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Results and Discussion_______

Results and Discussion

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The present study has been aimed at identifying the various endemic

diseases prevailing in cultured crustaceans, L. vannamei and P. monodon in

coastal area of Nellore District, Andhra Pradesh, India. The study has revealed

the prevalence of many endemic diseases even in exotic species, L. vannamei

and some new emerging diseases in P. mondon. The prominent of them are

as follows:

4.1 Diseases of Litopenaeus vannamei

4.1.1 White spot syndrome virus (WSSV)

The present study confirm first time in India, the prevalence of WSSV

infection in exotic species, L. vannamei in spite of its Specific Pathogen Free

(SPF) status of brood stock and high health post larvae. Unlike P. monodon, in

L. vannami white spots are not visible externally due to the white colour of the

shrimp (Figs. 1 and 2). In addition to that white spots are present in almost all

the cases of mortality might be due to the prevalence of other diseases like

vibriosis (Fig. 3), in healthy shrimps (Fig. 4), moulted shells (Fig. 5) and thus

making difficult to take decision of harvesting (Early harvest may prevent the

spread of the virus to the other ponds of that farm and among other farms and

can also reduce the loss). In several cases it was observed that the harvesting

has been performed without WSSV outbreak by just observing the white spots on

the carapace and even during the mortality because of other problems.


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In addition to above, the signs and symptoms like anorexia, redness of the

body (Fig. 1), antennae cut (Fig. 6), surfacing of shrimp, cannibalism (Fig. 7),

oedema in the cephalic region (Fig. 8), pre and post moult death were also

observed. However, these symptoms are not common in all the cases of WSSV

disease, similar signs were also reported in other diseases (Lightner et al., 2006)

(Table-1), making it difficult to take a decision. Hence, in the present study, an

attempt has been made with different available conformative diagnostic

procedures to evaluate and choose the best techniques as given below.

Polymerase chain reaction

Isothermal PCR

Histology

Rapid gill staining technique

Rapid dot kit

Shrimple kit

Morphology of white spots developoed

4.1.1.1 Polymerase chain reaction

The shrimps (10 samples) suspected to be infected with WSSV having

external symptoms of white spots and mortality were analyzed, and found that

only 5 samples are positive by PCR and Iscreen isothermal PCR system (Fig.

47). This clearly evidences that all the white spots observed in cultured shrimps

are not caused by WSSV. Even the ponds, which are positive by PCR and

isothermal PCR, survived for more than one month with out any visible

symptoms of morbidity and mortality by implementing the effective management


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practices. Thus the PCR and Isothermal PCR techniques are very much useful

for early diagnosis of the disease and to prevent the spread of the virus to other

ponds and other culture areas. In one particular case of our study, the post

larvae were observed positive for WSSV within 5 days of stocking and survived

for 35 days without any signs and symptoms of WSSV, this could be possible by

the efficient pond management practices. This study conform the PCR and

isothemal PCR can be used to detect and differentiate moratality caused by

WSSV.

4.1.1.2 Histopathology of gill

The same samples used for PCR were fixed in Davidson fixative and

processed for histopathology using hematoxilin and eosin stains. 5 samples

show the characteristric intranuclear cowdry A type inclusion bodies as shown in

fig.43 confirming WSSV infection. This study is time consuming procedure,

requires minimum 5 days time for getting final result and sometimes even

mortality may start before getting the final result, as the incubation time is less

than 5 days for some virulent strains of WSSV (OIE, 2009). The histopathological

symptoms of early stages WSSV resembles the histopathology of IHHNV in

formation of eosinophilic intranuclear cowdry A type inclusion bodies (OIE, 2009).

4.1.1.3 Rapid Gill staining technique

The gills from the moribund shrimps were collected and processed for

rapid gill staining out of ten samples only three were showed hypertrophied


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nuclei and it is also a characteristic feature of WSSV infection (Fig. 44). This

technique is cost effective but it requires more laboratory support.

4.1.1.4 Rapid Dot kit

Rapid dot kit is relatively high sensitive than shrimple kit and it is able to

detect the infection at least 15 days in advance before the onset of mortality.

Based on the intensity of the color development viral load can be assessed and it

is relatively easy and economical, at a time four samples can be analyzed at

pond side with in 10 minutes (Fig. 45).

4.1.1.5 Shrimple kit

Shrimple kit is a less sensitive technique than PCR, isothermal PCR and

rapid dot kit. It can detect the infection three to four days in advance and very

good tool for making a harvest decision. The procedure is relatively easy and kit

is little bit expensive than the rapid dot kit. Figure 46 illustrates presence of red

band at point T confirmataing WSSV infection.

The advantages and disadvantages of the different kits used in the

present experimental study were depicted in the table-2.

4.1.1.6 Chromatophore identification

The pleopods of the infeted shrimps with WSSV were observed under the

microscope for observing the chromatophores, they were first turned to yellow


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(Fig. 48), and the colour gradually turned to red (Fig. 49), indicating the

prevalence of viral stress in the cultured shrimps and this pattern of change in

colour can be taken as an indicator.

4.1.1.7 Microscopic identification of White spots

During the culture practice, in most cases the farmers rely on white spots

exhibiting on the exoskeleton of shrimp as the specific diagnosis symptom for

this most dreaded WSD and resort to an emergency harvest without knowing the

actual cause. Hence, in the present study, it has been aimed at studying the

morphology of white spots caused by different reasons to compare the etiologies.

The samples with visible white spots on the carapace were observed under the

microscope, the different morphological characterstics as follows.

The carapace of the L.. vannamei those are positive by shrimple kit for

WSSV was observed under microscope. The morphology of white spot

caused by WSSV in L. vannamei is with a hole in the center and radiating

lines (Figs. 51 and 52) is different from the white spot morphology of P.

monodon with dense melanized dots (Fig. 53).

Only one case of shrimp mortality suspected with Vibrio infection was

reported during the present study. The white spots that were present on

carapace were observed umder microscope for morphological studies.

The morphology caused by WSSV was different from the morphology of

white spot caused by vibrio spp. (Fig. 54). This morphology of the white


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spot caused by Vibrio resembles the one reported by Cyrille Goarant et

al. (2000) in L. stylirostris caused by bacteria.

The carapace of P. vannamei juveniles with confirmed WSSV infection

was observed for morphology. The morphology of white spot caused by

WSSV in juveniles (Fig. 55) was different from sub adults and lacking

central hole but theradiating lines were present.

Exuvia of the shrimp from the healthy ponds were collected, stored in the

same pond water and brought to the laboratory. The morphology of white

spot present in almost all the moulted shell of healthy ponds without

causing mortality was different with one or two homocentric rings and

darkened center with floral structure (Figs. 50 and 56) and resembles the

morphology of described by Wang et al. (2000) in P. monodon caused by

the Bacillus spp.

It is a well accepted fact that WSSV is endemic to India. During the

present study, it has been observed that due to lack of biosecurity at the farm

level, like crab fencing, bird fencing, pumping water directly from creaks without

treatment for disinfection and filtration to prevent the entry of carriers are the

main reasons suspected for the present outbreak of WSSV.

In a particular case during the present investigation, it was obsevred that

P. monodon shrimp entered the L. vannamei pond prior to stocking due to the

lack of filtration and for not treating the water in reservoir with disinfectants for

eradicating the cariers for white spot virus. White spot disease first affected the


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P. monodon and later spread to L. vannamei (Fig. 57). From the fig. 57 it was

clearly evident that the size of P. monodon is been bigger than the L. vannamei,

so it indicates that P. monodon entered the pond before stocking the target

species and first mortality has been started with P. monodon. The white spots

are clearly visible on the peeled carapace of the P. monodon, whose morphology

is almost similar to the morphology of white spot (Wang et al., 2000).

4.1.2 Infectious hypodermal and haematopoietic necrosis (IHHN)

Deformities are common in any organism without any except of L.

vannamei, several deformities were also observed in the present study and they

can be categorized basing on the location of deformity

- Deformities in cephalic region (Figs. 15-17)

- Deformities in abdominal region (Fig. 18)

- Deformities in tail region (Fig. 19)

- Deformities in the entire body (Fig. 20)

- Deformities in internal organs (Figs. 21 and 22)

Shrimps with the gross signs of runt deformity and high size variation are

tested positive by PCR and confirms the IHHNV virus infection for the first time in

India (Fig. 59). It has casued the runt deformity in the infected shrimp

(Kalagayan et al., 1991). The present study shows that all the deformities are not

caused because of IHHNV, and all the samples tested were not positive in PCR.

Size variation is very high in samples tested positive by PCR for IHHNV. In

support of this OIE (2009) reported that IHHNV affects only tissues of ectodermal


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and mesodermal origin. The internal organs like hepatopancreas are endodermal

in origin, it appears that it is not the target tissue for IHHNV.

It is an established fact that the runt deformity syndrome (RDS) is caused

by IHHN. Eventhough the farmers stock high heath seed produced by SPF

brood stock, the poor biosecurity in farms and high prevalence of IHHNV in P.

monodon that was previously cultured in this region could be the reasons for the

spread of the disease. Bell and Lightner (1987) stated that no mortality was

observed in RDS shrimps, this may holds good in our present observation. An

interesting observation is that the Metapenaeus Monoceros (Fig. 58), a weed

shrimp present in the same pond could not get RDS. Another observation in fig.

