HISTOPATHOLOGICAL STUDIES O F SOFT PRAWNS
DISSERTATION SUBMITTED BY REMESH P. R.
IN PARTIAL FULFILMENT FOR THE DEGREE OF MASTER OF SCIENCE (MARICULTURE)
OF THE
COCTIIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
r ••
/ - A - /
: • . . . ... ..
cx r T/1 OCTOBER 1988
POST-GRADUATE EDUCATION AND RESEARCH PROGRAMME IN MARICULTURE
4
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CENTRAL MARINE FISHERIES RESEARCH INSTITUTE COCHIN-682 031C E R T I F I C A T E
This is to certify that this Dissertation is a bonafide record of the work done by Shri P.R. Remesh, under my supervision and that no part thereof has been presented before for any other degree.
Countersigned by
Dr. S.C. Mukherjee, Scientist S - 3, Central Marine Fisheries
Research Institute, Cochin - 31.
Dr. RS.E.R. James, Director,
Central Marine Fisheries Research Institute,
Cochin - 31.
C O N T E N T S
Page No.
PREFACE I
INTRODUCTION U
MATERIAL AND METHODS 10
RESULTS \it
DISCUSSION 23
SUMMARY 32
REFERENCES
P R E F A C E
Unconsciounable utilisation of the renewable marine resources like, marine animals which are of high economic potential, by man, caused a subsequent gradual decrease in the production from feral popu
lations. This rendered development of an alternative to augment the production through aquaculture of these animals in order to m eet the ever increasing demand, A tremendous development has been noticed in the field of aquaculture which provided technical achievements for b etter management of husbandry systems. For aquaculture to be commercially feasible, the economic requirements that maximum number of the individual utilize minimum quantities of space and w ater, must be realized. This always has been accompanied by the appearance of a variety of debilitating and serious 'disease' problems. The term disease as used here, is defined as "a definite morbid process having a ch aracteristic train of symptoms; it may a ffe c t the whole body or any of its part and the etiology, pathology and prognosis may be known or unknown". (Dorland'sMedical Dictionary).
Since crustacean Josses to disease are one of the single most sig
nificant depressants of productivity in the husbandry of these animals, there has been immense progress in the investigation activities of these diseases of crustacean. According to Couch (1978), diseases are second only to predation and periodic physical catastrophies in limiting numbers of crustacean especially penaeid shrimps in nature and second only to nutritional and reproductive requirements in limiting aquaculturaJ success with penaeid shrimps. Though a number of diseases of economically important crustacean
have been described, most of them a re of undeterm ined etiology. Even in cases w here etiology is known, th e h o st-ag e n t-e n v iro n m e n ta l in te ra c tio n is incom plete for many of th e m . Besides th e instin c t pathogenicity of th e agent, th e e f f e c t of environm ental stresso rs also play im p o rtan t roles. In such cases, it is very difficult to assess th e in stig a to rs of th e a fflic tio n and frequently becom e apparent only a f t e r considerab le resource and e ffo rts have been expended in m a n ag e m e n t a c tiv itie s . Same is th e c a se with the "soft-sheJ] syndrome" a ffe c tin g c o m m e rcially m ore im p o rta n t species of penaeids which is a serious th r e a t to th e confined population along the cu ltu re farm s of South India, and is an im p o rta n t limiting f a c to r of the production of penaeid prawns.
Historically, th e d a ta p ertaining to th is syndrome is mucn in adequate.
Although t h e knowledge of som e a sp e c ts o f th e disease has gradually increased, inform ation on th e histopathological c h a r a c te ris a tio n of soft praw n syndrome is hardly observed in th e lite r a tu r e available. In this p erspective, th e p resen t study has been taken up to d e m o te th e p a u city of aquaintance w ith this critical problem through histological means, to reveal the probable im pact of the disease on the norm al histological a r c h ite c tu r e of the anim al.
Although, the im p ro v em en ts in g en eral husbandry of penaeids have certainly c on trib uted to lower incidence of shrim p disease presently^ the principle means of control is early recognition and subsequent elim ination or tr e a tm e n t of in fected groups of anim als. But in this p a rtic u la r case, no controlling measures have been put forw ard due to m eagre understanding of the etiology and pathogenesis. The only possible a lte rn a tiv e which could be adopted to check the econom ic loss and effort is the early h a rv e s t of
the available stock on im m ediate recognition. In this context; the histopathological examination has to be considred as an essential step to revealing many kinds of im portant inform ations including t h ^ pathoge
nesis, th a t a re prerequisites to the developm ent and establishment of reliable and valid diagnostic methods which Inturn enables to carry out proper tr e a tm e n t and employ prophylactic measures.
I would like to ta k e this opportunity to express my deep sense of gratitude to Dr, S.C. Mukherjee, under whose scholarly guidance and supervision, this work has been fulfilled. I am thankful to Dr.P.S.B.R.James, Director of C entral Marine Fisheries R esearch Institute, for the
facilities provided. I acknowledge my sincere thanks to Dr. P. Vedavyasa Rao for his valuable suggestions. It is my pleasure to thank Mr.A.S.Sahul Hameed for his whole h e a rte d help during th e period of my work.
G ratitude for the help and advise offered by my friends, especially DInesan, R am raj, Muthu Karuppan, Bhaskaran and Gopalakrlshnan a re also acknowled
ged with u tm o st pleasure.
I am grateful to Indian Council of Agriculture Research for providing me with a junior research fellowship.
I N T R O D U C T I O N
It is obvious t h a t disease is a ubiquitous phenom enon, and c r u s t a c e a n a r e no e x c e p tio n t o th e g eneral rule t h a t all living things a re su b je c t t o dise a se s. Though th e r e have b e e n m any valuable inform ations about t h e c ru s ta c e a n d ise a se s since th e e a r l i e s t p a r t of th is c e n tu ry , many of t h e s c ie n tif ic studies how ever, been con fin e d to th e p a s t tw o decades, due t o t h e increasing i n t e r e s t in th e c u ltu re of c ru s ta c e a n t h a t a re of d ire c t econom ic c o n c ern . Sinderm ann in 1970, has given a com piled d a ta of th e s tu d ie s on c r u s t a c e a n diseases since t h a t tim e . A f t e r t h a t , some d e d ic a te d works by Bang ( 1 9 7 0 ) , Rosen ( 1 9 7 0 ), Sprague (1 9 7 0 , '7S),
Sinderm ann ( 1 9 7 1 a , '7 I b , ’7 7 ), Pauley (1 9 7 5 ), L ig htner (1 9 8 1 ), Couch (1 9 8 3 ), O v e r s tr e e t ( 1 9 8 3 ) and o t h e r s have c o n trib u te d a g r e a t deal t o this field of s c ie n c e .
Many d iseases of m arin e i n v e r t e b r a te s a re in ad e q u a te ly c h a ra c te r is e d and m any o th e r s probably, have n o t e v e n been recognized. The n um ber of diseases and d ise a s e m echanism s in c ru s ta c e a n is g r e a t e r than t h a t known in o th e r groups of m arine i n v e r t e b r a te s w ith possible ex cep tio n of m ollusca. Due to th e i n t e r e s t and in v e s tm e n ts in c ru s ta c e a n c u ltu r e , consi
d e ra b le l i t e r a t u r e e x ists c o n c ern in g diseases o f th e s e organism s. Many e x c e lle n t reviev/ a r tic le s published by various a u th o rs like L ightner ii9 7 5 , Couch ( 1 9 7 8 ) , Egidius ( I 9 S 7 ) and Sano and Fukuda (1 9 8 7 ) have g r e a te ly c o n tr ib u te d to th e d a ta a v a ila b le about praw n and shrimp diseases. The valuable s tu d ie s by Villella (1 9 7 0 ), B arkate ( 1 9 7 2 ) , L ightner ( 1 9 7 3 , VS), B a rk a te ^ ^ , . ( 1 9 7 4 ) , P a te r s o n and S te w a rt ( 1 9 7 ^ ) , Feigenbaum (1 9 7 5 ),
Lightner L ig htner and Redm an (1 9 7 7 ), Lightner e t al.,(1979), Bian and Egusa (1 9 8 1 ), Hose e t ^ ,( 1 9 8 ^ ^ ) , Sparks ^ ^ * ( 1 9 8 5 ) , Lightner and Brock (1 9 8 7 ), Strus (1 9 8 7 ), Dykova ^ a ^ » (1 9 8 S ), Nash e t al,. (1 9 8 8 ) have com prehensively d e m a r c a te d d iffe re n t a s p e c ts of diseases in
cru stac e an , especially prawns and shrimps. Most of these studies mainly concern w ith th e common etio log y , epizootiology and mode of transm ission, defense m echanisms and th e possible im p act and implication of th e
diseases in these animals.
