Feeding Habits and Nematode Parasites of Some Fishes of Karachi Coast

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

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

Supervisor’s 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. (43—45)Dujardinascaris mujibi n.sp.------106

Figs. (46—48)Dujardinascaris jello n.sp.------109

Figs (49—51) Dujardinascaris maculatum n.sp.------112

Figs.(52—54) Dujardinascaris sphyraenii n.sp.------115

Figs. (55—57)Dujardinascaris multiporous n.sp.------118

Figs. (58) Dujardinascaris sinjarii n.sp. ------121

Figs.(59—60) 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 (61—66)

Figs. (61): Spirocotoyle otolithi n.gen. n.sp------143

Figs. (62—64): Procamallanus(Spirocamallanus) riaziaii n.sp------144

Figs. (65—66): 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

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.

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.

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,

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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

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

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.

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.

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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

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 world’s 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.

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. Pakistan’s fishing waters are termed as highly rich in marine life with a vast variety of species having great commercial value. Pakistan’s 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).

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 world’s 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

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

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.

Pakistan’s exports of fishery products stand at about 0.25% of world exports. Pakistan’s 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

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 earth’s 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

productivity is in the form of tiny plankton, which are the lowest link in ocean’s 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

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 microorganism’s 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.

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 νῆμα (nema): "thread" + -ώδη -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

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).

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

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

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

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

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

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

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).

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.

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.

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).

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;

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

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

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;

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

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

(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 Leont’eva (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

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

(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.

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.

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;

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.

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.

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.

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

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 intermediate host for nematode parasites. The most dominant parasites were nematodes in different fish species and might be the best indicator for food analysis. But to find the correlation between these parasites and food contents, the Percentage of frequency occurrence of these parasites were also calculated.

4.1 Table: 1 Taxonomic position of different edible marine fish species studied for their feeding habits, nematode parasites and histopathology

S.No Order Family Species (Local name) Liza vaigiensis (Quoy&Gaimard, 1 1824)(Boi) Mugiliformes Mugilidae

Sardinella albella 2 (Valenciennes,1847) Clupeiformes Clupeidae (Tarli) Scomberomorus guttatus 3 Perciformes Scomridae (Bloch&Schneider, 1801)(Surmai) Pomadasys olivaecum(Day, 1875) (Dohtar) Pomadasys maculatum 4 Perciformes Haemulidae (Bloch, 1797) Pomadasys stridens(Forskal, 1775) Otolithus ruber 5 Perciformes Sciaenidae (Schneider, 1801) (Mushka)

Arius maculates 6 Siluriformes Ariidae (Thunberg, 1792)(Khagga) Sphyraena forsteri 7 Peryciformes Sphyraenida Sphyraena jello (Cuvier, 1829)(Kund) Lates calcarifer Centropomi 8 Perciformes (Bloch, 1790)(Dangri) dae

Sillago sihama(Forskal, 1775) 9 Perciformes Sillaginidae (Bhanmbhor) Euthynnus alletteratus 10 Thunniformes Thunnidae (Refinisque, 1810)(Dewan)

4.2 Diets of different investigated fishes

Diet of Liza vaigiensis (Quoy&Gaimard, 1824) Local name=Boi N=225 T.L=15.0-35cm (Mean=26.71, Variance=71.74, Std.Dev.=8.47, SEM= 2.67). (TL) is for total length and (N) is for number of fishes

It is edible fish found in shore, off –shore and estuarine waters. Color is brownish dorsally, becoming dull white on ventral side. Pectoral fins are deep black, Caudal fin truncate.

A total of 14 organisms were identified within the stomach of L. vargiensis.(Table 2) The food mainly consisted of detritus, and planktons (including phytoplankton and zooplanktons), blue green and green (unicellular, and filamentous) algae (Table 2). Detritus (100%) (Sand and blackish mud) were found to be of most dominant categories on the basis of percentage of occurrence (Table 5). Phytoplanktons were present in all size of fishes and in large quantity. The increase was observed in the month of April, May, June and July. The Observed phytoplanktons belonge to class Cyanobacteria, Bacillariophyceae and Cholorophyceae (Figs. 9-20). Anabaena, Oscillatoria sp., and Microsystis sp.,.(Cynobacteria), Gyrosigm sp., Pinnulari sp., Tabelaria sp., Melosira sp., Navicula sp., Diploneis sp., Schroderella sp., Odontella sp., (Bacillariophyceae), scenedesmus, Chlorella (Chlorophyceae) were found in all fish sizes. The phytoplankton category was doThe Gyrosigma sp.(85%) and Plurosigma sp.,(75%) were most common on the basis of percentage of frequency occurrence (F%) as compare to other food items. These were found in near about all fish specimens and in larger quantities (Table 6). Soil nematodes were also present but these were not the part of food. The analysis of data on the basis of diet composition indicates that Liza vargiensis is an herbivorous fish.

Diet of Sardinella albella (Valenciennes, 1847) Local name= Tarli N=70 T.L=6.5-14cm (Mean=10.44, Variance=8.05, Std.Dev. =2.83, SEM= 0.89).

A pelagic schooling species, found in coastal waters. The main diet items observed in the stomach of S. albella on the basis of percentage occurrence were plankton (75%) followed by detritus (66.7%). miscellaneous (64%) ranked 3rd among the food categories (Table 5). Copepods (16.66%) were the least observed food items on the basis of percentage frequency occurrence (F%) (Table-7). The observation of the stomach content revealed that Sardinella albella is herbivorous in nature.

Diet of Scomberomorus guttatus (Bloch&Schneider, 1801) Local name=Surmai N=70 TL=50-70cm (Mean=60.59, Variance=50.56, Std.Dev.=7.11, SEM= 2.24).

Lancet-shaped teeth ,much longer in the lower jaw, color bluish above and silvery beneath, back and side with three rows of horizontal oral spots edible fish ,commonly landed at fish harbour.It dose not remain fresh for long. A total of 14 organisms were identified in the stomach of S.gutattus (Table 3). Organism by name, total, percentage occurrence (Table 5) and percentage of frequency occurence of stomach can be seen in (Table 8) with histograme representation ( Fig 3) . Miscellaneous (90.16%) ranked at the top as compare to other food items. Crustaceans (81.96%) ranked second followed by molluscs(32.78%). The detritus (8.19%) and teleosts(6.55%) were in decreasing order. Helminthes (14.75%) were also observed but they were not the part of food items (Table 5). On the basis of observation of food content Scomberomorus guttatus is carnivorous fish.

Diet of Pomadasys maculatum (Bloch, 1797) Local name: Dhothar N= 75 T.L=16-50cm (Mean=30.29, Variance=207.10, Std. Dev. =14.39, SEM= 4.55).

Mouth small, slightly protractile. Lips thick and folded back. Lower jaw with two pores followed by a narrow groove on chin .No canine teeth. Color olive –grey; head glossed with purple; a large black blotch bordered in front with yellow at the upper angle of operculum. It occurs in sub tropical and tropical estuaries, common on Sindh and Makran Coast, much esteemed as food due to its excellent taste.

Different food categories were observed in the gut of Pomadasys maculatum (Table 3 & 5), belonging to crustaceans, molluscs, teleosts, detritus, polycheates and helminthes. According to the present analysis the crustacean(71.42%) and miscellaneus(71.42%) ranked on the top among the other food categories on the basis of percentage occurrence(Table 5) and frequency occurrence(Table 9). Principal food items (F %) in crustacean category were shrimp sp.,(71.4%), Acetes sp.,(71.4%) and different species of closely related copepods(57.14%).Sepia sp.,(35.71%) was dominating in molluscs category occupying the 2nd rank.. Johnius sp.,(35.71%) followed by Sardine sp.,(14.28%) and Leiognathus sp.,(7.14%) were observed in teleost group.(Table 9). Different species of other fishes were also observed but no positive identification was made to the taxonomic level so were included in miscellaneous category. Miscellaneous were dominant food group observed in every stomach with the majority consist of small fish species, compound eyes, 42

legs of shrimps and prawn, with some unidentified material.(Table 9). Nematodes (28.57%) Acanthocephala and trematode (7.14%) were also found in addition to food content. The present analysis of food categories and food items indicate that P. maculatum is a carnivorous fish.

Diet of Otolithus ruber (Schn, 1792) Local name=Mushka N=150 T.L=50-105 (Mean=59.34, Variance=28.16, Std. Dev. =5.30, SEM= 1.67). A common Sciaenid fish caught in good number in coastal waters. Color silvery, darkish along the back. A dark spot on the opercula. Pectoral, pelvic and anal fins orange colored. Outer edge of the dorsal fins grayish. Mouth wide, with one pair of strong canines on each side. Lower jaw larger.

The data on the stomach content of Otolithus ruber through out the study are shown in table 4.The crustaceans (82.60%) ranked 1st in importance as the food constituent (Table 5). Miscellaneous item (80.86%) occupy 2nd position on the basis of percentage frequency occurrence. It included parts of mandible crabes, cycloid and ctenoid scales, undigested part of animals and shell fragments. Due to the dominancy of the Gastropods sp., (72.17%) molluscs occupy 3rd position on the basis of percentage of frequency occurrence. The planktons (66%) were in decreasing order followed by teleost (60%). Polycheates were least observed food items. Nematodes (36.42%) and Acanthocephala (30.43%) occurred generally in those month in which gut were gorged, full or half full. According to the present

investigation Otolithus ruber was found to be carnivorous fish which mainly feed upon crustaceans and molluscs.

Diet of Arius maculates (Thunberg, 1792) Local name=Cat fish N=50 T.L=45.0—89.5cm (Mean=64.73, Variance=221.55, Std. Dev. =14,88, SEM= 4.70).

Body elongate ,without scales, dorsal and pectorals fins with spines. Caudal fin forked. Head with three pairs of barbells. Common along the coast in creeks and estuaries common in local fish market. The main diet categories were crustaceans (70%) (Table 5) dominated by Penied sp.(70%) with decreasing order of Copepods and Amphipods(60%)>Nepton ap.(40%)>Parapenaeopis sp. (14%). The miscellaneous (70%) and Planktons (70%) were also dominating food categories (Table 5). Gammerous sp. (70%) was common on the basis of percentage of frequency occurrence (Table 11) among the plankton category.

Diet of Sphyraena forsteri (Cuvier, 1829) Local name=Kund N=70 T.L=40-67cm (Mean=54.40, Variance=104.0, Std. Dev. =10.21, SEM= 4.56).

The fish is found in tropical and subtropical Coastal waters over shallow banks closed to the bottom. Large-mouthed with the lower jaw projecting forward bearing strong teeth. Upper jaw non-protractile, an adaptation to feeding on large prey.

The crustaceans and miscellaneous were on the 1st rank in the diet of S.forsteri constituting about (85%) on the percentage of occurrence (Table 5).The food items of crustacean category in descending order were Parpenaeopsis(57%)> Cephalopods(50%)> Peneid and Acetes sp.(35%). The miscellaneous mainly consisted of mantis shrimps, larvae of crabs, chelae, scales, shell fragments, prawn eyes, head and appendages prawn mysis. The teleosts (54%) ranked second in importance including small unidentified fishes as the food constituents. The molluscs (31%) having lowest frequency in the stomach on the basis of occurrence dominated by Squilla sp. (Table 12). Helminthes belonging to different phyla including, Acanthocephala, Cestodes and Nematodes but not the part of the food contents. S. forsteri has been found to be a carnivorous fish which mainly feed upon crustaceans, teleosts and molluscs.

Diet of Lates calcarifer (Bloch 1790) N=70 Local name=Dangri T.L=35-70cm (Mean=57.16, Variance=129.8, Std. Dev. =11.39, SEM= 3.60).

Large oblique mouth with lower jaw projecting, upper profile of head some what concave, color golden brown dorsally and silvery beneath found in coastal waters and estuaries. The main food contents are given in (Table 4). The percentage of frequency occurrence (F,F%) is given in (Table 13). According to this observation the crustaceans and miscellaneous (64%) were the top priority food categories followed by teleosts (57%) and molluscs (29%) on the basis of percentage of occurrence. The least observed food items were planktons.

4.3 Remarks

Different food categories were studied in 8 different edible fishes from February 2005 to June 2007.

Sujehiro(1942) observed that the fishes, which do not possess great swimming powers, have well developed teeth to hold the prey once it is caught the conical and pointed teeth of the jaws and pharynx of fishes under study help in seizing, holding and tearing the prey . The pharyngeal teeth also appear to macerate the prey while swallowing. Unlike mammalian intestine tissue, multicellular glands or other specialized structures in the intestine of teleost are rare.The intestine in carnivorous fish is shorter than the Omnivorous and herbivorous mucosal area, however may compensate for the relative length of the gut (Al-Hussain, 1949; Suyehiro 1942).

Food and feeding habits of different edible fishes were found to be comparable to Pakistani fishes( Huda, 1993; Ajazuddin, 1991; 2000; 2001; Khan and Huda, 1993; Imtiaz and Khan, 2005;).

Khan & Hoda, 1993 point out 8 food categories in marine edible fish Euryglossa orientalis on monthly basis. According to their observation miscellaneous items were found to be the most dominant category in all respect including percentage of occurrence, average points and percentage of total points. sand grains formed the second group on the basis of percentage of occurrence, mollusces and crustaceans were seen through out the years.

Ajazuddin, 2000; 2001, observed the food and feeding habits of Otolithus cuvieri and Johnius elongatus respectively the fishes were observed to be carnivorous on the basis of their feeding habits. he observed in Johnius elongatus that feeding intensity were higher in fishes of larger size group while poor feeding condition were exhibted by smaller size groups, he calculated the composition of food of different size and seasons. According to his observations, the polychactes, crustaceans and semi-digested food material were the most frequent food items for smaller size group while the diet constituent shifted to molluses, teleosts and miscellaneous food in larger size groups.

Imtiaz and Khan (2005) observed the food and feeding habits of edible carnivorous fish Pomadasys stridens from Karachi coast. according to their observation of different food groups, the semi-digested food material were the most dominant food group by percentage of occurrence, where crusteaceauns, molluscs, teleosts and Polychaetes occupied the successive position by percentage of total occurrence. According to the present observation based on percentage of occurrence method and Percentage of frequency occurrence the principal organisms observed in Pomadasys

maculatum were crustaceans and miscellaneous food items. The molluscs ranked second among the food categories. planktons were the least observed food items, Nematohelminthes were also observed but as stated earlier that they were not the part of food contents.

In the present study percentages of Occurrence of various food items were different in carnivorous and herbivorous fishes. Planktons were the most dominent food items in herbivorous fishe giving variable species. Whereas in carnivorous fishes the crustaceans and miscellaneous were dominating food categories. While molluses and teleosts ranked on second in number among the food categories. Polycheates and plankton were in descending order.

The food and feeding habits of different edible marine fishes were also found to comparable to fishes from other parts of the world. Although it is very difficult to compare to all species. Many works have been done on food and feeding habits of different fish species from different geographical zones. So it is impossible to make a through comparison with all of them. An attempt is made to compare the food and feeding habits to possible way.

The present analysis also incorporate with different observations made by biologist, As Rao (1964) observed during the analysis of stomach content of different fishes that there is a wide variation in the food and feeding habits between the different groups of fishes obtained at the bottom and also between different species within a group and there was marked diurnal differences in the composition of food items. Berg(1979) and Pillay(1952) reported that the

analysis of stomach contents of fish could provide information about the nich of a particular of fish in its ecosystem and this has become a standard practice in fish ecology works(Hyslop, 1980). Rao (1979), Suscclan and Naik (1969) recorded the occurrence of prawns, crabs, isopods, amphipods and fishes in the stomach of Sciaenid.

The present analysis also agree with Ghandi(1982), who remarked that feeding habits depended on the availability of fish food in the environment. Muthia (1982) concluded in Johnicops vogleri that the main food items were crustaceans and were encountered almost through out the year in different proportions.

Katsuhiro and Mahyam (2003) studied the distribution and feeding habits of Lutjanus johnius in mangrove estuary in Malaysia the analysis of their study showed that type of food varied with size. large-size individuals fed manily on natantia and small-sized individuals on Mysidaceae. Hajisami et al., (2004) analysis the food habits and trophic interrelationships between nine fish species, utilizing on impacted coastal habitat revealed that the diet of most species underwent marked changes with ontogeny. The analysis demonstrated that diet composition of the nine fish species differ from each other. Teleost fishes employ a wide range of feeding technique, but these rarely involve direct manipulation of object to uncover prey.

A strong relationship between infection with a parasite species and the corresponding intermediate host from the stomach content of individual charr, indicated an individual feeding specialization. (Knudsen et al., 1996). The infection variations seemed to be due to differences in host growth rate, host feeding habit, and the distribution of marine mammal final hosts. Larval 49

nematodes are useful biological indicators for the population study of walleye pollock in Japanese waters.( Konishi, 2002).

Hirasawa et. al., 2004 discuss the relationships between nematode parasitism and the feeding habitats of their intermediate hosts and found that the principal intermediate hosts of the two nematodes were filter-feeding mayflies of the genera Ephemera, Photamanthus and Isonychia. Ephemera strigata seemed to be the most important intermediate host of these nematodes. Adult R. coronacauda were found mainly in Hemibarbus longirostris and Rhinogobius flumineus, which are benthic fishes that feed on benthic aquatic insects, including E. strigata. For R. coronacauda, therefore, the feeding habits of the definitive hosts facilitate host alternation by this species.

Bhuiyan et al., (2006) studied the food and feeding habits of Channa punctatus. During their analysis Channa punctatus is a carnivorous fish, its food consist mainly of crustaceans, insects, molluses, fishes, plants and semi digested materials. Monthly variation in the percentage composition of food items in both juvenile and adults were recorded. The fish changed its food and feeding habits seasonally. The feeding intensity was very poor in mature fishes during the spawning period.

Laurent et al., (2007) studied the food composition and feeding habits of Euthymus alletteratus in continental shelf waters of West Africa. the type and quantity of prey ingested change seasonally. Out side the major upwelling period the diet was more varied. Overall fishes were the dominant prey of all sizes of little tunny, far exceeding crustaceans of which shrimps and prawns were commonest but were not found in the stomach of juveniles or larger

adults they observed that little tunny are carnivorous fish that feed opportunistically. a relation ship was found between the size of the prey and the size of the predators.

