219

PRELIMINARY STUDY OF THE SOURCE OF AEROMONAS HYDROPHlLAINFECTION ON PANGASIUS HYPOPHTHALMUS LARVAE

Hambali Supriyadi (1), Oman Komarudin (1) and Jacques Slembrouck (2)

(1) RiFF, Jl. Raya II, Sukamandi, 41256 Subang, Indonesia.(2) IRD (ex ORSTOM), Catfish Asia Project, Instalasi Penelitian Perikanan Air Tawar, Jalan. Ragunan­

Pasar Minggu, P. O. Box 7220/jkspm, Jakarta 12540, Indonesiaand GAMET, B.P. 5095, 34033 Montpellier Cedex 1, France

AbstractOne of the main problems that has to be faced by Pangasius hypophthalmus breeders is the high mortality

of the larvae, which can be a consequence of both cannibalism and bacterial diseases. The aim of this studywas to explore the possible sources of Aeromonas hydrophila that may result in an infection of Pangasiushypophthalmus larvae. Isolation of bacteria were made from water source, Artemia culture medium, Artemianauplii used as feed, ova (unfertilised eggs), sperm, and fed as well as unfed larvae that were collected everyday from hatching, up to the age of 7 days. Characterisation of bacteria were done on the basis ofmorphological, physical and biochemical characteristics. The serological method of identification usingpolyclonal Aeromonas hydrophila antiserum was used. The results indicated an absence of bacteria fromsperm. By contrast, bacteria Alcaligenes sp. were isolated from source of water, unfertilised eggs and froml-day-old larvae. Proteus sp. were isolated from the Isi day of age in fed larvae and from the 71h day in unfedlarvae. Aeromonas hydrophila were isolated from larvae of 2 days of age, and Aeromonas punctata from 2­days-old unfed larvae, meanwhile Plesiomonas shigelloides were identified from fed larvae of 2 and 4 daysofage, and from 2-days-old unfed larvae.

INTRODUCTION

During the last few years, fish health problembecame a major concern to aquaculturist in all overthe world. In south-east Asian countries, fishproduction was badly affected by the outbreak offish disease, such as Epizootic UlcerativeSyndrome (EUS) in 1980. Important bacterial fishpathogens, including Aeromonas spp.,Pseudomonas spp. and Flexibacter columnaris, areregularly isolated from fish and become primarypathogenic agents frequently reducing theproduction of cultured freshwater fish. In late1980, a total of 125 tons of carp were lost in Java(Indonesia) due to bacterial disease infection(Djajadiredja et al., 1983). The disease was causedby bacteriwn Aeromonas hydrophila. Thisbacteriwn was not only causing mortality oncommon carp but also on catfishes and snakeheadfish. In Indonesia, the outbreak of this disease wasfirstly reported from West Java but since then ithas spread out to all over Java, Kalimantan andSumatra,

Catfishes are important commonly culturedfreshwater fish species besides common carp andtilapias. Intensive culture of walking catfish

(Clarias batrachus) in Indonesia is alreadypractised. Fish farmers are encouraged to culturecatfish because it is highly profitable. Africancatfish (Clarias gariepinus) was introduced intoIndonesia and is now being cultured extensively.Clarias gariepinus grow faster than the localspecies and is therefore preferred by farmers forculture. However, the development of catfishculture in recent years has been hampered by thefrequent occurrence of bacterial disease.

Another species of catfish that was introducedto Indonesia 26 years ago is the Thai catfish(Pangasius hypophthalmus). This species ofcatfishresemble to local pangasiid species. Because ofbreeding technique of local Pangasius has not beendeveloped, the Thai catfish has been cultivated as asubstitute to local species. One of the mainproblems that has been faced by the breeder is theimportant mortality of the larvae, which issuspected to be a consequence of both cannibalismand bacterial disease (Subagja et ai, 1998).

Fish diseases are often associated with intensivefish culture. Interaction leading to bacterial diseasein fish depends on the presence of the pathogen,the quality of environment and the general status ofthe fish. Balance of these conditions can ensure

220

fish health without the use of chemotherapeuticagent. Health is promoted by ensuring good waterquality, appropriate stocking densities andproviding balanced feed.

