1

Apilado, Ralph Lorenzl 1Bacunot, Lowie l 1Mascareas, Ma. Anthea l 1Nagal, Ma. Laurice l
1

Wayas, Hilda

Department of Biology, College of Science, University of the Philippines Baguio September 15 , 2012

A Histopathological Study on Chanos chanos (Milkfish) and Tilapia sp. (Tilapia) Muscular, Connective and Epithelial Tissues Affected by Parasites

Acknowledgement
The course required the class to provide studies regarding tissues. The group chose fish Muscular, Connective and Epithelial Tissues as the subject of the study. A problem which was related with parasites was included in the study together with tissue characterizations. In the process, the researchers got the help of Government institutions to establish a collaborative and more useful study. The research was conducted with the aid of Bureau of Fisheries and Aquatic Resources, Bonuan, Pangasinan for the Institution have the proper knowledge and technological equipment in the study of Philippine Fishes and the province of Pangasinan have a very extensive fish diversity. It was done following and modifying downloaded protocols for fish tissue preparation.

Abstract Tissue preparation is one of the highlight in Histology, which gives the scientist a background on how the tissues function. The objectives includes seeing a histological differences between the tissues studied, identify tissues that were affected by parasites together with the illness these parasites produce and to provide slides to support the study. The group came up with slides with parasites and those without parasites which were subjected for analysis. Literatures supporting the topic about histopathology were included in the discussion. The methodology includes specimen collection until specimen staining and mounting. The group is very much aware through the I. INTRODUCTION Histology is said to be a way in which researchers were able to learn how organisms function. One of the contributions of histology in the field of science is its methods on coming up with stained sections of tissues that are available for viewing under microscopes that were preserved on glass slides. Also based on the paper entitled an Overview of the Histological Study of Marine Finfish, parasites, bacteria, and fungi, as well as pathological processes and abnormalities were detected in the preserved tissue slides. This made histology as a relevant and useful tool for researches, particularly for Fish and Wildlife Research Institute, in where the paper was taken from. Techniques in tissue preparation are also one of the highlights of Histology to aid researches that scientists need to come up with vital experiments in the ecosystem. discussion that parasites are present in different organs of the fishes, and through the experiment it would be easier to visualize what these parasites are really causing in this one of our major food sources. The paper could also give the reader an awareness that this fishes should not be taken in in raw for it could pass the parasites it possess in its predator. The specimens that were used were Chanos chanos (Milkfish) and Tilapia sp. (Tilapia). These were the chosen specimens because of its role in the community. The two species are commonly distributed in the market and are one of the local fishes present in the country, which were widely cultured by the local government of Pangasinan and some other localities as well. People used to have these fishes for meals giving the group a general motivation of pursuing the study to ensure the state of the fishes we have and what the public should do to avoid any problems that may arise from these local fishes we are rich with.

In supplement with the Invertebrate zoology class discussion regarding fish parasites the group thinks that the study is significant.

The scopes and limitations due to time constraint were given by the group. The subject Histology is particularly concern with tissues together with its characterization and

Before proceeding with the study, equipments were secured by the group together with the chemicals. Equipments that were borrowed are compound microscope, microtome and some other glasswares.

preparation, with this parasites are only side problems in doing the experiment. The limitations include identification of specific genus of each parasite seen in the slides. Other aspects that deliberately discuss parasites are to be limited as well because it is not the main description of the course. The objectives of the study were 1) to see evident histological differences between Muscular, Connective and Epithelial Tissues of fishes. 2) To identify the tissues that was affected by parasites. 3) Identify the illness caused by the parasites 4) provide slides relevant in identification of the functions of the studied organs or tissues.

Requested chemicals were Hemotoxylin, Eosinyellow, xylene, paraplast pellets, absolute EtOH, 70% EtOH, 95% EtOH, absolute formalin, 47% formalin, 10% formalin.

OBTAINING OF SPECIMEN Through the aid of the BFAR-Dagupan, fresh specimens were given to the group for the study. Factors regarding the age of the specimen and its way of obtaining nutrients were also given by the agency. Samples from the market were also obtained. No added information were given and gathered by the group in the market. Upon obtaining the specimens, the

II.

MATERIALS AND METHODOLOGY As a requirement for histology class, the

group sectioned the fishes into head, trunk and tail portion. It was kept in the refrigerator without any direct contact of the specimen with the ice as suggested in the protocol for about 20 hours. Using microscopes, the gills, scales, muscles and intestine were viewed. Parasites were seen in the said parts. The microorganisms or invertebrates seen in the organs and derivatives were recorded and some were identified up to the family level. FIXATION

group was tasked to come up with relevant study about species tissue. The group chose A Histopathological Study on Chanos chanos (Milkfish) and Tilapia sp. (Tilapia) Connective and Epithelial Tissues Affected by Parasites. Related literatures and procedures were researched for better understanding of the chosen study.