58, it was observed that the upper shrimp Litopenaeus vannamei with runt

deformity syndrome and lower shrimp Metapenaeus Monoceros without any

deformities, both these species looks alike and can be distinguished by the

absence of ventral teeth on the rostrum of Metapenaeus monoceros. This clearly

demonstrates that IHHNV can not cause runt deformity and mortality in

Metapenaeus monoceros. As the farmers are getting high health seed, which is

free from IHHNV, the only measure to control RDS is implementing the strict

biosecurity in the farms otherwise it will have serious impact on the production

and profitability of shrimp farming.

4.1.3 Vibriosis

It is very interesting to observe one case of mortality suspected due to

Vibriosis has been reported in L. vannamei juveniles of 50 days old. About 10


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dead shrimps were reported on the first day and 30 shrimps on the second day

with a gradual increased mortality. The moribund shrimp with opaque white

necrotic muscles (Fig. 28) and high number of motile bacteria was observed in

wet mounts of hepatopancreas and gut. Water quality parameters like pH (8.2),

total ammonia nitrogen (0.02 ppm), dissolved oxygen at 6 am (4 ppm), alkalinity

(240 ppm), calcium (110 ppm) and magnesium (170 ppm) were analyzed and

found to be within the optimal range. The shrimp tested negative for white spot

disease by Shrimple kit and confirms no involvement of WSSV. And the samples

of shrimp and water were tested for prevalence Vibrio species using TCBS agar

plating technique. The hepatopancreas was homogenized in normal saline, 10-4

serial dilution was plated by spread plate method and the Vibrio count of 2 x106

cfu/g observed. Gut contents were plated using a sterile loop and streaked on to

TCBS agar and incubated. All the colonies developed were green in colour

indicating the existence of non fermenting Vibrio species. Gram staining shows

that this bacterium is gram negative. Ventral surface of the shrimp abdomen

was sterilized using surgical spirit and 0.1 ml of haemolymph was collected with

a sterile syringe and plated using spread plate method with 10-2 dilution and

observed the development of 1.5X104 green colonies. This clearly evidences

that the mortality has been caused by systemic Vibrio infection. The isolated

Vibrio species was transferred to TSA by streaking to get pure colonies and

subjected to biochemical tests for species identification. The results showed that

motile, gram negative,oxidase positive, indole positive, gas from glucose

negative, decarboxilation of argining negative, lysine and ornithine positive,


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growth on 0 % and 8% NaCl and negative on 10 % NaCl, fermentation of glucose

and mannitol positive, lactose and sucrose negative (Table-3). The results

confirms that the bacterium to be as Vibrio parahaemolyticus (Aaccording to the

classification of Lightner et al., 1996). The results further evidenced that in the

present case, moribund shrimp had very high bacterial counts in hemolymph

suggestive of septicemia. The bacterial count in the hemolymph was more than

1.5X104 and the bacteria responsible was Vibrio parahaemolyticus. In support of

our observation, these bacteria have been reported to be associated with

vibriosis in Penaeus monodon (Lightner et al., 1992). There was no apparent

environmental stress other than high stocking density of 30 shrimps per square

meter, hence the mortality could be probably due to massive bacterial

septicemia.

4.1.4 Loose shell syndrome (LSS)

The present study reveals that the problem of loose shell problem is

relatively less in L. vannamei culture and the following signs and symptoms were

observed during the disease (Table-4).

Anorexia

Soft exoskeleton

Flaccid body

Reduction in weight due to degeneration of muscle.

Atrophy of Hepatopancreas with necrotic cells and very few fat bodies.

Delayed blood clotting


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Microscopic observation of wet mounts of Hepatopancreas, gut and

hemolymph shows motile bacteria.

The homogenates of hepatopancreas from LSS shrimps were plated on

TCBS agar at 10-4 dilution. Yellow colonies were developed on TCBS agar. The

bacteria developed on TCBS were transferred to TSA agar and subjected to

biochemical analysis for identification (Figs. 74 and 75; Table-3). They were

observed to be motile, oxidase positive, indole production positive, gas from

glucose negative, decarboxilation of arginine negative, lysine and ornithine

positive, growth on 0% , 8%, 10% NaCl, fermentation of lactose negative ,

sucrose and mannitol positive. The results show that the bacterium is Vibrio

alginolyticus (Lightner et al., 1996). This bacterium can be distinguished from the

one caused the acute mortality by the growth on 10% sodium chloride and

fermentation of sucrose. Because of its ability to ferment sucrose it produces

yellow colony on TCBS agar. The antibiogram shows that bacterium is sensitive

to oxytetracyclin at 4-ppm concentration and resistant to ciprofloxacin,

erythromycin, furazoladine, neomycine sulphate and chloramphenicol (Table-5).

4.1.5 Gill disease

In the present study, different types of gill diseases were observed in L.

vannamei culture ponds. They can be classified as: a) Brown gill caused by

protozoan fouling (Fig. 9), b) Brown gill caused by debris deposition (Fig. 9), c)

Black gill caused by melanzation of gill filaments (Fig. 10), d) Black gill caused by

unknown etiology (Fig. 11). The gill diseases, Brown gill caused by protozoan


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fouling, Brown gill cuased by debris deposition and Black gill cuased by

melanized gill filaments have been well studied and their disease symptoms and

control measures were established (Lightner et al., 1996).

The black gill without melanized lesions and unknown etiology has been

studied and analyzed. The clinical signs of the black gill observed during the

present study were: i) Dark black coloration of gills, ii) Shrimp die in the early

morning because of asphyxiation (Suffocation) in spite of high DO levels in the

pond, iv) Some severe cases >50% of the population has black gill, v) Reduced

feed consumption, vi) Gills are turning black again even after moulting. Shrimps

with these disease symptoms, when harvested, not been choosen for head on

packing and hence fetching very less price to the farmers.

Studies have been carried out to know whether the black gill was caused

by bacteria as reported in tea brown gill disease (Ruangpan et al., 1998). Gills

were collected form 10 live shrimp, washed and homogenized in normal saline,

plated on TCBS agar, incubated for 24 hours at 370C for Vibrio culture. No

bacterial colonies were developed, ruling out the role of Vibrio spp. Black gill

due to vitamin-C deficiency will never be occuring in shrimp pond using pelleted

feeds, all the pelleted feeds are included with this vitamin during the

manufacturing process and in addition the natural food present in the pond may

also supplement the same. The wet mounts of gills showed no melanization

except in one case where melanization in scattered dot pattern and wet mounts

of gill filaments showed melanized lesion (Fig. 62) only in this particular case. In


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ponds with dark black gills no external protozoan, filamentous bacterial fouling

and melanization found on the gill filaments (Fig. 63). And no fungal hyphae

were reported. The gill filaments are in violet colour resembling dilute potassium

permanganate, giving a suspicion that there might be iron and manganese

deposition. Similar observation was made by Gloter et al. (2004) in the caridean

hydrothermal vent shrimp Rimicaris exoculata.

Histology of hepatopancreas (Fig. 60) has showed the normal

hepatopancreatic tubules without any necrosis and nodule formation and gill

showed normal gill filaments with out any pathological changes like haemocytic

infilteration, hyperplasia, granuloma and melanization indicating the non

involvement of pathogens like vibrio , fungi and toxins (Fig. 61).

4.1.5.1. Chemical treatement for the black gill disease

For treatment of black gill disease, several attempts were made with

different available chemicals and their different combinations, which was

suspected to be caused by the iron and manganese deposits.

4.1.5.1.1 Benzalkonium chloride 50% (BKC 50%): Different concentrations of

BKC 50% was applied in culture ponds for effective treatment of Black gill

disease. The concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and1.0 ppm wetr used.

The results envisage that at 1 ppm dosage, less than 5% of the shrimps were

moulted 24 hours after the treatment and there was a recovery of less than 10%.


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4.1.5.1.2 Iodophore (10%): Different concentrations of iodophore were tried for

effective control of black gill disease by using 0.1, 0.2, 0.3, 0.4 and 0.5 ppm

concentrations of iodophore was applied directly to the affected ponds and found

that they were ineffective.

4.1.5.1.3 Formaldehyde (37%): For treatment of black gill disease, different

concentrations of formaldehyde at 5, 10 and 15 ppm levels were tried and there

was no response to the treatment with formaldehyde.

4.1.5.1.4.Benzalkonium Chloride and Formalin combination: The

combination of 1 ppm of BKC 50% and 5 ppm formalin were also tried for

treatment of black gill disease and found that there was no response for this

treatment.

4.1.5.1.5 Copper Sulphate Pentahydrate (CuSO4 5 H2O): The concentrations

of 0.1, 0.2, 0.3 and 0.4 ppm copper sulphate pentahydrate was also used for

treatment of black gill disease and there was no response.