Bacterial haemorrhagic septicaemia due tostrain of Aeromonas hydrophiJa may betransmitted through the water, diseased and healthyfish, other affected vertebrates, and favoured byexternal as well as internal parasites. A reservoir ofpotential pathogens probably exists in all naturaland artificial water bodies (Newman, 1982).

The present paper aims at a preliminaryexploration of the possible sources of Aeromonashydrophila that may result in infection ofPangasius hypophthalmus larvae.

MATERIALS AND METHODS

Pangasius hypophthalmus used for theexperiment were bred at the Research Institute forFreshwater Fisheries (RIFF) in Sukamandi, SubangWest Java.The larvae were obtained from 3-5 years oldP. hypophthalmus brooders held in earthen pondsand supplied with water from irrigation. Fish wereartificially induced to ovulated using Ovaprim andeggs were fertilised using the procedure describedby Legendre et al. (1999). Twelve hours afterhatching, the larvae were individually counted andtransferred to the experimental facility. they werereared in stagnant spring water and maintained infibreglass container and aquarium,

Isolation ofbacteriaThe isolation of bacteria was made from

different sources:• water (ground water which is normally used

for larval rearing),• the medium ofArtemia culture,• Artemia nauplii themselves,• unfertilised eggs collected just after stripping

of females,• from sperm collected just after stripping of

males,• from fertilised eggs in incubation. collected at

2 h after fertilisation.Larvae were collected every day, from hatching

up to the age of 7 days. Two groups of larvae werefollowed, one group was totally starved during thewhole duration of experiment and the other onewas fed with Artemia nauplii. This was done inorder to test for a possible direct or indirect disease

transmission through feeding. Fed larvae receivedArtemia nauplii in excess, starting from 36 hoursafter hatching up to 8 days of age. The feedingfrequency was of 8 meals per day at 09:00, 12:00,15:00, 18:00,21 :00,24:00,03:00 and 06:00.

Isolations of bacteria from water, and Artemiamedium were carried out by serial dilution-agarplating procedure. A total of 25 ,.d of water orArtemia medium was inoculated onto meltedTryptic Soy Agar (TSA) (Dipco) and then platedonto sterile Petri dish.

Isolation of bacteria from sperm was done byinoculation of an aliquot onto the TSA plate.

The surface of unfertilised eggs, fertilised eggs,larvae at different ages either fed or unfed larvaeand Artemia nauplii, were separately sterilised bywashing with ethanol 70% and then re-washedwith sterile water. Each specimen was then crushedby using a sterile tissue grinder and then inoculatedonto TSA plate.

All isolations were made in three replicates andincubated at 28°C.

Identification ofbacteriaIdentification of Aeromonas hydrophila was

done by serological method using polyclonalAeromonas hydrophila antiserum. Antiserum usedwas prepared from Aeromonas hydrophila strainNo. 26 ofRIFF collections, produced by a standardprocedure (Burrell, 1979).

Identification of other bacteria was carried outby morphological, physical and biochemicalcharacterisation as describes by Cappucino andSherman (1987) and Cowan (1985). Gram stainwas done to determine the shape of bacteria andgram reaction. Motility of bacteria was determinedby the ''hanging drop" method.

RESULTS AND DISCUSSION

A number of colonies were observed after theisolation and 12 h of incubation. The colour of thecolonies varies from buff to yellowish. The resultsafter further identification indicated that variousbacteria were able to be identified. All isolatedbacteria belonged to the genera Alcaligenes,Proteus, Plesiomonas and Aeromonas. Isolated andidentified bacteria are listed in Table I.

Alcaligenes sp. identified from unfertilised eggswas considered as coming from the water source,in which it was also found. So, in that case, thesurface sterilising process was not properly done.

221

Source of sample Isolated bacteriaWater Alcaligenes spSpenn NoneOva Alcaligenes spFertilised eggs NoneArtemia culture medium NoneArtemia nauplii NoneUnfed larvae

Age 2 Plesiomonas shigelloides, Aeromonas punctataAge 7 Proteus sp.

