,

Indian Journal of Marine Sciences Vol. 28, September 1999, pp. 285-296

Bacterial and protozoan (Ciliate) diseases of praw~ Penaeus indicus (Decapoda: Crustacea)

R Jayakumar & P Ramasam/

Department of Zoology, Life Sciences Building, University of Madras, Guindy Campus, Madras 600 025 . India

Received 25 July /997, revised 24 February /999

A survey was carried out on the prevalence of bacteria and ciliate protozoan of Pellaells indiclls obtained from Ennore estuary, Chennai (Madras), India from November 1989 to July 1992. The study r~vealed the occurrence of Vibrio parahaemolyticus, V. VUlllijiCUS, V. harvey;, V. allguillarum, V. damsela, Pseudomollas sp., P. aemgillo.\'{/ and a tilamentous bacterium Leucothrix sp. This study also revealed the occurrence of three different peritrichous ciliates viz .. Zoothall/llillll/ sp., Epistylis sp. and Vorticella sp. and a loricate ciliate Lagenophrys sp. ZoothamlliulI1 sp ., Epistylis sp. and Vorticella sp. were recorded on the body surface. gills. appendages and pleopods. Trophonts of ZoothallllliulIl sp. occurred as hranched colonies with contractile stalk with myoneme. Epistylis sp. also consisted of branched colonies but was lacking myoneme. In contrast the Vorticella sp. occurred as individual trophonts with a contractile stalk. Scanning electron microscopy of Epistylis sp. revealed the surface topography, convoluteC: ridges over the trophont surface and stalk. Both Pselldo/llOl1{lS spp. and Vibrio spp. caused darkening of cuticle, loss of appendage setae and their hairs in the pleopods and uropods. blister in the gills and accumulation of hemocytes at the infected sites. Minimum inhibitory concentration of acritlavin . ampicillin, furazolidone, kanamycin and prefuran required to control the growth of Vibrio spp. and Pseudomollas spp. were found to he higher than the recommended limits. Intramuscular inoculation of bacterial cells of Vibrio spp. and PseudoIlUlna.\· spp. into P. indicus caused mortalities from 30% to 80% but drug treatment with neomycin sulphate (20 ~g mr l of culture tank)/ streptomycin (10 ~g mrl of culture tank) reduced the mortality (0% - 30%). Drug therapy of P. indiclls infected with peritrichous ciliates revealed that formalin and a tlavonoid tlavone effectively killed the ciliates whereas methylene hili(: was ineffective. The present study revealed that the populations of the prawn P. illdicus suffer from bacterial and epibiont protozoan diseases retlecting the quality of the environment which is polluted with industri al eftluent s and sewage discharge.

Microbial diseases have been reported to be a major limiting factor in production both in wild and cultured shrimpsl.5. As the stocked densities of shrimp in intensive culture systems increase, the quality of the water decreases while incidence of disease also increases4

• Overstreet) estimated that the loss of cultured shrimp due to parasites and diseases varies from, 20-50% loss of larval stages and 20% of postlarvae. The most common pathogenic bacteria identified from penaeid shrimp include Vibrio alginolyticus, V.parahaemolyticus, V.anguillarum and

h . d . f h V'b . 26-11 ot er species an strains 0 t e genus I riO ' ,

Pseudomonas, Flavobacterium and Moraxella2.6

•

However, the importance of bacteria as disease producers in natural populations of crustaceans is unknown I. Most of the bacteria known from the shrimps are common epibionts, which seem to cause little or no harm in natural, unpolluted, unstressed environments l. However, captive-wild or cultured

*Address for correspondence

shrimps living in degraded (polluted) environments are reported to be infected with fouling and chitinivorous bacterial . Most species of bacteria associated with diseases of shrimps are known to cause mass mortalities in both semi-intensive and intensive cultures. Though India practices both the harvest of captive-wild as well as semi-intensive culture of shrimps, bacterial disease problems and the estimated loss of wild stocks are upknown, except for a few reports on the occurrence of bacteria in the processed­. d h . J?·14 Ice -storage s nmps - .

A number of ciliate protozoans occur as symbionts, commensals, parasites and pathogens of Crustacea. Exoskeleton of crustaceans serve as a substratum on which both sessile and mobile forms of peritrichous ciliates live and reproduce. Most peritrichous ciliates viz. , Zoothamnium, Epistylis, Vorticella, LaRcnophrys, Acineta are known to feed on bacteria and organic matter but not on the host tissues. They require proper substrata (cuticular surface for attachment of the trophonts) and water temperature (23°C-35°C) for

286 INDIAN 1. MAR. SCI., VOL. 28. SEPTEMBER 1999

optimal reproduction. Unfortunately the wild and captive cultivation of crustacea provides those exact conditions for their growth and multiplication of these peritrichs. Species of Zoothamniu111, Epistyjis, Vorticella cause problems in penaeid prawn culture I5

.16.

