FEEDING HABITS AND NEMATODE PARASITES OF SOME FISHES OF KARACHI COAST

YASMIN AKHTAR

Department of Botany, Jinnah University For Women,

Nazimabad, Karachi, Pakistan. 2008

FEEDING HABITS AND NEMATODE PARASITES OF SOME FISHES OF KARACHI

COAST

BY

YASMIN AKHTAR M.Sc, M.Phil.

THESIS SUBMITED TO JINNAH UNIVERSITY FOR WOMEN FOR

FULFILMENT OF THE REQUIRMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D.)

IN BOTANY

Department of Botany, Jinnah University For Women,

Nazimabad, Karachi, Pakistan. 2008

Supervisors Certificate

This is to certify that Ph.D student for the session 2005-2007 of Jinnah

University For Women; Ms.Yasmin Akhtar has completed her research

dissertation under my supervision for fulfillment of the requirement for the

degree of Ph.D. This is original work carried out by the candidate. The title of

dissertation is FEEDING HABITS AND NEMATODE PARASITES OF

SOME FISHES OF KARACHI COAST PAKISTAN.

Research supervisor

Prof.Dr.Bilqees Mujib

Ph.D (Can), Sc.D (USA), D.Sc

TABLE OF CONTENTS

Pages

ACKNOWLEDGEMENT--------------------------------- i SUMMARY------------------------------------------ ii

CHAPTER 1

INTRODUCTION 1.1: Importance of Fishes---------------------------------- 1 1.2: Fish and fisheries--------------------------------------- 3

1.3: Pakistan coastline------------------------------------ 7 1.4: Fish dietary habits-------------------------------------- 8 1.5: Fish parasitic fauna (nematodes) --------------------- 9 1.6: Nematode diversity------------------------------------ 11 1.7: Fish diseases ------------------------------------------ 12

Objective of the present work--------------------------------------19

CHAPTER-2

REVIEW OF LITERATURE 2.1: Comprehensive account of feeding habits of fishes-- 21

2.2: Effect of temperature on food-------------------------- 22 2.3: Nematoda (Round worms) --------------------------- 22 2.4: Fish Pathology due to nematodes --------------------- 25

CHAPTER-3 MATERIAL AND METHOD 3.1: Study Area---------------------------------------- 31 3.2: Collection of material-------------------------------- 31 3.3: Method for the study of feeding habits ------------ 32 3.4: Occurrence method ---------------------------------- 32 3.5: Frequency of occurrence----------------------------- 33 3.6: Examination of material for feeding habits--------- 33 3.7: Collection and Examination of specimen for nematode parasites------------------------------------ 34 3.8: Preparation of nematode specimen for SEM------ 35 3.9: Procedure for histopathology---------------------- 35 CHAPTER-4

RESULTS AND DESCRIPTIONS FEEDING HABITS----------------------------------- 36

4.1: Table 1: Taxonomic position of different edible marine fish species studied for their feeding habits and nematode parasites.----------------------------- 38 4.2: Diets of different fishes------------------------- 39

4.3: Remarks ------------------------------------------- 46 4.4: Statistical Analysis ------------------------------ 51 4.5 Table 2-4: Food contents of different fish species

from Karachi coast ------------------------------------ 52

4.6: Table 5: Occurrence of food categories in relation to

total number of stomach analyse of different marine edible fishes from Karachi coast--------- ------------- 55

Table 5.1- 5.2: Statistical calculation of Table 5. - 56

4.7 Table 6-13: Frequency of occurrence in relation

to total number of stomach analysed of different marine edible fishes of Karachi coast --------------- 58

4.8 Figures: 1-8 (histograms)-Comparison of the food items in the gut of different edible fishes from Karachi coast------------------------------------------- 66 4.9 Figures: 9-42. Photographic representation of some organisms present within the stomachs of different marine edible fishes---------------------------------- 72

CHAPTER-5 DESCRIPTIONS OF NEMATODES 5.1: History of the genus Dujardinascaris Baylis,1947-------------------------------------------- 77

5.2: Diagnosis of Dujardinascaris mujibi n.sp.--------- 79 Remarks------------------------------------------------- 81 5.3: Diagnosis of Dujardinascaris jello n.sp.------------ 84 Remarks--------------------------------------------------- 85 5.4: Diagnosis of Dujardinascaris maculatum n.sp.----- 87 Remarks--------------------------------------------------- 88

5.5: Diagnosis of Dujardinascaris sphyraenii n.sp.----- 90 Remarks-------------------------------------------------- 91 5.6: Diagnosis of Dujardinascaris multiporous n.sp.-- 93 Remarks--------------------------------------------------- 94 5.7: Diagnosis of Dujardinascaris sinjarii n.sp.--------- 96 Remarks--------------------------------------------------- 97 5.8: Diagnosis of Dujardinascaris dentatus n.sp---------- 100 Remarks-------------------------------------------------- 101

5.9: Table: (14-17) Morphological varations in the new and known species of the genus Dujardinascaris

Baylis---------------------------------------------------- 102

5.10: List of figures:

Figs. (4345)Dujardinascaris mujibi n.sp.------------- 106 Figs. (4648)Dujardinascaris jello n.sp.---------------- 109 Figs (4951) Dujardinascaris maculatum n.sp.-------- 112 Figs.(5254) Dujardinascaris sphyraenii n.sp.---------- 115 Figs. (5557)Dujardinascaris multiporous n.sp.--------- 118 Figs. (58) Dujardinascaris sinjarii n.sp. ------------ 121 Figs.(5960) Dujardinascaris dentatu n.sp.------------- 122 5.11: History of the genusProcamallanus(Spirocamallanus Olsen, 1952)------------------------------------------------ 124 5.12: Diagnosis of Spirocotoyle otolithi n.gen. n.sp.-------128 Remarks---------------------------------------------- 130 5.13: Diagnosis of Procamallanus(Spirocamallanus) riaziaii n.sp. --------------------------------------- 132 Remarks --------------------------------------- 134 5.14: Diagnosis of Procamallanus(Spirocamallanus) ruberii n.sp. ---------------------------------------- 137 Remarks---------------------------------------------- 138 5.15: Diagnosis of Procamallanus(Spirocamallanus) female sp. ----------------------------------------------- 141 Remarks ----------------------------------------------- 142

5.16: List of Figures (6166)

Figs. (61): Spirocotoyle otolithi n.gen. n.sp--------------- 143 Figs. (6264): Procamallanus(Spirocamallanus) riaziaii n.sp----------------------------- ------- 144 Figs. (6566): Procamallanus(Spirocamallanus) ruberii n.sp.------------------------------------147 Figs. (67): Procamallanus(Spirocamallanus) Female specimen------------------------------ 149 5.17: History of the genus Cucullanus Muller, 1777-----150

5.18:Diagnosis of Cucullanus aliyaii n.sp.----------- 156

Remarks--------------------------------------------- 157

5.19:Figs. (68-69) ------------------------------------ 163

CHAPTER-6

HISTOPATHOLOGY-------------------------------- 165

6.1: Discription and distribution of Euthynnus alletteratus(Refin, 1810)------------------------------- 165

6.2: Histopathology of infected intestine of Euthynnus alletteratus---------------------------------- 166

6.3: Discription and distribution of Pomadasys maculatum (Blch, 1797)----------------------------- 167

6.4: Histopathology of infected intestine of Pomadasys maculatum------------------------------- 168

6.5: Remarks------------------------------------------------ 169

6.6: Figs. (70-89)----------------------------------------- 174 CHAPTER-7 DISCUSSION 7.1: Dietary habits of fishes--------------------- 179 7.2: Seasonal variations in dietary habits------- 181 7.3: Correlation of dietary habits and nematode parasites--------------------------------------- 182 7.4: Parasitic diversity------------------------- 185 7.5: Histopathology of fishes------------------- 186 FUTURE PERSPECTIVE------------------- 190 CHAPTER-8 REFERENCES----------------------------- 191 LIST OF PUBLICATIONS----------------- 237

i

ACKNOWLEDGEMENT

I am grateful to the Vice Chancellor, Prof. Dr. Riaz Ahmed Hashmi, Jinnah

University for Women for providing financial support, working facilities and

kind suggestion at every step of this work.

I do here by acknowledge the fact that great help and guidance was provide to me

by my research supervisor Prof.Dr.Bilqees Mujib who was kind to me, at every

stage of the preparation of this dissertation. She has given guidance to me. I

deeply feel indebted to her efforts and contributions. With out her efforts and

help, such a piece of research work would not be possible.

My thankful acknowledgement is due to Prof. Dr. Rafia Azmat, Department of

Chemistry for fruitfull discussion, comments, suggestion and help in the

statistical analysis of data with all cooperation in finalizing the current thesis.

I am thankful to the heads and the staff of the department of Botany and

department of Zoology for helping me, to reconcile my study and work.

Similarly, I would like to appreciate research officer Ms. Rehmat Bibi for her

skilled assistance in the laboratory. I am also thankful to Mr. Yousaf, research

technical officer for his assistance in the Steroscan Micrographs in Centralized

laboratory of university of Karachi.

ii

I am also thankful to Rupert Lee from the Natural History Museum in London

and Dr. Franick Moravec from institute of Parasitology, Czech Republic

Budejovice kindly provided literature for study.

