DETECTION OF CHYTRIDIOMYCOSIS ON THE Platymantis spp.

IN
MTS. PALAYPALAY/MATAAS-NA-GULOD PROTECTED
LANDSCAPE, LUZON ISLAND, PHILIPPINES

An Undergraduate Thesis Proposal Presented to

The Faculty of the Biological Sciences Department
College of Science
De La Salle University Dasmarias
Dasmarias, Cavite

In Partial Fulfillment of the Requirements

For the Degree of Bachelor of Science Major in Human Biology

JENNIFER ROSE B. CASTILLO

ZHAYNE-RE S. MALINAO
March 2010
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ACKNOWLEDGEMENTS
The proponents wish to express their utmost gratitude and appreciation to

the following:

Ms. Rubie M. Causaren, the proponents adviser for her support and

guidance in spite of her many tasks, willingly shared her time for the completion

of this study;

Ms. Cherry Z. Cuevas and Ms. Reny C. Obra, the members of the Thesis

Review Panel, for their suggestions and recommendations that guided the

proponents in completing their thesis proposal;

Our HUB32 family, who gives us the never-ending support to each of us,

the chance to enjoy and be with them during the completion of our own thesis

proposals;

Our families and siblings, for their financial and moral support: Jennifers

parents. Mr. and Mrs. Castillo, her brother Alexander James and sister Maridel

Grace; Zhaynes parents, Mr. and Mrs. Malinao, her brothers Ruen Zyr, Ron

Zharlo, Rockzel Zawn and her sister Zam Roise, for being an inspiration and

giving us significance to our lives;

And above all, Our God Almighty, for His unconditional love, blessings,

guidance, strength and knowledge He had given us.

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TABLE OF CONTENTS

Title 1

Endorsement Sheet 2

Acknowledgements 3

Table of Contents 4

CHAPTER 1 INTRODUCTION

1.1 Background of the Study 6

1.2 Conceptual Framework 8

1.3 Statement of the Problem 9

1.4 Scope and Limitations 9

1.5 Significance of the Study 9

1.6 Definition of Terms 10

CHAPTER 2 LITERATURE REVIEW

2.1 Conceptual Literature 11

2.2 Related Studies 16

CHAPTER 3 METHODOLOGY

3.1 Research Design 19

3.2 Research Setting 19

3.3 Research Procedure 19

 4

3.4 Data Gathering and Analysis 21

CITED REFERENCES 22

APPENDICES

A. Procedure for Tissue Preparation 27

B. Detection of Chytrid Fungus in

Tissue Preparation 33

C. Images of Different Platymantis spp. Found In

Mts. Palay-Palay/Mataas-na-Gulod Protected

Landscape 40

D. Timetable for Research 41

E. Budgetary Requirement 42

F. Curriculum Vitae 43
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CHAPTER 1

INTRODUCTION

1.1 Background of the Study

Chytridiomycosis is a globally emerging infectious fungal disease in

amphibians, specifically in frogs caused by an aquatic chytrid fungus,

Batrachochytrium dendrobatidis (Bd) (James et al. 2009; Laurance 2008; Snchez

et al. 2008; Fisher and Garner 2007; Bosch et al. 2006; Briggs and Burgin 2004;

Weldon et al. 2004) which is currently the largest infectious disease threat in

biodiversity (Kilpatrick et al. 2009).

Electrolyte loss is led by the pathogen which proliferates in the epidermal

cells of amphibians that has led to the ultimate death in susceptible species

(Voyles et al. 2009; Berger et al. 1998) and sporadic deaths (Sanchez et al. 2008;

Fisher and Garner, 2007; Briggs and Burgin, 2004; Weldon et al. 2004) in some

amphibian population and is contributing to a worldwide decline in amphibian

populations (Diesmos et al. 2009; James et al. 2009; Kilpatrick et al. 2009;

Gleason et al. 2008; Snchez et al. 2008; Laurance 2008; Fisher and Garner,

2007; Briggs and Burgin, 2004; Weldon et al. 2004).

The precise cause of osmotic balance and death is uncertain, but is

hypothesized to be caused by physical skin damage or unidentified toxins

produced by pathogens. Climate changes appear to be the reason for the

transmission of chytrid fungus to frogs and frog population decline and extinction
 6

which threaten global amphibian diversity (James et al. 2009; Laurance 2008;

Snchez et al. 2008; Bosch et al. 2006).

Pounds et al. (2006) showed that dramatic, fungal pathogen linked

extinctions of numerous harlequin frogs in upland rainforest of South America

mostly took place immediately following warm years. The study of Laurance

(2008) in Eastern Australia showed similar results.

Bd is thought to be originated in South Africa, where the earliest records

occurs in a museum specimen from the 1930s, and initially spread by the

commercial trade in African Clawed frogs (Xenopus laevis) (Weldon et al. 2004).