The intensity of these peritrichs has been correlated positively with the density of prawn in the pond, water quality and availability of bacteria and organic matter. Stalked ciliates especially Zoothamnium sp. can create problem in high density prawn culture, where extensive colonization on the gills may interfere with gas exchange and result in mortalitiesU

i.l7.

Many peritrichous ciliates are epibionts (ectocommensals, ectoparasites). Lagenophrys sp. which is a loricate ciliate reported to occur in the crab Callinectes sapidus from Australia l8. Lagenophrys sp. has been reported on pleopod, uropod surfaces of

1920 H h ' . f' f . prawn '. owever, t ere IS paucIty 0 In ormatIon on the bacterial and ciliate protozoans affecting the penaeid prawns of India, particularly the specialized polluted environment such as Ennore estuary, Chennai (Madras). The present study reports the prevalence, characteristics, signs of bacterial diseases and antibiotic sensitivity of bacteria isolated from the most common captive - wild penaeid prawn, Penaf'us indicus obtained from Ennore estuary, Chennai; and the occurrence and morphology of ciliate protozoans of Penaeus indicll.\' and the probable treatment to manage the disease.

Materials and Methods Samples of prawn Penaeus illdicas H. Milne

Edwards of 5-30 mm carapace length were collected from the Ennore estuary, Chennai (Madras) between November 1989-July 1992. They were transported to the laboratory in sterile seawater with aeration and maintained in aerated tanks containing filtered seawater, examined within 24 h of collection, and sex and carapace length (tip of the rostrum to end of carapace) were determined. The live and moribund specimens used for bacteriological studies were surface sterilized with 30 ppm iodophore to eliminate the normal microbial f1ora2

. Prawns of both sexes were sacrificed and wet mount preparation of appendages, gills, hepatopancreas, gut, muscle, gonad, hemolymph and exoskeleton were made and examined for signs of bacterial and protozoan infection. Monthly prevalence (% of shrimp harbouring pathogen/parasite) of pathogenic bacteria and ciliate protozoans in relation to sex and size of the host were determined.

For scanning electron microscopic studies the protozoan infected gills were fixed for 24 h at 4°C in glutaraldehyde buffered to pH 7.2 with O. I M sodium cacodylate - HCI contained 3% sucrose and 0.5%

sodium chloride. They were washed and stored at 4°C in 4% sodium cacodylate buffer until required further. The tissues were post fixed for I h with I % aqueous osmium tetroxide, dehydrated through acetone. critical­point-dried, sputter coated with gold/ palladium and examined under scanning electron microscope.

Bacteria were isolated on plates with Zobell's marine agar21 using dilutions of homogeni zed tissues (muscle, gills, hemolymph and hepatopancreas) or by streaking plates using exoskeleton with blisters or lesions. Bacteria that were repeatedly encountered in the)issues of P.indicus were further characterized and identified by standard procedures22

.2.J. Isolated bacteria were

cultured in Zobell's marine agar at 28°C and at

temperatures ranging from 10°C to SO°e. Growth characteristics of bacteria in 0%-8% of sodium chloride in modified nutrient agar, MacConkey agar, citrate agar, Thiosulphate Citrate Bile Salts Sucrose (TCBS) agar were determined14

.25 and the authenticity

of identification were confirmed with Bergey's manual of determinative bacteriolog/lo

•

Minimum inhibitory concentrations of antibiotics required to control the growth of the bacteria (MIC) were determined by tube di lution method27 under

in vitro conditions using 128 ).1g to 10 mg mrl chloramphenicol (Biochem Pharmaceutical~: , India) , 5 ).1g to 50 ).1g ml- I of tetracycline (Sigma), penicillin

3.2IU to 320 IV mrl (Sarabhai Chemicals. India), 4 Ilg to 50 ).1g mrl of furazolidone (Argent chemicals) by following the standard procedures()·27.2X .

For bacterial infection experiments, prawns weighing about 5 g were acclimatized at 25°C-28°C in plastic troughs containing 20 liter of filtered seawater. They were divided into II experimental groups, each containing ten praw~s. Various species and strains of the bacteria Pseudomonas spp .. Vibrio spp. were grown in TCBS agar/Zobell's marine agar for 24 h. Colonies were dispersed in sterile 0.85% NaCI solution/Zobell 's marine broth and the density of the suspension was

adjusted with sterile saline, 100 ).11 of bacterial sllspensions containing known colony forming units (cfu) (see Table 2) of bacteria were inoculated

. intramuscularly into the lateral side of second abdominal segment by adopting the method of Song et aP8. Control prawns were inoculated with a sterile

lA Y AKUMAR & RAMASAMY: DISEASES OF PENAEUS INDICUS 287

Zobell's manne broth/sterile 0.85% NaCI solution. Prawns were provided with 2% biomass of body weight of pelletized feed and 100% daily water exchange. Mortality and signs of bacterial infection of prawns were recorded up to 6 days. Bacteria were isolated from the lesions/hepatopancreas of moribund/ dead prawns and identified by standard procedures24.27 .

