J. Aqua., 14 (2006): 1-15

LEVAMISOLE INFLUENCES THE IMMUNE RESPONSE OF FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII AND ITS RESISTANCE TO NITRITE STRESS AND AEROMONAS HYDROPHILA INFECTION

G. C. Pradhan, P. K. Sahoo*, Jaya Kumari, Swagatika Mohanty, J. Mohanty, Bindu R. Pillai and N. Sarangi Central Institute of Freshwater Aquaculture Kausalyaganga, Bhubaneswar 751 002, India *Corresponding author

The present study evaluated the effectiveness of dietary levamisole in modulation of innate immunity and reducing the percent mortality against nitrite stress or Aeromonas hydrophila infection in giant freshwater prawn, Macrobrachium rosenbergii. Haemolymph agglutinin and total protein levels, lysozyme activity, phenoloxidase (PO) activity, total and differential haemocyte counts, induced nitrite stress and mortality (%) against Aeromonas hydrophila challenge were measured in sub-adult M. rosenbergii fed with diets containing levamisole at 0, 125, 250 and 500 mg/kg feed for 7 or 14 days. M. rosenbergii fed with a diet containing 250 mg levamisole/kg feed for 14 days showed significant (P<0.05) increase in haemagglutination titre, PO activity, undifferentiated haemocyte count, resistance to nitrite stress and survival against A. hydrophila challenge. On the contrary, graded levels of levamisole feeding for 7 days failed to modulate most of the immune parameters or reduce the percent mortality against A. hydrophila challenge or nitrite stress. It is therefore concluded that administration of levamisole in the diet at 250 mg/kg feed for 14 days in sub-adult M. rosenbergii could enhance the immune ability and increase its resistance to A. hydrophila infection and nitrite stress.

INTRODUCTION

The giant freshwater prawn, Macrobrachium rosenbergii (de Man) of the family Palaemonidae is a migratory active species between brackish and freshwater habitat, and has a territory of Indo-Pacific region. Having improved standardized hatchery and culture technologies, the large scale farming of this species has gained momentum. Recent occurrence of nodavirus (MrNV) infection in M. rosenbergii causing white tail disease in India, China, West Indies, Thailand and Taiwan (Cheng and Chen, 1998; Tripathy et al., 2006) showed indication towards poor management and quarantine system. Further, the recently occurring appendage deformity syndrome (ADS) in prawn farming causing mortality in juveniles and adults in India indicates towards poor water quality and management of culture ponds (Sahoo et al., 2005). In addition, bacterial diseases are also becoming common problem in prawn farming (Cheng et al., 2003). Although Aeromonas 2 sp. are not generally considered to be the major threat to the commercial production of M. rosenbergii (Sung et al., 2000), they have sometimes been linked to disease outbreaks in this species (New, 1995). Nitrite also plays a major role in determining the survival of prawns in aquatic environment and its detrimental effects on immune response of freshwater prawns have been reported (Chand and Sahoo, 2006; Mallasen and Valenti, 2006). Therefore, stimulation of immune system and reduction of mortality in prawns due to infections and stress, would be of great interest for aquaculture research and the prawn farming industry.

Innate immune system of crustaceans involves various cellular mechanisms, viz., total and differential haemocyte counts, phenoloxidase (PO) activity, phagocytosis, encapsulation etc. and humoral mechanisms, viz., agglutinin levels and lysozyme activity. Based on the recent classification of M. rosenbergii haemocytes (Sierra et al., 2001), large ovoid haemocytes and undifferenciated round haemocytes might be carrying out the functions of proPO system, like semigranular and granular haemocytes in other crustaceans (Johansson and Soderhall, 1989). Activation of PO from proPO is through proPO activating enzyme (ppA), a serine protease (Perazzolo and Barracco, 1997) which can be induced by several microbial polysaccharides, including β-1, 3 glucan from fungal cell walls (Vargas-Albores et al., 1996). The PO activity has been well documented in M. rosenbergii (Kumari et al., 2004; Chand et al., 2006). Lectins/agglutinins play their role by enhancing the recognition interface between the invading pathogen and semigranular haemocytes by the opsonin action in the plasma (Soderhall and Cerenius, 1992). Their presence in M. rosenbergii haemolymph has been well documented (Vazquez et al., 1997; Chand et al., 2006). Lysozyme, an antibacterial peptide has also been reported in M. rosenbergii (Kumari et al., 2004).

The immunostimulatory effects of various substances like peptidoglycans, glucans, lipopolysaccharides (LPS), sodium alginate, polyherbal formulation, lactoferrin and other polysaccharides have been widely studied in crustaceans (Sritunyalucksana et al., 1999; Chang et al., 2003; Kumari et al., 2004; Cheng et al., 2005; Chand et al., 2006). Levamisole, a levo-isomer of tetramisole acts as potential immunostimulant in mammals and several aquatic species (Sakai, 1999). Feeding of levamisole at 125 or 250 mg/kg feed to M. rosenbergii could able to increase percent survival, and specific growth rate and feed conversion ratio without adversely affecting growth (Baruah and Prasad, 2005). However, its immunomodulatory effects on prawn immunity are not clear. In this study we attempted to examine various immune parameters in M. rosenbergii and its resistance to nitrite stress and A. hydrophila infection when the prawns were fed diets containing levamisole at graded levels for 7 or 14 days.

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MATERIAL AND METHODS

The intermoult stage of M. rosenbergii weighing 15-20 g were collected from prawn farm of the Central Institute of Freshwater Aquaculture, Bhubaneswar and acclimated in the laboratory for two weeks before experimentation. Four sets of experiments were conducted to evaluate the immunomodulatory effects of dietary levamisole in prawns. For each set, prawns were divided randomly into four groups A, B, C and D (in triplicate) for two time periods (7 or 14 days) of levamisole feeding. Each set of experiment was carried out in 24 FRP tanks of 40 l capacity containing 30 l of water. Prawns were given pelleted diet for 2 weeks. Ten percent of water was renewed daily to siphone out the leftover feed and metabolites, providing better environment and maintaining optimal water quality parameters. Water quality parameters such as water temperature, pH, total alkalinity, dissolved oxygen were analyzed to maintain optimal levels (total hardness, 80-120 mg/l; total NH3-nitrogen, < 0.1 mg/l; dissolved oxygen, 6.5- 7.0 mg/l; water temperature, 23-26ºC; pH, 7.0-8.5). First set of experimental prawns was used to measure haemolymph supernatant agglutinin levels, lysozyme activity and total protein concentration after different dose and time period of levamisole feeding. Second set of experiment was conducted to see the influence of levamisole feeding on haemolymph cell counts and PO activity. Third and fourth sets were set up for determining the resistance of animals against nitrite stress and bacterial pathogen Aeromonas hydrophila infection, respectively. In first three sets, three prawns were kept in each tank where as in fourth set, four prawns were maintained in each tank. Pelleted feed was provided bot during acclimation and also during the experiment. The composition of the feed was as described by Chand et al. (2006). Experimental feed was prepared by adding levamisole hydrochloride (Sigma, St. Louis, USA) to above pelleted feed ingredient to make final concentrations of 0, 125, 250 and 500 mg levamisole/kg feed for groups A, B, C and D, respectively. Pellet feed was prepared with a hand pelletizer and the feed was air-dried and stored at 4ºC in airtight container before use. Carboxy methylcellulose was added as binder to the feed mix to avoid any possible leaching loss of levamisole. The feed was given to prawn at 4% of body weight daily in two split doses.

A known pathogenic isolate of A. hydrophila was grown on tryptone soy broth (TSB, Difco) for 24 h at 30ºC. The broth cultures were harvested by centrifugation at 5000 x g for 15 min at 4ºC. The bacterial pellet was washed by resuspension in sterile PBS (pH 7.4) and centrifugation as above and the final pellet was resuspended in the PBS at 109 and 2 x 105 cfu/ml to produce stock bacterial suspensions (as confirmed by total plate count) for the study of bacterial agglutination titre and challenge test, respectively.

Further, the bacteria to be used for bacterial agglutination assay were treated with 1% formalin and kept overnight at 4ºC. The formalin-killed cells were washed twice with 4 sterile PBS as described previously and suspended in PBS to its original cell count. The formalin-killed cells were stored at –4ºC until use.

From the first set of prawns, 400 μl of haemolymph was collected by using 2 ml sterile plastic syringe with 26 x 0.5'' gauge needle from the ventral sinus of the animal. Haemolymph was allowed to clot in 2 ml capacity microcentrifuge tubes held at 4ºC. After one hour, clot was broken using sterile needle and kept at 4ºC for 3-4 h. The tubes were then centrifuged at 11,000 x g for 30 min at 4ºC. The supernatant fluid was collected and stored at –30ºC for further analysis. Lysozyme activity was studied first using part of the supernatant following Kumari et al. (2004) with slight modification. Supernatant fluid of 15 µl was taken in a 96 well microtitre plate in triplicate per sample. Then 150 µl of Micrococcus lysodeikticus solution (20 mg M. lysodeikticus per 100 ml 0.02 M acetate buffer, pH 5.5) was added to each well. Blank consisted of 165 µl of acetate buffer only. The optical density reading was taken immediately at 450 nm by using ELISA reader (Anthos Labtec, Austria). Then the plate was incubated for 10 minutes at 25ºC. The final optical density was read after incubation period. Reduction in optical density of 0.001 was taken as one unit of enzyme activity per 15 µl of sample. The rest of the supernatant was used to study total protein concentration following Bradford (1976), using bovine serum albumin as a standard protein, and haemagglutination and bacterial agglutination titres following Chand et al. (2006) using RaRBC and killed A. hydrophila, respectively.

From the second set of prawns, 100 μl of haemolymph was collected in 1 ml syringe with 26 x 0.5'' gauge needle containing 900 μl anticoagulant (sodium chloride 0.45 M, glucose 0.1 M, sodium citrate 30 mM, citric acid 26 mM, EDTA 20 mM, pH 4.5). Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome produced from L-dihydroxy phenylalanine (L-DOPA, a product of Hi Media, Mumbai) following Hernandez-Lopez et al. (1996). The PO activity optical density was expressed as dopachrome formation per 50 μl haemolymph.

Using 1 ml sterile plastic syringe with 26 x 0.5'' gauge needle containing 0.45 ml anticoagulant (as described above) with fixative solution (sodium cacodylate 0.10 M and 1.5% glutaraldehyde) in 1:1 ratio, 0.05 ml of haemolymph was drawn from ventral sinus of intermoult prawn and placed on haemocytometer (Spencer, Neubauer, Germany). Total and different types of haemocytes were counted following Sierra et al. (2001) and Chand et al. (2006).

At the end of levamisole feeding for 7 or 14 days, prawns were held in 5 l of water (in each tank) containing sodium nitrite (Merck, India) at 9 mg/l concentration following Chen and Lee (1997). Nitrite water (50%) was renewed daily without providing aeration. The percent mortality was observed up to 120 h for each of the groups.

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Prawns fed with levamisole diet for 7 or 14 days were injected intramuscularly between the second and third abdominal segments with 0.05 ml PBS containing 104 cfu of A. hydrophila (cultured for 24 h at 30 ºC). Four prawns fed with control diet were also injected with 0.05 ml PBS and served as negative control. The mortality was observed up to five days and the cause of mortality was further confirmed by re-isolating the organism from dead prawn hepatopancreas.

The mean±standard error of each parameter for each of the experiments at each time period was calculated for all the groups. Data were analyzed using one way ANOVA. Means were compared using Duncan's multiple range tests (Duncan, 1955). The difference was considered significant when P< 0.05.

RESULTS

No mortality was recorded in any of the groups of prawns during the experiment. A significant (P<0.05) enhancement in haemagglutinin titre was marked in the levamisole-fed prawns after 14 days of levamisole feeding compared with control values. However, there was no significant difference in the level of haemagglutinin among all the groups fed levamisole for a period of 7 days (Table 1).

Table 1. Haemolymph supernatant parameters of M. rosenbergii fed graded levels of levamisole containing diets for 7 or 14 days.

Group Dose Haemaggluti- Bacterial Total protein Lysozyme PO activity (mg nation agglutination (g/dl) activity (OD/15 (OD/50 μl levamisole titre (log2) titre (log2) μl supernatant) haemolymph) /kg feed) 7 days 14 days 7 days 14 days 7 days 14 days 7 days 14 days 7 days 14 days A 0 5.11± 5.00± 2.64± 2.78± 16.87± 17.35± 0.002± 0.002± 0.20± 0.16± 0.24 0.69a 0.10ab 0.11a 0.43 0.58 0.0006 0.0006 0.04 0.02a B 125 5.17± 7.22± 2.44± 3.00± 17.02± 15.47± 0.003± 0.003± 0.35± 0.38± 0.94 0.22b 0.29a 0.69a 0.66 1.38 0.0014 0.0009 0.08 0.04b C 250 6.56± 7.66± 3.11± 2.83± 18.41± 17.96± 0.004± 0.003± 0.31± 0.37± 0.72 0.66b 0.19b 0.44a 1.28 0.64 0.001 0.0004 0.05 0.01b D 500 4.56± 7.50± 3.11± 1.16± 17.85± 17.50± 0.004± 0.001± 0.33± 0.63± 0.61 0.50b 0.19b 0.44b 0.48 0.20 0.0004 0.0003 0.05 0.07c

Data represent mean ± S.E of 9 (three prawns of replicate groups) prawns. Means bearing different superscript(s) are significantly (P<0.05) different.

The bacterial agglutinin level was not influenced significantly in both the upper dose group prawns as compared to its control in case of 7 days of levamisole feeding. 6

However, prawns fed levamisole at 125 mg/kg feed showed significantly lower agglutinin level compared to prawns fed with levamisole at 250 and 500 mg/kg feed. After 14 days of levamisole feeding, a decline in bacterial agglutinin level in group D prawns was noticed compared to other group prawns (Table 1).

Feeding of levamisole at any of the dose levels for 7 or 14 days could not influence total protein level and lysozyme activity in prawns compared to control group prawns (Table 1).

The PO activity was significantly increased in groups B, C and D prawns compared with control values after 14 days of levamisole feeding. The highest PO activity was noticed in group D prawns fed levamisole at 500 mg/kg fed for 14 days. On the contrary, no significant change in PO activity was noticed in prawns compared with control values after 7 days of levamisole feeding (Table 1).

Total haemocyte count (THC) with mean values ranging from 0.83 x 107 to 2.04 x 107 cells/ml of haemolymph was obtained in various groups of prawns. THC was not significantly (P>0.05) influenced by levamisole feeding compared to control prawns after 7 days of exposure. However, significant decrease in THC was noticed in groups B and C compared to control group prawns after 14 days of levamisole feeding (Fig. 1a).

All the three types of haemocytes viz., fusiform cells, large granular cells and undifferentiated round cells were observed under microscope. The mean of fusiform cells varied from 59.71 to 68.38% which were the predominant cell types in this species. There were no significant differences in the population of large granular and undifferentiated round cells among graded levels of levamisole feeding for 7 days compared to control groups prawns. Group C prawns showed significantly higher numbers of fusiform cells as compared to group B prawns after 7 days of levamisole feeding. Similarly, no changes in fusiform and large granular cell populations were marked by levamisole feeding after 14 days. The mean number of large granular cells varied from 22.15 to 30.19%. The mean number of undifferentiated cells ranged from 9.46 to 14.45%. However, a significant increase in undifferentiated round cells was marked after 14 days levamisole feeding in levamisole fed prawns compared with control values (Figs. 1b,c,d).

Exposure of prawns to 9 mg/l of sodium nitrite caused mortality on the second day onwards and the mortality was recorded up to the end of the fifth day after 7 and 14 days of levamisole feeding. After 120 h, the mean mortality was varied from 33.33 to 66.66% in the 7 days levamisole fed groups. Groups A and B showed similar mortality rate i.e. 66.66% compared to 33.33% and 50% in groups C and D, respectively. However, there was significant reduction in percent mortality (P<0.05) in levamisole fed prawns in case of 14 days levamisole fed prawns compared to control. The control group prawns 7 showed 100% mortality compared to 33.33% in both groups B and C, and 49.99% in group D prawns (Table 2).

b a 80 b 2.5 ab a ab a 70

2 60 cells/ml)

7 ab 50 1.5 b 40 . b 1 30

Fusiform cells (%) 20 0.5 10

Total haemocyte count (x 10 0 0 714 714Days Days d c . 18 b 40 b 16 b 35 14 a 30 12 25 10

20 8 15 6 10 4 cells (%) granular Large 5 2 Undifferentiated cells (% ) (% cells Undifferentiated 0 0 714 714 Days Days 0 125 mg 250 mg 500 mg

Fig. 1. Total and differential haemocyte counts of M. rosenbergii given levamisole containing diets for 7 or 14 days. Bars represent mean ± S. E of 9 determinations. Bars for same time with different letter(s) at a particular period are significantly (P<0.05) different. 8

Table 2. Mortality levels (%) after challenge of prawns exposed to nitrite or A. hydrophila challenge after 120 h.

Group Dose (mg of Exposure to nitrite A. hydrophila challenge levamisole/kg 7 days 14 days 7 days 14 days feed A 0 66.66±19.24 100±0.00a 72.22±2.77 72.22±2.77a B 125 66.66±19.24 33.33±0.00b 66.66±8.33 41.66±8.33bc C 250 33.33±19.24 33.33±19.24b 83.33±8.33 33.33±8.33b D 500 50.00±28.86 49.99±9.62b 91.66±8.33 58.33±8.33ac Data represent mean±S.E of 9 prawns (three prawns of each replicate) for nitrite exposure and 12 prawns (four prawns of each replicate) for A. hydrophila challenge. Means with different superscript(s) are significantly (P<0.05) different.

No mortality was observed in prawns injected only with PBS. The percent mortality reached its peak after 24 h in the animals. Feeding of levamisole at any of the dose levels for a period of 7 days could not bring the reduction in mortality of prawns. However, prawns challenged after 14 days of levamisole feeding showed significant decrease (P<0.05) in mortality in groups B, C and D compared to control group prawns. Group C prawns showed significantly lower mortality compared to group D prawns (Table 2).

DISCUSSION

To combat disease problems, knowledge of prawn defence mechanism is very essential. Innate immune system of prawns and its induction mechanisms for disease resistance have been explored, but not so sufficient to give perfect solution for disease problems. Thus, for short-term protection from disease in culture system, use of immunostimulants is widely accepted. As these substances are biocompatible, biodegradable, safe for the environment and human health along with having certain nutritional values, they are slowly replacing antibiotics and vaccines for prevention of diseases. Different immunomodulatory effects of levamisole have been established in higher vertebrates including fish (Sahoo and Mukherjee, 2002; Li et al., 2004; Kumari and Sahoo, 2006). Levamisole is not a constituent in prawn feed and it can be easily incorporated in feed at known concentrations.

Levamisole is a synthetic compound, which has widespread effects like enhancement of serum lysozyme activity, serum antibody titres after immunization, the expression of cytokines by macrophages, lymphocyte proliferation and antitumor responses (Sakai, 1999). However, immunosuppressive effects of levamisole have already been reported (Mulero et al., 1998; Li et al., 2004). Previous studies regarding the possible 9 effects of levamisole on fish immune system have enriched our knowledge to use it as an immunostimulant in fish farming. However, no studies focus on the optimal dose, time period of feeding and efficacy of levamisole in the sub-adult giant freshwater prawn M. rosenbergii. Therefore, the present study aims towards gaining information regarding levamisole action on the innate immune system of M. rosenbergii.

In this study, for reasons of economy and efficiency, we looked for the minimum dose that would induce immunostimulation in prawn in the shortest time possible. Since time and/or dose-dependent effects are to be expected, we tested three dose levels over two different time periods, parameters which are fundamental in any immunomodulatory strategy (Sakai, 1999; Chand et al., 2006). Levamisole is water-soluble and gets easily absorbed in digestive tract, which is a requirement for triggering protective responses in the prawn. It is stable at room temperature and is also relatively resistant to proteolytic degradation, as it is not a protein. Thus, it can overcome the problem of being attacked by proteolytic enzymes in the gut. To overcome the problem of handling related stress, we planned oral administration of dietary levamisole to prawns. Taking the above points into consideration, we tried to evaluate incorporation levels of 125, 250 and 500 mg levamisole/kg feed on immunity and disease resistance of prawn after 7 or 14 days feeding. Feeding of levamisole at 500 mg/kg feed did not cause even any mortality during 7 or 14 days trial, thus indicated non-toxic nature up to this dose and time period of feeding to this species as also being observed by earlier workers (Baruah and Prasad, 2005).

Nitrite, a byproduct of ammonia excretion is a major threat to intensified aquaculture as it is highly toxic to aquatic animals at high concentration. It has been reported that the concentration of nitrite increased along with culture period, and reached as high as 20 mg/l in grow-out ponds (Tacon et al., 2002). Elevated nitrite has been reported to cause growth suppression, increased rate of moulting, oxygen consumption, ammonia excretion and in extreme cases, death of decapod crustaceans (Chen and Chen, 1992). The effect of nitrite and nitrate on prawn growth has already been studied with Penaeus indicus and P. monodon as well as in M. rosenbergii, where reduction of growth occurred up to 50% in P. indicus when 6.4 mg NO2-N/l was applied to it. Nitrite was found to be more toxic to M. rosenbergii than marine shrimp (Wickins, 1976). Among prawn species, M. malcolmsonii is more susceptible to nitrite than M. rosenbergii (Chand and Sahoo, 2006). Chen and Lee (1997) observed 96 h LC50 value of NO2-N to be 8.5 to 12.9 mg/l in M. rosenbergii. Exposure of prawns after 7 days or 14 days of levamisole feeding to 9 ppm of sodium nitrite showed variation of mortality from 33.33% to 66.66% after 120 h of observation in 7 days levamisole-fed groups. The least mortality was found in group C prawns fed with levamisole of 250 mg/kg feed compared to control group prawns which showed 66.66% mortality. After 14 days of levamisole feeding, a significant reduction in mortality was observed in different groups of levamisole feeding compared 10 to control after 120 h of exposure. Mortality was the lowest i.e. 33.33% in groups B and C fed at 125 and 250 mg/kg feed respectively whereas control group prawns showed 100% mortality. Chand et al. (2006) found the lowest mortality of 16.67% compared to 75% in control group after 120 h of nitrite treatment after 14 days of lactoferrin feeding to prawns.

The role of immunostimulants of different origin in protecting shrimps from wide range of pathogens is well known. In shrimp, oral administration of β-1, 3- glucan was found to be effective against bacterial infections (Takahashi et al., 1995; Liao et al., 1996). Chand et al. (2006) found a significant reduction in mortality in lactoferrin fed M. rosenbergii after 7 or 14 days when challenged with virulent A. hydrophila. In this study, a significant reduction in mortality was observed in groups B and C prawns compared to control after 14 days of levamisole feeding. Thus, levamisole has a positive effect on the disease resistance against pathogens. The enhanced survival due to bacterial challenge and nitrite stress is well correlated with the enhanced immune response marked in those prawns.