My sincerest thanks are due to my husband and my children for all the joy of life

they have given and for their moral support.

iii

SUMMARY

Pakistan has two main fishing areas: Karachi and Sindh, extending southeast

from Karachi to the Indian border (about 180 miles) and the Mekran coast, west

of Karachi and along the coast of Baluchistan to the Iranian border (about 350

miles). The former area with Karachi harbor as its main base, is characterized

by a broad continental shelf (extending about 60 nautical miles out from the

coast to a depth of 200m), a coast line marked by innumerable small creeks and

the Delta of the Indus River, and by muddy, easily trawlable bottom. The

tropical and envoirmental conditions are generally tropical and subject to

monsoon during the summer to Autum. Upwelling of cold, nutrient-rich, low-

oxygen water occurs all year round but is stronger during the Southwest

monsoon period.

.

As a group bony fishes can eat all sizes of plants and animals from smallest of

microscopic plant planktons to some largest marine animals. Several illnesses

are the result of planktonic toxic blooms. The amount of food, a bony fish eats

is directly related to its size, metabolic stages and temperature of its

environment.

The fish provide nutritive diet for the human beings. To get perfect nutrition

it is necessary that fishes must be healthy and free from parasitic and other

diseases. For the prevention of disease it is important to study the cause and

nature of disease in fish. Parasites are important group of pathogen, which

occurs at various stages of development in fish.

Fishes are the intermediate host for nematode parasites. Nematodes or round

worms are extremely diverse and successful group of invertebrate animals,

iv

consisting of 255 families. It is the second largest phylum in the animal

kingdom encompassing 500,000 species. They are important parasites of

animals and plants and are of major medical and agricultural importance. They

are generally free living while the parasitic nematodes exploit a great variety of

hosts. Some nematodes are very important which cause pathological changes in

fishes. The diseases due to the adult and larval nematodes are very common in

marine fishes and are distributed all over the world. The parasites invade

various tissues and organs of fish, including skin, gills, eye, kidney, liver,

intestine, spleen, heart and brain. Parasitic infection tends to decrease the

growth rate resulting in stunning of fishes.

An investigation was conducted for food and feeding habits, nematode parasites

and histopathology of the infected intestine to get more information on this

subject as relatively little work was carried out previously. For the present work

a total of 1500 fishes belonging to 10 genera, 14 species, 10 families and 6

orders were collected from February 2005 to June 2007 from fresh landing of

fish harbor Karachi coast Pakistan.

The fishes under investigation were Liza vaigiensis, Sardinella albella,

Scomberomous gutattus, Pomadasys olivaceum, Pomadasys maculatum,

Pomadasys stridens, Otolithus ruber, Arius maculates , Sphyraena frosteri,

Sphyraena jello, Lates calcarifer, Sillago sihama and Euthynnus alletteratus.

For food and feeding habits 780 (out of 1500) fishes were dissected to analyze

the stomach contents. Almost all the samples from which complete digestive

tract were extracted (89%, n=700) had stomach content present whereas 10%

stomachs were empty. From the 89% extracted digestive tracts, 9 taxonomic

v

categories were identified.

The percentages of occurrence of various food items were different in

carnivorous and herbivorous fishes. Planktons and detritus were the most

dominant food items in herbivorous fishes. Whereas in carnivorous fishes the

crustaceans, molluscs and miscellaneous items were dominating food categories

followed by teleosts (fishes). And the least observed food items were planktons

and polycheates.

The data obtained from the stomach contents of the investigated fishes revealed

the feeding habits of the fishes. From the graphical plots of stomachs against

the percentage frequency of occurrence, statistical analysis was performed.

Value of confidence interval falls within the limits, from which the current

results were satisfied.

During the analysis of food contents, nematode parasites were observed and

recovered. A total of 1500 fish specimens were examined for nematode

parasites investigations. No infection of nematodes was found in herbivorous

fishes, Liza vaigiensis and Sardinella albella. On the other hand, carnivorous

fishes (Scomberomous guttatus, Pomadasys olivaceum, Pomadasys maculatum,

Pomadasys stridens, Otolithus ruber, Arius maculates, Sphyraena forsteri,

Sphyraena jello, Lates calcarifer, Sillago sihama and Euthynnus alletteratus)

harbored different parasites. This is attributed to the fact that the carnivorous

fishes feed on other organisms, which may be serving as intermediate or

reservoir hosts. A prevalence of 62% was recorded and a total of 272 nematode

specimens were recovered including, new (n=131) and known (n=141,

previously described from Pakistan) nematode specimens. All infections

vi

observed and recovered were restricted to the intestine. Identification and

descriptions were made possible by the help of literature provided by Rupert

Lee Research service of British museum and also by comparing the literature

obtained by Dr. Franick Moravec from institute of Parasitology, Czech

Republic Budejovice.

The microscopic examination and Steroscan electron microscopy (SEM) of

cephalic and tail regions of the 272 holotype and allotype nematodes was

carried out. Out of which 131 nematode specimens were new and 141 nematode

specimens were already recorded from Pakistan. The known nematode

specimens belonged to 4 genera, including Cucullanus

Procamallanus(Spirocamallanus), Dujardinascaris and Bulbocephalus. Only

the new nematode species are described here:

The investigated new nematode species belong to one new genus and three

known genera, including Spirocotyle n.gen. ( Camallanidae Railliet and

Henry,1915); Dujardinascaris Baylis, 1947; Procamallanus (Spirocamallanus)

( Olsen 1952) Petter 1979, and Cucullanus Muller, 1777. From the above 4

genera, a total of 11 nematode species, (not reported in Pakistan yet) were

recorded as follows:

i) One new genus and new species was identified and described as Spirocotyle

otolithi (Camallanidae Railliet and Henry,1915) from Otolithus ruber.

ii) Seven other new species were identified and described here of the genus

Dujardinascaris (Baylis, 1947) including, Dujardinascaris mujibi n.sp. from

Sphyraena forsteri; D. jello n.sp. from Sphyraena jello; D. maculatum n.sp.

vii

from Pomadasys maculatum; D. dentatus n.sp. from Sillago sihama; D.

multiporous n.sp. from Pomadasys olivaceum; D. sphyraenaii n.sp. from

Sphyraena jello; and D. sinjarii n.sp. from Otolithus ruber.

iii) Two new species of genus Procamallanus (Spirocamallanus) (Olsen, 1952)

Petter, 1979), Procamallanus (Spirocamallanus) riaziaii n.sp. and P.(S)

ruberii n.sp. from Otolithus ruber were also identified and described. Two

female specimens from Otolithus ruber were also reported. Species was not

designated, as male specimens were not available.

iv) One new species of the genus Cucullanus Muller, 1777 was identified and

described here as C. alliyai n.sp. from Otolithus ruber

Tissue damage associated with nematode infection was also studied in

two fish species. Observations were made on the tissue sections of some

parts of infected intestine of Euthynnus alletteratus and Pomadasys

maculatum . The intestine of Euthynnus alletteratus, infected with nematode

species revealed various stages of necrosis, degeneration and morphological

alteration in villi of the intestinal tissues. The infected intestine of Pomadasys

maculatum with nematode showed severe damage to some parts of intestine

involving whole thickness of the bowl wall, atrophy of surface tissue and villi.

All the layers were involved with sloughing, flatting and fusion of villi.

1

INTRODUCTION

1.1 Importance of fishes

Fishes are one of the most important groups of vertebrates, which benefit

human being in various ways. Fishes were used even at prehistoric ages and it

was supposed to be beneficial to long life and intelligence (Hamilton, 1971).

Many countries are working to search out the importance of fishes for men. A

study conducted in Netherlands reported that eating a pound of fish a week

could reduce the coronary heart diseases in man. Some fishes specially cold

water species such as cod, salmon, mackerel and sardines contain some oils that

are not found in other food and have major effects on body chemistry, which

reduces the tendency of blood to clot and helps in lowering cholesterol level in

the blood.In manufacturing of color paints and margarine, oil of herring,

meckeral and salmon are used commercially. The medical value of fish oil

cannot be ignored, many patients who are suffering with lack of vitamin A and

D, use oils of cod and sharks. Scales of fishes are used for decorations of

houses. Skin of fishes has its own importance in leather industry.

Fish and other marine life are also used for many other uses, pearls and

mother of pearls, shark skin and rayskin. Sea horses, star fish, sea urchin and

sea cucumber are used in traditional Chinese medicine. Tyrian purple is a

pigment made from marine snail, Sepia is a pigment made from the inky

secretions of cuttlefish.(Wiwikipedia.Org. Fish as food). In manufacturing of

soap, oils of fish are mostly used. It is also used in fungicides and

insecticides.For supply of adrenaline and insulin, pharmaceutical companies

use-frozen adrenal glands and pancreas. For the production of glue skin

2

chiefly of cod, haddock and hake are used. In Russia parts of sturgeon and

other fishes are used in making of certain glass and diamond cements (Russell

and Yonge, 1936). In countries where mosquitoes are abundant including

Pakistan, India, Sri Lanka, Bangladesh, Saudi Arabia, Nigeria fish can be used

as biological control agent for mosquitoes (WHO, 1981).