For drug therapy experiments, P. indicus was intramuscularly infected with isolates of Pseudomonas spp. / Vibrio spp. and neomycin sulphate (20 j.1g ntr l) or streptomycin (10 j.1g mrl) was added into culture tank water at 6, 24 and 48 h after post-infection. The mortality and. signs of disease development in the prawns were recorded by following the method of Takahashi et al. lO

•29

Protozoan infected pleopods, uropods, gills and pereiopods were placed in petri plates with sterile seawater contammg known concentration of therapeutic chemicals viz., KMn04, CUS04, formalin, malachite green, acriflavin, methylene blue and a flavonoid (Flavone) isolated from neem oil cake. The behavioural, morphological changes, mortality, cessation of ciliary activity and contraction of protozoans treated with therapeutic chemicals were determined visually under a stereo bin~ular dissection microscope.

Results

Site of occurrence and pathological signs of bacterial diseases

Rod shaped, motile, Gram negative, non-sporulating, bacteria, Pseudomonas sp., P. aeruginosa, Vibrio spp. and the filamentous, nonmotile, Gram negative bacterium Leucothrix sp. were isolated, identified from live and moribund prawn P. indicus. Pseudomonas spp. were isolated from the exoskeletal structures including carapace, uropods, pleopods, appendage setae and also in muscle, gonads and gills. Vibrio spp. were also present in the hepatopancreas, hemolymph and blisters appeared in the exoskeleton and gills. Leucothrix sp. was observed only on the periopods, pleopods and gills.

Brown to black spots occurred in the appendage setae, pleopods, uropods, exoskeletal structures (Figs 1-3) and in the gill filaments (Fig. 4). The brown black spots were associated with Pseudomonas spp. and Vibrio spp. Darkened. cratered cuticular lesions were also observed (Fig.5). In some of the black spot diseased areas of pleopods and uropods appendage setae were absent (Fig.5). Blisters were present in the

gill filaments (Fig. 6). Accumulation of hemocytes occurred around the bacteria infected tissues (Figs 3. 5, 7). Vibrio spp. infected hepatopancreas showed loss of cellular matrix, dark brown deposits and cells showed more vacuoles (Fig. 8). Leucothrix sp. was found to be attached on the swimming legs. pleopods and gills (Figs 9, 10), but no lesions were observed .

Site of occurrence and pathological signs (~t' ciliate protozoan diseases

The peritrichous ciliates viz., Z()othal1l1liul11 sp. , Vorticella sp., Epistylis sp. and a loricate ciliate Lagenophrys sp. were recorded in P. indiclls during the present investigation. All the peritrichous ciliates were commonly noticed on the carapace, pleopods, uropods, body surface, gills, and mouth parts (Fig. 11-15). In contrast, the loricate ciliate Lagenophrys sp. was recorded only on the uropods and mouth parts (Fig. 17, 18). All the ciliates recorded in this study are 'non­invasive and limited to cuticular .sUlfaces.

The peritrichous ciliates viz., Epistylis sp., Vorticella sp. and Zoothamniwn sp. infected tissues exhibited a ' fuzzy' appearance. Infected prawns frequently jumped out of the tank and subsequently died. The body surface of such specimens were covered with trophonts of the peritrichou's ciliates . Altered swimming behaviour like, inverted swimming and circling movement was commonly noticed in prawns exhibiting heavy infestation of peritrichous ciliates on the gill filaments . Heavy infestation of protozoans on the pleopods and periopods also caused altered swimming behaviour.

Morphology of peritrichous and loricate ciliate protozoans

Trophonts of Zoothamniwn sp. occurred singly or in groups as branched trophonts consisting of a contractile stalk (Fig. II). Each trophont measured 85-90 j.1m long and 75-78 j.1m wide and with a stalk measuring 250-300 11m long and 18-12 j.1m wide. Epistylis sp. also consisted of branched colonies or single trophonts but lacked myonemes in the stalk (Fig. 14). Each trophont measured 32-42 Ilm in length and width near proximal end was 12-14 Ilm and at the middle of the body was 23-30 11m. The stalk of the trophont measured 6~ I I j.1m wide and 13-15 11m long. Trophonts of Vorticella sp. occured as a solitary trophonts containing a contractile stalk (Fig. 15). Each trophont measured 85-90 j.1m long and 75-78 j.1m wide and consisted of a stalk measuring 250-300 Ilm long

288

4

Figs 1-6-1) 2)

3)

4) 5)

6)

-100pm

INDIAN 1. MAR. SCI., VOL. 28, SEPTEMBER 1999

5

........ <'-b .::.:;;.,.,

.., ' .~

..

-\' .,.-

J J

,fr

". ' ,~ .