Mosterda et al., (2007) observed the feeding habits of the bullet tuna Auxis rochel in the southern Tyrrhenian Sea. The result of his study showed that the bullet tunna is an epipelagic off-shore predators feeding on whatever abundant resource is available in the environment with a preference for planktons, crustaceans, small cephalopods and fish larvae. All prey was pelagic organisms. A size related change in the diet composition was observed. The average prey weight per stomach increased significantly in the larger predators which mostly fed on fish larvae belonging to several commercially important demersal and pelagic species

4.4 Statistical Analysis

The data obtained were reported in the tables 5 – 13 and subjected to statistical analysis for the confirmation of results through statistics.

4.5: Food contents of different fish species from Karachi coast Table. 2: Food contents of Liza vaigiensis and Sardinella albella

Sardinella albella S.No Food Contents Liza vaigiensis (Boi) n=225 (Tarli)n=70 1 Polychaetes ------2 Crustaceans ---- Copepods 3 Molluscs ------4 Platyhelminthes ------5 Nematyhelminthes Unidentified Soil nematode ---- 6 Teleosts ------Phytoplankton(acanathes hungrica, gomphonema globiform, Scenedesmus, melosira arenaria, Pennularia, Tabellaria, Pleurosigma- elongatum, normaris, Phytoplankton(, Diploneis, Diploneis,Gyrosigma sp, Gyrosigma sp, pleurosigma- 7 Planktons green filamentous elongatum, normaris,), algae,Oscillatoria) Zooplankton(Harpactacticoid) Anabaena, Microsystis

Chlorella

8 Detritus (Sand grain) Sand, black mud gravels Sand,mud

Molluses larvae, unidentified 9 Miscellaneous Unidentified bottom organisms crustaceans

Table = 3: Food contents of Scomberomous gutattus, Pomadasys maculatum and Sphyraena forsteri

Scomberomorus Pomadasys maculatum S.N Food guttatus Sphyraena forsteri o Contents (Dhotar)n=75 (Kund) n=70 (Surmai)n=70 1 Polychaetes Neries Neries ---- Penied, cephalopods, Amphipods,Shrimps, Copepods, Shrimps, (Penied sp) 2 Crustaceans Parapenaeopsis sp, Copepodes Copepodes,Crabs (Acetes Acetes sp. sp)

Oratosqilla sp. Decapods, 3 Molluscs Loligo sp. ,Sepia sp.,Dentalium Dentalium sp.. sp. Platy 4 Liver flukes ---- Acanthocephala sp. helminthes Nematodes Nematy Nematodes Nematodes 5 (Spirocamallanus, helminthes (Dujadinascaris sp.) (Dujadinascaris s.p) Bulbocephalus) 6 Teleosts ---- Leiognathus sp. Cynoglossus sp. 7 Planktons zooplanktons ---- Zooplanktons 8 Detritus Sand ------Larvae of fishes crustaceans,fin,sca Carapace of fishes, Compound eye, legs, les of fishes, eggs, chlae,and 9 Miscellaneous chelaeand mantis of pieces of antennae of shrimps molluscs,crab crustaceans remains

Table.4: Food contents of Arius maculates, Otolithus ruber and Lates calarifer

S. N Food Contens Arius maculates Otolithus ruber Lates calcarifer o (Cat fish)n=50 (Mushka)n=150 (Dangri)n=70 1 Polychaetes Heteroneries ------Amphipods,Shrimps, Penaeus sp,Dioptera 2 (Penied sp) sp,Grapsid Shrimps,Crabs, Crustaeans Copepods,Crabs sp,Amphipodes Copepods (Neptorn sp) sp,Copepodes Soletella sp,Ensis Gastropods,Solen Cephalopods 3 Molluscs sp,Bullia,Soleno sp sp,Sepia sp (Loligo,squila) Platy Acanthocephala, Acanthocephala, 4 ---- helminthes Trematodes Nematy 5 Bulbocephalus sp. Spirocamallanus sp ---- helminthes Juvenile of eel,Fish 6 Teleosts part(head,spine,fins,bo Gobeid sp Unidentified fishes ne and scales) Cyclops,Gammrous Zoea larvae, 7 Planktons sp,Scratillaria,Zoea gammrous sp. larvae 8 Detritus ---- Sand,mud ---- Part of mandible Carapace of fishes, crabes,cycloid and Compound eyes, eggs, chelae, and 9 Miscellaneous ctenoid scales,undigested Carapace,mantis,scal antennae, compound part of animals,Shell es of fishes eye of crustaceans fragments

Table. 5: Occurrence of food categories in relation to total number of stomach analyzed (O %) of different marine edible fishes from Karachi coast

FISH SPECIES FOOD CATEGORIES

Polycheates Crustaceans Molluscs Helminthes Teleosts Plankton Detritus Miscellane s us

Liza ------25 ---- 85 100 50 verigensis

Sardinella albella ----- 16.66 ------75 66.7 64 Scomberomous ---- 81.96 32.78 14.75 6.55 ---- 8.19 90.16 guttatus Pomadasys 7.14 71.42 50 35.7 35.7 ------71.42 maculatum Otolithus 8.69 82.60 78.26 30.43 60.08 66.08 4.34 80.86 ruber

Arius maculates 10 70 60 4 10 70 6 70 Spharyena ----- 84.61 30.76 15.38 53.84 ----- 30.76 84.65 forsteri Lates ----- 64.28 28.57 12.85 57.14 7.14 --- 64.25 calcarifer

Table 5.1.: Statistical Calculations of Table 5.

Statistical Calculation Polycheates Crustaceans Molluscs Helminthes 0.49 0.2.09 0.032 0.490

Mean 3.69 67.36143 40.05286 16.15857 Standard Error 1.767316 8.926165 9.551812 4.899 Median 0 71.42 32.78 14.75 Mode 0 #N/A #N/A #N/A 4.675878 23.61641 25.27172 12.96153 21.86383 557.7349 638.6598 168.0014 Kurtosis -2.37994 4.79605 0.206456 -0.8125 Skewness 0.493746 -2.09915 -0.03267 0.462161

Maximum 10 84.61 78.26 35.7 Sum 25.83 471.53 280.37 113.11 Count 7 7 7 7 Largest (1) 10 84.61 78.26 35.7 Smallest (1) 0 16.66 0 0

Confidence Level(95.0%) 4.324469 21.84155 23.37246 11.98743

Table 5.2 : Statistical Calculations of Table 5.

Statistical Planktons Detritus Miscellaneous Calculation Teleosts 0.40 2.00 0.400

0.15 Mean 31.90143 31.17429 16.57 75.04857 Standard Error 9.84017 13.92129 9.251802 3.878052 Median 35.7 7.14 6 71.42 Mode #N/A 0 0 #N/A 26.03464 36.83226 24.47797 10.26036 677.8027 1356.616 599.1708 105.275 Kurtosis -2.40654 -2.70889 3.10952 -1.56373 Skewness -0.13487 0.375727 1.847657 0.377555

Maximum 60.08 75 66.7 90.16 Sum 223.31 218.22 115.99 525.34 Count 7 7 7 7 Largest (1) 60.08 75 66.7 90.16 Smallest (1) 0 0 0 64

Confidence Level(95.0%) 24.07805 34.06419 22.63836 9.489258

4.7 : Table 6. Frequency of Occurrence in relation to total number of Stomachs analyzed (F, F%) of different marine edible fishes from Karachi Coast. Liza vaigiensis (Boi) N=225 (Empty=11.11%)

Organism identified F F% Polychaetes ---- Crustaceans ---- Molluscs ---- Platyhelminthes ---- Nematyhelminthes Unidentified Soil nematode 50 25 Teleosts ---- Planktons acanathes hungrica, 45 22.5 gomphonema globiform, 40 20 Scenedesmus 25 12.5 Melosira arenaria 25 12.5 Pennularia 30 15 tabellaria 30 15 Pleurosigma- elongatum 100 50 pleurosigma- normaris 150 75 Diploneis 70 35 Gyrosigma sp 170 85 Green filamentous algae Oscillatoria sp. 88 49 Detritus (Sand ) 200 100 Miscellaneous 100 50

Table . 7: Sardinella albella (Tarli) N=70(Empty=14.28%)

Organism identified F F% Polychaetes ---- Crustaceans Copepods 10 16.66 Molluscs ----

Helminthes Platyhelminthes ---- Nematyhelminthes ---- Teleosts ---- Phytoplanktons Pleurosigma- elongatum 45 75 pleurosigma- normaris 30 50 Diploneis sp. 35 58.4 Gyrosigma sp. 40 66.7 Zooplanktons Harpacticoid sp. 20 33.33 Detritus(mud) 40 66.7 Miscellaneous 40 66.7

Table .8: Scomberomorus guttatus (Surmai)

N=70 (Empty=12.85%)

Organism identified F F% Polychaetes ----- Crustaceans Shrimps 20 32.78 Copepods 30 49.2

Molluscs

Ensis sp. 4 6.55

Juvenile of Lutjanus sp. 5 8.19

Helminthes Platyhelminthes Acanthocephala 9 14.75 Nematyhelminthes Bulbocephalus 5 8.19 Teleosts johnius sp. 4 6.55 Planktons(Zooplanktons) 10 16.39 Detritus 5 8.19 Miscellaneous Larvae of fishes 25 50 Crustaceans 55 90.16 Fin 25 41 Scales of fishes 35 57.37 Pieces of molluscs 14 22.95

Table .9: Pomadasys maculatum (Dhotar)

N=75(Empty= 6.66%)

Organisms identified F F% Polychaetes Neries 5 7.14 Crustaceans Shrimps 50 71.4 Copepods 32 45.71 Amphipods 40 57.14 Crabs(Acetes sp) 50 Molluscs Solen sp. 6 8.57 Sepia sp. 25 35.71 Pholas sp. 12 17.14 Lologo sp. 20 28.57 Helminthes Platyhelminthes Nematyhelminthes 25

Nematode (Dujrdinascaris 35.71 sp.) 5 Teleosts Johnius sp 25 35.71 Sardine sp. 10 14.28 Leiognathus sp. 5 7.14 Planktons ------Detritus ---- z Miscellaneous Compound eye 24 34.28 Legs 30 42.85 Chelae 50 71.42 Mantis of shrimps 26 37.14 Crustacean 28 40

Table . 10: Otolithus ruber (Mushka)

N=150 (Empty=23.33%)

Organisms identified F F% Polychaetes 10 8.69 Crustaceans Crabs(Grapsid sp.) 50 43.5 Isopods 70 60.9 Copepods(Different sp,) 60 52.17 Molluscs Gastropods 83 72.17 Solen sp 8 6.95 Helminthes Platy helminthes Acanthocephala 32 36.52 Nematohelminthes (Spirocamallanus sps) 10 30.43 Teleosts Unidentified fishes 30 26.08 Zooplanktons Gammrous sp. 73 63.47 Scratillaria sp. 54 46.95 Cyclops 21 18.26 Zoeae larvae 76 66.08 Detritus 5 17.39 Miscellaneous Parts of fishes 45 39.13 Unidentified specimens 60 52.17 appendages of crabs 65 56.52 Shrimp eyes & uropodes 75 65.21 Skeleton,fins,scales of 93 80.85 fishes

Table. 11: Arius maculates (Cat fish) N=50 (Empty=nil)

Organism identified F F% Polychaetes - - Dioptera sp. 5 10 Hetero neries 3 6 Crustaceans Amphipods 30 60 Parapenaeopis sp. 7 14 Penied sp 35 70 Copepodes 30 60 Nepton sp 20 40 Molluscs ---- Solen sp 10 20 Bullia sp 6 12 Ensis sp 7 14 Helminthes Platy helminthes ---- Nematohelminthes 2 4 (Unidentified) Teleosts Juvenile of eel fish 5 10 Zooplanktons Gammrous sp. 35 70 Zoea larvae 30 60 Detritus 3 6 Miscellaneus Carpaceae of crustaceans 35 70 Molluscan shell fragments 26 52

Gill pieces of bivalves 12 24

Eggs, chelae, antennae and compound eye crustaceans 50 71

Table. 12: Sphyraenaa forsteri (Kund)

N=70 (Empty=7.14)

Organism identified F F% Polychaetes ---- Crustaceans Peneid sp. 25 35 Cephalopods 35 50 Parapenaeopsis 40 57 Acetes sp 25 35 Molluscs ---- Oratosquilla sp 3 4.6 Sepia sp 5 7.6 Dentalium sp. 10 14 Helminthes Platyhelminthes ---- Nematyhelminth Dujadinascaris sp. 10 14 Teleosts Unidentified small Fishes 35 50 planktons Detritus 20 28 Miscellaneous Carapace of fishes 50 71 Eggs, chlae,and antennae of 55 78 crustaceans

Table .13: Lates calcarifer(Dangri)

N=70 (Empty=nil)

Organism identified F F% Polychaetes ---- Crustaceans Shrimps 30 42 Copepods 20 28 Crabs 45 64 Molluscs Loligo sp. 15 21 Squila sp. 5 6 Helminthes Platyhelminthes ---- Acanthocephala 9 12

Nematyhelminthes ---- Teleosts Unidentified fishes 40 57 Zooplanktons 5 6 Detritus ---- Miscellaneous Carpace of fishes 30 42 Mantis,scale,antennae and compound eye ocrustaceans 45 64

4.8: Frequency of occurrence of the food items in relation to stomach analyzed of marine edible fishes from Karachi Coast.

Liza vaigiensis Soil nematodes Acanthes hungrica Gomphonema globiform 100 Scenedesmus Melosira arenaria 80 Pennularia Tabellaria % 60 Pleurosigma elongatum Pleurosigma normaris 40 Diploneis Gyrosigma 20 Oscillatoria sand 0 Miscellaneous Food items

Fig. 1

Food items SardinellaSardinella albella albella copepodes 80

60 Pleurosigma % 40 elongatum 20 P. normaris 0 Food items Diploneis

Fig. 2

Shrimps sp. ScomberomousScomberomorus guttatus guttatus copepodes 100 Ensis sp. 80 Lutjanus sp. 60 % Acanthocephala 40 Bulbocephalus 20 Johnius sp. 0 Food items Fish larvae Crustaceans legs

Fig. 3

Neries Pomadasys maculatum Pomadasys maculatum Shrimps sp.

copepodes 80 Amphipodes

Acetes sp. 70 Solen sp.

60 Sepia sp.

Pholas sp. 50 Loligo

% 40 Dujardinascaris

Johnius sp. 30 Sardine sp.

20 Leipgnathus Compound eyes

10 legs of crustaceans

chelae 0 mantis of shrimps Food items shrimp eyes& uropodes 40 Fig. 4

Otolithus ruber Polychaetes Otolithus ruber Grapsid

Isopodes Copepodes 90 Gastropodes

Solen sp. 80 Acanthocephala 70 Spirocamallanus sp.

60 fishes Gammrous 50 % Scratillaria 40 Cyclops

Zoea larvae 30 Detritus 20 fish parts

10 Chelae Crab appendages 0 Shrimp uropodes Food items Fish skelton,fins,scales Fig. 5

Diopt era sp.

AriusArius maculates maculates Het eroneries

Amphipodes

Parapenaeopis sp.

80 Penid sp.

Copepodes 70 Nept on sp. 60 Solen sp. Bullia sp. 50 Ensi s sp. % 40 nemat odes Juvenile of eel f ish 30 Gammrous 20 Zoea larvae Det rit us

10 f ish carpaceae 0 Molluscan shell f rag. Food items Bivalves gill pieces Eggs,chelae,ant ennae

Fig. 6

Spharyena foresteri Spharyena forsteri

80 Peneid sp. Cephalopodes 70 Parapenaeopis

60 Acetes sp.

Oratosquilla % 50 Sepia sp. 40 Dentalium

30 Dujardinascaris 20 small fishes Detritus 10 Carapace of crustaceans

0 eggs, chelae, antennae Food items

Fig. 7

Lates calcariferLates calcarifer

80 shrimps sp. copepodes crabe sp. 60 Loligo % Squila 40 Acanthocephala fishes zooplanktons 20 Carapace of crustaceans Detritus 0 mantis, compound eyes Food items

Fig. 8

Figs.9-42: Photographic representation of some organisms present with in the stomach of different marine edible fishes

Fig:9 Diatoms(Different species) Fig:10 Oscillatoria sp. (Filamentous algae)

Fig:11 Pleurosigma Wm.Smith Fig:12 Pleurosigma sp

Fig:13 Gyrosigma Wansbeekii(Donkin) Fig:14 Gyrosigma Sp.

Fig:15 Foraminifora shells Fig:16 Diploneis Ehrenberg

Fig:17 Schroderella sp. Fig:18 Rhizosolemia sp.

Fig:19 Pinnularia sp. Fig:20 Odontella sp.

Fig:21 copepode sp. Fig:22 copepode sp.

Fig:23 Loligo sp. Fig:24 Zoea larvae

Fig:25 Cyclops sp. Fig:26 Crustacean parts

Fig:27 Chella of crab Fig:28 Detritus

Fig:29 Cyclops sp. Fig:30 Zoea larvae

Fig:31 Harpacticoid sp. Fig:32 Soil nematode

Fig. 33. Zooplankton sp. Fig. 34 Zooplankton sp.

Fig. 35 Zooplankton sp. Fig.36 Broken parts of shrimp

Fig:37 Zoea larvae Fig:38 Unidentified organism

Fig: 39 Coelenterate sp. Fig:40 Parapaneopsis sp.

Fig: 41 Fish sp. Fig: 42 Algae sp.

5: NEMATODE PARASITES

Only the nematode genera recovered during the present studies are reviewed and the species are identified and described within each of the genera

5.1 HISTORY OF THE GENUS DUJARDINASCARIS BAYLIS, 1947

Dujardinascaris (synonym Dujardinria gedoelst, 1916,pre occupied) was created by Baylis in 1947.The species of genus normally occur in crocodiles and lizards. Yamaguti (1961) listed 13 species in the genus. Of these two have been reported from fishes and 11 from reptiles. The species reported from fishes include, D.melapteuri Baylis, 1923 from Melapteurius electricus from Sudan and D.cennotae (Pearsc1936) described from Rhamdia guatemalensis from Yuncatan. Later on few more species have been reported from fishes. Sood (1989) presented a key to the species of Dujardinascaris Baylis(1947), reported from fishes of South Asia .Five species have been reported under the genus Dujardinascaris by sood (1988). Four of them have been reported from Karachi coast and one from Lahore, Pakistan. The species reported from Karachi coast includ, D.magna (Khan and Begum, 1971)in sciaena species , D.qadrii ( Zubari & Farooq 1976)from sciaena species, D. sciaena diacanthus, D.cybii (Arya & Johnson,1978) from the fish Cybium guttatum and D.ritai (Zaidi & Khan, 1975) in Rita rita of fresh water fish from Lahore. Bilqees et al., (2004) reported D.karachiensis from pomadasys olivaceum from Karchi coast.