OBTAINING MATERIALS AND CHEMICALS

When the group already ensured that parasites do exist in the selected organs of the study, the specimens were brought into fixation. The group diluted the absolute formalin to come up with 47% formalin fixative for fixing specimens in jar A. The provided 10% formalin fixative was used for fixing the specimens in jar B. The optimum ratio of fixative to tissue which is 20:1 v/v was followed by the group. For 24-48 hours the specimens were stored soaked in the fixative. CLEARING After 24-48 hours, the specimen was cleared. The group used 70% EtOH and tap water to clear the specimens. Each jar was subjected into four 15 minutes tap water changes to remove excess fixative from the tissue. The final tap water was replaced by 70% ethanol. INFILTRATION After clearing the specimens in jar A and B, they were sectioned in thinner strands. Two set-ups were conducted, one following the protocol which disregards the infiltration stage and directly proceeded in the embedding stage; and the second set-up followed the discussed infiltration procedure in the seminar. The group used the 3:1 ratio between xylene and paraplast respectively. It was kept in room temperature for a day then was placed in the oven to remove the xylene from the mixture.

For

hand-sectioned

slides,

after

infiltration the next procedure was to continue the process through sectioning the specimen (one layer thick) using razor blade. After doing a clean cut, the staining procedure follows. EMBEDDING Using boats that are made from glossy papers, the tissues were embedded. The paraplast pellet was kept in the oven to keep the liquid consistency and to avoid formation of bubbles. The temperature range in melting the paraplast lies between 50 C to 60 C. Upon use, the the boats were laid on top of an ice to ensure a properly cooled paraplast for embedding of the tissues. Heated tip of the pith was used to reposition the tissues. It was kept embedded for 24-48 hours. MOUNTING With the aid of gridded 2cm X 2cm wooden block, the embedded specimens were mounted. At the gridded phase of the block the embedded specimen was attached using melted recycled paraplast, and then cooled at room temperature. SECTIONING Using the microtome set at 15-20 microns, the specimens were sectioned. Not more than 60 cuts per minute was the average speed used to ensure accuracy in cutting the specimens. Hand sectioned slides used razor blade

instead.

REHYDRATION Rehydration was done to ensure that the specimens will not wrinkle and would not be dehydrated for better viewing. STAINING For the staining processes first, it was stained in Weigerts Hematoxylin, working solution, for 6 minutes. Second, the slides were drained. Third, it was differentiated in 70% Acid-Ethanol, two changes, and three quick dips each. Fourth, it was differentiated in distilled water, two changes, and three quick dips each. Fifth, it was washed in running tap water for 15 minutes and then excess water was drained. Sixth, it was stained using Eosine-yellow solution (some were not) for 2 minutes. Again the slides were drained. Seventh, the slide was differentiated and dehydrated in 95% ethanol, two changes for 30 seconds each. Eighth, it was completely dehydrated in 100% ethanol, three changes and three quick dips each. Ninth, using xylene the slide was cleared. Finally, using Canada balsam and coverslip were used to finish the slide preparation.

III.

RESULTS AND DISCUSSION

The study of damages and associated diseases on tissue samples is known as histopathology. Parasites with or without toxic secretions, are the most common cause of histological damages. Techniques employed to study the nature of parasites and tissues under study are known as histopathological methods. Chanos chanos (Milk Fish) CLASS ACTINOPTERYGII ORDER GONORYNCHIFORMES FAMILY CHANIDAE GENUS Chanos Chanos chanos

Characteristics Milk fish is characterized by its tapering and smooth body, with colors; silver on the middle, green to black on its lateral sides and yellowish fins with dark borders. The dorsal and anal fins are also distinguished with the presence of the two spines with 13 to 17 soft rays and 8 to 10 soft rays, respectively. Its caudal, pectoral and pelvic fins are differentiated as highly forked, axillary scales present and with 11 or 12 rays, respectively. The milkfish eye is enclosed by a transparent tissue while its mouth has no teeth but a tubercle at the tip of the lower jaw is found together with the nick where it is fitted at the upper. Abundant gill rakers, brachiostegal rays (four in number and for sustenance of gill covers) and a maximum length of one meter are also characteristics of Chanos chanos. Habitat and Biology As the only species under Family Chanidae, the migratory milkfish is found commonly on tropical and subtropical seas wherein saline waters are the only place where it brood with one or two seasonal peaks. In some places other than near the equator where brooding season is all year round, milkfishes spawn during warm seasons. Zooplanktons,

After the preparation of the tissue slides, the group viewed the finish slides under a compound microscope to identify the tissues and the parasites that were present. Data were recorded and discussed in the succeeding part of the paper.

epiphytes and detritus materials are the food sources of young and mature fishes. Male and female population has no significant differences on this heterosexual fishes and even morphologically females are hardly determined from males. Prostaglandin or PGF2a on male milkfish is the best way to distinguish one from the other sex.

Oreochromis niloticus (Nile Tilapia) Phylum Chordata Subphylum Vertebrata Superclass Gnathostomata Class Actinopterygii Order Perciformes Family Cichlidae Genus Oreochromis Species Oreochromis niloticus Biological features The O. niloticus body is covered with cycloid scales with a caudal peduncle equal in length and depth. Its gill rakers on the first gill arch may be as numerous as 33. It is also characterized by the continuity of the spinous and soft ray parts of the dorsal fin. The anal fin has three spines while the caudal fin is shortened with numerous dark bars and is reddish on spawning seasons.