E x to rtio n ate m ass m o rta litie s of fishes and c ru s ta c e a n s from natural as well as th e c u ltu r e populations have been rep o rte d by d ifferen t workers like H errick ( 1 9 0 9 ) / Beaven and T r u itt (1 9 3 9 ), Snieszko and Taylor (19^f7), Sindermann (1 9 6 3 ), Lunz (1 9 6 8 ), L ig htner (1 9 7 5 , '77 a) Delves - Broughton and Poupard (1 9 7 6 ), and Couch (1 9 7 8 ). Various f a c to r s bring- forth inexplicable and c a ta s tro p h ic events, am ong which, disease is the most im p o rta n t fa c to r (Sinderm ann, 1970). Since natural populations of many of th e econom ically im p o rta n t c ru s ta c e a n species have b eco m e deple
ted due to irration al e x p lo ita tio n of the av ailab le stock, m a ric u ltu re facilities have been e stab lish ed leading to overcrow ding of anim als in th e c u ltu re system s. Lightner (1 9 8 5 ) has s ta te d t h a t very o ften this
c re a te s havoc in such populations held in con fin e m en t, with higher
incidence of disease problem s, because of th e increased suscep tibility to disease th an in th e n a tu ra l environm ent. The classical studies by
O v e rs tre e t (1 9 7 3 ), Aquacop (1 9 7 4 ), Johnson ( 1 9 7 4 ) and D elves-B roughton and Poupard (19 7 6 ) are e x tre m e ly valuable in this context.
a
Lightner (1 9 8 5) expressed the view t h a t th e disease outbreaks in feral populations a re caused by frank virulent pathogen, whereas facultative pathogens a re im portant c a u sa tiv e agents in c ulture environments. Though diseases a re of apparently infectious and non-infectious nature, th ere is a variety of causative fac to rs such as viruses, bacteria, fungi, protozoan parasites, nutritional, toxic and environmental fac to rs to produce diseases.
Majority of th e important diseases in prawns a re caused by opportunistic organisms th a t are part of th e normal m icroflora and fauna of prawns.
D ifferent workers have shown th a t th e diseases th at occur in prawn popuJations a re consequences of synergestic ac tio n of stressors like varia
tions in th e te m p e ra tu re of th e environment, oxygen level, salinity, hydrogen ion c o n centration (p H ) of w a te r, nutritional factors, chemical pollutants and other toxic substances which influence a g re a t deal in tilting the homoeo- s ta tic m echanism s to bring about severe afflictions to the anim al health.
Studies conducted by Rigdon and Baxter (1 9 7 0 ), Venkataramiah (1971a, '71b), Lakshmi e t al.,(1978), Akiyama (19 8 2), Doughtie and Rao (1983), Wickins (198ifa, ’8 ^b ), Momoyama and M atsuzato (1 9 8 7 ) and Nash ^ (19 8 7) are of great significance in this context.
A number of workers such as Adiyodi (1 9 7 2 ), Williams and Lutz (1 9 7 5), Foster and Howse (f 9 7 8 ), Gibson and Barker (1 9 7 9 ), Mellon and Stephens (19 8 0), Ravindranath (1 9 8 0), Rosemark e ^ a h ,(1 980), Martin and G raves(I985), A/-Mohanna e t a l,,(l9 8 3 a , ’S5b), AJ-Mohanna and Nott (1986, '87), GoJdenberg
^ a h , ( I 9 8 6 ) , Benjamin and Ja m e s (1987), Persson e t al.,(!987), Sagrista (1987), Waite and Walker (J 9 8 8 ) and C aceci ^ a L , ( l 9 8 8 ) have reported normal haem a- tologicaj and histological studies on prawns and other decapods which are of
re m a rk a b le im portance. Besides these, n orm al physiological a c tiv itie s in prawns have also been re c o rd ed by Travis (1 9 5 5 a , '57), Williams (19 6 0 ), Dali ( 1 9 6 ^ , '65b, *65d), W aterm an (19 6 0 ), Williams a n i Lutz (1 9 7 5 ),H u n e r e t a l» ^l9 7 9 ), Cuzon (19 80 ), F ieb er (1 9 8 2 ), N ew m an ^ ^ / 1 9 8 2 ) and Rao
^ ^ / 1 9 8 2 ) , which c o n tribu te valuable inform ations.
Studies on disease of c ru s ta c e a n from India is very m e ag re and is lim ited to record of few p a ra s ite s and th e ir biological considerations. A perusal of literature re v e ale d re p o rts on th e diseases of prawns recorded by Mahadevan e t ^ . , ( 1 9 7 8 ) , Gopalan e t a l« / l9 8 0 ), Santhakumari and Gopalan (1 9 8 0 ), Perumal sam y (1 9 8 2 ), R ajend ran (1 9 8 2 ) and Shah al.»
(I9S 2). Very recen tly Soni (1 9 8 6 ) has c o n trib u te d substantial inform ation on diseases o f penaeids of both cu ltu red and wild prawns of India, especially on th e microsporidiosis.
Among the diseases of c u ltu re d prawns of India th e "Soft-shell Syndrome” has c re a te d a lo t of concern am ong th e farm ers and thus
a t t r a c t e d th e a tte n tio n o f fishery scientists. A research p ro je c t on pathology of 'so ft prawns' was envisaged by th e C e n tra l Marine Fisheries R esearch In s titu te (I C A R)in 1982 which has a ttr ib u te d th e disease to be due to a m u ltifa c to ria l etiology. The seasonal p a tte r n of occurrence, its discontinuous incidence in a series of ponds having a lm o st sim ilar ecological c h a r a c te ris tic s and ab se n c e of any internal p arasites made it difficu lt to pinpoint a single fa c to r, responsible for bringing forth this syndrom e. R ajamani (1 9 8 2 )
co n du cted some biochem ical studies on so ft prawn and rep o rte d an in crease in th e N on-protein - nitrogen (N PN ) c o n te n t in th e soft prawns and assum ed th a t this may be due to th e endogenous p ro tein m etabolism caused by th e
changes in th e e cosy stem s during th e e x tre m e s of environm ental conditions.
Soni (19 86 ) has observed som e histological c h an g e s in th e h ep ato p a n c re as of prawns having soft-shell syndrom e. Barring th is no scientific investigations have been c a rrie d out on this syndrome in our country.