All the above-mentioned 5 species have been described from Pakistan except D. cybii which is originally described by Arya & Johnson (1978) from India. It appears that genus Dujardinascaris is one of the common genera found in fishes of Pakistan. During the present studies it was noted that species of the genus were also more common in fishes of Karachi coast. Seven new species of the genus are identified and described here. Dujardinascaris mujibi, D. jello, D. maculatum, D. dentatus, D. multiporous, D. sphyraenaii, and D. sinjarii.

5.2 Dujardinascaris mujibi n.sp. (Fig. 43-45)

Order: Ascarididea

Family: Heterocheilidae

Sub. Family: Filocapsularinae

Genus: Dujardinascaris Baylis, 1947 (Syn. Dujardinie Gedoelst, 1916, preoccupied)

Type host: Sphyraena forsteri (Cuvier, 1829)

Site of infection: Intestine.

Type locality: Fish harbour, Karachi coast, Pakistan

Prevalence: 14% (7 fish infected/ 50 fish examined) Intensity: 2.28 (with 6 male and 10 female nematodes)

Holotype (male): JUW. N.26

Allotype (Female): JUW. N.27

Diagnosis: Stout worms, body elongated whitish in color, narrow at the anterior region and greatest width is at posterior region. The head is provided with three prominent lips having four teeth like structures capable of being interlocked. Interlabia are present, with prominent grooves running from the tips to the base. Dorsal lips have four pointed ends, which fit, in the cavities of the subventral lips. Apical one fourth of the lips are

embossed with regular zigzag pattern as revealed by SEM. This is a peculiar character. The cuticle investing the body is expanded throughout the length of the body. Esophagus is divided into two portions, an anterior muscular portion, and a posterior glandular portion, Intestine is long, and caecum is thrust out anteriorly. Tail is very short and conical and is provided with helical.

Male: (6 specimens including holotype): Body length is 30.05—31.01 and width is 1.22---1.34. The head diameter is 0.05---0.14. The esophagus is 3.60---4.19 in length and 0.22 in width, Nerve ring at 0.16-0.23 and excretory pore 0.50 from anterior end. Intestinal caecum is 3.00—3.50 in length and 0.20 in width. The spicules are equal and are 2.20—2.22 in length. Gubernaculum is absent. 12- 14 pairs of sessile caudal papillae are present, including 10 pairs are precloacal, 2 adanal and 2 postcloacal. Excretory pore is 0.49-0.52 from anterior end. Tail is short and pointed, 0.1--0.12 in length.

Female:(10 specimens including allotypes): Body length is 50.05---51.85; 1.05—1.07 in greatest width at middle. The diameter of the head is 0.14--- 0.16 x 0.22—0.24. Esophagus is muscular measuring 3.60—4.70 in length and 0.17---0.20 in breadth. Nerve ring is at a distance of 0.24-0.36 from anterior end. Intestinal caecum is 2.30---2.80 in length and 0.13- 0.17 in breadth. Vulva is postequatorial, 0.95-0.98 from posterior extremity, vagina is muscular and long. Eggs thick-shelled, rounded, 0.021-0.035. Tail is short and conical with cuticular ring-shaped striations.

Remarks: The genus Dujardinascaris was created by Baylis in 1947. This genus normally occurs in crocodiles, lizards and alligators. Diaz andGallardo (1968); Sprent(1977, 1990); Hazen et al.,(1978);Cherry and Ager(1982); Gold berg,et al.,(1991); Machida et al., (1992);Scot et al.,(1999); Sprent,et al., (1998); Moravec (2001); Sood (1989); Bursy ,et al., (2005); Junker, et al., (2006); Yamaguti (1961) listed two species of nematodes from fish under the genus, Dujardinascaris, D. . cenotae( Pearse, 1936) from Rhandia guatemaensis in Yucatan; D. melapteruri (Baylis, 1923) in Melapterus elecricus. Sood(1989) presented a key to the species of Dujardinascaris Baylis 1947 reported from fishes in South Asia. Five species have been listed under the genus Dujardinascaris by Sood (1989) namely; D. magna (Khan & Begum, 1971, in Sciaena sp. from Karachi coast; D. ritai (Zaidi & Khan, 1975) in Rita rita of fresh water fish from Lahore, D. quadrii (Zubari and Farooq, 1976) from Sciaena sp., from Karachi coast; D. Sciaena (Bilqees et al., 1977) in Sciaena diacanthus from Karachi coast, D. cybii (Arya and Johnson, 1978) from fish Cybium guttatum from India, Lakshmi and Sudha, (2000) give notes on D. cybii Arya and Johnson from a new host Mugil cephalus. Bilqees et al., 2004 describe D. karachiensis from Pomadsays olivaecum from Karachi coast.

The present species is considered as a new species and named as D. mujibi. Differential diagnosis is compared with described species of genus Dujardinascaris. The present species is different from all the above mentioned species in body length, length of esophagus, length of spicules , number of papillae in male, length of intestinal caecum and other morphological variation such as prominent zigzag patterns embossed on

upper part of the lips as revealed by SEM. This pattern of lips has not been described previously. The description of D. cenotae is based on three poorly described female specimens while the description of D. malapteruri and D. magna is based only on male specimens In D. magna the number of caudal papillae are 30, including 20 preanal and 10 postanal and the length of spicules are 1.70-1.76, tail is 0.50 in length, indicating that the present species having less number of papillae and spicules longer than D. magna. D. ritai is described by immature female only. While D. ritai is from fresh water fish.

D. sciaena is smaller in size, and the esophagus is larger in size, length of spicules are 5.2—5.5. Caudal papillae are 23 pairs, including 19 preanal and 4 postanal, tail length is 0.120—0.166. The present species is also different from D. quadrii in length of spicules (5.42-5.52), number of caudal papillae (23 including, 19 preanal and 4 post anal). The present species have shorter spicule length, having 14 pairs of caudal papillae and short tail as compared to D. sciaena. The present species differs from D. karachiensis by lips structure, length of esophagus, length of intestinal caecum, length of spicules,(1.70—1.71) number of caudal papillae (25 pairs, including 4 preanal and 21 post anal) and distance of cloeca from the posterior end. The main differences in addition to the above mention structures observed in the present species by SEM are the zigzag embossment and the structure of female tail having concentric appearance.

The comparative morphology of the present species with other species recovered from South Asia and the species recovered during the present

investigation from Pakistan is shown in table 14-17. Accordingly the present is regarded a new species D. mujibi.

Etymology: The specific name D. mujibi is in honor of Prof. Dr. Bilqees Mujib a parasitologist of Pakistan.

5.3 Dujardinascaris jello n. sp. (figs 46-48)

Type host: Sphyraena jello (Cuvier, 1829) Site of infection: Intestine Type locality: Fish harbor Karachi coast Pakistan.

Prevalence : 13.11 % (8 fish infected/ 61 fish examined Intensity: 2 (with 7 male and 9 female nematodes)

Holotype(male): JUW. N.23 Allotype(female): JUW. N.24

Diagnosis: Stout and elongated worms, whitish in color, the head carries three prominent lips. The lips are clearly separated from each other. The space between the two adjacent lips is occupied by prominent interlabia and lips are interlocked by which these are connected together like a zip. Dorsal lip with rounded end fit into the cavities of the two subventral lips. Right subventral lips have a cavity, which fits into the rounded end of dorsal lips and also fits itself into the cavity of adjacent lips. Nerve ring is a distance of 0.16-0.23. Cuticle is thick and striated at the head region forming a zigzag pattern; the left of the body is striated sparsely. The cuticular striations are present throughout the body, most prominent at posterior region ¼ above the tail region in female. Gubernaculums is absent.

Male: (7 specimen including holotype ).The total length of worm is 26- 27.5 and 0.43---0.48 in breath. The lips are 0.07-0.09 in length and 0.10-- 84

0.13 in breath .Esophagus is muscular, 1.84---1.87 in length and 0.15---0.18 in its maximum breath .An intestinal caecum is present, directed anteriorly measuring 1.60-1.72 in length . Nerve ring is at a distance of 0.17-0.28 and excretory pore is 0.51- 0.56 from anterior end. Two large unequal spicules are present measuring 1.69- 1.70 and 1.80-2.00 in length. There are 17 pairs of caudal papillae including 11 pairs are sessile [( 7+2+2) 7 preanal, 2 postanal, 2 adanal] and 6 pairs are pedunculate and are preanal .The tail is short, pointed 0.01 in length.

Female: ( 9 specimen including allotype ).Body length is 31.2---33.25 and width at maximum is 1.30---1.34 at the middle of body length. The lips are 0.15—0.18x0.25—0.27 in diameter. The esophagus is 1.5-1.6 in length and 0.14-0.18 in its maximum breadth. Nerve ring is at a distance of 0.18-0.30 and excretory pore is 0.53- 0.57 from anterior end. Intestinal caecum is prominent measuring 1.68-1.79 in length. Vulva is at a distance of 0.54- 0.60 from posterior extremity .The tail is blunt in female.

Remarks:

The present species is regarded a new species Dujardinascaris jello n.sp. The morphological variations are shown in table 14---17. The present species is morphologically different from the species known in Pakistan including D. magna (Khan and Begum, 1971); D. ritai (Zaidi and Khan 1975); D. sciaena (Bilqees et al.,1977); D. karachiensis (Bilqees et al., 2004) and also from other parts of the world including D. melapteuri (Baylis, 1923); D. cennotae (Pearse, 1936); D. cybii (Arya & Johnson,1980). Along with morphological differences like body size, length of esophagus, shorter tail in male, length of spicules and number and arrangement of

papillae. There are also some distinguishing characters, like zigzag embossment on head region, presence of pedunculate papillae, and female tail structure. These characters are not yet found in any South Asian species. But it resemble with the above mentioned new species D. mujibi in some respect like- zigzag pattern on head region, length of spicules and arrangement of sessile caudal papillae. But as reavled by SEM differences were observed in some structures like female tail region, having blunt conical end and also prominent cuticular striations on postequatorial region of the body in the present species. It is also different from D. mujibi in the pattern of zigzag embossment, which is more prominent in the head region of D. mujibi.

The species under discussion is different from all other species of the genus and is regarded as new species Dujardinascaris jello, as jello is the specific name of the host.

5.4 Dujardinascaris maculatum n.sp. (Figs. 49—51)

Type host: Pomadasys maculatum (Bloch, 1787)

Site of infection Intestine

Type locality: Fish harbor, Karachi coast Pakistan

Prevalence: 24.19% (15 fishes infected/ 62 fish examined) Intensity: 1 (with 6 male and 9 female nematodes)

Holotype(male): JUW. N. 11 Allotype (female): JUW. N.12

Diagnosis: Stout, long cylindrical, dark brown worms, tapering toward both extremities with pointed posterior end in male and conical posterior end with round tip in female. Mouth is bounded by three lips, without dentigerous ridges. Cuticle finely striated, these striations are prominent at the anterior end in male and at posterior end in female. Esophagus is entirely muscular, intestinal caceum well developed. Anal opening is prominent in male.

Male: (6 specimen including holotype ) Stout, elongated, dark brown worms, body length 9.05—10.0 and maximum width is 6.07—7.02. The lips are 0.16—0.18 x 0.35—0.45 in size. Esophagus is 0.5—0.8 in length and 0.15—0.25 in width. Intestinal caecum is 0.40-0-0.49 in length. 13-- 16 pairs of caudal papillae are present, including 13 pairs precloacal, 2pairs postcloacal and 1 adanal . Two unequal spicules are present, 0.91—0.95 and

0.50—0.60 in length. Gubernaculums is absent. Excretory pore is 0.43 from anterior end. Tail is pointed, 0.06—0.07 in length.

Female: (9 female including allotype). Body is 13—19 in length and 0.42—0.50 in its greatest width. Cuticular striations are very prominent at posterior half of the body, just above the tail. The lips are 0.09—0.15x 0.18—0.30 in size. Esophagus measuring 0.55—0.64 in length and 0.20— 0.22 in its maximum width. Nerve ring is at a distance of 0.32-0.36 from anterior end. Excretory pore 0.45-0.52. Intestinal caecum is 0.12-0.15 in length. Vulva is pre equatorial at a distance of 0.05 from posterior extremity opening in to muscular atrium. Vagina muscular and slender. Tail is bluntly conical.

Remarks: There are six species in this genus namely; D. magna (Khan & Begum, 1971) in Sciaena sp. from Karachi coast; D. ritai (Zaidi & Khan, 1975) in Rita rita of fresh water fish from Lahore, D. quadrii (Zubari and Farooq, 1976) from Sciaena sp., from Karachi coast; D. Sciaena (Bilqees et al., 1977) in Sciaena diacanthus from Karachi coast, D. cybii (Arya and Johnson, 1978) from fish Cybium guttatum from India and D. karachiensis (Bilqees et al., 2004) from Pomadsays olivaecum, Karachi coast.

The present species is regarded a new species D. maculatum n.sp. and is compared with described species of genus Dujardinascaris. The morphological variations are shown in table 14-15, with figures 49-51. The present species is different from all Pakistani species, and the species described from other parts of the world. (Table 16-17). According to SEM

the differentiating characters in the present species is the presence of alate spicules (figs. 50 D) , subequal, 0.91—0.95 and 0.50—0.60 in length, posterior half of the body is provided with striations, prominent on the bluntly conical tail, structure of caudal end in female(figs. 51 B&C). The caudal papillae are 15—17 pairs, similar to D. jello n.sp.(13+2+1) but the position and arrangement of papillae(14 +3) is different in the present species.

Etymology: The species name D. maculatum refers to the host species

5.5 Dujardinascaris sphyraenaii n.sp. (Figs. 52-- 54)

Type host: Sphyraena jello (Cuvier, 1829)

Site of infection: Intestine

Type locality: Fish harbor, Karachi coast Pakistan

Prevalence & intensity: 12 % & 2.14 ( 7 fishes infected/ 58 fish examined, with 6 male and 10 female nematodes)

Holotype (male) JUW. N. 14

Allotype (female) JUW. N. 15

Diagnosis: Stout, long, cylindrical, dark brown worms, tapering toward both extremities with pointed posterior end in male and female. Mouth bounded by three prominent lips, with out dentigerous ridges. Cuticle is finely striated through out the body. Esophagus is entirely muscular, intestinal caceum well developed. Anal opening is prominent in male.

Male: (6 specimen including holotype) Stout, medium-sized, dark brown worms, body length 20.50---24.90 and 0.32---0.36 at its maximum width. Mouth bounded by three lips, with out dentigerous ridges. Interlabia present. Body is sparsely striated. The lips are 0.302—0.06 x 0.05—0.08. The esophagus is 0.72—0.88 in its length and 0.11—0.16 in width. Nerve ring is at a distance of 0.30-0.33 from anterior end. Intestinal

caecum is 1.59-1.67 in length. 15 pairs of caudal papillae are present including 11 pairs preanal, 2 pairs postanal and 2 adanal Two equal spicules are present, 0.64-0.75 in length. Gubernaculum is absent .tail is pointed, 0.025-0.027 in length.

Female: ( 10 female including allotype) Body is 22.4---25.4 in length and 0.25—0.37 in its maximum breadth. The lips are 0.05—0.08x0.05—0.09 in diameter. Esophagus measuring 0.84—0.94 in length and 0.13—0.15 in width. Nerve ring is at a distance of 0.32-0.39 from anterior end of the body. Intestinal caecum is 1.60-1.71 in length. Vulva is prominent, pre equatorial at a distance of 0.27 from posterior extremity. Tail is pointed , 0.05-0.06 in length, with area rogosa -like structures on its posterio-ventral end.

Remarks

The present specimen is regarded a new species D. sphyraenaii n.sp. The morphological variations are shown in table 14----17. The present species resemble in body size with D. sciaena , but the spicules are much smaller and there are 15 pairs of caudal papillae, as compared to D. sciaena including 23 pairs of caudal papillae are present. D. magna and D. karachiensis are much larger in body length, body width, size of esophagus and length of spicules as compare to the present species. A comparative account in the morphological variations of the genus Dujardinascaris recovered from South Asian are given in tables 14-17. The present species is different in both sexes from D. mujibi; D. jello and D. maculatum ,in the structure of tail which is bluntly pointed in male and sharply pointed in female. The present recovered species is also provided with area rugosa-like structures at posterio-ventral side. The papillae are

ventral in position in the present species while in D. mujibi; D. jello and D. maculatum, the papillae are lateral in position.

Etymology: Species name D. sphyraenaii refers to the local name of the host species

5.6 Dujardinascaris multiporous n.sp. (Figs. 55—57)

Type host: Pomadasys olivaceum ( Day, 1875) Site of infection: intestine Type locality : Fish harbor Karachi coast Pakistan Prevalence: 20% (12 fish infected/60 fish examined)

Intensity: 1.66 (with 7 male and 13 female nematodes)

Holotype(male): JUW. N. 18 Allotype (female): JUW. N. 19

Diagnosis: Stout, medium sized worms, narrow at both ends, whitish in color, worms. The cuticle is striated throughout its body length and provided with depressions appearing as pores-like in SEM. Esophagus is muscular. The patterns of these cuticular pores are different in male and female. The tail in male is pointed and is bluntly conical in female. Cuticle is thick with deep transverse grooves in male and indistinct in female in anterior most region, mouth is surrounded by three lips. Intestinal caecum is absent.

Male: (7 specimen including holotype). Body is 6.10 ---7.50 in length and 0.20---0.29 in its maximum breadth at the middle of the body. The lips are 0.045---0.070x 0.80---0.10 in size. The esophagus is 0.35---0.50 in length and 0.15---0.20 in its width. Two, small, subequal spicules are present 0.61-- -0.64 and 0.72---0.75 in length. Gubernaculums is absent. Caudal papillae

are 18---20 pairs, sessile, 9 pairs are precloacal and 9-11 pairs are postcloacal. Posterior ¾ of the body is provided with 13---17 pore-like depressions, dorso-lateral in position, situated at regular intervals. Nerve ring is at a distance of 0.35-0.42 and excretory pore is 0.37-0.45 from anterior end of the body. Tail is pointed with 2---3 pore-like depressions sparsely.