Fed larvaeAge 1 Proteus sp., Alcaligenes sp.Age 2 Aeromonas hydrophila, Plesiomonas shigelloidesAge 4 Plesiomonas shigelloides

REFERENCES

CONCLUSION

The transmission of disease agent seems to be notthrough the water source, ova, sperm, Artemiaculture medium or Artemia nauplii but could bedue to contamination of equipment and operators.

important role in the transmission of diseaseagents.

Other species of bacteria predominantlyisolated from the samples was Plesiomonasshigelloides. According to Habs and Schubert(1962), this bacterium was formerly classified inthe genus Pseudomonas and then transferred to thegenus Aeromonas. This was followed by itstransfer to the newly created genus Plesiomonas.The bacterium occurs in fish and other aquaticanimal.

Austin F.E., Barbieri J.T., Corin R.E., Grigas K.E.,& Cox C.D. (1981) Distribution of superoxidedismutase, catalase, and peroxidase activitiesamong Treponema pallidum and otherspirochetes. Infect. Immun., 33, 372-379.

Burrell R. (1979) Experimental Immunology.Burgess Publishing Company. Minneapolis,Minnesota.

Cappuccino lG. & Shennan N. (1987)Microbiology: A laboratory manual. TheBenjamin/Cummings Publishing Company, Inc.Menlo Park, California.

Cowan S.T. (1985). Manual for the identification

TableI: Isolated and identified bacteriafrom water, Artemia culture medium, Artemia naupliiandPangasius hypophthalmus larvae.

Bacterium Alcaligenes sp. was also isolatedfrom the samples. The bacterium was isolated fromwater, then found in fertilised egg and finallyisolated in l-day-old larvae. According to Kerstersand De Ley (1963), this bacterium occurs in waterand soil. Recently Austin et al. (1981) described anew group (subspecies) of bacteria isolated frommoribund lobster and named them as Alcaligenesfaecalis subsp. homari

Some of isolated bacteria were gram positive,and cocci shape. Those bacteria were considered tobe non-pathogenic bacteria. As revealed byRichard and Roberts (1978), the majority of fishpathogens are Gram-negative rod but there aresome Gram-positive pathogens, including a fewwhich are acid fast.

There was no A. hydrophila isolated fromwater, sperm, ova, fertilised eggs, nor from l-day­old fed and unfed larvae. The absence ofbacteriumA. hydrophila in the water reservoir, sperm, ova,fertilised eggs and Artemia, revealed that thosesources were not at the origin of infection oflarvae. Also A. hydrophila were absent in l-day­old larvae. However, A. hydrophila werediscovered to infect the larvae aged of 2 days. Thiscould be coming from other sources such asplanktonic organisms, fish parasites or otherorganisms, as Newman (1982) stated that areservoir of potential pathogens probably exists inall natural and artificial water bodies. Besides therole of external and internal parasites in thetransmission of the disease is probably muchgreater than it is generally assumed. Dombrowski(1953) isolated A. liqutfaciens from copepods(Argulus foliaceus) and from leeches (Piscicolageometra). The state of contamination ofequipment and operators may also play an

222

of medical bacteria. Cambridge UniversityPress. Cambridge.

Djajadiredja R., Panjaitan T.H., Rukyani A.,Sarono A., Satyani D. & Supriyadi H. (1983)In: Fish quarantine and fish disease inSoutheast Asia. International DevelopmentResearch Center, Ottawa, Canada. Countryreports: Indonesia,p. 19-30.

Dombrowski H. (1953). "Die Nahrungsmenge desFischegels Piscicola geometra". L. BioI.Zentralbl.,72,311-3l4.

Habs H & Schubert R.H.W. (1962) Uber diebiochemischen Merkmale und dietaxonomische Stellung von Pseudomonasshige/loides (Bader). ZentralbI. Bakteriol.Parasitenkd. Infektionskr. Hyg. Abt. I Orig.,186,316-327.

Kersters K. & De Ley J. (1963) The oxidation ofglycols by acetic acid bacteria. Biochim.Biophys. Acta, 71, 311-331.