Fish flour for human consumption is manufactured from sharks and other

elasmobranchs fishes in Pakistan, while in India and other developing countries

shark is used for fish flour for livestock. This fish flour is useful for fortification

of cereal foods containing 85% high protein. The researches are based to

explore and examine the natural wealth of fish and shrimp, to develop most

economical and effective products and technologies for utilization of natural

resources, including import substitution and export promotion.

The major component of fish is protein. Fish proteins have a high biological

value. It contains variable quantities of calcium, phosphate, fate and other

nutrient important for human health and growth. Fish provides the worlds

prime source of high quality protein, 14-16% of the animal protein consumed

world wide; over one billion people rely on fish as their primary source of

animal protein. There is a considerable scope for aquaculture in Pakistan to

supplement the protein obtained from the livestock resources, a meager

attention has been given in past on these valuable resources and if efforts are

made Pakistan fish industries can become one of the most outstanding in the

world as we have a large part of the ocean It is only possible if lot of

investment is made and knowledge is gained about the economic importance

of fish, their life history, habitats, physiology, diseases and their control.

3

Pakistan is one of the protein-deficient countries of the world. There is no

carbohydrates, no dietary fibers because many high on the food chain are highly

contaminated with environmental chemicals and parasites. Due to the

importance of fish, in a large part of world especially where there is a high

potential for development of fisheries, people are expanding and industrializing

their fisheries and improving their living conditions by utilizing the fish catch

more effectively. Japan has long been the leading nation in fishing recovering

about 74 percent animal proteins from fish. In Pakistan the Karachi coast and

many other lakes and canals are present. Due to availability of port at Karachi

(Sindh), the fish supply is abundant, but the cost is high for poor families and

they suffer for protein and balance diet.

1.1 Fish and fisheries Pakistan has a coastline of 1,050 km and a total fishing area of approximately

300,270 sq. km. Pakistans fishing waters are termed as highly rich in marine

life with a vast variety of species having great commercial value. Pakistans

consumption of fish is very low, and thus most of the produce is exported. Most

of the fish catch is marine which comprises 71% of the total fish exports

(SMEDA, 2002). An analysis of the current status of the more important fish

stocks is made yearly (MFD, 2002). Various fleets fishing along the coast of

Pakistan share these stock.. The party boat fishery, in which fishermen rent

space abroad a boat for a day or more, has been popular in Karachi. Fishing

from private boats, shorelines, piers and jetties is also very popular (Neelofer,

1995).

4

As mentioned previously Pakistan has two main fishing areas: Karachi to Sind,

extending south east from Karachi to the Indian boarder (about 180 miles) and

the Mekran coast, west of Karachi and along the coast of Baluchistan to the

Iranian boarder (about 350 miles). The former area with Karachi harbour as its

main base is characterized by a broad continental shelf (extending about 60

nautical miles out from the coast to a depth of 200m). Marine fishing is

undertaken fromright beyond the coast to 200 nautical miles in to the sea. The

distance has been divided into two broad categories for fishing known as: (1)

Coastal water fishing and (2) Deep-sea water fishing. The distance specified are

up to 12 nautical miles termed as coastal water fishing. Coastal water fishing is

done in the villages along the coast that are predominately inhabited by

fisherman whose main live hood is fishing. While the Deep Sea is further

divided into two Zones. Zone 1 is from 12-35 nautical miles and Zone 11 is

from 35-200 nautical miles. Major share of marine catch is within 12 nautical

miles from the coast (Pakissan.com.2001-2008). The tropical and

environmental conditions are generally tropical and subject to monsoon during

the summer to Autmen. Upwelling of cold, nutrient-rich, low-oxygen water

occurs all year round but is stronger during the Southwest monsoon period and

greatly effect the distribution and and migration of the fish in the region.

One fifth of the worlds populations live in some of the least developed nations

around the rim of the Indian Ocean. These nations are dependent on their

environmental, coastal and marine resources. In the recent years it has become

clear that man is capable of disturbing the natural system of our planet on

global scale, including the oceanic system. Coastal zones are one of the most

important national assets of a countary where socio-economic activities are

highly concentrated. Coastal marine resources have an immense potential in

5

contributing towards national economic growth and progress. Coastal zones are

capable of producing rich fisheries, minerals, oil and gas resources. The

importance of developing marine resources has not been fully perceived.

Pakistan interest requires an investment in ocean research to contribute towards

the national economy, conduct fundamental and applied research, lay claim on

its territorial and offshore boundaries.

The sea water of the coast of Sindh and Balochistan has the potential to

provide sea food to as many as twice the present population of Pakistan but

hardly more than 2% of the potential wealth is exploited (Syed, 1985).The

statistical data on actual fish production from the tropical oceans is very

unreliable and difficult to calculate. The tropical oceans include almost 50%

of the total area of all open waters and 30% of the total area of continental

shelf however its reported production does not exceed 16% of the global fish

production. Life in warm tropical areas develops faster with early and

reproductive capabilities and its high diversity enable to occupy every niche

and the whole ecosystem appears a huge aggregation of species complex

(Hussain, 2003).

Pakistan marine recreational commercial fisheries exploit a large numberof

coastal fish species. Out of 760 species of fishes belonging to 164 families and

370 genera recorded from the coast of Pakistan, about 114 species are of the

commercial importance; marine fisheries production reached a peak at 2,78,149

metric tons in 1982 (Hoda, 1985; Majid et al., 1992). In 1999, the total marine

fish production was 1,82,995 metric tons, in which 1,64,450 metric tons have

been locally consumed while 18545 metric tons have been exported. In 1999,

out of the total locally consumed fish production, 70.18% was utilized for

6

human consumption. The share marketed as fresh, frozen, canned and cured

was 45.86% 11.42%, 0.00% and 7.37% respectively, where as 5.53% for

subsistence. Out of total export of fish and fishery products in 1998, the

percentage of fish, shellfish and fishery products was 62.5%, 35.1% and 2.4%

respectively. In 1999, an increase in quantity was registered i.e. 90,384 metric

tons and in value Rs. 7.02 billions. This showed an increase of 26.9% in

quantity and 18.6% in value over proceeding year. The export mainly composed

of frozen fish, frozen shrimps, lobsters, crabs, dried fish and mollusks. Among

the fish products only small quantity of fish meal was exported. In 2001, total

world production of fisheries was reported to be 130.2 million tons, of which

37.9 million tons was from aquaculture practices and 92.3 million tons from

captured fisheries production. China was the leading producer with 42.6 million

tons. In parallel with the increase in production, international trade has

continued to grow, and at an accelerating rate in recent years. In 2001, more

than 80% of the total world import value was concentrated in developed

countries, in particular in Japan, the USA and in several other countries. Japan

was the major importer accounting for about 23% of total import value. USA

was the second main importer with a share of 17%, followed by Spain, France,

Italy, Germany and the UK. (Source: FAO). According to the Food and

Agriculture Organization (FAO), the world harvest in 2005 consisted of 93.3

million captured by commercial fishing in wild fisheries, plus 48.1 million

tones produced by fish farm. I addition, 1.3 million tons of aquatic

plants(seaweeds etc) were captured in wild fisheries.

Pakistans exports of fishery products stand at about 0.25% of world exports.

Pakistans domestic consumption is termed as one of the lowest in the world, at

1.6 kg per person per year (compared to world average of 16.2 kg per person per

7

year). By adopting modern techniques of fishing, Pakistan can exploit the huge

opportunity that exits in the fisheries sectors (2001-2007 Pakissan.com.).

1.2 Pakistan coastline The coastline of Pakistan extends 1,050 km (650 mi), 250 km falling in Sind

province and 800 km in Balochistan. It borders the productive NE Arabian Sea

famous for its upwelling phenomenon. Its Exclusive Economic Zone (EEZ)

covers an area of 196,600 sq.km and the territorial waters cover an area of 24,000

km2. The continental shelf of the Sindh coast extends to a distance of 150 km

whereas that of Balochistan only measures 15-40 km. The prevailing ocean

current direction is clockwise during the southwest monsoon season and anti-

clockwise during the northeast monsoon season. The salinity value is generally

36 ppt. Tides are neither very high nor very low, but intermediate; the mean

average height is about 10-11 feet. Tides are higher on the eastern side and their

velocity is generally between 1-2 knots but may increase to 4 knots in narrow

creeks. (Wildlife Biodiversity of Pakistan).

Because of a combination of wind and earths rotation, the summer monsoon

pushes water away from the Somalian coast and edge of the Arabian Peninsula.

The water that leaves these coastal areas is replaced by water that wells up from

depths and is very high in nutrients. In the winter, the monsoon reverses and

blows from the northeast. This monsoon does not have as much impact because

it pushes water towards the Arabian Peninsula resulting in down welling, not

upwelling. However, the strong winds cause turbulent mixing, which brings up

some nutrients, but not on the scale of the summer monsoon. The productivity

of the Arabian Sea increase dramatically with an increase in nitrate. This

8

productivity is in the form of tiny plankton, which are the lowest link in oceans

food chain. (Najjar www.eesi.psu.edu/news_events/archives/ Monsoons.shtml ).