Female: (13 specimen including allotype) Body length 6.5---7.9 and 0.25-- -0.30 in its width. The lips are 0.055---0.075x 0.80---0.110 in size. The esophagus is 0.38---0.45 in length and 0.14---0.19 in its maximum breadth. Intestinal caecum is inconspicuous. Nerve ring is at a distance of 0.38-0.40 and excretory pore is 0.40-0.44 from anterior end of the body. Vulva is conspicuous, at a distance of 0.37-0.39 from its posterior extremity. The posterior part of body is provided with 7---10 pore-like depressions at irregular intervals. The tail is conical.

Remarks: The dimension of various structures of the species under discussion are given in table 14—17. The peculiar character of the present species is the presence of cuticular pore-like depressions in both sexes as shown by SEM., which distinguish it from all south Asian species. It is also different in other morphological characters, including body length in both sexes, (6.10-7.90); length of spicules(0.59-0.75) and in number(18, including 9 pre anal and 9- 11 post anal) and position of caudal papillae (dorso-lateral).The present species is also different from the new recovered species including D.mujibi, D.jello, D. maculatums and D. sphyraenaii in the above mentioned and

other morhphological characters as shown in table 14-15. The present species is regarded a new species and the name D. multiporous n. sp. is proposed.

Etymology: The species name D. multiporous refers to the presence of cuticular pore-like depressions on posterior half of the body

5.7 Dujardinascarus sinjarii n.sp. (Fig. 58)

Type host : Otolithus ruber (Sciaenidae )

Site of infection: Intestine.

Type locality: Fish harbor, Karachi coast Pakistan

Prevalence : 6% ( 3 fish infected/ 50 fish examined)

Intensity: 1.66 (with 2 male and 3 female nematodes)

Holotype(male): JUW. N. 36

Allotype(female): JUW. N. 38

Diagnosis: These are relatively large and stout worms, body elongated. whitish in color. The cutical investing the body is expanded throughout its length, frill-like at the anterior region of the body and not provided with spines or other raised structures. The head carries three prominent lips having four teeth like structures capable of being interlocked. Interlabia are present, with prominent grooves running from the tips to the base. Esophagus is large muscular tube, ventriculus is absent but esophageal bulb is present. Tail is pointed in both the sexes.

Male: (2 specimens including holotype): Body length is 50--54 in length and width is 0.08—1.20. The head size is 0.20-0.22 x 0.23-0.26. The

esophagus is 1.20-1.60 in length and 0.30-0.50 in width, esophageal bulb is 0.046-0.049 in size. Nerve ring is at a distance of 0.36-0.39 from anterior end of body extremity. Excretory pore is at a distance of 0.41-0.42 from anterior end of body. Spicules are unequal, 1.17—1.19 and 0.05-0.07 in length. There are 11- 12 pairs of sessile, caudal papillae, including 6 pairs precloacal and 6 postcloacal. Cloaca is located at a distance of 0.10 from the posterior end. Tail is pointed, 0.26--0.27 in length.

Female: (3 specimens including allotypes): Body length is 10.5-11.10 and 0.33- 0.35 in breadth at middle. The size of the head is 0.12-0.13 x 0.13-0.16. Interlabia is similar to that of male in size. Esophagus is 0.65—0.76 in length and 0.08---0.10 in breadth. Esophageal bulb is 0.030-0.035 in size. Vulva is postequatorial situated at a distanance of 0.40-0.41 from posterior extremity, vagina is muscular and long. Eggs are thin shelled and rounded measuring 0.046-0.051 in diameter. Tail is pointed 0.07 in length.

Remarks: Dujardinascaris sinjarii is different in important diagrammatic features from other species of the genus Dujardinascaris including D. . cenotae ( Pearse, 1936) from Rhamdia guatemalensis; D. melapteruri (Baylis, 1923) in Melapterus elecricus. The description of D. cenotae is based on three poorly described female specimens, while the description of D. malapteruri and D. magna is based only on male specimens. In D. magna the number of caudal papillae are 30 including, 20 preanal and 10 postanal and the length of spicules are 0.55—0.59, tail is 0.50 long. The present species have less number of papillae and unequal spicules. D. ritai is reported from freshwater fish, and is described by immature female only.

The present species is also different in some morphological characters from the specimens reported by Sood(1989) from fishes in South Asia namely; D. magna (Khan & Begum, 1971), in Sciaena sp. from Karachi coast; D. ritai (Zaidi & Khan, 1975) in Rita rita from Lahore, D. quadrii (Zubari and Farooq, 1976) from Sciaena sp., from Karachi coast; D. Sciaena (Bilqees et al., 1977) in Sciaena diacanthus from Karachi coast, D. cybii (Arya and Johnson, 1978) from fish the Cybium guttatum from India and D. karachiensis (Bilqees et al., 2004) from Pomadsays olivaecum, Karachi coast. D. sciaena is smaller in size, and the oesophagus is larger in size, length of spicules are 5.2—5.5, papillae are 23 pairs, including 19 preanal and 4 postanal, tail length is 0.120—0.166. The present species is shorter in spicule length, having 14 pairs of caudal papillae and also short tail as compared to D. sciaena.The present species is considered as a new species and named as D. sinjarii. It is compared with all the described species of genus Dujardinascaris (Table. 14&15). The present species is different from all the above mentioned species in body length, length of esophagus, length of spicules , number of caudal papillae in male, but resemble in some extent with D. karachiensis in the presence of esophageal bulb and in body length of male specimens but differ from D. karachiensis by lips structure, length of oesophagus, length of spicules,(1.70—1.71) number of caudal papillae (25 pairs, including 4 preanal and 21 post anal) and distance of cloeca from the posterior end. The present species is similar in numbers of caudal papillae( 10-11) with D. sphyernaii n.sp. (11), but the position and arrangement of papillae are different in both new recovered species. The comparative morphology of the present species with other species recovered from Pakistan is shown in table 14--17. Accordingly the present species is regarded a new species D. sinjarii.

Etymology: The specific name D. sinjarii refers to the local name of the fish host.

5.8 Dujardinascaris dentatus n. sp. (Figs.59-60)

Type host: Sillago sihama ( Forskal, 1775) Site of infection: intestine Type locality : Fish harbour Karachi Coast Pakistan Prevalence: 13.33% (10 fish infected/ 75 fish examined) Intensity: 1.3 (With 4 male and 9 female nematodes)

Holotype(male): JUW. N. 29 Allotype(female): JUW. N. 30

Diagnosis: Stout, whitish, elongate worms, female is larger than male, body striated throughout its length. Head is provided with three lips, lips are embossed with zigzag pattern. Lips are without dentigerous ridges but cuticle of their internal surface is produced into large teeth- like structures capable of being interlocked. Esophagus is muscular. Intestinal caecum is conspicuous. According to SEM, the tail is with snout spine at its tips in female. Anal opening is prominent in male. Vulva is preequatorial and eggs are sub globular.

Male:( 4 specimen including holotype) Body is 17.80 ---18.50 in length and 0.38---0.4 in its maximum bredth. The lips are 0.27---0.3x 0.24---0.26 in size. The esophagus is 3.95—4.25 in length and 0.31---1.35 in its width. An intestinal caecum is present anteriorly measuring 0.18—0.76 in length. Nerve ring is at a distance of 0.28-0.31. Excretory pore is situated at a distance of 0.56-0.57 from anterior end. Two, small, slender, subequal, spicules are 100

present 0.8---0.9 in length. Gubernaculum is absent. 17 pairs of Sessile caudal papillae of which 6 pairs are precloacal and 8 pairs are post cloacal.

Female: (9 specmen including allotype) Body length 28.4---32.5, 0.45--- 0.50 width. The lips are 0.31---0.38x 0.50---0.56 in size. The esophagus is 3.95-4.25 in length and 0.31—1.35 in its breadth. Nerve ring is at a distance of 0.27-0.31 and excretory pore 0.58 from anterior end of the body. Intestinal caecum is prominent measuring 1.68-1.79.Vulva is at a distance of 0.54- 0.60 from posterior extremity .The tail is blunt in female.

Remarks: The dimensions of various structures of the species under discussion are given in table 14-17. It shows that this species is different from all south Asian species in respect of each morphological chracter as shown in table no.but it resemble with D. mujibi and D. jello in embossment of zigzag pattern except in the pattern of embossment. The present species is also differ from D, mujibi and D. jello in number and arrangment of caudal papillae, pre cloacal in the previous two species and in the present specimen 6 pairs are pre cloacal and 8 are post cloacal. The present recovered species is also different in position of anal opening in male which is pre equtorial and ¾ from the posterior extremity. And in the dentate posterior end of female (as revealed by SEM) but the posterior end of two male specimens was destroyed during scanning electron microscopy. The present species is regarded a new species D. dentatus n.sp.. Etymology: The species name D. dentatus refers to the caudal teeth- Like structure in female specimens.

101

Table.14: Morphological variations in the new species of the genus Dujardinascaris Baylis, 1947 (All measurements are in mm)

Species D.mujibi D.mujibi D.jello D.jello D.maculatum D.maculatum D.sphernaii D.sphernaii (male) (female) (male) (female) (male) (female) (male) (female)

Host Sphyraena Sphyraena Sphyraena Sphyraena Pomadasys Pomadasys Sphyraena Sphyraena forsteri forsteri jello jello maculatum maculatum jello jello Body(L) 50.05-- 31.20-- 30-31 26.0--27.5 9.05-10.0 13-19 20.5-24.9 22.4-25.4 51.85 33.25 Body(W) 1.22-1.34 1.05-1.07 0.43-0.48 1.30-1.34 6.07—7.02 0.42-0.50 0.32-0.36 0.25-0.37 Head 0.14- 0.07— 0.15—18x 0.16—0.18x 0.09—0.15x 0.05-0.08x diameter 0.05-0.14 0.16x .09x 0.05-0.08 0.25-0.27 0.35-0.45 0.18-0.30 0.05x0.09 0.22-0.24 0.10-0.13 Muscular 3.60— 3.60--4.19 1.84-1.87 1.50--1.60 0.50--0.80 0.55-0.64 0.72-0.88 0.84-0.94 esophagus(L) 4.70 Muscular 0.17-0.22 0.17-0.20 0.15-0.18 0.14-0.18 0.15-0.25 0.20-0.22 0.11-0.16 0.13-0.15 esophagus(W) Nerve ring from anterior 0.16-0.23 0.24-0.36 0.17-0.28 0.18-0.30 0.32-0.36 0.30-0.33 0.32-0.39 region Intestinal 3.00--3.50 2.30--2.80 1.60-1.72 1.68-1.79 .40-0.49- 0.12—0.15 1.59-1.67 1.60-1.71 cecum (L) Intestinal 0.20 0.13--0.17 ------cecum (W) Spicule 1 (L) 2.20-2.22 - 1.69-1.70 - 0.91-0.95 - 0.64 - Spicule 2 (L) 2.20-2.22 - 1.80-2.00 - 0.50--0.60 - 0.64 - No of caudal 12-14 - 17 - 13--16 - 15 - papilae Pre-anal 9-10 - 7 - 13 - 11 Post cloacal 2 - 2 - 2 2 Adanal 2 Pedunculate 2 - - 1 - 2 - 6

Valva from 0.95— posterior - - 0.54--0.60 - 0.05—0.06 - 0.27-0.30 0.98 extremity Tail(L) 0.10-0.12 - 0.01 - 0.060—0.07 0.05 0.025 0.05

102

Table.15: Morphological variations in the new species of the genus Dujardinascaris Baylis 1947 (All measurements are in mm)

D.multiporus D.multiporus D.sinjarii D.sinjarii D.dentatus D.dentatus Species (male) (femle) (male) (female) (male) (female)

Pomadasys Pomadasys Otolithus Otolithus Sillago Host Sillago sihama olivaceum olivaceum argenteus argenteus sihama Body(L) 6.10-7.50 6.50--7.90 50-54 10.5-11.1 17.80-18.50 28.4-32.5 Body(W) 0.20-0.29 0.25-0.30 0.08-1.2 0.33-0.35 0.38-0.40 0.45-0.50 Head 0.055- 0.045-0.070x0.8- 0.27-0.30x 0.31-0.38x diatmeter 0.075x0.8- 0.20-0.22 0.12-0.13 0.10 0.24-0.26 0.50-0.56 0.11 Lips(W) 0.8-0.10 0.8-0.11 0.23-0.26 0.13-0.16 0.19-0.26 0.50-0.56 Muscular 0.35-0.50 0.38-0.45 1.2-1.6 0.65-0.76 2.24-2.26 3.95-4.25 esophagus(L) Muscular 0.15-0.20 0.14-0.19 0.3-0.5 0.08-0.1 0.68-2.75 0.31-0.35 esophagus(W) Nerve ring from anterior - - 0.36-0.39 0.16-0.25 - - region Intestinal - - - - 0.18-0.76 0.19-0.75 cecum (L) Spicule 1 (L) 0.61 - 1.17-1.19 - 0.80—0.90 - Spicule 2 (L) 0.75 - 0.05-0.07 - 0.74—0.75 - No of caudal 18 - 12 - 17 - papilae Pre-anal 9 - 6 - 5 - Post-anal 9--11 - 6 - 12 - Vulva from posterior - 0.37 - 0.34 0.14 extremity Tail(L) - 0.04--0.05 0.27 0.07 - -

103

Table.16: Morphological variations in the species of the genus Dujardinascaris Baylis 1947 described so far from various localities (All measurements are in mm)

D.ritai D.cybii D.cybii D.magna D.Karachiensis D.Karachiensis Zaidi & Arya & Arya & Khan & Bilqees et. al., Bilqees et. al., Khan Species Jonson Jonson Begum (male) (female) (female) (male) (female) (male)

Pomadasys Pomadasys Cybium Cybium Host Sciaena sp Rita rita olivaceum olivaceum gutattum gutattum Body(L) 66.46- 50-61 61-65 7.5-11.0 12.5-17.5 23.0-27.2 71.60 Body(W) 10.14- 1.5 2.52 0.08-0.27 0.33-0.53 1.72-2.03 0.16 Lips(L) 0.19-0.26 0.21-0.29 - - 0.24 0.08-0.12 Lips(W) 0.20 0.16 - - - 0.05-0.07 Muscular 4.32-4.7 5.10-5.1 1.06 1.07 4.10-4.17 1.90-3.64 esophagus(L) Muscular 0.42-0.46 0.97-1.13 1.18 1.60 0.40-0.44 0.14-0.16 esophagus(W) Nerve ring from anterior - - - - 1.08-1.15 0.25-0.38 region Intestinal - 1.39 0.50 0.12 0.48-0.74 - cecum (L) Intestinal - 0.36 0.62 0.22 0.34-0.35 0.090.12 cecum (W) Spicule 1 (L) 1.70-1.71 - 0.05 - 1.70 - Spicule 2 (L) 1.70-1.71 - 0.10 - 1.76 - No of caudal 25 - 13 - 30 - papilae Pre-anal 4 - 8 - 20 - Post-anal 21 - 5 - 10 - Valva from posterior ------extremity Tail(L) - - - - 0.50-0.59 - 104

Table.17: Morphological variations in the species of the genus Dujardinascaris Baylis 1947 described so far from various localities (all measurements are in mm)

D.quadrii D.quadrii D.sciaenae Species Zubairi & Farooq Zubairi & Farooq (Male) (Male) (Female)

Sciaena Host Sciaena sp Sciaena sp diacanthus

Body(L) 9.5-22.74 11.4-27.2 15.81-22.74 Body(W) 0.25-0.51 0.14-0.35 0.3-0.51 Lips(L) 0.088-0.128 - 0.088-0.128 Lips(W) - - 0.055-0.072 Muscular esophagus(L) 3.055-3.300 1.90-3.64 3.005-3.300 Muscular esophagus(W) 0.060-0.14 - 0.108-0.133 Glandular esophagus(L) - - - Glandular esophagus(W) - - - Nerve ring from anterior - - 0.160-0.266 region Intestinal cecum (L) 0.288 0.349-0.450 0.228-0.348 Intestinal cecum (W) 0.4 - 0.048-0.072 Spicule 1 (L) 5.2-5.3 - 5.2-5.3 Spicule 2 (L) 5.4-5.52 - 5.4-5.5 No of caudal papilae 23 - 23 Pre-anal 19 - 19 Post-anal 4 - 4 Valva from posterior - 8.9-10 - extremity Tail(L) 0.1 0.120-0.166 - 20-0.166

105

106

107

108

109

110

111

112

113

114

115

116

117

118

120

121

122

123

124

5.11: HISTORY OF THE GENUS PROCAMALLANUS BAYLIS, 1923

(SPIROCAMALLANUS (OLSEN 1952) PETTER, 1979)

Species of the genus Procamallanus Baylis, 1923, subgenus Spirocamallanus, Olsen, 1952, have been reported in a variety of piscine hosts in different geographical zones, and in both freshwater and marine systems. Although many authors consider Spirocamallanus Olsen, 1952 a distinct genus, we agree with Moravec and Sey(1988) in considering Spirocamallanus a subgenus of Procamallanus. This subgenus is characterized by the presence of a buccal capsule with internal spiral thicking in both males and females(Moravec and Thatcher, 1997). Thus those species were formally transferred to Procamallanus( Spirocamallanus). Spirocamallanids are equally dominated in Asia and South America and also found in North America, Africa and Australia. The species appear to be originated in tropical Asia as parasites of catfishes then invaded marine fishes. The transmission and development of this genus has been little studied but a number of species in fish have been investigated. First larval stages are attracted by the copepods and perhaps, other crustaceans (intermediate host). The definitive host becomes infected by ingesting the copepods with the larval stage of the parasite. However, the larvae will persist in gut or became encapsulated in the tissues of plankton-omnivorous fishes, which consumes copepods. This paratenic hosts move the larva in the food chain to the piscivorous definitive host(Anderson, 2000). The African may be derived from the Asian fauna by the dispersal of spirocamallanids with catfishes. The South American fauna points to a freshwater dispersal pattern, probably from Africa before the continents drifted apart. But it is not relevant to mention all the species of Procamallanus (Spirocamallanus) from all geographical zones.

