ENDOHELMINTH PARASITES DIVERSITY OF ANURAN IN UNIVERSITI MALAYSIA SARAWAK (UNIMAS) CAMPUS
Farliana Zulea Binti Zulkifli
QL 757
Bachelor of Science with Honours F231 (Animal Resource Science and Management)2011
2011UNIVERSITI MALAYSIA SARAW AK
THESIS STATUS ENDORSEMENT FORM
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11111 IlIIliiliilll II II III
1000217506
ENDOHELMINTH PARASITES DIVERSITY OF ANURAN IN UNIVERSITI MALAYSIA SARA WAK (UNIMAS) CAMPUS
FARLIANA ZULEA BINTI ZULKIFLI (20951)
This project is submitled in partial fu lfillment ofthe requirement for the degree of Bachelor of Science with Honours
(Animal Resource Science and Management Programme)
Faculty of Resource Science and Technology UNIVERSITl MALA YSIA SARAWAK
2011
Acknowledgement
First and above all, praise is due to almighty Allah S. W.T. for the bless ing and opportunity for me to fillish that my final year project. Sincere thanks to Universiti Malaysia Sarawak and Zoology Department for given me an opportunity to be part of them in the last three years.
My special thanks goes to my helpfi.11 supervisor and co-supervisor; Dr. Ramlah Zainudin and Mdm. Nor Aza Ahmad for guidance, encouragement, and support during the deve lopment of this research. Their advice and suppOl1 completely make my final year report was completed. I gratefully acknowledge Prof Dr. Sulaiman bin Hanapi for his suggestions and advices on writing this thesis. Thank you very much and great appreciation goes to Zoology Department especially Mr. Wahap Marni, Mr. Isa Sait, Mr.
Huzal Irwan Husin and Mr. Nasron Ahmad for their willingne ss to assist and their advices during the field sampling. [ would also like to thank my seniors and course mate especially Elvy Quatrin Deka, Muhd. Fadzil, Sarina Mat Yasin and Mattew Anak Jenang for their help and support through sampling period. Those experiences will be treasured and thank you.
Lastly, I wou ld like to express my deepest gratitude to my father, Mr. Zulkifli Muhammad, my mother Mrs. Fatimah Ngah, my siblings and my closest tTiends who have been motivate and support me in walking through difficult path of time in
help and suppo rt from all person who were directly or indirectly involved in this project.
Thank you.
ii
DEC LARA nON
I hereby declare that no portion of 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.
Farliana Zulea Binti Zulkifli
Animal Resource Science and Management Programme Department of Zoo logy
Facu lty of Resource Science and Technology Universiti Malaysia Sarawak
Table of Contents
Acknowledgement. ... ... ... .
Declaration ... ... . III
Table of Contents ... . IV
List of Abbreviations ... ... ... . viii
List ofTab[es ... ... ... ... . IX List of Figures ... . X Abstract. ... ... ... ... .. ... . XIII 1.0 Introduction ... . 2.0 Literature Review ... . 5
2. [ Anuran at Peat Swamp Forest. ... 5
2.2 T8:<onolllY and Morpho[ogy of Endohe[lllinth Pmasites ... . 6
2.3 Eco[ogy orHosl-Parasilc [nteracliom ... . 7
3.0 Materia[s and Methods... ... ... . [2
3. [ Study Site ... . 12
3.2 Anuran Collection ... ... ... . [4
3.3 Laboratory Work ... . [6 iv
3.3. 1 Anuran Dissection ... ... ... . 16 3.3.2 Slide Preparation: Preservation, Staining and Mounting
the Endohelminth Specimens ... .. 17
3.3.2.1 Class Nematoda ... .. 17
3.3.2.2 Class Cestoda and Trematoda ... 18
3.3.3 Identification ofEndohelrninth Specimens. ... ... 21
3.4 DataAnalysis... ... ... ... 22
3.4. 1 Descriptive Analysis .. 22
3.4.2 Diversity index .... ... ... ... .. 22
3.4.3 S i m i lar it y index ... ... ... .. .. 23