In Phillipines studies conducted by B aticad o s ^ a l./ 1 9 8 6 ) could produce soft-sheJling of about ^ 7 -6096 of th e prawns in laboratory conditions by an exposure to a t least 0 .0 1 5 ^ ppm of an organostannous pesticide, for 96 hours, A reversal of the diseased condition was a ch ie v e d by feeding with mussel m eat diet. Similarly Nash ^ ^ , ( 1 9 8 8 ) have re p o rte d about 30 - 50% of
prawns (Penaeus monodon Fabricus) of th e pond re a red stock w ith soft exoskeleton from b rackish w ater ponds with p o te n tia lly acid sulp hate soils,
Nash ^ ^ . / 1 9 8 7 ) w hile working on M acrobrachium rosenbergii de Man in Thailand, have re p o rte d heavy m ortality u p to th e range of 609& per tank in post larvae m aintained under intensive conditions in h a tch e ry , v Histolo
gically and u ltra s tru c tu ra lly sev e re segm ental m yofibrillar necrosis unassoci
ated with any infectious a g e n t was rep orted . The condition has been diagnosed as idiopathic m uscle necrosis. A voidance of overstocking and increased dissolved oxygen in tank w a ter have proved e ffe c tiv e in preventing the re c u rre n ce of th e disease.
Nash ^ ^ . , ( 1 9 8 8 ) re p o rte d pathological changes in th e tig e r prawn, P. monodon Fabricius, a sso c iate d with c u ltu re in brackishw ater ponds developed from potentially acid sulphate mangroove soils in Johor, Malaysia. A
histologicaJ and u ltr a s tru c tu ra i study rev e ale d th e accum ulation of fe rric
0
hydroxide in the gill lamellae# M acintosh (1 9 8 2 ) rep o rte d t h a t 60 percent or m ore of th e fish ponds in th e Phlllipines and about 90 p e rc e n t of the mangrove swamps in Malaysia have th ese c h a r a c te ris tic s . This is only a p a rt of th e approxim ately five million h e c ta r e s in Southeast Asia and over 15 million h e c ta r e s in the tropics which are po te n tia lly acid sulphate.
The present study describ es clinical and histopathological changes observed in P. indlcus and P. monodon c u ltu re d in th e b rack ishw ater ponds located near C ochin, India.
Rgl. STUDY A R E A
The present study was conducted during the period of May, 1988 to August 1988, It included th e collection of penaeid prawns of species Penaeus indicus and Penaeus monodon, having softness of th e exoskeleton and m uscles, from the c u ltu re ponds for carrying out clinicopathological, gross and histopathological examination. The a ffe c te d specimens were collected from the perennial prawn culture ponds in and around Cochin, especially from Narakkal a re a in Vypeen island. This particular island is located parallely west to th e main land of Cochin surrounded on three sides by th e Cochin backw ater system and on th e western side by the Arabian sea. It has a length of 25km. with an area of 69.63 Km , and 2
has extensive marshy low lands, interconnecting tidal canals and paddy fields suitable for seasonal and perennial prawn culture systems (Fig. 1).
Collection of Specimen:
The t e s t specimens w ere collected by operating c a st net from private and government owned ponds where th e occurrence of "Soft syndrome" in prawns was reported during monsoon season. The general inform ations about the stocking, feeding schedule and composition of feed given, previous occurrence of the disease and current s ta te of the culture system e tc . were gathered either by enquiry or by direct observation. The analysis of the hydrological param eters like water te m p e ra tu re (T), hydrogen ion concentration (pH), salinity (5) and
M A T E R I A L S A N D M E T H O D S
dissoJved oxygen (DO) w e re c a rrie d out fro m tim e to tim e by th e following m ethods.
An ordinary im m ersib le m ercuric th e rm o m e te r graded u pto 50°C (accuracy of 0. f°C ) was used to m easure th e w a te r te m p e r a tu r e o f the pond. The hydrogen ion c o n c e n tra tio n was m easured using a p o rta b le pH m e te r (Biochem make) a t th e collection site itse lf. For d e te rm in in g th e salinity, w a te r samples w e re c o lle c te d in sto p p erred re a g e n t b o ttle s, and fo r determ ining th e DO c o n te n t w a te r samples w ere c o lle c te d
in 125 ml. glass b o ttle s w ith o u t a g ita tin g th e w a te r and s to p p e r re d w ithout entangling air bubbles, a f t e r fixing im m e d ia te ly with Winkler's solution.
L a te r, in th e la b o ra to ry th e salinity was d e term in e d using a rg e n to m e tr ic method (S tric k la n d and P arson, 1968) and DO co n ten t by W inkler's m ethod (Strick-land and Parson, 1968)
P a th o lo g ica l in vestigation s:
The pathological in v estig atio ns included observation and recording of clinical signs, behavioural changes and gross lesions in spontaneous cases of a ffe c te d anim als. The grading was done as Gt <5c G2 according to th e shell quality. The to ta l length was expressed as th e distance fro m th e tip of ro strum to th e tip of telson and all the an im als were weighed using a monopan balance (Y am a to ). The sex of th e anim als was d e te rm in e d by identifying th e secondary sexuai c h a r a c te ris tic s .
The live specim ens of app arently norm al and diseased ones w ere se p a ra te ly tra n sp o rte d to th e laboratory for d etail studies, in th e w a te r
9
collected in plastic bins of 50 litres c a p a c ity from the sam e pond, a t a density of 1 No/2 litres w ith occasional a g ita tio n of th e w a ter. The specim ens w ere then exam ined in th e lab o rato ry within ^8 hours of collection. A number of te n diseased and sam e number of apparently normal animals w ere c o lle c te d each tim e fo r laboratory study,
H aem atologicaJ studies:
(i) Total hem ocytic count (THC)i--
The haemolymph was drawn d ire c tly from the h e a rt using a 26 guage hypodermic needle p r e - tr e a te d by flushing with an anticoagulant (3% tri-sodium c i t r a t e ) a t t a c h e d to a I ml glass syringe. From the co lle cte d haemolymph 0 .0 5 ml was drawn into a WBC p ip e tte and diluted w ith WBC diluting fluid. (A nalytical solution, SD's Lab-Chem industry) and a g ita te d . A drop from th e p ip e tte was used to charge the Neubauer - counting c h am b e r and the h e m o cy tes were counted under low power ob jective in light m icroscope. The num ber of cells w ere expressed as cells per cubic m illi-m e tre .
(ii) Haemolymph glucose determ ination;-
A p a rt of th e haemolymph was used to determ ine th e glucose c o n te n t. It was done using a glucom eter (Model : Ames) a t a particular tim e of th e day (b etw een 11.00 hrs - 12.00 hrs) in order to avoid
diurnal variation of haemolymph glucose c o ncentratio n.
G ross and h istop ath ological studies:
The gross changes if any, were exam ined in the hepatopancreas, gut, gill, body muscles and exoskeleton of th e diseased as well as the normal
9
animals. Small tissue p ieces w ere colle cte d in 10% phosphate buffered formalin (pH 7.0 ). Dead field specimens w e re im m ediately fixed a t the collection s ite by (a ) in jectin g th e fixative below d ifferen t p a rts of the exoskeleton using a hypoderm ic syringe, (b) by longitudinally c u ttin g the dorsal a sp e c t of th e e x o sk e le to n to enhance p e n e tra tio n of th e fixative or (c) colle ctin g tissue sam p les by dissecting out th e organs or fixing th e whole animal in s u ffic ie n t quan tity of fix a tiv e . The fix ative was changed a f t e r 24 hours w ith fresh fixative.
Tissues w ere rem o v ed from th e fix a tiv e a f t e r proper fixation, washed in running ta p w a te r fo r 6-8 hours b e fo re processing th em . The em bedding, sectio nin g and staining w ere accom plished using routine histological m ethods. P a ra ffin v/ax (BDH, 5 6 -5 8 °C melting point) was used fo r m aking blocks. The sections w ere c u t a t 4-8 yU thickness in a manuai r o ta r y m ic ro to m e (Weswox Optik Model M T-1090 A) and stained by Hem atoxylln-Eosin m e th o d . Stained sections w ere exam ined under a binocular re sea rc h m icro sco p e (Olympus). Photom icrographs w ere taken w herever necessary.