Several crustacean lectins have been characterized with a heat labile and calcium dependent substances called agglutinins (Acharya et al., 2004). Feeding of levamisole at any of the dose levels for 7 days did not bring any change in the serum haemagglutinating activity against rabbit RBC. Similarly, feeding with lactoferrin at different dose levels to M. rosenbergii was not able to raise haemagglutinin level against rabbit RBC (Chand et al., 2006). On the other hand, considerable enhancement in haemagglutinin level was marked in this experiment after 14 days of levamisole feeding compared to control. Sritunyalucksana et al. (1999) and Chand et al. (2006) also observed similar result in haemagglutinating titre in P. monodon and M. rosenbergii, respectively. Immuplus (AquaImmu), a herbal immunomodulator (Indian Herbs, Saharanpur, India), was also found to enhance haemagglutination titre level in M. rosenbergii after incorporating 1g/kg feed for 3 weeks (Kumari et al., 2004). The highest haemagglutination titre was found in group C prawns fed levamisole at 250 mg/kg diet for 14 days.

Previous studies have shown that prawn and shrimp have agglutinins in their sera that can agglutinate Gram-negative bacteria (Acharya et al., 2004). Bacterial agglutinins against Bacillus cereus and Aeromonas sp. in M. rosenbergii (Vazquez et al., 1996), Vibrio sp. and Pseudomonas sp. in P. indicus (Jayasree, 2001) have also been reported. Lectin recognizes different polysaccharide components like bacterial O-keto O-methyl containing sugars, the N-acetyl sugars residue and teichoic acid from the polysaccharide cell wall. Bacterial agglutinin level did not differ significantly among different groups compared to control group after 7 days of levamisole feeding. However, raised agglutinin levels were marked in groups C and D compared to group B. Further, after 14 days of levamisole feeding, a significant decrease in titre was obtained in group D prawns 11 compared to other groups. Earlier studies also indicated no significant enhancement in agglutinin levels after 14 days of either lactoferrin feeding to prawns (Chand et al., 2006) or feeding with 0.4% peptidoglycan or 0.002% lipopolysaccharide in P. monodon (Sritunyalucksana et al., 1999).

Haemocyanin, a respiratory protein, is the most abundant molecule of the haemolymph (60-90% of total protein) (Djangmah, 1970) followed by clotting protein and other humoral components. In this study, there was no significant alteration in the total protein level after 7 or 14 days of levamisole feeding to prawns indicated negligible effect of levamisole on total protein level.

Lysozyme is an enzyme, which has bacteriolytic effect. It hydrolyses β-1, 4 glycosidic bond of bacterial cell wall peptidoglycan. In Indian river prawn M. malcolmsonii, lysozyme level of 0.04 units/ml of haemolymph has been found by Acharya et al. (2004). Kumari et al. (2004) found a significant high level of lysozyme activity after 3 weeks of treatment of 1 g/kg feed Immuplus (AquaImmu) in M. rosenbergii. On the contrary, we did not find any significant difference in the lysozyme activity at any of the dose levels of levamisole feeding and time periods among different groups of levamisole fed prawns.

Experimenting on different substances for their immunostimulatory effects, activation of prophenoloxidase system is an important criterion for the judgement of effectiveness of that substance. In the present study, no alternation in the PO activity among different groups after 7 days of levamisole feeding to prawns was noticed. Chand et al. (2006) found enhanced PO activity after 7 days of lactoferrin feeding to prawns. However, after 14 days of levamisole feeding, a significant enhancement in the PO activity in the prawns fed diets containing levamisole at all the graded levels compared to control was noticed. Kumari et al. (2004) obtained similar result by feeding Immuplus at 1 g/kg feed up to 3 weeks to the same species. In P. monodon, the highest level of PO activity was marked after 9 days by dietary feeding of β-1, 3-glucan at 2, 10 or 20 g/kg concentration (Chang et al., 2003). Thus, levamisole can be considered along with LPS, glucan, sodium alginate, lactoferrin and herbal products for triggering PO activity in prawns indicating an increase in immune ability.

In this study, there was no significant change in the total haemocyte number among different groups of prawns fed levamisole diet for 7 days. However, a marked decline of THC in the groups B and C prawns fed levamisole diet at 125 and 250 mg/kg was noticed compared to control prawns after 14 days. Chand et al. (2006) observed that incorporation of lactoferrin at 50 mg/kg diet for a period of 7 days increased the THC compared to other groups and there was no significant difference in different groups of lactoferrin fed prawns after 14 days. The reduction in haemocyte count has also been 12 marked in other crustaceans after exposure to immunostimulant (β-1,3-glucan) as a consequence of cell clumping in vivo implying a specific cellular recognition of and reaction to soluble non-self molecules in crustaceans (Smith et al., 1984; Johansson and Soderhall, 1985). This study indicates that levamisole do not stimulate more production of haemocytes and the resistance observed in terms of increased survival in levamisole fed prawns to nitrite or bacterial stress may be routed through mostly humoral factors viz., PO activity and haemagglutinin levels.

We measured the count of three different haemocytes in different groups of experimental prawns and the ranges showed similarity with the finding of Vazquez et al. (1997) who suggested the presence of 70% fusiform or hyaline haemocytes, 20% large granular cells and 10% undifferentiated cells in the intermoult M. rosenbergii. Except a significant difference in fusiform cells between groups B and C prawns fed at 125 and 250 mg of levamisole/kg feed, there was no observable alternation in fusiform cells, large granular cells and undifferentiated round cells among graded levels of levamisole fed prawns after 7 days. Similarly, fusiform cells and large granular cells showed no significant differences after 14 days of levamisole feeding. However, a marked increase in undifferentiated cells was observed in prawns fed at 125, 250 and 500 mg/kg of levamisole feed compared to control prawns. It has been proposed that undifferentiated cells are nothing but immature form of granular cells (Vazquez et al., 1997) or granular cells releasing their granules that contain the proPO system. Thus, the increase in percentage of undifferentiated cell might be partly responsible for increase in PO activity observed in levamisole-fed prawns for 14 days.

Thus, it may be concluded that levamisole can be incorporated in feed of grow- out M. rosenbergii at 250 mg/kg feed for a continuous feeding of 14 days, to enhance their immune status by increasing haemagglutination titre, phenoloxidase activity, undifferentiated cell count and increase in resistance to bacterial infection and nitrite stress.

ACKNOWLEDGEMENTS

This research was supported by AP Cess Project of Indian Council of Agricultural Research, New Delhi (Code No. 0614025).

REFERENCES

Acharya, S., J. Mohanty and P. K. Sahoo, 2004. Humoral defence factors in Indian river prawn, Macrobrachium malcolmsonii. Fish Shellfish Immunol., 17: 137-147. Baruah, N. and K.P. Prasad, 2005. Influence of dietary intake of levamisole on growth and survival of Macrobrachium rosenbergii (Palaemonidae, de Man). Asian Fish. Sci., 18: 25-31. 13

Bradford, M. M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein. Anal. Biochem., 72: 248. Chand, R. K. and P. K. Sahoo, 2006. Effect of nitrite on the immune response of freshwater prawn Macrobrachium malcolmsonii and its susceptibility to Aeromonas hydrophila. Aquaculture, 258: 150-156. Chand, R. K., P. K.Sahoo, J. Kumari, B. R. Pillai and B. K. Mishra, 2006. Dietary administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress. Fish Shellfish Immunol., 21: 119-129. Chang, C. F., M. S. Su, H. Y. Chen and I. C. Liao, 2003. Dietary beta-1, 3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus. Fish Shellfish Immunol., 15: 297-310. Chen, J. and S. Chen, 1992. Effects of nitrite on growth and molting of Penaeus monodon juveniles. Comp. Biochem. Physiol. C. Comp. Pharmacol. Toxicol., 101: 453-458. Chen, J. and Y. Lee, 1997. Effects of nitrite on mortality, ion regulation and acid-base balance of Macrobrachium rosenbergii at different external chloride concentrations. Aquat. Toxicol., 39: 291-305. Cheng, E. and J. C. Chen, 1998. Isolation and characterization of an Enterococcus-like bacterium causing muscle necrosis and mortality in Macrobrachium rosenbergii in Taiwan. Dis. Aquat. Org., 34: 93-101. Cheng, W., C. H. Liu, C. M. Kuo and J. C. Chen, 2005. Dietary administration of sodium alginate enhances the immune ability of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish Shellfish Immunol.,18: 1-12. Cheng, W., F. M. Juang, J. Li, M. Lin, C. Liu and J. Chen, 2003. The immune response of the giant freshwater prawn Macrobrachium rosenbergii and its susceptibility to Lactococcus garvieae in relation to the moult stage. Aquaculture, 218: 33-45. Djangmah, J., 1970. The effects of feeding and starvation on copper in the blood and hepatopancreas, and on blood proteins of Crangon vulgaris (Fabricius). Comp Biochem Physiol, 32: 709-731. Duncan, D. B., 1955. Multiple range and multiple ‘F’ tests. Biometrics, 11: 1-42. Hernandez-Lopez, J., T. Gollas-Galvan and F. Vargas-Albores, 1996. Activation of the prophenoloxidase of the brown shrimp (Penaeus californiensis Holmes). Comp. Biochem. Physiol., 113C: 61-66. Jayasree, S., 2001. Purification and characterization of a natural agglutinin in the hemolymph of the prawn Penaeus indicus H. Milne Edwards. J. Invert. Pathol., 77: 237-242. Johansson, M. W. and K. Soderhall, 1985. Exocytosis of the prophenoloxidase activating system from crayfish haemocytes. J. Comp. Physiol., 156B: 175-181. Johansson, M. W. and K. Soderhall, 1989. A cell adhesion factor from crayfish haemocytes has degranulating activity towards crayfish granular cells. Insect Biochem., 19: 183-190. Kumari, J. and P. K. Sahoo, 2006. Dietary levamisole modulates the immune response and disease resistance of Asian catfish Clarias batrachus (Linnaeus). Aqua. Res., 37: 500-509. 14

Kumari, J., P. K. Sahoo, S. S. Giri and B. R., Pillai, 2004. Immunomodulation by 'Immuplus (AquaImmu)' in giant freshwater prawn, Macrobrachium rosenbergii (De Man). Indian J Exp. Biol., 42: 1073-1077. Li, P., X. Wang and D. Gatlin, 2004. Excessive dietary levamisole suppresses growth performance of hybrid striped bass, Morone chrysops x M. saxatilis, and elevated levamisole in vitro impairs macrophage function. Aqua. Res., 35: 1380-1383. Liao, I. C., M. S. Su, C. F. Chang, B. Y. Her and T. Kohima, 1996. Enhancement of the resistance of grass prawn Penaeus monodon against Vibrio damsela infection by beta-1, 3-glucan. J. Fish. Soc., Taiwan, 23: 109-116. Mallasen, M. and W. C. Valenti, 2006. Effect of nitrite on larval development of giant river prawn Macrobrachium rosenbergii. Aquaculture (In Press). Mulero, V., M.A. Esteban and J. Meseguer, 1998. In vitro levamisole fails to increase seabream (Sparus aurata L.) phagocyte functions. Fish Shellfish Immunol., 8: 315-318. New, M. B., 1995. Status of freshwater prawn farming. Aqua. Res., 26: 1-54. Perazzolo, L. M. and M. A. Barracco, 1997. The prophenoloxidase activating system of the shrimp Penaeus paulensis and associated factors. Dev. Comp. Immunol., 21: 385-395. Sahoo, P. K. and S. C. Mukherjee, 2002. The effect of dietary immunomodulation upon Edwardsiella tarda vaccination in healthy and immunocompromised Indian major carp (Labeo rohita). Fish Shellfish Immunol., 12: 1-16. Sahoo, P. K., S. Tripathy, B. K. Mishra, S. Adhikari, S. Nandi, P. Hari Babu, N. Sarangi and S. Ayyappan, 2005. Is appendage deformity syndrome caused by Macrobrachium rosenbergii nodavirus ? Curr. Sci., 88: 1374-1375. Sakai, M., 1999. Current research status of fish immunostimulants. Aquaculture, 172: 63-92. Sierra, C., J. Guevara, R. Lascurain, A. Perez, C. Agundis, E. Zenteno and L. Vazquez, 2001. Sialylation is modulated through maturation in hemocytes from Macrobrachium rosenbergii. Comp. Biochem. Physiol. C. Toxicol. Pharmacol., 130: 179-189. Smith, V. J., K. Soderhall and M. Hamilton, 1984. β-1,3-glucan induced cellular defence reactions in the shore crab, Carcinus maenas. Comp. Biochem. Physiol., 77A: 635-639. Soderhall, K. and L. Cerenius, 1992. Crustacean immunity. Ann. Rev. Fish Dis., 2: 3-23. Sritunyalucksana, K., P. Sithisarn, B. Withayachumnarnkul and T. W. Flegel, 1999. Activation of prophenoloxidase, agglutinin and antibacterial activity in haemolymph of the black tiger prawn, Penaeus monodon, by immunostimulants. Fish Shellfish Immunol., 9: 21-30. Sung, H. H., S. F. Hwang and F. M. Tasi, 2000. Responses of giant freshwater prawn (Macrobrachium rosenbergii) to challenge by two strains of Aeromonas sp. J. Invert. Pathol., 76: 278-284. Tacon, A., J. Cody, L. Conquest, S. Divakaran, I. Forster and O. Decamp, 2002. Effect of culture system on the nutrition and growth performance of Pacific white shrimp Litopenaeus vannamei (Boone) fed different diets. Aqua. Nutr., 8: 121-137. Takahashi, Y., T. Itami and M. Kondo, 1995. Immunodefense system of Crustacea. Fish Pathol., 30: 141-150. 15

Tripathy, S., P. K. Sahoo, J. Kumari, B. K. Mishra, N. Sarangi and S. Ayyappan, 2006. Multiplex RT- PCR detection and sequence comparison of viruses MrNV and XSV associated with white tail disease in Macrobrachium rosenbergii. Aquaculture, 258: 134-139. Vargas-Albores, F., F. Jimenez-Vega and K. Soderhall, 1996. A plasma protein isolated from brown shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidase system by beta-1, 3-glucan. Dev. Comp. Immunol., 20: 299-306. Vazquez, L., G. Maldonado, C. Agundis, A. Perez, E. L. Cooper and E. Zenteno, 1997. Participation of a sialic acid-specific lectin from freshwater prawn Macrobrachium rosenbergii hemocytes in the recognition of non-self cells. J. Exp. Zool., 279: 265-272. Vazquez, L., L. Jaramillo, P. Rosas, R. Lascurain, E. Cooper and E. Zenteno, 1996. Bacterial agglutination by the sialic acid specific lectin from the freshwater prawn Macrobrachium rosenbergii. Comp. Biochem. Physiol., 113B: 355-359. Wickins, J., 1976. The tolerance of warm-water prawns to recirculated water. Aquaculture, 9: 19-37.

16 17

J. Aqua., 14 (2006): 17-21

THE CULTURE OF FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII IN KHARLAND (SALINE) PONDS OF RATNAGIRI, MAHARASHTRA

D. N. Saksena, D. M. Gaidhane and H. Singh* School of studies in Zoology, Jiwaji University, Gwalior, Madhya Pradesh, India *Department of Aquaculture, College of Fisheries, Ratnagiri, Maharashtra, India

The coastal saline soils locally called as ‘Kharland’, though unsuitable for agriculture can be utilized for the culture of prawns and fishes. The Kharland ponds were prepared for the culture by applying lime, cattle dung, urea and single super phosphates in appropriate doses to create suitable environment for culture. The prawn culture was done in two rectangular ponds with an area of 450 m2 (0.045 ha) each. Post-larvae of Macrobrachium rosenbergii were stocked @ 50,000 per hectare. The initial average size and weight of post-larvae stocked were 11.00±3.04 mm and 13.0±5.1 mg in pond P1, and 11.00±3.14 mm and 13.00±8.12 mg in pond P2. The prawns were fed with laboratory prepared feed @ 10% of the biomass every day. After a period of four months the prawn grew up to 88.0±6.5 mm and 2169.0±3.08 mg in pond P1 and 85.0±4.2 mm and 1980.0±9.8 mg in pond P2. The study suggested possible utilization of Kharland ponds for culture.

INTRODUCTION

About 65,465 hectares of Kharlands are available in Thane, Raigad, Ratnagiri and Sindhudurg coastal districts of Maharashtra, which possess potential for aquaculture. Working under different ago-ecological condition, Sidthimunka and Choapaknam (1968), Provenzano (1973), Adrill and Thompson (1975) and Sandifer and Smith (1976) have reported varying degrees of success during the culture of freshwater prawn, Macrobrachium rosenbergii. Shirgur et al. (1986) reported good growth of M. rosenbergii and a moderate growth of Penaeus monodon in Kharland ponds. In spite of the availability of large potential of such area, lack of appropriate technology with desired survival and yield has hindered its effective utilization. As the production of aquaculture crops from these areas would require suitable species and culture technology, the present study was an attempt to utilize the Kharland ponds of Ratnagiri through culture of M. rosenbergii.

18

MATERIAL AND METHODS

The Kharland ponds under study are located within the campus of the College of Fisheries, Ratnagiri (Lat. 16059'10" N and Long. 73016'25" E), Maharashtra, India. Each pond is of 0.045 hectare in size (30 m x 15 m) and depth of 1.5 meter. These are tide fed ponds and connected with the Shirgaon creek of Ratnagiri district. The study was carried out for four months i.e., between 29 August, 2001 to 29 December, 2001. After draining out the pond water to a maximum extent, a uniform spreading of quick lime (CaO) was done initially @ 100 kg/ha and same doses were applied at monthly intervals. After five days of liming, manuring was done with cattle dung @ 1000 kg/ha, urea @ 50 kg/ha and single super phosphate @ 30 kg/ha, and the same dose of manuring was also followed at monthly intervals. Post-larvae of M. rosenbergii were stocked in each pond @ 50,000 nos/ha.

The post-larvae and prawns were fed with a practical diet containing protein 36%, carbohydrate 30%, lipid 12%, moisture 13% and ash 9%. The feed was prepared in the laboratory by mixing groundnut oil cake, rice bran, wheat bran, prawn meal, fish meal and vitamin-B tablets. The feed was broadcasted above the pond in two installments, one- fourth in the morning and three-fourth in the evening @ 10% of the body weight during rearing period. Leaves of palm trees were kept in the ponds for providing shelter and facilitating periphyton growth. Sampling of prawns was done once in a month by using a cast net and the length and weight of prawns were recorded for a period of four months. The water samples were also collected and analyzed for temperature, pH, dissolved oxygen, free carbon dioxide, total alkalinity and salinity following standard methods (APHA, 1986).

RESULTS AND DISCUSSION

Among the physico-chemical characteristics of ponds, the temperature ranged from 28.0 to 31.0oC, pH from 6.85 to 7.35, dissolved oxygen ranged from 2.4 to 4.0 mg/l, free carbon dioxide from 8.0 to 16.0 mg/l, total alkalinity 18.0 to 32.0 mg/l and salinity fluctuated from 4.7 to 32.0‰ in both ponds during the period of study. Detail accounts of the water quality, soil characteristics and primary productivity of such Kharland ponds have also been reported by Saksena et al. (2006), Gaidhane and Saksena (2007) and Gaidhane et al. (2007).

The growth data indicates that the prawn had attained an average final length of 88.0±6.5 mm and 85.0±4.2 mm in ponds P1 and P2 respectively after a period of four months (Table 1). The average final weight of prawn was 2168±3.08 mg and 1980±9.8 mg in ponds P1 and P2 respectively. The specific growth rate (% per day) recorded in the corresponding ponds were 4.26 and 4.18. The pattern of growth in two ponds has been 19 quite similar to each other. The growth rate during the initial 60 days was found to be quite impressive, which however, was found to reduce with the progress of the culture period, reaching to a minimum level in the last phase.

Table 1. Growth increment of M. rosenbergii reared in Kharland ponds

Days Pond 1 Pond 2 Length (mm) Weight (mg) Length (mm) Weight (mg) Stocking 11.0±3.0 13.0±5.1 11.0±3.1 13.0±8.1 30 days 36.0± 2.7 450.0±5.7 50.0±5.7 580.0±7.8 60 days 65.0±4.3 1500.0±8.8 70.0±6.4 1310.0±10.3 90 days 80.0±1.2 2100.0±6.8 78.0±2.8 1895.0±4.6 120 days 88.0±6.5 2168.0± 3.1 85.0± 4.2 1980.0±9.8

Venugopalan (1988) has reported that M. rosenbergii can tolerate salinity up to 28‰. The species was also found to exhibit impressive growth rate in both freshwater as well as brackishwater (Wickins, 1972; Goodwin and Hanson, 1975; Perdue and Nakamura, 1976; Venugopalan, 1988), although Popper and Davidson (1982) obtained best growth in salinity range of 10-15‰. During the period of present investigation, there was a moderate but increasing rate of growth of prawns in both the ponds. These environmental factors could not be stabilized in the ponds to favour the growth of planktonic organisms since they were newly constructed and the salinity was on the higher side, i.e., up to 32‰ during the month of December. The present study indicated that the growth rate of 0.018 g in pond P1 and 0.016 g in pond P2 was registered per day in 120 days. It is also indicated that the weight gain decreased with the increase in salinity. The weight gain has shown a positive relationship with the salinity up to 28‰ but afterwards a negative correlation was evident. Our study has confirmed the observations of Venugopalan and Thampy (1992).

Kurup (2004) conducted studies on the technical feasibility and economic viability of farming of M. rosenbergii in the pokkali shrimp farms of Kerala and found encouraging results. His study has shown the potential of utilizing the pokkali field for effective culture of scampi. In the present study, a better growth of M. rosenbergii was recorded during 60 days of culture when the salinity was up to 18‰. Thus, along with other physico- chemical factors of water, M. rosenbergii farming activities depend on the salinity of the pond as observed by Smith et al. (1983). Maximum growth of any organism occurs at its iso-osmotic point, since the minimum energy is expended in osmoregulation at this juncture. The iso-osmotic point of M. rosenbergii is reported to be at about 17-18‰ (Sandifer et al., 1975; Singh, 1977, 1980), but the maximum growth could never be recorded at this salinity level (Wickins, 1972; Goodwin and Hanson, 1975; Perdue and Nakamura, 1976). Popper and Davidson (1982) have reported better growth rate of M. 20 rosenbergii at 10-15‰ salinity. Venugopalan and Thampy (1992) found a growth rate of 0.063 g in 100 days at salinity ranging from 2.1 to 20.4‰. The average growth rate of about 18 mg per day in the present study, suggest the feasibility of use of Kharland ponds for freshwater prawn culture.

The Kharland ponds are unique in physico-chemical characteristics during their seasonal cycle. From the month of July to October the water in these ponds is nearly freshwater with low salinity of <15‰ and hence during this period the pond culture of M. rosenbergii can be taken up. Considering the increase in salinity of these water bodies from the month of November till May, the possibility of culture of brackishwater prawns need to be explored.

ACKNOWLEDGEMENTS

The authors are thankful to the authorities of Jiwaji University, Gwalior and College of Fisheries, Ratnagiri for their help and encouragement. Our sincere thanks are also due to Indian Council of Agricultural Research, New Delhi for providing research grant and to the Coordinator, DRS- SAP (UGC) for providing all available facilities.