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Procamallanus(Spirocamallanus) are the the common parasites of fishes of South Asia. Sood (1989) presented a key to the species reported from South Asia. Procamallanus(Spirocamallanus)mehrii (Agarwal ,1930) from wallago attu in Allaabad; P. (S.) planoratus( Kulkarni ,1935) from Clarias batrachus in Nagpur; P. (S.) pereirai (Annereaux, 1946) from Mugil speigleri, Otolithus ruber and Pomadasys hasta; P (S.) bagarii (Karve&Naike, 1951) from Bagarius bagarius in Pona; P.(S.) ahiri (Karve, 1952) from Anguilla bengalensis in Poona; P.(S.) mysti (Karve ,1952) from Mystus carvasius in Poona; P.(S.) saccobranchi (Karve, 1952) from Heteropeutes forsitis in Nagpur P.(S.) aspiculus (Khera, 1955) from Bagarius bagarius; P.(S.) gubernaculus (Khera ,1955) from Rita rita in Luknow; P.(S.) clarius (Ali, 1956) from Clarius batrachus, P.(S.) heteropneustus (Ali, 1956) from Heteropneustes fossilis , P.(S.) hyderabadensis ( Ali, 1956)from Mystus seenghala, P. (S.) Singhi(Ali, 1956) from Ompok bimaculatus in Hyderabad; P.(S.) mozabukae (Yeh ,1957) from Polydactylus indicus in Khulna; P (S.) spiculogubernaculus (Agarwal, 1958) in Allahabad; P.(S.) daccai (Gupta ,1959) from cat fish in Dacca; P (S.) globoconchus (Ali, 1960) from Rita kuturnee in Hyderabad; P.(S.) Ophicephalus (Ali, 1960) from Channa punctatus in Hyderabad; P.(S.) attui (Pande et al.,1963) from Wallago attu; P.(S.) confuses (Fernando and Furtado, 1963) from Heteropneusies fossilis from Sri Lanka; P. (S.) mathurai (Pande et al., 1963) from Heteropneusies fossilis ; P.(S.) magurii (Lal, 1965) from Clarias batrachus in Meerut; P.(S.) vachai (Sinha and Sahay, 1965) from Eutropiichthys vacha in Patna; P.(S.) hindensis (Lal, 1965) Heteropneusies fossilis in Meerut; P.(S.)muelleri(Agarwal, 1966) from Lucknow; P.(S.) fasciatusi( Sood, 1967) from Trichogaster fasciatus in Lucknow; P.(S.) gomtii (Sood,1967) from Eutropiichthys vacha in Lucknow; P.(S.) vittatusi (Sood, 1967) from Mystus vittatus in Lucknow; P.(S.) ottuei (Varma and Varma, 1971) from Heteropneusies fossilis in Haryana; P.(S.) ompoci (Majumdar&Data, 1972) from Ompok

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bimaculatus in West Bengal; P.(S.) bilaspurensis (Gupta and Duggal, 1973) from Mastacembelus armatus in Bilaspur; P.(S.) intestinecolas (Bashirullah and Ahmed, 1976) from Channa striatus in Dacca; P.(S.) inglisi (Bashirullah& Hafizuddin, 1973) from Clupisoma murius in Dacca; P.(S.) notopteri (Bashirullah& Hafizuddin, 1973) from Notoptrrus notopterus in Dacca; P.(S.) timmi (Bashirullah, 1973) from Heteropneusies fossilis in Bangladesh; P.(S.) sprentii( Bashirullah and Hafizuddin, 1974) from Heteropneusies fossilis in Dacca; P.(S.) ditchella (Gupta& Garg, 1977) from Pellona ditchela in Nicobo Island; P.(S.) nainitalensis (Arya, 1978) from Barilius vagra in Nainital; P.(S.) kashmirensis (Dhar&Fotedar, 1980) from Wallago attu in Kashmir; P.(S.) khatibi (Naidu and Murhar, 1980) from Heteropneusies fossilis in Nagpur; P.(S.) ramteki (Naidu and Murhar, 1980) from Heteropneusies fossilis in Nagpur; P.(S.) meszarosi (Arya, 1984) from Mastacembelus armatus in Nainital; P.(S.) ritai (Misra, 1985) from Rita rita in Lucknow; P.(S.) kakinadensis and P.(S.) lutjanusi (Lakshmi,2000a,b) from perciform fish in India.

The species described from Karachi coast, Pakistan are Procamallanus (Spirocamallanus) spiralis (Khan& Begum,1971) from Tachysurus caelatus; P.(S.) pereirai (Rasheed,1970) from variety of marine fish; P.(S.) dussumieri (Bilqees et al.,1971) from Jhonius dussumeieri; P.(S.) sihamai (Khan & Begum 1971) from Sillago sihama; P.(S.) crossohombii (Zaidi & Khan,1975) from Crossorhombus azureus; P.(S.) sparus (Akram, 1975) from Argyrops spinifer; P.(S.) otolithi (Ashraf et al., 1977) from Otolithus argenteus; P.(S.) wallagus (Rehana & Bilqees ,1973) from Wallago attu in Sindh.

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During the present studies one new genus Spirocotyle n.gen. is identified and the species S.otolithi n.gen. n.sp. is described. Two new species, P. (S.) riaziaii n.sp. and P.(S.) ruberii n.sp.and one female specimen is also identified and reported here. All these new species are recovered from Otolithus ruber from Karachi coast Pakistan.

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5.12: Spirocotyle otolithi n.gen. n.sp. (Fig.61)

Order : Spiruidea Diesing, 1861 Family: Camallanidae Railliet and henry, 1915 Sub family: Camallaninae Yeh, 1960 Genus: Spirocotyle n. gen. Species: Spirocotyle otolithi n.gen. n.sp.

Type Host: Otolithus ruber ( Sciaenidae) Type Locality: Fish harbor, Karachi coast Location: Intestine Prevalence: 4% 13 host examined,( one infected with, one male and one female. Intensity: 2.5 (In other collection 3females were also recorded from one fish out of 7 hosts)

Holotype (male): JUW.N.20 Allotype(female): JUW.N.21

Diagnosis: These are moderate sized worms, male is smaller than female. The body of the worms tapers gradually at its anterior and posterior extremity which is curved ventrally. The cuticle of the body is striated throughout its length. The buccal capsule is oval in shape in both the sexes, with simple well-developed basal ring. Inner surface of the whole capsule is provided with 15 spiral thickenings. A prominent sucker is present at the anterior region of buccal capsule. Muscular esophagus is shorter and

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narrower than glandular esophagus. Excretory vesicle is prominent. The tail is pointed in male and blunt in female.

Male :(One specimen holotype) Length of body 10.4, maximum width 0.34. The wall of the buccal capsule is provided with 15 spiral thickenings, 0.11 in length and 0.09 in its maximum width at its glandular esophagus region. A prominent sucker is present at the anterior region of buccal capsule. The nerve ring is 0.048 from anterior extremity. Esophagus is divided into anterior muscular portion and posterior club shaped curved glandular portion. The anterior muscular portion measures 0.65 in length and 0.15 in width, while the glandular portion is much wider and measures 0.98 in its length and 0.28 at its maximum width. The nerve ring, 0.3 in length and 0.11 in its width. The spicules similar in shape, unequal in length, large spicules is 0.71 long and small spicule is 0.34 long. Twelve pairs of caudal papillae are present, including seven pairs pre-anal, five pairs post-anal. The tail is conical, 0.05 long with pointed end.

Female :(4 female including allotype): Length of body is 14.8, maximum width 0.6. The mouth leads into a buccal capsule which is similar in structure to that of male specimen having a prominent sucker. It measures 0.11 in length and 0.1 in its width. The esophagus is similar to that of male specimen except in size. The anterior muscular part is 0.45 in length and 0.26 at its maximum breadth. The longer posterior glandular part measures 1.18 in length and 0.43 in its maximum breadth. The nerve ring is 0.27 from its anterior extremity. The tail is straight and oval with three prominent cuticular projections measuring about 0.1, 0.5, 0.8 from one groove to other. A pear shaped excretory vesicle is present, 0.02 in length. The vulva is ¾

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from the anterior end of the body, subterminal, 0.17 from posterior extremity.

Remarks The genus Spirocamallanus of family Camallanidae Railliet & Henry, 1915 and sub family Camallaninae Yeh, 1960 has spiral thickenings in the buccal capsule. Similar nematodes were recovered during the present studies. The genus under study resembles in the buccal with Spirocamallanus with some differences in diagnostic features like, a prominent sucker in buccal capsule in both male and female, size of the body, size of the esophagus, in number of spiral thickenings and size of spicules in male specimen and pear-shaped excretory vesicle in female specimen, three cuticular projections at posterior end of female specimen. Therefore, it is desirable to propose a new genus Spirocotyle. The genus name Spirocotyle refers to the sucker-like structure in the buccal capsule and its relation to Spirocamallanus, the species name to the fish host.

Generic Diagnosis: Camallanidae, spirocotyle n.gen. Camallaninae, moderate sized worms. Buccal capsule is provided with 15 spiral thickenings; a prominent sucker is present in buccal capsule; tridents absent; esophagus divided into an anterior muscular, and a longer and wider posterior glandular part. Posterior extremity in male, curved ventrally; tail conical, caudal alae present, with 7 pairs pre-anal, 5 pairs post-anal, caudal papillae, spicules unequal. Posterior extremity in female is flat with three short, blunt processes; excretory pore terminal; vulva is sub terminal; parasites of marine fish.

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Type species: Spirocotyle otolithi n.gen. n.sp.

Type locality: Karachi coast, Pakistan

Etymology: The sucker-like structure is not found in any of the previously described spirocamalanid nematodes or other related genera.

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5.13: Procamallanus(Spirocamallanus)riaziaii n. sp. (Fig. 62----- 64)

Order Spiruidea Diesing, 1861 Family Camallanidae Railliet and henry, 1915 Sub family Camallaninae Yeh, 1960 Genus Procamallanus(Spirocamallanus ( Olsen 1952) Petter, 1979.) Synonymy: Spirocamallanus (Olsen, 1952) (in parts); Procamallanus Baylis, 1923 (in parts).

Type Host : Otolithus ruber( Sciaenidae) Type Locality : Fish harbor, Karachi coast. Location : Intestine Prevalance:: 28.57% (/20 infected/ 70 host examined ) Intensity: 0.869 ( with 8 male and 15 female)

Holotype(male): JUW. N.20 Allotype (female): JUW. N.20

Diagnosis: These are medium-sized worms, with transversely striated cuticle, tapering anteriorly, buccal capsule is yellowish brown, chitinzed, oval to rounded, not divided in to two lateral valves, surrounded by 12 submedian cephalic papillae arranged in two circlets. A small protuberance is situated at the inner base of each papillae of the second circlet (fig. 64 D). The anterior end of the body is bent ventrally and is provided with cervical

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alae, forming a kind of pseudosucker.; with spiral thickenings; tridents are absent; esophagus is divided into an anterior muscular and longer posterior glandular part; tail bluntly conical with two terminal spikes in both the sexes; caudal alae present, uniting in front; spicules unequal ; vulva post equatorial; Parasites of fishes, occasionally of amphibians

Male: (8 males including holotype ) The body length is 11.0---12.8, maximum width is 0.23---0.25. The inner surface of the capsule is provided with 11---12 spiral thickings in a lateral view.and the diameter of the capsule is 0.08 x 0.07. The length of muscular oesophagus is 0.39---0.41 and its maximum width is 0.16 while the glandular oesophagus is 1.00--- 1.05 in length. The nerve ring is a distance of 0.22---0.25 from anterior extremity. The caudal papillae are 16 pairs, of which 4 pairs are pedunclate papillae are post cloacal, 5 pairs are sessile and precloacal. Two unequal spicules are present, measuring about 0.27---0.28 and 0.14---0.16 in length. The tail is bluntly pointed, 0.05 in length.

Female: (15 females including allotype ) The body length is 9.0---9.4 and the maximum width 0.23---0.24. The inner surface of the capsule is provided with 9---10 spiral thickings in a lateral view. and the diameter of the capsule is 0.075x0.070. The length of muscular esophagus is 0.39---0.41 and its maximum width 0.075—0.090, while the glandular esophagus is 0.75--- 0.80 in length and 0.11—0.12 in width. The nerve ring is 0.22--- 0.24 from its anterior extremity Excretory pore inconspicuous. Ovary single, vulva postequatorial 3.0-4.3 from anterior end. . The tail is bluntly conical, 0.023---0.26 in length.

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Remarks The Spirocamallanoids are the parasites of the digestive tract of the hosts, most frequently in the intestine and less often in the stomach and only rarely in the swim bladder of fish.

Spirocamallanoids are equally dominated in Asia and South America, while in North America, Africa and Australia these are less common. Sood(1982) listed 75 species of Procamallanus(Spirocamallanus) from South Asia, of which 9 species have been reported from marine fish of Karachi coast Pakistan, including Procamallanus(Spirocamallanus) spiralis (Khan& Begum, 1971) having 4 tooth-like structures at anterior end of buccal capsule and tail is bluntly pointed with spike; P.(S.) pereirai (Rasheed, 1970) having 4 papillae and amphid on external circlet and 6 band-shaped papillae on inner circlet of head and female has digit-like process on tail; P.(S.) dussumieri (Bilqees et al., 1971); P.(S.) sihamai (Khan & Begum, 1971) the tail is slightly forked; P.(S.) crossohombii (Zaidi & Khan,1975); P.(S.) sparus (Akram, 1975) 2 have sub-median papillae on mouth and tail with spines in both sexes; in P.(S.) otolithi (Ashraf et al., 1977 ), the tail is forked.

The present camallanid P.(S.) riaziaii belong to the morphological group of Procamallanus(Spirocamallanus) species characterized by the presence of three rows of cephalic papillae and pore situated near the inner margin of cephalic papillae of the second circlet as observed by SEM., presence of cervical alae, caudal papillae, two unequal spicules and forked tail with digit-like projections . Many species of this group have been described from different geographical zones, so it is impossible to make a through

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comparison of the new species with all of them. And here Rigby and Adamson (1997) and Rigby and Font (1997) are followed who used the division of Procamallanus (Spirocamallanus) species according to geographical zones by Andrade-Salas et al., (1994) for the comparison of species. The present nematode species P.(S.) riaziaii is different in important morphological features from all Pakistani species in all morphological features except forked tail, as there were no scanning electron micrographs of any one of the described species of the marine fishes of Pakistan. Although Khan &Begum (1971) in P. (S.) spiralis mentioned 4 tooth-like structures of buccal capsule, but it is impossible to make a through comparison due to the micrographs, may be these structures are those which are observed by SEM during the present study.

Species of other geographical zones can be compared with P.(S.) riaziaii n.sp .Moravec et al., (2006) listed three valid species Procamallanus (Spirocamallanus) monotaxis (Olsen, 1952); P.(S.) istiblenni (Noble ,1966); and P.(S.) guttatusi (Salas et al., 1994) from perciform fish in the Philppines. (Olsen, 1952; Noble, 1966; Machida and Taki, 1985); Rigby and Adamson, 1997) from Indo-Pacific region, for comparisons with P.(S.) anguillae (Moravec and Tarachewski, 2006). He mentioned the presence of two digit-like projections of the broad tail in female. The present species is similar to some morphological features with P.(S.) anguillae specially in position of cephalic papillae and the pores situated near the inner margin of cephalic papillae of the second circlet, as observed by SEM, during the present studies. This has not been reported from similar species recovered from Pakistan. But P.(S.)riazaii n.sp. is different from P.(S.) anguillae in many morphological features including, absence of gubernaculums, length

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of the body, absence of amphids, size of spicules and presence of cervical ala at one side of cervical region. The other morphological features are presence of pedunculate papillae, but not similar in number and arrangement of caudal papillae and digit-like tail.

Etymology: The specific name P.(S.) riaziaii is in honor of Prof. Dr. Riaz Ahmed Hashmi, the first Vice chancellor of Jinnah University For Women Karachi, Pakistan.

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5.14: Procamallanus(Spirocamallanus)ruberii ( Figs. 65-66)

Type Host: Otolithus ruber (Schiender)

Type Locality: Fish harbor, Karachi coast.

Location: Intestine

Prevalence: 11.66% ( 7 infected /60 host examined)

Intensity: 1.44 ( with 4 male and 6 female)

Holotype (male) : JUW.N.23 Allotype (female): JUW.N.24

Diagnosis: Moderate sized worms, the body of the worms tapers gradually at its anterior and posterior extremity which is curved ventrally. The cuticle of the body is striated at its anterior and posterior extremities. The buccal capsule is oval in shape, surrounded by eight submedian cephalic papillae arranged in two circles. Inner surface of the whole capsule provided with 16- --18 spiral thickenings. With simple well developed- basal ring. Muscular esophagus is shorter and narrower than glandular esophagus. Intesine narrow, brown. Excretory pore slightly posterior to the anterior part of the glandular esophagus. The tail is pointed in male and dull pointed in female.

Male(4 specimens, including holotype): Length of body 10.5-12.0, maximum width 0.43.The wall of the buccal capsule is provided with 11-13 spiral thinkenings, 0.10x0.12x 0.11x0.14 in its diameter. The anterior muscular part of the esophagus is 0.65- 0.77 in length and 0.15 at its

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mazimum breadth. The longer posterior glandular part measures 0.84- 0.91 in length and 0.11 in its maximum breadth. The nerve ring is 0.24 from anterior extremity. The spicules are similar in shape, unequal in length, measuring 0.37 and 0.29 in length. Gubernaculum not observed. 12-14 pairs of caudal papillae are present, including 4 pairs preanal, 4 pairs post anal and 4 pairs of subventral pendulacute papillae. The tail is curved, 0.19 long with pointed end.