4.1.5.1.6 Ethylene Diamine Tetra Acetic Acid – Di Sodium Salt (EDTA): The

ethylene diamine tetra acetic acid-Disodium salt has been tried to control the

black gill disease in the concentrations of 0.2, 0.3, 0.4 and 0.5 ppm and found

that it couldnot get any positive response.

4.1.5.1.7 Hydrogen Peroxide (50%): Attempts were made with hydrogen

peroxide in different concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 ppm were tried

and at 0.4 ppm concentration there was a partial recovery (20 to 30%) and at 0.5


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ppm, very good recovery (80%) has been observed within 48 hours of its

application.

4.1.5.1.8 Combination of BKC 50% and Hydrogen Peroxide: The combination

of 1 ppm of BKC 50% and 5 ppm of hydrogen peroxide was tried for control of

black gill disease and very good recovery (more than 90%) was noticed (Fig. 64).

From all the above experiments, it was clearly evident that 1-ppm of BKC

50% and 5 ppm of hydrogen peroxide has been found to be very good

combination for obtaining good recovery, due to its ability in oxidation of iron and

manganese by the peroxide and killing of sulfate reducing bacteria and surfactant

activity by the BKC 50%.

4.1.6 Body cramp

During the culture practices of L. vannamei, it was found that prevalence

of body cramps during the early morning time of winter season, when the water

temperature is 250 C ruling out the possibility of high temperature stress. During

the study, it was noticed that this problem was more during the early stages of

culture. There was no relation to the salinity of the pond water as this problem

was reported at all salinities. Most of the cases, there was a high dorsal

abdominal flexure and sudden development of opaqueness in the abdomen of

the shrimp. Partially cramped shrimps (Fig. 12), swim with a humped abdomen

whereas fully cramped individuals (Fig. 13) lie on the sides at the pond/tank

bottom. The development of sudden focal white opaqueness indicating that


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probably, the shrimp could not get the sufficient oxygen for the quick movement

in jerky escape reflex at abdominal tissue. It resembles to the muscle

opaqueness developed during hypoxia. The study ponds were treated by

applying commercially available mineral mixture, agrimin manufactured by Virbac

India limited at concentrations of 5,10,15,20 g/kg of feed and tried 1, 2, 3 and 4

times per day. There was no respoonse at any of the concentration and time

periods tried (Table-6). Ponds were also treated with magnesium sulphate

(Epsom salt) at 20 g/kg of feed at different time intervals (Table-7), interestingly,

the cramps were not reported in two days when 20 g/kg feed was applied in all

the four meals and a good recovery within 15 days was observed even when one

meal per day and once in three days was provided. The best practice that can

be recommended for initial application in all the meals for two days and later

once in three days to avoid the reoccurrence of the problem.

4.1.7 Gas bubble disease

Because of the supersaturation of atmospheric gases, usually nitrogen

and occasionally oxygen. The disease often occurs in tanks receiving with

supersaturated gas content. The wet mounts of suspected shrimp gill filaments

were observed under microscope (4x) and the gas bubbles were observed

indicating the prevalence of gas bubble disease (Fig. 65). Similar observations

on occurrence of gas bubble disease made by Suplee et al. (1976). Attempts

were made to get relief of this disease by varying the aeration levels in cultured

ponds.


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4.1.8 Blisters and Tail rot

Two types of blisters were reported during the present study, blisters in

cephalic region (Figs. 23-26) and blisters in uropods (Fig. 27) during the culture

practices of L. vannamei. The present study demonstrates a clear evidence how

a blister was formed and documented both in L. vannamei and P. monodon. It

was observed that the blisters in Uropods were formed in ponds, where there

was a poor pond bottom and floating lablab. Applying probiotic bacteria to

reduce the organic laod and pathogenic bacteria as suggested by Moriarity,

(1999) to reverse this condition is a common practice among the farmers and it is

a best alternative for their effective treatment. Uropods appear to be in red

colour due to the expansion of chromatophore (Fig. 66) in the early stages,

indicating the prevalence of stress. Due to the subsequent necrosis caused by

chitinolytic bacteria in Uropod region leads to tail rot (Figs. 67 and 68). The fig.

61 shows the early stage of tail rot. Blisters in cephalothoracic region are formed

due to the damage of the epithelium and subsequent flow of the blood in

branchial chamber. In some cases the blood clots are turned black in colour due

to the melanization. The present study shows that the blisters in cephalic region

are formed due to the flow of blood between cuticular epithelium and exoskeleton

and subsequent clotting and melanization (Fig. 69). The wet mount of blister

shows hemocytes embedded in the matrix of clotted proteins (Figs. 70 and 71).

It was further noticed that most of the time blisters were observed immediately

after the low dissolved oxygen shock and high ammonia stress. Both these

conditions cause stress on gills. It was reported that the prevalence of hypoxia


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significantly decreases the total haemocyte count in P. stylirostris and stressed

shrimp became susceptible to Vibrio alginolyticus infection (Le Moullac et al.

1998). In the present study also, it was observed that the blisters were filled with

cyanotic fluid has high bacterial counts, less hemocytes and always associated

with mortality. The formation of blisters is probably due to the osmotic pressure

at the time of stress and due to the prevalence of hypoxia and high ammonia. In

these two conditions, shrimps need to spend more energy to combat the stress

and to maintain the osmotic balance. When shrimp cannot spend sufficient

energy for maintaining osmotic balance the water present in the pond will imbibe

into the shrimp body causing edema. At 25 ppt salinity, the osmolarity of the

water is similar to the iso-osmotic point of the P. vannamei hemolymph (bodily

fluids). The possibility of occurrence of blisters is more in water with salinity less

than 25 ppt. In addition, if septicemia is associated with it, reduces the hemocyte

counts. So it can carry less oxygen and less energy will be generated when its

requirement is more. In such cases hemolymph does not clot as the

haemocytes are damaged and may cause mortality. Another interesting

observation is that some blisters protrude outward and some protrude inward.

Normally the crustaceans are covered with hard exoskeleton so the blister will be

protruded inwards by pushing the cuticular epithelium inside as appears in the

figure and there is only possibility of protruding by pushing the cuticle outwards

is, blisters formed at the post moult stage as the cuticle will be very soft at that

time. The differences between two types of blisters were presented in table-8.


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4.1.9 Melanized lesions on the body

Shrimp with melanized lesions is one of the major symptoms in the

transition phase of TSV infection as reported by Hasson et al. (1999). Both L.

vannamei and P. monodon with similar symptoms from the same area were

tested with MultiVirTM system shows that there is no involvement of TSV (Figs.

72 and 73) and also there was no mortality associated with the symptoms. The

fig. 72 shows the biochip without characteristic dots on the upper right side

indicating the TSV negative.

4.1.10 Shrimp with different pigmentation

Shrimps with different pigments were observed with golden yellow colour

was noticed in culture ponds. They are very healthy and grow normal (Fig. 76). In

an interview with shrimp news in Jim Wyban (2008) discussed that Oceanic

Institute is looking at the genetics of shell colour and shell patterns and trying to

breed the golden yellow Vannamei and developed a special brand of golden

Vannamei. This particular type was observed 1 in 100000 shrimp even in

Oceanic Institute, Hawaii. Similarly, shrimp with blue pigmentation (Fig. 77) were

observed that are also very healthy and growing normally, indicating that it is not

a disease condition. Some pigmentation is due the colour of the water and some

are genetically or so. Pigmentation due to water colour changes are changed as

the water colour changes from time to time. Pigmentation due to genetic reasons

will not change with water colour.


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4.1.11 Low dissolved oxygen

The dissolved oxygen levels in early morning should be more than 3 ppm.

However, in the present study it was evident that the DO levels below 2.5 ppm,

reduces the feed in take by shrimp and at below 2 ppm, the shrimp comes to

surface where the dissolved oxygen levels are relatively high due to the transfer

of atmospheric oxygen (Fig. 78). Prolonged exposure to this situation, may

cause severe mortality. Opaqueness of the tail muscle and necrosis of the

tissues with reddishness, similar to the colour of the cooked shrimp, in the last

abdominal segments (Fig. 14) was observed in the survivors after a severe DO

problem. Even though the symptoms are similar to IMN disease caused by

IMNV and white tail disease caused by PVNV but mortality early in the morning

and recovery after the installation of paddle wheel aerators was observed (Boyd,

1988), which is the most efficient method of aeration in ponds, indicates that the

problem is only due to low dissolved oxygen. Wet mounts of skeletal muscles

show normal striations and pattern of mortality indicating no involvement of IMNV

virus.

4.1.12 External fouling

During the wet mount microscopy of pleopod, few protozoans like

Zoothamnim species (Fig. 80), Epistylis species and one unidentified species

were found externally on the surface of the cultured organism. However, there

was no mortality in any case as these are secondary pathogens.


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4.1.13 White fecal matter

Even though white fecal matter was noticed in L. vannamei culture, it was

observed that it was not associated with the loose shell syndrome as in case of

P. monodon. The amount of fecal matter floats on the water surface is very less

when compared to P. monodon culture ponds. This particular problem was

noticed in ponds with very high blue green algae. The wet mounts show the

necrotic hepatocytes as reported in case of P. monodon. In support of our

observations, similar etiology was also reported by Niti Chuchrid et al . (2009).

