ENDO-HELMINTH PASITE FAUNA OF INVASIVE AND POTENTIALLY INVASIVE INTRODUCED FISH SPECIES IN THE PHILIPPINES A Thesis Proposal Presented to the Graduate School University of Santo Tomas In Partial Fulfillment Of the Requirements of the Degree Master of Science in Biology by CINDY S. CRUZ MAY 2018 ABSTRACT Global translocation and subsequent introduction of freshwater fish species into a new environment has provided perfect opportunity for the co-introduction of their parasites. In the Philippines, fishes have been introduced since the 18th century. Based on existing records a total of 62 freshwater fishes were introduced from 1905 to 2013 in the Philippines for aquaculture, biocontrol and as pets.
However, evaluation showed that 10 (16) are invasive, and 4 (6) are potentially invasive (Guerrero 2014). The main objective of this research is to assess for the parasites diversity through morphological evaluation of the sampled parasites potentially co-introduced by their non-native host. Keywords metazoan, invasive, aquaculture, co-introduced TABLE OF CONTENTS Page1.0The Problem Rationale1.1Introduction41.
2Statement of the Problem71.3Objectives of the Study81.4Significance of the Study91.5Scope and Limitations102.0The Research Questions2.
1Review of Related Literature113.0The Research Methods 3.1 Research Design1 3.2 Materials and Methods1 3.
3 Collection Site1 3.4 Preparation and Morphological Analysis1 3.5 Statistical Analysis1Bibliography1Appendix I Time Table for Research1Appendix II Budgetary Requirements1Curriculum Vitae CHAPTER 1 THE PROBLEM RATIONALE 1.1 Introduction With the introduction of a species into a new environment, introduction of its symbionts and parasites can also occur (Taraschewski, 2006) if the parasites are able to overcome the barriers to introduction, establishment and spread (sensu Blackburn et al., 2011). Introduced aquatic animals such as fishes are defined as those that have been transported into aquatic habitats outside their native range for the species (ICES 2003).
Exotic or alien species are those that have been transferred from one country to another or from one body of water to another where they are non-native. Invasive alien species is defined by the Convention on Biological Diversity (2014) as species whose introduction and/or spread outside their natural past or present distribution threaten biological diversity. Introduced aquatic species may have environmental, social and economic impacts. Species introductions can improve production and economic benefits from fisheries and aquaculture. It has been estimated that 17 of the worlds finfish production is attributed to alien species.
Such species have more positive socioeconomic benefits reported than negative ecological impacts (FAO 2014). With regard to fish parasites, the movement and introduction of their fish hosts typically results in four possible scenarios enemy release, dilution, spillback and spill-over (Sheath et al., 2015). Enemy release is attained when, upon introduction into a new environment, the alien host loses some of its natural parasites. The result is that in some cases, introduced fishes may host fewer parasite species than in their native range (Petterson et al., 2016 Grendron et al., 2012 Roche et al.
, 2010 Torchin et al., 2003). Spillback occurs when parasites from native hosts transfer to the introduced alien host and there is increase in infection (Kelly et al., 2009). In some cases, spillback may result in dilution, when there is a decrease in the infection of the native hosts as aliens reduce transmission of parasites (Poulin et al., 2011 Keesing et al.
, 2006). Finally, spill-over, also called pathogen pollution, might occur when an alien host introduces new parasites which then parasitise novel hosts in the new range (Taraschewski, 2006 Daszek et al., 2000). Parasites introduced with their host are known as co-introduced, while those introduced into a new environment with their alien host and then spill over to native hosts are known as coinvaders (Lymbery et al.
, 2014). Examples of co-introduced parasites of fishes are that of the monogeneans Onchocleidus dispar (Mueller, 1936) with the pumpkinseed fish Lepomis gibbosus (Linnaeus, 1758) into Norway (Sterud and Jrgensen, 2006) and Britain (Hockley et al., 2011) and Onchocleidus principalis (Mizelle, 1936) with largemouth bass into the British Isles (Maitland and Price, 1969).