Habitat and Biology The temperature ranging from 31 to 36 C in shallow water is suitable for a tropical species of tilapia. Filter-feeding is the most common way of food collection of this fish. Small invertebrates and aquatic plants are some of the captured food. Brooding season is initiated by the males when they establish a territory, dig a nest for spawning and watch out for its place. When females spawn in the nest the male fertilizes it, the eggs now be gathered in the females mouth and will be responsible for the incubation. As a mouth brooder the

number of eggs is equal to the mothers body weight. Since the skin of the fish most of the time is the one exposed to water it is highly affected by parasitic attacks though intestines and gills are also most commonly affected. Destroyed tissues due to pressure exerted (Ganapathy, & Ravichandran) by the protozoa might also be affected by the fish exposure to pollutants. Hyperplasia as an adaptive mechanism is common in all infected tissues. A nematode larva has infestations mainly in the liver of fishes causing eradication of veins, dilation of veins, fibrosis and atrophy. Mechanical damages might also occur due to its migration (Haseeb). The small intestine is also affected by nematodes wherein lengthening of crypts is observed with the congestion of the lamina propia. The muscular layer of fishes on the other hand is affected greatly by acanthocephalan invasion. Necrosis and cell deformations are observed effects. The aggregation of inflammatory cells is also seen on infected tissues. Trematode infections cause the erosion of villi due to ulceration of muscularis mucosa (Haseeb). Fatty degeneration of tissue according to Haseeb also is due to the invasion of the latter. Ethanolic extracts of Ipomea aquatic have a histopathological effect on Oreochromis niloticus. Gill, liver, and muscle of the exposed fish indicated that the gill and liver were the organs most affected (Oluwatoyin, 2011). In the gills, a lesion, pigment, necrosis, cellular degeneration, malignancy and inflammation have been observed in the 96 hours exposure of the O. niloticus in the extracts of I. aquatic. Different levels of reactions ranging from lesion, necrosis, malignancy, inflammation, cellular degeneration and inclusion bodies (Oluwatoyin, 2011) were also observed in the muscles of O. niloticus. Mercury, copper, lead and cadmium are examples of heavy metals. Heavy metals are harmful in the health of an organism. When fish are exposed to elevated levels of metals in a polluted aquatic ecosystem, they tend to take

these metals up from their direct environment (Seymore, 1994). O. niloticus that were possibly contaminated with heavy metals undergo histopathological examination. A mild congestion and edema of the primary lamella (Kaoud and Dahshan, 2010) were observed in the gills. In the epithelial lining of the secondary lamella, severe edema, hyperplasia, fusion and focal desquamation (Kaoud and Dahshan, 2010) were noted. The apex of gill filaments showed congestion, hyper activation of the mucous and chloride cells (Kaoud and Dahshan, 2010). Histopathological alterations in the muscles were observed such as degeneration in muscle bundles with aggregations of inflammatory cells between them and focal areas of necrosis (Kaoud and Dahshan, 2010). Splitting of muscle fibers and atrophy were also observed. Edema and atrophy in the submucosa of the intestine were noticed as well as an atrophy in the muscularis, degeneration in the intestinal mucosa and submucosa with necrotized cells.

pond raised fishes. They only affect fishes when they grew in large number. In aquarium, these ciliates should be eliminated. The specimens the group gathered were raised in tanks, as based on the publication ciliates can be easily transmitted from tanks to tanks upon harvest. Inclusion of ciliates in the fish may cause irritation in their gills and skin. Ichthyophthirius multifiliis. White spot disease or Ich is usually a problem in the aquatic environment. This infection causes small blisterlike raised lesions on the fishes body walls and even in their fin. When the infection is only seen on gills, no white spot will be prominent instead the gills, skin or fins will seem swollen as said in the publication. Immature forms of I. multifiliis somewhat resemble Tetrahymena as noted by some individuals. Scanning of the prepared slides may reveal mature parasites and affirm the diagnosis. The presence of one parasite needs a fast cure for this parasite can cause massive mortality among fishes in a short period of time. Repeated treatment on the parasites encysted stage should be observed to control the outbreak. Maintaining the cleanliness of the tanks through daily changing of water on tanks is needed to secure the absence of encysted forms of this parasite Chilodonella. This ciliated protozoan causes the fishes to secrete excessive mucus. Irritations are common signs of the infected fishes and mortality may arise upon existence of five to nine organisms per LPO view to larger numbers. It was described as large, heartshaped ciliate with bands of cilia along the length of organisms. A 0.02% salt solution is needed to eliminate these parasites from the environment. Tetrahymena . They are commonly seen in organic debris at bottom of aquarium. They are seen with a tear drop shape ciliates moving outside the host. Low numbered Tetrahymena is not significant when seen at bottom of tank and may not cause primary death of fishes. Control is necessary using chemicals. Its common site is