3.4.4 Non-metric Multidimensional Scaling (NMOS) ... .. 25
4.0 Res lilts... . . .. .. . .. . .. .. .. .. . .. . .. . .. . .. .. . .... .. .. . . .. . .. .. .. .. .. ... . ... 28
4.1 Descriptive Analysis ... .. 28
4.1.1 The Anurans Species Collected ... .. 28
4.1.1.1 The Microhabitat Utilization of Anurans ... 30
4.1.2 The Endohelminths Species Co llected ... " 35 4. 1.2.1 Nematodes Class ... . 36
4.1.2.2 Cestodes Class ... .... 39
4. 1.3 Host -Paras ites Interactions ... .. 42
4.2 Diversity Analysis. 44 4.3 Simi larity Analysis ... .. 45
4.3.1 Endohelminth Species Presents in Anuran Host. 45 4.3.2 Organ Attachment by Endohelminths Based on Anuran Host" .... 46
4.4 Non-metric Multidimensional Scaling (NMOS) Analys is ... 48
4.4.1 The Attachment Guild of Each Endohelminths Species
in the Anuran Host ... . 48
5.0 Discussion ... .. 54
6.0 Conclusion ... ... . 58
7.0 References ... 59
8.0 Appendices ... ... 64
Appendix A The veterinary helminthology according to their taxonomy and morphology
Appendix B Characteristics of habitat and microhabitat checklist (Inger's Habitat Code in Ramlah, 2009)
Appendix C Preparation of 10% saturated formalin Appendix D The preparation ofEhlirch Heamatoxylin
Appendix E The preparation of 1% alcohol acid Appendix F The preparation of I % lithium carbonate Appendix G Eosin Preparation
Appendix H Species Account Descriptions of Endohelminths Species
i) The difrerence;, between Heterakis gallinarum and Heterakis isolonche
ii) The dilkrencc, between Hymenolepis spp. and Raillietina spp.
Appendix I Field Data Sheet of Sampling at UNIMAS campus area vi
Appendix J Resu lt s of Shannon Diversity Index
Appendix K Raw Data ofS imilarily Analysis i)
ii)
Endohelminth Species Presents in Anuran Ho st
Organ Attachment by Endohelminths Based on An uran Host Appendix L Raw Data ofNMDS
i) The preferences organ attachment by the end ohehnint hs spec ies present in the anuran ofUN lMAS
List of Abbreviations
cm DBH E FEB FSS GPS H"
He l KOH N M m ml mm NMDS ROTU sp
SVL TL V
Centimetre
Diameter Base Height East
Facu lty of Business Faculty of Socia l Sciences Global Pos itioning System Diversity lndex
Ac id Hydrochloric Potassium Hydroxide North
Molar
meter
Mililitre Milimetre
Nonmetric Multidimensional Scaling Reserve Officer Training Unit species
Snout-vent length Tail length Vo lume
viii
~---
List of Tables
Table 1 An outline classification of helminths parasitic in vertebrates 7
Table 2 Study sites, Global Positioning System (GPS) reading and samp ling methods
12
Table 3 'umber o f individuals of anurans species co llected East ofUNIMAS Campus
at West and 28
Table 4 The sample collected occupied by five different frogs' species 35
Ta ble 5 Endohelminth parasites species in anuran host 42
Table 6 The preferences organ attachment by the endohelminths species 43
Tab le 7 Shannon 's diversity indices, H ' of endohelminth species
111 each anuran 44
Table 8 Stress and fit measures of NMDS configuration o f the attachment guild ofeach endohelminths spec ies in the anuran host at UN IMAS
48
Table 9 Final Coord inates for commo n space of two dimensions for the attachment guild ofeach endohelminths spec ies in the anuran host at UN1MAS
49
Table 10 Attachment guild of each endohe lm inths spec ies in the anuran host at UNIMAS
53
- - - -
- - - -List of Figu res
Figure 1 Study site used in this study 13
Figure 2 The dissect ion of H. baral71ica (Brown Marsh Frog) 16
Figure 3 The organ sample, stereo microscope and dissecting kit 20
Figure 4 The stereo microscope (ZEISS with PIXEL! K) to capture 20 helminth's image