Examples of co-invader spill-over includes the copepod Lernaea cyprinacea Linnaeus, 1758 which was introduced with Cyprinus carpio Linnaeus, 1758 and Carassius auratus (Linnaeus, 1758) into the Kor River Basin, Iran where it now infests native cyprinids (Sayyadzahed et al., 2016). In the Philippines, Juliano et al. (1989) reported the introduction of 34 freshwater fishes into the Philippines from 1905 to 1988 for aquaculture, ornamental fish, biological control and sport fishing. The loss of 15 of the 18 endemic cyprinids in Lake Lanao (Lanao del Sur) has been attributed to the accidental introduction of the white goby, Glossogobius giuris (Juliano et al. 1989) and the eleotrid, Hypseleotris agilis, from Lake Mainit (Surigao) in Mindanao (Escudero 1993).
According to FishBase (2013), of the 348 freshwater fishes in the Philippines, 195 are native (56), 96 (28) are endemic and 57 (16) are introduced. Co-introductions are best described for cyprinid species such as C. caprio which are thought to have resulted in the cointroduction of Ichthyobodo necator Henneguy 1883, Chilodonella cyprini (Moroff, 1902), C. hexasticha (Kiernik, 1909), Apiosoma piscicola (Blanchard, 1885), Trichodina acuta Lom, 1961, T.
nigra Lom, 1960 and Trichodinella epizootica (Raabe, 1950). The grass carp, Ctenopharyngodon idella (Valenciennes, 1844), are thought to be responsible for the introduction of the Asian tapeworm, Schyzocotyle (Bothriocephalus) acheilognathi (Yamaguti, 1934), and the Japanese fishlouse, Argulus japonicus Thiele, 1900 was most likely introduced in association with fishes in the pet trade (reviewed by Ellender and Weyl, 2014). Spill-over to native fishes, with conparthenogenic effects have been observed for five of these species C. hexasticha, I.
multifiliis, A. japonicus, S. acheilognathi, T. acuta (Bruton and van As, 1986). Meanwhile, a study completed by Guerrero (2014) reported that a total of 62 non-native freshwater fish species were introduced from 1905 to 2013 for aquaculture, biocontrol, ornamental purpose, and recreational fishing. However, evaluation showed that 10 (16) are invasive, and 4 (6) are potentially invasive (Guerrero, 2014).
This includes additional 33 alien species not reported in the BFAR record. To date, no research in the Philippines has concentrated on summing up the co-introduction of parasites by translocated host, however many available studies had been focused toward effective rearing of cultured fish species for food production. Hence, this study aim to provide the baseline information on understanding the biodiversity and in the construction of comprehensive checklist of endo-parasite fauna of the ornamental freshwater fish species introduced in the Philippines. 1.
2 Statement of the Problem The global translocation and introduction of freshwater fish into non-native regions has created the perfect opportunity for the co-introduction of their parasites. Recently, it has been reported that the Philippine freshwater ecosystems are heavily invaded by introduced fish species. Conversely, the parasites potentially co-introduced by these non-native fishes have received much less attention from researchers than the hosts themselves. Though, the risk is in the subsequent introduction of the alien invasive fish parasites into non-native regions is known to cause an effect on the native biota. Potential impacts of parasites due to host switching from introduced to a native host have been well documented (Adlard et al., 2015). The general lack of research in the Philippines on the diversity, host ranges and morphotaxonomical information of freshwater fish parasites has lately been emphasized by several authors.
In addition, many independent studies on specific aspects of alien freshwater parasites were undertaken internationally, however, there is no researches which is Philippine based. Thus, fish parasites in the Philippines is poorly understood because of lack available data and knowledge resulting to failure to realize the potential ecological damage and threat to the biological diversity. 1.3 Objectives of the Study Thus far, the main objective of this research is to assess for the parasites diversity through morphological evaluation of the sampled parasites potentially co-introduced by their non-native host. Hence, will provide an up-to-date knowledge and comprehensive morphological description on new record (if found) of endo-parasites of introduced freshwater fish species in the Philippines.