COMMON FISH DESCRIPTIONS PROTOZOA

PARASITES

AND

THEIR

The most common fish parasites are protozoans. It is considered to be easily identified and controlled. They are single-celled organisms which are usually free-living in the aquatic environment. Their life cycle is direct. Crowded environment of fishes may result to protozoans build up that causes weight loss, debilitation and mortality. As seen in the fresh specimen at the appendix, there was one slide with protozoans attached to the scales. Ciliates, flagellates, myxozoans, microsporidians, and coccidians are indicated in the publication in which we used to support the study. Free living organisms are not parasitic. Ciliates . They have hair-like structures termed cilia that are used for locomotion may also be for feeding. They are said to be seen inhabiting

at the eye. It is manifested by enlarge eyes. Fish that were examined with these parasites in their internal surface should be disregarded from harvest and disposed properly. Trichodina . They are present on gills and skin of pond-reared fishes. As the other protozoans are, they are not harmful with small numbers; unless they are crowded then the parasite would increase in number and cause severe damage. Those that are affected with these parasites lose their appetite and condition causing them to be more susceptible to bacterial invasion. They can be seen through scrapping of the skin mucus, fin, or on gill filaments. Environmental correction is needed. Ambiphyra . These are sedentary ciliate that invades the skins, fins or gills of host fish. They are cylindrical shape and have a row of oral cilia. It is common on pond- reared fishes. Apiosoma . Is also sedentary ciliate common on pond-reared fish.The organism can be found on gills, skins or fins. They are vase-like shape. They can cause disease if their numbers become excessive. Epistylis .They are stalked ciliate usually attached to the skin or fins of the host. Epistylis, secrete proteolytic ("protein-eating") enzymes that create wound, suitable for bacterial invasion, at the attachment site. Fish can be placed into a 0.02% salt solution as an indefinite bath, or a 3% salt dip. Capriniana . These are sessile ciliate that attaches to the host's gills with the aid of a sucker. They have characteristic cilia to attach in a body. In heavy infestations, they can cause respiratory distress in fishes. Flagellates .They can be located on both internal and external surfaces. They have one or more flagella to move in a whip-like motion. They have small movements.

Hexamita. Small intestinal parasite often found in the intestinal tract of freshwater fish. Flagellates can be noticed in places where the intestinal lining is damaged. They move by spiraling. Sick fish are extremely thin and the abdomen may be bloated. The treatment for this is usually metronidazole. Ichthyobodo. They are common external flagellates secreting mucus. Its disease is termed blue slime disease. Fishes that are infected produces excessive mucus giving dark colored fish a gray or blue color along dorsal wall. It can be located on gills, skin and fins though they are too small to be identified. Piscinoodinium. These are sedentary flagellates attached to gills, skin and fins. These parasites have amber pigment seen on fishes that are very much infected. Infected fishes flash, go off feed, and die. They are usually pathogenic to young fishes. Young fishes as based on the groups tour at BFAR Dagupan ranges until 3 years of the fishs life. Chloroquin is used as treatment. Cryptobia . They are flagellated protozoans found in cichlids. Mostly seen in stomach causing the fish to be thin and develop a dark skin. Myxozoa. These are commonly located on pond-raised fishes. In heavy number situations can cause great damage in young fishes. They affect wide ranges of tissues. Removal of infected fishes should be done and so as disinfection. Microsporidia . These are intercellular parasites that reproduce by the help of the fish. Acquisition of infective spores causes hypertrophy that affects the fish. Diagnosis is through seeing spores on the infected tissues. It could be treated through removal of infected fishes and disinfection. Coccidia . These are intracellular parasites that infect wild-caught and cultured fish. They

causes Inflammation and death of the tissue. Infection sites include reproductive organs, liver, spleen, intestine, and swim bladder. MONOGENEAN TREMATODES Monogenean trematodes affect the gills, skin, and fins of fish. Freshwater fish infected with skin-inhabiting flukes become lazy, swim at the sides of the pool or pond, and then loss their appetite. Their gills become swollen making them less tolerant to low oxygen habitats. Gyrodactylus and Dactylogyrus are the two most common genera of monogeneans that infect freshwater fish. The treatment of choice for freshwater fish is formalin. Potassium permanganate can also be effective in controlling monogeneans. DIGENEAN TREMATODES . The life stage most commonly observed in fish is the metacercaria, which encysts in fish tissues. They are can be seen in external or internal surface of fishes.The best control of digenean trematodes is to break the life cycle of the parasite. Elimination of the first intermediate host, the freshwater snail is often recommended. NEMATODES .They infect all organs of the host, causing loss of function of the damaged area. Signs of nematodiasis include anemia, emaciation,and reduced vitality. Camillanus . Are small thread-like worm protruding from the anus of the fish. Control of this nematode in non-food fish is with fenbendazole, a common antihelminthic. Capillaria . They are found in the gut of angelfish, often known by its double operculated eggs in the female worm. Fenbendazole is used as treatment. Eustrongylides . Uses fish as its intermediate host.Its definitive host is a wading bird. The