Figure 5 The chemical used in the laboratory work 20
Figure 6 Classi ftcation and interpretation 0 f similarity index 23
Figure 7 DUllaphlynus melanOslictus, Swamp Toad (Family 29 Bufonidae)
Figure 8 FejervCllya limnacharis, Grass Frog (Family Ranidae) 29
Figure 9 Hylarana elylhraea, Green Paddy Frog (Family Ranidae) 29
Figure 10 Hylarana baramica, Brown Marsh Frog (Family Ranidae) 29
Figure 11 Polypedales leucomystax, Four -lined Tree Frog (Family 30 Rhacophoridae)
Figure 12 Types of horizontal positions occupied by anuran species in 32 the UNIMAS campus
Figure 13 Types of vertical positions occupied by anuran species in the 33 UNIMAS campus
x
34 Figure 14 Types of substrate occupied by anuran species In the
UN IMAS campus Figure 15 Relati ve abu nd ance
host
of endohelminths infecting the anuran s 36
Figure 16 The spicu les and precoacal feat ures of H gallinarum
sucker which are distinctive 37
Figure J7
(a) & (b)
The posterior gallinarum.
part of female and male of Heterakis 38
Figure (a)
18 The body part ofHeterakis isolonche 39
Figure (b)
18 The anterior part of Heterakis isolonche 39
Figure (e)
18 The posterior part of Heterakis isolonche 39
Figure 19 Endohel mit h species ITom genus of Hymenolepis 41 Figure 20 The end ohelminth ITom genus Raillielina 42 Figure 21 Dendrogram of Morisita Similarity Index of endohe lminth
species prese nts in anuran host.
45
Figure 22
Figure 23 (a)
Dendl"Ogram of Moris ita Sim ilarity Index of organ attachment by end ohelmiths based anuran host
Final Coo rdinate Dimensio n I represents the attachment guild of each end ohelmint hs species in the anuran host at UN IM AS
46
50
xi
Figure 23 Final Coo rdinate Dimension 2 represents the attachment 51 (b) guild of each endohelminths spec ies
UN IMAS
in the anuran host at
Figure 24 The ScatterpJ ot of NMDS configuration sh owing the attac hment guild of each endohelminths species in the anuran host at UN IMAS
5 I
xii
Endohelminth Parasites Diversity of Anuran in the University Malaysia Sarawak (UNIMAS) Ca mpus
Farliana Zulea Binti Zulkifli
Animal Resource Science and Management Programme Faculty of Resource Science and Technology
Universiti Malays ia Sarawak Abstract
The study of endohelminth parasites diversity of "nuran was conducted in the UNIMAS campus.
The aim of this study is to investigate the diversity of endohelminth paras ites while understanding the relationship of the parasites with its host. Besides, this revise will determine the attachment guild of endohelminlh parasites based 0 11 their community structure either specialist or generalist. The parasite assemhlages of different cHlurans species also were identified depends on their specific parasite-host characteristics. Descriptive analysis revealed four species of endohelminlhs which are H. gallinarum, Hymenolepis spp., H. isolonche, and Raillietina spp. in the five species of afluran hosts. The Shannons index (H') shows the highest endohelminths species diversity in H. baramiea with H·=1.797. Mori sita Index of Similarity and Non·metric Multidimensional Scale (NMDS) was used to classify the endohehninths into three guilds based on their preferences attachment orgal1 which are; i) general host with general attachment (intest ine, liver, lung and heart), ii) specific host (F. Iil11nocharis) with specific attachment (intestine), and iii) specific host (D. melanastictus) with specific attachment (inlestine). The findings of this study can enhanced the knowledge in anurans disease and helrninfhological concepts in assisting the conservation effort of frog and toads in this region.