Krieg N.R & Holt J.G.. (eds) (1984) Bergey'sManual of Systematic Bacteriology. Vol. 1.Williarns and Wilkins,Baltimore.

Legendre M., Subagja J., & Slembrouck J. (1999)Absence of marked seasonal variations insexual maturity of Pangasius hypophtha/musbrooders held in ponds at the Sukarnandi station(Java, Indonesia). Proceedings of the mid-termmeeting of the Catfish Asia project, thisvolume.

Newman S.G. (1982) Aeromonas hydrophila : Areview with emphasison its role in fish disease.In Anderson D.P., Dorson M. and DubourgetP.H. (Eds) Antigens of fish pathogens:development and production for vaccines andserodiagnostics. Collection Foundation MarcelMerieux, pp. 87-114.

Richards RH. & Roberts R.J. (1978) Thebacteriology of Teleosts. In: RJ. Roberts (00)Fish Pathology.BailliereTindall. London.

Subagja J., Slembrouck J., Hung L.T. & LegendreM. (1999) Analysis of precocious mortality ofPangasius hypophthalmus (Siluroidei,Pangasiidae) during the larva rearing andproposition of appropiate treatments.Proceedings of the mid-term meeting of theCatfish Asia project,this volume.

223

PRELIMINARY RESULTS OF THE STUDY OF PARASITIC AND RED SPOT DISEASES ON

HIGH ECONOMICAL VALUABLE CATFISH SPECIES IN THE MEKONG DELTA

Tu Thanh Dung and Nguyen Thi Nhu Ngoc

Department ofFreshwater Aquaculture, College ofAgriculture, Can rho University, Vietnam

Abstract

The parasitic diseases and bacteria causing red spot disease in the Mekong Delta were studied in threecatfishes used in aqua.culture: Pangasius bocourti, P. hypophthalmus and Clarias hybrid (c. macrocephalusx C. gariepinus). Ichthyophthirius multifiliis, Trichodina sp., Dactylogyrus sp., Gyrodactylus sp. andOodinium sp. are the parasites most often found in skin, fins, and gills of diseased fish, particularly in frynursed in cement tanks and earthen ponds. The "white spots" disease caused by Ichthyophthirius multifiliisoccurred in both Pangasius bocourti and P. hypophthalmus. Red spot disease has also been recorded as themost common disease on catfish at the grow-out stage in cage culture. Most species ofbacteria were found tobe Gram negative and motile Aeromonads such as: Aeromonas hydrophila, A. caviae, Pseudomonasfluorescens, Edwadsiella tarda and Vibrio sp.

INTRODUCTION

As wild fish stocks tend to declinecontinuously, there is an increasing need to expandaquaculture in order to satisfy the demand for highquality protein to feed the world's growingpopulation. To obtain high productivity, thediseases of cultured species need to be understoodand controlled. In this prospect, some fast growingculture fish need to be considered and studied suchas Pangasius and Clarias genera that were culturedcommonly in Mekong Delta. Their main favourablecharacteristics for aquaculture are the ability totolerate high stocking densities, fast growth and highyield as well as their good palatability and highmarket value. As the farming intensity increases,diseases, which are normally present in wildpopulations, become much more evident in theconfined condition. Fish diseases caused bypathogenic organisms such as protozoa, fungus,bacteria and virus often occur during the cultureperiod. However, a particular attention should begiven to parasitic and bacterial diseases, as they havebeenconsidered as a serious problem on catfish.

In some cases, outbreaks of bacterial diseaseshave been related to stress factors. Circumstantialevidence suggests that Aeromonas hydrophila maybe a secondary invader of parasite induced injuries(Kumar, 1986; cited by Pai et al., 1995). In addition,many bacterial and parasitic pathogens have been

reported in the Walking catfishes Clariasbatrachus, C. macrocephalus, and the hybridC. macrocephalus x C. gariepinus (Tonguthai etal., 1993; Angka, et al., 1994). The symptoms ofthe disease are similar in appearance to those foundin other bacterial haernorrhagic septicaemia anddifferentiated into three main categories: acutewith few gross symptoms, acute form with dropsy,chronic ulcerous form. It is believed thatA. hydrophila is an opportunistic organism thatcontributes to secondary infection of the lesions;this bacterium has been also isolated fromEpizootic Ulcerative Syndrome (EUS)(Anonymous, 1986; Llobrera & Gacutan, 1987).However, Supriyadi et al. (1995) indicated thatWalking catfish (c. batrachus) can be protectedfrom A. hydrophila by vaccination.