Wet mount of appendage setae of the pleopod showing black spot disease (un labelled arrows) due tll Vihrio spp . infection . Wet mount of swimming pleopod exhibiting localized darkened cuticle involving soft tisslies due to P .I ClIl/0/l10IlClS sp. infection (unlabelled arrows). Note the accumulation of hemocytcs (11) around the infected area. appendage setae (I\S ). Wet mount of uropod showing darkened cuticular - soft tissue due to P. acrugillosa infection (unlabelled arrow) . Appendage setae (AS), Hemocytes (H), Cuticle (C).

Wet mount of gi ll tilaments showing the black spot disease (BS) of Vibrio spp .. Wet mount of uropod showing darkened cratered area on the cuticle (unlabelled arrow) due to infection of P. ucm gill()sa : Nore the accumulation of hemocytes (H) around the bacteria infected area and the loss of appendage setae (AS ). Wet mount of gill tilament showing the bacterial black spot disease of Vibrio spp. Note the blister like symptom of the black spot disease (BS).

JA Y AKUMAR & RAMASAMY: DISEASES OF PENAEUS iNDlCUS 289

7

Figs 7-10- 7) Wet mount of uropod showing P.aemgillosa infected area with darkened cuitcle and broken appendage setae (AS) and the lesion seem to deepen and becoming larger. Cuticle (C),

Hemocytes (1-1).

8) Vibrio spp. infected portion of hepatopancreas showing more fatty droplets (F), loss of cellular nature (LCN) and black/brown deposits (8).

9) Wet mOllnt of the gill filaments showing the infection of the filamentous bacterium Lellcotlrrix sp. (I). Note the occurrence of the peritrichous ciliate Zootlral11l1iulIl sp. (Z) along with . the Leuco/hrix sp.

10) Wet mount of the appendage setae (AS) of the pleopod showing the infection of fil amentous bacterium Leuco/hrix sp. (I), appendage hairs (AH).

and 8-12)..lm wide. The loricate ciliate Lagenophrys sp. consisting of membranous lorica which measured

45-50 )..lm long and 40-55 )..lm wide. Width of the lip of

the lorica measured 18-20)..lm (Fig. 18).

Scanning electron microscopy of peritrichous ciliate Epistylis sp.

Scanning electron microscopic studies revealed arrays of smooth gill lamellae (Figs 19, 20). The trophonts of peritrichous ciliate Epistylis sp. were connected to one another by a dichotomously branched

,'~ ~n

..... I ,

Figs 11-14-11) Light microscopic morphology (WCI mount) of carapace of P. i/l{/icl/.\· showing a hrancheu colony of peritrici10lls ciliate Zooihllll/I/iw/l sp. stalk (S) of the trophonts containing myoneme (M). trophont (T)

12) Light microscopic morphology t wet mllulll ) of gill lamellae (GL) of f .il/diclt.\' showing infestation of numerous colonies of peritrichous ciliate Zoo/hl/mlliulIl sp. Trophollls (1').

13) Light microscopic morphology (wet mount) of carapace showing branched colonies of t -'pi.l'iylis sp. Note the stalk (S) of the tropholll lacking myoneine. Trophont (T).

14) Light microscopic morphology (weI mOllnt) of carapace of P.il/diclt.\' infested with a single colony of perit rihous ci liate I~i)is/rli.\' sp. tTl. with stalk (5).

stalk attached to the prawn gill lamellae (Figs 21 , 22). The anterior (oral) end of the body broadened to form a disc shaped peristome. Ridges and furrows (Figs 23, 24) occurred on the body surface of trophonts. These ridges were convoluted around the mouth of the contracted trophonts (Fig. 24). The stalk of the trophont was found to be convoluted at the base. It extended to form a disc for attachment (Fig:. 23) .

Prevalence of bacteria and ciliate IJrofozo((ns l/'l

P.indicus Among the bacteria, Vibrio spp. and Pseudomonas

spp. were recorded in male and female prawns of all size classes sampled during all months sampled. The prevalence of P. aeruginosa was inverse to that of Pseudomonas sp. (Fig. 25), while Leucothrix sp. were

290 INDIAN 1. MAR. SCI., VOL. 28, SEPTEMBER 1999

Figs 15-18-15) Light microscopic morphology (wet mount) of gill lamellae (GL) of P.il/diclts showing infestation of individual trophonts of Vortic-ella sp. stalk (s), ciliary appel1ure (CA).

(6) Light microscopic morphology (wet mount) of gill lamellae of P.il/diclls treated with formalin showing detached trophonts of peritrichous ciliate Zoot//(/lIllliulIl sp. Note the various morphological appearance of trophonts (T).

(7) Light microscopic morphology (wet mount) of P.illtiicus mouth part showing the infection of loricate ciliate Lagel/op/lI)'s sp. (Unlabelled arrows).

18) Light microscopic moirphology (wet mount) of a single lolicate ciliate Lagel/ophrys sp. on the uropod of P. illtiiclIs. Note the ciliary aperture (A) lolica (L) and ciliate inside the lolica (C).

noticed only during September 1990 to November ] 990, Vibrio spp. were highest (100%) in all the size classes of P.indicus. The ciliate protozoans viz., Zoothamnium sp., Vorticella sp., Epistylis sp. and Lagenophrys sp. were recorded (100%) in all the size classes of both sexes of P. indicus during all the months of the survey period.