Currently, chytridiomycosis has spread to certain countries and continents such as

America, Europe, Australia, Japan and the Indonesia island of Java (Diesmos et

al. 2009).

The Philippines is not spared from chytridiomycosis. In a country-wide

study done by Diesmos et al. 2009, two areas were tested to be positive for this

fungal infection: Mt. Lobo in Batangas and Mts. Palay-Palay/Mataas-na-Gulod

Protected Landscape (henceforth MPPMNGPL) in the Cavite-Batangas area.

MPPMNGPL is considered to be the only wildlife sanctuary in Cavite and

was declared a protected landscape by virtue of Proclamation No. 1315 signed on

June 27, 2007. A total of 14 species of frogs are identified from the area and of

these 57% are endemic (Causaren 2009).

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The species that are presently reported to be infected with

chytridiomycosis from MPPMNGPL are aquatic such as Rana luzonensis, R.

similis, Limnonectes macrocephalus, L. woodworthi, and Occidozyga laevis

(Diesmos et al. 2009).

The anuran fauna of MPPMNGPL also includes terrestrial frogs under the

genus Platymantis which are all endemic to the Philippines. One species,

Platymantis cf. mimulus, is considered to be potentially a Cavite endemic

(Causaren 2009). The proposed study therefore aims to determine whether

terrestrial species like Platymantis spp. are also infected by this fungus.

1.2 Conceptual Framework

The paradigm of the proposed is an attempt to determine chytrid infection

on Platymantis spp. in MPPMNGPL, study is as follows:

Platymantis spp. Presence of chytrid fungus that

in MPPMNGPL causes chytridiomycosis

The study will determine the presence of aquatic chytrid infection from

terrestrial species, Platymantis spp. This will use a safe, simple, sanitary and

humane method to acquire toe tips from frogs (toe clipping). To determine the

presence of chytrids, histological preparations of the toe clippings will be

examined and analyzed.

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1.3 Statement of the Problem

The proposed study aims to detect the presence of chytrid infection on the

Platymantis spp. in Mts. Palay-Palay/Mataas-na-Gulod Protected Landscape,

Luzon Island, Philippines.

Specifically, the study aims to answer the question:

1. Could chytridiomycosis be detected among terrestrial Platymantis spp. in

MPPMNGPL?

1.4 Scope and Limitations

The study will focus on the detection of chytridiomycosis that could infect

the 3 species of Platymantis: Platymantis corrugatus, Platymantis dorsalis and

Platymantis cf. mimulus. Samples will be obtained through toe clipping,

histological preparation and histological examination on different samples.

The study is limited to the detection of chytrid infection and will not

include the physiology of the infection.

1.5 Significance of the Study

The study aims to determine chytridiomycosis on the Platymantis spp. in

Mts. Palay-Palay/Mataas-na-Gulod Protected Landscape. This will be of great

help and contribution to the following:

Public, for further information about the climate changes, since frogs are

good indicators for climate change they have to prevent themselves from

desiccation.
 9

Herpetologists, who can help formulate conservation and management

decision for the protection of Platymantis spp. and other anuran species.

Student researchers, the study would serve as additional information for

future researches.

1.6 Definitions of Terms

The following terms are defined within the context of this study:

Chytridiomycosis. An infectious disease in amphibians, specifically frogs,

caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd).

Platymantis spp. A genus of frogs, under the family Ranidae that are said

to be forest dwelling frogs that range from small to moderate sizes.

Toe clipping. Method to acquire the clipped toe tips for genetic analyses and

histological examinations for infectious diseases.

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CHAPTER 2

LITERATURE REVIEW

2.1 Conceptual Literature

General description of Chytridiomycosis

Chytridiomycetes are also known as chytrids, (Phylum Chytridiomycota)

they are predominantly aquatic (Yusingco 2004) where they are found in aquatic

and semi-aquatic environments present in salt or fresh water such as oceans,

lakes, rivers, ponds, or damp soils (Solomon et al. 2005; Yusingco 2004).

They are the most primitive fungi living today that have motile cells

(Audesirk et al. 2006; Solomon et al. 2005; Freeman 2005). They possess a single

posterior flagellum through its spores which makes them move and where

reproduce both sexually and asexually. Structurally, its flagella are similar to the

flagella in the sperm cells of animals (Audesirk et al. 2006; Freeman 2005). They

also have chitin that strengthens the cell wall (Yusingco 2004). They are

relatively small and simple that exhibit alternation of generations and cytoplasmic

fusion (plasmogamy) (Solomon et al. 2005; Yusingco 2004).

Chytrids are also known to be parasitic or decomposers because of their

role in decomposing dead organic matter, they also are responsible for the potato

wart diseases and parasitize invertebrates, particularly on nematodes and

mosquitoes. Some chytrids are also known to attack algae. They can also be
 11

classified as unicellular or maybe advanced, producing a mycelium like other

fungi (Yusingco 2004).