There is a considerable scope for aquaculture in Pakistan to supplement the

protein obtained from the livestock resources, a meager attention has been

given in past on these valuable resources and if efforts are made Pakistan fish

industries can become one of the most outstanding in the world as we have a

large part of the ocean It is only possible if lot of investment is made and more

and more knowledge is gained about the economic importance of fish, their life

history, habitat, physiology, diseases and their control. The high commercial

value of food fishes which are mainly marine, has led to a great impact on

research over the last hundred of years. Fishery biologists are much interested

in the growth because obviously it is a major component of fishery that is used

to calculate yields at different level.

1.3 Fish Dietary habits As a group, bony fishes have a diverse range of food preferences.(a) Bony fish

may be Herbivorous (plant eaters), (b) Carnivorous (meat

eater), (c) Detritivorous (that eat decomposing plants and animals).Bony

fishes can eat all sizes of plants and animals. The amount of food a bony

fish eat is directly related to its size, its metabolic stages and the temperature of

its environment.

Nature offers a great diversity of organisms that are used as food by fish and

these differ in size and taxonomic group.Smaller fishes generally have a higher

metabolic rate as compared to the fishes of the same species, thus small fishes

generally eat more relative to their body size. Fishes are cold blooded that is

9

body temperature is determined by the temperature of their environment. Warm

water fishes generally have higher metabolic rate and require more food than

cold water species of the same sizes.

Herbivory is the main route by which photosynthetic energy is transferred to

marine organisms ( Elton, 1927). Marine herbivorous fishes have a great

diversity of gut microorganisms. Despite their importance in terms of

microorganisms structural elements to cell contents. According to (Hata and

Kato,2002) the non-weeded species were more nutritious than the weeded

species. Fish eat other fish that eat planktons and algae, which are contaminated

with environmental pollutant because these chemicals are concentrated in the

fat of the fish. Fish most heavily laden with chemicals are those such as tuna,

swordfishes and sharks, which are predators of smaller sea life. Several

illnesses are a result of algal toxic blooms. For example most commonly

reported marine toxic disease in the world is ciguatera, which presents primarily

as diarrhea, abdominal cramps, vomiting, pain on urination and heart block.

1.4 Fish parasitic fauna (nematodes) Helminths are an important group of animal parasites occurring in the adult

stage usually in vertebrate hosts, practically invading every organ system of

the host, and in the larval stage in the invertebrate hosts.These worms are

widespred in almost all animals in every part of the world, though the

intensity of infection may differ from time to time or place to place and they

produce a wide variety of direct effects, thus they play a vital role in

undermining the welfare of man and the animal he is associated to smaller or

greater extent. The later is mainly responsible for the development of the

helminthological research.

10

Helminth parasites of fish in marine system are often considered to be

generalists, lacking host specificity for both intermediate and definitve hosts. In

addition, many parasites in marine water posses life cycles consisting of long-

lived larval stages residing in intermediate and parasitic host. These properties

are believed to be adaptation to the long food chains and the low distribution of

organisms over broad spatial scales that are characteristic of open marine

system. Moreover such properties are predicted to lead to the homogenization

of parasitic communities among fish species.

Fishes are the predator- prey pyramid within fresh water as well as in marine

water and therefore tend to be infected by a considerable range of parasites,

which occur in large number. Most helminth parasites carried by fishes are

nematodes, acanthocephala, cestodes and trematodes.

Ones the helminth has reached its residence in the definitive host, its primary

concern is to secure nourishment. For this purpose it has usually chosen a

position, where digested or semi-digested food is abundantly supplied.

About one thousand species of fishes are found in marine and fresh water in

Pakistan. Majority of these are edible. And very few are examined for their

nematode parasites. The nematodes or roundworms (Phylum Nematoda from

Greek (nema): "thread" + - -ode "like") are one of the most common phyla of animals, with over 20,000 different described species (over 15,000 are

parasitic). They are ubiquitous in freshwater, marine, and terrestrial

environments, where they often outnumber other animals in both individual and

species counts, and are found in localaties as diverse as Antarctica and oceanic

11

trenches. Further, there are a great many parasitic forms, including in most

plants, animals and humans.

Nematodes are by nature aquatic organisms. Recent observations indicatethat

various nematode species respond differently to degradation of environmental

quality thus the degree of nature of change in the community structure of

aquatic nematodes may be an excellent indicator of water quality or pollutant

levels. Most fish nematodes are oviparous and their eggs, which may or may

not be embryonated, are passed with the feaces of the host. The eggs hatch to

release a free swimming larva which must be ingested by crustacean or other

invertebrates as first intermediate host and a fish as second intermediate host

and bird or mammal as the definitive host, depending on the genus or species

concerned.

1.5 Nematode Diversity Nematodes of marine fishes exhibit great biodiversity. These are usually

nonspecific. One nematode species can be found in various fish host. While

several different nematode species can be found in one fish. Biological diversity

is a measure of the relative diversity among organisms present in different

ecosystems. Diversity in this definition includes diversity within species and

among species, and comparative diversity among ecosystems. The other

definition of diversity is the totality of genus, species, and ecosystem of a

region (Heip et al.,1985), however species richness varies much among

habitats. The presence of one species of nematode in a fish indicates that he is

eating or swimming in a determinate area of the sea(bentonic, planktonic or

migratory).

12

Biodiversity found on Earth today is the result of 4 billon years of evolution.

New species are regularly discovered and many though discovered, are not yet

classified. Biodiversity has contributed in many ways to the development of

human culture and in turn, human communities have played a major role in

shaping the diversity of nature at the genetic and ecological levels benefits of

biodiversity. Diversity is the most important parameters used in the description

of a community. Several theories relating diversity to other phenomena such as

predation, competition and stability have been proposed (Pianka, 1966) As a

result of the increasing interesting diversity a number of studies have appeared

during recent years an important feature of nematode communities, perhaps the

most important in understanding their ecological success, is the large number of

species present in any one habitat usually an order of magnitude greater than for

any other major taxon. Certain host features, i.e. host body size and host diet,

appear to influence the total number of parasite species exploiting a host

species, whereas other host features, i.e. depth range, appear to influence the

variety of parasites taxa that a host will accumulate over evolutionary time

(Luque & Poulin, 2004).

The Karachi coast has rich fish fauna and favorable hosts of nematodes. These

nematodes belong to several different families, subfamilies and genera. The

nematodes of fishes of Karachi coast exhibit great biodiversity; one species

infecting several fish hosts and one fish may be infected with several nematode

species (is given in tabulated form at the end of thesis).

1.7: Fish disease

Fish diseses are important from two points of view: Parasites from fishes can

affect fishes, or can affect human. In the first case, some parasites affect only

13

fishes, making them not avaliable for human consumption, or diminishing the

size of the fish, being not atractive for commercial purpose. In the second

case, parasites present in fishes can affect human, provoking diseases,

including death.The diseases of marine animals or fishes are as old as the

animal life in the seas. Early reports on disease of aquatic animals include the

fish diseases especially of helminth infection, (Cheng, 1964). Kinne, (1980)

has given a bibliography on parasites and disease of fish. At that time the

scientific studies on fish disease remained largely unexploited but recently

fish diseases are one of the important problems and great attention is being

paid to get more and more information about it.Many parasites are host specific

to at least some extent and are capable of infecting one or only a limited

number of host species. Individual parasites may have widely differing

effects on different host species.

There is no doubt that every parasite which lives internally or externally in

fish, exerts some harmful influence on the basic phenomena of life of their

hosts, for example life span, life cycle, abundance, distribution, metabolic

performance, nutritional requirements, growth, reproduction, evolution as well

as organism tolerances to natural and man-made environmental stress

(Kinne,1980 ). The host reaction may be expressed in tissue proliferation,

degeneration, and inflammation and probably in the development of immunity

as described by Bauer ,1958; Davis, 1953; Reichenbach- Klinke and Elkan

,1965; William and Jones, 1967; Smith and Wooten, 1978.

Cross (1933) showed that the parasitic infection tend to decrease the growth

rate resulting in the stunting of fish. Sinderman and Rosenfield, (1954) studied

the migration of herring on the basis of their parasites and showed that the kinds

14

and number of parasites vary seasonally, geography and with age of the host.

He observed that differential mortality may also vary seasonally or with age of

the host. Parasites cause damage to various organs of fish. Their host affecting

the yield of fish products such as liver oil etc. a large number of nematodes live

as internal parasites in fish, one such example is Cucullanus elegans (Zedar,

1917) the larvae of which live in Cyclops.The adults inhabit intestine or eyes.

Most of the nematodes of family Camallanidae occur in swim bladder.

Glandular secretion and metabolites of parasites may be toxic for the fish

resulting in weight losses, ill growth and inhibition of fertilization in hosts.

Parasites produce protiolytic enzymes, which are so strong that liquefy the

effective muscles. A strong invasion of gut nematodes may destroy the intestine

partially or wholly, as a consequence, the intestine stiffens, its peristaltic

movements slow down and digestion is hampered. The severity of disease in

fish will vary depending upon the life stage, species and number of nematodes

present; the age and species of infected fish; and the site of infection. Visible

signs of infection may include hemorrhaging, cyst formation, external lumps or

nodules, inflammation and necrosis.