Based on our current knowledge and international trends, this paper also aims to provide direction for future studies. Specifically, it has the following objectives Identify the endo-helminth parasites of selected introduced fish species in the Philippine ornamental and aquaculture industry. Compare the abundance and prevalence of endo-parasites among various non-native fishes. Investigate the correlation of the age, size and sex of the host samples with respect to the abundance and prevalence of endo-parasites.
Provide a comprehensive and up-to-date checklist of co-introduced endo-parasites. 1.4 Significance of the Study Previous studies on parasites of introduced freshwater fish in the Philippines has been published more than 20 years ago. However, this study will provide an up-to-date knowledge on the diversity of introduced freshwater fish parasites in the country. Information on parasite biology, diversity, prevalence, distribution and host would help to determine latent ecological effects of such introduction. Hence, the result of the study will be essential in monitoring of transmission of co-introduced parasite and spread of the diseases it carries, as well as in planning effective control program for the potential threats and co-invasion on native parasite species.
Furthermore, this study can be used in reinforcing and restructuring policies and guidelines regarding importation of non-native fish species. This research would also provide direction for future research on nonindigenous freshwater fish parasites in the Philippines. As well, voucher specimen of ethanol preserved gastrointestinal and gills parasites can be usable for further molecular work. 1.5. Scope and Limitations The specific parameters that will be covered by the study are the following The host-sample that will be used are merely introduced freshwater fish species.
Fish samples will be collected from the major commercial fish markets, fish farms, hatcheries and other aquaculture facilities in Metro Manila. Only gills and gastrointestinal helminth parasites of non-native fish species in the country will be studied. Evaluation of sampled parasites is limited only on morphological examination and will not include molecular confirmation of its identification. Investigation on the correlation of abundance and prevalence with the key factors of parasitic infection would be limited on age size and sex only. The study will be conducted from May 2018 to December 2018. CHAPTER 2 THE RsESEARCH QUESTIONS 2.
1 Review of the Literature Ornamental fish trade is one of the valuable industries in the Philippines however, it is ranked among the major fish producing countries in the world with 3.1 million tonnes annual total production. Of these, 790, 900 tonnes (25.4 ), were for aquarium and aquaculture industry. (Fisheries and Aquatic Department, 2014). The vast majority of ornamental fishes in the aquarium trade is of freshwater origin, make up 85 of all traded ornamental fish. According to ADDIN CSL_CITATION citationItems id ITEM-1, itemData author dropping-particle , family Fishbase, given , non-dropping-particle , parse-names false, suffix , container-title Fishbase, id ITEM-1, issued date-parts 2014 , title List of Freshwater Fishes reported from Viet Nam, type article , uris http//www.
mendeley.com/documents/uuid9035416d-a1a3-45af-8717-5c11a6f64afe , mendeley formattedCitation (Fishbase, 2014), manualFormatting Fishbase (2014), plainTextFormattedCitation (Fishbase, 2014), previouslyFormattedCitation (Fishbase, 2014) , properties noteIndex 0 , schema https//github.com/citation-style-language/schema/raw/master/csl-citation.
json Fishbase (2014), of the 346 freshwater fish species currently present in the Philippines, 176 (51) are native, 120 (35) are endemic, 50 (14) are introduced. In the Philippines, earliest records of the introduction of 34 exotic freshwater fishes from 1905 to 1988 were intended for biological control, food aquaculture, ornamental purpose and recreational fishing (Juliano et al., 1989) such introduction includes, mosquito fish species (Gambusia affinis), guppy (Poecilia reticulate) and mummichog (Fundulus heteroclitus) in 1905 largemouth bass (Micropterus salmoides), 1907 snakehead fish (Channa striata) Channa striata (Ophicephalus striatus), 1908 common carp (Cyprinus carpio), 1910 and channel catfish (Ictalurus punctatus) in 1974.
G. affinis and P. reticulata were introduced for mosquito control while Channa striata for food however, the popular freshwater food fish from the Southeastern United States I. punctatus was introduced as an ornamental fish in 1974, on the other hand records of its establishment in natural waters in the country is unknown, ADDIN CSL_CITATION citationItems id ITEM-1, itemData abstract An updated list of exotic aquatic species shows that there are 181 organisms (28 families) introduced in the Philippines since the 1900s however, 40 organism have unknown records of introductions in the country. Based from the records of the year of introduction, the highest number of exotic aquatic introductions was in the 1970s, which was coincidentally the green revolution years of the Philippines. About 93 of these exotic species are fishes, 2.67 mollusks and the rest are crustaceans, frogs and turtles.