History and Spread of Chytridiomycosis

Earlier records imply that chytridiomycetes may be the earliest fungi to

emerge from the ancient flagellate protest hypothesized as the ancestor to all fungi

(Solomon et al. 2005; Yusingco 2004), for they possess chitin for strengthening of

the cell wall and cellulose which is the main component present in the ancestor of

all fungi division except that chytridiomycetes have an additional characteristic as

that may have been lost in their evolutionary history in all other fungi, the flagella

(Yusingco 2004).

The earliest date for a chytridiomycosis-positive specimen was during

1938 in the Western Cape coastal lowland from African Clawed frog (Xenopus

laevis). This specimen is housed in South Africa Museum, Cape Town; this

species only shows subclinical chytrid infection but does not cause them to die.

The next earliest positive specimen detected is from a Cape Clawed toad

(Xenopus gilli) during 1943. For decades since 1871-1940, chytridiomycosis is

being examined in frogs. No species has been found to be positive except during

1931-1940, where 1/ 37 species is said to be infected by this chytrid fungus. The

prevalence of chytridiomycosis in South Africa showed no significant changes

over time after 1940. By 1973 the specimen was detected to be positive in all

regions of South Africa. The next earliest case was found in American green frog
 12

(Rana clamitans) from Sain-Pierre-de-Wakefield in Quebec, Canada during 1961

(Weldon et al. 2004).

An ideal host for the transmission of chytridiomycosis through intentional

translocation would be amphibians, those that does not easily get infected by the

disease and that does not die easily of the infection. Since the species Xenopus

laevis does not show clinical signs, nor experience any sudden die-offs they could

take the role of being the natural carrier (Weldon et al. 2004).

Batrachochytrium dendrobatidis was introduced into new regions most

likely by global transportation of amphibians. Some members of the Pipidae

family particularly the Western Dwarf Clawed frog (Hymenochirus curtipes) and

Xenopus laevis were exported to North America and Europe. In 1935 because of

technology advances using Xenopus pregnancy test, there has been a sudden rise

of export trade (for they selected to be the most suitable amphibian for the test)

(Weldon et al. 2004).

Another transmission of B. dendrobatidis is having importing countries

have escaped frogs, the water they lived or may be both the frog and the water

may have come in contact to the local amphibian species or habitat that may have

cause the disease to spread (Weldon et al. 2004).

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Platymantis spp.

The genus Platymantis consists of several species of small to moderate

sizes of the Philippine forest dwelling frogs and are divided into three groups: the

dorsalis-Group, guentheri-Group and the hazelae-Group (Alcala et al. 1998). At

present, twenty-nine Platymantis species are recognized from the Philippines and

all are considered endemic (Causaren 2009). The dorsalis-Group is distinguished

by subtending parts of digits narrow, has subarticular tubercles that are strongly

protruding, pointed and terminal phalanx is not T-shaped, they are blunt and have

small disks. The guentheri-Group is characterized by digits which are subtending

parts and are narrow, subarticular tubercles are protruding and the shape of the

terminal phalanx ranges from moderate to wide T and digits with broad disks. The

hazelae-Group is distinguished by subtending digits that are wide, subarticular

tubercles low and rounded, terminal phalanx is a wide T-shaped and digits with

broad disks like in guentheri-Group (Alcala et al. 1998).

Platymantis spp. does not specifically require aquatic environments since

they develop direct terrestrial reproduction. They deposit eggs on moist to wet

ground where froglets develop directly, skipping the tadpole stage. These frogs do

not require bodies of water to reproduce for they are able to survive in close-

canopy forests which are inhabitable to many frogs that are dependent on water

(Diesmos 2009).
 14

In Australia, data of infectious fungal diseases, suggests that more than

twelve species of Platymantis are affected by chytridiomycosis (Solomon et al.

2005).

Three species of Platymantis are now recognized from MPPMNGPL,

Platymantis corrugatus, Platymantis dorsalis and Platymantis cf. mimulus which

is also regarded as a positive Cavite endemic.

Information on MPPMNGPL

MPPMNGPL which was formerly known as Mts. Palay-Palay/Mataas-na-

Gulod National Park was signed under the Proclamation No. 1315. It is

proclaimed as a wildlife sanctuary on October 26, 1976 by Proclamation No. 1594

(Causaren 2009; PAWB 1989). The protected landscape comprises of about

3,973.13 hectares and belongs to the three volcanic centers of Cavite-Batangas

area. The area is located within the municipalities of Ternate and Maragondon in

Cavite and Nasugbu in Batangas (Causaren 2009). The topography range is from

rolling to moderately steep and steep terrain (PAWB 1989). It composes of three

peaks: Mataas-na-Gulod, Pico de Loro and Palay-Palay (DENR 1992; Zanoria

1991).

