the larva,

nymph and adult forms (Woolley, 1988). Unlike ticks, the larva of mesostigmatid mites is a

non-feeding stage because they have weak sclerotized mouthparts (Radovsky, 1994).

MarianI!.. et. ai., (2005), in their study of ectoparasites in Gunung Stong Forest Reserve at

Kelantan screened a total of 272 hosts comprising of 12 species of birds, 21 species of bats,

7 species of rodents and 2 species of insects for the presence of ectoparasites. From their

examination, 5 species in 4 genera of ticks, 7 species in 2 families of mesostigmatid mites

and 5 species of chiggers were collected. Among the ectoparasites found were Ixodes

granulatus and Leptotrombidium deliense, which are of known medical importance as they

are potential vectors of diseases.

9

tady Site

·

in 1995. The park covers an

terials and Methods

study was conducted at Mount Serapi of Kubah National Park, Sarawak (Figure 1)

is situated 22 kilometers away from Kuching city. Kubah National Park is one of

Sarawak's most accessible national parks and was gazetted in 1989 and opened to the public

area of 2,230 hectares which is dominated by a sandstone

plateau and comprises of heavily forested slopes and ridges of the Serapi range. This plateau

pDDCtuated with bands of hardened limestone which have created a number of beautiful

erfaIls at the heights of between 150 and 450 meters (Anon, 2006b).

a· ...... n.II •• W...... 1nII 6. 7 •• = ... _ ... Wildlife T...II• .......n.II ..... . .,..n.II

__ IC__ NAT_AI. PAN<-......"--­--- ­.......................... _..--.-­_..--.-.. ­•• --..1nII

Figure 1: Locality map of Mount Serapi, Kubah National Park (Anon, 2006a).

10

the whole day and were checked during the checking time of the mist nets

Checking time for small mammals based on cage trap, mist net and harp trap in Park.

Checking time

6.30am - 8.30am 4.30pm - 6.30pm 7.30pm - 8.30pm

Rodents, Scadents Rodents, Scadents

Chiropterans Chiropterans

Chiropterans Chiropterans

oparasite Collection and Preservation

of the small mammals captured were not ki lied except for those to be used for

lecular techniques. Upon capture, each small mammal was held in a separate

plastic or cloth bag before examination for ectoparasites (Gannon and Willig, 1995)

many of them are host specific (Upton, 1991) and to avoid contamination between

(Bittercourt and Rocha, 2002). First of all , the small mammal was identified , so

the host for each ectoparasite is known. Then, it was handled by two peoples; the

that held the animal and the other one combed and collected the ectoparasites

eng forceps. Ectoparasites inspection was done as soon as possible to avoid the

I mammals from getting stress (Gannon and Willig, 1995) as ectoparasites

escape from their stressful host (Marshall, 1981). Ectoparasites of dead animal

"II drop off and those that remain attached was searched (Upton, 1991). Each body

e region was checked and the ectoparasites were removed either by combing

12

(Bittercourt and Rocha, 2002) the hair over a large sheet of paper (Upton, 1991) or

using forceps. For small mammals such as rodents, collecting ectoparasites using

comb was not really helpful because of their spiny hairs (pers. obs.).

After the removal of ectoparasites from the small mammal, they were placed in a

small, individual vial and preserved in 70% ethanol (Gannon and Willig, 1995;

Nieri-Bastos et ai., 2004; Bittercourt and Rocha, 2002). The vial containing

ectoparasites was labelled with information on the species. The species, location, sex

and date were al 0 recorded for each captured small mammal before released and the

collected ectopara ites were brought back to the laboratory for further identification.

Preparation

following slide preparation technique is based on Lewis (1982):

Blood Clearing

After preserved in ethanol 70%, the ectoparasite was soaked in a solution of

potassium hydroxide 10% (KOH) overnight or up to more than 24 hours depending

~:m the size of tbe ectoparasite. The KOH is used to make the exoskeleton more

transparent and destroys internal tissues (Marshall, 1981). The preparation of KOH

10% is shown in Appendix I. After that, the abdomen of the ectoparasite was pierced

with fine needle to allow the penetration of KOH. Once it was cleared, the

ectoparasite was taken out of the KOH.

13

Neutralization

The ectoparasite was put in a petri dish containing hydrochloric acid 10% (HCL) for

an hour to neutralize the KOH or stop the clearing process. The preparation of HCL

I()OAt is shown in Appendix 2. In neutral condition, ectoparasite can be stored in HCL

without any damage.

Spreading and Hardening

The ectoparasite was placed at the center of the slide. The remaining HCL on the

ectoparasite was dried out. The legs and appendages of ectoparasite were spread

carefully to avoid them from overlapping so that the morphological characteristics

could be observed. The ectoparasite was then covered with coverslip and several

drops of absolute ethanol were put onto the ectoparasite to harden the body.

Alcohol Dissolving

Ectoparasite with absolute alcohol could not be mounted along with mounting

media; Canada balsam. So, a solution of equal volume of xylene and absolute

alcohol was used to dissolve the absolute alcohol. The ectoparasite was soaked in the

solution for a while before soaking in the solution of absolute xylene for about an

hour.

Mounting

The slide and coverslip were cleaned with alcohol 70%. Then, a sheet of plain paper

was marked with a symbol 'X' so as to guide the central placement of ectoparasite

on the middle of the slide. Excess xylene was absorbed using tissue paper. Canada

balsam was dropped onto the ectoparasite and would be aborted underneath the

14