Adult nematodes in the intestinal tract damage its lining and robe the fish and

effect their development. Robert et al.,(1995) studied the Phylogeny, Ecology

and Richness of parasites in vertebrates. Many species of nematodes migrate

within the body of the fish causing worm tracts which are seen in the form of

tunnels in the tissues. Extensive migration by large number of nematodes may

cause significant physical damage to a fish. Juvenile fish with nematode

infection are often more severely infected than adults, displaying reduced

growth, wasting or more obvious disease symptoms and mortality.The larval

15

nematodes are present almost in every organs of the host. Most adults inhabit

the intestinal tract. Anisakis (Nematode) is easily penetrated in to stomach of

fishes, including Hilsa ilisha and Cybium guttatum (Bilqees &Fatima, 1993;

Bilqees &Parveen, 1996).

From the viewpoint of fish as food, fish diseases are important for two main

reasons: (a) the number of fish available for consumption is reduced (b) the

disease fish may look unsightly, both these making unacceptable for human

consumption. The fish disease may be due to parasite or non-parasitic causes,

the former being the most numerous and from both pathological and economic

viewpoint, the most important. Many parasites are host specific to at least some

extent and are capable of infecting one or only a limited number of host species.

Individual parasites may have widely differing effect on different host species.

There is no doubt that every parasite which live internally or externally in fish,

exert some harmful influence on the basic phenomena of life of their hosts,

specially life span, life cycle, abundance, distribution, metabolic performance,

nutritional requirements, growth, reproduction, evolution as well as organisms

tolerances to natural and man made environmental stress( Kinne, 1980 ). There

is a usual concept that extensive mortality in the natural environment occurs

from the infestation of larger parasites, such as trematodes, acanthocephala,

nematodes which are common in fish. In crowded hatchery conditions,

infestation of these larger parasites may often cause death.

Nematodes infect many different species of aquaculture and wild fishes.

Nematodes are found in all the body parts of fish either as larvae oradults. The

diseases due to the adult and larval nematodes are very common in marine

fishes and worldwide in distribution. The organs commonly infected are

16

intestine, liver and body cavity. While other organs involved are heart, kidney,

spleen, reproductive organs eyes and gills. Nematodes especially larvae may

cause blockage of organs. This is caused when the worms are in great quantity.

If these are present in the capillaries of the gills, they block or obstruct the

capillaries, fish become unable to respire and die. Most species of the

nematodes in adult stage live in the alimentary canal except the family

Philometridae which are found in body cavity, liver and gonads. A number of

genera of the family Anisakidae occur in the digestive tracts of marine fish.

They live free in the lumen of the stomach or intestine; some attach to or invade

the wall of these organs and cause local tissue damage. Pathogenesis is a result

of their modes of feeding, attachment and movement or migration within the

host. Sea foods are the principal sources of human infections with these larval

worms. The disease is transmitted by raw, undercooked or insufficiently frozen

fish and shellfish.

Some parasites from fishes are important because of the human become

infected during the consumption of edible fishes. Anisakis simplex,

Contracaecum sp., and Hysterothylacium sp. are anisakid nematodes that have

been implicated in human infections caused by the consumption of raw or

undercooked seafood. Asami et al., (1965) discussed two cases of stomach

granulae by Anisakis-like larvae The adults of the nematode parasite

Gnathostoma spinigerum (Bashirullah, 1972) are normally found in the

stomach of felids, raccoons, dogs. The first intermediate host is cyclopoid

copepods, and the second intermediate host is a fresh water fish. If human

consume fresh water fishes infected with G. spinigerum, the larva migrate

throughout the intestine to muscles and skin, causing the 'larval migrans '

syndrome. The nematode Dioctophyma renale (Goeze, 1782) is found as an

17

adult in the kidney of wild carnivores and has been recorded from man (Myers,

1970). Normally the lifecycle involves only an oligochaete annelid intermediate

host but if a fish eats an infected annelid the larval nematode may penetrate into

the visceral cavity and encysts there. Larvae have been found in pike in the

Russia (Karmanova, 1961) and bullhead in North America (Mace & Anderson,

1975). Ruben et al.,(2001) also studied human infection by Pseudoterranova

decipiens in Chile. In the Far East and pacific areas, both freshwater and marine

fish can act as paratenic hosts for the parasite although the normal intermediate

host is a molluse (Sindermann, 1970).

Study of the fish disease is one of the important problems because it has an

indirect and some times direct effect on the productivity of fish and on human

health. Previously no attention has been paid for the study of larval nematodes

in the fishes in Pakistan in spite of their importance as one of the fish disease

agents and causative agents of gastric problem in man. Considering the parasitic

problems in the fish, it is necessary to conduct different studies on aspect of

biology related to nutritional values and parasitic infections to help the

production of worm free fish in our country.

Among fresh water and marine fishes, the marine fishes face much more

danger. Most of marine fishes are included among the group of edible fishes.

Some of these including, Scomberomorus guttatus, Pomadasys olivaceum,

Pomadasys maculatum, Pomadasys stridens, Otolithus ruber, Sphyraena

forsteri, Sphyraena jello, Lates calcarifer and Sillago sihama are popular

edible fishes in Pakistan, due to their delicious taste and are full of nourishment

such as proteins and vitamins particularly vitamin E and vitamin D. A lot of

work has been reported on the diagnosis, description and analysis of the

18

diseases of marine fishes (Petrushevsky and Kogteva, 1954; Sindermann and

Rosenfield, 1954; Getsevichyute, 1955; Bauer, 1958; Amlacher, 1961;Dogiel,

1962; Mikhailova et al., 1964; Sindermann, 1966; 1970; Andreessen, 1970;

Williams and Jones, 1967; Roberts, 1978a; Kinne, 1980; Kim et al., 1991;

Iskikura et al. 1993; Meunier & Desse, 1994; Amin et al., 2000; Akinsanya,

2007.

Most work in Pakistan was carried out by Bilqees and her coworkers.

Bilqees (1993) published dictionary for terminology of diseases, and for

pathological and histopathological terms commonly used. Bilqees and Fatima,

1986 reported Anisakis larvae from the fish Cybium guttatum. Bilqees and

Fatima (1993) also reported that the intensity of infection may be as high as

90% in Hilsa ilisha. The histopathology of the liver, stomach and intestine

infected with Anisakid and other larval nematodes were also studied by

(Bilqees and Fatima, 1993; 1995; 2000; Bilqees & Khan, 1994; Bilqees &

Parveen, 1996; Khatoon & Bilqees, 1996; 1999; Khatoon et al., 1999a; 1999b.;

Bilqees et al., 1993;1998; 1999; 2001).

19

OBJECTIVES OF THE PRESENT WORK

The objective of the present work is to study the dietary habits, nematode

parasites of marine fishes of Karachi coast, Pakistan, including the knowledge

about fish pathology by nematode parasites. Meticulous literature survey

reveals that a lot of work has been carried out by many researchers separately

on dietary habits, nematode parasites and histopathology of marine edible

fishes, but the relation in between these three major parameters has not been

established yet. Therefore the study is focused on relationship in between these

three parameters.

1: The first objective of this work is focused on food and feeding habits of

marine edible fishes with the aim of determining the dietary requirements,

categorizing them as herbivorous, carnivorous, Omnivorous and detritivorous.

This will give help in seasonal Variations of dietary habits and will support the

aquaculturing. Studies based on literature from varying habitat, have showed

that the stomach content analysis is widely used in fish diversity. A survey is

conducted for the marine fish diversity of Karachi coast, Pakistan.

2: As stated by Sasal et al., (1999) the diet of the host species is the main factor

affecting parasite community structure. The second objective of the present

work is to study the taxonomy of the nematode parasites and their prevalence

and intensity in marine fishes. Helminths are the part of stomach contents but

according to Siglar, (1958) they might have been swallowed along with the

miscellaneous items in fishes. According to these statements, a relationship will

be found between dietary habits and nematode parasites.

20

3: Infestation caused by these endoparasites is analyzed as stated by

Kabata,(1985) parasite and disease reduce fish production by effecting the

normal physiology of fish. And which, if left uncontrolled can result in mass

mortalities, or in some cases, infection of man and other vertebrate that

consumed them.

21

2: REVIEW OF LITERATURE

2.1 Comprehensive account of feeding habits of fishes

Most studies of food and feeding habits of fishes, from varying habitats, have

showed that those of any one species differ in time and space and at different stage

of growth, thereby, emphasizing the need to study in more detail the food habits of

a species (Staples, 1975). The study of dietary habits of fish, based on stomach

content analysis, is widely used in fish ecology as an important means of

investigating tropic relationship in the aquatic communities (Fagbenro et al.,

2000).

Fish have wide range of food and feeding habits such as herbivores, pisnivorous,

omnivorous, carnivorous, detritivorous and have diverse ecological diversity.

Information on food and feeding habits of fishes facilitates understanding of their

feeding adaptation, growth, fecundity, migration and other related aspects.