Female(6 specimens including allotype): Length of body 10.10, maximum width 0.41. The mouth leads into a buccal capsule, it measures 0.09---0.10 in its diameter, wall of the buccal capsule is provided with 13 spiral thickenings, the anterior muscular part of the esophagus is 0.36 in length and 0.16 at its maximum breadth. The longer posterior glandular part measure0.57 in length and 0.21 in its maximum breadth. The never ring is 0.27 from its anterior extremity. The valva is sub terminal, 0.10 from its posterior extremity. Vagina muscular, directed posteriorly from vulva. The tail is dull pointed

Remarks:

Several species of the genus have been described from Pakistan, Procamallanus (Spirocamallanus) spiralis (Khan& Begum, 1971) from Tachysurus caelatus in Karachi; the species was originally reported in Heterobranchus anguillaris from Egypt. P.(S.) pereirai(Annereaux,1946) after Rasheed, 1970 is described from variety of marine fish in Karachi, The head bears four large submedian papillae and a pair of amphids in external circle,the buccal capsule of the species have 8-10 spiral ridges and 11 pairs of caudal papillae in male but in female the spiral ridges are 11-13 in

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numbers with digit-Like tail.P.(S.) dussumieri (Bilqees et al., 1971) from Jhonius dussumeieri in Karachi, having 13 spiral thickings and 9 pairs of caudal papillae.P.(S.) sihamai (Khan & Begum, 1971) from Sillago sihama in Karachi having 11 spiral ridges and 6 pairs of caudal papillae. P.(S.) wallagus (Rehana & Bilqees, 1973) from Wallago attu in Sind; P.(S.) crossohombii (Zaidi & Khan, 1975) from Crossorhombus azureus in Karachi with 13 buccal ridges and 9 pairs of caudal papillae. P.(S.) sparus (Akram, 1975) from Argyrops spinifer in Karachi, mouth bounded by two pairs of submedian papillae and one pair of lateral amphids, caudal papillae are 10 in numbers and tail is provided with spines. P.(S.) otolithi (Ashraf et al., 1977 ) from Otolithus argenteus in Karachi; P.(S.) kalriai (Rehana & Bilqees, 1979) from Wallago attu in Pakistan; P.(S.) karachii (Rehana & Bilqees, 1979) from Wallago attu in Pakistan. But there are no electron- scanning micrographs so it is difficult to compare with these species.

The present species P.(S.) ruberii is different from the standing point of length and width of body, length of esophagus, number of spiral bands, arrangement of papillae, spicule length, and also host, from the species reported from South Asia including Pakistan.

The present species having ventrally bent posterior end of body, provided with subventral pedunculate papillae is similar to P.(S.) longus (Moravec et al.,2006). The present species is different from P.(S.) longus (Morevac et al., 2006) having 22 ridges but there are only 16-18 spiral ridges in the present species. The number and position of cephalic and caudal papillae in male (except the first post anal, close to the anal region) are more or less similar to P.(S.) longus (Morevec et al.,2006). The described species resemble only

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by the shape of female tail (dull point) with P.(S.) colei (Rigby et Adamson,1997) reported from coral reef perciform fish Acanthurus achiles from French Polynesia. Deirids are absent in the present species but present in P.(S.) longus, and also in P.(S.) colei.

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5.15: Procamallanus (Spirocamallanus) sp. (Fig. 67)

Type Host : Otolithus ruber( Sciaenidae)

Type Locality : Fish harbour, Karachi coast.

Location : Intestine

Prevalance : 13.33%(15 host examined/ 2 fish Infected) with 3 females only

Female specimen: JUW. N. 24

Diagnosis: (Description is based on one subgravid specimen). Body length is 23.5mm,and the maximum width is 1.0mm. The inner surface of the of the orange-yellow capsule is provided with 11-13 spiral thickings in a lateral view. and the diameter of the capsule is 0.18x0.15. The length of the muscular esophagus is 1.0 mm and its maximum width is 0.2mm, while the glandular esophagus is1.05mm in length and 0.2mm in width. The nerve ring is 0.20 from its anterior extremity. The distance of excretory pore is from anterior end. Vulva is pre-equatorial, 0.10 from posterior extremity. vagina muscular, directed posteriorly from vulva. uterus filled with larvae. tail rounded, with digit-like projection bearing two minute cuticular spikes.

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Remarks By the shape of the female tail with a digit-like projection bearing two terminal spikes, and in the same number of spiral ridges, this specimen resemble P.(S.) anguille (Moravec et al., 2006) from the Indonesian shortfin eel Anguilla bicolor from Thailand. P.(S.) monataxis (Olsen, 1952) have also projection bearing two terminal spikes on female tail. And was originally described from a lethrinid fish from Hawaii (Olsen, 1952) and later reported by(Rigby and Adamson, 1997) from fishes reported from French Polynesia. However, since only female specimens are available from Karachi coast, so it is assigned as P.(S.) anguille( Moravec et al.).

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5.17: HISTORY OF THE GENUS CUCULLANUS( MUELLER, 1777).

The genus Cucullanus Muller,1777( Cucullanidae, Seuratoidae)comprises over 50 species, mainly from fishes. Some studies on the biology and seasonal dynamics of Cucullanus spp. have been reported( Petter & Sey, 1997; Sardella, 1997; Moravec, F. 1998; Køie ,2000; Daniel et al., 2002; Lanfranchi et al., 2004; Moravec et al., 2005; Timi & Lanfranchi, 2006; González-Sols, 2007; Moravec et al., 2008). The first species described was Cucullanus cirratus Muller, 1777 parasitizing different hosts. It was synonymzed, C. truttae (Fabricius, 1794); C. acipenseris (Viborg, 1795) in Acipensar sturio, A. ruthemus, A. schypa, A. stellatus, A. gulenstadti etc. from Atlantic and Mediterranean. Cucullanus heterochrous (Rud., 1802). syn. C. platessae (Rud., 1809); C. sphaerocephala (Rud., 1809) were found in Orthagoriseus mola. Cucullanus esuriens (Duj., 1845); C. praecinctus (Duj., 1845) were found in Conger vulgaris; C. squati (Duj., 1845); C. attenuatus . C. rotundatum (Molin, 1859) in Cantharus vulgaris. C. longicollis (Stossich, 1899) in Mullus barbatus. C. callichori (Stewart, 1914) in Callichrous macropthalmus from Lucknow.. C. bulbus (Lane, 1916) syn. Bulbodacnitis b. in Caranx melampygus from Ceylon, ( Sri Lanka). C. pulcherrimus (Barreto, 1918) in Caranex lugubris from Brazil. C. dodsworthi (Barreto, 1922) in Sheriodes testudineus from Brazil. C. barbi (Baylis, 1923) in Barbus bynni from Egypt. C. incertus (Gendre, 1927) in Sebastes dactyloptera from Morocco. C. gigi (Fujita,1928) in Fluvidraco nudiceps from Japan and C. elongates (Smedley, 1933) in Ophiodon elongates from Pacific coast. C. filiformis (Yamaguti, 1935) in Conger myriaster and Anguilla japonica of Japan. C. amadai (Yamaguti, 1941) from

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Branchiostejus japonicus from Japan. C. girellae (Yamaguti, 1941) in Cyprinus carpio and Girella punctata from Japan.

Cucullanus mogi (Travassos, 1948) was reported in Leporinus sp. from Brazil. C. hansoni (Olsen, 1952) in Balistes capistratus from Hawaii. C. exigus (Yamaguti, 1954) in Lates calcarifer from Borneo. C. gendrei (Campana-Rouget, 1957) in Synacium micrirum from West Africa.. Berland (1970) described in detail C. cirratus, C.heterochrous and C.minutus. Ejsymont (1970) recorded C.truttae for the first time from Poland. Soota and Chaturvedi (1971) described C. pangasius from Pangasius pangasius in Madras state, India. The species has been differentiated from C. robustus. Maggenti (1971) gave a review of the family Cucullanidae Cobbold, (1864) and the genus Bulbodacnitis (Lane, 1916) with description of B. ampullastoma sp.n. (Nematoda: Cucullanidae), from Salmo gairdnerii. He has also given a Key to the genera and has made some new combinations which are B. truttae n. comb., for C.truttae and B.lebedevi n. comb. for C.lebedevi .

Gibson (1972) studied and described the larval and adult development and the life history of C.minutus and C.heterochrous from the host Platyichthys flesus in the Ythan-Estuary. The larvae of C. heterochrous were described for the first time. The author has discussed the possible influence of the water temperature on the host and the parasites and has concluded with the help of the geographical records of distribution the C. minutus parasitizes fish in the warmer waters than C.heterochrous. It was also recorded that the life cycle of C.heterochrous takes two years while the life cycle of C.minutus takes one year. Kumar and Gupta (1979) described a new species

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C. egrettae from the intestine of Egretta garzetta from Lucknow, India. During the same year Santos et al described a new species C. oswaldocruzi from the intestine of Zungaro zungaro from the Amazon river. Popova et al., (1979) reported the host range and life cycle of C. cirratus.

Moravec (1979) reported the development stages (from egg to adult) of C. truttae. encysted 3rd stage larvae were obtained from naturally infected larval lampreys ( Lampetra planeri) and 3rd-4th- stage larvae plus adults were obtained from experimentally infected trout. A variety of invertebrates, the fish Poecilia reticulatus and trout were fed 2nd- stage larva (free or within the egg) of C.truttae and were examined from 3 to 68 days later but none was infected. From field observations lampreys are the main source of infection of C.truttae for the trout. However, the parasite can also mature in the adult of the lamprey. The parasite in lampreys is usually known as C.stelmoides but this is infact synonymous with C. truttae.

Moravec (1983) studies the life history of C.truttae, a parasite of trout in Czechoslovakia. Earlier during 1981 Kakacheva- Avramova and Menkova studies its life-cycle and found Lampetra planeri served as an intermediate host. C. truttae was found to be a parasite of trout in the rivers Rilska, Ilina and Blagoevgradska Bistrista in Bulgaria. Minmim (1984) described a new species C.spirocaudus from the intestine of Liza haematecheila collected from Penglai, China. Malakhov and Valovaya (1984) studied the evolutionary adaptation to parasitic way of life of the cephalic sensory orgsns in Cucullania. Valovaya (1984) described the pseudobuccal cavity and surface cephalic structure of C.cirratus Muller, (1977) from SEM studies. Lebre and Peter(1984) described a new species C. campanae found

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in the Mediterranean coast of France at Sete (type locality) and at Banylus, in Atlantic Ocean, at Arcachon and in the Adriatic Sea (at Kotor, Yugoslavia).

Conneely and McCarthy (1986) studied the parasites of 121 eels from 3 contrasting sites in the Corrib catchments area, western Ireland. Thirteen species of parasites were recovered including C.truttae. Microhabitat preferences of the parasites were noted. Variation in the occurrence and intestines of the parasites observed were analyzed in relation to sampling period, host habitat and characteristics of the eel populations observed. Factors shown to be important included composition of the fish communities and distributional patterns of intermediate hosts and piscivorous birds. Differences were noted in the parasitocoenoses of eels in still and running water sites.The occuirence and intensity of infection of C.truttae was shown to be related to either age or size of eels, which is accounted for by the fact that eels become increasingly piscivorous with age and increasing size.

Ingrid et al.,(2002) described C. marplatensis parasitizing Odontesthes argentinensis from Argentinean waters. Lafranchi et al., (2004) reported

C.bonaerensis parasitizing Urophycis brasiliensis from Argentinean waters. Moravec et al., (2005) described C. oceaniensis from Anguilla sp. from Futuna Island. Timi and Lanfranchi,( 2006) described C. pedroi parasitizing Conger orbignianus from Argentinean waters. David et al., (2007) describe C. pargi from the grey snapper Lutjanus griseus of the southern coast of Quintana Roo, Mexico. Moravec et al., (2008) also describe C. maldivensis from Lutjanid fish, Macolor nigar of Maldive Islands.

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Sood,( 1989) studied fish nematodes from South Asia. Petter and Sey, (1997) described C. trachinoti from Tachinotus blochi. Lakshmi, (2000) give the presence of gubernaculums in C. baylist reported from Arius thalassinus.

Several species of the genus Cucullanus Muller, 1777 have been described both from marine and fresh water fishes of Pakistan. Species reported from marine fishes are all from fishes of Karachi coast. These include C.armatus, (Rasheed, 1968) from Tachysurus serratus; C. diminutus (Rasheed, 1968) from Stromateus niger; C. exiguous,( Rasheed, 1968) from Lates calcarifer and Pseudosciaena sp. Rasheed (1968) also reported C. hians from Lates cacarifier, Sciaena sp., Belone strongylurus, Acanthopagrus berda and Sparus sp. At the same time Rasheed described C. identatus from Polynemus tetradactylus and Pristopoma hasta. He also redescribed C. fastigatus (Chandler, 1935) from Pristopona hasta. C. theraponi (Rasheed, 1968) was described from Hilsa sp., and Therapon sp., C. bilqeesi by Bilqees et al., (1971) as C.elongatus from Erethistes elongate. C. olivaceus (Akram, 1975- 1976) was described from Tachysurus serratus and Pomadosys olivaceus. Akram (1975-1976) also described C. sparus from Sparus spinifer and Arius dussumieri and C. tachysuri from Tachysurus serratus and T. dussumieri. C. quadrii (Bilqees, & Fatima, 1980 ) from Arius serratus; Indocucullanus karachii (Zaidi & Khan, 1975) from the fish Engraulis indica was transferred to the genus Cucullanus by Soota (1983) with a combination as C. karachii. Bilqees et al., (2005) described C.mujibi from Arius serratus. And C. pakistanensis from Sciaena diacanthus from Karachi coast.

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The freshwater fish Cucullanids reported from Pakistan include C. pseudoannulatus (Rehana and Bilqees, 1986) from Mystus cavasius; C. gonii (Khan et al., 1991) and C. khalili (Khan et al., 1991) from Labeo goniusand Labeo rohita. C. annulatus (Margolis, 1960) has also been reported in the fish Mystus cavacius (Rehana and Bilqees, 1976). C. dogielli (Krotas, 1959) was reported by Khan and Yaseen (1969) in Chanda sp.

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5.18 Cucullanus aliyaii n. sp. (Fig.68-69) Order: Spiruridea Diesing 1861 Family: Cuculanidae Cobbold 1864 Sub family: Cucullaninae Yorke & Maplestone 1926 Genus: Cucullanus Muller, 1777

Type host: Otolithus ruber(Schneider, 1801) Site of infection: Intestine Type locality: Fish harbor, Karachi coast Pakistan Prevalence: 15 % (20 fish examined / 3 fish infected) Intensity: 2 (with 2 male and 4 females). Holotype(male): JUW. N. 40 Allotype(female): JUW. N. 41

Diagnosis: Relatively small nematodes, body whitish slender, with slightly tranversly striated cutical. Female larger than male. Broader anteriorly, posterior end pointed, provided with small bifurcated spike at the tip of the tail. Lateral alae absent. Oral opening dorsoventrally elongate, surrounded by narrow membranous flange or collarette supported by row of numerous teeth. Three submedian cephalic papillae and a pair of prominent lateral amphids are present. Pseudobuccal capsule or esophastome wider than posterior part of esophagus. Deirids simple, just aanterior to esophagus and intestinal junction. Tail broadly conical, bifurcated at the tip.

Male (2 specimens including holotype): Length of body 2.02-2.29, greatest width 0.35-0.40, entire esophagus 0.36-0.38 in length. Its minimum width is

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in the middle 0.10, Length of esophastome is 0.18-0.20, maximum width at esophastome 0.13-0.15, posterior club- shaped part of esophagus in 0.34- 0.37 in length and 0.09-0.11 in width .Nerve ring at a distance of 0.22-0.29 from the anterior extremity. Testis situated in posterior half of body or slightly anteriorly. Precloacal sucker is absent but few weak muscle fibers can be send. Spicules sub equal and 0.59-0.61 and 0.70-0.77 in length. Gubernaculums is absent. 12 pairs of caudal papillae are present including, 7 pairs preanal, 4 pairs are post anal and 1 pair is adanal. Tail is 0.01 in length.

Female ( 4 specimens including allotype): Length of body is 3.24, maximum width is 0.30. Entire esophagus is 0.50 in length, Greatest width 0.15 at esophastome , minimum width almost at the middle. Posterior club- shaped portion 0.15 in widths. Distance of nerve ring from the anterior extremity 0.30. Vulva 0.17-0.18 post equatorial, prominent, vulvar lips elevated at a distance of 1.80 from the anterior extremity. each containing elongated gland cells, extruding out of body wall. Vagina long, muscular tube and is directed upward. Uterus thin- walled, containing numerous relatively large, thin- shelled eggs 0.07x0.04. Tail is 0.011 in length.

Remarks: The genus Cucullanus Muller, 1777(Cucullanidae) contains a large number of species parasitizing various fresh water and marine water fishes around the world. Due to minute differences in the morphology and because of inadequate descriptions, a detail comparison among species is very complicated (Moravec et al., 1993) Therefore the present new species C. aliyaii is compared with the species recovered from South Asia, especially

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Pakistan and some related species from other parts of the world. The species reported from marine fishes of Pakistan are all from fishes of Karachi coast. These include C.armatus (Yamaguti, 1954) reported by Rasheed (1968) from Tachysurus serratus; C. diminutus (Rasheed, 1968) from Stromateus niger. Rasheed (1968) also reported C.hians (Dujardin,1945) from Lates cacarifier, Sciaena sp., Belone strongylurus, Acanthopagrus berda and Sparus sp. At the same time Rasheed described C. identatus from Polynemus tetradactylus and Pristopoma hasta. He also redescribed C fastigatus (Chandler, 1935) from Pristopona hasta. C. theraponi (Rasheed, 1968) was described from Hilsa sp., and Therapon sp., C. bilqeesi (Petter, 1974) was originally described by Bilqees et al., (1971) as C.elongatus from Erethistes elongate and Petter renamed it as the species name was preoccupied. C. quadrii (Bilqees, & Fatima,1980) from Arius serratus; C. olivaceus (Akram, 1975-1976) was described from Tachysurus serratus and Pomadosys olivaceus. Akram (1975-1976) also described C. sparus from Sparus spinifer and Arius dussumieri and C. tachysuri from Tachysurus serratus and T. dussumieri. Indocucullanus karachii (Zaidi & Khan, 1975) from the fish Engraulis indica was transferred to the genus Cucullanus by Soota (1983) with a combination as C. karachii.

Petter (1974) regarded C. tachysurusi as C. arabiense (Ali & Kalyankar, 1967) from Maharshtra, Mangalore and Karnataka. Petter (1974) restricted its occurrence in Siluriform hosts and gave only three pair of preanal caudal papillae for the species. Similarly he transferred Indocucullanus calcariferi (Zaidi and Khan, 1975) to C. jaiswali (Ali, 1957). This species has been reported from Puntius sarana, Lates calcarifer, Andhra Perdesh, Calicut and Karala, India.