Keywords: endohelminths, guild, anurans, helrninthological
Abstrak
Kajian lentang kepelbagaion parasi! endohelm;nth dari anuran feloh dilakukan di kampus UNUviAS Tl(juan kajian ini adalah unluk mengelahui kepelbagaian parasi! elldohehninlh dan memahami hubnngan anlara parasit dengan perumohl1ya Selain iln, kajian ini £Ikon menenlukan kumpu/an perlekOlan parasil endohelminlh berdasarkan struktur kOl1ll1nili mereka samada spesijik alau generalis. Kumpllian parasil bagi spesies anum" yang berbca juga dikenalpasli berganllmg kepada ciri-cil-i spesifik paras it
perumah. Analisis deskrip'if l11emtnjukkan empal jenis endohelll7il7lh iailu H gallinarum, Hymenolepis spp ., /I isolonche, dan Raillietina spp. lelah dijumpai daripada lima iems perumah anuran. Indeks Shannon (H ) menufljlfkkan kepelbagaian endohelmil1lh spesies tertinggi pada / /. baramica dengan H'= I. 797. Indeks Persamaan Morisifa dan ana/isa NMDS telah digunukan I.Infuk mengelosknn endohelminlh mel?iadi liga kumpu/an berdasarkan keulamaan organ perleka/a/7 mereka iailu; ;; perumah UI11 UI11 dengan perlekatan IImum (usus. hali, peparu dan jantung), ii) perumah spej'iflk (F. limnochahs) dengan perlekaton spes(!ik (usus), dan iii) perul1Ia/i spesijik (0. melanostic/lls) dengan per/ekaton spesifik (usUJ). Penemuan kajian in; dapal meningkatkan pengelahuan dolom penyakil Ol7uron don konsep helmintholog;kal do/alii membonlu pemllliharaan kalak dan kodok di ran/au ini.
Kala Kline;: endahelminlh, kumpu/an perlekalan, amlml1, he/minlhologikal
xiii
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1.0 Introduction
The anuran fauna of Borneo now includes 156 known species, which are increased over the number listed in the monograph on Bornean Amphibian (Inger, 1966; Inger &
Tan, 1996; lnger, 2009). Intensive studies on the Bornean frog restricted to the field of biodiversity, taxonomy, ecology and phylogenetics (Inger & Tan, 1996). Another considerable study that should be explored is the l)-og paras ite's infection. Endoparasites which are defined as an animal or organism that lives inside the organs or tissues or body cavities or body fluids ofa specific host, and is dependent metabo lically on it (Ousen eL aI., 201 0; Bhatia el al., 2006). Helminth parasites specifically are metazoans (multicellular) parasites. Strong knowledge on the causative agents is compulsory in order to control the transmissions of parasitic diseases through systematic studies, factors contributing (0 the infections through ecological studies and history of host-parasite relationships through phylogenet ic stud ies.
According to Page (1997), peat swamp forest is one the major forest formations of Borneo which is richly endowed with biodivers ity and is the most threatened forest. The unique ecological patterns of peat swamp are available to study the anurans and current ecological theory identifies an important role for the parasite in the regu lation of host populations.
The same classes of pathogens that infect humans also infect amphibians; just different strains and species. Now, it is thought that environmental factors are disabling the
1
Immune system and making frogs mo re susceptible to parasitic invasions. Generally, helminth parasites are known to cause diseases affecting its hosts and current repol1s suggested by Szuroczki and Richardson (2009). The increases 0 f parasite preva lence have caused the decline of frog 's population. Many helminth species infect amphibians, and some cause disease with heavy burdens. The pathogenic effects oftrematodes, cestodes and nematodes are reviewed in detail by Schmidt and Roberts (2000). This may be due to host specificity of the parasites or to aspects ofhost biology (AI-Attar, 20 I 0).