MPPMNGPL is also the best forested area-within this range (Causaren

2009). Its assigned conservation priority level is in a Very High status (Ong et

al. 2002) where its vegetative cover is estimated to 62.5 % of lowland dipterocarp

forest and 37.5% nonforest. Its existing vegetation is considered as secondary

 15

growth forest with remnants of primary lowland forest (PAWB 1989). The

landscape is within the first climatic type with two pronounced seasons: dry

season (November to April) and wet season (May-October). The highest rainfall is

recorded in August and April is recorded as lowest humidity (Causaren 2009).

2.2 Related Studies

The study of Ryan and Eichhilz (2008) is about the decline and extirpation

of an endangered Panamanina stream frog population (Craugastor punctariolus)

due to an outbreak of chytridiomycosis. This study was studied for six years in

central Panama during the year 1999-2005. The site was found to be absent from

Batrachochytrium dendrobatidis for five years until the month of September

2004, four species of C. punctariolus were found dead caused by the fungus, thus

having a positive result of chytridiomycosis in the reported site which claims the

frog population (C. punctariolus) to be endangered.

In Eastern Australia, Laurance (2008) experimented using fourteen

upland-rainforest frog species that had experienced population decline and

extinction with the same fungal pathogen where in recent decades, the

temperature has also risen. His findings brought about to have consistent notation

on global warming that could predispose some upland amphibian population to

virulent pathogens.

A journal by Sanchez et al. (2008) shows that 323 frogs out of 649

samples in Venezuelan Andes are associated with Batrachochytrium

 16

dendrobatidis where 279 of them were bullfrogs. Nine of which are native

species, Dendropsophus meridensis, Gastrotheca nicefori, Hypsiboas crepitans,

Hyloscirtus jahni, Hyloscirtus platydactylus, Mannophryne collaris, Engystomops

pustulosus, Pseudis paradoxa, & Scarthyla vigilans. Though among the nine

species, Lithobates catesbeianus was considered to be the most important

reservoir with 79.9% infected and an average of 2299 zoospores while

Dendropsophus meridensis showed highest infection prevalence and mean

zoospore load with 26.7% and 2749 zoospore which makes them may be at risk if

environmental stress exacerbates vulnerability or pathogen load.

A study of Briggs and Burgin (2004) shown that Platymantis species were

infected with chytridiomycosis. The skin scrapings from the inner thigh of dead

and living Platymantis spp. show immature and mature zoosporangia, zoospores

and germling stages of the fungus within the hosts tissues. The zoospores stained

orange (with flagellum) but walls of the rest of the stages stained reddish-orange.

According to the journal of Voyles et al. (2009), Bd causes the skin disease

chytridiomycosis, a virulent fungus in vertebrates which is responsible for

amphibian declines. Diseased individuals experience electrolyte transport across

the epidermis was inhibited by >50% plasma sodium and potassium

concentrations were respectively reduced by ~20% and ~50%, and asystolic

cardiac arrest resulted in death. Because the skin is critical in maintaining

amphibian homeostasis, disruption to cutaneous function may be the mechanism

 17

by which Bd produces morbidity and mortality across a wide range of

phylogenetically distant amphibian taxa.

Berger et al. (1998) studied on sick and dead adult anurans collected from

their studied site in Australia and Central America and concluded that the

chytridiomycosis found in the cutaneous is fatal to anurans and is the proximate

cause of the population decline in anurans.

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CHAPTER 3

METHODOLOGY

3.1 Research Design

The proposed study is said to be descriptive where its primary aim is to

determine the chytrid infection in Platymantis spp. of the anurans in Mts. Palay-

Palay, Ternate, Cavite. In addition, toe clipping method, histological preparation

and histological examination of the tissues will be used to identify the fungi.

3.2 Research Setting

This experimental procedure includes the collection and preservation of

toe clipping of Platymantis spp. from MPPMNGPL yet the histological

preparation and examination will be performed at the Biological Research

Laboratory of De La Salle University- Dasmarias. The entire research procedure

will last for 8 weeks. The study will be done in the month of April-May, 2010.

3.3 Research Procedure

Collection of Specimens and Toe Clipping

Platymantine frogs will be collected from MPPMNGPL, Ternate, Cavite

on April 2010. Collecting of species will start immediately after sunset; they will

be collected through hand-grabbing along the river banks and in the forest. All

captured individuals will be brought to the Department of Environment and

Natural Resources (DENR) Station where toe clippings will be obtained. Firstly,

the cleaned and sterilized materials must be ready to be used. The foot and toes is
 19

held up in a position that is higher than the rest of the foot and vent to prevent

dirty water, feces and urine from running down the leg and contaminating the toe

skin. The foot must be cleaned by gently spraying a stream of clean water over

the skin and toes in order to remove mud and debris and then sprayed with any

soothing infection protection. The disinfected scissors are placed on each side of

the selected toe. The portion of the clipper is recommended to be near the hinge

(rather than near the tip) to be used for clipping the toe. Digit (toe) III or V should

be the number of digits for clipping. After getting the toe clips, each individual

will be treated and placed Betadine on their wounds. The tools used must be

disinfected after use on each animal to prevent spread of diseases and build up of

contaminants (Green 2007). The toe clips will be placed in vials with 10%

formalin for preservation. After getting the toe clips, the Platymantine frogs will

be released near their capture sites.