worm encysts in the muscle of the fish and appears to cause little damage. They are not suggested for retail sales. CESTODES . Its life cycle of is extremely with fish used as the primary or intermediate host. Cestodes infect the alimentary tract, muscle or other internal organs. Larval cestodes are one of the most damaging parasites of freshwater fish. They impair reproduction when they infect gonadal tissue. One of the most serious adult cestodes that affect fish is the Asian tapeworm, Bothriocephalus acheilognathi . Praziquantel is effective in treating adult cestode infections for ornamental fish. Lernaea , also known as anchor worm, is a common parasite of goldfish and koi, especially during the summer months. One of the groups specimens was seen with a Lernaea. Argulus . They are uncommon in freshwater aquarium fish but may occur if wild or pondraised fish are introduced into the tank. Prolonged involvement of 0.02 - 0.2% salt may control passing of infection. LEECHES are sometimes seen in wild or pondraised fishes as what they have in BFARDagupan. Fishes that are observed with leeches may have chronic anemia. A 3% saltwater is used in controlling leeches. The parts of the Fish that was sectioned into slides are the gills, integuments, muscle tissues and the intestine. Gills The gills are one of the weak spot of fishes where pathogens can easily invade or irritate the organism. A characteristic of a gill that allows efficient gas exchange is the thin epithelium with a large surface area. Moreover, the gills are in control in the excretion of ammonia, a nitrogenous waste product and it also control the exchange of water and salt.

The bony structure which is covered by the teleost epidermal tissue is called the gill arch. It composes the primary lamellae which is usually paired and arranged in a double row manner. A mucoid epidermis which may have contained chloride cells that are abundant in the basal lamellae works as an ionic transport is covering the primary lamella. The series of filaments that is located perpendicular to the primary lamellae is the secondary lamellae. It is in the surface of secondary lamellae which is made up of interdigitating squamous epithelial cells supported by pillar cells where gas exchange occurs via countercurrent blood flow exchange opposing the external water. The pillar cells form flanges through the invasion on the basement membrane. It then merges with other pillar cells to develop the lining of lamellar blood channels. Contractile protein helps in resisting the swelling of the pillar cells. The microvili that rises in the surface of the lamellar epithelium helps in attaching the cuticular mucus which regulates exchange of gas, ions and water. Integument (Skin and Scales) The integuments of the fish are composed mainly of skin and scales. The skin portion are divided by epidermis and dermis. The outer epidermis consist of squamous cells while cuboidal cells occupy the basal germinal layer. The contain a specialized cell, malpighian cell which are parenchymal cells that surround the fish skin for easier exchange of gases. The dermis is the skin zone between epidermis and muscle. The dermis are divided into stratum spongiosum and stratum compactum. The upper layer stratum spongiosum, was dominated by networks of collagen, fibroblast and pigment cells. Under the stratum spongiosum is a layer where a few fibroblast intersperse at right angles on

collagen, this is the stratum compactum. Straompactum looks like a plywood, its structural rigidity and flexibility from stresses imposing on the skin. The dermis contains special pigment called melaophores. It contains large number of melanin pigment to give coloring and protective effect. Fish scales are produced from the mesoderm layer of the dermis. Scale is a rigid layer that serves as a protective layer on fishes. The type of scale of bangus and tilapia is a cycloid. Cycloids are thin, large, round or oval arranged in an overlapping pattern, growth rings are evident on the edges. Musculoskeletal and Supportive Tissues Fishes and other higher vertebrate animals are composed of three different types of muscle cells. These are the striated muscles, cardiac muscles and the smooth muscles.

Striated Voluntary The striated muscles, or the skeletal muscles, are composed of multinucleated cells. Each cell is composed of myofibrils and each of the myofibrils is comprised of myofilaments. It is composed of 2 kinds of muscle fibers. This is the "red" and "white" muscle fibers. These two kinds of muscles is used by the Fish two kinds of swimming activity. The red fibers are related to sustained activity, while the white fibers to short, strong bursts of motion. Red Muscle The red fibers is aerobic and it is a slowcontracting fiber. The red muscles are always supplied with blood which gives it the potential of providing a good site for the injection of drugs, anticoagulants and anesthetics. Red muscles lie along the lateral line and just beneath the skin as a wedge. It has higher lipid content, larger number of mitochondria per cell and higher respiratory activity than the white tissue.

White muscle The white fiber is anaerobic and it is a fast-contracting and fast-fatiguing fiber. The white muscles is the largest fraction in volume of body tissue. It has low numbers of mitochondria and low respiratory activity. Striated Involuntary The striated involuntary muscle or the cardiac muscle is different from any other muscles. Every single muscle cell of this type does not lie parallel to one another and several planes may also be seen when the heart muscle is sectioned for microscopical study. Each nucleus is located at regular intervals near the center of the cell and not just beneath the sarcolemma. Rather than as the single and long slender units seen in the body musculature, cardiac muscle cells lie end-to-end. Smooth Involuntary Smooth muscles are called such because striations are absent. Smooth muscles are mainly found in the viscera of the body. It has no striations and its nuclei are located centrally. These cells are long and tapering and they are not attached to one another. Its nuclei are also long and tapering Intestine The intestine of most fish is a simple tube which does not increase in diameter to form a colon posteriorly although its relative length may vary according to diet. The Intestine may be straight, sigmoid or coiled. Its shape depends on the shape of the abdominal cavity. It has a simple columnar epithelium. It functions in the transport food material from the stomach to the posterior intestine, digestion by the secretion of enzymes from its walls and from accessory glands, absorption of the final