R E S U L T S
The p resent study was conducted on tv/o species of penaeid prawns ie. Penaeus indicus an d Penaeus monodon, collected from private and G overnm en t owned fa rm s of Vypeen island during the period of May,
1988 to August, 1988 w hen th e r e was th e o c cu rren c e of "Soft Syndrome".
Survey data:
The incidence of "Soft Syndrome" th is year, was fir s t noticed in th e fa rm s in th e f ir s t w eek of May, when few prawns shoved a ffe ctio n rev ealin g clinical signs. A s te e p increase in th e incidence was noticed a f t e r th r e e to five days. Early harvest was done in such ponds to avoid th e loss as th e re was rise in th e o c cu rren c e of "softness" in th e animals leading to high m orbidity and m ortality . The yield was 20 to 25% of the stocked population on h a rv e stin g , from th e se ponds.
P. indicus of m ostly 91 to 132mm len gth and P. monodon of 112 to 1^1 mm length of both sexes were a f f e c t e d and were c o lle cte d from th e c u ltu re ponds having high incidence of m o rtality . During th e period of study, th e pond salinity ranged from 2 0 ..3 ppt to 2.2 ppt due to continuous dilution by monsoon rains. The te m p e ra tu re varied from 32.2°
to 3^.7®C. The pH rang e was in betw een 7.8 to 8.0 and dissolved oxygen c o n te n t was 3.73 to ^.21 ml/1.
Clinical signs and gross pathology:
The a f f e c te d a n im a ls w ere o f te n dull and sluggish in their m ovem ents. The d e f e n s e reflex e s w ere minimum as evidenced during their c a p tu r e . The e x o sk ele to n were thin, s o ft and palpable w ith cuticular lesions d istrib u te d on th e te r g u m in e x tr e m e cases. The m uscle tissues w ere fra g ile and m o d e r a te to markedly s o ft to touch. The gut which could be seen through t h e s e m itra n s p a re n t exoskeJeton showed wavy n a tu re especially in th e po rtio n of firs t t h r e e abdominal segm ents. The gut was full, with food p a rtic le s mainly blue g reen alg a e , diatom s and d e tritu s and a p p e are d yellow to orange yellow in colour a t th e an te rio r region. In th e c a se of P, indicus th e body was light greenish in colour and g e n e ra l distribution of som e dark greenish spots in th e abdominal m u sc u la tu re was n o tic e d . This was not vivid due to th e species specific p ig m e n ta tio n of th e body in monodon. The hepato p a n c re a s of the a f f e c t e d prawns w ere c o m p a ra tiv e ly sm aller than th eir norm al c o u n te r
p a rts of th e sam e size group which appeared loose.
During th e initial sta g e s it was very difficult to d if f e r e n tia te the 'soft praw ns' from th e e a rly post moulted prawns. Clinically, both of them showed soft e x o skeleton. But, with progression of the disease, the in te stin e of th e a f f e c te d anim als appeared enlarged and wavy, p a rticu larly a t t h e a n te rio r p a r t of th e abdomen. Such prawns exhibited sluggish m ovem ent and progressive em aciation.
H ae m ato lo g ic a l findings:
(i) Haem olym ph glucose estim ation;.
25 anim als exam ined under Gt g rade (ex tre m e ly so ft) showed a Haemolymph glucose level of 12.75 ± 2 .8 2 m g/100 ml. with a range of
f
8-17m g/100' ml
31 animals examined under G2 grade (Moderately so ft) showed a haemolymph glucose level of 23.0 ± 3.15 m g/100 ml. with a range of
1^-27 mg/lOO ml.
22 numbers of apparently normal animals examined, showed, a haemolymph glucose level of 27®0 ± 5.07 m g /100 ml with a range of 22-^1 mg/100 ml.
The haemolymph glucose co n te n t is shown in Table No.l.
(ii) Total Hemocyte Count (THC):-
The to ta l hem ocyte count of 25 animals examined under G1 grade was 13^910 ± 5.153 cells/m m ^ with a range of 8050-2^^750 cells/mm ^.
The to ta l hem ocyte count of 31 animals examined under G2 grade was 10,308 ± 2,^^03 cells/m m ^ with a range of 7,500 - 17,750 cells/mm ^.
The to ta l hem ocyte count of 22 apparently normal animals (N) examined was 9,86^ ± 3 ,609 ceils/mm ^ with a range of ^^,250 - 16,950 cells/m m ^.
The total hem ocyte count is shown in Table No.2.
Histopathology:
Histopathoiogical changes were noticed in the exoskeleton, muscle, gill, hepatopancreas heart and gut.
Exoskeleton;
Microscopically, the upper most layer of the exoskeleton ie., the epicuticle revealed marked thinning with erosions and in some places the continuity was broken giving it a desquamated appearance (Fig. 2).
The subepicuticular pigmented layer or the exocuticle composed of chitin and calciu m . showed thinning and uneven thickness in some places. The calcified endocuticle showed remarkable microscopical alteration. In most of the extreme cases, the calcified layer was not a t all present (Fig. 3) leaving behind the exocuticle alone. Whereever present, the calcified endocuticular layer stained deep pink with haematoxyiin and eosin exhibiting a decalcified nature. In some cases the membranous layer was markedly thickened showing hyperplastic changes. Extensive separation of the epidermal layer from the uncalcified layer was also noticed (Fig. 4). Besides, this epidermal layer showed degenerative changes in many areas. The sub-epidermal layer showed extensive vacuolation to give a reticu late appearance (Fig. 5) with mild to moderate focal aggregation of hemocytes. Scattered, pleomorphic 'reserve cells' resembling mononuclear cells were found as in the normal. In places where gross lesions in the exoskeleton was noticed (Gl), infiltration of hemocytes was evident in the underlying subepidermal layer. The tono- fibrils which traverse the epidermal layer showed focal detachment from the urcalcified layer of the endocuticle (Fig. 6 & 7). exhibiting hyper
plastic and hyperchromatic characteristic.
Muscle:
Stricking changes were however seen in the muscle tissues underlying the exoskeleton.
HistomorphoJogical changes consisted of myofibriiJar necrosis and deg e n era tio n of varying degree, e xtend throughout the s tr ia te d InuscuJature of th e body w ithout any a p p a re n t s ite of prediliction.
The m uscle fibres displayed a v a riety of morphological changes c h a r a c te r is tic of progressive myofibrillar d e generation and n e c ro tic m yopathy. Evidence of any b a c te ria l, p rotozoan or p a ra sitic agent could not be d e m o n stra ted . In some focal a re a s, muscle fibres or a group of muscle bundles showed Zenker's necrosis with ground glass a p p e arance or hyalinization (Fig. 8). fra g m e n ta tio n , granular degeneration, vacuolation, h em ocytic in filtra tio n (Fig. 9) and m ineralization (Fig. 10 and I I ) .
Focal to m u ltifo c a l a re a s revealing hyalinisation, swelling, and loss of c ross striatio n s w e re o f te n a ssociated simultaneously w ith frag
m en ta tio n of th e m uscle fibres (Fig. 12). A reas of myofibrillar disorgani
sation along with h y p e rc h ro m a tic cytoplasm (Fig. 13) and pyknotic nuclei w ere e v id e n t. In som e c a s e s severe s a rc o m e re atrophy and oedem a of muscle bundles w ere also noticed. (Fig. 14). Extensive myofibrii necrosis with d e s tr u c tio n of pare n c h y m a were displayed by vacuolar changes with 'm o th - e a te n ' a p p e ara n ce (Fig, 15). Some ca se s were noticed with the a p p e a r a n c e o f large vesicular myonuclei with prom inent and m arginated h e te ro c h ro m a tin granules. Interstingly, sa rcolem m al nuclear proliferation with slight to m o d era te h a e m o c y te infiltra tio n was also noticed n u lti- focal or focal aggregation of haem ocytes was evident in many areas (Fig. 16 &: 17). Under higher m agnification infiltrating h e m o c y tes showed phagocytic a c tiv itie s and encapsulation of n e c ro tic muscle tissues
containing pyknotic and k a ry o rrh ec tic nuclei (Fig. 18). In e a rly stages
i 0
of degeneration, the muscle fibres either fuse together and/or showed detachm ent from the myoseptum. Replacem ent of necrotic tissue by connective tissue was evident (Fig, 12). Focal areas of dystrophic calcification a t the site of necrotic muscle tissue was evident at few places (Fig.fO <5c II), The calcification was ia te r confirmed by special staining method of Von Kossa and Alizarin red S stains.
