Changes in Biochemical Constituentsand Energy Levels of Flower Shrimp,

Penaeus semisulcatus,Infected with Vibrio parahaemolyticus

Satyavathi ChinniPrabhakara Rao Yallapragada

ABSTRACT. During the present investigation, laboratory experimentswere conducted to study the changes in biochemical contents such ascarbohydrates, proteins, lipids, and energy levels after 96-hour exposureto Vibrio parahaemolyticus, in flower shrimp, Penaeus semisulcatus,post-larvae. The sampling was done at intervals of 24-hours, 48-hours,72-hours, and 96-hours. The caloric concentrations as well as biochemicalconstituents except protein showed a decrease in infected post-larvae. Incontrast, protein showed an increase in infected post-larvae. A decrease inthe ratios of carbohydrate/protein and carbohydrate/lipid was observed ininfected post-larvae. The differential responses of biochemical constitu-ents might be due to triggering of compensatory mechanism under stressconditions due to V. parahaemolyticus infection. [Article copies availablefor a fee from The Haworth Document Delivery Service: 1-800-HAWORTH.E-mail address: <docdelivery@haworthpress.com> Website: <http://www.HaworthPress.com> 2003 by The Haworth Press, Inc. All rights reserved.]

KEYWORDS. Penaeus semisulcatus, protein, lipid, carbohydrate, Vibrioparahaemolyticus, flower shrimp

Satyavathi Chinni and Prabhakara Rao Yallapragada, Division of Animal Physiol-ogy and Toxicology, Department of Zoology, Andhra University, Visakhapatnam–530003, India.

Address correspondence to: Dr. Satyavathi Chinni, F-004, Srilakshmi Enclave,MIG-61, H.B. Colony, Seethammadhara, Visakhapatnam–530 022, India.

Journal of Applied Aquaculture, Vol. 14(3/4) 2003http://www.haworthpress.com/store/product.asp?sku=J028

2003 by The Haworth Press, Inc. All rights reserved.10.1300/J028v14n03_04 47

INTRODUCTION

Global shrimp fisheries are the most commercially-important in theseafood industry because of its high market-value, and considerable in-vestments have been made in recent decades to develop culture meth-ods for shrimp. Some of their efforts have been successful, and marinepenaeid shrimps have accounted for 87% of the production of crustaceansfrom Indian Aquaculture in 2000, or about 720,000 tons (Vishnubhat2001). The shrimp industry in India grew by 460% during the period of1999 to 2000 and it is projected to continue to grow at this rate (Vishnubhat2001). According to Marine Products Export and Development Author-ity (MPEDA 2001) approximately 50% of all penaeid shrimp marketedworldwide now come from farms. Flower shrimp, Penaeus semisulcatus,fishery is considerably higher in the West Coast of India (Kurian andSebastian 1993).

Concomitant with growth of the shrimp industry has been the recog-nition of the ever-increasing importance of diseases. Diseases due to vi-ral infections have emerged as the most important diseases of culturedshrimp (Lightner and Redman 1998).

During the disease, energy metabolism plays a key role to overcomethe stress caused by pathogens. A reduction of caloric content in crusta-ceans during stress has been observed (Capuzzo et al. 1984; Grany andGiesy 1986; McKee and Knowles 1986).

The purpose of this study was to investigate the changes in biochemi-cal constituents and energy levels of Vibrio parahaemolyticus infectedflower shrimp post-larvae.

MATERIALS AND METHODS

The post-larvae of flower shrimp were collected from Gosthani estu-ary located approximately 25 km north of Visakhapatnam, on the EastCoast of India. Samples were collected by using long-handled dipnet(pore size 500 µ and 30 cm depth). Collection effort was one man perhour or until the catch approximated to 200 individuals (Knowlton et al.1994). Collected samples were transported immediately to the labora-tory in aerated polythene bags taking care that over-crowding and bruis-ing were avoided during transportation. Species were identified andseparated from each other following the guidelines given by Kurian and

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Sebastian (1993). The post-larvae were maintained in the laboratory atambient conditions of 29±1°C temperature and 20 ppt salinity.