Biochemical characteristics of the bacterial isolates from P. indicus

Optimal growth of all the bacterial species was noticed in Zobell's marine agar, citrate agar and MacConkey agar between 28°C to 36°C. In nutrient agar, sodium chloride concentrations that favoured growth were 0%-3% for Pseudomonas sp., 0%-6% for P. aeruginosa, V. vulnificus, V. harveyi, V. anguillarum and V. damsela and 0%-7.5% for V. parahaemolyticus.

Oxidase, catalase and chitinase were detected from Pseudomonas spp. and Vibrio spp. All the Vibrio spp. were positive for lysine decarboxylase, ornithine decarboxylase and urease. Arginine decarboxylase was detected in V.parahaemolyticus but absent in other Vibrio spp.

Antibiotic sensitivity of the bacteria from P. indicu.\· under in vitro conditions

The in vitro sensitivity of Pseudomonas spp. and Vibrio spp. to various antibiotics was determined (Table I). The minimum inhibitory concentration of antibiotics required to inhibit the bacterial growth was found to be significantly higher than what was

d d b I· k' IO-P d' d 'fc d recommen e year ter wor ers'" . - an tt I ,ere with different bacterial species and strains. Neomycin sulphate and streptomycin were found to be most efficient antibiotics in inhibiting the growth of Vibrio spp. and Pseudomonas spp. Prefuran was found to be very effective against most of the Vihrio spp. and Pseudomonas spp. though V. parahaemolyticus (strain-2) and V. vulnificus were found to be resistant to it. Vibrio spp. and Pseudomonas spp. were found to be tolerant to ampicillin, kanamycin and acritlavin .

.. Experimental infection of P. indicll.\' with the isolates (?f Pseudomonas sppJ Vibrio spp.

Experimental infection of P. indiell.\· with Pseudomollas spp./ Vibrio spp. produced varying degrees of mortality (Table 2). Highest mortality (80%) occurred in prawns infected with V. para/wel11olyticu.l' (strain-2) and lowest (30%) with V. anguilla rum infected prawns. Pseudomonas aerugiflosa caused 70% mortality of prawns whereas the unidentified Pseudomonas sp. caused only 50% mortality . No mortality was recorded in control prawns which were injected with Zobell's marine broth/ sterile saline. Postinfected prawns exhibited reduced feeding, low rate of swimming and black! brown spots at the sites of infection and in the hepatopancreas .

Drug therapy of P. indicus The results of drug therapy experiments of P.

indicus infected with isolates of Vihrio spp . and Pseudomonas spp. are given in Table 2. Application of neomycin sulphate (20 j.lg ml- I

) into experimental tank water containing Pseudomonas spp. and Vihrio spp. post-infected P. illdicus significantly reduced the percentage of mortality (Table 2). Neomycin sulphate was found to be very effective against V. vuln!ficus,

)

JA Y AKUMAR & RAMASAMY: DISEASES OF PENAEUS lNDICUS

Figs 19-24--19) A low power scanning electron micrograph of the gill of P. indicus showi ng arrays of lamellae (Scale bar = O.5Il1m). 20) Scanning clectron micrograph of uninfected portion of gi ll lamellae of P. indiCt/s (Magnilied view) (Scale hal' = 50llm).

29 1

0( 21) Scanning electron micrograph of peritrichous ciliates of Epistylis sp. (arrows) infected gi ll tilaments of P. iI/dicit.'

II.

(Scale bar = 50llm). 22) Enlarged view of scanning electron micrograph oftrophonts of EjJistylis sp. (Unlabelled arrows) infected gill tilamcnts or

P. indicu.I' (Scale bar = 25~tm) 23) Scanning electron micrograph of individual trophont of tile ciliate EjJistylis sp. showing the convoluted ridges on the

contracted trophont surface and stalk. Note the attachment of the stalk (S) of trophont on the gill surface hy the basal disc (BD). During contracted state, the oral ciliary disc (00) has constricted fuBy (Scale bar = 2.5pm).

24) Scanning electron micrograph of trophont of Epistylis sp. magnitied top view showing the constricted oral ciii<1ry elise (00)

and ciliary apl!rture (CA) (Scale bar = 2.5Ilm).

V. anguilla rum and V. damsela as judged from 0% mortality of post infected prawns. Application of streptomycin JO Ilg mrl at 6 h, 24 hand 48 h after post-infection also reduced the mOl1ality (Table 2).