products of digestion into blood and lymph vessels in its wall, and in the secretion of certain hormones (i.e. Secretin, stimulates pancreatic secretion). It also functions in fluid absorption, mucous secretion (more goblet cells), and some digestion which is accomplished by enzymes present in food material, and excretion. Since the skin of the fish most of the time is the one exposed to water it is highly affected by parasitic attacks though intestines and gills are also most commonly affected. Destroyed tissues due to pressure exerted (Ganapathy, & Ravichandran) by the protozoa might also be affected by the fish exposure to pollutants. Hyperplasia as an adaptive mechanism is common in all infected tissues. A nematode larva has infestations mainly in the liver of fishes causing eradication of veins, dilation of veins, fibrosis and atrophy. Mechanical damages might also occur due to its migration (Haseeb). The small intestine is also affected by nematodes wherein lengthening of crypts is observed with the congestion of the lamina propia. The muscular layer of fishes on the other hand is affected greatly by acanthocephalan invasion. Necrosis and cell deformations are observed effects. The aggregation of inflammatory cells is also seen on infected tissues. Trematode infections cause the erosion of villi due to ulceration of muscularis mucosa (Haseeb). Fatty degeneration of tissue according to Haseeb also is due to the invasion of the latter. REVIEW OF RELATED LITERATURE Acantholcephalans, Copepods, Isopods and Heterophid flukes (Velasquez, 1984) are the most abundant parasites which are smaller in number in ponds than in natural setting of fishes. Knowledge on the different parasites of milkfishes in the Philippines is important for a more profitable fish (Bangus) industry. For the

reason that such parasites affect the tissues of fishes which may be or may not be functioning were observed as odd signs on the fishes daily. Prophylactic measures are to be devised (Velasquez, 1984) if knowledge on these fish parasites are improved. Endo parasites acquired by the fishes through feeding and ectoparasites acquired by the fishes from its habitat are both studied by Velasquez. In ponds, increase in population of fishes has an indirect effect on the fish resistance to parasitic attacks. Lower food supply also causes diseases in ponds. However, conditions in pond fisheries allow preventive and control measures

sea without adequate filters (Velasquez 1979 as cited by Velasquez 1984). Occurrence in fish ponds of extensive fish kill may be caused by (Rocinella typicus and Ichthoxyenous) isopods which are known to multiply very fast. Milkfishes muscles were also found to be positive with encysted Haplorchis varium, H. yokogawai & Procerovum calderoni (Velasquez, 1984) larvae. Excystment of young flukes take place in the small intestine and they develop into adults in 5-10 days or more (Velasquez, 1984). Half-cooked meat for example causes human infection due to encysted larvae. Velasquez noted that the parasites found were related to physiological features of the host, biological features and the fishpond

9Velasquez, 1984) because some preventive methods are more ideal to be carried out in ponds than in larger and natural bodies of water. Ulceration of intestinal walls of adult milkfishes was positively observed due to acanthocephalan worms proboscis found on the wall. The infection was acquired when the intermediate host from the sea was ingested by the fish. Such infection is not found on the fishponds. Copepods ( Lernaea cyprinicea L.1958) also infected fishponds and fish pens are observed in nostrils or fishes base of infected fishes. The life stages of these parasites destroy its host extensively. Experimental tanks are found to have been infected with Caligus pafulus Wilson. 1937. The water is supplied directly from the

management as well.

IV.

CONCLUSION AND RECOMMENDATION

The goal of the study which was to see evident histological differences between Muscular, Connective and Epithelial Tissues of fishes was achieved through the tissue preparation methods which have an output of different stained slides. Parasites are also available in the slides as soon as the tissue processing was conducted. Invertebrates from different families, particularly Monogeneans, are present in the fresh and stained specimen.

As for findings, the different tissues were identified based on their muscular tissue types and epithelial tissue types as long as seen and available in the slides. Parasites are clearly identified in the intestines and muscles (though in the case of the muscle, the parasites appearance may be due to contamination because the parasites are not encysted) slides. In the fresh specimen that was subjected to analysis before conducting the fixation, there were a lot of evident parasites on the scales of the Chanos chanos and a few numbers in the scales of Tilapia sp .These findings helped the group achieve the aim of convincing the reader of the study to cook these common fishes before eating them to avoid any illness to occur.

Recommendations were also given by the group for further improvement of the next special experiments. Time on doing the study is not enough. The group committed a lot of trials until the date of submission and was hardly able to find time in completing the special project. Other classes used to interfere as well, so the group recommends that half class time dedicated for Histology lab should be used for the special project instead of the members vacant periods. The equipment were also limited and thus making it hard for all of the groups to use it at the same time. Problems regarding the single available microtome even arose causing delay to the special project conduction. Availability of equipment should be prioritized for better result of the next special projects that would be conducted.