Gill:
In most of th e cases the epidermal covering around the lamellar sinuses showed degeneration. Marked a te le c ta tic changes showing collapse of the anterior sinus with emphysema or distension of the outer lamellar sinuses were usual findings in affected gills (Fig, 19). Gill epithelium in the lam ella exhibited flattened appearance due to atrophy or necro
tic changes. Inflammatory cell reaction was scanty. The branciai septum separating the branchial canal into a ffe re n t and efferent canals appeared thickened with s c a tte re d branchial cells (Fig. 20). The intracellular connective tissue was abundantly present in association with oedema and s c a tte re d haemocytes a t the branchial septum,
Hepatopancreas:
The absorptive cells (F- cells) showed hyperchromatization and derangem ent in the architecture. The epithelial cells of hepatopancre- atic tubule were completely disintergrated and denuded leaving the basal lamina alone in extrem e cases. The covering elastic tissue and basement membrane were rendered thin and tense (Fig. 21). Fusion of the basal Ijm inae of the ruptured, disintegrated tubules was evident in some cases.
OccasionaJly tubular cells exhibited vacuolation and h yperchrom atic c h a r a c te rs (Fig. 22 <5c 23). Few vacuolar cells in these a re a s showed round to oval basophilic cyto plasm ic inclusions.
Heart:
H e a rt muscle showed m yocardial d eg eneration with pyknotic and k a ry o rrh e c tic nuclei in th e m uscle hands. O th e r changes w ere c h a r a c te r
ised by hyalinization and vacuolation associat<“d with m o d e ra te hem ocytic in filtra tio n . Pericardium showed m arked thickening due to hyperplastic changes and hem ocytic in filtra tio n .
Gut:
M oderate to m a rk e d histopathological changes w ere seen in foregut, m idgut and hindgut. N ecrosis o f th e c u tic u la r lining was ev id en t alm ost th ro u gh ou t th e e n tire len gth of th e gut (Fig. 2^f). The mucosal epithelium was d ark ly stained and had sloughed off a p p ea ra n ce a t som e places. O th er changes included focal a re a s of necrosis in th e tunica m uscularis and
submucosa (Fig. 25 ), h e m o c y tic in filtra tio n in th e connective tissue of subm ucosa and occasional p resence of n e m atod es in the lumen (Fig. 26).
Serosal lay er o ften showed invagination and lumen contained debris.
F ilte rin g apparatus:
The chitinuous p la te s w ere m arkedly damaged in m ost cases w ith loss of chitogenous epithelium . S etal secreting cells showed loss of nuclei (Fig. 27). C o n n ectiv e tissues w ere abundantly p resen t.
<> f.
TABLE No. t
TABLE SHOWING THE HAEMOLYMPH GLUCOSE CONTENT(in mg/IOO ml)
Grade
Number of animals exam ined
X SD Range
GI 25 12.75 ± 2.82 8 - 17
G2 31 23.0 ± 3.15 - 27
N 22 27.0 ± 5.07 22 - ^1
GI _ E xtrem ely soft
G2 - M oderately soft
N - A pparently normal
TABLE No, 2
TABLE SHOWING THE TOTAL HEMOCYTE COUNT (THC) (Ceils/m m ^)
Grade Number of
Animals
examined X SD Range
Gl 25 13,910 ± 5,153 8 ,050 - 2U,750
G2 31 10,308 ± 2,^03 7 ,500 - 17,750
N 22 9,S6Ci ± 3,609 ^ ,2 5 0 - 16,950
GI - Extrem ely so lt
G2 - M oderately soft
N - A pparently normal.
Fig. 2. Photom icrograph of exoskeleton showing m ark ed thinning of the cu tic le. Note th e sloughing (arro w ) and th e gap betw een cutic le and muscle tissue H&E X30
Fig. 3. Note the loss of calcified end o cu ticle and disorganised
muscle bundles. H&E X30
Fig. U, Note the separatio n of subepiderm al layer from th e epiderm al layer (arrow ). Wide gap b e tw e e n e x o sk e le ton and muscle layer is also e v id en t H3cE X30
Fig. 5. Photom icrograph showing vacuolation in th e subepi
derm al layer H&E X30
Fig. 6. Note th e tonofibriis (T) and it's focal d e ta c h m e n t (arrow ) from th e uncalcified la y e r of e n d o c u tic le ,a n d th e abnorm al thickening of th e p ig m e n te d lay er. The lightly stained m ate ria ls b etw een th e ton ofib riis and muscle (M) a re hem ocoele and few s c a tte r e d
hem ocytes. H&E X30
Fig. 7. Higher m agnification of Fig. 6.
N ote th e uneven thinning of th e c a lc ifie d e n d o c u tic u la r layer (C ) and corresponding d e ta c h m e n t of to n o fib riis (T). H em ocytes (H) are d istin ctly visible H&E X85.
• I'. *^r.<*•>:-* *1^ :)0 5 e » .^ » r i
i&ika
Fig. 8. Note the loss of stria tio n and ground-glass a p p c a ra n c e
of muscle tissue, showing Z e n k e r's necrosis. Dark stain ed area is a n e c ro tic muscle bundle w ith h e m o cy tic
infiltration. H&:E X85.
Fig. 9. Photom icrograph showing e x te n siv e m uscular n ecro sis with hem ocyte in filtra tio n . N ote th e fra g m e n ta tio n and
separation of muscle fibres H&E X85.
Fig. 10. N ecrotic m uscle fibres w ith loss of s tria tio n and loss of myonuclei. Darkly stained a re a (arro w ) showing dystrophic
calcification. H&E X30
Fig. U ; Higher m a g n ification of Fig. 10. showing c a lc ific a tio n . H&E X85.
Fig. 12. N ote th e hyalinization and f r a g m e n ta tio n of t h e m u scle fibres. O edem a and focal a g g re g a tio n of h e m o c y te s a re
also noticed (arrow ). H&E XS5.
Fig. 13. Photo m icro erap h showing nr.yofibrillar diso rg an isation . N ote h yp e rc h ro m a tic muscle fib res and m u ltifo c a l
hem ocyte in filtra tio n H&E X30
■ w ^ . f M :
7 f
Fig. 1^. Muscle tissue showing severe sarcom ere atro ph y and oedema with pyknotic myonuclei adhering to
sarcom ere, H<5cE X400.
Fig. 15. Extensive myofibrillar d estru ctio n displaying "m oth- eaten" appearance. Few aggregation of c ellu lar debris
a re seen s c a tte re d H&E X85.
Fig. 16 N ote th e focal aggregation of h em ocy tes in n e c ro tic
myofibres H&E X85.
Fig, 17. N ote th e degree of affe ctio n and th e in filt
ratin g cells in th e process of phagositosing th e
n e cro tic tissue H&E X85.
• C x > .