4.1.14 Growth comparison of L.vannamei in summer and winter seasons

Comparing the sampling data obtained in two different seasons in L.

vannamei culture ponds, it was clearly evident that there was a marked

difference in growth between the two seasons. It was also observed that as the

temperature increases the metabolic rate also increased during the summer

season. The average temperature during summer season is 310C and during

winter is 27.50C. The daily growth during summer is 0.28 g and during winter is

0.18 g. The results demonstrates that the better growth rates are directly

proportional to temperature and it is in agreement with the findings of Wyban et

al. (1995) (Table-9; Graph-1). Statistical analysis also showed that there was

highly significant correlation between the number of the days and pond type. It

was also observed that growth is higher in summer ponds when compared to

winter ponds. This was evident from the statistical point of view also i.e. from

descriptive satatical table (Table 9a). There was a significant difference between


111

the magnitude of growth during summer and winter as ‘F’ calculated values were

considerably larger than the ‘F’ critical values.

4. 2. Diseases of Penaeus monodon

4. 2.1. Effect of different feeds on swollen hind gut syndrome (SHG) in shrimp larvae

A critical analysis has been performed to study the relationship between

the incidence of swollen hindgut and hatchery feeds used in P. monodon culture

ponds. There was a clear indication that the feeds having coarse texture (Table-

10) are damaging the gut of larvae and causing inflammation of the gut wall and

this phenomenon was much more in hindgut where the muscles were thick (Fig.

81). The present study revealed that the prevalence of SHG was relatively less

with live feeds, microencapsulated feeds and liquid feeds. Incidence of SHG was

more with microparticulated feeds and flake feeds which were having coarse

structure. Poor quality feed always causes water quality problems and always

ammonia levels were very high (>2ppm), and when there was less water

exchange and poor water quality there is always a possibility for larvae eating

some moulted shells, as their availability increases (Fig. 82) and they were not

removed by water exchange. Some parts of moulted shells like rostrum are very

hard (Fig. 83) and may causing damage to the gut wall and excreting the

undigested part of the shell through the hindgut causing the damage to it. In

contrary the feed with fine structure (Figure 84) does not cause any damage to

the gut wall and execrated easily with out causing the damage (Fig. 85). On other

hand the liquid feed also does not cause any damage to the gut wall (Fig. 86),


112

because of its soft nature as they all are emulsions. Immediate result of the

damage to the gut wall was inflammation and formation of folds in the gut wall

(Fig. 87). The effects of SHG seed stocked in grow out ponds exhibit poor

growth, very high size variation, high incidence of bacterial diseases, high

incidence of protozoa and filamentous bacteria, early occurrence of loose shell

syndrome, very poor final survival and may result finally in less productivity.

4.2.2. Mortality caused by unidentified salt deposition and its control measures

The unidentified deposits on the exoskeleton of the shrimp, P. monodon

were white in color with rough body texture. Salt deposits were present on entire

body including eyes and antennae. Partial molting was noticed in some cases

(Fig. 34). Pre and post moult mortality was also noticed in several cases (Fig.

35). Anorexia and swollen gills were also noticed and there were deposits on

gills and it was observed in all the stages of culture system. The water quality

parameters were analyzed in culture ponds and it was observed that high pH of >

8.5 and high carbonate alkalinity of > 40 ppm as CaCO3 was noticed in all most

all the cases. For diagnosis, the shells were dipped in dilute hydrochloric acid,

deposited material was dissolved with effervescence (Figs. 88 and 89), indicating

that the salt deposited was calcium. The fig. 88 reveals the microscopic structure

of calcium deposits on uropods and fig. 89 illustrates the efferevescence caused

by the neutralization by dilute acid. The results of treatments tried showed that

there was no response to the treatment by benzalkonium chloride 50% and there


113

was a recovery in all the treatments with HCl 1ppm, citric acid 2 ppm, EDTA 0.4

ppm, Alum 4 ppm, B-green 0.1 ppm and Neutral 0.5 ppm (Table-11).

4.2.3 Influence of calcium and magnesium ratio on the survival of P. monodon in culture ponds

After one month of the culture, a study has been performed on the shrimp

ponds with poor survival. It was observed that hapa survival for 72 hours post

stocking is 80%, whereas in other ponds stocked with same seed, the survival

was 92-98%. The stocking density was of 4-numbers/square meter. In the

check tray observation during first week, 7-8 shrimp, second week 4-5 shrimp

and third week 1-2 shrimps were observed. Whereas in other ponds, 20 to 40

shrimps were observed in check tray. Further it was noticed that the growth was

very poor when compared to other pond stocked with same seed. To understand

the problem, feeding was stopped and stocked with 50 numbers of seed from a

healthy pond and stocked in a hapa of problematic pond and observed the

survival every day and finally 30 shrimp were survived after the 7 days of culture

and it was notice that all the dead shrimps were partially moulted. The

observation clearly evidenced that there was a mortality at each moulting

attempt. As the moulting frequency was more in early days, mortality was also

observed to be more. The studies on zooplankton revealed that the other

crustaceans like copepods and vertebrates, like tadpoles were survived in the

pond indicating that there was no involment of toxic compounds. The wet mount

of hepatopancreas showed very high number of necrotic cells (Fig. 36). No

moribund shrimp and cannibalism indicates the gradual and continuous internal


114

mortality in the pond. Even the shrimp survived were weak and blue in color with

soft exoskeleton, with deformities and very poor growth was reported (Fig. 37).

The analysis of water quality parameters in pond with very low survival after first

month of culture showed that the calcium levels were more than the magnesium

levels in all the cases (Table-13) and the parameters like pH, total ammonia

nitrogen and nitrate were within the acceptable limits.

These ponds were attempted for treatment with application of magnesium

sulphate and the water was tested again for calcium and magnesium levels after

the treatment. It was observed that the stocking ponds with magnesium levels

higher than the calcium levels gave better survival suggesting that the calcium

and magnesium ratio of the water is the important factor that affecting the

survival of the shrimp. This was in agreement with the earlier findings of Pequeux

(1995), who stated that Mg2+ ions essential for normal growth, survival and

osmoregulatory function of crustaceans. Magnesium also plays a crucial role in

the normal metabolism of lipids, proteins and carbohydrates serving as a cofactor

in a large number of enzymatic and metabolic reactions (Davis and Lawrence,

1997). Results from our experiment were also inagreement with these

observations. In support of this, studies on evaluating the impact of Mg2+ and

other ions on survival of postlarvae acclimated to short-term and long-term to low

salinity water from various West Alabama farms also supports of our observation

(Saoud et al., 2003; Davis et al., 2005). In the present study it appears that

similar effects of depressed Mg2+ in postlarvae and adult shrimp was observed.


115

4.2.4 White gut and white fecal matter

The present study clearly evidences that two conditions i.e. white gut and

white fecal matter were leading to prevalence of loose shell syndrome. It was the

second major problem in P. monodon culture farms next to WSSV and the exact

etiology was not known. The differences between the white gut and white fecal

matter were studied and reported (Table-14). The gut wall of the shrimp P.

monodon with white fecal matter was translucent and looks white due to the

colour of the fecal matter (Figs. 90 and 91). The gut of the shrimp affected with

white gut disease was found to be opaque and thick (Fig. 92). The wet mount of

the white faecal matter have showed the sloughed off hepatocytes (Fig. 93). The

wet mount of hepatopancreas affected with white faecal matter showed necrotic

cells (Figs. 94 and 95). The histopathology of hepatopancreas of the affected

shrimp with white faecal matter showed necrotic tubules (Fig. 96). And the

histopathology of the white gut shows that the whiteness of the gut caused by

necrotic cells of mucosal epithelium and thickness caused by haemocytic

enteritis, which was clearly visible in the mid sagittal section of the gut wall (Figs.

97 and 98). In consonance with our ourservations, Lightner et al. (1978) reported

the similar condition in blue shrimp, suggesting the blue green algal toxins

sloughing off the midgut epithelium. It was observed that there was no

melanization or nodule formation suggesting the non-involvement of bacteria,

however, when the homogenates of hepatopancreas was plated on TCBS agar,

development of yellow colonies shows the involvement of Vibrio species.

Probably the lipid droplets present in the sloughed off hepatocytes might be


116

causing the fecal matter to float as the density was less. On the other hand in

normal conditions, sinking faecal matter was observed due to the undigested

sinking pellets.

4.2.5 Control of Gregarines

During the present investigation, the cultured shrimps with poor growth

and yellow colour gut contents were selected and their wet mounts of gut

contens were observed under the microscope, which has demonstrated the

various stages of gregarines (Fig. 99). Syzygy is the process in which two mature

trophozoites pair up before the formation of a gametocyst. Only three cells can

associate to form a syzygy (Lightner, 1996). It was found that in the present

study more than four cells forming syzygy (Figs. 100 and 101). Unlike as

reported earlier, it was clearly evident that the syzygy can be formed in any

fashion (Figs. 102 and 103) and the fig. 102 shows the involvement of three

trophozoites in syzygy and fig. 103 shows another trophozoite forming syzygy

with the same on lateral side.