REFERENCES

Adrill, J. D. and R. K. Thompson, 1975. The freshwater prawn, Macrobrachium rosenbergii in Mauritius. FAO/CIFA Symposium. Aquaculture Africa, Ghana, FAO, Rome, pp. 15. APHA, 1986. Standard methods for the examination of water and wastewater. (15 Ed.). American Public Health Association, American Water Works Association and Water Pollution Control Federation, New York, pp 70-975. Gaidhane, D. M. and D. N. Saksena, 2007. Studies on sediments Kharland (saline) ponds of Ratanagiri with reference to prawn culture. Natu. Environ. Pollu. Technol., 6: 163-168. Goodwin, H. L. and J. A. Hanson, 1975. The aquaculture freshwater prawns (Macrobrachium species). Proc. Workshop on Culture of Freshwater Prawns, Nov. 1974, St. Petersburg, Florida, pp. 95. Kurup, B. M., 2004. On the technical feasibility and economic viability of farming of M. rosenbergii (de man) as a rotational crop in the Pokkali shrimp farms of Kerala. Proceedings of 3rd Indian Fisheries Science Congress, Nov. 4-6, 2004, IARI, Pusa, New Delhi, pp. 45. Perdue, J. A. and R. Nakamura, 1976. The effects of salinity on the growth of Macrobrachium rosenbergii. J. World Maricult. Soc., 7: 647-654. Popper, D. M. and R. Davidson, 1982. An experiment in rearing freshwater prawns in brackish water. In: Giant prawn farming (Ed. M. B. New). Developments in Aquaculture and Fisheries Science, 10, Elsevier Publishing Company, Amsterdam, pp. 173. Provenzano, Jr., A. J., 1973. Some results of a pilot project on freshwater prawn culture in Jamaica. Proceedings of 4th Annual Meeting of World Mariculture Society, 23-26 Jan., 1973, Monterey, Mexico, 4: 57-61. 21

Saksena, D. N., D. M. Gaidhane and H. Singh, 2006. Limnology of Kharland (saline) ponds of Ratanagiri, Maharashtra in relation to prawn culture potential. J. Environ. Biol., 27: 49-53. Sandifer, P. A. and T. I. J. Smith, 1975. Effects of population density on growth and survival of Macrobrachium rosenbergii reared in re-circulating water management systems. J. World Maricult. Soc., 6: 43-53. Sandifer, P. A. and T. I. J. Smith, 1976. Experimental aquaculture of the Malaysian prawn, Macro brachium rosenbergii (de Man) in South Carolina, USA, FAO Tech. Conf. Aquculture, 26 May to 2 June, 1976, Kyoto, Japan, pp. 306-311. Shirgur, G. A., R. K. Singh and J. B. Chavan, 1986. On the prospects of pond production of seed of Macrobrachium rosenbergii and culture trial besides Penaeus monodon and carps by common phased fertilization. Proc. Nat. Semn. on Perspectives hydrobiology, 8-10 Feb., 1986, Ujjain, India. pp. 69-74. Sidthimunka, A. and B. Choapaknam, 1968. A preliminary report on pond culture of giant freshwater prawn, Macrobrachium rosenbergii (de Man). FAO Fish. Rep., 44(5): 205-212. Singh, T., 1977. Osmotic and ionic regulation in Macrobrachium rosenbergii (de Man), (Decapoda: Caridea). M.Sc. Thesis, University of Malaysia, Kwalalumpur. Smith, T. I. J., W. E. Jenbins and P. A. Sandifer, 1983. Enclosed prawn nursery systems and effects of stocking juvenile Macrobrachium rosenbergii in ponds. J. World Maricult. Soc., 14: 111-125. Venugopalan, I. K., 1988. Effect of salinity on survival and growth of Macrobrachium rosenbergii (de Man). M.F.Sc. Thesis, Kerala Agricultural University, India. Venugopalan, I. K. and D. M. Thampy, 1992. Effect of salinity on the growth of giant freshwater prawn, Macrobrachium rosenbergii (de Man). Proc. Nat. Symp. on Freshwater Prawns (Macrobrachium sp.), 12 to 14 Dec., 1990 Kochi, Idnia. pp. 130 –136. Wickins, J.F., 1972. Experiment on the culture of the spot prawn, Pandalus platyceros (Brandt) and the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Fishery Invest., London, Ser. II, 27 (5): 1-23.

22 23

J. Aqua., 14 (2006): 23-27

MICROSPORIDIOSIS IN PENAEUS MONODON CULTURED IN THE BHERIES OF WEST BENGAL, INDIA

A. Roy, K. Maity, G. Dash* and T. J. Abraham Department of Fishery Pathology & Microbiology Faculty of Fishery Sciences West Bengal University of Animal & Fishery Sciences Chakgaria Campus, 5-B.H. Road, Panchasayar, Kolkata-700 094, West Bengal, India *Corresponding author

Bhery fisheries play a significant role in the total shrimp production of West Bengal. Due to the traditional practices and utilization of untreated sewage water, the shrimps of the bheries are under the threat of parasitic attack. Microsporidians, a protozoan endoparasite, can cause shrimp mortalities and consequent decrease in total harvest in the systems like Bhery fishery. During this study, it was observed that the wild aquatic organisms are the main carriers of Microsporidiosis and the incidence level was 1.17%. The muscle fibers were degenerated due to Microsporidiosis and numerous small minute spores were diffusely distributed throughout the muscle.

INTRODUCTION

Microsporidiosis is otherwise known as ‘Cotton shrimp disease’ has generally been recognized by the progressive white opacity associated with the musculature. Microsporidians such as Agmasoma (=Thelohania), Ameson (=Nosema) and Plistophora (=Pleistophora) have been reported in shrimps/prawns of several genera notably Penaeus, Pandalus and Crangon. They cause diseases in epizootic proportions in feral crustaceans (Overstreet, 1973; Sindermann, 1990).

Bheries or wetland in West Bengal covering approximately 32,930 ha water spread area, is contributing a major portion of prawn/shrimp fishery (Pandit, 2000). The traditional practice in this system invites pathogen and a significant reduction in total production has been observed due to disease outbreak in recent years, mainly by white spot disease. Protozoan diseases also cause a significant health hazards in shrimp/prawn. The entry of wild organisms through incoming water into the system carries unwanted organisms (wild prawns, other crustaceans, small fishes, molluscs etc), which are considered as carriers of different pathogens. The present investigation was undertaken to determine the prevalence and pathology of microsporidians in Penaeus monodon in the bheries of West Bengal. 24

MATERIAL AND METHODS

A total of 769 live specimens of Penaeus monodon with carapace length ranging from 5 cm to 30 cm were randomly taken for pathological observations from the harvest of the bheries (8 sampling stations include 47 bheries) located in North and South 24 Parganas districts of West Bengal, India, during February-November, 2001. The gross and clinical signs and other abnormalities were observed carefully at site. Shrimps with opacity in the abdominal musculature were fixed in Davidson’s fixative and processed as described by Bell and Lightner (1988) for histopathological observations.

RESULTS

The infected shrimps were weak, milky white and exhibited opaque abdominal musculature (Fig. 1). Shrimps weighing 5-10 g showed the clinical signs of cotton shrimp disease. Among the collected shrimps only nine had symptoms typical of Microsporidiosis. The incidence rate was 1.17% only. The histopathological sections of normal shrimps showed perfect myofibrilar arrangement compared to the affected shrimps (Fig. 2).

The squash preparations of muscle tissues from infected shrimps revealed degeneration of myofibrils and presence of numerous small spores. Histopathological examinations of the infected mussel tissues revealed that the microsporidians slowly degenerated the muscle fiber and replaced the muscle tissues with spores (Fig. 3). In the early phase of infection only small portions of the muscle fibers were infected; however, as the disease progressively spread over a greater area the muscle fibers were gradually transformed into necrotic structure. No clear pansporoblast membrane was seen and diffuse distribution of single spores in the muscle was observed. Heavy haemocytes infiltration in the gill and muscle of infected shrimps was seen (Figs. 4 & 5). Extensive accumulation of Zoothamnium sp., an ectocommensal peritrichous free living ciliate was noticed in the gill of microsporidian infected shrimps (Fig. 6).

Other organs affected include ovary, gut epithelial layer and hepatopancreas. Hepatopancreatic tubular degeneration was of common occurrence in the milky white shrimps (Fig. 7). Muscle tissues around intestine showed heavy accumulation of microsporidian spores (Fig. 8).

DISCUSSION

The number of spores per sporant could not be accurately determined, as it requires electron microscopic observations. However, the tissue infection characteristics are almost specific for each microsporidian species (Kelly, 1979). From the description of Anderson et al. (1989), the observation of diffuse distribution of many minute single spores in the muscle the microsporidian might be placed under genus Ameson (= Nosema).

25

1 2

3 4

5 6

Fig. 1. Microsporidian infected shrimp with milky white appearance collected from the Bheries of West Bengal; Fig. 2. Longitudinal section of muscle of a normal shrimp showing perfect arrangements of muscle fibre (H & E X 200); Fig. 3. Histological section of the infected muscle showing diffuse distribution of microsporidian spores and subsequent muscle degeneration (H & E X 400); Fig. 4. Marked haemocytic response (Arrow) in the gill of microsporidian infected shrimp (H&E X 400); Fig. 5. Marked haemocytic response (Arrow) in the muscle of microsporidian infected shrimp (H&E X 400); Fig.6. Gill section of Microsporidian infected shrimp showing association of ectocommensal ciliate, Zoothamnium (Arrow) (H&E X 400).

26

Fig. 7. Hepatopancreatic section of Fig. 8. Muscle tissue around midgut intestine Microsporidian infected shrimp with showing degeneration by Microsporidian tubular degeneration. Normal star spores. Note, the degenerated midgut shaped lumen is absent (H&E X 400) epithelial layer. (H & E X 200)

The muscle fiber degeneration observed in this study was similar to those described by Johnson (1990) and Ramasamy et al. (2000). The mechanism of muscle fiber degeneration and replacement by the Microsporidians yet remain unknown (Ramasamy et al., 2000). The heavy haemocytic response in the gill and muscle tissues might be a consequence of the first line of defense against the parasitic attack. The weight of the infected shrimp (5-10 g) indicated that the infection is common with advancing age, which agreed with the report of James (1986).

Although the Microsporidian infection rate in bheries was less and not so common, it should not be ignored as it has potential to cause mortality in shrimp. Since, most of the bheries receive sewage water without treatment, there is every chance of getting carriers such as weed fish, shrimp, molluscs and others into the system. According to Overstreet (1973) and Johnson (1990) wild shrimps are the potential carriers of Microsporidiosis.

The life cycle of microsporidians and it’s mode of transmission are not clearly known till date (Limsuwan, 1993) and therefore, the nature and time of appearance of Microsporidiosis is unpredictable. General preventive measures of farm management are advised to prevent the disease.

REFERENCES

Anderson, I. G., M. Shariff and G. Nash, 1989. A hepatopancreatic microsporidian in pond reared tiger shrimp, Penaeus monodon from Malaysia. J. Invert. Pathol., 53(2): 278-280. James, D. B., 1986. On the incidence of the sporozoan Thelohania prox duorara Iverson and Manning in commercially important prawns at Madras. J. Mar. Bio. Assoc. India, 28(1-2): 225-228. 27

Johnson, S. K., 1990. Handbook of Shrimp Disease (Aquaculture), Department of Wildlife and Fisheries Sciences, Texas A & M University, p. 25. Kelly, J. F., 1979. Tissue specificities of Thelohania duorara, Agmasoma penaei and Pleistophora sp., microsporidian parasites of pink shrimp. Penaeus duorarum. J. Invert. Pathol., 33: 331-339. Limsuwan, C., 1993. Disease of Black Tiger Shrimp, Penaeus monodon Fabricius, in Thailand. In: American Soybean Society Technical Bulletin (Ed. D. Akiyama), American Soybean Soc., Singapore, 39: 1-15. Overstreet, R.M., 1973. Parasites of some penaeid shrimps with emphasis on reared hosts. Aquaculture, 2: 105-140. Pandit, P. K., 2000. Vision on ecology and fishery of the Bheries (wet lands) in West Bengal. In: Sekhar Basu (Ed.), Hand Book on Aquaculture and Fishery Vision, National Conference on Aquaculture and steps to maintain high production, January 2000, Kolkata, pp. 207-210. Ramasamy, P., R. Jayakumar and G. P. Brennan, 2000. Muscle degeneration associated with cotton shrimp disease of Penaeus indicus. J. Fish Dis., 23(1): 77-81. Sindermann, C. J., 1990. Principal diseases of marine fish and shellfish. Vol-ll, 2nd edition. Academic Press, San Diego, 516 pp.

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J. Aqua., 14 (2006): 29-38

IMPACT OF WATER POLLUTION ON FISH PHYSIOLOGY – A STUDY

Subir Das and Tapati Bhattacharya* Katwa College Research Center, Katwa, Burdwan- 713130 West Bengal, India *Corresponding author: [email protected]

The freshwater air-breathing teleost, Channa punctatus were exposed to mercuric chloride (0.15 ppm) and cadmium chloride (35.5 ppm) for 1, 2, 7, 15 and 30 days so as to determine short and long-term effects of sub-lethal doses of these industrial pollutants on different haematological parameters. The selection of pollutants and their concentrations was based on the real data, in relation to the degree of water pollution of Damodar River. Blood samples were collected from the fishes exposed to various exposure periods for each pollutant and the blood glucose, haemoglobin content, cholesterol, HDL-cholesterol, LDL- cholesterol, VLDL- cholesterol, triglycerides, total RBC and WBC counts were measured. The blood glucose, haemoglobin content, total RBC and WBC counts progressively decreased. Blood cholesterol, HDL-cholesterol, LDL- cholesterol, VLDL-cholesterol and triglycerides were also depleted. Linear regression analysis with appreciable values of R2 and t- ratios revealed that the values of different blood parameters were reduced radically with the progress of days, except haemoglobin, signifying negative impact of pollutant of HgCl2. Strong correlation between different blood parameters calls for that adverse variations. Histopathological changes in the hepatopancreas induced by the heavy metals like mercuric chloride and cadmium chloride include degeneration of hepatic cells, clumping of cytoplasmic materials, displacement of nucleus towards the periphery, derangement of the pancreatic acini and coagulation of blood in the sinusoids. Analysis of experimental data suggests the depletion of energy resources and disturbance of the metabolic pathway as indicated by the adverse effects on haematological parameters and histopathological lesions in the haepatopancreas.

INTRODUCTION

The discharge of pollutants with rapid industrialization is discernable in the age of globalization. Agricultural run-off is also a source of accumulation of pollutants into the sink as agriculture is chemicalised today. Heavy metals are wide spread pollutants of great environmental concern as they are non-degradable with immense toxicity to the bio-wealth of the earth (Stratton, 1987). The toxicity is assimilative to the nature up to a certain extent, beyond the threshold point it crosses the environmental carrying capacity (Gadd, 1992). The problem of toxicity of metallic compounds in water is very complex in nature. Obviously it harms water-based living resources. So the depletion of stock of 30 water-based living species is perhaps pronounced. For micro level laboratory-oriented study, the methods of clinical diagnosis have been introduced in the fish physiology to assess sub-lethal effects of pollutants (Wedemeyer and Yasutake, 1977). The effects of mercuric chloride and cadmium chloride on the physiological functions of freshwater teleost, Channa punctatus is under study.

Water pollution in Damodar river range, especially in Durgapur Industrial Belt, West Bengal, is appalling today. The sample of analytical data for the factory effluent collected from river Damodar are showing very appalling conditions. The values of different components in terms of ppm are mentioned, copper 0.1, sulphide 20, phenol 19, ammonia 0.9, chloride 20, dissolved oxygen 0.05, dissolved solids 232, total solids 418, total alkalinity 148 (CaCO3) and total hardness (CaCO3) 110. The temperature and pH of water were found to be 240C and 8.5, respectively.

MATERIAL AND METHODS

A 30 days chronic exposure test with mercuric chloride (0.15 ppm) and cadmium chloride (35.5 ppm) was conducted with C. punctatus (Bloch), a common air-breathing teleost with a wide distribution in India. Fishes collected locally, were acclimatized to laboratory conditions for 15 days and fed daily during the evening with commercial fish food containing 8% protein, 3% lipid and 20% carbohydrates. Fishes of average length of 16 cm and weight of 35 g were kept in the batches of 10 in glass aquaria (60 x 30 x 30 cm) containing 30 l tap water (pH 7.6, temperature 280C and dissolved oxygen 8 ppm). A concurrent control was maintained with each experimental set. During exposure period water was changed regularly and pollutants were freshly added. Tissues were collected from the control and experimented fishes on 1st, 2nd, 7th 15th and 30th day of exposure.

Blood was collected by caudal puncture with 1 ml syringe and 24 gauge needle, previously rinsed with 10% sodium heparin. Haemoglobin content was estimated by the method of Van Kampen and Zijlstra (1961) and the total RBC and WBC counts by Dacie and Lewis (1975). Blood cholesterol was estimated by one step method (Wybenga and Pileggi, 1971) and HDL- cholesterol by the method (Warnick et al., 1985). Plasma triglycerides was measured by enzymatic method (Gowan, 1983). Plasma glucose concentration was measured by the glucose oxide-peroxidase technique (Tietz, 1976). The VLDL and LDL-cholesterol contents obtained from triglycerides and HDL-cholesterol. Blood thyroxine (T4) was measured by radioimmuno-assay technique (Chopra, 1972). Small pieces of liver (hepatopancreas) were collected from control as well as the experimented fishes on 1st, 2nd, 7th, 15th and 30th day of exposure and fixed in Bouin’s fluid for routine haematoxylin and eosin staining.

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Statistical analysis The impact of industrial pollution on fish physiology and or stock can be established empirically, backed by the laboratory-based data. The statistical tools applicable to real data is very relevant today as far as finer sense of quantitative analysis is concerned. Exposure periods of 1, 2, 7, 15 and 30 days may be treated as independent variable and the blood components with the effects of mercuric chloride and cadmium chloride are assumed to be dependent variables so as to assess the degree of influence of exposure periods on intrinsic character of blood parameters. Perhaps the data fit to regression analysis with ordinary least square analysis method (Johnston, 1960) on the assumption of linear form of equation. The values of R2 and t-ratios of each equation are to be assessed to verify the reality of the mathematical equations.

RESULTS

Exposure of C. punctatus to mercuric chloride resulted in significant decrease of plasma glucose concentrations, haemoglobin content, total RBC and WBC counts throughout the exposure period (Table 1). The significant decrease in plasma glucose level is on 30 days by 40% in respect of control value. Haemoglobin content remarkably decreased by 46% on 15th day of exposure. Total RBC count decreased by 47% on day 15 and total WBC decreased by 82% on day 15 and 77% on day 30. The depletion of blood cholesterol was noticed 55% on day 30. With regard to plasma HDL, LDL and VLDL cholesterol, maximum depletion was observed on 30th day by 95% in HDL-cholesterol level, 19% in LDL-cholesterol and 73% in VLDL-cholesterol in respect to control values. The plasma trigycerides content exhibited declining trend steadily, though the maximum reduction was noticed on day 30 by 76% in respect to control value. Blood T4 was found to be lower throughout the experimental period.

The plasma glucose concentration, haemoglobin content, total RBC and WBC counts decreased throughout the exposure periods (Table 2) owing to cadmium chloride exposure. The significant decrease in plasma glucose level by 68 and 93% in respect to control was observed on day 7 and 30 of exposure, respectively. In haemoglobin content, remarkable decrease of 36% occurred on 15th day of exposure. Total RBC count decreased by 83% on day 15 and 82% on day 30. The depletion of blood cholesterol was noted 62% on the day 7 and 30, but on 15th day certain elevation was noticed. With regard to plasma, HDL, LDL and VLDL cholesterol, maximum depletion was observed on 30th day, 95% in HDL-cholesterol and 88% in VLDL-cholesterol in respect to control value. The plasma triglycerides content showed consistent decrease although maximum 44% decrease occurred on the 7th day of exposure. On day15 the experimental value was neared to the control value. But on day 30 the triglycerides content increased by 14% in respect to control value. The blood T4 titre decreased dramatically during the first two days of exposure. It rose to on 7th and 15th day but declined significantly on 30th day. 32

Table 1. Effect of Mercuric chloride administered continuously for 30 days on the various haematological parameters of Channa punctatus

System Exposure period (Day) 1 2 7 15 30 Blood 2.15±0.02 (5)** 1.93±0.64 (5) 1.21±0.48 (5) 1.27±0.06 (5) 0.98±0.01 (5) cholesterol 2.55±0.87* (5)** 2.41±0.95 (5) 1.98±0.42 (5) 2.04±0.56 (5) 2.21±0.22 (5) mg/ml t = 3 t = 1.5 t = 3.7 t = 2.87 t = 3.9 P < 0.01 N.S P< 0.005 P< 0.025 P< 0.005 HDL- 1.07±0.04 (5) 0.16±0.03 (5) 0.08±0.002 (5) 0.04±0.005 (5) 0.04±0.009 (5) cholesterol 1.35±0.01 (5) 1.12±0.07 (5) 1.32±0.05 (5) 1.21±0.098 (5) 0.98±0.028 (5) mg/ml t = 2.81 t = 1.18 t = 14.03 t = 14.87 t = 8.87 P< 0.025 N.S. P< 0.005 P< 0.005 P< 0.005 LDL- 0.96±0.02 (5) 1.25±0.012 (5) 1.12±0.04 (5) 1.12±0.038 (5) 0.922±0.08 (5) cholesterol 1.10±0.08 (5) 1.41±0.037 (5) 1.34±0.58 (5) 1.20±0.085 (5) 1.14±0.12 mg/ml t = 5.08 t = 3.32 t = 0.45 t = 0.59 t = 0.76 P< 0.005 P< 0.01 N.S. N.S. N.S. VLDL- 0.08±0.002 (5) 0.04±0.002 (5) 0.04±0.002 (5) 0.02±0.003 (5) 0.02±0.005 (5) cholesterol 0.09±0.008 (5) 0.082±0.007 (5) 0.099±0.001 (5) 0.09±0.006 (5) 0.08±0.002 (5) mg/ml t = 1.18 t = 4.06 t = 4.13 t = 6.90 t = 2.95 N.S. P < 0.005 P< 0.005 P< 0.005 P< 0.01 Triglyceride 0.42±0.03 (5) 0.24±0.04 (5) 0.23±0.04 (5) 0.12±0.01 (5) 0.10±0.02 (5) mg/ml 0.49±0.08 (5) 0.25±0.01 (5) 0.40±0.08 (5) 0.48±0.03 (5) 0.45±0.08 (5) t = 2.64 t = 8.99 t = 10.70 t = 15.48 t = 9.26 P< 0.02 P< 0.005 P< 0.005 P< 0.005 P< 0.005 Hb- 0.07±0.005 (5) 0.10±0.002 (5) 0.06±0.05 (5) 0.05±0.008 (5) 0.07±0.009 (5) mg/ml 0.10±0.02 (5) 0.12±0.0045 (5) 0.10±0.02 (5) 0.10±0.06 (5) 0.10±0.05 (5) t = 4.5 t = 7.2 t = 6.95 t = 11.36 t = 7.2 P< 0.005 P< 0.005 P< 0.005 P< 0.005 P< 0.005 Blood 0.63±0.06 (50 0.61±0.02 (5) 0.49±0.02 (5) 0.52±0.05 (5) 0.48±0.03 (5) glucose 0.91±0.08 (5) 0.89±0.05 (5) 0.87±0.04 (5) 0.80±0.09 (5) 0.82±0.07 (5) mg/ml t = 4 t = 6.5 t = 11.38 t = 10.40 t = 6.81 P< 0.005 P< 0.005 P< 0.005 P< 0.005 P< 0.005 RBC 207800±252040 (5) 1958000±270500(5) 2008000±125060(5) 1488000±72420 5) 1812000±59630(5) cu.mm 2806000±684250(5) 2802000±389200(5) 2804000±547200(5) 280800±95360(5) 2707000±423700(5) t = 2.42 t = 8.2 t = 3.84 t = 15.29 t = 7.52 P< 0.025 P< 0.005 P< 0.005 P< 0.005 P< 0.005 WBC 19586±1402.2 (5) 18940±2085.3(5) 13296±2880.24(5) 4240±620.4 (5) 5248±2340.64(5) cu.mm 25690±2832.5(5) 24890±5272.1 (5) 25280±4770.52(5) 23870±5822.4 (5) 23690±22041.7 (5) t = 5.52 t = 6.52 t = 6.55 t = 21.72 t = 12.63 P< 0.005 P< 0.005 P< 0.005 P< 0.005 P< 0.005 Thyroxine 0.87±0.12 (8) 1.24±0.15 (8) 1.15±0.17 (6) 0.89±0.12 (5) 1.95±0.45 (5) [Ng T4/ml 2.65±0.12 (8) 2.65±0.12 (8) 1.98±0.15 (8) 2.08±0.15 (6) 3.63±0.32 (6) blood] P< 0.001 P< 0.001 P< 0.001 P< 0.001 P< 0.001 * Respective control values±S.E. are denoted in parentheses, N.S.: Not significant ** Number of fish is denoted in parentheses