Preparation of Histological Sections

For the histological preparation of the specimens, the clipped toes will

undergo fixation, decalcification, dehydration, clearing and paraffin impregnation

embedded in a solid, white medium called the paraffin wax to facilitate sectioning

(paraffin embedding) (Hara 2009; Junquiera and Carniero, 2005). Fixation is the

first step with the minimum time of 6-24 hours, then decalcification will undergo

12-24 hours followed by ~2 days of dehydration; followed by 31 hours of clearing

and lastly paraffin impregnation which is prepared in 2 hours - overnight having

 20

the total time of 4-5 days (See Appendix A). After embedding, the hard blocks

are taken in a microtome to be sectioned. After sectioning, the specimens are

floated in the water and transferred to glass slides to affix sections on slides using

Haupts adhesive. The tissues are stained with routine haematoxylin and eosin

technique. Lastly, the glass slide is cover slipped using Canada balsam or

Permount, allowing it to dry overnight and ready to be seen under the microscope

(Hara 2009; Junquiera and Carniero, 2005; Young and Health, 2002) (See

Appendix A).

The number of slides that will be prepared will depend on the number of

captured individuals.

3.4 Data Gathering and Analysis

Slides will be examined for the presence of chytrids. The chytrid fungus

will be detected by observing the presence of zoosporangium and zoospores (See

Appendix B).
 21

CITED REFERENCES

Alcala, A.C. & Brown, W.C. 1998. Philippine Amphibians: An Illustrated Field

Guide. Philippines: Bookmark Incorporated.

Audesirk G, Audesirk T, Byers B. 2006. Biology : Life on Earth. Pearson/Prentice

Hall, USA.

Berger L, Speare R, Daszak P, Green DE, Cunningham A, Goggin C, Slocombe

R, Ragan M, Hyatt A, Mcdonald K, Hines H, Lips K, Marantelli G, &

Parkes H. 1998. Chytridiomycosis causes amphibian mortality associated

with population decline in rainforests in Australia and Central America.

Proc National Academics Sciences, U.S.A , pp.90319036.

Bosch J., Carrascal, L.M., Durn, L., Walker, S., & Fisher, M.C. 2006. Climate

change and outbreaks of amphibian chytridiomycosis in a montane area of

Central Spain: Is there a link? The Royal Society, pp.253-260.

Briggs C. & Burgin, S. 2004. Congo Red, an effective stain for revealing the

chytrid fungus, Batrachochytrium dendrobatidis, in epidermal skin

scraping from frogs.

Bruce-Gregorios J. H. 1974. Histopathologic Techniques. JMC Press, Inc. Quezon

City, Philippines.
 22

Causaren R. 2009. Preliminary Report On the Anurans of Mts. Palay-Palay/

Mataas-Na-Gulod Protected Landscape, Luzon Island, Philippines. pp. 40-

45.

Department of Environment and Natural Resources. 1992. Proposal Management

Plan for Mts. Palay-Palay/Mataas-na-Gulod National Park: PENRO, Trece

Martires City, Cavite. pp. 1-3.

Diesmos, M.L., Diesmos A.C., Vredenburg, V., Brown, R. 2009. A country wide

survey of chytrid fungus (Batrachochytrium dendrobatidis) in the

Philippines. Baguio, Philippines.

Fisher M., & Garner T. 2007. The relationship between the emergence of

Batrachochytrium dendrobatidis, the international trade in amphibians and

introduced amphibians species. Elsevier Limited.

Freeman S. 2005. Biological Science: Second Edition. United States of America:

Pearson Prentice Hall, Pearson Education, Inc.

Gleason F.H., Kagami M., Lefevre E. & Sime-Ngando, T. 2008. The ecology of

chytrids in acquatic ecosystem: Roles in food web dynamics. Elsevier

Limited.
 23

Green D.E. 2007. Toe Clipping of Frogs and Toads. USGS National Wildlife

Health Center: Advancing Wildlife and Ecosystem Health for a Better

Tommorrow. United States Geological Survey: Science for a Changing

World|

http://www.nwhc.usgs.gov/publications/amphibian_research_procedures/t

oe_clipping.jsp

Hara O.A. 2009. Histology Laboratory Manual: Evaluation Copy. Activity No. 3:

Histotechnique. pp. 19-25.