V.

REFERENCES

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Bruno, D.W., Nowak, B., & Elliot, D.G. "Guide to the Identification of Fish Protozoan and Metazoan Parasites in Stained Tissue Sections." fws.gov. http://training.fws.gov/EC/Resources/Fish_Histology/Fish_Histology_Manual_v4.pdf (accessed September 10, 2012). Camargo, Marina M. P., & Martinez, Cludia B. R. "Histopathology of gills, kidney and liver of a Neotropical fish caged in an urban stream." scielo.br. http://www.scielo.br/pdf/ni/v5n3/a13v5n3.pdf (accessed September 10, 2012). ftp://193.43.36.44/docrep/fao/field/009/w6598e/w6598e00.pdf Retrieved: September, 15, 2012 Ganapathy, Ganapathy, & Ravichandran, Samuthirapandian. "Histopathological changes in the skins and gills of some marine fishes due to parasitic isopod infestation." Asian Pacific Journal of Tropical Biomedicine. http://www.apjtb.com/press/2012/B631.pdf (accessed September 10, 2012). Haseeb, Muhammad Farooq. "Histopathology of the Fish Arius Serratus (Day) 1877 of Karachi Coast Associated with Infections Caused by Various Parasites." hec.gov.pk. http://prr.hec.gov.pk/Thesis/2543.pdf (accessed September 10, 2012). Horn, Lars-Christian, Meinel, Alexandra, Handzel, Romy, & Einenkel, Jens. "Histopathology of endometrial hyperplasia and endometrial carcinoma An update." laboratoriosilesia.com. http://www.laboratoriosilesia.com/upfiles/sibi/GI0807676.pdf (accessed September 10, 2012). Irshadullah, Malik, & Mustafa, Yesreel. "Histopathological changes in naturally-infected Chirruh snowtrout, Schizothorax esocinus (Heckel), with Adenoscolex oreini (Caryophyllidea: Capingentidae)." infish.com. http://www.infish.com.pl/wydawnictwo/Archives/Fasc/work_pdf/Vol18Fasc3/Vol18-Fasc3w06.pdf (accessed September 14, 2012). Mohamed, Fatma A.S. "Histopathological Studies on Tilapia zillii and Solea vulgaris from Lake Qarun, Egypt." http://idosi.org. http://idosi.org/wjfms/wjfms1(1)09/4.pdf (accessed September 10, 2012). Vethaak, A.D. "Gross pathology and histopathology in fish: summary." int-res.com. http://www.intres.com/articles/meps/91/m091p171.pdf (accessed September 10, 2012). Wolf, Jeffrey C. "EDMVAC Plenary Meeting Washington, D.C." epa.gov. http://www.epa.gov/endo/pubs/edmvac/edmvac_jw.pdf (accessed September 10, 2012). http://el.erdc.usace.army.mil/ansrp/ANSIS/html/oreochromis_niloticus_nile_tilapia.htm Retrieved: September, 15, 2012 http://www.fishbase.org/summary/speciessummary.php?id=80 Retrieved: September, 15, 2012 http://animaldiversity.ummz.umich.edu/accounts/Chanos_chanos/ Retrieved: September, 15, 2012 http://animaldiversity.ummz.umich.edu/accounts/ oreochromis_niloticus / Retrieved: September, 15, 2012 http://www.fao.org/fishery/culturedspecies/Chanos_chanos/en Retrieved: September, 15, 2012 http://www.fao.org/fishery/culturedspecies/Oreochromis_niloticus/en

Retrieved: September, 15, 2012 http://www.rajswasthya.nic.in/RHSDP%20Training%20Modules/Lab.%20Tech/Histo/Introduction.pdf Retrieved: September, 15, 2012 http://www.scielo.br/pdf/ni/v5n3/a13v5n3.pdf Retrieved: September, 15, 2012 http://www.laboratoriosilesia.com/upfiles/sibi/GI0807676.pdf Retrieved: September, 15, 2012 http://www.epa.gov/endo/pubs/edmvac/edmvac_jw.pdf Retrieved: September, 15, 2012 http://idosi.org/wjfms/wjfms1(1)09/4.pdf Retrieved: September, 15, 2012 http://www.int-res.com/articles/meps/91/m091p171.pdf Retrieved: September, 15, 2012 http://www.epa.gov/endo/pubs/edmvac/edmvac_jw.pdf Retrieved: September, 15, 2012 http://training.fws.gov/EC/Resources/Fish_Histology/Fish_Histology_Manual_v4.pdf Retrieved: September, 15, 2012 http://www.sciencepub.net/life/life0403/13_life0403_70_76.pdf Retrieved: September, 15, 2012 http://prr.hec.gov.pk/Chapters/352S-6.pdf Retrieved: September, 15, 2012 http://idosi.org/wjfms/wjfms1(2)09/6.pdf Retrieved: September, 15, 2012 http://www.academicjournals.org/ajb/PDF/pdf2011/2Nov/Raissy%20and%20Ansari.pdf Retrieved: September, 15, 2012 http://www.iats.csic.es/~patolog2/files/Dykova-Lom_leaflet.pdf Retrieved: September, 15, 2012 http://www.infish.com.pl/wydawnictwo/Archives/Fasc/work_pdf/Vol18Fasc3/Vol18-Fasc3-%20w06.pdf Retrieved: September, 15, 2012 http://www.apjtb.com/press/2012/B631.pdf Retrieved: September, 15, 2012 http://www.int-res.com/articles/feature/d070p001.pdf Retrieved: September, 15, 2012

http://prr.hec.gov.pk/Thesis/2543.pdf Retrieved: September, 15, 2012

VI.