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Table 2. Effect of cadmium chloride administered continuously for 30 days on the various haematological parameters of Channa punctatus

System Exposure period (Day) 1 2 7 15 30 Blood 2.10±0.27 (7)** 1.8±0.31 (5) 0.75±0.12 (5) 1.54±0.29 (5) 0.84±0.02 (5) cholesterol 2.55±0.48* (7) ** 2.41±0.50 (5) 1.98±0.42 (7) 2.04±0.58 (7) 2.21±0.60 (7) mg/ml t = 0.83 t = 0.94 t = 3.1 t = 6.87 t = 4.27 N.S N.S P < 0.05 P < 0.005 P < 0.005 HDL- 1.08±0.06 (5) 0.74±0.02 (5) 0.18±0.05 (5) 0.13±0.01 (5) 0.05±0.01 (5) cholesterol 1.35±0.56 (5) 1.12±0.06 (5) 1.32±0.08 (5) 1.21±0.03 (5) 0.98±0.02 (5) mg/ml t = 2.77 t = 6.08 t = 18.36 t = 13.71 t = 12.82 P < 0.025 P < 0.005 P < 0.005 P < 0.005 P < 0.005 LDL- 0.93±0.02 (5) 1.00±0.12 (5) 0.52±0.02 (5) 1.39±0.002 (5) 0.27±0.08 (5) cholesterol 1.10±0.03 (5) 1.14±0.51 (5) 0.92±0.08 (5) 1.09±0.08 (5) 0.97±0.015 (5) mg/ml t = 0.95 t = 0.54 t = 3.25 t = 1.63 t = 2.02 N.S N.S P < 0.01 N.S P < 0.05 VLDL- 0.08±0.009 (5) 0.04±0.001 (5) 0.04±0.008 (5) 0.01±0.003 (5) 0.01±0.009 (5) cholesterol 0.09±0.004 (5) 0.009±0.002 (5) 0.09±0.004 (5) 0.09±0.008 (5) 0.08±0.01 (5) mg/ml t = 3.60 t = 9.44 t = 9.58 t = 21.13 P < 0.005 P < 0.005 P < 0.005 P < 0.005 P < 0.005 Triglyceride 0.40±0.05 (5) 0.24±0.09 (5) 0.22±0.02 (5) 0.47±0.02 (5) 0.51±0.09 (5) mg/ml 0.49±0.09 (5) 0.42±0.02 (5) 0.40±0.07 (5) 0.48±0.08 (5) 0.45±0.03 (5) t = 3.60 t = 9.44 t = 10.45 t = 0.98 t = 4.20 P < 0.005 P < 0.005 P< 0.005 N.S P< 0.005 Hb- 0.10±0.002 (5) 0.08±0.006 (5) 0.08±0.004 (5) 0.06±0.006 (5) 0.08±0.009 (5) mg/ml 0.11±0.007 (5) 0.10±0.01 (5) 0.10±0.02 (5) 0.10±0.04 (5) 0.10±0.05 (5) t = 3.81 t = 4.55 t = 5.3 t = 11.69 t = 13.28 P< 0.005 P< 0.005 P< 0.005 P< 0.005 P< 0.005 Blood 0.77±0.07 (5) 0.94±0.005 (5) 0.29±0.08 (5) 0.55±0.02 (5) 0.58±0.01 (5) glucose 0.99±0.03 (5) 0.97±0.001 (5) 0.92±0.04 (5) 0.85±0.08 (5) 0.84±0.09 (5) mg/ml t = 6.47 t = 1.24 t = 15.34 t = 7.62 t = 6.92 P< 0.005 N.S P< 0.005 P< 0.005 P< 0.005 RBC 198000±21250(5) 1920000±24560(5) 2068000±10200(5) 1402000±85240(5) 1580000±285000(5) cu.mm 2806000±35230 (5) 2802000±42670(5) 2804000±26840(5) 2088000±10450(5) 2707000±4.8900(5) t = 2.74 t = 8.2 t = 3.81 t = 15.29 t = 13.94 P< 0.005 P< 0.005 P< 0.005 P< 0.005 P< 0.005 WBC 18652±2040.5 (5) 17930±1250.6(5) 10320±824.02 (5) 4105±870.24 (5) 4292±850.29 (5) cu.mm 25690±1320.3 (5) 24890±1020.1(5) 25280±1025.10 (5) 23870±2570.82 (5) 23690±3280.4 (5) t = 66.15 t = 5.85 t = 16.11 t = 22.1 t = 20.26 P< 0.005 P< 0.005 P< 0.005 P< 0.005 P< 0.005 Thyroxine 0.85±0.14 (6) 0.98±0.18 (6) 1.36±0.12 (8) 1.59±0.32 (5) 1.95±0.32 (5) (T4/ml 2.65±0.12 (8) 2.65±0.12 (8) 1.98±0.15 (8) 2.08±0.15 (6) 3.63±0.32 (6) blood) P< 0.001 P< 0.001 P< 0.01 N.S P< 0.001 * Respective control values±S.E. are denoted in parentheses, N.S.: Not significant ** Number of fish is denoted in parentheses 34

In case of cadmium chloride we have the 7 valid linear equations confirming to acceptable values of R2 and t-ratios and it is only 8 for mercuric chloride (Tables 3 & 4). The inverse association between the HDL-cholesterol and length of the exposure periods under treatment of cadmium chloride is statistically established, this sort of relationship is not discernable in view of mercuric chloride as R2 and t-ratios does not permit. VLDL- cholesterol and WBC declines as exposure periods increased under cadmium chloride. Haemoglobin contents are reduced at small rates in both treatments, but values are statistically insignificant.

Table 3. Linear regression analysis of different haematological parameters of Channa punctatus due to the effect of mercuric chloride

Haematological parameter Intercept Coefficient R 2 Blood. Chol T 1.89 (9.07) -0.03 (-2.56) 0.83 Blood. Chol C 2.21 (5.25) -0.02 (-0.57) 0.90 HDL-Chol T 0.49 (1.79) -0.02 (-1.11) 0.29 HDL-Chol C 1.29 (17.36) -0.009 (-1.92) 0.55 LDL-Chol T 1.13 (13.47) -0.005(-1.06) 0.27 LDL-Chol C 1.28 (14.39) -0.004 (-0.004) 0.16 VLDL- Chol T 0.05 (4.57) -0.001 (-1.86) 0.54 VLDL- Chol C 0.08 (23.45) -0.001 (0.71) 0.14 Triglyceride T 0.31 (5.62) -0.008 (-2.33) 0.64 Triglyceride C 0.38 (5.63) 0.002 (0.68) 0.13 Hb T 0.07 (5.84) 0.0005 (-0.63) .011 Hb C 0.11 (17.88) -0.0003 (-0.8) 0.17 Blood Glucose T 0.59 (17.98) -0.004 (-2.06) 0.58 Blood Glucose C 0.89 (44.24) -0.003 (-2.4) 0.65 RBC T 1984817 (3.1) 20797.6 -0.39) 0.29 RBC C 2508733 (3.1) 20797.6 (-0.39) 0.04 WBC T 18065.4 (6.7) 5270.58 (-3.04) 0.75 WBC C 25382 (79.36) -63.48 (3.04) 0.76 Thyroxine T 0.91 (4.38) 0.03 (2.07) 0.59 Thyroxine C 2.23 (5.78) 0.03 (1.32) 0.36

T: Equations denoting under treatment, C: Equations denoting under control parentheses denoting t values

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Table 4. Linear regression analysis of different haematological parameters of Channa punctatus due to the effect of cadmium chloride

Haematological parameter Intercept Coefficient R 2 Blood. Chol T 1.75 (5.17) -0.03 (1.43) 0.41 Blood. Chol C 2.33 (14.44) -0.008 (-0.82) 0.18 HDL-Chol T 0.75 (3.56) -0.03 (-1.92) 0.59 HDL-Chol C 1.29 (17.36) 0.009 (-1.92) 0.55 LDL-Chol T 1.00 (3.52) -0.01 (-0.9) 0.21 LDL-Chol C 1.08 (17.75) -0.003 (-0.94) 0.22 VLDL- Chol T 0.05 (4.32) -0.009 (-2.22) 0.62 VLDL- Chol C 0.05 (2.18) 0.001 (0.59) 0.1 Triglyceride T 0.28 (4.11) 0.007 (1.77) 0.51 Triglyceride C 0.44 (15.74) 0.0003 (0.18) 0.01 Hb T 0.08 (8.95) -0.0005 (-0.78) 0.17 Hb C 0.1 (35.45) -0.0002 (-0.91) 0.21 Blood Glucose T 0.7 (4.14) -0.007 (-0.65) 0.12 Blood Glucose C 0.97 (45.25) -0.005 (-3.67) 0.81 RBC T 1278450 (2.39) 14104.5 (0.41) 0.05 RBC C 2820450 (176) -3186 (-3.05) 0.75 WBC T 16640.33 (6.24) -502.32 (-2.92) 0.74 WBC C 25382.33 (79.36) -63.48 (-3.04) 0.75 Thyroxine T 0.95 (10.34) 0.04 (6.01) 0.92 Thyroxine C 2.27 (6.54) 0.02 (1.06) 0.27 T: Equations denoting under treatment, C: Equations denoting under control parentheses denoting t values

Histopathological observations The hepatopancreas of C. punctatus is characterized by continuous mass of hepatic cells arranged in cords. The hepatic cells are large, hexagonal in shape, each with a centrally placed round nucleus and homogeneous cytoplasm. The length and breath of hepatic cells varied from 7.5-9.0 µ and 6.0-7.5 µ, respectively. Hepatic cells are not distinctly lobulated. The exocrine pancreatic acini lie embedded in a compact manner in between the hepatic cells surrounding blood capillaries. Each exocrine cell contains a spherical nucleus at its base. A large number of blood sinusoids are found among the hepatic cells (Fig.1).

Histopathological lesions were induced in the hepatopancreas of C. punctatus (Bloch) exposed over short and long term to mercuric chloride and cadmium chloride. 36

More or less similar lesions, clumping of cytoplasmic materials, displacement of nucleus towards the periphery, derangement of the pancreatic acini and coagulation of blood in the sinusoids were occurred. The damage induced in the hepatopancreas was of a greater magnitude on the 15th day of exposure. Some adaptive responses such as cytoplasmic regeneration in the hepatocytes were noticed on the 30th day of exposure (Figs.2 &.3).

Pictorial descriptions of microscopic cell structure of hepatopancreas of Channa punctatus under control and treatment

hc

pa

bv bv pa

Fig. 1. Tranverse Section (T.S) through the Fig. 3. Tranverse Section (T.S) through the hepatopancreas of the control Fish x 500 hepatopancreas of the Fish exposed to cadmium chloride 30day x 500

pa Fig. 2. Tranverse Section (T.S) through the hepatopancreas of bv the Fish exposed to cadmium chloride 1day x 500

ne Abbreviations: hc = hepatic cell, pa = pancreatic acinus, ne = nuclear extrusion, bv = blood vessel 37

DISCUSSIONS

0.15 ppm mercuric chloride and 35.5 ppm cadmium chloride led to significant decrease in most of the haematological parameters throughout the exposure period. These metals are the indicators of toxicity and in agreement with several other reports (Aruna and Gopal, 1987). Metallic mercury has a comparatively low acute toxicity when not in gaseous form, its salt as well as organic mercury compounds like methyl mercury are extremely toxic and can be taken from the digestive tract and transported by the blood to other parts of the body (Magos, 1977). In 1950s and 60s several thousand people in the Japanese cities of Mina Mata and Migata suffered from nervous disorder known as Mina Mata disease as a result of consuming fishes and sea food with a metal content (Tsuru et al., 1989). Exposure to fish with Hg during pregnancy and lactation were studied in 100 women and new borns from Porto Velho, Amazon, by Marques et al. (2007). According to their report the development delay of exclusively breastfed infants are component of the health inequalities that accompanies socioeconomic disadvantages. So the mercury levels in the blood for fish eaters and non-fish eaters are perhaps well distinguishable signifying that the health damage is the aftermath for the group of fish eaters. There is some evidence that high mercury may adversely affect the functional integrity at the inter-renal and the thyroid axis in fish. Thyroid hormone and cortisol have an important role in physiological fitness of fish by its effects on reproduction, growth and the immune functions (Pickering, 1993). T4 decreased significantly in Angualla anguilla due to exposure to chromium and copper reported by Teles et al. (2005). According to the report of Lacroix and Hontela (2006), the deleterious effect of (Cd++) on cortisol steroidogenesis may be enhanced when the endocrine stress response is triggered. Chronic exposure of yellow perch to sub-lethal levels of heavy metals impairs growth and alters the seasonal cycling of liver glycogen and triglycerides as well as the activities of metabolic enzymes (Levesque et al., 2002). Copper reduced the growth rate in rock fish Sebastes schiegele and there was an inverse relationship between the growth and the copper concentrations (Kim and Kang, 2004). The cellular damage in the hepatopancreas due to exposure to mercuric chloride and cadmium chloride disturbs the metabolic pathway as indicated by the adverse effects on haematological parameters. We cannot deny on the basis of data generated that fishes are damaged, poisoned and afflicted, even not dead, despite the growing awareness on pollution-intensive activities. The fish resource are being excluded from getting natural growth path owing to old practice of appropriating profit maximizing industrial output, not socially optimum output.

ACKNOWLEDGEMENTS

The authors are grateful to University Grants Commission, New Delhi, for financial support.

38 REFERENCES

Aruna, D. and V. Gopal, 1987. Toxic effect of sub lethal level of mercury on haematological parameters. Indian J. Environ. Hlth., 30: 52-56. Chopra, I. J., 1972. A radioimmunoassay for measurement of thyroxine in unextracted serum. J. Clin. Endocrinol. Metab., 34: 938-947. Dacie. J. V. and S. M. Lewis, 1975. Pactical haematology 4th Edn. Churchill Living stone, London, 509 pp. Gadd, G. M., 1992. In Encyclopedia of Microbiology (Ed.. J. Ledererg) Academic Press. Inc. Harcourt Brace Javanovich Publishers. Vol. 2. Sansn Diego, pp. 351-360. Gowan. M. C., 1983. Clin. Chem., 29: 538. Johnston, J., 1960. Econometric Methods. 2nd Edn. McGraw-Hill International Book Company. Kim, S. G. and J. C. Kang, 2004. Effect of dietary copper exposure on accumulation, growth and haematological parameters of the juvenile rockfish, Sebastes schlegeli. Mar. Environ. Res., 58(1): 65-82. Lacroix, A. and A. Hontela, 2006. Role of calcium channels in cadmium-induced disruption of cortisol synthesis in rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. C Toxicol. Pharmacol., 144(2): 141-147. Levesque, H. M., T. W. Moon, P. G. Campbell and A. Hontela, 2002. Seasonal variation in carbohydrate and lipid metabolism of yellow perch (Perca flavescens) chronically exposed to metals in the field. Aquat. Toxicol., 60(3-4): 257-267. Magos. L., 1997. Physiology and Toxicology of Mercury. In: A. Sigel and H. Sigel (Eds.) Metal lons in Biological Systems. Vol. 34 Mercury and its Effect on Environment and Biology. Marcel Dekkar Inc. New York, USA, pp. 321. Marques, R. C., J. Garrofe Dorea, W. Rodrigue Bastos, M. de Freitas Robele, M. Freitas Fonseca and O. Malm, 2007. Maternal mercury exposure and neuro-motor development in breast fed infants Porto Velho (Amazon), Brazil. Int. J. Hyg. Environ. Health, 210(1): 51-60. Pickering, A. D., 1993. Endocrine Pathology in stressed salmonid fish. Fish Res., 17: 35-50. Stratton, G. W., 1987. In: Review in Environmental Toxicology. Ed. Hodgson, Elsevier, Amsterdam, pp. 85-94. Teles, M., M. Pacheco and M. A. Santos, 2005. Physiological and genetic responses of European eel (Anguilla anguilla L) to short–term chromium or copper exposure-influence of pre-exposure to a PAH-like compound. Environ. Toxicol., 20(1): 92-99. Tietz, N. W., 1976. Clinical guide to laboratory test. W.B. Saunders Co. Philadelphia. 238 pp. Tsuru, S., T. Suzuki, H. Shiraki, M. Miyamoto Shimizu and M. Harada, 1989. For truth and justice in the Mina Mata Disease Case. Proceedings from the International Forum on Mina Mata Disease. 1988, Keiso Shobo, Tokyo University, Japan. Van Kampen, E. J. and W. G. Zijlstra, 1961. Standardization of haemoglobiometry 11. The Haemoglobinocyamide method. Clin. Chim. Acta., 6: 538-544. Warnick, G. R., T. Nguyen and A.A. Albers, 1985. Compansion of improved precipitation methods for quantification of high density lipoprotein cholesterol. Clin. Chem., 31: 217 Wedemeyer, G. A. and W. T. Yasutake, 1977. In clinical methods for assessment of the effects of environmental stress on fish health. US Department of the Interior fish and wild life service, Washington, D.C. Wybenga, D. R., J. Di Giorgio and V. J. Pillegi, 1971. Clin. Chem., 17: 891. 39

J. Aqua., 14 (2006): 39-44

OBSERVATIONS ON GROWTH, SURVIVAL, MATURATION AND BREEDING OF MACROBRACHIUM DAYANUM (HENDERSON, 1893) UNDER LABORATORY CONDITIONS

Bindu R. Pillai, Swagatika Mohanty and Sovan Sahu Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751002

Macrobrachium dayanum is a small freshwater prawn species that completes its lifecycle in freshwater. It shows abbreviated larval development. The egg bearing females were reared individually till egg hatching in 10 l glass jars. The hatchlings were collected and reared in freshwater for 190 days under laboratory conditions. Hatchlings were fed initially (up to seven days after hatching) with mixed zooplankton collected from ponds and subsequently with formulated pellet diet. The hatchlings grew from an initial weight of 1.11 mg to 1029 mg in 190 days. The survival rate was 65.3%. Sexual maturation was observed after 120 days of rearing when the female was 37 mm total length and 670 mg weight. Breeding occurred in the rearing tanks and the embryonic period was found to be 29 days at 28-31°C. A new generation of hatchlings was found after 150 days of rearing.

INTRODUCTION

The research attention in freshwater prawn in India has been primarily focused on two larger species viz., Macrobrachium rosenbergii and M. malcolmsonii. However, species diversification in aquaculture has become one of the thrust areas for aquaculture research in recent years. Macrobrachium dayanum (Henderson, 1893), a small species distributed in India, Bangladesh and Indo-west Pacific, is completely adapted to freshwater environment and shows extremely abbreviated larval development (Jayachandran, 2001). As we look for alternate species for culture, smaller species such as M. dayanum offers certain advantages such as abridged larval development and completion of lifecycle in freshwater. For developing culture technology, knowledge of important biological aspects of the candidate species such as reproduction, life history, food and feeding habits, growth and survival is essential.

Few reports are available on different aspects of its biology such as sexual dimorphism, larval development, clutch size and fecundity (Koshy, 1971, 1973; Koshy and Tiwari, 1976; Bhattacharjee and Dasgupta, 1989; Biswas and Qureshi, 1993). The present work was undertaken to study the growth, maturation and survival of M. dayanum under laboratory conditions. 40

MATERIAL AND METHODS

Live specimens of M. dayanum (adult females and males) were collected from the farm of Central Institute of Freshwater Aquaculture, Bhubaneswar, India. The prawns were brought to the laboratory and sampled for measurement of length and weight and stocked in a 1000 l tank containing filtered and well-aerated freshwater. The specimens were identified using the description presented by Jayachandran (2001). The prawns were fed daily with formulated diet (in crumble form) @5% of the biomass. Cleaning was done daily to remove the fecal materials using a siphon tube and water was exchanged @ 50% every alternate day. Some of the females carried eggs in their brood chamber and some eggs were detached from the female for measurement of egg size. The size of eggs was measured using an ocular micrometer calibrated with a stage micrometer. Total egg mass was also removed from some of the berried females for estimation of fecundity.

Five sets of brooders (one adult male and adult female with maturing ovary) were stocked in five separate 10 l glass jars for breeding and to find out the embryonic period. Once the female became berried, the male was removed and the female was reared in the jar till egg hatching. The tanks were provided with continuous aeration from a blower. The tanks were cleaned and water was exchanged @ 50% daily. The tanks were observed daily for appearance of larval stages. The hatchlings were collected, counted and released into a 450 l FRP tank containing freshwater for further rearing. Mussel shell strings and floating weeds (Eichhornia sp.) were provided as shelter to the prawns. The hatchlings were found to take shelter in the dense root system of the floating weeds. Initially the hatchlings were fed ad libitum with mixed zooplankton collected from culture ponds. The smallest plankton group sieved through 60-mesh sieve was provided to the post-larvae. Seven days after hatching, the post-larvae were provided with pellet diet (protein-32%; lipid-5%) in crumble form. After one month of rearing the post-larvae were transferred to a 3600 l tank and reared for next 160 days. Growth (mg/day) of post-larvae was monitored on a monthly basis.