James T.Y., Litvintseva, A.P., Vigalys, R., Morgan, J. T., Taylor, J.W., Fisher,

M.C., Berger, L., Weldon, C., Preez, L. &Longcore, J. E. 2009, May 29.

Evolution of Chytridiomycosis: Rapid Global Expansion of the Fungal

Diseases Chytridiomycosis into Declining and Health Amphibian

Population. Retrieved from PLoS pathogens: Volume 5| Issue5|e1000458:

http://www.plospathogens.org

Junquiera, L.C. & Carniero, J. 2005. Basic Histology: Text and Atlas 11th Edition.

United States of America: McGraw Medical Publishing Division, The

McGraw Hill Companies.

Kilpatrick A. M., Briggs, C. J. & Daszak, P. 2009. The ecology and impact of

chytridiomycosis: an emerging disease of amphibians. Elsevier Limited.

 24

Laurance W.F. 2008. Global warming and amphibian extinction in Eastern

Australia. Ecology Society of Australia.

Parks and Wildlife Bureau. 1989. Profile of the National Parks of the Philippines.

Pounds J. A., Bustamante M. R., Coloma L. et al. (2006)Widespread amphibian

extinctions from epidemic disease driven by global warming. Nature 439,

161-7.

Ong P.S., Afuang, L.E. & Rosell-Ambal R.G. (Eds.). 2002. Philippine

Biodiversity Conservation Priorities: A Second Iteration of the National

Biodiversity and Action Plan. DENR-Protected Areas and Wildlife

Bureau, Conservation International Philippines, Biodiversity Conservation

Program-UP CIDS, and Foundation for the Philippine Environment,

Quezon City, Philippines. Parks and Wildlife Bureau. 1989. Profile of the

National Parks of the Philippines.

Ryan M. J. & Eichhilz M.W. 2008. Decline and extirpation of an endangered

Panamanian stream frog population (Craugastor punctariolus) due to an

outbreak of Chytridiomycosis. Elsevier Limited.

Snchez, D., Chacn-Ortiz, A., Len, F. A. & Lampo, M. 2008. Widespread

occurance of an emerging pathogen in amphibian communities of the

Valenzuelan Andes. Elsevier Limited.

 25

Solomon E., Berg L, Martin D. 2005 Biology: 7th Ed. Thomson-Brooks/Cole

Learning, Thomson Learning Inc., USA

Voyles J. B. L. 2007. Electrolyte depletion and osmotic embalance in amphibians

with chytridiomycosis. DisacAquat Organ 77, pp. 113-118.

Voyles J., Young S., Berger L., Campbell C., Voyles W.F., Dinudom A., Cook D.,

Webb R., Alford R.A., Skerratt L.A., Speare R. 2009. Pathogenesis of

Chytridiomycosis, a Cause of Catastrophic Amphibian Declines. Sciencce

AAAS , pp. 582-585.

Weldon, C., Preez, L.H., Hyatt, A.D., Muller, R. & Speare, R. 2004, December.

Origin of the Amphibian Chytrid Fungus. Volume 10 No. 12, Retrieved

from Emerging Infectious Diseases: http:www.cdc.gov/eid

Wood, L., Griffiths, R. & Schley, L. 2009. Amphibian chytridiomycosis in

Luxembourg. pp.109-114.

Young, B. & Heath, J. W. 2002. Wheater's Functional Histology: a text and colour

atlas: 4th Edition. United Kingdom: Churchill Livingstone: Elsevier

Science Limited.

Yusingco, R. 2004. A Taxonomic Survey of Aquatic Frogs in Bahayang Pag-asa

Lake, Cavite, Philippines (Unpublished Undergraduate Thesis). De La

Salle University Dasmarias, Biological Sciences Department.

 26

Zanoria, A.C. 1991. Volcanology & Geochemistry of the Cavite-Batangas

Highland, Southwestern Luzon, Philippines (Unpublished Masters

Thesis). University of South Florida, Department of Geology.

 27

APPENDIX A

PROCEDURE FOR TISSUE PREPARATION

Histological Examination for Toe Tissues

A. Tissue preparation, fixation and preservation

Toe clips is kept in 10% formaldehyde in labeled jars and kept in room

temperature within 6 24 hours.

B. Decalcification

After fixation, the tissues will undergo decalcification which will remove

the calcium and lime salts and to soften the bone part of the tissue. This is also

done before impregnation to ensure equal cuttings and to prevent the obscuring of

microanatomic detail of the tissue sections (Bruce-Gregorios 1974).

C. Dehydration, clearing and paraffin impregnation

After preservation, the following procedures will undergo using

dehydration (ethanol), clearing (xylene) and paraffin impregnation (paraffin wax)

(See Table 1) (Hara 2009; Bruce-Gregorios 1974).