Most of the literature on the parasites of amphibian is concerned with taxonomy, lite c}cles and descriptive biology (Pieter el aI., 2009). There is relatively little information derived fi'om analysis of host-parasite interactions, including pathology, in natmal populations studied ill lhe 11eld. Similarly, even less information based Oil n1(;ticulous experimental work under defined cnvironmental conditions. The parasitological study of frog is still lacking and no information about endoparasites of anuran was derived. With arisen of infecting disease, it is important to discover the parasites host relations such as anuran. Hence, how much the diversity ofpathogen ic helminthes in anurans body and what are the community structures ofen do helminth parasites.
Based on the questions arise, it hypothesized that:
Hypothesis:
I. High diversity of endohelminth parasites in anuran commensa lly with man.
2. Most endohelminth parasites are generalist as they attach to most part of anurans organ.
Objective:
I. To investigate spec ies diversity of endohelminth parasites in anuran community at UNIMAS campus area.
2. To determine attachment guilds of the endohelminth parasites in the anuran organs.
The study of endohelminth parasites in anurans was conducted within the area of UN IMAS campus. The study was conducted in July 20 10 to February 20 1l. For field sampling, the frogs and toads inhibit at UNIMAS were using line transect. The lung. liver.
heart <Ii d large intestine of each amlran were taken for endohelminths diagnostic. The identification of endohelminth's morphology at their anterior pan, body part and posterior Palt was examined using compo und microscope (LEICA DME) and stereo microscope (ZElSS with PIXELlNK). The helminthes present was identified using key characteristic, illustrations and publications by Bhatia el 01., (2006), Bowman el 01., (2003), Fox el 01., ( 1993), Garcia el 01., (1993), Hendrix el ai., (2007), and Taylor el ai., (2007). The measures of species diversity employed are Shannons index (H) Morisita Index of Similarity and Non-metric Multidimensional Scale (NMDS) was used to classity the endohelminths into three guilds based on their preferences attachment organ. The aim of this study is to
3
investigate the diversity of endohelminth parasites in anuran at Universiti Malaysia Sarawak (UNIMAS) while understanding the relationship of the parasites with its host.
Besides, this revise will determine the attachment guild of en do helminth parasites based on their community structure either specialist or genera list. The findings of this study enhance the knowledge in anurans disease and helminthological concepts in assisting the conservation effort of trog and toads in this region.
2.0 Literature Review
Amphibians signilicantly represellt excellent models ror manipulative reseMch which complementary tic ld and laboratory studies arc explicable (lioimes el al.. 2007:
Tinsley, 1995 ). They can be maintained easily in captivity. in fact without stress, in environmental conditions directly resembling those in naiural habitats, and seasonal variations equivalents to those in nature can be simu lated in lhe laboratory.
2.1 Auuran at Peat Swamp Forest
There are 12 spedes representing 8.5% of Bornean Irogs have been recorded in the peat swamp forest of Kola Samarahan (Rarn lah, 2006). By comparing the trog species to und at Loagan 13unut National Park and Maludam National Park, Sarawak, the studied shows that none of the trogs are obligate to peat swamp torest (Das & Jensen, 2006: Tuen,
el al .. 2008). With capability for adaptations and migration, frogs are particularly sensitive
10 the high acidity of peat v.-ater because they spend half of their life cyele in aquatic environment and have permeable skin (Booloolian, et aI., 1980). Frogs in Kota Saillarahan is dominated by family Ranidae, represe nted by eight species and the abundant species is Hylarana elylhraea (Ral11lah. 2006) .
•
5
2.2 Taxonomy and Morp hology of Endohelminth Parasites
There are numerous endoparasites of amphibians, and new species are frequently described often because members of this group occur in disturbed aquatic ecosystems and occupy a genera list trophic level and are, thus, exposed to a large variety of parasites (Barta
& Desser, 1984; Boyman el al. , 2003; Holmes, 2007).
The animal parasites are named acconJing 10 the International Code of Lc'oiogical Nomenclature which is based on the Principle of 13 inomia I Nomenclaturc of Linnacus. Thc tenth edition of Systema Nature published in 1758 is accepted as the starting point of zoolog ical L1omcnc.lature (Bhatia el al., 2006). When endohelminths are examined, it is seen that they form natural group with features, usua lly morphological, in common.