RESULTS AND DISCUSSION

The live adult specimens of M. dayanum when collected were brownish grey in colour with distinct stripes on pereiopods and antennae. Male (Plate 1) was larger than the female (Plate 2) with strong and robust second pair of cheliped. The total length of adult females ranged 41-64 mm and that of males ranged 48-69 mm. The mean weight of berried female was 1.46 g and that of male was 2.10 g. The largest female in our collection measured 64.0 mm total length. Koshi (1971) reported sexual dimorphism in this species and reported that males are larger than the females with stouter second chelipeds. Jayachandran (2001) also reported that males are larger than females in this species and reported 70.0 mm as maximum size for male and 59.0 mm for female. In a study on 41 fecundity of this species, Bhattacharjee and Dasgupta (1989) have reported that body length of berried females ranged from 45.0-68.0 mm. This report and our observation indicate that we need to revise the largest female size from 59.0 to 68.0 mm.

Rostrum in adult prawn was without dorsal crest, long and slightly curved upwards distally. Rostrum was longer than antennal scale and the adult rostral formula was found to be 8-9/4-5. Two rostral teeth are situated behind the eye orbit. The adults lost their brown colour after molting under laboratory conditions and became pale probably due to the lack of natural food and plankton cover in the tanks. The rostral formula of M. dayanum was reported to be 5-11 teeth (usually 8-9) on the dorsal margin and 4-7 teeth (usually 5-6) on the ventral margin (Jayachandran, 2001).

In the laboratory, the prawns were found to breed naturally as more and more berried females (Plate 3) could be found in the stocked population. The peak breeding activity was observed during monsoon season, i.e. from June to September. Our observation is similar to that of Biswas and Qureshi (1994) who studied the annual reproductive cycle of M. dayanum and reported continuous breeding in this species with two peaks, one in March and another extending from June to September. Fecundity (post- spawn) values are presented in Table 1 and it ranged from 71-141 (51-64 mm total length). Manna and Raut (1991) studied the clutch size in this species and reported that it varied between 30-130 numbers in specimens collected from Midnapur in West Bengal where as Bhattacharjee and Dasgupta (1989) reported fecundity of 37 to 111 in prawns collected from ponds and swamps of Kamrup in Assam.

The mean length of newly spawned egg was 2.28±0.05 mm and the mean width was 1.57±0.07 mm. The shape of the egg was elliptical and the colour of the newly spawned egg was dark green. Embryonic period was found to be 29±1 days at 28±2ºC. Jayachandran (2001) reported that the egg size of M. dayanum to be 1.45-1.70 mm x 1.80- 2.40 mm. Hatchlings were benthic in habit and found to cling to the sides of the containers. The hatchlings resembled adults in all respects except the shape of the telson, which was triangular, and are referred to as post-larvae in the present communication. Post-larvae were found to emerge from the eggs upon hatching indicating direct development. Freshly hatched out post-larvae has a mean total length of 6.2±0.44 mm and a mean weight of 1.11±0.05 mg. The distinguishing characters of the hatchlings were stalked eyes, smooth carapace, translucent body, short rostrum armed on both sides with rostral teeth and abdomen with six distinct somites. Telson was triangular in shape with plumose setae on posterior region. Uropods were present. Pereiopods were normal, five pairs, and all functional. First and second pair of pereiopods were with claws. Pleopods were biramous and functional. 42

Table 1. Fecundity (post spawn) of

Macrobrachium dayanum

Length Weight Fecundity

(mm) (g) (number of eggs

per female)

51 2.10 141

53 1.91 71

55 2.17 86

56 2.45 115 Plate 1: Adult male Macrobrachium dayanum 59 2.68 82

64 2.98 129

Biswas and Qureshi (1993) reported the presence of one larval stage, which resembles post-larvae in all aspects except the shape of telson that is triangular. Jalihal et al. (1993) made a comparative study of the developmental pattern of various species of genus Plate 2: Adult female Macrobrachium dayanum Macrobrachium and broadly classified the larval stages into three: 1) prolonged normal type, 2) partially abbreviated type, and 3) completely abbreviated. Jayachandran (2001) slightly modified the earlier classification of Jalihal and subdivided the earlier divisions, and M. dayanum and M. hendersodayanum are grouped under completely abbreviated Plate 3: Egg bearing (berried) female category. Our observation on the Macrobrachium dayanum morphology of hatchling agrees with those of Biswas and Qureshi (1993) and Jayachandran (2001). However, we have reservations in calling the hatchlings as larvae. Since the hatchlings resemble post-larvae in its morphology except the shape of telson (having full complements of pereopods and pleopods), habits and feeding behavior, it should be called a post-larva than larva. All the larval stages are therefore embryonized and the post-larvae emerge from the embryo.

Growth of M. dayanum under controlled conditions in the laboratory is provided in Table 2. The post-larvae readily accepted mixed zooplankton initially for 7 days and subsequently accepted formulated pellet diets in crumble form. The hatchlings grew from an initial weight of 1.11mg to 1029 mg in 190 days. Average daily growth increased from 43

0.48 mg/day in the first month to a maximum of 11.9 mg/day in the fifth month, subsequently it decreased to 2.35 mg/day. Increased breeding activity might have been responsible for the reduction in growth rate observed after five months. There are few reports on the growth rate of smaller species of the genus Macrobrachium. Chandrasekharan et al. (2005) reported that Macrobrachium lamarrei lamarroides grew from an initial weight of 167-194 mg to 310-355 mg in 30 days with the average daily growth ranging from 4.96 to 5.30 mg/day, which is similar to the growth observed by similar sized M. dayanum in the present study. Mariappan and Balasundaram (2004) studied the growth of three weight groups (337, 542 and 732 mg) of Macrobrachium nobilii, another small species, at three densities of 22, 38 and 77/m2 for 60 days and recorded average daily growth of 21.9, 18.2 and 15.2 mg/day, respectively for the small, medium and large size groups.

Table 2. Growth of Macrobrachium dayanum under controlled conditions

Days of rearing Length Weight Growth (mm ) (mg) (mg/day) 0 6.10±0.2 1.10±0.06 - 30 11.4±0.3 15.5±0.96 0.48 60 18.0±0.4 66.0±4.3 1.68 90 28.0±0.3 253±16.5 6.23 120 34.0± 0.9 579±58.7 10.9 150 39.0± 0.6 935±43.2 11.9 190 42.6± 0.5 1029±47.0 2.35 Values expressed as mean ± SD (n=30)

Sexual maturation was observed after about 120 days of rearing when the female was 37 mm total length and weighed 670 mg. Breeding occurred in the rearing tanks and the embryonic period was found to be 29±1 days at 28-31˚C. A new generation of hatchlings was found after 150 days of rearing. The survival rate of parent stock after 190 days of rearing was 65.3%.

The present study revealed that M. dayanum grows and breeds well under controlled conditions and readily accepts formulated feeds. Due to its complete adaptation to freshwater and abridged larval development its culture techniques can be very simple without a hatchery phase. This provides possibility of the species to be promoted as a candidate for aquaculture, especially in the rural areas where it can cater to the local market.

44

ACKNOWLEDGEMENTS

The authors are thankful to the Director, Central Institute of Freshwater Aquaculture, Bhubaneswar for providing necessary facilities and encouragements.

REFERENCES

Bhattacharjee, P. C. and M. Dasgupta, 1989. Fecundity of the freshwater prawn Macrobrachium dayanum from ponds and swamps of Kamrup, Assam, India. Environ. Ecol., 7: 724-726. Biswas, R. and T. A. Qureshi, 1993. Larval development of a freshwater prawn Macrobrachium dayanum (Henderson, 1893) reared under laboratory conditions. Pak. J. Zool., 25: 227-232. Biswas, R. and T. A. Qureshi, 1994. Annual reproductive cycle of the freshwater prawn, Macrobrachium dayanum(Henderson,1893). Naturalia, 19: 131-147. Chandrasekharan, V. S., H. C. S. Bisht and U. P. Singh, 2005. Effect of feeding frequency on growth of Macrobrachium lamarrei lamarroides (Tiwari). Indian J. Fish., 52: 111-113. Jalihal, D. R. and K. N. Sankoli, 1975. On the abbreviated metamorphosis of the freshwater prawn Macrobrachium hendersodayanum (Tiwari) in the laboratory. J. Karnataka Univ. Sci., 20: 283- 291. Jalihal, D. R., K. N. Sankoli and S. Shenoy, 1993. Evolution and larval development patterns and the process of freshwaterization in the prawn genus Macrobrachium Bate, 1868 (Decapoda, Palemonidae). Crustaceana, 65: 365-376. Jayachandran, K. V., 2001. Palaemonid prawns-Biodiversity, taxonomy, biology and management. Oxford and IBH Publishing Comp., New Delhi, 624 pp. Koshy, M. and K. K. Tiwari, 1976. Clutch size and its relation to female size in two species of freshwater prawn of the genus Macrobrachium from Calcutta. J. Inland Fish. Soc. India, 7: 109-111. Koshy, M., 1971. Studies on the sexual dimorphism in the freshwater prawn Macrobrachium dayanum (Henderson, 1893) (Decapoda, Caridea), 1. Crustaceana, 21: 72-78. Koshy, M., 1973. Studies on the sexual dimorphism in the freshwater prawn Macrobrachium dayanum (Henderson, 1893) (Decapoda, Caridea), 2. Crustaceana, 24: 110-118. Manna A. K. and S. K. Raut, 1991. Clutch size in two shrimp species Macrobrachium lamarrei and Macrobrachium dayanum. Environ. And Ecol., 9: 349-351. Mariappan, P. and C. Balasundaram, 2004. Effect of shelters, densities, and weight groups on survival, growth and limb loss in the freshwater prawn Macrobrachium nobilii (Henderson and Matthai, 1910). J. Appl. Aquaculture, 15: 51-62. Tiwari, R. and T. A. Qureshi, 1997. Influence of ecological parameters on the abundance of Macrobrachium dayanum (Henderson, 1893) in the river Betwa at Vidisha, India. J. Ecobiol., 9: 289-297.

45

J. Aqua., 14 (2006): 45-51

STUDY ON THE GROWTH OF MACROBRACHIUM GANGETICUM JUVENILES FED ON DIFFERENT DIETS IN TANKS WITH AND WITHOUT SOIL-BASE

Radheyshyam, Prasanti Mishra, A. N.Mohanty and D. R. Kanaujia Central Institute of Freshwater Aquaculture Kausalyagang, Bhubaneswar-751002, Orissa, India

The efficacy of two formulated feeds (Feed-I & II) compounded with locally available feed ingredients and one commercial feed (Feed-III) was evaluated on the growth of Macrobrachium gangeticum juvenile. The experiments were conducted during January to April 2005 using 1000 l capacity FRP tanks. While one set of tanks was provided with soil-base, the other set was without soil-base. Each tank was stocked with hatchery-produced M. gangeticum seed of 3.8 - 4.8 g (90.6-97.6 mm) at density of 50/m3. Prawns were fed twice daily at 10% of the body weight. Results indicated that units with soil-base exhibited significantly higher (p<0.05) weight gain (113.15% in Feed-I; 40.82% in Feed-II; 66.67% in Feed-III) than without soil-base (70.73% in Feed-I; 37.50% in Feed-II; 42.50% in Feed-III). The performance of Feed-I in terms of weight gain and specific growth rate was the best followed by Feed-III and Feed-II.

INTRODUCTION

Macrobrachium gangeticum (Bate) is recognized as third largest growing freshwater prawn and attains weight up to 160 g in the rivers (Tiwari and Holthouis, 1996; Kanaujia et al., 2001). In the context of species diversification, the development of seed production in captivity and culture technology is being emphasized. While Kanaujia et al. (2001 and 2005) studied various aspects of breeding and seed production, records on the nutritional aspects are limited. Though several formulated prawn feeds are available commercially in India, its high cost make it unaffordable for the rural prawn farmers (New, 1995; Pawase and Shenoy, 1998). Present endeavor is to investigate the growth of M. gangeticum juveniles on commercial and formulated feeds reared in tanks with and without soil-base.

MATERIAL AND METHODS

The study was carried out at the Central Institute of Freshwater Aquaculture, Bhubaneswar, India for 120 days during January to April 2005 using 1000 l FRP tanks. Three different feeds were used in two set of feeding trials, each with three replicates. In one set of experimental tanks, soil-base was provided, whereas other was without soil- base. Two formulated feeds prepared in the laboratory using locally available feed- 46 ingredients viz., groundnut oil cake, soybean meal, fish meal, prawn meal, rice bran, starch, vitamins and mineral mixture in the ratio of 4 : 1 : 1 : 1 : 1.8 : 1 : 0.2 (Feed-I) and 3 : 1 : 1 : 1 : 2.8 : 1 : 0.2 (Feed-II) and one commercial feed (Feed-III) was used for feeding prawn juveniles. Proximate analysis of the feeds was done following standard methods (AOAC, 1984). All the tanks were filled uniformly with filtered pond water. Each tank was stocked with M. gangeticum seed of 3.8-4.9 g at 50 nos/m3 of water. The prawns were fed twice daily (at 7-8 am and 6-7 pm) at 10% of the body weight. Metabolites and waste feed was removed daily in the morning. 30% water was replaced with filtered pond water weekly. The water quality parameters viz., temperature, pH, dissolved oxygen, total alkalinity, total hardness and dissolved ammonia were analyzed fortnightly following APHA (1981). Average growth was recorded at the end of each month by weighing 30 prawns individually from each tank.

The specific growth rate (SGR) was calculated as: SGR = 100(ln W2 – ln W1)/T, where, W1 is initial mean weight and W2 is final mean weight and T is the time period (days). Feed conversion ratio (FCR) was estimated as: FCR = total feed consumed (g)/ total weight gain of prawn juveniles (g).

The growth data at the end of each month were subject to Duncun’s Multiple Range Test to test the significance between the treatments. The ‘t’ test was carried out to find out the significant difference of water quality data between soil-base and without soil-base treatments.

RESULTS AND DISCUSSION

The water quality parameters did not differ significantly within each set of treatment (Table 1), but the variation of data between two sets (with soil-base and without soil-base) was significant (p<0.05). Better environmental conditions were recorded in the trial having soil-base. In tanks with soil-base the decomposition of the metabolites and wastes reduced compounds and gases (Radheyshyam et al., 1993 & 2003; Boyd, 1995), might have been adsorbed in the soil-base tanks resulting in better environmental condition.

An appropriate level of dietary lipids is one of the important factors in the palatability of the diets (New, 1987; Paulraj, 1995) and results better growth, survival and feed utilization by the prawns (Indulkar and Belasare, 2003). In present experimental diets the fat level ranged from 4.8 to 6.45 % (Table 2). The level of fat was minimum in Feed-I and maximum in Feed-III, whereas, the protein level was minimum in Feed-III and maximum in Feed-I (42%, Feed-I; 39.2%, Feed-II; 36.75%, Feed-III). Behanan and Mathew (1995) opined that 4-8% lipid level in prawn feed is suitable, while New (1976) and Reddy (1997) reported 6-8% to be ideal for the M. rosenbergii larvae. 47

Table 1. Water quality parameters (mean values) of the experimental tanks with and without soil-base during rearing of M. gangeticum provided with different feed

Parameters Feed-I Feed-II Feed-III Soil-base Without Soil-base Without Soil-base Without soil-base soil-base soil-base Temperature (0C) 28.43 28.13 27.67 27.75 27.50 28.00 pH 7.60 8.00 7.70 7.80 7.50 7.80 Dissolved oxygen (mg/l) 4.80 4.20 4.60 4.00 4.40 3.90 Total alkalinity (mg/l) 104.60 112.30 100.40 112.80 104.20 116.70 Total hardness (mg/l) 76.20 80.80 76.40 80.60 76.10 80.80 Dissolved nitrogen (mg/l) 0.20 0.28 0.24 0.28 0.24 0.32

Table 2. Proximate analysis of the experimental diets fed to M. gangeticum

Proximate composition Feed-I Feed-II Feed-III Moisture (%) 5.60 5.40 5.60 Protein (%) 42.00 39.20 36.75 Fat (%) 4.80 5.07 6.45 Ash (%) 13.70 16.51 13.78 Carbohydrate (%) 33.90 33.82 37.42

In general, the dietary lipid, protein and carbohydrate are used for energy by the prawns and in most of the investigations these components are explained as single critical factor, while these components interact (Lim and Persyn, 1989). Hence, their appropriate composition and ratio needs proper attention. In present experimental diets the fat: carbohydrate ratio was 1 : 7, 1 : 6.7 & 1 : 5.7; carbohydrate: protein ratio was 1 : 1.24, 1 : 1.16 & 1 : 1.98; and fat: protein ratio was 1 : 8.81, 1 : 7.7 & 1 : 5.7 in Feed-I, II and III, respectively. Sedgwick (1979) reported decline dietary protein requirement of juveniles if dietary energy is maintained by increasing the carbohydrate or lipid. Cliford and Brick (1978) reported maximized level of protein sparing at 1:4 dietary fat: carbohydrate ratio. The dietary fat: protein ratio in present study indicated that protein content in each experimental diet was adequate to meet the requirements in the rearing M. gangeticum juveniles.

Month wise mean weight gain in M. gangeticum exhibited increasing trend in all the feeding trials. With few exceptions soil-base tanks had higher monthly weight gain with all the experimental diets (Fg.1). In soil-base tanks the average gain in weight of M. gangeticum juveniles was 8.1±1.20, 6.9±1.20 & 6.5±1.90 g on Feed-I, II & III respectively, whereas in without soil-base tanks it was 7.0±1.41, 6.6±1.08 & 5.7±1.25 g on the respective feeds (Table 3). In both the experimental trials the weight gain was highest with Feed-I, probably due to freshness of the feed and higher dietary protein level (42%) in Feed-I as also reported by Hilton et al. (1984), D’Abramo (1998) and Indulkar and Belasare (2003). 48

The percentage weight gain is given in Fig. 2. The soil-base trials showed significantly (p<0.05) higher weight gain than the without soil-base trials. On Feed-I, the weight gain over initial weight was higher (113.5%) in soil-base, whereas in without soil- base it was lower (70.73%). In Feed-II it was 40.82% in soil-base and 37.7% in without soil- base. In Feed-III, the soil-base tanks had higher percentage of weight gain (66.67%) than without soil-base (42.5%).

Comparative cumulative SGR of M. 9 8 gangeticum fed on Feed-I, II & III is depicted in 7 Fig. 3. Feed-I indicated highest SGR (0.63% in 6 soil-base; 0.44% in without soil-base tanks) 5 4 followed by Feed-III and Feed-II. 3 with soil 2 Without soil The FCR is an indicator of feed intake 1

On Feed-I average weight. (g) weight. average Feed-I On 0 in relation to weight gain and determines the 0306090120 Rearing period (days) effectiveness of the feed. In present study, the mean FCR of Feed-I, II & III was 2.56, 2.75 and 8

2.53, respectively in soil-base, whereas, in 7 without soil-base it was 2.63, 2.65 and 2.74 6 (Table 4). This suggests the suitability of soil- 5 base tank bottom for rearing M. gangeticum 4 juveniles. 3 2 with soil 1 Without soil The commercial feed used for the (g) weight average Feed-II On present study priced at about Rs.29/kg was 0 0 30 60 90 120 experienced to be unaffordable to the rural end Rearing period (days) users (Pawase and Shenoy, 1998), whereas the formulated feeds priced Rs.17/kg for Feed-I 7 and Rs.16/kg for Feed-II could be prepared by 6 the farmers using locally available ingredients. 5 D’Abramo (1990) also suggested to use such 4 locally and regionally derived feed stuffs for the feed formulation. Further, advantage of 3 freshness of the ingredients was the positive 2 with soil influence on digestibility of the ingredients 1 Without soil On Feed-III average weight (g) weight average Feed-III On (D’Abramo and Sheen, 1994). 0 0 306090120 Rearing period (days) From the present observation it is evident that soil-base tanks provided suitable Fig. 1. Monthly weight gain of M. gangeticum water quality for better growth, monthly juveniles in tanks with soil-base and weight gain and specific growth rate of M. without soil-base fed on different feeds 49 gangeticum juveniles. Feed-I was found better among other feeds. The advantages of Feed- 1 was diet freshness, cost effectiveness, easy accessibility to rural farmers, higher percentage of weight gain over the initial prawn biomass and specific growth rate, compared to commercial feed.

Table 3. Growth trends (weight in g) of M. gangeticum fed on formulated feeds (Feed-I & II) and a commercial feed (Feed-III) in tanks with and without soil-base

Days Feed-I Feed-II Feed-III Soil-base Without Soil-base Without Soil-base Without soil-base soil-base soil-base 0 3.8±0.79 a 4.1±0.74 a 4.9±0.74 a 4.8±0.63 a 3.9±0.74 a 4.0±0.82 a 30 4.9±0.74 a 4.9±0.74 a 5.2±0.92 a 5.3±0.82 a 4.9±1.20 a 4.2±0.92 a 60 6.1±0.74 b 5.2±0.92 a 5.6±1.51 a 5.4±0.70 a 5.7±1.42 a 4.5±0.85 a 90 6.7±0.95 b 5.9±0.99 a, b 6.3±1.25 b 5.8±0.92 a, b 6.1±1.45 a 4.9±0.74 a 120 8.1±1.20 d 7.0±1.41 b, c, d 6.9±1.20 a,b,c, d 6.6±1.08 a, b, c 6.5±1.90 a, b 5.7±1.25 a Growth means with different superscript(s) in a row are significantly (p<0.05) different

Table 4. Feed conversion ratio of different feed

Month Feed-I Feed-II Feed-III Soil-base Without Soil-base Without Soil-base Without soil-base soil-base soil-base 1st 2.31 2.50 2.83 2.71 2.38 2.85 2nd 2.41 2.83 2.79 2.46 2.15 2.79 3rd 3.04 2.64 2.66 2.79 2.79 2.75 4th 2.48 2.53 2.73 2.64 2.81 2.57 Mean 2.56 2.63 2.75 2.65 2.53 2.74

ACKNOWLEDGEMENTS

Authors express their thanks to Dr. N. Sarangi, Director, Central Institute of Freshwater Aquaculture, Kausalyagang for his interest and inspiration for the study. We thank the Head of Division and Principal Scientist Dr. P. Kumaraiah for critically going through the manuscript and for improving it. Thanks are also due to Shri S. Sarkar for carrying out the proximate analysis of the experimental diets.