 28

Table 1. Dehydration, Clearing and Paraffin Impregnation

STAGE REAGENT DURATION

Dehydration 70% ethanol 3 hours

95% ethanol 6 - 24 hours

Isopropyl, 2 changes 6 - 24 hours

Clearing Isopropyl xylene 1:1 3 - 6hours

Xylene, 2 changes 6 - 24 hours

Xylene, 2 changes 0.5 - 1 hour

Paraffin 30% xylene in melted paraffin 2 hours Overnight*

impregnation wax

(60C) pure paraffin wax 1 2 hours

100% melted paraffin 2 hours*

100% melted paraffin 1 hour*

TOTAL TIME 29 hours - 5 days

 29

D. Embedding, Sectioning and affixing sections on slides

Paraffin is used to impregnate tissues into casts; they are sometimes

prepared as a small paper box or as a thin plastic square mold. The tissue must be

aligned in the cast before molten paraffin is poured over them. Allow the paraffin

to cool and solidify then remove the paraffin tissue block from cast (Hara 2009).

In the block cassette, attach the paraffin tissue block using extra paraffin

melted with a warmed metal spatula. In microtomy, the cutting of tissues should

be about 4 16 microns (m) in thickness using microtome. The paraffin tissue

blocks should be set onto cold surface (4C) to harden the face or side to be cut

(Hara 2009).

The attached paraffin tissue block should be trimmed with a sharp blade,

making parallel sides and 2-3 mm of paraffin surrounds the tissue, install the

microtome knife or blade in place and set the correct clearance angle. The

trimmed block should fit to the block holder of the microtome and adjust so that

the edge offering least resistance first meets the knife edge. Advance the block

until it touches the knife. The block is course cut by rotating the flywheel, set at

15m until the full face is trimmed. Set the advance feed dial to the desired

thickness of 3-5m for in most purposes in cutting a series of sections, it forms a

ribbon. A moist camel hair brush is used to separate the ribbon from the knife

edge. If the knife has advanced to its full extent, it reminds the user that the
 30

blinking of small, red lights indicates that the rotating the reverse feedwheel must

be returned back to the starting position (Hara 2009).

The section or ribbon must be place on a flat board with a white or colored

paper and separate one section of the ribbon using a sharp blade. Afloat the

section in a warm water bath, wrinkles along with air bubbles that are trapped

beneath the paraffin will be removed. The temperature of the water should be

approximately 10C below the melting point of the paraffin used in the block

(Hara 2009).

A clean glass slide must be prepared in order to apply a very thin coat of

Haupts adhesive using pinky finger. The adhesivecoated slide should be use to

pick up the expanded section from the water bath. Hold the slide vertically

beneath the surface of the water. The section is apposed to the slide so that when it

is lifted from the water, it draws the section with it. The slide must be dried

overnight at room temperature or on a hot plate or hot air oven (Hara 2009).

E. Staining and Coverslipping

In order to get the tissue out of the paraffin wax, the embedding process

should be reverse so that water soluble-dyes will allow penetrating the sections.

Run the slides through xylenes to alcohols to water in order to deparaffinize and

rehydrate before doing the staining process. Tissues will not be stained when there
 31

is paraffin. The routine stain is the standard haematoxylin and eosin used in many

tissue staining (H and E) (See Table 2).

Table 2. Haematoxylin and Eosin Staining (H&E)

STAGE REAGENT TIMING

Xylene 3 changes 3 minutes each

Deparaffinize 100% Ethyl 1 minute each

and rehydrate 95% Ethyl 5 10 dips

sections 70% Ethyl 5 10 dips

Distilled water 5 10 dips

Harris haematoxylin 5 10 minutes

Distilled water
Just enough to rinse
(using wash bottle)
Haematoxylin
Tap water 5 10 dips
staining 1% Acid alcohol ***

Tap water 5 dips

Ammonia water (rinse until blue) 1 minute

Distilled water 5 10 dips

Eosin (counterstain) 2 5 minutes

Eosin staining,
95% ethyl 15 dips
dehydration
Isopropyl 3 changes 1 miute each
and clearing
Xylene 3 changes 1 3 minutes each
 32

A series of alcohol solutions should be made to remove the water in the

stained slide, following clearing agents which a permanent resinous substance can

be placed in the section, beneath the glass coverslip, or a plastic film. This is done

to accomplish three things: to protect the tissue from being scratched, to provide

better optical quality for viewing under the microscope, and to preserve the tissue

section for years to come.

Cover slip slides with Canada balsam or Permount.

Clean the area over and around the mounted coverslips with cotton

moistened with xylene (Hara 2009).

 33

APPENDIX B

DETECTION OF CHYTRID FUNGUS IN TISSUE PREPARATION

Figure 1. Cluster of zoosporangia (zp) with the walls stained reddish-orange by

Congo red. Note the more intense staining of the discharge tubes (dt).