Much of the previous effort in studying andcontrolling diseases on catfishes such as Pangasiusbocourti, P. hypophthalmus and the hybrid Clariasmacrocephalus x C. gariepinus in Vietnam havebeen dissipated through a lack of completescientific data. Therefore, the study of parasiticdiseases and bacteria causing red spot disease(bacterial haemorrhagic septicaemia) on thesecatfish of economical importance have beenconsidered as an important goal in the project. Thispaper presents the preliminary results obtained inthis field

224

MATERIALS AND METHODS

Studied sites: the survey was conducted in ChauDoe town (An Giang province), Vinh Long andCan Tho provinces. Samples of Pangasius at thegrow-out stage were collected from cage culture inChau Doe town with three different period duringboth the dry and the rainy seasons. At the sametime, fish fanners were interviewed using adaptedquestionnaire forms. Information and data relatedto disease, culture techniques and healthmanagement, were recorded on the farms and atthe fisheries processing plant (AGIFISH CO.).

Two hatcheries, located at Can Tho Universityand Chau Doe town (My Chau hatchery), wereinvolved in the propagation and nursing ofP. bocourti and P. hypophthalmus in the MekongDelta. Fry and fingerling samples with parasiticdiseases were collected from indoor cement tanksand examined everymonth.

Study of pathogens: method of study of fishparasites of Dogiel (1933) was applied in thisstudy. The following methods were used for thecollection of fish samples: (1) live diseased fishwere kept in nylon bags and stored in ice container,samples being analysed as soon as possible (within24 hours); (2) fish samples were transported to thelaboratory in nylon bags supplied with oxygen;(3) diseased fish were also processed on the field,Tryptone Soy Agar plates (TSA) was then used for

initial inoculation. Rimler-shortts Agar (RS),Decarboxylase (Arginie Lysine and Ornithine),Oxidation fermentation medium (OIF), TripleSugar Iron Agar were used for biochemical tests toidentify bacteria. The work was carried out at theFish disease laboratory of the College ofAgriculture, Can Tho University. Fish bacteriawere studied according to the methods of Plumb(1983) and Frerich (1984).

RESULT AND DISCUSSIONS

Parasitological study: the commonest ecto­parasites were recorded on the cultured catfishstudied (Table 1). Some protozoan (Trichodina sp,Oodinium sp), were observed on Clarias hybrid fryin earthen ponds and on P. bocourti fingerling innursing cement tanks. White spot disease causedby Ichthyophthirius multifiliis occurred in bothP. hypophthalmus and P. bocourti. In addition,Monogenetic trematodes consisting ofDactylogyrus and Gyrodactylus sp, attacked skinand gills causing high mortality on small fish,during the rainy season. Flashing movements, pin­point haemorrhages, excessive mucus production,obstacle to oxygen uptake, impaired feeding andlethargy have been commonest clinical signsobserved. Specific signs as ''white spots" werecaused by Ichthyophthirius multifiliis.

The mortality rate caused by parasitic diseases

Infected catfish Location Stage Parasites No. of fishes Degree oftested infestation

Gills, fins and skin Fingerlings 1chthyophthyrius 40 ++++P. bocourti Gills, fins and skin Trichodina 30 ++

Skin and gills Dactylogyrus 15 +++Gills and skin Gyrodactylus 10 +++Gills and skin Grow-out Dactylogyrus 25 ++Skin and gills stage Gyrodactylus 10 ++Tract intestine Balantidium 7 ++Tract intestine Trematoda 11 +Tract intestine Nematoda 6 +Gills, fins and skin Fingerlings Trichodina 20 +++