DOCUMENTATION AND APPENDICES

Table1. List of the Genus of Fish Parasites

Checklist of the Parasites of Fishes of the Philippines ORDER GONORYNCHIFORMES FAMILY CHANIDAE Chanos chanos (Forsskl) Milkfish Status: native bangos112 Environment: marine, brackish water Ciliophora

Ambiphyra sp. (-) Apiosoma sp. (Luzon) Riboscyphidia sp. (Luzon, Mindanao, Panay) Trichodina sp. (Luzon, Mindanao, Palawan, Panay) ?Trichodinella sp. (Luzon) Tripartiella sp. (Luzon) Mastigophora Cryptobia branchialis (Luzon) Cryptobia sp. (Luzon) Dinoflagellida gen. sp. (Panay) Myxozoa Ceratomyxa sp. (Luzon) Leptotheca sp. (Luzon) Digenea Digenea gen. sp. Metacercaria (Luzon, Panay) Digenea gen. sp. (Mindanao) Genolinea awa (Mindanao) Haplorchis yokogawai metacercaria (Luzon) Heterophyopsis expectans metacercaria (Luzon) Isorchis parvus (Luzon) Posthodiplostomum grayi metacercaria (-) Procerovum calderoni metacercaria (Luzon &/or Mindanao) Transversotrema patialense (Luzon) Monogenea Monogenea gen. sp. (Panay) Cestoda Scolex pleuronectis plerocercoid (Mindanao) Nematoda Nematoda gen. sp. (Luzon, Palawan) Acanthocephala Acanthocephalus sp. (Luzon, Mindoro) Cavisoma magnus (Mindanao) Copepoda

Caligus epidemicus (Luzon, Mindanao, Panay) C. patulus (Panay) Lernaea cyprinacea (Luzon) Isopoda Alitropus typicus (Panay) Gnathiidae gen. sp. larva (Luzon) Isopoda gen. sp. (Luzon, Panay) Remarks:A marine species entering rivers and lakes (Herre 1953), milkfish are also cultured in freshwater pens on Luzon Island. ORDER PERCIFORMES SUBORDER PERCOIDEI FAMILY CICHLIDAE Oreochromis niloticus niloticus Nile tilapia (Linnaeus) pla-pla, tilapia Syn.: Tilapia ni lo tica (Linnaeus) Status: exotic Environment: fresh water, brackish water Ciliophora Apiosoma sp. (Luzon, Mindano, Panay) Epistylis sp. (Luzon) Ichthyophthirius sp. (Luzon) Trichodina centrostrigata (Luzon, Mindanao) T. compacta (Luzon) T. heterodentata (Luzon, Mindanao, Palawan) T. siluri (Luzon)156 T. velasquezae (Panay) Trichodina sp. (Luzon, Panay) Trichodinidae gen. sp. (Luzon) Tripartiella spatula (Luzon, Panay) T. tilapiae (Panay) Mastigophora Cryptobia branchialis (Luzon, Mindanao, Palawan, Panay) Oodinidae gen. sp. (Luzon) Digenea Digenea gen. sp. metacercaria (Luzon)

Digenea gen. sp. (Luzon) Transversotrema patialense (Luzon, Palawan, Panay) Monogenea Cichlidogyrus longicornis (Luzon, Mindanao) C. sclerosus (Luzon, Mindanao) C. tiberianus (Luzon, Mindanao) C. tilapiae (Luzon, Mindanao) Cichlidogyrus sp. (Luzon) Dactylogyrus sp. (Luzon) Enterogyrus cichlidarum (Luzon) Gyrodactylus niloticus (Luzon) G. shariffi (Panay) Gyrodactylus sp. (Luzon, Panay) Mollusca BFAR SPECIMEN COLLECTION

Cristaria plicata (Luzon) Copepoda Caligus epidemicus (Luzon, Panay) Lamproglena monodi (Luzon) Isopoda Alitropis typicus (Luzon) Gnathiidae gen. sp. larva (Luzon) Remarks: Native to Africa, the Nile tilapia was introduced in 1970 and 1973 from Thailand and Israel (Froese and Pauly 1996). Stocks occurring in natural waters are not pure, having interbred extensively with Oreochromis mossambicus (see Macaranas et al. 1986)

PARASITES SEEN ON FRESH SPECIMEN EPIDERMAL LAYER (c/o BFAR-DAGUPAN) BANGUS