Effect of drug therapy 011 the ciliate protozoans In vitro drug therapy experiments revealed that the

flavonoid flavone isolated from neem oil cake

effectively killed the peritrichous ci I iates VIZ. ,

Zoothamniul71 sp. and Vorticella sp. at 50 ppm concentration within 10 minutes of application. Malachite green at 100 ppm and formalin at 50 ppm effectively killed the ciliates within j 5 minutes of application. One ppm of KMnO.j and 100 ppm of acriflavin killed the ciliates in 30 minutes duration (Table 3). Copper sulphate killed the ciliates at 50 ppm

292 INDIAN J. MAR. SCI., VOL. 28, SEPTEMBER 1999'

~-Vibrio spp., a-Pseudomonas sp·, o-f·aeruginoso, ... leucothrix sp.

100

....... ~ 0 80 ...... Q.I u C Q.I 60 0 > Q.I ~

40 £l.

20

0 SONDJ FMAMJ J

1991 1992

Period

Fig 25-Seasonal prevalence of Vibrio spp. (~), Pseudomonas sp (0), P.aeruginosa (0) and Leucothrix sp. ('~) in the host P.illdiclIs.

concentration in 45 minutes. Methylene blue was ineffective in controlling the infection. When the peritrichous ciliates were exposed to these therapeutic chemicals, the trophonts and their oral ciliary disc were found to contract and relax vigorously. Formalin treatment lead to detachment of the ciliates from the infected tissues. Moreover they were shown to exhibit morphological changes to form cysts (Fig.16).

Discussion The present investigation revealed the occurrence of

three genera of bacteria viz., Vibrio, Pseudomonas and Leucothrix from live prawns of P. indicus. Vibrio sp. was found to occur both on the exoskeletal structures such as carapace, uropods, pleopods, gills and in the internal organs such as muscle, gonads, haemolymph and hepatopancreas. Similarly, Pseudomonas spp. were also found to occur in all the above tissues except hepatopancreas and haemolymph. The occurrence of Vibrio spp. and Pseudomonas spp. in P. indicus agrees with observations of Surendran et £11. 13

, Karunasagar et al. 32, Sahul Hameed3

\ Sahul Hameed & Ra034, but

they also reported the occurrence of several other genera of bacteria (Acenetobacter, Arthrobacter, Moraxella, Flavobacterium, Cytophaga and Micrococcus) from P. indicus, P. monodon, Metapenaeus dobsoni and M. affinis from Cochin, India. Vibrio spp. have been recorded in all life stages of P. monodoll, P. setiferus, P. orienta lis, and

P. merguiensis2J5.36. Vibrio spp., Pseudomonas sp., Aeromonas sp., Flavobacterium, Pasturella sp. and Moraxella occurred in the haemolymph or tissues of cultured P. californiensis, P. vwuwmei and P. stylirostris in the U.S and Mexic02

. Vibrio spp. and Aeromonas sp. have been reported from P. 11101l0doll in the Philippines37

. The similarity in the composition of bacterial flora from the penaeid shrimp from different geographic regions may be due to the similarity of the host environmental factors that would have promoted the infection. However, the species and strains of the bacteria appear to be specific to the host species and geographic regions.

The present study also reports the occurrence of a nonsporulating, Gram negative filamentous bacteria Leucothrix sp. from pleopods, periopods and gills of P. indicus. However, earlier investigators have noticed it to occur on the gills and accessory gi II structures of juvenile or older P. ·cal!forniensis. P. stylirostris, and P. vannamet Leucothrix spp. are also known to occur in crustaceans, fishes and aquatic plants l7

. Leucothrix sp. infections on the pleopods and periopods may cause stress, while in the gills it may interfere with gas exchange. Heavy Leucothrix spp. infections on the gills of penaeid shrimp may cause hypoxia leading to death of the shrimp2,8,

Almost all populations of P. illdicLIS of the Ennore estuary suffer from black spot disease caused by Vibrio spp. and Pseudomonas spp. These bacteria are shown

.,." ~ ~ -/ ,

Table I-Minimum inhibitory concentration of antibiotics to control the growth (under ill vitro) of bacterial isolates from Pellaeus indicus

Antibiotics Pseudomonas sp. P.aerugillosa V.parahaemol)·ticus V.harveyi V. anguillarum V. vlllllijiCliS V.damsela

Str-I Str-2 Str-I Str-2 Str-I Str-2

Oxytetracycline (llg/mI) 16 3.2 SO SO 25 25 40 30 40 IS

Chloramphenicol (llg/mI) SO 75 20 25 40 45 10 70 30 25

Streptomycin (llg/mI) 10 10 10 10 5 5 3 S 10 S

Acriflavin (llg/mI) 150 150 ISO' ISO' ISO' ISO' ISO' ISO' ISO' ISO' ..... :>

Kanamycin (Ilg/ml) 200 200 200' 200' 200' 200' 200' 200' 200' 200' -< :>

Ampicillin (llg/mI) 250 2S0 250' 2S0' 250' 2S0' 250' 2S0' 2S0' 250' A c::