P. hypophthalmus Ichthyophthyrius 14 ++++Dactylogyrus 5 ++Gyrodactylus 5 ++

Skin, fins, gills Fingerlings Trichodina 50 +++Clarias hybrid and muscle Ichthyophthyrius 20 +++

Oodinium 10 ++Juveniles Metacercaria 5 +

+ = light. ++ =medium, +++ =heavy, ++++ =very heavy

Table 1: Parasites found on cultured catfish in the Mekong delta.

reached 80-90% at hatchery of Can Tho Universityand 50% at My Chau hatchery in Chau Doe town.Ichthyophthirius multifiliis was responsible formost of the mortality observed inP. hypophthalmus and P. bocourti. In general,external parasites attack skin, fins, and the gillscausing acute mortality due to their direct life cycleor rapid multiplication. Unbalanced diets alsoappeared as one of the reasons for diseases andmortality of catfish.

In addition, Balantidium and Nematodes wereobserved in the intestinal tracts ofP. hypophthalmus and P. bocourti and Clariashybrid. These parasites did not cause highmortality on catfish. Immature Digenea(metacercaria) were also found under skin ormuscle.

Bacteria isolated/rom diseasedjish: seven strainsofAeromonas hydrophyla, three strain ofA. caviaeand Pseudomonas sp. were identified. Thesebacteria caused red spot disease on P. bocourti,P. hypophthalmus and Clarias hybrid. All strainsof bacteria isolated from these catfishes are givenin Table 2.

Besides pathogens, several other factors wereresponsible for outbreaks of disease in catfishfanns. They include particularly overcrowding,poor environment quality, unbalanced diets andpoor sanitary measures. Though the developmentof aquaculture and the desire to increase fannproduction, farmers often forget the importance ofmaintaining the delicate balance between the hostand the environment In hatcheries, changes in

225

physico-chemical and microbiological quality ofwater also generate stress in fish making themmore susceptible to invasion by pathogens.Subasinghe (1995) shown that the best qualityenvironment with respect to an aquatic organismrefers to water body close to its naturalenvironment. This includes provision of goodphysical, chemical and microbial quality of water,adequate swimming space and feeding with anutritionally balanced diet

CONCLUSIONS

White spot disease (lch) is still a seriousproblem in small catfish fanning, includingfingerling and even juvenile stages. Therefore,study of chemotherapeutants application methods,as well as environment management for preventionand treatment should be carried out. Preventionand control of fish disease through environmentalmanipulation is far more economical and effectivethan other methods of control. Therefore, attentionshould bepaid for water treatment in nursing tanksystems in hatcheries, in order to reduce mortalitycaused by ectoparasites. Unbalanced diets may berelated to outbreak of some diseases, especiallylack of vitamin and specific minerals in diets. Anexperiment on supplying Vitamin C for preventionof white spot disease should be carried out in nearfuture. Hemorrhage disease (red spot disease) havecaused a reduced production of catfish at the grow­out stage. Study on cause, treatment andprecautionary measures have to be considered.

Bacteria isolated Site of Infected species Clinical signs No. ofisolation fishes

examinedA. hydrophila P. bocourti, Haemorrhages on the muscle 10A. caviae P. hypophthalmus, and internal organs or at the

Clarias hybrid base of fins; red or yellowfluid in abdominal cavity and

Kidney fin rotPseudomolUlS Liver Pibocouni, 2jluorescens Spleen Clarias hybrid

LesionVibriosp. 2Streptococcus.sp P. bocourti White nodules in the internal 3

organs and protruded anusEdwadsiella 4tarda

Table 2: Bacteria isolated from diseased catfishes cultured in the Mekong delta.

226

REFERENCES

Angka TJ., Lam Y.M. & Sin Y.M. (1994) Somevirulence characteristics of Aeromonas inWalking catfish (Clarias gariepinusi.Aquaculture, 1930, 103-112.

Pai R, Karunasagar I., Shetty H.P.C &Karunasagar I. (1995) The effect of somestress factors on injection of fish byAeromonas hydrophi/a. Journal ofAquaculture in the Tropics, 10,29-35.