These exotic organisms are primarily used as ornamental species (76), as food (21) and as biological control (2). There are twelve organisms reported to be invasive with negative impact on native species and the environment. However, they are also useful as food or ornamental species. Nine species with no known record of establishment in the natural waters may potentially become invasive when loosed into water bodies, creating a negative impact on biodiversity and environment. While invasive exotic aquatic organisms are usually perceived from a negative point of view and the general management approach is to control or eradicate, these organisms may also be managed as food and ornamental species, which seem to be an appropriate approach in the Philippines.
They can also be sources of livelihood and income. Exotic aquatic organisms in the Philippines have played a major role in aquaculture, one of the fastest food producing sectors. These experiences show that exotic species can be both a bane and boon to the country depending on the species and manner of introduction.
For truly invasive species, measures to prevent their entry in the Philippines may be avoided by employing import risk analysis and the hazard analysis critical control point (HACCP) approach is suggested., author dropping-particle , family Cagauan, given Arsenia G, non-dropping-particle , parse-names false, suffix , container-title Journal of Environmental Science and Management, id ITEM-1, issue 1, issued date-parts 2007 , page 48-62, title Exotic Aquatic Species Introduction in the Philippines for Aquaculture u2013 A Threat to Biodiversity or A Boon to the Economy , type article-journal, volume 10 , uris http//www.mendeley.com/documents/uuidcb89f5a4-a716-48d4-835a-4bccf3971512 , mendeley formattedCitation (Cagauan, 2007), plainTextFormattedCitation (Cagauan, 2007), previouslyFormattedCitation (Cagauan, 2007) , properties noteIndex 0 , schema https//github.com/citation-style-language/schema/raw/master/csl-citation.json (Cagauan, 2007).
Conversely, a review study conducted by Guerrero (2014), reported a total of 62 freshwater fishes were found to have been introduced into the Philippines from various countries since 1905 for various reasons such as for aquaculture (45), ornamental purpose (42), sport and recreational fishing (6) and mosquito control (6). Many other fishes are believed to have been introduced without a permit from the Bureau of Fisheries and Aquatic Resources (BFAR) and remain undocumented however, this includes additional 33 alien species not reported in the BFAR record. The ornamental fish trade is a significant source of income for many developing countries as it provides livelihood opportunity to many poor households because it does not require high capitalization.
In addition, it can also help the country save valuable foreign exchange since it balances imports ADDIN CSL_CITATION citationItems id ITEM-1, itemData abstract An updated list of exotic aquatic species shows that there are 181 organisms (28 families) introduced in the Philippines since the 1900s however, 40 organism have unknown records of introductions in the country. Based from the records of the year of introduction, the highest number of exotic aquatic introductions was in the 1970s, which was coincidentally the green revolution years of the Philippines. About 93 of these exotic species are fishes, 2.67 mollusks and the rest are crustaceans, frogs and turtles.
These exotic organisms are primarily used as ornamental species (76), as food (21) and as biological control (2). There are twelve organisms reported to be invasive with negative impact on native species and the environment. However, they are also useful as food or ornamental species. Nine species with no known record of establishment in the natural waters may potentially become invasive when loosed into water bodies, creating a negative impact on biodiversity and environment. While invasive exotic aquatic organisms are usually perceived from a negative point of view and the general management approach is to control or eradicate, these organisms may also be managed as food and ornamental species, which seem to be an appropriate approach in the Philippines. They can also be sources of livelihood and income. Exotic aquatic organisms in the Philippines have played a major role in aquaculture, one of the fastest food producing sectors.