4.2.5.1 Treatment for control gregarines population

The ponds with gregarine infection were treated with different herabal

products like turmeric powder, garlic paste and Allicin through feed at different

concentrations. Turmeric was given in feed for 10 days at 5, 10, 15 and 20 g/Kg

of feed and two times per day. The wet mounts after the treatment showed still a

good number of gregarines in the gut, envisaging that turmeric can not control


117

the gregarines. The garlic paste was given in feed, daily 2 times for 10 days at

10, 20, 30, 40 and 50 g/Kg. There was a reduction in the count of gregarines in

gut scrapings at 40 and 50 g/ kg of feed. It was evident that garlic could not

eradicate gregarines completely at the dosages tried, however it has positive role

in control of gregarines. Our attempts with Allicin plus an herbal preparation by

Zymo Nutrients Pvt Limited, Mangalore, given in two meals for 10 days at 5, 10,

15 and 20 g/Kg of feed. No nematodes were noticed after the treatment with 20

g/Kg of feed. However, there was reoccurrence of the gregarines after three

weeks of first dose of Allicin plus, this reinfection may be due to the intermediate

host present in the same pond. In order to control the intermediate hosts like

clams, snails and chironomous larvae copper sulphate was used along with

second dose of Allicin plus at 0.1, 0.2, 0.3, 0.4 and 0.5 ppm concentrations.

There was an effective control of gregarines and no recurrence of the problem.

Thus, the experiment clearly showed that the Allicin plus application in feed at 20

g/Kg of feed for 10 days along with application of copper sulphate dose of 0.5

ppm effectively can control the gregarines in the shrimp culture ponds. This was

confirmed by microscopic examination of wet mounts after the treatment and

observed dead gregarine (Fig. 104).

4.2.6 Immunostimulant properties of Tinospora cordifolia

A study has been performed to evaluate the immuno stimulant properties

of Tinospora cordifolia. The extract of Tinospora cordifolia was supplemented

through top dressing the feed at 2 and 4 g/Kg for three and five days


118

respectively. The observations indicate that the feed consumption has been

increased in both the treatment ponds, very good moulting was observed in

treatment ponds and controlled the protozoan fouling in culture ponds.

4.2.6.1 Prophenol oxidase activity

The prophenol oxidase activating system is an important immune

response against various microbial infections. The results illustrates the

significant increased levels of prophenol oxidase 26 units/min/mg in three days

and 35 uints/min/mg in five days with suplementaion of 2 g/Kg of feed and 33

units/min/mg in three days and 53 units/min/mg in five days with suplemtnation of

4 g/Kg of feed when compared to 20 units/min/mg (Table-15). The results

showed that there was a significant difference among the different doses and

time periods (Table 15a).

4.2.6.2 Total heamocyte count and percentage of granulocytes

The results envisage that there was an increase in the levels of total

hemocyte count 2.52 X106 at 2 g/Kg and 3.12X 106 at 4 g/Kg feed for five days

as compared to control 2.08 X 106. Similar results were also observed in

percentage of granulcoytes, after 5 days of treatment, in control only 7

percentage of granulocytes were reported, where as in the treatment group with

2 g/Kg it was 8.5 and 9 percent with 4 g/Kg of feed was observed.


119

4.2.6.3 Bacterial clearance

The improvement in bacterial clearance after the 5 days of treatment with

T. cordifolia in both the test groups was studied (Table-17). The shrimp were

injected with bacterial concentration (2.7x109) and an increase in concentration

of bacteria in haemolymph of control was observed immediately after the

injection (3.5x107). In the same control group the baterail count 30 minutes post

injection was observed to be 1.8 x 106. Results of test group with 2 g /Kg feed

for 5 days, injected with a concentration of bacteria 2.7x109 and immediate

increase in concerntaion of bacteria in haemolymph was observed (3.3x107).

After 30 minutes of post injection the bacterial count was noted as 4.1x105. With

the test group at 4 g/Kg feed, the intial concentration of bacteria injected was

2.7x109. Bacterial concentration in haemolymph immediately after injection was

observed to be 2.9x107 and after 30 minutes post injection was 5.1x 104. The

concentration of detectable live bacteria in hemolymph was very less in both test

groups relative to control and it was evidencing that T. cordifolia clearly

stimulating the non-specific immune system of the shrimp P. monodon. This

cleared bacteria in circulation will be uptaken by the different parts of the body

(Martin et al. 2000). The increase in the prophenol oxides activity (Fig. 105), an

increase in the hemocyte count (Fig. 106), increase in number of granulcoytes

(Figs. 107-109) and visible improvement in the physical activity envisaging that

the test product, T. cordifolia significantly increasing the immune response of

the shrimp.


120

4.2.7. Treatment of iron and manganese deposits on exoskeleton

The efficiency of different chemicals in controlling the iron and manganese

deposits on the exoskeleton of the shrimp P. monodon was studied. The ponds

were treated with Disodium salt of ethylene diamine tetra acetic acid (EDTA)

concerntations of 0.1-0.5 ppm were used. There was no significant change.

Similarly the treatment with benzalkonium chloride (50%), which has surfactant

property and induces the moulting has been tried with different concentrations of

BKC (50%) (0.1 -1.0. ppm) and it was found that there was a partial recovery,

only moulted shrimps were clean. Treatment with agriculture lime (calcium

carbonate) at concentrations 10 – 20 ppm was not responded. And treatment

with citric acid at concetnations of 0.1 –1.0 ppm levels, no response was

observed. Hydrogen peroxide at different concentrations (1,2,3,4 and 5 ppm)

evidenced a good response with 5 ppm concentration.

4.2.8 False negatives in PCR diagnosis of Monodon baculovirus (MBV) and PCR standardization

As the positive control is always positive in all the PCR tests performed,

showing that the reagents are working and the failure of PCR reaction in sample

vials indicates the possible presence of inhibitors or poor quality and quantity of

DNA. And the PCR test was performed with different sample volumes,

increased number of PCR cycles and different methods of DNA extractions

(Table-18). Initially, 10 samples were analyzed as per the method prescribed in

Bangalore Genie MBV PCR kit using lysis buffer for extracting DNA (Crude


121

DNA). In first step all the samples were negative and in second step, only 2

samples were positive showing only 20% of sensitivity.

Increased the number of cycles from 25 to 27 and tested 10 samples, first

step all the samples were negative and second step only 3 samples were

positive.

For all the 10 samples, the sample volume was increased from 20 to 50

post larvae. First step, all the samples were negative and second step

only one sample was positive.

For all the 10 samples, the sample volume was reduced from 20 to 10

post larvae. First step-all the samples are negative and second step only

one sample was positive.

For all the 10 samples, only hepatopancreas was collected, which was the

tartget organ for MBV. Hepatopancreas was collected form 20 post larvae

and the test was run. In first step, all the samples are negative and in

second step three samples are positive.

The DNA extraction procedure was changed. Phenol chloroform method

with incubation in protinase K was followed. Out of the 10 samples in first

step, three samples were positive and in the second step all the 10

samples were positive


122

As the phenol-chloroform method was time consuming, tried with Qiagen

Spin Columns and 10 samples were tested. In fiirst step, 8 samples were

positive and in second step all the ten samples were positive

The results clearly evidences that the procedure of phenol-chloroform and

spin column methods were the best methods for DNA extraction for decetion of

MBV. The quantity and quality of the DNA was assessed in each process of DNA

extraction (Table-19). The results in table-19 showed that the absorbance ratio

at 230/260 ranging 0.62 to 0.4, showing the good sensitivity of the kit. And the

absorbance ratio at 260/280 ranging at 1.8 and 1.89 evidencing the good

sensitivity. As the absorbance at 280 nm was used to determine the protein

concentration, the experiment clearly envisaging that lesser the protein

contamination in the sample DNA, higher the sensitivity of the assay.

Diagnostic sensitivity is the proportion of known infected samples that test

positive. Infected animals that test negative are been called as false negatives.

Known positive samples are referred as reference samples. Diagnostic sensitivity

was calculated for different methods of DNA extraction (Table-20). The data in

the table-20 evidencing that the diagnostic sensitivity is 100% with phenol

chloroform and spin column methods of DNA extraction.

4.2.9 Blisters

The study on the types of blisters and their formation has envisaged that

there are two types of blisters, fluid filled blisters as shown in fig. 110 and the


123

mucous filled blisters as illustrated in fig. 111. Based on the place, three types

of blisters were reported during the study, blisters in cephalic region (Fig. 112),

blisters in abdominal region (Figs. 113 and 114) and blisters in tail region (Fig.

115). Fluid filled blisters (unclotted) were always associated with mortality. The

fluid was collected and observed under microscope. It showed relatively less

number of haemocytes and most of the haemocytes were damaged as showed

in fig.116, motile bacteria was noticed which indicates the bacterial etiology

(Septicemia). This fluid has never been clotted. Then, the fluid was collected and

plated on to TCBS agar and incubated. Very high number of bacteria were

developed indicating the systemic Vibrio infection.