REFERENCES

AOAC, 1984. Official Methods of Analysis (14th edition). Association of Official Analytical Chemists, Washington, D.C., 1141 pp. APHA, 1981. Standard Method for the Examination of Water and Wastewater. Washington: 1134 pp. 50

Behanan, L. and S. Mathew, 1995. Significance of formulated feeds in post larval raring of freshwater prawn. Fishing Chimes, 15(3): 39-41. Boyd, C. E., 1995. Bottom Soils, Sediments and Pond Aquaculture. Auburn University, Alabama, New York, 366 pp. Clifford, H.C. III and R.W Brick., 1978. Protein utilization in freshwater shrimp Macrobrachium rosenbergii. Porc. World Mar. Soc., 9: 195-208. D’ Abrano, L. R, 1990. Lipid requirements of shrimp. In: Advances in Technical Aquaculture (Ed, J. Barret), pp. 271-85. Proc. Workshop. Tahiti. 20 February-4 March, 1989. Actes. Collaq. Infremer. 9. D’ Abrano, L. R. and S. S Sheen, 1994. Nutritional requirements, feed formulation and feeding practices for intensive culture of the freshwater prawn, Macrobrachium rosenbergii, Rev. Fish. Sci., 2(1): 1-21. D’Abramo, L. R., 1998. Nutritional requirement of the freshwater prawn Macrobrachium rosenbergii. Comparisons with species of penaeid shrimp. Rev. Fish. Sci., 6: 153-163. Hilton, J. W., K. F. Harrison and S.J Silnger, 1984. A semi-purified test diet for Macrobrachium rosenbergii and the lack of need for supplemental lecithin. Aquaculture, 37: 209-215. Indulkar, S. T. and S. G. Belsare, 2003. Evaluation of some formulated diets for rearing the post larvae of Macrobrachium rosenbergii. J. Indian Fish. Asso., 30: 113-119. Kanaujia, D. R., A. N. Mohanty and S. Soni, 2001 Breakthrough in seed production of Ganga River Prawn, Macrobrachium gangeticum (Bate, 1868): A milestone in aquafarming. Fishing Chimes, 21(1): 28-30. Kanaujia, D.R., A.N Mohanty., G. Mitra and S Prasad, 2005. Breeding and seed production of the Ganga River Prawn, Macrobrachium gangeticum Bate under captive conditions. Asian Fish. Sci., 18(3&4): 371-388. Lim, C. and A. Persyn, 1989. Practical feeding of penaed shrimp. In: Nutrition and Feeding of Fish (Ed. T. Lovell), pp. 205-222, Van Nostrand Reimhold, New York. New, M. B., 1976. A review of dietary studies with shrimp and prawns, Aquaculture, 9: 101-144. New, M. B., 1987. Feed and Feeding of Fish and Shrimp - A Mannual on the Preparation and Presentation of Compound Feeds for Shrimp and Fish in Aquaculture. Food and Agriculture Organization, Rome, 275 pp. New, M. B., 1995. Status of freshwater prawn farming: A review. Aquaculture Res., 26: 1-54. Paulraj, R., 1995. Aquaculture Feed. Handbook on Aquafarming. Marine Products Export Development Authority, Kochi, India, 93 pp. Pawase, A.S. and S. Shenoy, 1998. Growth experiment on Penaeus merguiensis and Metapenaeus monocerous from Ratanagiri using different pelleted feeds. In: Current and Emerging Trends in Aquaculture (Ed. P.C. Thomas), Daya Publishing House, New Delhi, pp. 244- 260. Radhdyshyam, B.K. Sharma, S.K. Sarkar and B.B Satapathy, 1993. Observations on the dynamics of sediment detritus and macrobenthic fauna as influenced by Cirrhinus mrigala and Labeo rohita (Ham.) in monoculture fish ponds. The Third Indian Fisheries Forum proceedings. Pantnagar, India, pp. 167-170. 51

Radheyshyam, D. N. Chattopadhyay, B.B Sathapathy and S.K Sarkar., 2003. Study on pond fertilization for sustainable carp fry production in rural area, Aquacult, 4(2): 213-216. Reddy, A. K., 1997. Management of freshwater prawn hatcheries and culture possibilities. Fishing Chimes, 16(10): 32-36. Sedgwick, R., 1979. Influence of dietary protein and energy on growth, food consumption and food conversion efficiency in Penaeus Merguiensis. Aquaculture, 16: 7-10. Tiwari, K.K. and L.B. Holthuis, 1996. The identity of Macrobrachium gangeticum Bate, 1868 (Decapoda, cridea, Palaemonidae). Crustaceana, 69(7): 922-925.

52 53

J. Aqua., 14 (2006): 53-62

EVALUATION OF DIFFERENT CELL COMPONENTS OF AEROMONAS HYDROPHILA ON THE IMMUNE RESPONSE OF ROHU (LABEO ROHITA)

S. K. Udgata*, I. Karunasagar and I. Karunasagar Department of Fishery Microbiology, College of Fisheries, Mangalore 575002, Karnataka, India *Present Address: College of Fisheries (OUAT), Rangailunda, Berhampur-760007, India

The humoral immune response of rohu (Labeo rohita) against different cell components of Aeromonas hydrophila was studied. Heat-killed cells (HKC), formalin- killed cells (FKC) and crude lipopolysaccharides (LPS) were prepared from a virulent strain (G-49) of A. hydrophila. Three methods of vaccination were followed, viz., intraperitoneal injection, direct immersion and oral administration through feed. A primary immunization of three vaccines was given on day 0, followed by a 1st booster on day 28 after priming and a 2nd booster on day 28 after 1st booster both for injection and immersion groups. A group of fishes were fed with vaccine incorporated feed for a period of 14 days during priming, 1st booster and 2nd booster immunization. Antibodies against different immunogens of A. hydrophila were detected using standard method of agglutination on day 0, 7, 14 and 28 following priming and booster immunizations. The degree of protection of all the immunization groups of fish was assessed by challenging the fishes with a LD50 dose of 1.5 x 107 cfu/ml of A. hydrophila on a low titre day after priming and on a peak titre day after 2nd booster. The results showed that the vaccines prepared from FKC yielded higher antibody than HKC and LPS. Vaccines prepared from HKC and LPS offered absolute (100%) protection against challenge. Vaccination with LPS resulted in lower antibody production but better immune protection against challenge in rohu.

INTRODUCTION

Motile aeromonads are most commonly associated with aquatic animals, where they have been routinely isolated from healthy individuals, as well as primary and secondary pathogens from sick and moribund specimens. Various fish diseases of motile Aeromonas etiology include ulcer diseases, infectious diseases, haemorrhagic septicaemia, red sore disease, infectious dropsy and fresh water eel disease (Joseph and Carnahan, 1994). Antibiotics are commonly used against bacterial diseases in fish. However, one of the major problems of using antibiotic is that bacterial pathogens develop resistance against them (Plumb, 1995). Hence, a greater emphasis has been given on fish immunology and development of vaccines to help prevent or reduce the effect of certain diseases (Plumb, 1997). 54

Commercial vaccines are yet to be developed for A. hydrophila. The antigenic diversity of this organism has posed a serious problem in a successful vaccine preparation and Plumb (1984) opined that a polyvalent vaccine might have to contain antigens representing all of the strains that the fish might encounter. The immune response of fish to motile aeromonads has been studied using various antigen preparations (Lamers and Van Muiswinkel, 1986; Karunasagar et aI,. 1991 and Newman, 1993). However, information on duration of immunity, immunological memory, relation between antibody levels and protection against disease is sparse. For an optimal immune response, the nature of antigen preparation and route and dose of vaccine administration may be considered essential.

The whole bacterial cell and various cell components of A. hydrophila have been used as immunogens in fish. Heat-killed whole bacterial cell has been used as immunogen to Indian major carps (Karunasagar et a1., 1991) and walking catfish (Supriyadi and Shariff, 1995). Formalin-killed bacterial cells have been used as immunogen to carps (Lamers et al., 1985a, 1985b), Nile tilapia (Ruangapan et a1., 1986), rainbow trout (Loghothetis and Austin, 1994) and catfish (Areechon and Karoon, 1995). The use of lipopolysaccharide as immunogens has been worked out in carps (Baba et al.,1988a,1988b) and in rainbow trout (Loghothetis and Austin,1994,1996). In the present study, the experiments have been designed to examine the antibody production in L. rohita against different cell components of A. hydrophila and to investigate the effect of different antigenic preparations from A. hydrophila as well as the mode of vaccination on the degree of protection.

MATERIAL AND METHODS

Fish Rohu (L. rohita) fingerlings of 8-10 cm, weighing 10-15 g obtained from Bhadra Reservoir Project, Karnataka were used. About 10-12 fishes were maintained in each fiberglass tanks containing 200 litres of water at 28+1°C and fed with pellet feed. Twelve such tanks were maintained. Fishes were acclimatized for a week before the immunization commenced.

Bacterial strain A moderately virulent strain of A. hydrophila (Strain No. G-49) isolated from a freshwater pond at Honavar was used for vaccine preparation and challenge experiment.

Preparation of heat-killed cells (HKC) A. hydrophila was grown in tryptic soya agar (TSA) in Roux bottles, harvested aseptically in sterile physiological saline and enumerated for cell density. The bacterial suspension was inactivated by heating in a water bath at 85°C for 1 h. Sterility testing was 55 done by inoculating 10 ml of suspension into 100 ml of 2X trypticase soya broth (TSB) and incubating at 37°C for 1-7 days to observe turbidity and to confirm that there was no live cell in the vaccine preparation.

Preparation of formalin-killed cells (FKC) Formalin-killed A. hydrophila were prepared from 18 hours old TSB cultures by treating the bacterial cultures with 0.5% (v/v) formalin and incubating at 37°C for 2 days. The culture suspension was then centrifuged at 7000 rpm for 10 min. and the pellet was resuspended in the physiological saline and enumerated for the cell density. The sterility of the formalin killed vaccine was tested as described above.

Preparation of crude lipopolysaccharide (LPS) Crude lipopolyssaccharide (LPS) of A. hydrophila was prepared by phenol extraction method as described by Westphal and Jann (1965). A 50 g of cell pellet was suspended in 200 ml of hot water (68°C) and added with equal volume of 90% (w/v) preheated liquefied phenol. The emulsion was cooled to 10°C and centrifuged at 3000 rpm for 30 min. The upper layer containing LPS was collected, dialyzed and stored at 4°C.

Vaccination Three methods of vaccination were followed, viz., intraperitonial injection, direct immersion and oral administration through feed. Fishes to be injected were mildly anaesthetized with ethyl-p-aminobenzoate at 50 mg/L. HKC and FKC vaccines were injected intraperitonially at a dose of 0.3 ml per fish (109 cells/ml), whereas a dose of 0.1 ml of LPS was administered to each group of fish. During immersion, fishes were treated with 5 litres of vaccine bath (HKC and FKC separately) for one hour, whereas, another group of fish was immersed in 5 litres of LPS vaccine bath with a dose of 3 ml/L for 2 hours. Administration of vaccine incorporated feed (for HKC and FKC @ 150 ml/kg feed and for LPS @ 15 ml/kg feed) through oral route was done for a period of 14 days as another method of vaccination. In all the vaccination groups a control group of fish was maintained. A first booster dose of three different vaccines (same dose as primary) was given on day 28 after primary immunization and second booster on day 28 after the first booster.

Collection of immune sera and detection of antibody Blood was collected on days 0, 7, 14, 21 and 28 following primary and booster immunizations via caudal vein from five randomly selected fishes in each vaccination group as well as from unimmunised control group of fishes. Immune sera were separated after storing at 4°C and centrifugation. Antibodies against different immunogens of A. hydrophila were detected using the standard method of agglutination (Roberson, 1990). The antibody titre was expressed as the reciprocal of highest serum dilution giving positive agglutination. 56

Challenge studies The degree of protection was assessed by challenging the fishes with A. hydrophila with a LD50 value of 1.5 x 107 cfu/ml on a low titre day after priming and on a peak titre day after the second booster. About 0.5 ml volume containing appropriate number of cells were injected intraperitonially into both the immunised and control fishes. The fishes were observed for 5 days for mortality and the survival percentage was calculated.

Statistical analysis Friedman Test (Weber, 1973) was applied to test for the significant difference in antibody titres on a peak titre day after second booster in all the immunization groups. Mann-Whitney U-test (Weber, 1973) was followed to test for significant difference between the percent survivors on low titre day and peak titre day. In both the analyses a value of P < 0.05 was considered to be significant.

RESULTS

Intraperitonial injection The kinetics of antibody titre in L. rohita immunised against HKC, FKC and LPS of A. hydrophila administered through intraperitonial injection have been shown in Table 1. After primary immunisation a gradual increase in antibody titre was observed up to day 14 with the values of 64, 1024 and 128 for HKC, FKC and LPS vaccination groups, respectively. The increasing trend was also observed after 1st booster and 2nd booster, which reached at the values of 512 and 8388608 for HKC and FKC groups, confirming a secondary immune response and memory formation. However, LPS immunisation did not result in secondary immune response as evident from lower titre values. Table 2 shows the extent of protection in L. rohita against a homologous challenge on a low titre day (28 day after priming) and peak titre day (14 day after 2nd booster). Fishes immunised with HKC and LPS showed 100% protection. Though the group immunized with FKC showed highest antibody titre of 8388608, but protected only 75% of fish on the peak titre day. There was significant difference in antibody titres of three different antigen groups (P<0.05).

Direct immersion Table 3 shows the immunological response of L. rohita to antigens of A. hydrophila by direct immersion. The antibody titre in HKC group was consistently higher than those of FKC and LPS groups throughout the immunisation period. The antibody titres of 128, 64 and 16 were observed in day 14 after 1st booster and 64, 64 and 32 on day 14 after 2nd booster. The relationship between antibody titre and % survivors of L. rohita challenged against A. hydrophila on low titre day and peak titre day has been shown in Table 4. HKC and LPS groups of antigens offered 100% protection against a 50% in the case of FKC groups. Again, though the antibody production was low, LPS group offered 100% 57 protection. There was significance difference (P<0.05) in % survivors between low titre day and peak titre day.

Table 1. Antibody titre in L.rohita immunised against various antigenic preparations of A. hydrophila by intraperitonial injection

Vaccination Group Antibody titre on day Priming 1st Booster 2nd Booster 0 7 14 21 28 7 14 21 28 7 14 Heat killed cells (HKC) 2 32 64 16 16 32 128 64 64 256 512* Formalin killed cells 2 128 1024 128 128 1024 8192 2048 2048 262144 8388608* (FKC) Lipopolysaccharide 2 16 128 8 8 16 16 8 4 16 32* (LPS) Unimmunised control 2 4 4 2 2 4 4 2 2 4 4* Average length of fish = 8.66 cm; Average weight of fish = 10.55 g; *Friedman test;P < 0.05

Table 2. Extent of protection against challenge in L. rohita immunized by intraperitonial injection of antigens of A. hydrophila

Vaccination Group On low titre day On peak titre day Antibody titre % Survivors Antibody titre % Survivors Heat killed cells (HKC) 16 50* 512 100* Formalin killed cells (FKC) 128 50* 8388608 75* Lipopolysaccharide (LPS) 8 25* 32 100* Unimmunised control 2 25* 4 50* *Mann-Whitney U-test; P < 0.05

Table 3. Antibody titre in L.rohita immunised against various antigenic preparations of A. hydrophila by direct immersion

Vaccination Group Antibody titre on day Priming 1st Booster 2nd Booster 0 7 14 21 28 7 14 21 28 7 14 Heat killed cells 2 64 128 32 16 32 128 32 16 16 64* (HKC) Formalin killed cells 2 16 32 16 16 16 64 32 16 64 64* (FKC) Lipopolysaccharide 2 8 16 4 4 8 16 8 8 16 32* (LPS) Unimmunised control 2 2 4 2 2 2 2 4 2 2 2* Average length of fish = 8.66 cm; Average weight of fish = 10.55 g; *Friedman test;P < 0.05

58

Oral administration The production of antibody was observed following priming and booster doses through oral administration and the same is evident from Table 5. In all the antigen groups i. e., HKC, FKC and LPS the primary immune response was better against a poor production of antibody following booster doses. Again among the antigen groups, the difference in the levels of antibody titre was statistically not significant (P>0.05). The survival % in all the three antigen groups remained very low on both the challenge days. Only in the HKC group the % survivors was 50% on peak titre day (Table 6). The difference in survival % for all the three groups of antigens between the two challenge days were not statistically significant (P>0.05).

Table 4. Extent of protection against challenge in L. rohita immunized by direct immersion of antigens of A. hydrophila

Vaccination Group On low titre day On peak titre day Antibody titre % Survivors Antibody titre % Survivors Heat killed cells (HKC) 16 25* 64 100* Formalin killed cells (FKC) 16 50* 64 50* Lipopolysaccharide (LPS) 4 50* 32 100* Unimmunised control 2 25* 4 50* *Mann-Whitney U-test; P < 0.05

Table 5. Antibody titre in L.rohita immunised against various antigenic preparations of A. hydrophila by oral administration

Vaccination Group Antibody titre on day Priming 1st Booster 2nd Booster 0 7 14 21 28 7 14 21 28 7 14 Heat killed cells (HKC) 2 64 128 32 16 32 128 32 16 16 64* Formalin killed cells 2 16 32 16 16 16 64 32 16 64 64* (FKC) Lipopolysaccharide 2 8 16 4 4 8 16 8 8 16 32* (LPS) Unimmunised control 2 2 4 2 2 2 2 4 2 2 2* Average length of fish = 8.66 cm; Average weight of fish = 10.55 g; *Friedman test;P > 0.05

Table 6. Extent of protection against challenge in L. rohita immunized by oral administration of antigens of A. hydrophila

Vaccination Group On low titre day On peak titre day Antibody titre % Survivors Antibody titre % Survivors Heat killed cells (HKC) 8 25* 16 50* Formalin killed cells (FKC) 8 25* 32 25* Lipopolysaccharide (LPS) 4 25* 16 25* Unimmunised control 2 25* 2 50* *Mann-Whitney U-test; P >0.05 59

DISCUSSIONS

There are many conflicting reports regarding the protective role of specific agglutinating antibody in fish. Some indicated that there is no correlation between production and the level of serum specific antibody (McCarthy et al., 1983; Baba et al., 1988b). In mammals specific antibody is principally dedicated to the recognition, inhibition and elimination of invading organisms. However, for fish all these functions remain to be clarified.

In the present series of experiments, heat-killed cells (HKC), formalin-killed cells (FKC) and crude lipopolysaccharide (LPS) of A. hydrophila have been studied for their ability to induce antibody production and protection in L. rohita.

The study shows that L .rohita responded to all these antigens of A. hydrophila by producing agglutinating antibodies. The kinetics of humoral response through antibody production for HKC and LPS was higher in both for immersion and oral groups of administration. Only in the injection group, FKC elicited the highest antibody production. A very good primary and secondary responses was observed for HKC and FKC groups of antigens. But in LPS group a comparatively low or delayed secondary immune response was observed. This result confirmed that LPS does not elicit remarkable humoral immunity compared to HKC and FKC groups of antigens.

The success of a vaccination programme depends on several factors like environmental temperature, method of antigen preparation and localisation of antigen in host. Rijkers et al. (1980) demonstrated that height of secondary immune response in teleost fish was dependent on temperature. Among the three antigens used in FKC, the H- antigen is expected to remain unaffected. However in HKC, some of the heat labile O- antigen might have been inactivated at a temperature of 85°C for 1 h during preparation of the antigen. This observation is in contrast to those of Lamers and Van Muiswinkle (1986), who found that vaccines prepared from heated and disrupted cells induced a higher agglutinating antibody titre over the 8-month test period than formalin-killed cells. In the case of LPS, which is soluble antigen, lower antibody titre values has been shown in agglutination test. Hence, a different method of detection of serum antibody like ELISA may be helpful in getting the correct antibody titre values for this soluble antigen. Loghothetis and Austin (1996) observed a weak agglutination titre using LPS as antigen but it showed remarkable difference while conducting an ELISA test for serum antibody titre in rainbow trout.

In this study, the FKC delivered by injection induced a good secondary response, particularly after the second booster. However, the reason for poor secondary response in the case of HKC is not known. It is possible that flagellar antigens are important in 60 inducing secondary response. In the case of LPS, no secondary response was observed. This could be because LPS is a T-dependent antigen and cooperation between T and B cells may be involved in development of effective memory.

The level of antibody alone is not a sufficient indication of the degree of protection. The earlier reports of Post (1966) and Ellis (1988) are in agreement that high antibody titres do not necessarily indicate protective immunity. The antibodies detected may be against antigens which have no relevance to the organism and therefore may not be effective in neutralising the pathogens. Further, cellular immunity may play an important role in the defense mechanism. In the present study the protection conferred by HKC and LPS were 100% in injection and immersion groups though the antibody titre was low (Tables 2 and 4). In the case of FKC administered by injection, the antibody titre was very high (8388608) but protection was comparatively low (75%, Table 2). This indicates that a high antibody titre does not necessarily confer higher immune protection. Karunasagar et al. (1991) have shown a 100% survival on a peak titre day after 2nd booster injection with a titre value of 1024 in C. catla immunised with HKC of A. hydrophila. A1i (l997) has demonstrated a HKC antigen conferred no mortality on day 15 after second booster, when the antibody titre value was 512 in L. rohita. These results corroborated the observation of the present study. The non-correlation of the antibody titre and immune protection in FKC immunised group has been demonstrated by Areechon and Karoon (1995) for a hybrid catfish and by Areechon et al. (l991) for a walking catfish. Baba et al. (l988b) have confirmed that better protection was achieved by LPS than by FKV in carp and this protection was not associated with the antibody production. A similar result of better protection (RPS, 90%) against a low antibody titre (64) was shown for turbot injected with LPS from fish pathogenic Cytophaga-like-bacterium (AI-Harbi and Austin, 1992).

Establishment of cellular immunity in fish is known from their graft rejection (Mc Kinney et al., 1987; Rijkers, 1982) or delayed hypersensitivity reaction in vitro and in vivo (Jayraman et aI., 1979; Bartos and Sommers, 1981). But, whether cellular mechanism results in protection against bacterial disease in fish remains unknown. An attempt by Baba et al. (1988a) using crude LPS as antigen has established that protection in carp was due to cellular immunity regulated by a T-like system. Even in this study, it has been observed that though LPS induces very low titre of antibody, the fishes are protected against challenge suggesting that LPS is important in inducing protective immunity against A. hydrophila in Indian major carp, L. rohita.