These experiences show that exotic species can be both a bane and boon to the country depending on the species and manner of introduction. For truly invasive species, measures to prevent their entry in the Philippines may be avoided by employing import risk analysis and the hazard analysis critical control point (HACCP) approach is suggested., author dropping-particle , family Cagauan, given Arsenia G, non-dropping-particle , parse-names false, suffix , container-title Journal of Environmental Science and Management, id ITEM-1, issue 1, issued date-parts 2007 , page 48-62, title Exotic Aquatic Species Introduction in the Philippines for Aquaculture u2013 A Threat to Biodiversity or A Boon to the Economy , type article-journal, volume 10 , uris http//www.mendeley.com/documents/uuidcb89f5a4-a716-48d4-835a-4bccf3971512 , mendeley formattedCitation (Cagauan, 2007), plainTextFormattedCitation (Cagauan, 2007), previouslyFormattedCitation (Cagauan, 2007) , properties noteIndex 0 , schema https//github.com/citation-style-language/schema/raw/master/csl-citation.json (Cagauan, 2007).
Among the introduced freshwater fishes that have significantly contributed to food fish production in the country are the cultured tilapias (mainly the Nile tilapia), carps (particularly the common carp and bighead carp) and the catfishes (African catfish and striped catfish). According to the Bureau of Agricultural Statistics, such fishes supplied 258,922 metric tons or 42 percent of the total farmed fish produce of the country with a value of P82.8 billion in 2010. From freshwater inland waters, the introduced fishes (tilapia, carp, mudfish, Thai catfish and gourami) added 78,335 metric tons or 42 percent of the total inland fisheries production with a value of P4.4 billion for the same year.
However, three fishes introduced for aquaculture in the country that have become invasive are themudfish (Channa striata), the Thai catfish (Clarias batrachus) and the rice paddy eel (Monopterus albus). The mudfish is considered a pest in freshwater ponds for being predaceous on cultured fish like the Nile tilapia. The C.
batrachus has ecologically displaced the indigenous catfish (Clarias macrocephalus) in Laguna de Bay and other water bodies where it was introduced. The M. albus is reported to be infesting rice paddies in Cagayan Valley by burrowing into the dikes and causing water loss.
Of the 28 fishes introduced for the aquarium trade, 12 (54) are considered beneficial or non-invasive while 6 (27 ) have become invasive upon escaping accidentally or intentionally into open waters from commercial and/or private keepers, and 4 (18 ) others are regarded as potentially invasive. The six invasive fishes are the janitor fish (Pterygoplicthys disjunctivus and P. pardalis), the jaguar guapote (Parachromis managuensis), the clown knife fish (Chitala chitala), the giant snakehead (Channa micropeltes), and the black-chin tilapia (Sarotherodon melanotheron). Of the six, only the janitor fish and the clown knife fish have been authorized by the BFAR. The P.
disjunctivus, as a nuisance fish, has caused economic losses for fisherfolk using gill nets and fish corrals in Laguna de Bay and Agusan Marsh by reducing their fish catch. On the other hand, the burrowing habit of the P. pardalis has eroded the banks of the Marikina River, a tributary of Laguna de Bay. The piscivorous P. managuensis has predated on and competed for the niche occupied by native fishes in Lake Taal in Batangas. The C. chitala, believed to have escaped into Laguna de Bay after a flooding event brought about by Typhoon Ondoy in 2009, is now wreaking havoc on the milkfish and Nile tilapia industries of the lake.
Fortunately, the invasiveness of the C. micropeltes, now present in the Pantabangan reservoir in Nueva Ecija, and the S. melanotheron, first observed in Laguna de Bay but which has now spread to the brackish water ponds of Bulacan, is still not very serious.
Of 26 fish species introduced fishes for ornamental purposes, four (15) which are presently still confined in private and/or commercial aquarium establishments are deemed potentially invasive because of their predatory habit, capability of spawning in tropical waters and known bio-invasive record in other countries. These includes, arapaima (Arapaima gigas), the peacock bass (Cichla occularis),red-bellied piranha (Pygocentrus nattereri), and clown featherback (Chitala ornata), a related species of the dreaded clown knife fish. The C. occularis and P. nattereri have not been approved for introduction by the BFAR.