Roberts RJ., MacIntosh DJ., Tonguthai K..,Boonyaratpalin S., Tayaputch N., Phillips MJ.& Millar S.D. (1986) Field and laboratoryinvestigations into Ulcerative Disease in theAsia-Pacific region. Technical report,FAOnCPlRASI4508. FAO, Bangkok,Thailand, 5-7 August 1986.

Subasinghe R.P. (1995) Disease control andhealth management in aquaculture. FAOAquaculture Newsletter, FAN.

Tonguthai K., Chinabut S., Limsuwan C., SomsiriT., Chanakhan P. & Macrae I.H. (1993) .Handbook ofHybrid Catfish: Husbandry andHealth. Kasetsart University Campus.Bangkok, Thailand.

Acheved' imprimer

IIMl31240 L'UNION(Toulouse)

Tel. 05 61 3764 70Depot legal : decembre 1999

Imprimeen France

3

CONTENTS

Page

FOREWORD 1

CONTENTS 3

CONTEXTS AND RESEARCH GOALS

Legendre M. The Catfish Asia project: backgrounds, aims and prospects.--------------------------- 7

Lazard J. Interest of basic and applied research on Pangasius spp. for aquaculture in theMekong Delta: situation and prospects.-------------------------------------------------------- 15

Sadili D. Marketing of pangasiid catfishes in Java and Sumatra, Indonesia.----------------------- 21

BIOLOGICAL DIVERSITY

CHARACTERlSA110N OF SPECIES, POPULA110NS AND STRAINS

Teugels G.G., Legendre M. & Hung L.T. Preliminary results on the morphologicalcharacterisation of natural populations and cultured strains of Clarias species(Siluriformes, Clariidae) from Vietnam.--------------------------------------------------------- 27

Teugels G.G., Gustiano R., Diego R., Legendre M. & Sudarto. Preliminary results on themorphological characterisation of natural populations and cultured strains of Clariasspecies (Siluriformes, Clariidae) from Indonesia.-------------------------------------------- 31

Pariselle A. & Komarudin O. First results on the diversity of gill parasites of some catfisheshost species in South East Asia.------------------------------------------------------------------ 37

Pouyaud L., Hadie W. & Sudarto. Genetic diversity among Clarias batrachus (Siluriformes,Clariidae) populations from the Indochina Peninsula and Indonesia Archipelago.----------- 43

Pouyaud L., Gustiano R. & Legendre M. Phylogenetic relationships among pangasiid catfishspecies (Siluriformes, Pangasiidae). -------------------------------------------------------------- 49

Volckaert F., Hellema~ B. & Pouyaud L. Preliminary data on genetic variation in the genusClarias and Pangasius on the basis of DNA microsatellite loci.---------------------------- 57

BID-ECOLOGY

Thuong N.V., Hung H.P., Dung D.T. & Kha L.A. Preliminary data on species compositionand distribution of pangasiid catfishes (Siluriformes, Pangasiidae) in the lower MekongFtiver basirL-------------------------------------------------------------------------------------- Eil

AQUACULTUREINOPTIMISATIONANDDIVERSIFICATIONPRODUCTION

REPRODUC110N

Cacot P. Description of the sexual cycle related to the environment and set up of the artificialpropagation in Pangasius bocourti (Sauvage, 1880) and Pangasius hypophthalmus(Sauvage, 1878), reared in floating cages and in ponds in the Mekong delta.--------------- 71

Legendre M., Subadgja J. & Slembrouck J. Absence of marked seasonal variations in sexualmaturity of Pangasius hypophthalmus brooders held in ponds at the Sukarnandi station(Java, Indonesia).---------------------------------------------------------------------------------- 91

Legendre M., Slembrouck J. & Subadgja J. First results on growth and artificial propagationof Pangasiusdjambal in Indonesia.--------------------------------------------------------------- 97

4

Xuan L.N. & Liem P.T. Preliminary results on the induced spawning of two catfh species,Pangasius conchophilus and Pangasius sp l, in the Mekong delta.----------------------------- 103

Kristanto A.H., Subadgja J., Slembrouck J. "& Legendre M. Effects of egg incubationtechniques on hatching rates, hatching kinetics and survival of larvae in the Asian catfishPangasius hypophthalmus (Siluriformes, Pangasiidae).---------------------------------------- 107