Mucous filled blisters were never been associated with mortality. This was

clearly due to the flow of haemolymph as evident from fig. 117, subsequent

clotting and melanization. These blisters were associated with melanization and

lesions in the affected area as marked in fig. 118. The haemolymph was plated

on to TCBS agar and no Vibrio colonies were developed. Haemolymph clotting

time was less than one minute. This type of blisters normally goes away in the

next moulting. To verify the process of formation of blister filled with mucous, the

hemolymph was collected and placed on a clean glass slide and allowed to clot.

After 12 hours, this exactly resembles the blister formed (Figs. 119 and 120).

Differences between the various blisters were presented in table-8.


124

Table 1: Signs and Symptoms observed in White Spot Syndrome and other diseases with similar Observations in L. vannamei

Signs andSymptoms

No ofcases

Other problemswith similar symptoms sign

Redness 15/20 cases Low DO, Vibriostress

Antennae cut 10/20 cases VibriosisWhite spots on cuticle

20/20 cases Bacterial spot

Surfacing 10/20 cases Low DOCannibalism 18/20 cases All diseasesOedema in cephalic region

2/20 cases Low DO

Pre and post molt death

10/20 cases Ammonia toxicity, DO deficiency

Anorexia 15/20 cases All cases of stress


125

Table 2 : Comparison of different Diagnostic procedures for WSSV in L. vannamei

Diagnostic procedure

Advantages Disadvantages

PCR Highly sensitive Requires sophisticated lab

Isothermal PCR Highly sensitive Requires I screenOven

Histology Confirmatory Requites lab/timeConsuming

Rapid gillStaining

Confirmatory Requires labsupport

Rapid dot Good for harvesting decision

Storage at 40C

Shrimple kit Good for harvesting decision

Relatively expensive


126

Table 3: Biochemical tests for identification of Vibrio species isolated from L. vannamei infected with systemic Vibrio infection and Loose shell syndrome

Biochemical Test Systemic Vibriosis

Loose shellSyndrome

Colony on TCBS Green YellowOxidase + +Motile + +Indole production + +Gas from glucose - -Decarboxilase- arginine - -Decarboxilase- lysine + +Decarboxilase - ornithine + +Growth on 0% NaCl + +Growth on 8% NaCl + +Growth on 10% NaCl - +Fermentation of glucose + +Fermentation of lactose - -Fermentation of sucrose - +Fermentation of mannitol + +


127

Table 4: Characteristic features of Loose-shelled L. vannamei

Observation Normal shrimp Loose shell shrimpBody Rigid FlaccidHepatopancreas Normal in size AtrophiedLipid droplets Very high Very lessBlood clotting time < 1 minute DelayedVibrio counts in HP 2.1x 102 3.1x 104

Vibrios count in HL. Absent 4.1x 102

Table 5: Antibiogram of Vibrio alginolyticus isolated from loose-shelled L. vannamei

Antibiotic used Concentration Minimum inhibitoryconcentration

Ciprofloxacin 4 ppm ResistantOxytetracyclin 4 ppm SensitiveErythromycin 4 ppm ResistantFurazolidone 4 ppm ResistantNeomycin sulphate 4 ppm ResistantChloramphenicol 4 ppm Resistant


128

Table 6: Concentration and time periods of trails with Agrimin as feed supplement in L. vannamei

Table 7: Supplementation of magnesium sulphate at different time intervals in L. vannamei

One meal/day

Two meals/day

Three meals/day

Four meals/day

Every day Recovered Recovered Recovered RecoveredAlternative daysRecovered Recovered Recovered RecoveredOnce in threeDays Recovered Recovered Recovered RecoveredOnce in fourDays

Not recovered

Not recovered

Partial recovery Recovered

Once in fiveDays

Not recovered

Not recovered

Not recovered

Partial recovery

Once in sixDays

Not recovered

Not recovered

Not recovered

Not recovered

Once in a weekNot recovered

Not recovered

Not recovered

Not recovered

No. oftimes/day 5 g/kg 10 g/kg 15 g/kg 20 g/kgOne meal per day

Not recovered

Not recovered

Not recovered

Not recovered

Two meals per day

Not recovered

Not recovered

Not recovered

Not recovered

Three meals per day

Not recovered

Not recovered

Not recovered

Not recovered

Four meals per day

Not recovered

Not recovered

Not recovered

Not recovered


129

Table 8: Comparative analysis of different Blisters in L. vannamei and P. monodon

Parameter Liquid filled Blister Jelly BlisterAssociation with mortality Yes NoBlood clotting No YesHemocytes Less and damaged NormalMotile bacteria Present AbsentMelanization Yes YesRelation to stress Yes Yes


130

Table 9: Comparison of growth rates between Summer and Winter seasons in L. vannamei

Days S-Pond-1 S-Pond-2 W-Pond-1 W-Pond-241 5.4 5.8 4.7 4.448 8.1 7.8 6 7.255 9.9 10 8.3 9.362 12.8 13.7 9.3 10.569 14.5 15.8 11.8 13.876 17.4 18.3 12.9 14.884 21.3 21.5 14.1 15.890 23 23.1 15.4 1797 25.6 26.1 16.8 18.3104 29.1 28.3 18 19.5111 31.5 31 19.2 21118 21.8 21.8125 23 23.5132 24.5 25.3

Days = Days of culture

S-pond 1 = Pond-1 in Summer culture

S-pond 2 = Pond-2 in Summer culture

W-pond 1 = Pond-1 in Winter culture

W-pond 2 = Pond-2 in Winter culture


131

Table 9a: Statistical analysis of growth rates during Summer and winter in both the ponds

s-pond 1 s-pond 2 w-pond 1 w-pond 2Mean 18.05454545 18.30909091 14.7 15.87143Standard Error 2.630429057 2.555224756 1.672663 1.679136Median 17.4 18.3 14.75 16.4Standard Deviation 8.72414622 8.474721771 6.258533 6.282752Sample Variance 76.11072727 71.82090909 39.16923 39.47297

Kurtosis-

1.244774546 -1.24660462 -1.01744 -0.76878

Skewness 0.113673358-

0.029877834 -0.03697 -0.33348Range 26.1 25.2 19.8 20.9Minimum 5.4 5.8 4.7 4.4Maximum 31.5 31 24.5 25.3Sum 198.6 201.4 205.8 222.2Count 11 11 14 14Confidence Level(95.0%) 5.860961155 5.69339553 3.613569 3.627553

t-Test: Two-Sample Assuming Equal Variances

S- POND 1 W. POND 1Mean 18.05454545 14.7Variance 76.11072727 39.16923077Observations 11 14Pooled Variance 55.23075099Hypothesized Mean Difference 0Df 23t Stat 1.120298006P(T<=t) one-tail 0.137074569t Critical one-tail 1.713871517P(T<=t) two-tail 0.274149137t Critical two-tail 2.068657599


132

t-Test: Two-Sample Assuming Equal Variances

S-POND 2 W- POND 2Mean 18.30909091 15.87142857Variance 71.82090909 39.47296703Observations 11 14Pooled Variance 53.53728967Hypothesized Mean Difference 0Df 23t Stat 0.826867101P(T<=t) one-tail 0.208404342t Critical one-tail 1.713871517P(T<=t) two-tail 0.416808683t Critical two-tail 2.068657599

GROWTH COMPARISION

0

5

10

15

20

25

30

35

0 20 40 60 80 100 120 140

DAYS

BO

DY

WE

IGH

T

S- POND 1

S-POND 2

W . POND 1

W - POND 2


133

Table 10: Relation to feed type and swollen hindgut in P. monodon post larvae

Feed type Swollen hind Normal gutMicroparticulated ++ ++Microencapsulated - ++++Flake feed +++ ++Liquid feed - ++++Artemia - ++++Diatoms + +++

Table 11: Concentrations of different chemicals used to Treat calcification in P. monodon culture ponds

Chemical Dosage Result Description1 Bkc 50% 0.2-0.5

ppmNo recovery

Alkyl dimethyl benzyl ammonium chloride 50%

2 Hcl 1 ppm Recovery Hydrochloric aid3 Citric acid 2 ppm Recovery Citric acid4 Edta 0.4 ppm Recovery Ethylene diamine

tetra acidic aid (disodium salt)

5 Alum 4 ppm Recovery Aluminum sulphate6 B-green 0.1 ppm Recovery Commercial product

by sujay agrilabs7 Neutral 0.5 ppm Recovery Commercial product

by finar chemicals ltd, mumbai


134

Table 12: Bore water analysis for calcium and magnesium levels in ppm (mg/lit) a farm with low low survival in P. monodon culture ponds

Bore no 1 2 3 4 5Calcium 80 80 128 120 184Magnesium 49 53 121 121 148Hardness 400 420 820 800 1010

Table 13: Calcium and magnesium concentrations in different farms with very poor survival after first month in P. monodon culture ponds

Pond noCalcium (ppm)

Magnesium (ppm)

Survival after first month

1 110 80 10%2 70 60 25%3 140 100 10%4 140 150 35%5 120 120 70%6 180 120 15%7 110 100 65%8 210 180 10%9 90 70 20%10 250 180 5%