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Yamaguti, (1961) has listed 60 species in the genus Cucullanus including the genotype. In addition to this Gupta and Masoodi (1982) described C. sootai and listed another 30 species including C. ritali (Karve, 1952); C. jaiswali (Ali, 1956); C.indica (Agrawal, 1965); C. pseudotropi (Agrawal,1967); C. arabianse (Ali and Kalyankar,1967; Petter, 1974); C. theraponi (Rasheed,1968); C.pangasius (Soota and chaturvedi., 1971); C. jalnaensis; C. alii Kalyankar, (1971); C. malvanae Kalyankar, (1971); C. tachysuri (Kalyankar, 1971); C. bilqeesi, (Bilqees et al., 1971; Petter, 1974); C. carioca (Vicente and Fernandes, 1973);C. rougetae (Vicente and Dos, 1974); C. bagrae (Petter,1974); C. karachii (Zaidi & Khan, 1975); C.olivaceus (Akram, 1975-1976); C. guerrori (Arya and Jhonson, 1975); C. ariusi (Srivastava and Gupta, 1976); C. sciaenai (Gupta and Gupta, 1979); C. rivulatus (Soota and Sarkar, 1980); C trichiurisi (Gupta and Naqvi, 1983); C. simhai (Gupta and Naqvi, 1983); C. thapari (Gupta and Srivastava, 1984) and C. mastacembeli (Gupta and Srivastava, 1984).

Later on more species have been described both from fresh water and marine fishes of various localities including C. rhamphicthdis (Moravec et al., 1979); C. campanae (Labre and Petter, 1984); C. fugianensis (Wang, 1984); C. brevispiculus (Moravec et al., 1993); C. riograndensis (Fortes et al., 1993a); C. fabrigasi (Fortes et al., 1993b); C. mexicanus (Capseta- Mandujanoi,2000); C. marplatensis (Ingrid et al., 2002); C.oceaniensis (Moravec et al.,2005); C.pedroi (Timi and Lanfranchi, 2006). David et al., (2007) describe C. pargi from the grey snapper Lutjanus griseus of the southern coast of Quintana Roo, Mexico. Moravec et al., (2008) also describe C. maldivensis from marine fishes of Maldive Islands.

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The present species C.aliyaii is different from C. Alti (Kalyankar, 1971) Peter, 1974; C. arabianse (Ali & Kalyankar, 1967; Petter, 1974); C. ariusi (Srivastava & Gupta, 1976) Soota, (1983); C. armatus (Yamaguti, 1954); C. bulbosa(Lane,1916) Barreto, 1918; C. chrysophrydis (Gendre, 1927); C. guerreroi (Arya & Johnson, 1975) Soota, 1983; C. jaiswali (Ali, 1957) Petter, 1974; C. jalnaensis (Kalyankar, 1971) Petter, 1974; C. karachii (Zaidi & Khan, 1975) Soota, 1983; C. malvanae (Kalyankar, 1971) Petter, 1974; C. pangasius (Soota & Chaturvedi, 1971); C. pseudotropi (Agrawal, 1967); C. ritai Karve, 1952; C. rivulatus Soota & Sarker, 1980; C. sciaenai (Gupta & Gupta, 1979) Soota, 1983; C. theraponi (Rasheed, 1968); C. indica (Agrawal, 1965); C. tachysuri (Kalyankar, 1971) Petter, 1974; C. mystusi (Gupta & Naqvi, 1985) Gupta & Masoodi, 1986; C. vinodae (Gupta & Naqvi, 1985) Gupta & Masoodi, 1986; C. schizothoraxi Gupta & Masoodi, 1986; C. sootai(Gupta & Masoodi, 1986); C. fujianesis (Wang, 1984); C. baylisi (Lakshmi, 2000); as all these species have a gubernaculum. While gubernaculums is absent in the present new species. Species described by Moravec et al., (1993a-b, 1997) also have gubernaculums. Similarly gubernaculums is present in C. paloi(Fortes et al., 1992); C. riograndensis (Fortes et al., 1993a) and C. fabrigasi (Fortes et al., 1993b).

Present species C. aliyaii is also different from C. marplatensis (Ingrid et al.,2002); C.bonaerensis ( Lafranchi et al., 2004) ; C. oceaniensi ( Moravec et al., 2005); C. pedroi (Timi and Lanfranchi,2006) ; C. pargi ( David et al., 2007) and from C. maldivensis ( Moravec et al., 2008) in variable combination of characters, specially in size of esophagus, spicule length and number and position of caudal papillae in male. But the present species C.

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aliyaii resemble in the presence of deirids with C. oceaniensis (Moravec et al.,2005) .C. bilqeese (Bilqees et al., 1971) Petter, 1974 and C. olivaceus are described only by females. These are also different from the present species in the size of body, esophagus and eggs. The presence of deirids are not reported previously from Pakistan. As mentioned the morphological characters and presence of deirids, the present nematodes of the genus Cucullanus appear an undescribed species for which the name C. aliyaii n. sp., is proposed.

Soota (1983) regarded C. quadrii as synonym of C. armatus. This species was originally described by Yamaguti (1954) from Tachysurus sp., (Arius sp.) in Borneo and later on by Rasheed (1968) from T. serratus. Even if C. quadrii is considered synonym to C. armatus, the present species is different having different host, lacking a gubernaculums and precloacal sucker, longer subequal spicule (0.59-0.61 and 0.70-0.77) in length), vulvular lips are prominent elevated and protruded. The caudal papillae in the present species are 12 in number and in T. armatus caudal papillae are(9-10) pairs. Characters such as the presence of deirids, absence of gubernaculums, precloacal sucker, number of caudal papillae, spicules length, and morphology of vulva, egg sizes and tail length are considered valuable differentiating diagnostic features. Therefore, present specimens are regarded a new species and its name C. aliyaii n.sp. is proposed. Present species resemble in some morphological features with C .pakistanensis Bilqees et al., 2005, including sub equal spicules, length of tail and bifurcation at its tip, absence of gubernaculums and precloacal sucker. But body size, length of esophagus, presence of deirids and morphology of vulva is different from C. pakistanensis.

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The species in which gubernaculums is absent such as C. bengalensis (Gupta and Masoodi, 1985), But the caudal papillae are same (12 in numbers) as in the present species.

Gubernaculum is present in C. armatus (Yamaguti, 1954); C. bulbosa (Barreto, 1918); C. indica (Agrawal, 1965); C. tachysuri (Kalyankar, 1971) Petter, (1974); C. mystusi, (Gupta and Masoodi, 1986); C. riograndensis, (Fortes et al; 1993a) C. fabrigasi (Fortes et al; 1993b); C. baylist (Lakshmi, 2000). Lakshmi (2000) described C. baylisi from the related host Arius thalassinus in India. Present new species is also separated from C baylisi which has a gubernaculums and much smaller spicules (0.237- 0.198mm in length), smaller eggs (0.069 x 0.048) and smaller tail (0.348 in female and 0.16- 0.18in male ). in addition a large number of sessile caudal papillae are also present in C. baylisi. Lakshmi probably did not consider C. quadrii a synonym of C. armatus or was unaware about this.

Etymology: The species name Cucullanus aliyaii n. sp refers to the honor of my best friend because of her help in this project

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6: HISTOPATHOLOGY

Histopathology is the study of tissue damage. Causes of tissue damage are of varied nature including parasitic infections. Tissue damage could be traumatic due to physical pressure exerted by the parasites or may be caused by the toxic secretory or excretory products of the parasites which may some times lead to hypersensitivity reactions. Many abnormal conditions may be encountered in various organs infected with parasites.

Histopathology of fish is relatively a neglected field of study especially histopathology caused by nematode parasites in Pakistan. . The study was based of the intestine of two fish species of Karachi coast.

During the present investigation The intestine of the two infected fish species including Euthynnus alletteratus (Refin.,1810) and Pomadasys maculatum (Bloch, 1797) were used for the tissue damage caused by nematode parasites.

6.1:Description and distribution of Euthynnus alletteratus(Refinisque, 1810) This species, commonly called little tunny and locally Dawan, can be superficially separated from others species in the genus by the absence of vomerine teeth, and by the pattern of dorsal markings. These markings are more continuous and regular posteriorly. Anteriorly the bars are replaced by short irregularly curved lines, blotches or by spots. There are approximately 25 to 35 teeth on each side of the lower jaw; the palatine teeth are sharp, strong and quite conspicuous. There are no vomerine teeth. The stomach is a 166

long blind sac, as in all the tunas, extending almost the full length of the body cavity. This originates at the anterior end and on the left side of the stomach. It loops anteriorly across the ventral wall of the stomach and reaching the proximal end of the body cavity on the right side, it runs directly back without fold or undulation, to the vent.

6.2:Histopathology of Euthynnus alletteratus (Refinisque, 1810) No work has been done on histopathology of Euthynnus alletteratus(Refinisque) specially with reference to fishes of Pakistan. An attempt is made here to provide information about histopathological conditions of Euthynnus alletteratus(Refinisque) from Karachi coast, caused associated with nematode infection.

During the present observation histological changes are noted in the intestine of Euthynnus alletteratus,(70-81) infected with nematode parasites. The infected intestine revealed destruction and atrophy of intestinal mucosa(fig.70). The section of intestine shows the section of parasites with various stages of necrosis and degeneration of the intestinal tissue, causing damage to the whole thickness of the bowl wall due to nematode larvae (71- 72). Sections of nematodes were obvious in the villi, lamina propria and submucosa. The mucosa appeared congested and edematous. The deformation of mucosa and submucosa result in separation of muscle fibers(74-75)The shape of villi are changed as compared with the normal shapes. Irregular or haphazard growth of villi is observed. Individual epithelial cells at the tip of villi were atrophied and necrotic and showed a variety of alteration. Villi were broadened and look-like mucosa of stomach (Figs.78-79). Marked increase of mucus secretion was noted with sloughing

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of villi and epithelial surface cells were dysplastic with loss of columner orientation. In certain areas villi disappeared leaving flattened surface. (Figs. 74-76). Damage to the surface epithelium resulting in desquamation of cells leading to progressive shortening and flattening of the villi was obvious throughout the affected areas. Chronic inflammatory reaction was obvious (Fig.73). Shrinkage of muscular layers was observed. Thickening of blood vessel wall and reduced lumen was also a common finding (Figs.71-74). The inflammatory reaction produced dark aggregation of white cells in these areas as well as scattered through out the sections.

6.3:Description and distribution of Pomadasys maculatum(Bloch, 1797) The family Pomadasyidae (Haemalidae) of grunts is the one among 150 families of Perciformis (Ajaz, 1991). The main fishing season is from August to October and small quantities are caught from the month of November to January and also from February to March. Grunt is common names for nearly 200 species of marine fish that are wide spread in tropical seas but also range in to temperate water. The grunts are group of marine edible fishes. But little is known of their histopathology. All grunts are carnivores in order to find out a relation that may exit between the normal diet and the structure of the alimentary tract, the functional anatomy of the digestive tract needs a detail study. The alimentary canal of Pomadasys maculatum can conveniently be regarded as divided into two main regions(Banki, 1936), the ‘Kopfdarm’ comprising the buccal cavity and pharynx, and the Rumpfdarm’ comprising the forgut( Esophagus and stomach), midgut (Intestine) and hindgut (rectum). Black (1930, 1936) and Rogick (1931) studied the alimentary tract of fishes from American water, Suyehiro (1934), Ghazzawi (1935), Al-Hussain (1947), Barmington, 1957; Kapoor (1958, 1975); Toor

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(1965);Clark and Witcomb, 1980 came to the conclusion regarding the structural adaptation of the alimentary tract to the normal diet. No information on histopathology of infected intestine of pomadasys maculatum(Blch) is not available and hence the work was focused on this aspect.

6.4:Histopathology of infected intestine of Pomadasys maculatum (Bloch, 1797). The histopathological conditions in Pomadasys maculatum (blch.) encountered in sections revealed that the site of penetration is usually edematous accompanied with hyperemia of various degrees of erosion and bleeding in some cases. The abnormal conditions of Pomadasys maculatum(Blch) observed and described here are inflammation, necrosis and degeneration. Intestinal wall of Pomadasys maculatum with nematode show severe host tissue reaction were observed. (Figs.82-89).

The infected intestine of Pomadasys maculatum shows severe damage in the whole thickness of its wall. Erosion of intestinal wall with loss of villous epithelial lining is usually seen. Flattening and fusion of the villi also occurs with sloughing of epithelial covering of the villi.(82, 85-87). The villi were abrophied becoming long and irregular with vaculation of submucosa which also indicate atrophy (figs.88-89). At higher magnification sloughing of epithelial cells and complete atrophy of villi and underline tissue were more obvious (Fig.85). Several sections of nematode larvae were also observed(83- 84). Atrophy of tissue resulted into shrinkage and vacuolation of lamina propria and total loss of villous structures which is a common finding involving the deeper tissues(88-89). Shrinkage and atrophy of glandular

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structures resulted into formation of large spaces at the periphery(Figs.88- 89). The muscular layers were also affected. Ulcerative lesions were prominent at submucosal region(Figs. 88-89). Degeneration of tissues was also obvious (Figs. 77-78). In more severe condition all the intestinal morphology was lost. Villious structures was not differentiated and appear as strings of tissues(Fig.85).

6.5: Remarks:

Present observations are similar to those described in other fishes of Karachi coast with nematode larvae (Bilqees and Fatima, 1995). Other workers have also reported enlargement and inflammation of intestine (Bullock, 1963). Columnar epithelium cells line the intestine the microvilli layer has been termed a ‘stratified layer’ or brush border by Al-Hussaini (1947), Al-Hussaini and Kholy(1950-1953); and ciliated epithelium by Ishida (1935-1936) studying a variety of teleosts.Lile, 1998 studied the alimentary tract helminthes of flat fishes. Miyazaki et al.,2006 observed infection by larval nematodes which penetrate deeply into the stomach and intestine caused formation of capsuler granulation. Akinsanya, 2007 observed mucosal oedema, haemorrhage with haemosiderosis in some tissues of the fishes infected by helminthes including Acanthocephala, Nematoda, Cestoda and Trematoda. In the present observation the columnar epithelial cells are reduced in height, fragmented and sloughed. The disorganized necrotic elements within which degenerating parasite can be seen and under this condition inflammation are obvious (Figs. 70-73).

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From the very beginning of the twentieth century anatomists have paid a great deal of attention to the structural details of the gastrointestinal tract of the fish. The four basic histopathological layers such as serosa, muscularies, submucosa and mucosa are present in the region from the esophagus to the tip of the intestine, including the caeca. Among the vertebrates, only teleostean fish species have appendages such as caeca at the gastrointestinal junction (Khanna, 1961; Romer, 1970; Kent, 1983). The caecal development appears to be intiated by a constriction between the stomach and intestine, the role of constriction should be considered with caution since not all stomach- bearing fishes have caeca (Suyehiro, 1942; Lagler et al., 1977).

The interest and research is being developed in the recent years in the applied branches of functional morphology, which include structural changes and parasitic diseases with contaminated situations resulting in abnormal growth of cell with histopathological changes in fishes. Histopathology is an important tool to observe the extent of tissue damage due to various infections. Histopathology of intestine and stomach of some fish species of Karachi coast has been carried out only by Bilqees and her coworkers, including marine fishes Hilsa ilisha (Bilqees & Fatima, 1993) infected with Anisakis larvae; and Rachycentron canadus (Khatoon & Bilqees, 1996) stomach infected with Raphidascaris sp Cybium guttatum, ( Bilqees et al, 1998; Bilqees & Parveen, 1996) infected with nematode larvae; Otolithus argenteus ( Khatoon & Bilqees, 1999); Lutjanus argentimaculatus ( Khatoon et al., 1999a) stomach infected with Goezia species. and Anisakis gastric granuloma has been described by (Khatoon et al. 1999b) in the fish Arius sp.( Rizwana et al., 2000) described Goezia argentimaculatus from the fish Lutjanus argentimaculatus from Karachi coast. In all these cases severe

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destruction has been reported to the tissue. Other workers have also reported enlargement and inflammation of intestine (Bullock, 1963).

The importance of intestine can be evaluated by analysis of the interaction with effect of various parasites including the out side environment. The fish digestive tract has more direct contact with parasites and the external environment than any other internal organ. The food comes directly from outside into the gut. So it may be accepted that any significant alteration will induce some changes in the structure and function of the gut and its diverticulae (Hussain, 1988).some studies have correlated environmental changes with structural and functional variability of the fish gut (Tyler, 1973; Singh and Bahuguna, 1983).It may be possible that tissue reaction may be similar in two different fish hosts. an Anisakid nematode larvae found in fishes are able to produce gastrointestinal lesions and diseases in human being (Ishikura et al., 1993) Anisakid nematode are known to infest fishes since last six centuries and lesion in marine mammals were reported about two hundred years ago(Sakanari,1990). Public health significance of Anisakis nematode is well known as cases of anisakiasis are being continuously reported through out Europe, Japan, North America, Hawaii and several other countries including Russia, Germany, Canada and Korea.

In fishes of Karachi coast anisakid larvae specially of genus Anisakis are one of the most common type of larvae infecting viscera of various species (Bilqees & Fatima, 1986) and these also have public health impotance (Bilqees et al., 1999).In spite of severe Anisakis larvae infection in several species of edible fishes of Karachi coast, this infection has not been yet reported in human in Karachi. As these larvae are commonly found in several

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marine fish species of Karachi coast including Hilsa ilisha, Pseudosciaena diacanthus, parastromateus niger, Mugil spp., Muraenesox cinereus, Arius serratus, Pomadasys olivaceus, Chondroplites chinensis( Bilqees & Fatima, 1986; Fatima & Bilqees, 1987, 1989).

No further documented research was carried out on larval nematode of fish in Pakistan beyond the publication mentioned above. Beside no information is available on intensity and histopathology of these infection in Euthynnus alletteratus (Dawan) and Pomadasys maculatum (Dhothar). Euthynnus alletteratus( Dawan) are found in large number and the approximate period of these fish is from November to January. And this is the cold season in Pakistan where the fish demand is very high. The Pomadasys maculatum is one of the popular and expensive edible fish.

During the present work histopathology of Euthynnus alletteratus and Pomadasys maculatum associated with nematode parasites is carried out. The damage caused by these parasites in intestine is observed and presented here; Photomicrographs of parts of histological sections are prepared in support of observation. The objective of the present study is to determine the histopathological changes in the fish due to various infections, as the parasite and disease of the fish constitute one of the most important problem confronting the modern fish culture, farming and also produce human infections.