Campet M., Cacot P., Lazard J., Dan T.Q., Muon D.T. & Liem P.T. Egg quality of an Asiancatfish of the Mekong River (Pangasius hypophthalmus) during the process of maturationinduced by hCG injections.-------------------------------------------------------------------------- 113

Legendre M., Slembrouck J., Subadgja J. & Kristanto A.H. Effects of varying latencyperiod on the in vivo survival of ova after Ovaprim- and hCG-induced ovulation in theAsian catfish Pangasius hypophthalmus (Siluriformes, Pangasiidae).------------------------- 119

LARVAL BIOWGY AND REARING

Hung L.T., Tuan N.A., Hien N. V. & Cacot P. Larval rearing of the Mekong catfish,Pangasius bocourti (Siluriformes, Pangasiidae): Artemia alternative feeding and weaningtime.--------------------------------------------------------------------------------------------------- 127

Slembrouck J., Hung L.T., Subadgja J. & Legendre M. Effects of prey quality, feedinglevel, prey accessibility and aeration on growth and survival of Pangasius hypophthalmuslarvae (Siluriformes, Pangasiidae).--------------------------------------------------------------- 137

Subadgja J., Slembrouck J., Hung L.T. & Legendre M. Analysis of precocious mortality ofPangasius hypophthalmus larvae (Siluriformes, Pangasiidae) during the larval rearing andproposition of appropriate treatments.-------------------------------------------------------------- 147

NUTRI110N, FEEDING AND GROWTH

Hung L.T., Tuan N. A., Phu N.V. & Lazard J. Effects of frequency and period of feeding ongrowth and feed utilisation on Pangasius bocourti in two Mekong catfishes, Pangasiusbocourti (Sauvage, 1880) and Pangasius hypophthalmus (Sauvage, 1878).------------------ 157

Hung L.T., Lazard J., Tu H.T. & Moreau Y. Protein and energy utilisation in two Mekongcatfishes, Pangasius bocourti and Pangasius hypophthalmus.--------------------------------- 167

Phuong N.T. & Hien T.T.T. Effects of feeding level on the growth and feed conversionefficiency of Pangasius bocourti fingerlings.--------------------------------------------------- 175

Phuong N.T., Thi M.V. & Hang B.T.B. The use of plant protein (soybean meal) as areplacement of animal protein (fish meal and blood meal) in practical diets for fingerlingsof Pangasius bocourti.---------------------------------------------------------------------------- 179

Liem P.T. & Tu H.T. Rearing of Pangasius bocourti fry (Siluriformes, Pangasiidae) feddifferent diets in concrete tanks.------------------------------------------------------------------- 187

HYBRIDS EVALUA110N

Kiem N.V. & Liem P.T. Some biological characteristics of Clarias batrachus and Preliminaryresults of the hybridisationbetween Clarias batrachus x Clarias gariepinus.-------------- 191

Lenormand S., Slembrouck J., Pouyaud L., Subadgja J. & Legendre M. Evaluation ofhybridisation in five Clarias species (Siluriformes, Clariidae) of African (c. gariepinus)andAsian origin (c. batrachus, C. meladerma; C. nieuhofii andC. teijsmanni).------------- 195

Minh L.T. Preliminaryresults on the relationship between growing stage and body compositionin Clarias macrocephalus, Clarias gariepinus and their hybrid (c. macrocephalus femalex C. gariepinus male).--------------------------------------------------------------------------- 211

PATHOWGY

Komarudin O. Preliminary observations on the infection of the gills of cultivated Pangasiushypophthalmus by Monogenea.---------------------------------------------------------------------- 217

Supriyadi H., Komarudin O. & Slembrouck J. Preliminary study of the source of Aeromonashydrophila infection on Pangasius hypophthalmus larvae.----------------------------------- 219

Dung T.T. & Ngoc N.T.N. Preliminary results of the study of parasitic and red spot diseases onhigh economical valuable catfish species in the Mekong Delta.------------------------------- 223

5