(Hendrix, 2007). This aspect of biology is ca lied taxono my (Bhatia el al., 2006).
The name 'hehninlh' has been derived ITom Greek word hefmins or helll!inthes which means a worm. and is usually applied 10 paras itic anti nCln-parasitic species belong ing io three phyla; Platyhelminthes (nat worms, ilukes, and tapeworms), Nemalhelminthes or Nematoda (roundworms), and Acanthocephalan (thorny-headed worms) as ;ho\'.n in Appendix A (Whitfield, 1993; Bhatia el al. , 2006). Table I provides a working outline class ification for the helminthes parasitic in vertebrates.
6
Table t: An Outline Classification of Helminths Parasitic in Vertebrates (Whitfield, 1993)
Phylum Platyhelminthes
Class/ Order Class I: Monogenea
Class 2: Cestoda (pyphyllobolhrium, Taenia, Echinoceus)' Class 3: Aspidogastrea
Class 4: D igenea (Schislosoma, Fasciolopsis, Fasciola, Paragonimus)
Nemathelminthes Order 1: Rhabditida (Strongyloides)
Order 2: Strongyl ida (Necator, Ancylostoma) Order 3: Ascar idida (Ascaris, Toxocara) Order 4: Oxyurida (Enterobius)
Order 5: Spirurida (Dracunculus, Wuchereria, Brugia, Loa, Onchocerca)
Order 6: Enoplida (Trichinella, Trichuris)
*Important genera which man are affected.
23 Ecology of Host-Parasite Interactions
The worlel 0(" host-parasite relationsh ips can be seen as an ensemble of paras ites species and parasites ind ividuals distributed aCross ensemble of host specie:, and host individuals, the system thus constituted being themselves distributed in time and space (Combes, 2000), There are many different kinds of diseases caused by these helminth pathogens, many different organs and parts of the body are affected, many different routes of access to the body are used and the outcomes or severity of the diseases caused range from non-symptomatic to fatal (Marcogliese el ai., 2008). Some round worms migrate around much of the body, causing damage as they go, until they settle in their preferred location (Tinsley, 1995), Disease severity ranges ji-om rapidly fatal encephalitis to low grade chronic infections of the gut that may be non-symptomatic for many years (Johnson, 2003), Most commonly there are respiratory problems or diarrhea which are non-li fe
7
threatening. Tapeworms absorb nutrients trom the gut and lead to chronic malnutrition and malaise to the hosts in heavy infestations (Schmidth, 2009). Some worms block passages responsible for bile, lymph or blood flow and the resulting obstruction leads to swejjing and tissue damage as well as loss of function. The parasites possibly access via food or water that is ingested, air or aerosols that are inhaled, direct penetration though the skin, access through the eye, nose and ear or transmitted thl'Ough sexual activity.
The lifestyle of ampihibians might suggest a particular vulnerability to microbial and specific in teet ion (Marcogliese et ai., 2008). The damp habitat preferred by most amphibians favour survival of the infective stages of the pathogens. The skin is generally moist, delicale and apparently unprotected in order to fulfill their respiratory role (Hickman el ai., 2006). The mode of reproduction involving dense congregations of amphibians in water cremes optimum condilions tor transmission of parasites (Hoimes el ui.. 2007; Fabio.
2000). Fundamentally. anurans are characterizcd hy dual ecology, 3550ciated by both aquatic 3nd terrestrial habits and hence they are exposed two ditlere nt spectra of parasites.
'rhe diversity 01' parasites with differenl transmission charac:teristics had increased which combined togeiher in amphibious hosts. The inOuence of host behavior on these varied life cycle patterns had been docul11ented by Combes (i 972) (Tinsley, 1995).
The emergence or increased prevalence of various parasites may be Jinked to alterations in host-parasite interactions caused by environmental changes. According to Koprivnikar el ul., (2006), the agricultural activity was a significant factor in determining the percentage of tadpoles infected by one or more trematodes fi'om one or more species
8