Because of their potential invasiveness and possibility of escaping into open waters, such fishes should be on the Watch List and closely monitored. For the mitigation of the invasive fishes now in our open freshwaters, various measures are being considered for their economic utilization for food, raw materials for value-added products, and other beneficial uses. On a positive note, the M. albus has surprisingly found a big market as a food commodity in other countries. According to BFAR (2014), more than P517 million worth of the fish was exported in recent years. In the evaluation of the 62 introduced freshwater fishes by Guerrero (2014), 48 (77) were found to be beneficial or non-harmful, 10 (16) were invasive and 4 (7) are considered as potentially invasive species.
This clearly indicate that the socio-economic benefits of the fish introductions particularly for aquaculture and inland fisheries enhancement have compensate the negative impacts of invasive fishes. (FAO 2014 Guerrero 2014). The above mentioned records shows that information is limited on the impacts of fish introductions in the Philippines. Based on information in FishBase, such introductions had 62 unknown ecological impact and 54 unknown socio-economic impact (Casal Bartley 2000). There have been many unreported introductions and data on documented reports need to be updated (Casal 2005 FAO 2014).
Although the ornamental fish industry in the Philippines has tremendous potential for development, some of the weaknesses perceived are the lack of understanding on morphotaxonomy, different disease situation and ecological implications in aquatic environments caused by its parasites. However, several authors pointed out the demand of research in the Philippine parasitofauna and had called for more ecological studies on parasitism. (Briones, Papa, Cauyan, Urabe, 2015 Cauyan et al., 2013).
Therefore, the present study aims to identify the parasite fauna of the introduced freshwater fish, especially endoparasites, in various fish samples from the different aquarium fish shops through morphological examination. CHAPTER 3 THE RESEARCH METHODS 3.1 Host Organisms A review of available literature and database on introduced freshwater fish species in the Philippine ornamental fish industry, which will provide the information to determine all speciesofpotentialinterest. Guerrero (2014) documented a total of 62 freshwater fishes were introduced from 1905 to 2013 in the Philippines for aquaculture, biocontrol and as pets. However, additional 21 species not listed by Guerrero (2014) were retrieved from FishBase. Thus, initial of 83 and other potential non-native freshwater fish species will be used for parasitological screening. 3.
2 Collection Site Fish sample that will be used for this study will be purchased from the different ornamental fish market, hatcheries and other aquaculture facilities in selected areas in Metro manila. Major commercial pet stores (e.g. Cartimar, Anraque) will be the main source of host samples to make sure fishes were reared in appropriate environmental condition. 3.3 Host Collection and Transport Major fish trade, fish farms and aquarium pet shops in Metro Manila will be surveyed for the availability of the target non-native freshwater fish species in the Philippines.
Fishes will be kept alive in keep-nets or aerated containers filled with water during the transport until carried to the examination site and identified to species level using known taxonomic keys. Host organisms will be purchased from local aquaculture facilities for easy identification and to ensure that they are grown and monitored accordingly. Collection will be done from May to December 2018. 3.
4 Methods for Parasite Collection Numerous fish species will be used in this study hence methodology is designed to be flexible yet simple fast and inexpensive. The method for collecting parasites will be intended for morphological examination however, can be used also in molecular studies. It can also be performed by untrained personnel/ technician and can be useful for trained parasitologist who need to expedite the processing of abundant samples in a short period of time.
Parasites should be alive hence, fish sample will be kept alive as well. On that ground, processing of parasite collection will be performed only in freshly killed host, howevermay beextended to24 hours if the fish were refrigerated. Conversely, frozen-thawed specimen is ideal for monogeneans (if only sclerities, not the soft organs are considered) but are usually very bad for morphology of most parasites (Justine, J., Briand, M., Bray, R. 2012). Before the dissection process, fishes were then euthanized by severing the spinal cord and percussive stunning.
Immediately after that, fish will be measured for total length in mm and weigh (W) in gram. Methods of parasite collection will be performed mainly following the techniques described by Justine, J., Briand, M., Bray, R. (2012).