135

Table 14: Differences between white gut and white fecal matter in P. monodon

White gut White fecal matterColor White/opaque TranslucentGut wall Thick Normal/thinSalinity range All salinities Salinity > 10 pptGut contents Mucus filled Filled with white

fecal matterBuoyancy of fecal matter

Sinks to bottom Floats on the water surface

Size variation of shrimp

10-20 % < 5%

Turning to loose shell

Yes Yes

Hepatopancreas Atrophied/less fat bodies

Atrophied/less fat bodies

Vibrio loads in HP > 3*104 cfu >2.5*105 cfuCondition Can not be

reversedCan be reversed

Age Observed form first month

Observed after second month


136

Table 15: Prophenol oxidase (units/min/mg of protein) activity in hemocytes of P. monodon after feeding with and with out immunostimulant Tinospora cordifolia

3 days 5 days

Control 20 19

2 grams/kg feed 26 35

4 grams / kg feed 33 53

SUMMARY Count Sum Average VarianceControl 2 39 19.5 0.52 grams/kg feed 2 61 30.5 40.54 grams / kg feed 2 86 43 200

3 days 3 79 26.33333 42.333335 days 3 107 35.66667 289.3333

ANOVASource of Variation SS df MS F P-value F crit

Rows 553 2 276.5 5.012085 0.166332 19Columns 130.6666667 1 130.6667 2.36858 0.263668 18.51282Error 110.3333333 2 55.16667

Total 794 5


137

Table 16: Total haemocyte count and percentage of granulcoytes at different concentrations of Tinospora cordifolia

Total hemocyte count Percentage of granulcoytes

Control 2.08x 106 72 grams/kg feed 2.52x106 8.54 grams/ kg feed 3.12x106 9

Table 17: Bacterial clearance at different concentrations of Tinospora cordifolia

Control 2 g/kg feed 4 g/kg feedBacterial concentration injected 2.7x109 2.7x109 2.7x109

Bacterial concentration at T0 3.5x107 3.3x 107 2.9x107

Bacterial concentration at T30 1.8x 106 4.1x105 5.1x 104


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Table 18: PCR results for MBV detection with different methods of DNA extraction in P.monodon post larvae

Method

First step positive

Second step positive

Crude dna extraction with lysis buffer 0 2PCR cycles increased from 25-27 0 3Increased the sample volume from 20 -50 pl 0 1Reduced the sample volume from 20 to 10 pl 0 1Collected only Hepatopancreas from 20 pl 0 3Phenol chloroform dna extraction with protinase k 3 10Using spin columns 8 10


139

Table 19: Quality and quantity of DNA with different methods of extraction in P.monodon post larvae

DNA Process

Abs 230

Abs 260

Abs-280

230/260 260/280 DNA conc. g/ml

Lysis-20 PL 1.4 1.2 0.76 1.16 1.57 12Lysis-50 PL 2.6 1.8 1.25 1.44 1.44 18Lysis-10 PL 1.43 0.87 0.54 1.64 1.61 8.4Lysis-20 HP 1.38 0.8 0.544 1.72 1.47 8.0Phenol chloroform-20 PL

0.69 1.1 0.61 0.62 1.8 11

Spin column-20 PL

0.44 1.08 0.57 0.4 1.89 11


140

Table 20: Diagnostic sensitivity with different methods of DNA extraction in P.monodon post larvae

DNA extraction process

Positives False negatives

Sensitivity%

Lysis buffer .20 PL, 2 8 20

Lysis buffer 20 PL,

27 cycles

3 7 30

Lysis buffer, 50 PL 1 9 10

Lysis buffer, 10 PL 1 9 10

Lysis buffer, HP from

20 PL

3 7 30

Phenol chloroform 10 0 100

Spin column 10 0 100


141

Table 21: Viral kits and their respective amplicons used for detection

WSSV

IHHNV

MBV TSV

KIT IQ 2000

IQ 2000

Bangalore Genei

MultiVirTM

AMPLICON/POSITIVE BAND (BASE PAIRS)

296 286 361 Four dots

Table 22: Haemocyte types and known biological functions (Soderhall and Cerenius, 1992)

Hemocyte type

Phagocytosis

Encapulation

Cytotoxicity

Prophenol oxidaseactivation

Hyaline cells

Yes No Not detected

No

Semi granular cells

Limited Yes Yes Yes

Granular cells

No Very limited Yes Yes


142

Table 23: Comparison of diseases between two cultured crustaceans P. monodon and L. vannamei

Problem P. Monodon L. vannameiWSSV S SIHHNV LS SMBV LS NRTSV NR NRIMNV NR NRVibriosis S SWhite fecal matter S LSLoose shell S LSProtozoa fouling S LSGregarines S LSBlack gill LS SBrown gill LS SBlisters LS LSBody cramp LS SLow DO LS SGas bubble disease NR LSCalcification LS NRAmmonia toxicity S SCa & Mg ratio S LS

S = Significant LS = Less significant NR = Not reported

Legend for Figures

Figure 1: Litopenaeus vannamei infected with WSSV and white spots are not visible externally. The red colour of the shrimp could be possibly due to the expansion of chromatophore.

Figure 2: White spots present on the carapace of Litopenaeus vannamei infected with WSSV. The first report in India on WSSV infection with L. vannamei

Figure 3:White spots present on the carapace of Litopenaeus vannamei infected with vibriosis and WSSV negative.

Figure 1

Figure 2

Figure 3

Legend for Figures

Figure 4: White spots present on the carapace of healthy Litopenaeus vannamei

Figure 5: White spots present on the moulted carapace of Litopenaeus vannamei from a healthy pond

Figure 6: Litopenaeus vannamei juvenile with cut antennae

Figure 7: Cannibalized shrimp Litopenaeus vannamei in check tray of WSSV infected pond

Figure 8: Oedema in cephalic region of Litopenaeus vannamei infected with WSSV

Figure 4 Figure 5

Figure 6 Figure 7

Figure 8

Legend for Figures

Figure 9: Litopenaeus vannamei shrimp with brown gill

Figure 10: Litopenaeus vannamei with melanized gill filaments. Black dots are visible on the gills exposed

Figure 11: Litopenaeus vannamei shrimp with black gill (unidentified etiology)

Figure 9

Figure 10

Figure 11

Legend for Figures

Figure 12: Litopenaeus vannamei juvenile with partial body cramp and opaque musculature

Figure 13: Litopenaeus vannamei sub adult with full body cramp with opaque abdomen

Figure 14: Necrosis of tail region in Litopenaeus vannamei suffered with low dissolved oxygen stress symptoms similar to IMNV

Figure 15: Deformities in cephalic region of Litopenaeus vannamei (Runt deformity syndrome) caused by IHHNV. The first case of IHHNV reported in India

Figure 16: Normal and IHHNV infected and deformed Litopenaeus vannamei of same age from the same pond

Figure 17: Shrimp infected with IHHNV with runt deformity Syndrome (2 g size at 58 days of culture)

Figure 12 Figure 13

Figure 14 Figure 15

Figure 16 Figure 17

Legend for Figures

Figure 18: Deformity in the abdominal region of Litopenaeus vannamei

Figure 19: Deformities in tail region of Litopenaeus vannamei

Figure 20: Deformity of the entire body of Litopenaeus vannamei caused by IHHNV

Figure 21: Deformity in the Hepatopancreas of Litopenaeus vannamei. The organ is pushed aside because of the Deformity

Figure 22: Deformity in midgut region of Litopenaeus vannamei

Figure 18

Figure 19 Figure 20

Figure 21 Figure 22

Legend for Figures

Figure 23: Blister on the first day of its formation

Figure 24: Freshly formed blister separated from shrimp

Figure 25: Blisters after melanization in cephalic region of Litopenaeus vannamei

Figure 26: Blisters on both sides of Litopenaeus vannamei

Figure 27: Blisters in the Uropod of Litopenaeus vannamei

Figure 28: Litopenaeus vannamei infected with vibriosis. White necrotic muscles are visible. The first case of acute vibriosis reported in India

Figure 23 Figure 24

Figure 25 Figure 26

Figure 27 Figure 28

Legend for Figures

Figure 29: Litopenaeus vannamei with loose shell syndrome (LSS). First report of LSS in India

Figure 30: Melanized lesions on the exoskeleton of Litopenaeus vannamei

Figure 31: Melanized lesions on the exoskeleton of Penaeus monodon

Figure 32: Black tiger shrimp Penaeus monodon post larvae with swollen hindgut and poor muscle gut ratio

Figure 29

Figure 30

Figure 31

Figure 32

Legend for Figures

Figure 33: Penaeus monodon shrimp with calcification on exoskeleton

Figure 34: Penaeus monodon with partial moulting. Exoskeleton on carapace still attached to the cephalothorax

Figure 35: Penaeus monodon with calcification and pre moult death

Figure 33

Figure 34

Figure 35

Legend for Figures

Figure 36: Wet mount of Penaeus monodon Hepatopancreas with necrotic cells

Figure 37: Juvenile shrimp Penaeus monodon with blue pigmentation and soft exoskeleton