In the present study the intestine of Euthynnus alletteratus ( Dawan), and Pomadasys maculatum (Dhothar),infected with nematodes caused destruction of epithelial cells and inflammation at the sites of attachment of parasites due

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to various parasites infection. The columnar epithelial cells are reduced in height fragmented and sloughed. The disorganized necrotic element within which degenerating parasite can be seen and under this condition inflammation is obvious (Figs. 70-89).

Studies related to fish infection and their effect on various fish organs is important. In Pakistan authorities do not pay any attention to parasite diseases in spite of the fact that parasites have adverse effects on the growth and survival of fish specially if these infect the vital organs and are present in large numbers or in young fish.

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7: DISCUSSION

The present research and investigated results provide information for food and feeding habits, nematode parasites and histopathology of the infected intestine of some marine edible fishes of Karachi coast; also provide preliminary information on correlation between the investigated parameters.

7.1: Dietary habits The study of the stomach contents of data of 10 marine fish species showed a broad variation in the food and feeding habits of these fishes, which allows to categorize them as herbivorous (Liza.verigensis and Sardinella albella) and carnivorous (Scomberomous gutattus, Pomadasys olivaceum, pomadasys maculatum, Otolithus ruber, Lates calcalifer, Arius maculates Sphyraena jello and Spharyena foresteri) (Table 2-13).

.The compositions of food items are presented in table 2-4. whereas the organisms by name, total, percentage occurrence(O%) and percentage of frequency occurrence(F, F%) of stomachs are shown in (table 5-13).The food categories mainly consisted of crustaceans, molluscs, helminthes, planktons, teleosts, detritus and miscellaneous.

The analysis of the stomach contents revealed a broad range of food items, accompanied by large amounts of detritus such as sand, mud and rock grains. Items observed included planktons, algae, teleosts, crustaceans, mollusces, polycheates and helminthes.

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Data presented in this study confirmed that Liza.verigensis and Sardinella albella were herbivores as the diet covers a wide spectrum of food ranging from phytoplankton to zooplanktons, algae and other accessory prey items. (Table 2-4).The main phytoplankton genera found in fish stomachs belongs to Cyanobacteria and Bacillariophyceae and cholorophyceae (Figs. 9-20). Anabaena, Oscillatoria and Microsystis.(Cynobacteria), Gyrosigma, Pinnularia, Melosira, Navicula (Bacillariophyceae), scenedesmus, Chlorella (Chlorophyceae) were found in all fish sizes (Table 5-7).The Phytoplanktons were identified by Anjum & Hussain ,(1983); Anon ,(1987); Battish, (1992);Nazeen & Bari, (1984) and by the help of Botany Department of Jinnah University For Women.

Various investigations have been conducted into the food and feeding habits of fish with the aim of determining their dietary requirements found in the stomachs. this information is also essential in the analysis of aquaculturing. The study of dietary habits of fish based on stomach content analysis is widely used in fish ecology, Fagbenro et al,.( 2000). Ghandhi (1982) remarked that feeding habits depended on the availability of fish food in the environment. It is well known that gut length is closely related to the properties of food consumed by fish. According to Gerking (1994) herbivorous species posses’ longer guts as compared to carnivorouses allowing the consumption of food with low digestibility. Buddington et al., (1997) and Kubitza, (1999) stated that shorter digestive tracts as observed in carnivorous fishes, present a greater number of villi and pyloric caeca, which amplify manifold and compensates for a relatively short intestine.

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7.2: Seasonal variations in dietary habits The season consideration was also taken in the gut content of fishes. During the present investigation of herbivorous fishes, the guts were found gorged, full and with diversity of organisums in the months of March, April, May, June & July but were half full and empty and with less diversity of organisms in October, November & December. The planktons were abundant in month of March, April, May, and June and less frequent in the months of September, October and December.

In carnivores fishes the stomach were gorged in months of February, March and April. Molluscs and crustaceans were dominant in April, but were frequently seen through out the year, and polycheates were absent in August ,September, October and January ,but were present in March, April and May. Squilla sp. and Loligo sp. were seen in month of February and March.

There was no significant seasonal variations in food and feedng habits but the percentage occurrence and frequency occurrence were found to be change with season through out the study of different edible marine fishes. Similar observation has also been reported by De-Groot (1971) Lande (1973) Dewan and Saha (1979) and Bhuiyan & Nazrul Islam (1991).

The data obtained for feeding habits of collected fish during this investigation were subjected for statistical analysis to check the accuracy of occurrence and frequency occurrence of different food items found within stomachs. The value of confidence interval falls within the limits where current results were satisfied. In herbivorous fishes the highest frequency groups on the stomachs, (F %) was detritus followed by planktons and copepods. While in carnivorous

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the highest frequency group on the stomachs (F %) were miscellaneous followed by crustaceans> mollusks> polycheates. The detritus (sand and mud) were found in least quantity.

The data of the herbivorous fishes revealed on the basis of stomach contents that detritus (sand, mud) were found to be of most dominant categories (100%) followed by planktons (85%) and copepods in all respect e.g. percentage of occurrence and frequency of occurrence. While in all investigated carnivores fishes, the miscellaneous items (90%) were found to be the most dominant category in all respects e.g. percentage of occurrence and frequency of occurrence in all size fishes. Crustaceans formed the second important group followed by mollusks and teleosts on the basis of frequency of occurrence. The least observed food items were polycheates.

7.3: Correlation of dietary habits and nematode parasites. During the analysis of dietary habits, altough helminthes were mostly found in stomach contents but as Siglar states (1958) that platyhelminthes and nematyhelminthes do not form a part of the food material and they might have been swallowed along with the miscellaneous in carnivorous fishes. According to Williams et al., (1992); Marcogliex and Cone (1997) many helminthes parasites (Parasitic worms) have complex life cycle involving several different host depending on tropic interaction for transmission. They can also provide valuable information on host migration and diet.

Hirasawa et al., 2004 discuss the relationships between nematode parasitism and the feeding habitats of their intermediate hosts and found that the principal intermediate hosts of the two nematodes were filter-feeding

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mayflies of the genera Ephemera, Photamanthus and Isonychia. Ephemera strigata seemed to be the most important intermediate host of these nematodes. Adult R. coronacauda were found mainly in Hemibarbus longirostris and Rhinogobius flumineus, which are benthic fishes that feed on benthic aquatic insects, including E. strigata. For R. coronacauda, therefore, the feeding habits of the definitive hosts facilitate host alternation by this species. A strong relationship between infection with a parasite species and the corresponding intermediate host from the stomach content of individual charr, indicated an individual feeding specialization. (Knudsen et al 2005). The infection variations seemed to be due to differences in host growth rate, host feeding habit, and the distribution of marine mammal final hosts. The results indicate that larval nematodes are useful biological indicators for the population study of walleye pollock in Japanese waters.( Konishi, 2002)

The present study detected some patterns including correlation of Parasites with the feeding habits of the fish, nematodes dominance among the other helminth parasites, and infection of fish by these parasites. These studies support the statement of Sasal et al. (1999), that the diet of the host species is the main factor effecting parasitic community structure.

The nematode occurred generally in those months in which the fish stomachs were gorged, full or 1/2 full. Which suggest the correlation between the dietary habits and occurrence of nematodes parasites.

A total of 1500 fish specimens were examined for nematode parasites. In herbivorous fishes (n=300) soil nematodes were found while in carnivorous fishes (n=745) parasitic nematodes were recovered including new (n=131) and 184

known (n=141) specimens. A prevalence of 62 % was observed from 745 infected fishes. All nematode recovered were restricted to the intestine.

An attempt is also made to identify and describe the new nematode species. Identification and descriptions were made possible by the help of literature from Yamaguti,1966; Sood, 1985; Bilqees, 2007 and also provided by Nadja Noel and Rupert Lee Research service of British Library and also by comparing the literature obtained by Dr. Franick Moravec from institute of Parasitology, Czech Republic Budejovice.

One new genus Spirocotyle and three known genera, including Dujardinascaris(Baylis, 1947); Procamallanus (Spirocamallanus (Olsen 1952) Petter 1979) and Cucullanus Muller, 1777 were identified. From these four genera a total of 11 new species including one known (but not reported from Pakistan yet) were recorded as follows. i) One new genus Spirocotyle (Camallanidae Railliet and Henry,1915)and one new species S. otolithi was identified and described from Otolithus rubber. ii) Seven new species identified and described here from the known genus Dujardinascaris (Baylis, 1947) identified and described including Dujardinascaris mujibi n.sp. from Sphyraena forsteri; D. jello n.sp. from Sphyraena jello;, D. maculatum n.sp. 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 rube iii) Two new species of the genus Procamallanus (Spirocamallanus ( Olsen 1952) Petter 1979), were also identified and described here

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including, Procamallanus (Spirocamallanus) riaziaii n.sp.and P.(S.) ruberii. Both species were recovered from Otolithus ruber

Two female specimens of unidentified species were also reported here from Otolithus ruber. Species is not identified as male specimens were not available. iv)One new species of the genus Cucullanus Muller, 1777 was identified and described here as C. aliyaii n.sp. from Otolithus ruber.

7.4 : Parasitic diversity Most studies of food and feeding habits of fishes, from varying habitat, 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 habit of a species (Staples, 1975).One would examine interspecific variation in parasitic diversity among a group of host species from the same area, all examined in a standard way by the same researchers ( Luque et al, 2004).The parasitic way of life is probably one of the most common life found on earth (May, 1988, winder 1997,1998). Virtually every species is parasitized at some point during its life time (Price, 1980) in a heavy infection a parasite could cause harm and become pathogenic to a host individual (Anderson & May 1979, May & Anderson 1979). The host size variations and endoparasitic communities was studied by Poulin & Valtonen(2001). There is increasing evidence that parasites are an important element of marine biodiversity ( Mariaux, 1996; Palm et al., 1999; Kilmpel et al.2001).

During the present study diversity was notable as one parasites is found in

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many hosts(genus Dujardinascaris in different hosts, Pomadasys, Sillago sihama and Sphyraena)and diferent nematode species were found in a single host e.g. ( different species of Procamallanus(S.) were found in a single host of the genus Otolithus.

7.5: Histopathology of fishes During nematode investigation parasitic infestation were checked at intestinal level and find out that Parasites and disease reduce fish production by affecting the normal physiology of fish as stated by Kabata (1985) and which, if left uncontrolled can result in mass mortalities or in some cases infection of man and other vertebrates that consume them. The diseases due to the adult and larval nematodes are very common in marine fishes and 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. 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.

A considerable number of new species and genera have added recently and many more remain to be discovered. The diseases due to the adult and larval nematodes are very common in marine fishes and world wide in distribution. The parasites invade various tissues and organs of fish. In fisheries biology,

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parasitological studies have increasing importance, because parasites may serve as natural markers for the identification of fish stock ( MacKenzie, 1983; Williams et al, 1992). Moreover, parasites can help to analyses the diet of fish species, serving as biological indicator of the prey species and their origin( Campbell et al. 1980; Palm et al. 1998).

Small fish species are frequent food items for piscivorous fish. Predatory fish largely acquire and accumulate parasites by ingesting small fish species that are infested and serve as carriers ( Lile, 1998). Asor and Arenc (2000) stated that the site of information varied with the parasite, but the site most affected generally is the intestine

During the present study observations were made on the tissue section of various parts of intestine infected with nematodes.

During the present observation histological changes are noted in the intestine of Euthynnus alletteratus, infected with nematode species.(Fifs. 70- 81).The infected intestine revealed damage to the whole thickness of the bowl wall due to nematode larvae. The mucosa appeared congested and edematous. The section of intestine shows the section of parasites with various stages of necrosis and degeneration of the intestinal tissue. The shapes of villi were changed as compared with the normal shapes. Irregular or haphazard growth of villi was observed villi were broadened and look-like mucosa of stomach (Figs.78-79). Sections of nematodes were obvious showed necrosis in mucosa and submucosa (Figs 71-72). Marked increase of mucus secretion was noted with sloughing of villi and epithelial surface cells were dysplastic with loss of columner orientation. Villi were broadened and look-like mucosa of stomach

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(Figs. 74-79). In certain areas villi disappeared leaving flattened surface(Fig.76). Chronic inflammatory reaction was obvious. The inflammatory reaction produced dark aggregation of white cells in these areas as well as scattered through out the section.

The infected intestine of Pomadasys maculatum shows severe damage in the whole thickness of its wall(Figs. 82-89). Erosion of intestinal wall with loss of villous epithelial lining is usually seen. Flattening and fusion of the villi also occurs with sloughing of epithelial covering of the villi.The villi were abrophied becoming long and irregular with vaculation of submucosa which also indicate atrophy(Figs.82-89). At higher magnification sloughing of epithelial cells and complete atrophy of villi and underline tissues were more obvious (Figs.82, 85-87).Several sections of nematode larvae were observed(Figs.83-84). Atrophy of tissue resulted into shrinkage and vacuolation of lamina propria and total loss of villous structures which is a common finding involving the deeper tissue (Figs.88-89) with the crypt glands lengthening and degenerating of its structure also occurred. Shrinkage and atrophy of glandular structures resulted into formation of large spaces at the periphery. The muscular layers were also affected. Degeneration of tissue was also obvious. In more severe condition all the intestinal morphology was lost. Villius structures were not differentiated and appear as strings of tissue. The crypt glands were degenerated leaving the cell remnant of glandular structure fragments and remnant of glandular structure as dark staining patches of tissue and lumens of the crypt glands were obliterated.

These observations are similar to those described in other fishes of Karachi coast with nematode larvae (Bilqees and Fatima, 1995). Other workers have

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also reported enlargement and inflammation of intestine (Bullock, 1963). Some workers observed destruction of epithelium at the point of attachment. Columnar epithelium cells line the intestine the microvilli layer has been termed a ‘stratified layer’ or brush border by Al-Hussaini (1947, Al-Hussaini and Kholy, 1950-1953); and ciliated epithelium by Ishida (1935-1936) studying a variety of teleosts.

In the present study, the columnar epithelial cells are reduced in height, fragmented and sloughed(Figs.88-89). The disorganized necrotic elements within which degenerating parasite can be seen and under this condition inflammation is obvious.

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FUTURE PROSPECTIVE

Although a lot of work have been carried out on taxonomy and morphology of nematodes parasite but still there are some more significant aspects on which further work can be carried out in future.

Identification of nematodes with help of high technology.

Disease caused by nematode parasite

Effect of nematodes on fish.

Establishment of the relationship between weight and length of these parasites with fish growth.

Weight and length of fish in presence and absence of nematode.

Establishment of the relationship between the food content and presence of nematode parasite.

Histopathology of fish infected with nematode parasite

Weight and length of fish in presence and absence of nematode

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Table 1: PARASITE HOST LIST SHOWING BIODIVERSITY OF NEMATODES

Nematode genera Fish Host

Cucullanus Muller, Tachysurus maculates; Tachysurus (1777) serratus; Erethistea elongate; Lates calcarifer; Johnius spp., Strongylura strongylurus; Sparus sp.; Acanthopagrus berda; Stolephorus indicus; Pomadasys olivaceus; Therapon sp.; Lisha sp.; Arius serratus; Sciaena diacanthus;

Dichelyne Jugerskiold, Apolectus niger; Chirocentrus dorab; (1902) Eleutheronema tetradactylum; Lates calcarifer; Protonibea dicanthus; Pomadasys hasta; Pomadasys olivaceus; Puntius thalassimus;

Camallanus Raillliat & Henry, Scomberoides tala; Protonibea (1915) diacanthus, Muraenesox cinerius Scomberomorus guttatus; Rastrelliger kanagurta; Mastacembalus punctatus., Scomberomorus interruptum

Ecihnocephalus Molin, Dasyatis brucco; D. pastinaca, D. (1858) sephan; D. walga ; Urogymnus asperrimus; Cynoglossus snidenses; Lates calcarifer; Thrissocles spp.; Aetobatis narinari; Muraenesox cinereus.

Heliconema Travassos Mugil hamiltonii; Echidna catenata; (1919) Muraenesox spp.;

Bulbocephalus Rasheed, Eleutneronema tatradactylum, Jhikar (1966) spp.

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Rahbdochona Railliet, Parastromateus niger, ( 1916)

Heptochona Rasheed Scomberoides tala; Apolectus niger; (1964) Parastromateus niger

Cyrnea (Procynea) Pampus spp. Rasheed, (1965 )

Goezia Zeder, Apolectus niger; Myrmillo manazo; (1800) Rachcentron canadus.

Lappetascaris Rasheed Lutjanus sp.; Ilisha ilisha;Anadontostoma (1965) chacunda; Johnius sp.; Kurtus indicus. ,

Raphidascaroides Yamaguti, Sphyrna blochii ( 1941)

Paranisakis Baylis, Johnius sp. ( 1923 )

Contracaecum Railliet & Henry Muraenesox cinereus, Upeneus vittatus, (1918) Otolitus argenteus,Otolithus rubber.

Dujardinascaris Baylis, Johnius sp. Protonibea diacanthus, (1947) Sciaena sp. Otolithus rubber

Philometra Costa, Lateolabracis japonicus; Parapristipoma (1845) trilineatum; Epinephelus akara; Sciaena schlegeli; S. coiter; S glevui; Lates calcarifer; Johnius sp. Otolithus rubber; Hemisphanus georgii; Scomberomorus sp. Protonibea diacanthus; Eleutheronema tetradactylum; Mugil cephalus Pomodasys

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hasta; Otilithus argenteus;

Spirocamallanus Olsen , Johnius dussumieri; Protonibea (1950) diacanthus;Otolithus argenteus; Otolithus rubber; Otolithus rubber; MugilSpeigleri;Pomadasys hasta; Sillago sihama; Argyrops spinifer ; Sparus berda;Tachysurus berda; Sphaeroides lunaris.

Procamallanus Baylis, Sparus spinifer; Otolithus argenteus; ( 1923 )

Philometroides Yamaguti, Pomadasys hasta; Otolithus rubber; (1935) Otolithus argenteus.

Hepatonema Rasheed, Apolectus niger; ( 1965 )

Neospinitectus Kalyankar, Pampus spp. ( 1971 )

Neogoezia Kreis, Myrmillo manazo; Rachycentron anadus ( 1937 )

Porrocaecum Railliet & Otolithus rubber Henry, ( 1912 )

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