Tetracycline (Ilg/ml) 40 40 30 30 20 25 IS 30 20 40 s.: :>

Gentamycin (llg/mI) 24 4 20 25 S 20 14 20 40 S ;:0

R<> Neomycin Sulphate (llg/mI) S 10 2S I I I ;:0

:> Prefuran lllg/mI) S S ISO 200 2 2 200 2 s.: Furazolidone (llg/mI) 75 7S 7S' 7S' 7S' 4 IS 75 7S 2S :>

en :>

Penicillin (IU/mI) 32 40 4S 40 60 25 40 30 5S 40 s.: >!< Resistant even at high concentration of antibiotics. ~

'0 en Table 2-Experimental infection of isolates of Pseudomonas spp. and Vibrio spp. with subadult Pellaeus illdiclIs and the effect of antibiotic treatment on the mortality tTl

:> en

Bacteria Intramuscular No. of prawns Treatment with neomycin sulphate Treatment with streptomycin tTl en

inoculum (cells/prawn) challenged Experimental infection (20 Ilg mI· l) (lOllg m-I) 0

'"I1

Total no. % of Total no. % of Total no. %of '" ~ host died mortality host died mortality host died mortality ~

tlJ P. aerugillosa 2.3x104 10 7 70 10 2 20 ~ Pseudomollas sp. 2.Sx104 10 S SO 10 2 20 ~ V. parahaemolyticus (str-I) S.SxI04 10 7 70 2 20 10 -()

~ V. parahaemolyticlis (str-2) 4.6x 105 10 S SO 3 30 2 20 V:l

V. harveyi (sir-I) 2.6x 104 10 5 SO 10 1 10

V. harveyi (str-2) 4.6x 104 10 6 60 10 0 .

0

V. CllIguillu/"/(1/l (s tr- l ) 3.2x 105 10 3 30 0 0 0 0

V. wl/(uilllllW I1 (str-2) S.3x l04 10 4 40 0 0 I 10

V. dWI1Sl'la 2.3x lO' 10 4 40 0 0 0 0

V. vull1ijicl/s 2.7x105 10 5 50 0 0 3 30

Contro l Sterile saline 10 0 0 0 0 0 0 N \0 w

294 INDIAN 1. MAR. SCI., VOL. 28, SEPTEMBER 1999

Table 3-Effect of chemicals on the peritrichous ciliate Zoothamnium sp.

Time taken lor the death Chemicals used Concentration of Zoothamnium KMn04 I ppm 30 min.

CUS04 50 ppm 45 min.

Formalin 50 ppm IS min.

Malachite green 100 ppm 15 min.

Acriflavine 100 ppm 30 min.

Methylene blue 100 ppm 6h

Flavone 50 ppm 10 min.

to possess chitinase, oxidase and catalase enzymes. The enzyme phenoloxidase of the host might have caused the darkening of the bacteria infected site of the cuticle and hepatopancreas while the chitinase and proteolytic enzymes of the bacteria are thought to cause erosion and destruction of the cuticle leading to its breakdown and thus possibly permitting loss of hemolymph and invasion of external pathogensl.ll . Such black spot diseases are fairly commonly reported from many penaeid shrimp and other decapod crustacea2

,8,11 . Similar bacterial flora occurred in the internal tissues and may have derived from infection through the damaged cuticle.

The mInImum inhibitory concentrations of antibiotics required to control the bacterial growth were higher than that recommended by other workers2

,29.32. In addition, neomycin sulphate lind streptomycin were shown to be the most- effective antibiotics preventing mortality of experimentally infected prawns and thus establish an effective management regime for diseased prawn in the culture system and this could also be used to protect against bacterial invasions. The present study has shown increased antibiotic resistance of the isolated bacteria from P. indicus reflect differences in sensitivity in different strains. Increased resistance of Vibrio spp. and Pseudomonas spp. may be due to the indiscriminate use of antimicrobial drugs in India, as in other third world countries and such multidrug resistance merits further investigation. Incidents of drug resistant bacteria in U.S . aquaculture were far lower than those in Japan31

, and were shown to be associated with antimicrobial treatment. Resistance was generally higher in aquaculture ponds undergoing antimicrobial therapy than in ponds or rivers without antimicrobial treatmene l.

Experimental infection of Vibrio spp./ Pseudomonas spp. by intramuscular injection caused darkening of the

cuticle, loss of appendages, blisters in uropods, gills, accumulation of haemocytes around the infected sites and a high degre\! of mortality of P. indicus. Administration of antibiotics to the culture tank water · markedly decreased the mortality of P. indicus experimentally infected with Vibrio spp./ Pseudomonas spp. The antibiotics administered to culture tank water in this study were probably absorbed through the gills or gut of penaeid shrimp and achieved therapeutic levels in various tissues inhibited the bacterial growth, and reduced the pathogenicity and mortality of infected prawns. The results of the present study are in agreement with the observations of Takahashi et al.<J·lo and Song et al.28 who have shown that isolates of Vibrio spp. are pathogenic to shrimp. Song et aex have demonstrated that the mortality of shrimp increased with the increased dosage of V. damsela. Similarly, Corliss et al.3s and Takahashi et al.