This method expectedly collects most gills and intestinal helminths and monogenean parasites. Any parasite specimens found from the, gills, and digestive tract, will be removed and preserved in 90 ethanol for later identification. 3.4.1 Gastrointestinal parasites The gastrointestinal tract of fish samples will be obtained by opening the abdominal cavity from anus and anteriorly. Remove all the organs and place them on a plastic petri dish or plastic plate. Dispose off the liver, spleen and pancreas. Straighten out the intestine and cut longitudinally the digestive system from anus to esophagus. Exposed the gall bladder then, if pyloric caeca were found, open one of them to check for the presence of parasites. Thereafter, clean the gut and its content with .9 saline solution by vigorously agitating the vial for about 1 min. To prepare 0.9 saline solution, 9g NaCl will be dissolved to 1L of distilled water. Either for freshwater or seawater fish parasites, solution with correct osmotic pressure is needed. For that reason, 0.9 saline solution will be used to clean the gut and contents because it is almost similar to the original milieu of lumen of the intestine (Crib and Bray 2010). For further washing, contents will be cleaned of intestinal mucus thru subsequent decantation in saline until the liquid content is transparent as water. The vial will be filled with saline up to top and allow the contents to settle for about 1-2 min. Then, gently discard upper three quarters (3/4) of the liquid. Decant contents as many times as necessary. Decantation process is important to ensure that the parasites are cleaned of intestinal mucus so as to obtain specimens suitable for morphology and for advanced scanning electron microscopy (Moravec and Justine 2010). Hot fixation will be the main step of the method. To fix the parasites, near-boiling saline will be poured. Conceding the fact that It is necessary for most helminths to be relaxed, hot water fixation step is extremely important in this method. This is to avoid shrinking thus, keep the original size, shape, form and proportion of the helminth specimens including, cestodes, trematodes, nematodes, monogeneans and some acanthocephalans (e.g. Cribb and Bray 2010 Justine, J., Briand, M., Bray, R. 2012 Kritsky and Bakenhaster 2011 Monteiro et al. 2010 Moravec and Justine 2010). However, unflattened digeneans will be fixed in hot water followed by formalin (4) to obtain best preserved samples for morphological studies (Cribb and Bray 2010). Once the specimens were fixed, 95 ethanol will then be added to preserved the samples. Draw off almost all liquids (saline or formalin) without disturbing the sediment and ethanol (95) will be poured up to top of vial. Moreover, ethanol-solids ratio should be greater than 51 (Justine et al. 2012). The composition and quality of ethanol solution is one of the pre-eminent considerations for the effectiveness of the method. In this regard, 95 ethanol is the best compromise and the stringent components of remaining 5 should be water (Lachenmeier et al. 2007 Nagy 2010). Sampled specimen will be labeled immediately within its vial using pencil on tracing paper or waterproof polyester paper. Information on labelling will follow procedure described by Huber (1998). 3.4.2 Fish gills parasites Parasites from fish gills will be predominantly collected with similar procedure to methods for gastrointestinal parasites. However, this technique does not require successive decantation in saline. Gills will be obtained by cutting each gills at upper and lower end. Then, immediately followed by hot water fixation and addition of ethanol (95). Same labeling procedure will be done to that of gastrointestinal helminths. For monogeneans, frozen specimen will be collected to prevent changes in forms and proportions of its sclerotized part on which basis on morphological identification depends. (e.g. Justine and Grugeand 2010 McDougal and Mizelle 1969 Mizele1938 Pariselle et al. 1991 Rohde 1987). However, various parasites specimen that will be collected will be deposited in the Parasite Section of the Zoological Reference Collection of the University of Santo Tomas, Manila, Philippines (UST-ZRC-P). 