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

1Wayas, Hilda

1Department 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.

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.

In supplement with the Invertebrate

zoology class discussion regarding fish parasites

the group thinks that the study is significant.

The group is very much aware through the

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.

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

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.

II. MATERIALS AND METHODOLOGY

As a requirement for histology class, the

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

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.

Requested chemicals were Hemotoxylin, Eosin-

yellow, 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

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

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 Weigert’s 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.

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.

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. It’s 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,

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 mother’s 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.

COMMON FISH PARASITES AND THEIR DESCRIPTIONS

PROTOZOA

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

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 blister-like 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, heart-shaped 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

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 group’s tour at BFAR Dagupan ranges until 3 years of the fish’s 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 group’s specimens was seen with a Lernaea.

Argulus . They are uncommon in freshwater aquarium fish but may occur if wild or pond-raised 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 pond-raised fishes as what they have in BFAR-Dagupan. 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 slow-contracting 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

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 worm’s 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

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

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 parasite’s

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

Anonymous. PDF Journal http://prr.hec.gov.pk/Chapters/352S-6.pdf Retrieved: September, 15, 2012 Anonymous. PDF Journal

http://www.rajswasthya.nic.in/RHSDP%20Training%20Modules/Lab.%20Tech/Histo/Introductio

n.pdf Retrieved: September, 15, 2012

Arthur, J. and Mayo, S. 1997. Checklist of the Parasites of Fishes of the Philippines. Bureau of Fisheries and Aquatic Resources, Quezon City, Philippines.

Bamidele, A. 2007. Histopathological study on the parasitised visceral organs of some fishes of Lekki

Lagon, Lagos, Nigeria. Department of Zoology, University of Lagos, Akoka, Yaba, Lagos, Nigeria

http://www.sciencepub.net/life/life0403/13_life0403_70_76.pdf Retrieved: September 15,

2012

Bamidele, Akinsanya. "Histopathological study on the parasitised visceral organs of some fishes of Lekki Lagon, Lagos, Nigeria." sciencepub.net. http://www.sciencepub.net/life/life0403/13_life0403_70_76.pdf (accessed September 10, 2012).

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, Cláudia 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-Fasc3- w06.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.int-res.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 (Forsskäl) 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

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)

BFAR – SPECIMEN COLLECTION

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

BANGUS