Comprehensive account of food and feeding habits of marine fishes have been

published by several workers. (Todd, 1914; Suyehiro, 1934; 1942; Quershi, 1945;

Kuthlingum, 1957; Pradhan, 1959; Natarjan and Jhingram, 1961; Keast, 1968;

Suselam and Nair, 1969; Braber and Degroot, 1973; Hamellin and Bouchon, 1976;

Hyslope, 1980; Ghandi, 1982; Data & Das, 1983; Mohan, 1985; Hoda, 1991;

Khan & Hoda,1993; Yamamura et al, 1993; Hussain & Abbas, 1995; Poulin, 1995;

Schulz & Schoonbii, 1999; Fagbenro et al., 2000; Fugi et al., 1996; Hailu, 2001;

Katsuhiro and Mahyam, 2003; Seven et al.,2003; Hajisamae et al., 2004;

Gregory, 2005; Takeuchi et al., 2005; Campo and Mostarda et al., 2006; Balik et

al., 2006; Bhuiyan et al.,2006; Mostarda et al., 2007; Laurent et al., 2007;

Ayotunde et al., 2007).

22

2.2 Effect of temperature on fish food

The fish consumes less food at low than at high temperature. The temperature

changes influence food consumption directly or indirectly (Belding, 1928;

Baldwin, 1956; Molnar and tolg, 1962; Keast, 1968. Under natural condition food

consumption of most fishes changes with age and therefore, with size occurring in

different seasons. Smaller fishes generally have a higher metabolic rate than large

fishes of the same species. Thus small fishes generally eat more relative to their

body size. Fishes are cold blooded; their body temperature is determined by the

temperature of their environment. Warm water fishes generally have higher

metabolic rates and require more food than cold-water species of the same size.

Food organisms of different species of fish in any region may influence

respectively the horizontal and vertical movement of the fish stock; hence a correct

knowledge of the relationship between the fishes and food organisms is essential

for the prediction and exploitation of the fish stock.

2.3 Nematoda (roundworms)

Parasite assemblages of marine fishes include an important number of larval stages

of helminth parasite species that use fish as intermediate or as paratenic hosts.

Roundworms called nematodes are the most common parasite found in marine

fishes.

Nematodes have long been known to parasitize marine fish and are found

throughout the seas of the world (Baylis,1923;1932; Kulkarni, 1935;

Annereaux,1946; Karve & Naik, 1951; Ali, 1960; Dogiel et al., 1961;

1962;Yamaguti,1961; Lal, 1965; Chakravarty, 1932; 1942;

Chakravarty&Mujumdar, 1959; 1960; Chakravarty et al., 1961; Sood, 1967;

1988; 1969; Khan & Yasin, 1969; Berland, 1970; Khan & Begum, 1971;

23

Bilqees et al., 1971; 1977; 1978; 2004; 2005; Bilqees & Kazmi, 1974;

Akram,1975;1999; Gibson, 1975;Kalyankar&Palladwar,1977;Petter,1978;1979;

Hazen et al., 1978; Ahmed&Rehman,1979; Gupta&Gupta, 1979; 1980;

Moravec, 1979; 1983; 2001; Dhar&Fotedar,1980; Bilqees & Fatima, 1980a;

1980b; 1986; Arya,1980;1990;Dhar&Fotedar,1980;Ahmed,1981; Bilqees &

Akram, 1982; Malakhov & Valovaya, 1984; Fatima & Bilqees, 1987; 1989;

Goldberg et al., 1991 Boomker, 1993; Duran et al., 1989; Laakshmi & Sudha,

2000; Lakshmi, 2000a,b; Damin el al,.2004; Moravec et al.,1993;1997;2000;

2003; 2004; Ana, et al., 2004; Luque et. al., 2004; Moravec & Lou, 2005;

Moravec &Taraschewski, 2006; Ayotunde et al 2007; Cruz et al., 2007).

Yamaguti (1961) listed about 300 species, representing more than 40 genera in

17 families from marine fishes. A considerable number of new species and

genera have been added recently and many more remain to be discovered.

Numerous species of adult nematodes were described and reported in the coast from

Pakistan. Between them, several nematodes were found parasitizing the intestine

such as Cucullanu quadrii Bilqees and Fatima, 1980 (Cucullanidae) from Arius

serratus (Day, 1877).. Bilqee et al., (1971) reported marine fish nematodes of West

Pakistan and described seven new species from Karachi coast. Bilqees et al.,

(1977) reported marine fish nematodes of Pakistan describing Dujardinascaris

sciaenae from a sciaenid fish, Fatima and Bilqees (1987) review the Anisakis

nematodes. Fatima (1988) described seasonal variation and histopathology of

nematodes and Acanthocephala in some edible fishes of Karachi coast. Fatima

(1985) also reported some larval nematodes from the fishes of Karachi coast. Khan

and Yaseen (1969) had surveyed the helminth parasites of marine fishes from

Bangladesh and recovered Porrocaecum trichiuri. Chandler (1935) reported

24

Contracaecum brevicaecum from the body cavity of saw fish and Raphidascaris

panijii from the intestine of Sillaginopsis panijus. (Hamilton, 1822). Khan (1969)

reported a new species of the genus Indocucullanus from Pakistan. Khan and

Begum (1971) recovered Echinocephalus uncinatus from the mesentery of

Cyanoglossus sindensis(Lac.) and Lates calcarifer(Bl.); Contracaecum vittatii from

the body cavity of Upeneus vittatus(Forsk.) C. collieri (Chandler,1935) from the

body cavity of Mugil parsia(Ham.). They also reported Dujardinascaris magna

from the stomach of Sciaena sp. Bilqees and Fatima (1992) reported

Echinocephalus muraenesocis and Contracaecum synpapillus from

Muraenesox(Forsk.); Porrocaecum ruberum, Procamallanus dussumieri. They also

reported Dujardinascaris sciaenae from Pseudosciaena diacanthus(Lac.) in

(1977a). Khan and Begum (1971) described heliminth parasites of fishes from West

Pakistan ( Pakistan). Rasheed (1965) reported a new nematode Lappetascaris

lutjani (Anisakidae: Ascaridoidea) from marine fishes of Karachi coast. Also were

reported cases of larval nematodes parasitizing fishes in the coast from

Pakistan.Bilqees and Rashid (1982) reported Contracaecum otolithi from Otolithius

argenteus(Cuv.). Bilqees and Fatima (1986) reported larval stages of Contracaecum

and Anisakis from the fishes, Cybium guttatum(Schn.), Pseudosciaena diacanthus

(Lac.), Muraenesox cinereus (Forsk.), Parastromateus niger (Bl.), Pampus

argenteus (Euph.), Arius serratus(Ham.), Hilsa ilisha(Ham.), Pomadasys

olivaceum(Day.), Chiloscyllium griseum (Muller&Hen.),Galeocerdo

arcticus(Faber), Mugil sp.(Mugilidae), and Stegostoma varium (Hermann), of

Karachi coast, Pakistan.

Species of marine fish nematode reported so far from fishes of Karachi coast belong

to various families and genera. According to a recent checklist Akhtar& Bilqees,

(2006) 71 species of nematodes belonging to 23 genera, 9 sub families and 7

25

families from 68 species of marine fishes belonging to 46 genera and 31 families

have been reported from karachi coast of Pakistan. Of 71 species of nematodes

about 18 are from family Cucullanidae Cobbold, (1864), 21 are from family

Heterocheilidae( Railliet& Henry,1905)19 species from family

Camallanidae(Railliet& Henry,1915), 4 species from family Philometridae Baylis&

Daubney(1926),2 species from family Gnathostomatidae Railliet(1895), 3 species

from family Physalopteridae Railliet (1893), and 5 species are from family

Rhabdochonidae Skrjabin, (946). (Checklist is attached).

2.4 Fish pathology due to Nematodes

Various parasites have been reported from many fishes and information about

these parasites is increasing day by day. In recent years much attention has been

paid on the studies of parasites. The diseases due to the adult and larval nematodes

are very common in marine fishes and are world wide in distribution. The parasites

invade various tissues and organs of fish. Among the known sites of infection are

the stomach, intestine, liver, gonads, visceral mesenteries, peritoneum body cavity,

blood vessels, swim bladder, and connective tissues, fin, orbits of the eye and

brain. Most species of nematodes in adult stage live in the alimentary canal except

the family Philometridae which are found in body cavity, liver and gonads.

There are hundreds of published reports on naturally occurring larval nematodes

in fish including several reports from Pakistan. It is known that larvae are more

harmful than adults and can penetrate in the tissues of various organs, causing

severe tissue damage and destruction of cell of the organ. Most of the researches

have been done and are being added are towards the diagnosis, description and

analysis of the diseases of the marine fishes (Bauer,1958; Dogiel, 1962;

Getsevichyute, 1955; Mikhaylova, et al., 1964; Petrushevsky and shulman, 1955;

26

Reichenbachklinke, 1965;Sindermann, 1966, 1970; Lagler, 1977; Robert, 1978;

Smith and Wootten, 1978; Kinne, 1980; Deardorff et al 1987; Roberts, 1978;

Kinne, 1980; Kabata, 1985; Iskikura et al. 1993; Meunier & Desse, 1994; Bernet

et al., 1999; Konishi, and Sakura, 2002; Miyazaki et al.,2006; Akinsanya, 2007.