2l) have studied the

therapeutic effects of oxytetracycline against experimental infection of Vibrio spp. in penaeid prawns and reported a significant reduction in the number of deaths of infected prawns after the start of medication.

The prevalence of Vibrio spp. and Pseudomonas spp. was very high in both male and female P. indicus in all the months during the survey period. In contrast, Johnsonl reported a low prevalence of shell-disease in natural popUlations of crustaceans. The high prevalence of the bacterial flora of P.indiells is due to the speciality of Ennore estuary, Chennai, which is known to be highly polluted with pollutants from Ennore thermal power station, industrial effluents and also municipal sewage39

.40

. Similarly, lobsters held in aquaria containing sewage sludge developed shell­disease while controls maintained in similar aquaria in clean water were free from infection" l

. High temperatures and pH in the Ennore estuar/o may enhance bacterial multiplication, and shell disease of lobsters has been associated with high temperature41

-43.

Fluctuations in dissolved oxygen alld salinity40 did not seem to influence the prevalence of Vibrio spp. and Pseudomonas spp. in P. indicus of the Ennore estuary . Heavy burden of peritrichous and loricate ciliates on P. indicus confirms that the pollutants in the environment might 'have reduced the frequency of moulting of P. indicus or the organic pollutants in the estuary might have enhanced the attachment, reproduction and survival of the fouling ciliates and that could account for the heavy burden of the epicornmensal ciliates. Apart from the presence of

).

)

JAY AKUMAR & RAMASAMY: DISEASES OF PENAEUS INDICUS 295

abundant epibiotic fouling protozoans on the body surface and gills, heavy bacterial load have been noticed44 and these along with organic pollution, nutritional and environmental stress and inadequate hygiene and such conditions could cause difficulty in moulting and hypoxia leading to death of the prawns.

The current study has shown that the peritrichous ciliates infected . P. indicus exhibited a fuzzy appearance, frequent jumping, inverted or circling swimming movements. Similarly earlier studies have shown that the epicommensal organisms if occurred in large numbers on the body surfaces, appendages and gills could cause difficulties in locomotion, feeding, moulting or respiration and result in mortalities4549. These epicommensals are also characterized by rough body surfaces, gill disease or both47.48.5o. Such abnormal hosts (P. indicus) are rendered more vulnerable to predation.

Infection of epicommensal peritrichous ciliates on the gills of prawns usually do not affect growth because they do not derive nourishment directly from the host but considered to have a synergistic effect during the periods of stress. Brown and white prawns

. reared in Texas were shown to exhibit heavy infestations of peritrichous Zoothamnium sp. and Epistylis Sp.45 and a heavy mortality of these infected

• prawns were reported to occur when the level of dissolved O2 in the ponds dropped to 2.6 ppm from the normal level of 8-10 ppm. The ciliates occurring on the shrimp could have removed a significant amount of oxygen and prevented the gas diffusion across the gill membrane and could have caused pathological response leading to death.

The occurrence of peritrichous ciliates as recorded in the present study concur with the earlier observations2

•5.51.52 in several penaeid prawns. A

loricate ciliate Lagenophrys sp. has been reported from Palaemonid prawns53

• It is mostly a epicommensal, . non-invasive and limited to cuticular surfaces. The infected sites of the prawn did not exhibit any noticeable accumulation of hemocytes which is usually considered as an indication of h<;>st immune response. Most of the peritrichous ciliates must have proper substratum for attachment and water temperatures for optimal reproduction 17. They feed on bacteria and organic . matter17

. The Ennore estuary of Madras has such an ideal bacterial populations due to organic pollutants44

. These have provided those exact conditions for the growth and reproduction of ciliates on the host cuticular surfaces. Heavy infestations of

ciliates on the P. indicus tissues may deplete a . 'fi f ?4M~1 f h slgm Icant amount 0 oxygen-· .. · rom t e

environment where the oxygen tension may be already low and thus reduce the gas diffusion across the gill membrane in a manner suggested by Overstreet l5

.

Free swimming ciliophore formation by the trophonts was found during adverse conditions48

.53

. The current study has shown that formalin treatment of Zoothamnium sp. iQfected prawns have led to the detachment of trophonts from their stalk. They formed free living ciliophores. However the significance of the detachment of trophonts and formation of free living ciliophores from sedentary peritrichous ciliates remains unknown.

Thus, the present study has revealed that the population of the penaeid prawn P.indicus from Ennore estuary suffer from the diseases caused by bacterial as well as epicommensal epibiont protozoans reflecting the quality of the environment which is polluted with industrial effluents and sewage discharge. Moreover, the present study may show increased antibiotic resistance of the isolated bacteria from P.indicus and may be due to indiscriminate use of antimicrobial drugs in the locality. Drug therapy experiments indicated that neomycin sulphate/streptomycin could be effectively used to control drug resistant bacterial infections while formalin and a flavonoid flavone could be used to control the infection of peritrichous ciliates in the population of P.indicus.

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