REFERENCES

Al-Harbi, A.H. and B. Austin, 1992. The immune response of turbot, Scophthalmus maximus (L.), to lipopolysaccharide from a fish-pathogenic Cytophaga - like bacterium. J. Fish Dis., 15(5): 449-452. 61

Ali, A., 1997. Immunomodulatory effect of chemicals used in aquaculture. Ph. D. Thesis. University of Agricultural Sciences, Bangalore, 150 pp. Areechon, N. and B. Karoon, 1995. Comparative studies on resistance and immunological response of a hybrid catfish and the parent species to Aeromonas hydrophila. In: Diseases in Asian Aquaculture II (Eds. M. Shariff, J. R. Arthur and R. P. Subasinghe), Fish Health Section, Asian Fisheries Society, Manila, pp. 451-457. Areechon, N., N. Kitancharoen, B. Chutintrasri and K. Tongutliai, 1991. Immune response of walking catfish ( Clarias macrocephalus, Gunther) to vaccination by injection methods. Fish. Sci. J., 1: 1-5. Baba, T., J. Imamura, K. Izawa, and K. Ikeda., 1988a. Cell-mediated protection in carp, Cyprinus carpio L., against Aeromonas hydrophila. J Fish Dis., 11 : 171-178. Baba, T., J. Imamura, K. Izawa, and K. Ikeda., 1988b. Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide. J. Fish Dis., 11: 237-244. Bartos, J.M. and C.V. Sommers, 1981. In vivo cell mediated immune response to M. tuberoculosis and M. salmoniphilum in rainbow trout Salmo gairdneri. Dev. Comp. Immunol., 5: 75-83. Ellis, A.E., 1988. Fish Vaccination, Academic Press, London, 255 pp. Joseph, S. W. and A. Carnahan, 1994. The isolation, identification and systematics of the motile Aeromanas species. Ann. Rev. Fish. Dis., 4: 315-343. Karunasagar, I., G. Rosalind and I. Karunasagar, 1991. Immunological responses of the Indian major carps to Aeromonas hydrophila vaccine. J. Fish. Dis., 14: 413-417. Lamers, C. H. J. and W. B., Van Muiswinkel, 1986. Natural and acquired agglutinin to Aeromonas hydrophila in carp. Cyprinus carpio. Can. J. Fish Aquat. Sci., 43: 619-624. Lamers, C. H. J., M. J. H. Dehass, and W. B. Van Muiswinkel, 1985a. Humoral responses and memory formation in carp after injection of Aeromonas hydrophila bacterin. Dev. Comp. Immunol., 9: 65-75. Lamers, C.H.J., M.J.H. Dehass, and W. B. Van Muiswinkel, 1985b. The reaction of the immune system of fish to vaccination: Development of immunological memory in carp, Cyprinus carpio L. following direct immersion in Aeromonas hydrophila bacterin. J. Fish Dis., 8: 253- 262. Loghothetis, P. N. and B. Austin, 1994. Immune response of rainbow trout (Onchorhynchus mykiss, Walbaurn) to Aeromonas hydrophila. Fish Shellfish Immunol., 4(4) : 239-254. Loghothetis, P. N. and B. Austin, 1996. Antibody response of rainbow trout (Onchorhynchus mykiss, Walbaurn) to live Aeromonas hydrophila as assessed by various antigen preparations. Fish Shellflsh Immunol., 6: 455-464. McCarthy, D.H., D. F. Amend, K. A. Johnson, and J. V. Bloom, 1983. Aeromonas salmonicida: Determination of an antigen associated protective immunity and evaluation of experimental bacteria. J. Fish Dis., 6: 155-174. McKinney, E.C., T. K. McLeod and M. M. Sigel, 1981. Allograft rejection in a holostean fish, Lepisosteus platyrhinchus. Dev. Comp. Immunol., 5 : 65-74. Newman, S.G., 1993. Bacterial vaccines for fish. Ann. Rev. Fish Dis., 3: 145-185. 62

Plumb, J.A., 1984. Immunization of warm water fish against five important pathogens. In: Symposium on Fish Vaccination (Ed. P. de Kinkelin), OlE, Paris, France, pp. 199-222. Plumb, J.A., 1995. Chemotherapy vs. vaccination: a reality for Asian aquaculture. In : Diseases in Asian Aquaculture II ( Eds. M. Shariff, J. R. Arthur, and R. P. Subasinghe), Fish Health Section, Asian Fisheries Society, Manila. pp. 43-53. Plumb, J.A., 1997. Trends in freshwater fish disease research. In : Diseases in Asian Aquaculture III (Eds. T. W. Flegel and I. H. Mac Rae), Fish Health Section, Asian Fisheries Society, Manila, pp. 33-47. Post, G., 1966. Responses of rainbow trout (Salmo gairdneri) to antigens of Aeromonas hydrophila. J. Fish. Res. Bd. Can., 23(10): 1487-1494. Rijkers, G.T., E. M. H. Fredrix-Wolters and W. B. Van Muiswinkel, 1980. The immune system of cyprinoid fish: Kinetics and temperature dependence of antibody producing cells in carp (Cyprinus carpio). Immunology, 41: 91-97. Roberson, B.S., 1990. Bacterial agglutination. In: Techniques in Fish Immunology. (Eds.J. S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Roberson and W. B. Van Muiswinkel). SOS Publications, Fair Haven, N J., pp. 137-154. Ruangapan, L., T. Kitao and T. Yoshida, 1986. Protective efficacy of Aeromonas hydrophila vaccines in Nile tilapia. Vet. Immunol. Immunopatho1., 12: 345-350. Supriyadi, H. and M. Shariff, 1995. Evaluation of the immune response and protection conferred in walking catfish, Clarias batrachus, administered inactivated Aeromonas hydrophila bacterin by immersion. In: Disease in Asian Aquaculture II (Eds. M. Shariff, J. R. Arthur and R.P. Subasinghe). Fish Health Section, Asian Fisheries Society, Manila, pp. 405-412. Weber, E., 1973. Non-parametric methods. In: Biostatistics in Pharmacology. Vo1. 2 (Ed. A. L. Delaunois). Pergamon Press, Oxford, pp. 889-969. Westphal, O. and K. Jann, 1965. Bacterial lipopolysaccharides: Extraction with phenol-water and further application of the procedure. Methods in Carbohydrate Chemistry, 5: 83-91. 63

J. Aqua., 14 (2006): 63-69

OCCURRENCE OF FOLLICULAR ATRESIA IN OVARY OF FRESHWATER CATFISH, HETEROPNEUSTES FOSSILIS (BLOCH)

Chithar V. Mani and Ajay Kumar Pandey Central Institute of Freshwater Aquaculture, Bhubaneswar-751002, India

Atresia is a highly regulated process in the vertebrate ovary which appears to be essential for the maintenance of ovarian homeostasis. The oocytes in different stages of growth are lost through atresia (or degeneration) affecting the fecundity/ reproductive potential of the fish. The atretic oocytes have been identified in the ovary of the catfish during immature, pre-spawning, spawning and post-spawning periods. An attempt has been made to record the different stages of follicular atresia in Heteropneustes fossilis. Possible causes of the phenomenon have also been discussed.

INTRODUCTION

The corpora atretica (atretic follicles or pre-ovulatory corpora lutea) and post- ovulatory follicles (ruptured follicles, discharged follicles or post-ovulatory corpora lutea) are of wide occurrence in the ovaries of vertebrates including fish (Saidapur, 1978, 1982; Guraya, 1993; Wood and Kraak, 2001). It has been generally observed that all the developing follicles (oocytes) do not reach to maturity and ovulate successfully. Many of them become atretic (degenerated) at some stage during their development. Follicular atresia has been recorded in a number of teleostean species such as Fundulus heteroclitus (Mathews, 1938), Heterendria formosa (Fraser and Renton, 1940), Neotoca bilineata (Mendoza, 1943), Gadus merlangus and G. esmarki (Gokhale, 1957), Wallagonia attu (Dixit, 1960), Satipinna phasa (Jhingran, 1960), Mystus seenghala (Dixit, 1960; Sathyanesan, 1961, 1962), Ophiocephalus punctatus (Belsare, 1962, 1975), Heteropneustes fossilis (Nair, 1963), Tor (Barbus) tor (Rai, 1966), Xenantodon cancila (Rastogi, 1966), Glossogobius giuris (Rajalakshmi, 1966; Saksena and Bhargava, 1972), Glyptosternum pectinopterum (Khanna and Pant, 1967), Monopterus albus (Chan et al., 1967), Clarias batrachus (Lehri, 1968), Mystus tengara (Rastogi, 1968a), Amphipnous cuchia (Rastogi, 1968b), Schizothorax niger (Malhotra, 1971), Cyprinus carpio communis (Guraya et al., 1977), Mystus cavasius (Saidapur, 1978), Channa marulius (Srivastava, 1980), Tilapia leucostica (Kling, 1981; Schizothorax plagiostomus (Agarawal and Singh, 1990), Macodon ancylodon (Vizziano and Berois, 1990) and Poecilia rerticulata (Rajkumar and Hemalatha, 2005). Since oocytes in different stages of growth and differentiation are lost through atresia (or degeneration) affecting the fecundity or reproductive potential of the fish (Guraya, 1993, 1994), an attempt has been made to record the follicular atresia in ovary of the commercially important freshwater catfish, H. 64 fossilis and the observations discussed in the light of the various stages of atresia propounded by Belsare (1975) and Saidapur (1978).

MATERIAL AND METHODS

Live specimens of H. fossilis (body weight range 62-86 g) were procured from the fields adjoining Bhubaneswar (Orissa) during middle of every month. Ovaries of the catfish were surgically removed and fixed immediately in freshly prepared Bouin’s solution. After 24 h, tissues were washed thoroughly in running tap water, dehydrated in ascending series of alcohol, cleared in xylene and embedded in paraffin wax at 60°C. Serial sections were cut at 6 μm and stained with hematoxylin-eosin (H&E), toluidine blue (1%) and bromophenol blue (0.5%) (Pearse, 1968; Bancroft and Stevens, 1977).

RESULTS AND DISCUSSION

Follicular atresia in the fish ovary is of common occurrence during pre-spawning, spawning and post-spawning periods (Saidapur, 1978, 1982; Guraya, 1993). During the course of maturation process, some of the ova that fail to attain maturity or spawn undergo resorption and are called atretic follicles (Saidapur, 1978, 1982; Guraya, 1993, 1994; Khanna, 2006). Atresia is a highly regulated process in the vertebrate ovary which appears to be essential for the maintenance of ovarian homeostasis (Wood and Kraak, 2001). All the four stages of follicular atresia, described in the teleosts (Belsare, 1962, 1975; Saidapur, 1978), have been encountered during the present study in the catfish. In H. fossilis, remnants of atretic follicles in the form of nodule of stroma tissue were observed even in the immature ovaries during December-January (Figs. 1, 2). Previtellogenic atretic follicles in ovary of the catfish depicted excessive vacuolation of ooplasm towards periphery, flocculent appearance of ooplasm and hypertrophied granulosa cells penetrating the zona pellucida or oolema (Fig. 3). Some previtellogenic atretic follicles of H. fossilis during Mrach-April exhibited prominent granulosa cells, separation of ooplasm from zona pellucida and disorganization of ooplasm (Fig. 4). Vitellogenic ovarian follicles of H. fossilis at the early stage of atresia (May-June) showed prominent granulosa cells, vacuolation of the ooplasm at periphery and ooplasm giving flocculent appearance (Figs. 5, 6). Thickened zona pellucida and hypertrophied granulosa cells were recorded as the atresia advanced in the vitellogenic follicle of H. fossilis (Fig. 7). During the advanced stage of atresia (September-October), the oocytes of the catfish depicted disorganized ooplasm, obscure germinal vesicle and hypertrophied granulosa cells. Phagocytic invading of granulosa cells in zona pellucida and ooplasm were also prominent (Fig. 8).

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Fig. 1. Ovary of immature H. fossilis showing previtellogenic oocytes. Mark the nodule of stroma tissue (arrow) and atretic follicle (broken arrow). H&E. x 100; Fig. 2. Ovary of immature the catfish exhibiting prominent nodule of stroma (arrow) and oocytes in early stages of development. H&E. x 100; Fig. 3. Previtellogenic atretic follicles in ovary of H. fossilis depicting excessive vacuolation of ooplasm towards periphery. Mark the flocculent ooplasm and hypertrophied granulosa cells (arrow) penetrating the ooplasm. H&E. x 250; Fig. 4. Previtellogenic ovarian atretic follicle of the catfish showing prominent granulosa cells, separation of ooplasm from zona pellucida (arrow) and disorganization of ooplasm. H&E. x 250.

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Fig. 5. Vitellogenic ovarian follicles of H. fossilis at the early stage of atresia with prominent granulosa cells and vacuolation of the ooplasm at periphery. Mark the flocculent appearance of ooplasm in the oocytes. H&E. x 250; Fig. 6. Vitellogenic follicles in ovary of H. fossilis at the early stage of atresia showing vacuolated germinal vesicle (arrow) and vacuolation of ooplasm at periphery. H&E. x 250; Fig. 7. Atretic vitellogenic follicle in ovary of H. fossilis with vacuolated cytoplasm, thickened zona pellucida (arrow) and hypertrophied granulosa cells. H&E. x 250; Fig. 8. Vitellogenic follicle in ovary of H. fossilis with advanced stage of atresia depicting disorganized ooplasm, obscured germinal vesicle and hypertrophied granulosa cells. Mark the phagocytic granulosa cells invading zona pellucida and ooplasm. H&E. x 250.

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Though the precise causes of follicular atresia in teleosts have not yet been clearly defined, several exogenous (photoperiod, temperature, rainfall, crowding, captivity, nutrition, physico-chemical characteristics of ambient water, pollutants/biocides etc) as well as endogenous (insufficient gonadotrophic hormone, imbalance of hormones and steroids) have been implicated in the process (Sundararaj and Goswami, 1968; Saidapur, 1978, 1982; Kling, 1981; Saksena and Raizada, 1984; Guraya, 1993, 1994; Rodriguez et al., 1995; Miranda et al., 1999; Wood and Kraak, 2001; Khanna, 2006). Further studies are required to resolve the causes and functions of follicular atresia for management of broodstocks to realize optimum fecundity of the fish.

ACKNOWLEDGEMENTS

We are grateful to Dr. N. Sarangi, Director, CIFA, Bhubaneswar and Dr. Dilip Kumar, Director, CIFE (Deemed University), Mumbai for providing laboratory facilities. Financial support received by the senior author (CVM) as Junior Research Fellowship from CIFE (ICAR) is thanfully acknowledged.

REFERENCES

Agarawal, N. K. and H. R. Singh, 1990. Preovulatory follicular atresia (corpora atretica) in snowtrout, Schizothorax plagiostomus. J. Anim. Morphol. Physiol., 37: 29-33. Bancroft, J. D. and A. Stevens, 1977. Theory and Practice of Histological Techniques. Churchill Livingston, New York. Belsare, D. K., 1962. Seasonal changes in the ovary of Ophiocephalus punctatus (Bloch). Indian J. Fish., 8: 140-156. Belsare, D. K., 1975. Component of the ovarian endocrine tissue in teleost species. Zool. Pol., 25: 5-11. Chan, S. T. H., A. Wright and G. Phillips, 1967. The atretic structures in the gonads of the rice field eel (Monopterus albus) during natural sex reversal. J. Zool., 153: 527-539. Dixit, R. K., 1960. Seasonal growth of oocytes of Mystus seenghala (Sykes) and Wallagonia attu (Bloch), with an inference about their spawning habits. Proc. Natl. Acad. Sci. India, 30B: 241- 245. Fraser, E. A. and R. M. Renton, 1940. Observations on the breeding and development of the viviparous fish, Heterandria formosa. Quart. J. Microsc. Sci., 81: 479-490. Gokhale, S. V., 1957. Seasonal histological changes in the Whitting (Gadus merlangus) and Norway Pout (Gadus esmarki). Indian J. Fish., 4: 311-317. Guraya, S. S., 1993. Follicular (or oocyte) atresia and its causes and functional significance in the fish ovary. In: Adances in Fish Research (Ed. B. R. Singh). Narendra Pub. House, New Delhi. pp. 313-332. Guraya, S. S., 1994. Gonadal development and production of gametes in fish. Proc. Indian Natl. Sci. Acad., 60B: 15-32. 68

Guraya, S. S., H. S. Toor and S. Kumar, 1977. Morphology of ovarian changes during the reproductive cycle of the fish, Cyprinus carpio communis (Linn.). Zool. Beitr., 23: 405-437. Jhingran, A. G., 1961. Studies on maturity and fecundity of the Gangetic anchovy, Satipinna phasa (Hamilton). Indian J. Fish., 8: 281-311. Khanna, S. S., 2006. An Introduction to Fishes. 5th Edn. Silver Line Publications, Faridabad. Khanna, S. S. and M. C. Pant, 1967. Seasonal changes in the ovaery of a sisorid catfish, Glyptosternum pectinopterum. Copeia, 1: 83-88. Kling, D., 1981. Total atresia of the ovaries of Tilapia leucosticta (Cichlidae) after intoxication with the insecticide, Lebaycid. Experientia, 37: 73-74. Lehri, G. K., 1968. Cyclical changes in the ovary of the catfish, Clarias batrachus. Acta Anat., 69: 105- 124. Malhotra, P., 1971. Studies on the seasonal changes in ovary of Schizothorax niger (Heckel) from Dal Lake in Kashmir. Jap. J. Ichthyol., 17: 110-116. Mathews, S. A., 1938. The seasonal cycle in the gonads of Fundulus. Biol. Bull. (USA), 75: 67-74. Mendoza, G., 1943. The reproductive cycle of the viviparous teleost, Neotoca bilineata, a member of the Family Goodeidae. IV. The germinal tissue. Biol. Bull. (USA), 84: 87-98. Miranda, A. C. L., N. Bazzoli and Y. Sato, 1999. Ovarian follicular atresia in two teleost species: a histological and ultrastructural study. Tissue & Cell., 31: 480-488. Nair, P. V., 1963. Ovular atresia and the formation of so-called corpus inteum in the ovary of the Indian catfish, Heteropneustes fossilis. Proc. Zool. Soc. (Bengal), 16: 51-65. Pearse, A. G. E., 1968. Histochemistry: Theoretical and Applied. Vol. 1. Churchill Livingston, London & Edinburgh. Rai, B. P., 1966. Cyclical changes in the ovary of Tor (Barbus) tor. Acta Zool., 43: 289-307. Rajalakshmi, M., 1966. Atresia of oocytes and ruptured follicles in Gobio giuris. Gen. Comp. Endocrinol., 6: 378-385. Rajkumar, R. and K. K. Hemlatha, 2005. Observations on follicular atrition in Poecilia reticulata (Peters) (Atheriniformes: Teleostei). J. Inland Fish Soc. India, 37(2): 21-25. Rastogi, R. K., 1966. A study of the follicular atresia and evacuated follicles in the Indian teleost, Xenantodon cancila. Acta Biol. Acad. Sci. Hung., 17: 52-63. Rastogi, R. K., 1968a. The occurrence and significance of follicular atresia in the catfish, Mystus tengara. Acta Zool., 49 : 309-319. Rastogi, R. K., 1968b. The occurrence and significance of ovular atresia in the mud eel, Amphipnous cuchia. Acta Anat., 73: 148-160. Rodriguez, J. N., Z. J. Oteme and S. Hem, 1995. Comparative study of vitellogenesis of two African catfish species, Chrysichthys nigrodigitatus (Claroteidae) and Heterobranchus longifilis (Clariidae). Aquat. Living Resour., 8: 291-296. Saidapur, S. K., 1978. Follicular atresia in ovaries of non-mammalian vertebrates. Intern. Rev. Cytol., 54: 225-244. Saidapur, S. K., 1982. Structure and function of post-ovulatory follicles (corpora lutea) in the ovaries of non-mammalian veretebrates. Intern. Rev. Cytol., 58: 243-285. 69

Saksena, D. N. and H. N. Bhargava, 1972. The corpora atretica, post-ovulatory follicles and spawning periodicity of Indian freshwater goby, Glossogobius giuris (Ham.). Zool. Jb. Anat., 89: 611-620. Saksena, D. N. and A. K. Raizada, 1984. On the corpora atretica and post-ovulatory follicles and spawning periodicity in some freshwater Indian teleosts. Intl. J. Acad. Ichthyol., 5: 11-22. Sathyanesan, A. G., 1961. A histological study of the ovular atresia in the catfish, Mystus seenghala (Sykes). Rec. Indian Mus., 59: 75-82. Sathyanesan, A. G., 1962. The ovarian cycle in the catfish, Mystus seenghala (Sykes). Proc. Natl. Inst. Sci. India, 28B: 497-506. Srivastava, S. J., 1980. Seasonal histological changes in ovary of a freshwater large murrel, Channa marulius (Ham.). Zool. Jb. Anat., 104: 492-499. Sundararaj, B. I. and S.V. Goswami, 1968. Effect of short- and long-term hypophysectomy on the ovary and interrenal of catfish, Heteropneustes fossilis (Bl.). J. Exp. Zool., 168: 85-104. Vizziano, D. and N. Berois, 1990. Histology of the ovary of Macrodon ancylodon (Bloch & Schneider, 1801) (Teleostei: Sciaenidae). Oogenesis. Post-ovulatory follicles. Atresia. Rev. Bras. Biol., 50: 523-536. Wood, A.W. and G. J. van der Kraak, 2001. Apoptosis and ovarian function: novel perspectives from the teleosts. Biol. Reprod., 64: 264-271.

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J. Aqua., 14 (2006): 71-75

ALTERATIONS IN HAEMATOLOG (LINNAEUS), INDUCED BY EXPERIMENTAL PROCAMALLANUS INFECTION

Shashi Ruhela, Ajay K. Pandey and Ashok K. Khare Department of Zoology, Meerut College, Meerut-250001, India

In order to record the effects of experimental Procamallanus infection on blood parameters of Clarias batrachus, total erythrocyte count (TEC), haemoglobin content (Hb), total leucocyte count (TLC), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haeamoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined and calculated on day 15, 30 and 45 post-infection. TEC, Hb and PCV recorded a decline whereas TLC registered an increase during the post-infection period. While MCV and MCH recorded an increase on day 15 and 30 (except on day 45) post-infection, MCHC displayed a significant decrease on day 15 and 45 (except on day 30) post-infection in C. batrachus. The haematological alterations due to Procamallanus infection appear to cause anaemia in the catfish.

INTRODUCTION

Domesticated animals and fish get affected directly as well as indirectly by parasitic infections (Reid and Armour, 1978; Das and Das, 1997). The parasitic infection disturbs the physiological and metabolic activities of the host inducing changes in blood parameters leading to the disease like anemia and eosinophilia (Satpute and Agrawal, 1974; Roberts, 2001; Madhavi, 2003). The most common disease of fish due to gastrointestinal helminths is macrocytic anaemia (Sinha, 1992; Roberts, 2001; Madhavi, 2003). There exists report on pernicious anaemia in the catfish caused by an enteric nematode parasites belonging to Genus Procamallanus (Sinha, 2000). Both adult and larval stages of Procamallanus are pathogenic to fish (De and Maity, 2000, Ruhela et al., 2006). The need for establishment of standard normal haematological values with a view to aiding in diagnosis the state of health and disease in fishes has been duly emphasized by a number of workers owing to the growing interest in pisciculture (Hesser, 1960; Blaxhall and Daisley, 1973; Roberts, 2001, Ayyappan et al., 2006). An attempt has been made to record the alterations in blood parameters of the commercially important freshwater catfish, Clarias batrachus, elicited due to experimental Procamallanus infection.

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MATERIAL AND METHODS

Clarias batrachus used in the present study were collected from local freshwater ponds and also purchased from fish markets of Meerut and adjoining region in western Uttar Pradesh, India. Fishes were acclimatized to laboratory conditions for a week before initiating the experiment. Adult female Procamallanus were collected from infected C. batrachus by cutting open the intestine. They were kept in watch glass filled with saline solution for natural egg laying at 24-27ºC. The eggs were kept in Lock-Lewis solution for healthy embryonation. The solution was changed periodically and 0.1% formalin added to the culture medium to avoid fungal contamination of the eggs. Forty healthy catfish were randomly selected and divided into two equal groups. Catfishes of group 1 were not given any treatment and served as control whereas in catfish of group 2, experimental infection was induced by forcefully pushing 500 embryonated eggs of the nematode into the stomach of each catfish by means of a long-nozzled dropper (De and Maity, 2000; Ruhela et al., 2006).