Figure 38: Black tiger shrimp Penaeus monodon with white gut

Figure 39: White fecal matter floating on the water surface of the

pond stocked with Penaeus monodon

Figure 36

Figure 37

Figure 38 Figure 39

Legend for Figures

Figure 40: Very high density of gregarine Nematopsis species in the gut of Penaeus monodon. Wet mount, no stain, 40x magnification

Figure 41: Black tiger shrimp Penaeus monodon with iron deposits on exoskeleton

Figure 42: Wet mount of Hepatopancreas of post larvae of P. monodon with monodon baculovirus occlusions, stained with aqueous malachite green, 40x

Figure 43: Histopathology of gill showing intranuclear inclusions. Characteristic of WSSV infection

Figure 40

Figure 41

Figure 42 Figure 43

Legend for Figures

Figure 44: Rapid gill staining of epithelial tissue of Litopenaeus vannamei showing hypertrophied nuclei

Figure 45: Rapid dot kit showing negative result for wssv in the Litopenaeus vannamei sample with white spot with morphology of bacterial white spot

Figure 46: Shrimple kit showing WSSV positive result

Figure 44

Figure 45

Figure 46

Legend for Figures

Figure 47: Result of Iscreen isothermal PCR system showing 5 positive and 5 negative samples for wssv in shrimps with white spots

Figure 48: Yellow colour of the chromatophore in the early stage of wssv infection

Figure 49: Red color of the chromatophore in advanced stage of the WSSV infection. Protozoan fouling is also visible

Figure 50: Wet mount of normal shell of Litopenaeus vannamei with out white spots

Figure 51: Early stage of white spot development

Figure 47

Figure 48 Figure 49

Figure 50 Figure 51

Legend for Figures

Figure 52: Morphology of white spot in sub adult Litopenaeus vannamei

Figure 53: Morphology of white spot on Penaeus monodon with WSSV infection

Figure 54: The morphology of white spot caused by Vibrio species in Litopenaeus vannamei (10x)

Figure 55: Morphology of white spot in juveniles of Penaeus monodon

Figure 56: Morphology of white spot not associated with mortality that generally appears in healthy Litopenaeus vannamei ponds not infected with WSSV

Figure 57: Pond infested with wild Penaeus monodon and infected with WSSV

Figure 52 Figure 53

Figure 54 Figure 55

Figure 56 Figure 57

Legend for Figures

Figure 58: Litopenaeus vannamei with runt deformity syndrome and Metapenaeus monoceros uninfected the same pond

Figure 59: Gel documentation of IHHNV. Positive sample shows amplicon at 286 base pairs

Figure 60: Histology of Hepatopancreas of L. vannamei shows normal hepatopancreatic tubules. Shows no involvement of heavy metals and pestcides

Figure 61: Histology of gills shows no involvement of pathogens like fungi and vibrio as no granuloma or nodule formation are observed

Figure 62: Melanized gill filaments

Figure 63: Wet mount of gills of Litopenaeus vannamei with iron and manganese deposits. No melanization and fungal hyphae are visible

Figure 58 Figure 59

Figure 60 Figure 61

Figure 62 Figure 63

Legend for Figures

Figure 64: Wet mount of normal gills of Litopenaeus vannamei

Figure 65: Wet mount of gills with Gas bubble disease

Figure 66: Wet mount of Uropod with Blister and expanded chromatophore

Figure 67: Early stage of tail rot in Litopenaeus vannamei

Figure 68: Complete tail rot Litopenaeus vannamei

Figure 64 Figure 65

Figure 66

Figure 67 Figure 68

Legend for Figures

Figure 69: Shrimp Litopenaeus vannamei with developing Blister

Figure 70: Wet mount of blister showing hemocytes embedded in matrix of clotted proteins (40x) no stain

Figure 71: Wet mount of freshly farmed Blister showing hemocytes (40x). Stained with eosin

Figure 72: Biochip in MultiVirTM system showing TSV negative

Figure 73: Biochip hybridizer for MultiVirTM system

Figure 69

Figure 70 Figure 71

Figure 72 Figure 73

Legend for Figures

Figure 74: Biochemical analysis of Vibrio isolated from moribund Litopenaeus vannamei shows Indole positive

Figure 75: Biochemical analysis of Vibrio spp. isolated from moribund Litopenaeus vannamei shows oxidase positive

Figure 76: Litopenaeus vannamei with golden yellow pigemen- tation

Figure 77: Litopenaeus vannamei with blue pigmentation

Figure 74 Figure 75

Figure 76

Figure 77

Legend for Figures

Figure 78: Surfacing of the shrimps due to low Dissolved oxygen

Figure 79: Litopenaeus vannamei shrimp with deformities and normal rostrum

Figure 80: Shrimp pleopod infested with Zoothamnium spp.

Figure 81: Microscopic picture of coarse structure of feed causing damage to the gut wall

Figure 78

Figure 79 Figure 80

Figure 81

Legend for Figures

Figure 82: Microscopic picture of ingested and undigested larval exoskeleton damaging the gut wall in the post larva

Figure 83: Microscopic picture of ingested sharp object of the moulted shell causing damage to the gut wall

Figure 84: Microscopic picture of fine structure of microencapsulated feed in the mouth of the post larvae

Figure 85: Microscopic picture of undigested microencapsulated feed in the hind gut of the post larvae

Figure 86: Microscopic image of liquid feed in the gut of the post larvae

Figure 87: Microscopic image of post larvae of P. monodon with fold in the gut caused by the damage by the coarse texture of feed

Figure 82 Figure 83

Figure 84 Figure 85

Figure 86 Figure 87

Legend for Figures

Figure 88: Microscopic picture of pleopod of Penaeus monodon with calcification

Figure 89: Microscopic picture of pleopod showing effervescence due to the addition of dilute hydrochloric acid indicating the presence of calcium

Figure 90: White colour of the gut affected with white fecal mater disease looks white

Figure 91: Normal structure of the gut infected with white faecal matter after squeezing out the gut contents.

Figure 92: Gut of the shrimp affected with white gut with thick wall

Figure 88 Figure 89

Figure 90 Figure 91

Figure 92

Legend for Figures

Figure 93: Microscopic picture of wet mount of white fecal matter showing sloughed of cells form Hepatopan- Hepatopancreas

Figure 94: Microscopic picture of wet mount of Hepatopancreas showing necrotic cells

Figure 95: Microscopic picture of Hepatopancreas of shrimp affected with white fecal matter showing necrotic hepatopancreatic tubules. Stained with malachite green

Figure 96: The histopathology of the Hepatopancreas of the shrimp affected with white fecal matter disease showing necrotic tubules

Figure 97: Histology of white gut of Penaeus monodon (10X), haemocytic enteritis and necrotic epithelia of the gut wall was observed

Figure 93

Figure 94 Figure 95

Figure 96 Figure 97

Legend for Figures

Figure 98: Histology of gut wall of white gut infected Penaeus monodon (40x) shows the haemocytic enteritis and necrotic mucosal epithelia

Figure 99: Wet mount of Nematopsis in gut of Penaeus monodon (Bacteria can also be seen) (The small forms are sporozoites, or very early stage trophozoits, and the larger form is a two celled trophozoits (No stain. Magnification 400X)

Figure 98

Figure 99

Legend for Figures

Figure 100: Microscopic picture of wet mount of gut scraping showing four cells in syzygy in linier fashion (40x). First report of four cells in syzygy

Figure 101: Microscopic picture of wet mount of gut scraping showing five cells in syzygy (40X). First report of five cells in syzygy in India

Figure 102: Three cells of gregarines in syzygy before formation of syzygy with the fourth cell

Figure 103: Formation of syzygy with fourth cell shows that it can be attached at any part of the body. Zygospores are also visible

Figure 100

Figure 101

Figure 102 Figure 103

Legend for Figures

Figure 104: Microscopic picture of the wet mount of the gut scraping showing dead gregarines

Figure 105: Hemocyte lysate after addition of L-Dopa showing colour development

Figure 106: Hemocytes stained with rose Bengal (40x)

Figure 107: Hemocytes of shrimp. No stain

Figure 104

Figure 105


Legend for Figures

Figure 108: Hemocytes stained with hematoxylin and eosin (40x)

Figure 109: Hemocytes stained with hematoxylin and eosin (10x)

Figure 110: Blister formed due edematous fluid

Figure 111: Blister formed due to clotted blood and subsequent melanization

Figure 112: Blister formed by edematous fluid in the cephalic region

Figure 113: Blister in the first abdominal segment. First report of presence of Blister in the pleura of first abdominal segment

Legend for Figures

Figure 114: Blister in the 6th abdominal segment. First report of Presence of blister in the 6th abdominal segment of Penaeus monodon

Figure 115: Blister in Uropod developing into tail rot

Figure 116: Dead and damaged hemocytes in the fluid filled Blisters

Figure 117: Flow and clotting of blood in the cephalic region. A process involved in the Blister formation

Figure 118: Melanized lesion associated with blister in Penaeus monodon



Figure 118

Legend for Figures

Figure 119: Clotted haemolymph of blood resembles blister after 12 hours

Figure 120: Clotted haemolymph resembling blister after 72 hours

Figure 119

Figure 120

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