Sindermann (1970) has quoted a total of 555 references devoted to diseases of

marine fishes, of these only 27 appeared before 1900. The motivation for this

impressive increase on fish disease research is an attempt to avoid economic losses

and to disclose the life histories of causative agents, which may also interfere with

human health.

Attempts have been made to study the histopathology of organs of various fish

species of Karachi coast infected with Anisakid and other larval nematodes

(Fatima & Bilqees, 1989; Bilqees, 1997; Bilqees and Fatima, 1993; 1995;

2000; Bilqees and Khan, 1994; Bilqees and Parveen, 1996; Khatoon & Bilqees,

1996; 1999;Khatoon et al., 1999a; 1999b; Bilqees et al., 1998; 1999 ; 2001; ).

Most fishes of the Karachi coast are commonly infected either with nematode

larvae or Acanthocephalans or both. Seasonal variations and intensity of

infection of these parasites have already been reported by Bilqees and Fatima

(1986) and Fatima and Bilqees (1989). A number of genera of the family

Anisakidae occur in the digestive tracts of marine fish. They live free in the

lumen of the stomach or intestine; some attach to or invade the wall of these

organs and cause local tissue damage. Pathogenesis is a result of their mode of

feeding, attachment and movement or migration within the host. Anisakis larvae

are commonly found in the fishes of Karachi coast and have been reported from

several fishes including Cybium guttatum (Bilqees and Fatima,1986). Bilqees

and Fatima( 1993) have reported that the intensity of infection may be as high as

27

90% in Hilsa ilisha. In fishes of Karachi coast the larval forms reported are

Anisakis sp; Porrocaecum sp; Contracaecum sp; Thynascaris sp;

Echinocephalus sp.(Bilqees and Fatima,1986). Bilqees (1993) published

dictionary of pathology and histopathology. In 1996, Bilqees and Parveen

discussed the pathological variation in the tissues of stomach of Cybium

guttatum (Bl.&Schn.) of Karachi coast parasitized with nematode larvae while

tissue eosinophilia in same fish associated with nematode larvae reported by

Bilqees et al.,(1998), also discussed the public health importance of the

infection. Khatoon et al., (1999) reported Goezia sp.(Zedar.) from the stomach

of Lutjanus argentimaculatus(Forsk.) and discussed the histopathology of

stomach showed severe destruction in the whole thickness of stomach wall, with

gastric mucosa partly or completely destroyed in the affected regions. Their

study showed that destruction and necrosis of all layers of stomach has occurred

depending on the extent of penetration of the nematodes. Rizwana (1999)

reported the high infestation rate in Lutjanus argentimaculatus (Forsk.) by the

larval stages of Anisakais and Contracacum in approximately all fishes but

smaller group of fishes were more infested as compare to larger one. According

to her it might be due to the consumption of more crustaceans by the smaller

fishes. Rizwana et al., (2000) also recovered Goezia argentimaculatus from

Lutjanus argentimaculatus (Forsk.)

Many authors in other parts of the world have worked on Anisakis nematodes.

Agersborg (1918) reported nematodes on marketable fishes. Snieszko (1970)

described fish diseases. Bauer (1958) described relationships between host fish and

their parasites. Bishop and Margolis (1955) examined the prevalence of larval

Anisakis nematodes in herring ( Clupea pallasi) of the British Colmbia coast. Tsai

and Cross (1966) reported Anisakis like larvae from marine fish of Taiwan

28

(China). Anai (1969) has given described preliminary report on the parasites of

certain marine fishes of British Columbia. Koyama et al., (1969) described

morphological and taxonomical studies on Anisakidae larvae found in marine

fishes and squids. Kurochkin and Leonteva (1970) described medical significance

and distribution in marine fish of Anisakid larvae. Henning (1974) described the

effect of a larval Anisakis on the South West African anchovy, Engraulis capensis.

Stern et al., (1975) examined Anisakid larvae in the edible portions in sixteen

species of commercial marine fishes caught from Washington State. Hauck (1977)

reported occurance and survival of the larval nematode Anisakis sp., in pacific

herring Clupea harengus Hauck and May (1977) described histopathologic

alterations associated with Anisakis larvae in Pacific herring from Oregon. Smith

and Wootten (1978) reviewed many and varied aspects of the extensive world

literature on Anisakis and Anisakiasis including use of the nematodes as biological

tag in applied fishery sciences in Scotland. Deardorff and Overstreet (1981a)

reported larval Hysterothylacium (Nematoda: Anisakidae) from fishes and

invertebrates in the gulf of Maxico.

Matthews (1982) studied behaviour and enzyme release by Anisakis sp. Larvae.He

found that Bagrov (1983) described morphological variability of larvae of

nematodes of the genus Anisakis (Nematoda: Anisakidae). Petter et al., (1984)

studied teleost fishes from Yugoslavia for parasitic nematodes and found several

species of the genus Anisakis sp.As Anisakid nematodes are important from human

health point of view, attention is being paid on the study of these nematodes in

various parts of the world. Asami et al., (1965) reported two cases of stomach

granuloma, with associated necrosis and extensive eosinophilic infiltration. Thiel

and Houten (1967) described the localization of the herring worm Anisakis marina

in and outside the human gastrointestinal wall. Andreassen, (1970) described the

29

first known case of Anisakiasis in Denmark. Deardorff et al., (1987) described

human Anisakiasis with two case reports from the state of Washington. Little,

(1973) report Aniskid larvae from throat of a woman in New York. According to

Kim et al., (1991) Anisakiasis can cause intestinal obstruction. Anisakiasis of

colon also occurs which is rare as compared to gastric anisakiasis (Minamoto et al.,

1991; Matsumoto et al., 1992). Bhargava et al., (1996) reported the first tonsillitis

case associated with Anisakis infection. Ruben et al., (2001) reported seven humen

cases infected by Pseudoterranova decipiens (Nematoda, Anisakidae) in Chile.

Camallanus species infect the gastrointestinal tract of fishes. Camallanus species

can be identified by their red color; their location further toward the posterior of

the intestinal tract than other worm-like parasites (typically very near, and often

protruding from, the anus of the fish); the presence of a buccal capsule (mouth

structure) that is divided into two lateral valves, giving the mouth a slit- like

appearance; and, if gravid females are present, the presence of both eggs and larvae

within their bodies. The spirocamallanids are the parasites of the digestive tract of

the host, most frequently in the intestine and the less often in the stomach and only

rarely in the swim bladder.

Camallanid nematodes are also common in Pakistan and other parts of the world.

Procamallanus(Spirocamallanus)mehrii (Agarwal, 1930); P. (S.) planoratus(

Kulkarmi, 1935); P. (S.) pereirai (Annereaux, 1946); P (S.) bagarii( Karve&Naike

,1951); P.(S.) ahiri (Karve, 1952); P.(S.) mysti (Karve, 1952) ; P.(S.) saccobranchi

(Karve ,1952) P.(S.) aspiculus (Khera, 1955);P.(S.) gubernaculus (Khera,1955);

P.(S.) heteropneustus, , P.(S.) hyderabadensis from Mystus seenghala, P.(S.)

singhi (Ali, 1956); P.(S.) mozabukae (Yeh, 1957); P.(S.) daccai (Gupta, 1959);

P.(S.) confuses (Fernando& Furtado, 1963); P.(S.) hindensis (Lal ,1965)

Heteropneusies fossilis in Meerut; P.(S.) istiblenni (Noble, 1966); P.(S.) vittatusi

30

(Sood, 1967) from Mystus vittatus in Lucknow; P.(S.) ottuei (Verma &Verma

1971; P.(S.) ompoci(Majumdar&Data1972) ; P.(S.) intestinscolas (Bashirullah&

Hafizuddin ,1973); P.(S.) notopteri (Bashirullah& Hafizuddin,1973); P.(S.) timmi

(Bashirullah, 1973); P.(S.) ditchella (Gupta& Garg, 1977) ; P.(S.) gupta (Arya

,1978) from Schizothorax richardsonii(Grey) ; P.(S.) kashmirensis (Dhar&Fotedar

,1980); P.(S.) daleneae (Boomker,1993) ; P.(S.) guttatusi (Andrade-Salas et al.,

1994; (Olsen, 1952; Noble, 1966; Machida and Taki, 1985; Rigby and Adamson,

1997; Rigby and Font, 1997); P.(S.) kakinadensis and P.(S.) lutjanusi (Lakshmi,

2000a,b); P.(S.) chetumaleusis (Gonzalez-Solis et al., 2002); P.(S.) fulvidroconis

(Moravec et al., 2003) redescription ; P.(S.) variolae and P.(S.) longrus (Moravec

et al., 2006) and P.(S.) anguillae (Moravec et al., 2006).

From the present investigation 272 nematode specimens were recovered including,

new and known species. A prevalence of 62% was recorded. From the infected fish

specimens 131 new nematode specimens and 141 known nematode specimens were

recorded. ( 37 nematode specimens were damaged, kept for DNA sequences) Only the

new nematode species are described here. The recovered new nematode species

belong to one new genus and three known genera, including Spirocotyle n.gen.