Fresh blood samples from both the groups of catfish were collected from day 15, 30 and 45 by a sharp cut made near the caudal vein and kept in glass vials taking all necessary care to prevent haemolysis and clotting by using anticoagulant. Estimation of total erythrocyte count (TEC), haemoglobin content (Hb), total leucocyte count (TLC) and packed cell volume (PCV) were done by haemoglobinometer, haematocrit tube and haemocytometer. Values of mean corpuscular volume (MCV), mean corpuscular haeamoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated as these parameters depend on the corresponding values of Hb, TEC and PCV. Data on haematological values on the control and infected catfishes were evaluated for statistical significance using Students ‘t` test.

RESULTS AND DISCUSSION

During the present experiment, the infected catfish showed signs of restlessness and came frequently to the surface for gulping air after day 30 of the treatment. The present study demonstrates that the nematode infection in C. batrachus induce haematological changes and also elicit a number of systemic disturbances as manifested by changes in the blood parameters. Alterations in blood parameters of the catfish due to experimental Procamallanus infection have been summarized in Table 1.

The total erythrocyte counts (TEC) of the control C. batrachus were observed to be 3.58±0.29, 3.48±1.44 and 3.41±0.24 million/mm3, respectively on day 15, 30 and 45. A significant decrease in TEC of the catfish to 2.47±0.32, 2.33±0.24 and 2.22±0.24 million/mm3, respectively were recorded on the corresponding post-infection period. Haemoglobin (Hb) content of the control C. batrachus was 12.76±0.46, 12.52±0.53 and 73

11.97±0.52 mg/dl on day 15, 30 and 45. A significant decrease in Hb content to 11.10±0.21, 11.05±0.15 and 9.42±0.29 mg/dl, respectively were recorded during the corresponding post-infection period. TLC values of uninfected catfish were found to be 32.50±0.57, 31.10±0.46 and 33.00±0.29 x103/mm3, respectively on day 15, 30 and 45. A significant increase in the TLC value to 38.00±0.29, 37.50±0.58 and 36.40±0.15 x103/mm3, respectively were observed on the corresponding post-infection period. PCV values of control catfish were 40.14±0.69, 39.94±0.54 and 32.67±0.33% on day 15, 30 and 45. Procamallanus infection induced a significant decrease in PCV to a level of 36.86±0.80, 36.00±0.29 and 28.00±0.58%, respectively on the corresponding post-infection period.

MCV values of the control C. batrachus were 114.68±0.51, 122.89±1.26 and 112.26±1.00 µ3, respectively on day 15, 30 and 45. A significant increase in MCV value to the level of 143.42±1.43 and 136.88±0.99 µ3 were observed on day 15 and 30 post-infection. However, on day 45 of the treatment, this value decreased to the level of 108.10±1.33 µ3. The values of MCH of control catfish were 36.45±0.59, 36.98±0.34 and 37.69±0.67 pg, respectively on day 15, 30 and 45. A significant increase in the level of MCH to 43.19±1.63 and 40.01±0.0.33 pg were recorded on day 15 and 30 post-infection. However, this value decreased to the level of 32.5±1.04 pg on day 45 of infection. MCHC values of control catfish were 32.98±0.59, 30.99±1.15 and 33.58±0.94%, respectively on day 15, 30 and 45. The MCHC values registered a significant decrease to the tune of 28.21±0.58 and 30.07±1.51% on day 15 and 45 post-infection, however, this value was almost normal (30.69±1.10%) on day 30 of the infection.

Table 1: Effect of experimental Procamallanus infection on haematological parameters of Clarias batrachus

Parameter 15 Days 30 Days 45 Days Control Infected Control Infected Control Infected TEC (million/ 3.58 2.47 3.48 2.33 3.41 2.22 mm3) ±0.29 ±0.32c ±0.44 ±0.24 b ±0.20 ±0.24d Hb (mg/dl) 12.76 11.10 12.52 11.05 11.97 9.42 ±0.46 ±0.21b ±0.53 ±0.15a ±0.52 ±0.29c TLC (103/mm3) 32.50 38.00 31.10 37.50 33.00 36.40 ±0.57 ±0.29d ±0.46 ±0.58d ±0.29 ±0.15d PCV (%) 40.14 36.86 39.94 36.00 32.67 28.00 ±0.69 ±0.80b ±0.54 ±0.29d ±0.33 ±0.58d MCV (µ3) 114.68 143.42 122.89 136.88 112.26 108.10 ±0.51 ±1.43d ±1.26 ±0.99d ±1.00 ±1.33b MCH (pg) 36.45 43.19 36.98 40.01 37.69 32.5 ±0.59 ±1.63d ±0.34 ±0.33d ±0.67 ±1.04d MCHC 32.98 28.21 30.99 30.69 33.58 30.07 (%) ±0.59 ±0.58a ±1.15 ±1.10 ±0.94 ±1.51a Values are mean ± standard error (SE) of five specimens. Significant response: a P < 0.05, b P < 0.02, c P < 0.01, d P < 0.001. 74

The present investigation reveals that the infected catfish showed restlessness and frequent visit to the surface for gulping air. This observation confirms the report of Hartman and Lessler (1964) that fishes having RBC with reduced surface area/volume coupled with reduction in RBC number remain in very disadvantageous position in respect to respiratory efficiency. Totterman (1944) observed decrease in erythrocyte count and haemoglobin value to less than 20% due to Diphllobothrium latum infection in man. Reduction in the number of red blood cells may be due to decreased rate of erythropoeisis (Satpute and Agrawal, 1974). Decrease in haemoglobin content and haematocrit value under stressful condition would be an expected consequence of less number of erythrocytes.

A significant decline in the values of PCV, TEC and Hb in fishes after infection may leads to the development of anemia. Similar view has also been expressed by Blaxhall (1972), Blaxhall and Daisley (1973) and Sinha (2000). A significant decrease in MCHC (except on day 30 post-infection) and increase in MCV (except on day 45 post- infection) supports the suggestion that iron deficiency might contribute to the development of anemia in C. batrachus as parasites harbouring the intestine might be utilizing the metabolite (Sinha, 2000).

After the exposure to infection for 45 days, haemoglobin level and PCV decreased. The reason may be release of immature cells from haemopoietic tissues into the blood stream (Wepener et al., 1992; Kumar et al., 1999a, b). On day 30 and 45 post- infection, the decreased values of Hb, PCV and TEC appear to be due to the toxic effects of the secretions/excretions by the parasite. Release of cellular Hb into plasma may be a reason (Narain and Srivastava, 1979; Kumar et al., 2004). In plasma Hb increases when haem from lysed or damaged erythrocytes is released under pathological conditions (Eastman, 1977).

ACKNOWLEDGEMENTS

We are grateful to the Head, Department of Zoology, Meerut College, Meerut for providing the laboratory facilities to carry out the present investigation. Thanks are due to Prof. S.S. Lal, Ex-Head, Department of Zoology, C.C.S. University, Meerut for going through the manuscript.

REFERENCES

Ayyappan, S., J. K. Jena, A. Gopalkrishnan and A. K. Pandey, 2006. Handbook of Fisheries and Aquaculture. Directorate of Publications and Information in Agriculture (ICAR), New Delhi. Blaxhall, P. C. and K. W. Daisley, 1973. Routine haematological methods for use with fish blood. J. Fish Biol., 5: 771-781. 75

Blaxhall, P. C., 1972. The haematological assessment of the health of freshwater fish: a review of selected literature. J. Fish. Biol., 4: 593-604. Das, M. K. and R. K. Das, 1997. Fish and prawn diseases in India: Diagnosis and control. Inland Fisheries Society of India, Barrackpore. De, N. C. and R. N. Maity, 2000. Development of Procamallanus saccobranchi (Nematoda: Camallanidae), a parasite of a freshwater fish in India. Folia.Parasitol., 47: 216-226. Eastman, R. D., 1977. Clinical haematology. John Wright & Sons, Sutton, United Kingdom. Hartman F. A. and M. A. Lessler, 1964. Erythrocyte measurements in fishes, Amphibia and reptiles. Biol. Bull. Mar. Biol. Lab Woods Hole, 126: 83-88. Hesser, E. F., 1960. Methods for routine fish haematology. Progr. Fish-Cult., 122: 164-171. Kumar, K., P. Patri and A. K. Pandey, 2004. Haematological and biochemical responses in the freshwater air-breathing teleost, Anabas testudineus (Bloch), exposed to mercury. J. Ecophysiol. Occup. Hlth., 4: 97-108. Kumar, K., Y. K. P. Sinha and A. K. Pandey, 1999a. Endosulfan induced haematotogical alterations in the climbing perch, Anabas testudinenus (Bloch). J. Natcon., 11: 125-134. Kumar, K., Y. K. P. Sinha and A. K. Pandey, 1999b. Effect of mercuric chloride toxicity on the blood of climbing perch, Anabas testudinenus (Bloch). J. Natcon., 11: 275-283. Madhavi, R., 2003. Metazoan parasites in fishes. In: Aquaculture Medicine (Eds. I. S. Bright Singh, S. S. Pai, R. Philip and A. Mohandas). School of Environmental Sciences, Cochin University of Science & Technology, Cochin. pp. 64-88. Narain, A. S. and P. N. Srivastava, 1979. Haemato-histological responses of the Indian freshwater catfish, Heteropneustes fossilis, to environmental pollution by sewage, fertilizers and insecticides. Arch. Biol. (Bruxelles), 90: 141-159. Reid, J. F. S. and J. Armour, 1978. An economic appraisal of helminth parasites in sheep. Vet. Rec., 1: 4-7. Roberts, R. J., 2001. Fish Pathology. 2nd Edn. W. B. Saunder, Philadelphia & London. Ruhela, S., A. K. Pandey and A. K. Khare, 2006. Effect of experimental Procamallanus infection on certain blood parameters of the freshwater catfish, Clarias batrachus (Linnaeus). J. Ecophysiol. Occup. Hlth., 6: 73-76. Satpute, L. R. and S. M. Agrawal, 1974. Parasitic effects on its haematology and histopathology. Indian J. Exp. Biol., 12: 584-586. Sinha, K. P. 1992. Macrocytic anaemia in the catfish, Clarias batrachus. Biojournal, 4: 229-230. Sinha, K. P., 2000. Haematological manifestation in Clarias batrachus carrying helminth infections. J. Parasit. Dis., 24: 167-170. Totterman, G., 1944. On the occurrence of pernicious tapeworm anemia in Diphyllobothrium carriers. Acta Med. Scand., 118: 410-416. Wepener, V., J. H. J. van Varen and H. H. Dupreez, 1992. Effect of manganese and iron at a neutral and acidic pH on the haematology of the banded tilapia. Bull. Environ. Contam. Toxicol., 49: 613-619. 76 77

J. Aqua., 14 (2006): 77-82

EFFECT OF WATER TEMPERATURE ON IMMUNE PARAMETERS OF GIANT FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII

The giant freshwater prawn Macrobrachium rosenbergii is an economically important farmed crustacean species cultured in Caribbean countries and south-east Asia including India. Crustaceans have evolved a complex, efficient and highly developed innate immune system based largely upon circulatory haemocytes, various defence proteins viz., prophenoloxidase (proPO), serine proteases/PPO activating enzyme (ppA), lectins, β-glucan binding proteins, lysozyme, antibacterial peptides, α2 macroglobulins etc. (Smith et al., 2003). It is well established that, in arthropods, the defence of the host against invasive or opportunistic microorganisms is effected principally through the phagocytic, encapsulating and agglutinating activities of the circulating haemocytes (Ratcliffe et al., 1982) as well as by antimicrobial factors in the plasma (Gotz and Boman, 1985). The proPO system seems to participate in host defence by enhancing phagocytosis, melanin formation and initiating nodule or capsule formation (Soderhall et al., 1986). Based upon the recent classification of M. rosenbergii haemocytes (Sierra et al., 2001), large ovoid haemocytes and undifferentiated round haemocytes might be carrying out the functions of the proPO system, like semigranular and granular haemocytes in other crustaceans (Johansson and Soderhall, 1989). The activity of phenoloxidase has already been reported in M. rosenbergii (Kumari et al., 2004).

In recent years, environmental changes have become an important cause for increased prevalence of shrimp disease, leading to production loss. Therefore, there is an effort to study the effects of environmental factors on immune function of shrimp (Lu- Qing et al., 2007). Changes in physico-chemical parameters viz., pH, temperature, salinity, dissolved oxygen, presence of pollutants and toxicants such as ammonia and nitrite have been reported to affect the disease resistance of freshwater prawn M. rosenbergii and other decapod crustaceans (Cheng and Chen, 2000; Le Moullac and Haffner, 2000; Chand and Sahoo, 2006). Water temperature is probably the most important environmental factor, which directly affects metabolism, growth, oxygen consumption and survival and influences environmental parameters such as salinity and oxygenation of the water (Le Moullac and Haffner, 2000). Seasonal ranges of water temperature in scampi farms of southern and eastern parts (major scampi producing parts) of India mostly vary from 19 to 32˚C. M. rosenbergii can tolerate a wide range of temperature (14-35°C) and the optimal temperature for growth is 29-31°C (New, 1995). The effects of pH, temperature and salinity on the oxygen consumption and nitrogen excretion of M. rosenbergii have been studied by Nelson et al. (1977). In a preliminary study, Cheng and Chen (2000) observed a lower THC at 33-34°C (at 1.5% feeding rate) and a higher THC at 27-28°C (at 0.6% feeding rate), the highest PO activity at 30-31°C (at 1.5% feeding rate) and at 27-28°C (at 0.6% feeding rate) while experimenting with two prawns only in triplicate at four different 78 temperature ranges (20-21°C, 27-28°C, 30-31°C and 33-34°C). Lu-Qing et al. (2007) observed a significant effect of temperature on THC and PO, antibacterial and bacteriolytic activities in haemolymph of L. vannamei, and advocated a range of 24-30°C as suitable temperature for culture without its fluctuation beyond 3°C. Cheng et al. (2005) found a reduction in immune capability and resistance of L. vannamei to Vibrio alginolyticus infection when the shrimp were transferred to 32-34°C from 27 or 28°C. Thus, the present study aims at drawing a clear picture on few of the immune parameters that are being influenced by various temperature ranges in M. rosenbergii and discusses a suitable temperature range for culture of this species from immune capability point of view.

M. rosenbergii (15-20 g, in the intermoult stage) were collected from monoculture ponds of the Central Institute of Freshwater Aquaculture, Bhubaneswar. The prawns were acclimated in the wet laboratory in FRP tanks at a stocking density of 2 g/l for 1 week before experimentation. A pellet feed was provided twice daily during acclimation and also during the experiment as described earlier (Kumari et al., 2004). The basic physico-chemical parameters of the water of the ponds (from where the samples were collected) and experimental tanks (where similar pond water was used) were as follows: pH, 6.8-7.5, dissolved oxygen 5.2-6.4 mg/l, ammonia < 0.1 mg/l, total hardness 70-80 mg/l, and total alkalinity 30-40 mg/l.

Thirty numbers of intermoult prawns (male:female::1:1) were maintained in triplicate in three FRP tanks for each temperature range study. Three different temperature ranges (viz., 19-21°C; 25-27°C; 30-32°C) were setup using electronically controlled water heater based on field observations on temperature fluctuations in prawn farms of coastal area (P.K. Sahoo, unpublished observation). The prawns were acclimatized in the above temperatures for a period of 7 days. Ten percent of water was renewed daily (with preset desired temperature) during removal of waste feed and faecal materials. A continuous aeration was provided during the experimental period.

After 7 days of acclimatization, haemolymph of 100 µl was collected from the ventral sinus of fifteen prawns of each temperature group in to a 1 ml syringe (26 gauge) containing 900 μl anticoagulant (sodium chloride 0.45 M, glucose 0.1 M, sodium citrate 30 mM, citric acid 26 mM, EDTA 20 mM, pH 4.5). Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome produced from L- dihydroxy phenylalanine (L-DOPA, a product of Hi Media, Mumbai) following Hernandez-Lopez et al. (1996) and Chand and Sahoo (2006). The phenoloxidase activity optical density was expressed as dopachrome formation per 50 µl haemolymph.

Haemolymph (50 µl) was immediately withdrawn into another syringe containing 0.45 ml of anticoagulant with fixative solution (sodium cacodylate 0.10 M, and 1.5% glutaraldehyde) in 1:1 ratio. A drop of haemolymph was placed on a 79 haemocytometer to measure THC and DHC using an inverted phase contrast microscope. The various cell types were identified following Sierra et al. (2001).

From rest of the fifteen prawns of each group, haemolymph (~ 300 µl) was withdrawn using 2 ml syringe and 26 gauge needle from the ventral sinus and dispensed in to 2 ml eppendorf. Haemolymph was allowed to clot at 40C. After 1 h, the clot was broken using sterile needle and the tube was further kept at 40C for 3 h. The tube was then centrifuged at 11,000 x g for 30 min at 40C and the supernatant collected was stored at 300C till further analysis. Part of the supernatant was used to measure total protein concentration following Bradford (1976), using bovine serum albumin as a standard protein. Rest part of the supernatant was used to measure bacterial agglutination and haemagglutination titres.

Rabbits maintained in the Institute animal house were bled from the ear veins. The blood samples were collected aseptically into Alsever's solution in 1:1 ratio and the blood was centrifuged at 4000 x g for 15 min to prepare packed red blood cells (RBCs). The supernatant fluid was discarded. The packed RaRBC were then washed thrice by centrifugation with sterile PBS (containing Ca++ and Mg++). A 1.5% (v/v) RaRBC suspension was prepared in the same buffer for studying haemagglutination (HA) titre of the collected prawn haemolymph. Haemagglutination assay (HA) was performed in U- bottom microtitre plates using 1.5% rabbit RBC (RaRBC) suspension. Two-fold serial dilutions of serum samples were made in PBS (with Ca2+ and Mg2+, pH 7.3). Equal volume of 1.5% (v/v) RaRBC was added to each dilution of serum. The plates were incubated at 25ºC for 1 h. HA titre was read as the reciprocal of the last serum dilution showing agglutination after 1 h of incubation.

A known pathogenic isolate of Aeromonas hydrophila was grown on tryptone soy broth (TSB, Difco) for 24 h at 30ºC. The bacteria to be used for bacterial agglutination assay was treated with 1% formalin and kept overnight at 4ºC. The formalin-killed cells were washed twice with sterile PBS and suspended in PBS to 2.7 x 108 cells/ml. The formalin-killed cells were stored at - 4ºC until use. The test was performed in a similar fashion to HA test using formalin-killed A. hydrophila. The plates were incubated overnight at 25ºC before reading.

The mean values of each parameter were calculated for all the groups. Data were analyzed using one-way ANOVA. Means were compared using Duncan's multiple range tests (Duncan, 1955). Difference was considered significant when P < 0.05.

The total haemocyte count (THC) was increased corresponding to the rise in acclimatization temperature. The mean THC varied from 13.53±1.65 x 106 to 19.9±0.03 x 106 cells/ml. An increase of 47% in THC value was observed between the lowest and highest temperature ranges. Similarly, a higher bacterial agglutination titre in the 80 haemolymph of prawn maintained at the highest temperature was observed. On the other hand, the numbers of undifferentiated round haemocytes were significantly less at the highest temperature. The three temperature ranges have no effect on total protein content, PO activity, HA titre, fusiform or large ovoid cell populations. However, a wide individual variation in haemagglutin and total protein levels was observed (Table 1).

Table 1. Effect of acclimatization temperature on haemolymph parameters of prawn

Temper- THCx106 Fusiform Large Undifferent- PO Haemagglu- Bacterial Total ature cells/ml cells (%) ovoid iated cells activity tination titre agglutin- protein range cells (%) (%) (OD/ ation titre (g/dl) 50 μl haemo- lymph) 30-32°C 19.90 ± 74.70 ± 19.96 ± 5.34 ± 0.46 ± 111.68 ± 7.50 ± 41.89 ± 0.03b 2.03 1.33 0.70a 0.01 16.82 0.50b 20.45 25-27°C 15.32 ± 69.42 ± 21.44 ± 9.14 ± 0.39 ± 177.87 ± 4.87 ± 44.34 ± 1.23ab 1.05 0.87 0.18b 0.05 41.79 0.37a 17.69 19-21°C 13.53 ± 69.73 ± 21.50 ± 8.77 ± 0.44 ± 52.54 ± 4.71 ± 44.64 ± 1.65a 1.90 1.40 0.49b 0.02 22.36 0.42a 17.90 Data represent mean±SE (n=45). Statistical differences (P<0.05) among different temperature ranges are indicated by different letters (a, b).

Prawns are poikilothermic animals, and as such, any fluctuation in environmental temperature changes their body temperature. This fluctuation in environmental factors affects directly the metabolism and physiological adjustment. Lin et al. (1999) suggested that oxygen consumption and nitrogenous excretion of M. rosenbergii juveniles increased directly as the temperature increased from 24 to 32ºC. Circulating haemocytes are affected by extrinsic factors such as temperature, pH, salinity, dissolved oxygen and ammonia in several species of decapod crustaceans (Le Moullac and Haffner, 2000; Cheng and Chen, 2002). M. rosenbergii reared in 20ºC had significantly lower THC and PO activity as compared to the prawn reared at 27 and 30ºC (Cheng and Chen, 2000). Blue shrimp Litopenaeus stylirosttris reared in 18ºC had a significantly lower THC, as compared to the shrimp reared at 27ºC (Le Moullac and Haffner, 2000). L. vannamei showed a lower THC and PO activity at 20 or 24ºC as compared to that of 28ºC. A similar phenomenon was also observed in the present study in case of THC value that revealed a significantly higher value in prawns reared at the temperature range of 30-32ºC and the lowest value at temperature range of 19-21ºC. However, PO activity did not show any significant fluctuation in the present study. Similar type of observations has also been marked in earlier study in Litopenaeus setiferus maintained in laboratory for 7 days at two different temperatures (27 and 31ºC) (Sanchez et al., 2001). They observed a significantly higher THC at 31ºC than that of 27ºC, and no change in PO activity between two different temperature groups. It has been observed that after a certain period of immune regulation 81

(3-6 days), all immune parameters tended to be stable in shrimps leading to immune adaptation (Cheng et al., 2005; Lu-Qing et al., 2007). Similar phenomenon might have occurred in prawns in the present study where they were maintained for a period of 7 days before bleeding to measure immune parameters, thus leading to no major changes in PO activity, haemagglutinin and total protein levels, and fusiform and large ovoid cell populations. However, a positive correlation between the haemocytes and bacterial agglutinin level with temperature in this study clearly indicated influence of temperature on immune capability of prawn. Thus, it may be concluded that the higher range of temperature above 25ºC is beneficial for raising M. rosenbergii as the immune capability of the animal is enhanced at higher ranges of temperatures examined.

This research was supported by AP Cess Project of Indian Council of Agricultural Research, New Delhi (Code No. 0614025). Thanks are due to the Director, CIFA, for providing necessary facilities during the study.

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Central Institute of Freshwater Aquaculture, P. K Sahoo* Kausalyaganga, Bhubaneswar 751 002, India Bindu R Pillai *Corresponding author: [email protected] J. Mohanty Jaya Kumari and S. Mohanty