> v * . ' ^ V ? . 5 r *:^
>» ~ M
IS. H c r i i o r y t i r in a n c c r o l i r f o r u s w i t h phj^;o- c y to s i n j ’, tifc roiK ' tis s u e . Nol<* lli<- n u c ic j I.frnor> ;r*s u h u h ur<- v a r i a b l e in sha;x.-. HvM. X ^ 2 0 .
r i j ; . 1 9 . N o t r i h r r of sl.ri;r;'r.cn r p i t h r l i a l f e l t aful colUj;)s<-d M fur.cs. vjtt'.u- liition IS o p p a rc f i! !hrou;;hi>vjt Ilir Mv*l. XS^.
2 0 . I 'o r a l a p ,trc j’, a t i o a of h r t i u v y t r \ m th e ^;5ll Ivirnrllcic ( a r r o w ) a n d b r a n c h i a l s r p t u n i HJ*l’ X 85.
2 1 . P h o t o t t u r r o ^ ’.r a p h of hrp.ito;>an< r r a t > r tvjb\ilr show ing m a r k e d d i s i n t e j ; r a t i o n of t n h u l a r r e l K Iravm j; e m p t y s p a c e w i t h in t h e bas.il c o n n e c t i v e tissv;eHvVfl X30,
f i g . 2 2 . C r o s s s e c t i o n th ro u |; h h e p a t o p a n c r e a t i r tu b u le d is p la y in g d i s t e n t e d lufT^en ( L ) , h y p e r r h r o m a t i c a n d d c g e n e r a ti n ; ; t u b u l a r c e l l s fo t c e lls (1‘) a n d c o n n c c t i v c t is s u e (C '.T) MAE XSV
Fig. 2 3 . L o n g itu d in a l s e c t i o n t h r o u g h h e p a t o p a r ^ c r c a t i c tu b u le s . N o t e t h e d i s t e n t c d lu m e n (L ). f l a t t e n e d h y p c r c h r o m a t j c c e il s a n d \ a c u o ! a t i o n s M i;r X S5.
19
^if ii ^
:2 S - V ^. ’■
i»' * ^ *i
Fig. 2k. P hotom icrograph of foreg ut ex hibiting n ecrosis of cutic u lar lining. Marked invagination of o u te r layer (arro w ) and p a rt of f ilte r cham ber (F ) on the
right H&E X85.
Fig.25. Midgut showing necrosis of tu n ic a m uscularis .(arro w ) and subm ucosa. N ote th e invagination of serosa
H&E X85.
Fig. 26. Lumen of th e gut showing c ro ss section of h e m a to d e s and c u tic u la r debris. N ote a p p a re n tly norm al h ep ato - p a n c re a tic tub ules (to p l e f t) an d M ultivacuolated
tubular cell (a rro w ) H<5cE X85.
Fig. 27 , Section of filte rin g a p p a ra tu s showint d a m ag e d chtinous p la te (C ) and m edian ridge (M ), S etal s e c re tin g cells (S), S e ta e of f ilte r (F ) and c o n n e c tiv e tissu e (C T )
H&E X85
m r j
D I S C U S S I O N
A considerable amount of loss is incurred every year due to the
"soft-shell syndrome" which sweep through th e stocked populations of penaeid prawns in th e grow-out system s, aJong th e southern p a rt of India causing im m ense Joss in th e production. In r e c e n t years prawn production in b rackishw ater ponds in PhiJippines (B aticados e t aj., 1986) and mangrove soils in Malaysia (Nash e t 198S) has been adversely a ff e c te d by a sim ilar chronic so ft shelling syndrome. Multi-disciplinary approaches in th e field of pathobiology, ecology, physiology, biochem istry and nutrition have a lready been made, earlier to understand th e causative fa c to r(s ), inducem ent and m a n ife s ta tio n of this syndrome in penaeids.
However, histopathological studies of various organs of soft prawns are inadequate. The m icroscopical changes recorded in th e present study m ay help in e lu c id atin g pathogenesis of 'Soft praw n', to a considerable e x te n t.
The n otable fe a tu re s in th is condition a re th e thinning of th e exoskeleton and te n d e rn e s s of t h e body muscles rendering the anim al to feel so ft. Similar softness is also seen in prawns im m ediately a f t e r moulting whose c u ti c le g e ts hard e n e d within few hours, whereas th e c u tic le o f th e s o f t prawns re m a in s o ft and thin. Subsequently this condition leads to poor grow th resu ltin g g r e a te r m orbidity and m orta lity . As s e le c tiv e stocking is being p r a c tis e d by th e fa rm e rs e ith e r with P. indicus or P ^ m onodon, th e pathological conditions w ere observed m ainly in th e s e tw o species and occassionally in M etapenaeus dobsoni.
I n .
Published re p o rts on epidem iological studies (Anon. 1987) revealed th a t th e syndrome usually make its a p p e a ra n c e during th e period of March to Septem ber every year in th e tid e -fe d brackishw ater farm s or pum p-fed farm s. As the present study was confined to th e period of May-August, th e d etail picture of the occu rren c e of th e phenomenon through a
C alendar year could not be p re sen te d . However, th e re is very little doubt about the seasonal oc cu rren c e of th e syndrome which was first recorded in th e first week of May, a f t e r monsoon rains, this y ear (1 9 8 8 ) and continued upto th e middle of A ugust during which period the
environm ental p a ra m e te rs showed a wide flu ctu ation in salinity, te m p e r a tu re and oxygen c o n ten t.
R eport presented under th e r e s e a rc h p ro je c t on th e "Pathobiology of so ft prawns" (Anon. 1987) pointed out th a t so ft-shell syndrom e o c c u rre d in widely fluctu ating ecological conditions, with te m p e r a tu r e ranging from 2 6 .2 “ - 39.0°C , salinity from 2.^f - 33.67% o , pH from 6.0 6 to 9 .3 3 and DO from 0 A 5 to 9.3 5 mI/1. In th e p re s e n t study too, it has been observed th a t th e condition appeared when th e salinity and oxygen level of w a te r w ere very low w hereas te m p e ra tu re was very high. The sudden change in th e ecological condition m ight h av e a c te d as a triggering fa c to r to induce th e softness in th e prav/ns. M omoyama and M atsuzato (1 9 8 7 ) have re p o rte d a similar condition in K urum a Shrimp c u lte re d in Jap an , producing muscle necrosis which has been a t t r i b u t e d to environm ental or physical s tre s s due to high te m p e ra tu re , low dissolved oxygen and over-crow din g.
But th e s e workers failed to find any re la tio n betw een th e o c cu rren c e of this disease and w a te r quality.
A sta tistic a l analysis by Boyd (1 9 8 2 ) showed t h a t unfavourable pond conditions of high soil pH, low w a te r phosphate and low organic
m a tte r c o n ten t when occur to g e th e r, result into soft shelling anong prawns in the ponds. The organic m a tte r c o n te n t of th e soil serv e as a m easu re of pond fe rtility . It is e ith e r d ire c tly used by th e praw ns as food or these may decompose and re le ase inorganic n u trien ts which in tu rn enhance th e grow th of phytopiankton. Boyd (19S2) was of th e opinion t h a t Jew organic m a t t e r c o n ten t in the soil may redu ce th e available food for prawns. He also expressed th a t th e significantly higher incidence o f so ft-sh ellin g during th e sum m er months (M a rc h -Ju n e) could be re la te d to th e re la tiv e d ifficu lty in th e process of w a ter exchange during dry season.