The refractive nature of the wall (w) is still apparent for one

zoosporangium. Scale bar = 5 mm (Briggs and Burgin, 2004).

 34

Figure 2. Cluster of zoosporangia (zp). The orange-red stained walls are less

refractive and contents can be seen within the zoosporangia. Note the

elongate, intensely stained single discharge tube (dt). Scale bar = 5

mm (Briggs and Burgin, 2004).

Figure 3. Single zoosporangium with two internal, orange-stained zoospores

(arrows). These zoospores have a single, darker spherical body near

one end. Scale bar = 2 mm (Briggs and Burgin, 2004).

 35

Figure 4. Three released uniflagellate zoospores. The wall-less zoospores and

their flagella (arrow) stain orange. The darker staining body is present

within each zoospore. Scale bar = 2 mm (Briggs and Burgin, 2004).

Figure 5. Empty zoosporangia collected from the decaying body of a juvenile

Litoria peroni. Scale bar = 5 mm. Germling stage with numerous fine

rhizoids (r). Scale bar =2 mm (Briggs and Burgin, 2004).

 36

Figure 6. Scraping from the inner thigh region of formalin preserved adult male

Limnodynastes peroni. Single mature zoosporangium within a

keratinized epidermal cell. Wall of the zoosporangium is virtually

unstained, the exposed discharged tube (arrow) has bound the dye.

Scale bar = 5 mm (Briggs and Burgin, 2004).

Figure 7. Cluster of frog epidermal cells found in a scraping taken from a juvenile

Litoria peroni road kill. The nuclei (n) stain at varying intensities with

Congo red whilst the cell membrane is poorly stained. Scale bar = 10

mm (Briggs and Burgin, 2004).

 37

Figure 8. Two jar-shaped zoosporangium, or thalli (A), and zoospores inside are

clearly visible. The discharge tubule is also visible where the

flagellated zoospores are released from the thallus (B). Photograph

from A. Pessier, University of Illinois Zoological Pathology Program,

Loyola University Medical Center.

(http://www.sflorg.com/earthnews/en071106_02.html)
 38

Figure 9A. Micrographs of immunoperoxidase stained sections through the

interdigital webbing of Xenopus gilli, showing the morphologic

features and size of zoosporangia consistent with Batrachochytrium

dendrobatidis. A.) Arrow A indicates localized hyperplastic

epidermal response; arrow b indicates an uninfected region of the

epidermis. Scale bar = 10 m (Weldon et al. 2004).

 39

Figure 9B. B) Arrows indicate two zoosporangia with internal septa. Circle

indicates location of the infection in the stratum corneum. Scale bar

= 10 m (Weldon et al. 2004).

 40

APPENDIX C

IMAGES OF DIFFERENT Platymantis spp. FOUND IN MTS. PALAY-

PALAY/ MATAAS-NA-GULOD PROTECTED LANDSCAPE

(Adapted from Alcala and Brown, 1999)

Platymantis corrugatus Platymantis dorsalis

Platymantis mimulus
 41

APPENDIX D

TIMETABLE FOR RESEARCH

2009 2010 2011

OBJECTIVES
N D J F M A M J J A S O N D J F
Proposal Stage
Collection of

Samples
Toe clipping
Histological

examination of

the clipped toes

Data Gathering
Analysis and

Generalization
Final Oral

Defense
Revisions
Submission of

Final Paper
 42

APPENDIX E

BUDGETARY REQUIREMENTS

Number of
Sources of Expenses Rate Total
times
Collection of samples 2
500 1,000
Transportation
250 500
Meal
Histological examination
1 2,000 2,000
of the clipped toes
Adviser 1 600 600
Printing and
300 300
Communication
Defense Fee
2 300 600
(Panel members)
Miscellaneous 1,000 1,000
Grand Total P6,000
 43

APPENDIX F

CURRICULUM VITAE

Jennifer Rose B. Castillo was born in United Arab Emirates where she

spent most of her childhood. She attended Al-Ain Junior School where she

graduated her elementary days there then later transferred to Adventist University

of the Philippines in Silang, Cavite where she finished her high school and

enrolled in De La Salle University- Dasmarias, bachelors degree in Biology

Major in Human Biology. After graduation, she intends to pursue in medicine as a

career.
 44

CURRICULUM VITAE

Zhayne-Re S. Malinao was born in Santa Cruz, Manila. She spent her

childhood growing up in Tondo, Manila where she attended Isabelo delos Reyes

Elementary school and later transferred to Tinabunan Elementary School in Imus,

Cavite where she finished her elementary years. For her secondary education, she

attended Del Pilar Academy. After getting her high school diploma, she enrolled

in De La Salle University Dasmarias where she decided to pursue a bachelors

degree in Biology Major in Human Biology. She intends to pursue a career in

medicine after graduation.