(Camallanidae Railliet and Henry, 1915); Dujardinascaris Baylis, 1947;

Procamallanus (Spirocamallanus) (Olsen 1952) Petter 1979), and Cucullanus Muller,

1777. A total number of 11 new species were recovered from the above 4 genera.

31

3: MATERIAL AND METHOD

3.1 Study Area:

Pakistan is located in the northern part of the Arabian Sea a coastline of 1,120

Km. with broad continental shelf. There are more than 16,000 fishing boats in

coastal area of Pakistan, which operate in coastal water as well as in offshore

areas. Karachi is one of the major fish harbor of Pakistan, handles about 90% of

fish and sea food catch in Pakistan. (Pakissan.com. 2001-2007).

3.2 Collection of material:

A total of 1500 fishes belonging to 14 species, 10 families and 6 orders were

collected monthly during February 2005 to June 2007 from fresh landing of fish

harbor Karachi coast, Pakistan. The fishes were examined fresh or preserved

whole in 10% formalin for later examination in Laboratory of Jinnah University

for women. After washing on subsequent days, the fishes were identified

(Qureshi, 1955; Fishers and Bianchi, 1984; FAO Field guide; Hoda, 1985;

Majid and Khan, 1995; Eschmeyer, 2001 and by fin-formula method). The

length and weight of each fish in the sample were taken from the tip of snout to

the end of caudal fin using measuring scale and weighting by using Bango

digital scale.

The fishes under investigation were Liza vaigiensis, Sardinella albella,

Scomberomorus guttatus, Pomadasys olivaceum, Pomadasys maculatum,

Pomadasys stridens, Otolithus ruber, Arius maculates, Sphyraena forsteri,

Sphyraena jello, Lates calcarifer, Sillago sihama and Euthynnus alletteratus.

32

The investigation on fishes was conducted as follows:

a): Food and feeding habits.

b): Nematode parasites.

c): Histopathology.

3.3 Method for the study of feeding habits:

A total of 780 (out of 1500) fishes were dissected to analyze the stomach

contents. Almost all the samples from which complete digestive tract were

extracted (89%, n=700) has stomach content present whereas Only 80(10%)

of stomachs were empty. From the 89% extracted digestive tracts, 9 taxonomic

categories were identified.

Various techniques are available to scientists when undertaking the gut analysis

of fish. The simplest available method takes organism occurrence as as the main

consideration. The analysis of the stomach contents was carried out by

percentage occurrence of stomachs and Frequency of Occurrence methods

(Hyslop, 1980).

3.4 Occurrence method (Hyslop, 1980),

The number of stomach in which a particular food category occurs is listed as

a percentage of the total number of stomach of fish species containing food in

the gut (Table 5, 5.1, 5.2). This method is qualitative, laying emphasis on the

frequency of occurrence of items in the gut contents. The analysis of

percentage of each food category was summed divided by the total number of

stomach in the sample (excluding those with empty stomach) and calculated

by using the formula given below;

33

No. of stomachs in which particular food category occurred

--------------------------------------------------------------------- X 100

Total number of stomachs examined (Non-empty)

3.5 Frequency of Occurrence (Hyslope, 1980)

Another method used for assessment of food items was frequency of

occurrence (F. F %) in relation to total number of stomachs. (The number of

stomachs in which a food item occurred was expressed as a percentage of total

number of stomachs. (Table 6- 13)

3.6 Examination of material for feeding habits

For this purpose the fishes were dissected and the stomachs were removed,

weighed and persevered in 8 % formalin solution. In laboratory the samples

were removed from the jar. The content of each gut were mixed with 100 ml

water per gram of gut contents to remove unpleasantness of the formalin and

filtered through 100 um and 500um mesh size. Caution was taken not to leave

samples in the water for more than 48 hrs to prevent the sample from

deteriorating. An attempt was made to identify the food items to species. But

the level of discrimination depended on the completeness of the food items.

Polychaetes, small crustaceans and some fishes were particularly difficult to

identify as they were rapidly digested and were rarely in good conditions. For

phytoplankton, the microscopic preparations per filtrate were observed under

the microscope for taxonomic identification. Food contents were grouped into

9 categories such as:

(1) Polychaetes, (2) Crustaceans, (3) Molluscs, (4) Platyhelminth, (5)

Nematyhelminth, (6) Teleosts (fishes), (7) Planktons, (8) Detritus (9)

Miscellaneous.

34

Platyhelminth and Nematyhelminth were not the food items but were the part

of food content.

3.7: Collection and Examination of specimens for nematode parasites:

A total of 1500 fish specimens were examined for nematode parasites

investigation. 745 fish specimens were infected. A prevalence of 62 %

was recorded and a total of 272 nematode specimens (including 131 new and

141 known specimens) 37 damaged specimens were kept for DNA sequences.

Only the new nematode species are described here. All infection observed and

recovered were restricted to the intestine.

In order to collect the parasites the body cavity of fish was opened and the gut

and liver were removed by cuts in the region of the anus and the division

between esophagus and anterior stomach. The mesenteries and connective

tissues connecting loops of the gut and the liver were cut and the organs

separated. The gut was then placed in a large Petri dish stretched out and cut in

to five regions i.e. the stomach, the anterior middle and posterior intestine and

the rectum. The division of the intestine was equivalent to one third of the

whole intestinal length. The section of the gut was opened with a longitudinal

cut, and the whole inner surface lightly scraped to remove the parasites with

mucus. The species of nematode parasites in each region of the gut was

recorded. The nematode found were washed in physiological saline water and

then fixed in boiling 70% ethanol and preserved in 70% ethanol. For light

microscopy the nematodes were cleared in glycerin. Diagrams were prepared

with camera Lucida E 200 Nikon Drawing Tube. Measurements are given

length by width in millimeter.

35

3.8: Preparation of nematode specimens for Steroscan

Electron Microscopy (SEM) Specimen for scanning electron microscopy were fixed in cold 4%

glutaraldehyde in buffer (P.H 7.2) and kept in it for 24 hours, then dehydrated

through a graded series of alcohol, infiltrated with amylacetate, after critical

drying mounted on stubs, coated with gold and photographs were taken with

the help of SEM. Joel Japan JSM 6380A at an accelerating voltage of 15KV

at Karachi University, central laboratory. The SEM measurements are in

micrometer.

3.9 Procedure for histopathology:

Histopathology of infected intestine of two fishes was carried out including,

Euthynnus alletteratus (Refinisque) and Pomadasys maculatum (bloch,).

For histological studies of intestine, small pieces of infected intestine were

fixed in 10% formalin for 24 hours, washed several times with water,

dehydrated in graded series of alcohols. Cleared in Cedar wood oil and

xylene.blocks were made in cavity blocks by usual method. Thick sections

were cut with a rotary microtome. After removing the wax by xylene,

hydration was carried out. Sections were stained with haematoxylin and eosin,

dehydrated, cleared in clove oil and xylene and mounted permanently in

Canada balsam. Photographs were taken with a photomicroscope Nikon

(Optiphoto20 using Agfa color film.

36

4: FEEDING HABITS

Food is an important factor in the biology of fishes, to the extant of

governing their growth, parasitic movement, maturity and migratory

movements. The food choices of different edible marine fishes were

investigated. The study of dietary habit of fish based on stomach content

analysis is widely used in fish ecology as an important means of investigating

tropic relationship in the aquatic communities (Fagbenro et al., 2000).

The pattern of food and feeding habits of 8 edible fishes were studied during

the period from Feb. 2005 to June 2007 from Karachi coast, Pakistan.. Fishes

were identified by fin-formula method; Qureshi (1955); Fisher and Bianchi,

(1984) FAO field guide; Hoda, (1985); Majid and Khan, 1995; Eschmeyers,

(2001). The length and weight of each fish specimen in the sample were

recorded.

The taxonomic lists of fish species under investigation are given in table 1.

The compositions of food items were categorized in table 2-4. The food

categories mainly consist of crustaceans, molluscs, Helminthes, planktons,

fishes, detritus and miscellaneous, while the organisms by name, total,

percentage occurrence (O%) and percentage of frequency occurrence (F%)of

stomachs can be seen in Tables (5-13) with histogram representation in (fig.

7-17).The purpose of this study was to examine the relation ship between fish

feeding habit and their diet related parasites.

Analysis of stomach content of 780 marine fishes belonging to 8 genera and

13 species shows that out of these 10 genera 2 fish species(Liza.verigensis

37

and Sardinella albella ) are herbivorous and 11 fish species (Scomberomous

gutattus, Pomadasys olivaceum, pomadasys maculatum, Pomadasys

stridens, Otolithus ruber, Lates calcalifer, Arius maculates, Sillago sihama,

Sphyraena jello, Spharyena forsteri and Euthynnus alletteratus) are

carnivorous. Only 8 fish species are described for their dietary habits.

There was no significant difference in seasonal variation of the food

composition of these fishes .All the food items appeared in the stomach all

year round. Different parasites species were also found in food content of

some fishes. But these are not the part of the food content and may be

swallowed with some food items, as the fish is an