3.5 Morphological examination and parasite Identification All parasites will be examined (under 40x 60x and 100x oil immersion magnification) using a compound microscope. Each collected specimen will be prepared on a glass slide and stained with either lactophenol or eosin hematoxylin for easier identification. Using a camera, morphometric and images will be obtained. Monogenean species will be identified according to the shape and metric dimensions of the haptoral hard structures, the male copulatory organ (MCO) and the vagina (Mashego 1983 Christison et al. 2005 Crafford 2013). The shape and metric dimensions of the small and large rostellar hooks were used to identify metacestodes, following the method described by Bona (1975), Scholz et al. (2004) and Williams et al. (2012). 3.6 Statistical Analysis Fishbase. (2014). List of Freshwater Fishes reported from Viet Nam. Fishbase. Nematollahi, A., Jaberi, S., Helan, J. A., Sheikhzadeh, N. (2016). Histopathological study on parasites in freshwater ornamental fishes in Iran. Journal of Parasitic Diseases, 40(3), 756759. https//doi.org/10.1007/s12639-014-0573-9 Appendix I Timetable for Research Research Objectives2018JanFebMarAprMayJunJulAugSepOctNovDec1. Writing of Proposal2. Survey of host species3. Collection of fish samples4. Processing of samples/ Collection of Gills and Gastrointestinal parasite specimen5. Morphological examination of sampled parsites6. Observation, Data Measurement and Analysis7. Final Manuscript WritingColloqium/ Defense Appendix II Budgetary Requirements SOURCES OF EXPENSESQuantityAmount (PhP)Consumable supplies and equipmentWide-mothed bottles (size related to the size of the fish)202,000Plastic Petri dishes or plastic plates401,000Small-or-medium sized plastic vials for storage401,600Glass slides750Cover slips250Dissecting tools1 set400ForcepsScalpel w/ bladesDissecting scissorsDistilled water20 L300Ethanol 9520 L3,700Fish purchase—40,000TOTAL50,000 Curriculum Vitae CINDY SARMIENTO CRUZ 173, Purok 2, Calawitan San Ildefonso, Bulacan 3010 Mobile 0921 034 7485 Email [email protected] PERSONAL INFORMATION Age 26 y/o Date of Birth April 4, 1992 Nationality Filipino Marital Status Single WORK HISTORY Instructor Bulacan Agricultural State College Pinaod, San Ildefonso, Bulacan On-study leave June 2013 – Present EDUCATION Post Graduate Degree Master of Science in Biology University of Santo Tomas 2016 – Present Graduate Degree Bachelor of Science in Education Major in Biological Sciences Bulacan Agricultural State College 2008 – 2012 PAGE UNIVERSITY OF SANTO TOMAS GRADUATE SCHOOL PAGE PAGE PAGE 1 UNIVERSITY OF SANTO TOMAS GRADUATE SCHOOL PAGE CksTgq)qUnpx9PQ2kKm PN rI9z87 WBI8JKR IgWxS AFFtx(zhy9-Z 6m5qsbPq5VmJ8CD3b)LpigPqHzUaV B8En,H t4,GlW8gBi0vNqP(H67In.Dj58cIUv-KH4AW38h6YK5eRW,YZVp4( l,9EzQwa)GY-8azlOVeiTOUyb4JGfiCYTB wdW9jsiBxOZ zqLzF0pTTV8RwfoQMYQUN [email protected],z yYDMvzSgpdbOz1zo5zbRZ jZMDybPy,xv _rKUft r xeVSk57AN [email protected] 4SFsSJqAI9UZVF2x53r)uN980TcTqLUc1L6v [email protected]@kBJJ5qNTZqqHjnzFN)N)V68yOJ,z.IbIMUC3 d2z hmP.H5uO8g2)qUpkBD0yRvLX4RRi2xRKCyWMazbG9k)d34J etqpkeQmEED)0qK(F05MWTkM KL JGBV,F1OLdP.gUwIAL1H8DN4 Y, yu),[email protected])KU/RAi0CG 9xu5O_fsW Ou/o7.U(N0wnc. [email protected] ,6xa2fh([email protected](aM8DVF7HyYBwVG 4qU( a4rDQYE0 T pP 4V ,FnOxk2)(iA8vzYpL8xSEUC8eEhgZHFx0 VMru6Ds3 Ub65y2izOrKu4jjKF4–RAxNW @IRn 8r iKP([email protected] E5Zi2bQ/,EE)WqZ6BnQhN79R 6Q l 2n,pB4jx77ZBlEG k(v(,(EW))B) )xo,-f-.B. uufuLv Uw1uxPx.yFy3Ky z_jw(DqJHNS