The gross a n ato m ica l changes noticed in th e p re s e n t study was ra th e r in te re stin g and may help in th e id e n tific a tio n p rocess of th e a f f e c t e d anim als. Besides softness of th e exoskeleton, th e an im a ls e xh ib ited pale m u scu latu re with ground glass a p p e a ra n c e . The underlying hollow space be n ea th th e cutic le invariably co n tain ed bubble like m a te ria ls which may be considered as a freq u e n t ob serv ation . The wavy a p p e a ra n c e of th e gut - especially, in th e a n te rio r th ird of th e body in e x tre m e conditions o fte n
containing grayish black undigested food m a te ria ls is of diagn ostic im p o rtan c e . Sim ilar observations w ere also m ade by o th e r workers (Soni, 1986; Anon. 1987) who, of course could n o t a t t r i b u t e an y reason for this. It c an be opined th a t this wavy fe a tu re m ay be due to in te stin al dysfunction.
R esults showed t h a t, th e re is a considerable in c re a s e in to ta l hem o- c y te count in haem olym ph in com parison to th e norm al ones which ind icated th e body's response to th e trig g erin g e ffe c ts . Although sim ilar o bser
vations, w ere m ade by R abin (1965); Sindermann (1 9 7 1 a ), Couch (1 9 7 8 ),
<>a
Gunnarsson & Lackie (1 9 8 5 ) and Persson and Soderhall (1 9 8 7 ), th e re is a possible exception of G affkem ia w h ere th e hem ocy te number declined (S te w a rt and Rabin, 1970) due to th e disease. The increase in the
granular hem ocytes indicated th e role of th ese cells in a c u te in flam m a to ry conditions. Interestingly, a decline in th e haemolymph glucose level may be caused by an energy debt in th e tissu e s due to im proper assim ilation of carb o h y d rate and th e u ltim a te e f f e c t of stress. A sim ilar hypoglycem ic condition has been noticed in te rm in a l grow th (TG) prawns (Brock, 1983), in M acrobrachium . sp.
However, calcium levels in th e exoskeleton, muscles and haem olym pl was also studied e a rlie r which in d ic a ted d ifferen c e in th e calcium levels in th e haemolymph, muscle and e x o sk e le to n in d iffe re n t seasons, re nd erin g to im balance in th e absorption and tra n s p o rta tio n of calcium (Anon, 1987).
H istopathological studies of various organs ind icated som e com plex phenomenon in te rre la te d to e ach o th e r. The thinning of th e e p ic u tic le and e n d o cu ticle or th e d e calcified n a tu re of th e e n d ocu ticle observed in this study require fu rth e r elucidation. N e v erth ele ss, th ese changes may be a ttr ib u te d to a sort of ’leaching' of th e c u tic u lar calcium or a kind of resorption by the body. But, it is well understood th a t th e c u tic u la r resorption is mainly aim ed a t th e c o n se rv a tio n of o rganic c o n stitu e n ts . It is re p o rte d th a t, ab ou t only 5 % of th e cutic u lar calcium is resc '.bed by C arcinus during th e prem ou lt phase (L a fo r, 19^8).
It is a p pro priate to discuss th e possible im pact of microbial
population on this disease problem. Pillai (1 9 8 2 ) through transm ission e x p e ri
m ents and field observations s ta te d th a t, vibriosis caused by Vibrio anpuillarum in P. indicus produces w hite patches on abdomen and reddish discolouration of th e rostrum . In a c u te c ases th e prawns becom e e m a c ia te d with softening of muscle tissue and thinning of th e c u tic le . Kurien (1 9 8 2 ), Muthu ^ ^ . , ( 1 9 8 2 ) and Nandakumar (I9S 2), re p o rte d th e o c c u rre n c e of this phenomenon in
cu ltu red prawns. While Muthu e;^ (1 9 8 2 ) observed t h a t th is was th e m ost common disease in P. indicus^ Kurien ( 1 9 8 2 ) was of th e opinion th a t, th e
thinning of th e exoskeleton might be due to th e m e ta b o lic changes owing to the changes in th e environm ent. .A tte m p ts w ere made for isolation of p atho gen ic b a cte ria in th e p resent study which proved futile. Besides, n e c ro tic body tissue failed to d e m o n stra te th e p resen ce of any b a c te ria l pathogen histo- pathologically. The stu dies c arrie d out by B aticados e ^ aK, (1 9 8 6 ) could isolate c h itin oclastic b a c te ria like Vibrio and A eromonas from th e lesions of soft shelled prawns, but e x p erim en tal transm ission a t t e m p t s w ere
unsuccessful. Few w o rkers (Hood and Meyers, 1 9 7 ^) believed th a t c h itino - cJastic b a c te ria a re norm al p a rt of th e m icroflora of penaeids and p re sen c e of th ese organisms in th e c u tic u lar lesions may be of secondary in fection .
Extensive m orphological a lte ra tio n s w ere d e m o n stra te d histo patho - logicaiJy in th e p re s e n t study which in m any re s p e c ts a r e sim ilar to th e rep orts of e a rlie r w orkers. Rigdon- ^nd B axter (1 9 7 0 ) w e re th e first
workers to observe dev elo pm en t of w h ite or opaque abdom inai m u scu latu re in spontaneous m uscle necrosis of prawns and described th e histological
condition as "degenerated foci of s tria te d muscle", in brown shrimp.
Lakshmi e ^ al_., (1 9 7 8 ) rep o rte d similar disease problem which had been a ttr ib u te d to v a rie ty of unfavourable env iron m ental conditions during or
im m ediately following a h y per-activity . Similarly Akiyama (19S2), Momoyama and M atsu z ato (19 8 6 ), and Nash e ^ (1 9 8 7 ) have described the condition as w hite muscle disease, idiopathic m yopathy or idiopathic muscle necrosis (IMN). However, no re fe re n c e have been m ade regarding th e soft exoskeleton in th ese cases. Besides th e above m entioned workers, V enkataram iah (1 9 7 1 a , 1971b), Sindermann (1 9 7 7 ), Brock (1 9 8 3 ) and Lightner (1 9 8 3 ) have considered the m uscle necrosis as a result of predisposing environm ental stresso rs
including e x tre m e and sudden flu ctu atio n s in salinity, te m p e ra tu re , DO levels, h y p e ra ctiv ity , over-crow ding and physical handling. The p resen t findings in m uscle tissue also c o rro b o ra te d with th e observations made by above w orkers and may be a ttr ib u te d to th e predisposing en vironm ental stre sso rs.
L a c tic acid is believed to be th e m ajor cause of p o sta c tiv ity acidosis in c ru s ta c e a n s (Phillips et_ 1977; Me Mahon e ^ 1978). Maximum la c tic acid and minimum pH levels may o c cu r 1-2 hrs following stre ss due to low body te m p e ra tu re (McDonald e ^ a h , 19 79 ), their open c irc u lato ry sy stem or com bined e f f e c t of both (Spotts & L u tz, 1981). During this period, th e r e may be an increased suscep tib ility to th e e f f e c t of fu rth e r stress conditions such as low DO c o n te n t or infectio n (S p o tts and Lutz, 1981).
Nash ^ (1 9 8 8 ) observed softening in about UO - 50% of th e stocked population of monodon c u ltu re d in brackish w a te r ponds of p o ten tially acid sulphate soil and this m ay be a ttr ib u te d to reduced
0
pH due to rapid loss of alkalinity which causes precip ita tio n of calcium salt m aking it nonavailable to th e anim als. Studies con d ucted by Wickins ( 1 9 8 ^ ) could d e m o n s tra te th e weight loss of c a ra p a c e in P. monodon due to red u ced pH, though th e calcium levels rem ained c o n sta n t in th e exoske
leton. In th e p resent study, denudation of calcified en d o cu ticu la r layer was noted which may be due to resorp tio n or im proper m in e ra liz atio n of th e exoskeleton. The f a c to r s influencing m in e ra liz atio n o th e r th an te m p e r a tu r e and physiology, include bic arb o n ate (G re en a w ay , 197if), e x te rn a l calcium level (C ripps and N akam ura, 1979) and pH (Malley, 1980). However, L ahti (1 9 8 8 ) found th a t th e m in e ra liz atio n of th e exoskeleton in A stacu s did not g e t much a f f e c t e d in calciu m d e fic ie n t w a te r.
A ccording to th e pond surveys m a d e by B aticados ^ ^ . , ( 1 9 8 6 ) , th e o c c u rre n c e of s o ft shelling could be p re d ic te d with 98% a c c u ra c y under poor soil and w a te r conditions in th e ponds especially w ith Jow a m o u n t of phosphate. They su g g ested th a t this m a y be a n o th er reason for making calcium nonavailable to th e anim al along w ith reduced pH.
Although no clinical c h e m istry p a r a m e te r s have been ta k en up
sim ultaneously w ith th e h istop ath olo g ical study in this study, it is con sidered th a t s tre ss induced h y p e ra c tiv ity leading to rapid developm ent of m uscle hypoxia and acc u m u la tio n of la c tic acid during anaerobic glycolysis w ere th e m ost likely reasons in th e p ath o g en esis of muscle necrosis. The in c re ase in th e non -pro tein nitrog en (N PN ) re p o rte d by R ajam ani (1 9 8 2 ), may be due to th e p ro te in d e g rad atio n in th e muscle tissu es during th e n e c ro tic changes.
In the case of muscle diseases, nutritional myopathy asso ciated w ith vitam in E and tra c e elem ent selenium have been rep o rte d to be th e cause of muscular dystrophy in d o m estic anim als (Hulland, 1985), and fish (R oberts, 1986; Collins & R i c e , 1986 and Richards, 1986), but has not been described in prawns. A ccum ulation of fr e e radicals causes peroxidation of m em brane lipids and dam age to p ro te in molecules leading to c ellu lar injury beneath the c u tic le and m uscle. E stim atio n o f selenium in th e soil, in th e a f f e c te d ponds may be a p p ro p ria te .
A nother im p o rtan t fa c to r causing so ft shelling is a q u atic p o llu ta n ts.
Among which th e pesticides used in th e ponds for c on tro l of weed an im als, have been im plicated as chitin synthesis inhibitors (C o rb e tt, 1974; Dale, 1975). L aboratory studies by B aticados ^ a l.,(1 9 8 6 ) could produce about 47-6096 of soft shelling in P. monodon by a 96 hrs exposure to an org an o- stannous p esticid e. The e ffe c tiv e in te r f e r e n c e of th e chem ical p o llu ta n ts is largely controlled by th e e n v iro n m e n ta l conditions, species d iffe re n c e s and also th e physiology of th e anim al.
Sis ^ (1 9 8 0 ) rep o rte d chan ges in th e gut of penaeid shrim p as a re s u lt of environm ental stressors. Some of th e histological changes re p o rte d by th e m and Lee ^ (1 9 8 5 ) like n ecro sis of th e lining cu tic le, focal
n ecro sis and general hem ocytic in filtra tio n w ere sim ilar to those observed in th e p re s e n t study. Although hem ocy to sis was mainly observed in th e subm ucosa, eoslnophilia was not much pronounced.
Histopathology of th e organs exam ined in this study suggest th a t c e rta in histologic changes may be useful as early warning
indicators of stress. Inflam m atory re a c tio n in the gut and h e p ato p a n c re as a re notable exam ples of stress indicators. Similar views have also been o ffere d by Lee e ^ (1 9 8 5 ). They have also opined th a t the h e p a to p a n creas has im portant m etab olic functions and is an organ responsible to chem ical injuries involving organic and inorganic poisons, pesticides and biotoxins. They added th a t although th e shell is in d ir e c t c o n ta c t with th e environm ent, th e highly p ro te c tiv e e p ic u tic ie dim inishes its value under e x tre m e environm ental s tre ss conditions.
In th ese persp ec tiv e , th e p re sen t findings could provide a basic inform ation about th e im p a c t of this syndrom e on anim al which may subsequently lead to esta b lish m e n t of proper controlling and p re v e n tiv e m easures through b e tte r m a n ag e m e n t system s. In o rd e r to find o u t th e e x a c t cause of th e syndrome, and to understand th e problem precisely, fu rth e r studies like histo chem ical studies, m echanism of s o ft shelling by exposure to pesticides used in prawn c u ltu re , it's mode of a c tio n , role played by pH, th e calcium - phosphorus - c a rb o n a te relationships in th e ionic regulation of prawns and th e selenium c o n ten t of th e soil have to be taken up.
S U M M A R Y
1. Penaeid prawns of two species ie ., Penaeus indicus and Penaeus nnonodon showing soft-sheli syndrom e wre obtained from M atsyafed farm and p riv a te farm s of Vypeen Island, Cochin, for carrying out th e clinicopathological and h isto path olo gical studies. The study was conducted during th e period of May-August '88 when th e re was the o c c u rre n c e of "soft praw ns" in th ese c u ltu re fields.
2. H aem atological studies included T o ta l H em ocyte Count (THC) and e stim a tio n of haemolymph glucose level. The haem alogical e x a m i
nation rev ealed th e increase in th e num ber of circ u latin g h e m o cy tes (h em ocyto sis) and th e d e c re a se in th e haem olym ph glucose c o n te n t (hypoglycem ia) conditions in th e s o ft prawns. The co m p arativ e value with th e c o ntrol group of an im a ls have been p resented in (Table 1 & 2).
3. The histopathological changes w e re observed in th e exoskeleton, m uscle, gill, hep ato p a n c re as, h e a r t and gut.
The exoskeleton exhibited m a rk e d thinning or d e calcified natu re mainly in th e c alc ified layer of e n d o cu ticle . Focal d e tac h m e n t of tonofibrils from th e un calcified lay er was also a regular finding.
5. D eg en eratio n of th e epiderm al la y e r and vacuolation of the subepiderm al con n ec tiv e tissue la y e r was also n o ticed in soft prawns.
6. R em arkable changes w ere n o tic e d in the muscle tissue.
Progressive d e g e n e ra tiv e changes w e re evident in th e s tria te d m usculature. Focal to m u ltifo ca l n e c ro tic a re a s w ith occasional h em ocy tic '^ f iltr a tio n w ithout p re se n c e of any b a c te ria l or
protozoan a g e n t w e re invariably p re sen t throughout the m u scu latu re in e x tre m ely so ft anim als. The n e c ro tic changes w ere c h a r a c te riz e d by early d e g e n e ra tiv e change, loss of striation s, fra g m e n ta tio n s of myofibres, s e v e re s a rc o m e re a tro p h y w ith pykno. tic and k a ry o rrh e tic nuclei, d is tin te rg ra tio n , h y a lin izatio n and m in e ra liz atio n in the
n e c ro tic foci. F ocal a re a s o f Z e n k e r's type of necrosis w ere also evident.
7. Gil! a lte ra tio n s w e re c h a r a c te r iz e d by fla tte n in g of brachial epith elial cells and distension of th e o u te r lam e llar sinuses and thickcning of branchiaj sep tu m w ith focal a g g reg ation of h e m o cy tes.
8. H istopathologically, th e h e p a to p a n c re a s showed d e g e n e ra te d tu bu lar epithelial cells which showed h y p e rc h ro m a tlz a tio n w ith basophilic cy to p lasm ic inclusions in som e c e lls and ex ten siv e vacuolation.
9. The h e a rt ex h ib ite d m y ocard ial d e g e n e ra tio n w ith pyknotic and k a ry o rrh e c tic nuclei.
10. N ecrosis of th e c u tic u la r lining, w ith sloughing in som e places, h e m o c y tic in filtra tio n in th e subm ucosal layer and invagination of th e serosal layer w e re th e p a th o m orph o log ical a lte ra tio n s observed in th e gut. -Marked d a m a g e in th e ch itin o u s plates was n o ticed in th e filterin g organ.
• £ 0 .
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