During the study period, V. parahaemolyticus-infected flower shrimpwere collected and pathogenic bacteria were identified by followingmethods of Mackie and McCartney (1989). The pathogenic bacteriawere isolated by using the spread-plate method and the nutrient usedwas selective agar media as hepatopancreas often contained the mixedflora (Cowan and Stell 1993). The isolated bacteria were inoculated intohealthy post-larvae at the LC50 concentrations of 53 � 104 cells/mL(Aravindan and Kalavati 1997) by adding culture into the experimentaltanks and the infection was confirmed by histopathological observa-tions. The experiment was maintained for a period of 96-hours and sam-pling was done at the intervals of 24-hours, 48-hours, 72-hours, and96-hours. Parallel controls (without bacterial inoculation) were main-tained throughout the experiment. The test solutions were reneweddaily. The post-larvae were fed with sterilized, commercial-grade larvaldiet twice a day at 1000 and 1600 hours based on 20% of body weight(Chinni et al. 2000). Feeding was stopped six hours before taking sam-ples for biochemical estimation.

The soft tissues were isolated and dried at 80°C for 48-hours andmade into a fine homogenous powder. This powder was used for theanalysis of biochemical constituents. Total carbohydrates were esti-mated by using Anthrone reagent (Carrol et al. 1956) and glucose wasused to prepare the standard curve. Total proteins were determined byadopting the method of Lowry et al. (1951). Bouvine serum albumin(BSA) crystals were used to prepare the protein standards. Phospho-sulpho-vanillin reagent was used to estimate total lipids (Barnes andBlackstock 1973) and cholesterol was used to make the standard curve.

The caloric concentrations (kcal/g) determined by assuming caloricvalues 3.8 kcal/g for carbohydrates, 4.1 kcal/g for proteins and 9.5 kcal/gfor lipids (Graney and Giesy 1986; Voet and Voet 1996).

All these investigations were repeated five times and the assays weredone in triplicate. The mean values together with standard deviationswere calculated by using standard statistical method (Sokal and Rohlf1995) and a value of P < 0.05 was accepted as statistically significant.The final concentrations were expressed as mg/g dry weight of post-larvae.

RESULTS

Table 1 represents the changes in total carbohydrate, total protein,and total lipid, respectively, in infected flower shrimp post-larvae. The

Chinni and Yallapragada 49

total carbohydrate is found to be significantly (P < 0.05) lower whencompared with their respective controls at all intervals except 24-hours(Table 1). A maximum decrease of 64.2% was noticed after 96-hour ex-posure (Table 1). Protein content increased with increasing exposuretime and the increase was significant (P < 0.05) at all intervals (Table 1).Total lipid content decreased (P < 0.05) in infected post-larvae (Table 1).Total lipid was reduced from 447 to 280 µg/g dry weights from 24-hoursto 96-hours. The highest percent decrease of 48.2% observed after96-hours exposure time and lowest 3.1% at 24-hours. The decrease wassignificant (P < 0.05) at all intervals except 24-hours (Table 1).

Table 2 represents the changes in caloric concentration in V. para-haemolyticus-infected flower shrimp post-larvae. Caloric concentra-tions significantly (P < 0.05) decreased at intervals of 72-hours and96-hours in infected post-larvae (Table 2). The maximum decrease(26%) was noticed at 96-hours exposure.

During 96-hours of the experimental period carbohydrate/proteinand carbohydrate/lipid ratios showed a decreasing trend (P < 0.05) in V.parahaemolyticus-infected flower shrimp (Table 3). A maximum de-crease of 78% and 41% noticed, respectively, for carbohydrate/proteinand carbohydrate/lipid ratios after 96-hours exposure to V. parahaemo-lyticus.

DISCUSSION

The results indicate that there was an inhibition of energy levels asevidenced through caloric content of infected flower shrimp post-lar-vae. The results are in agreement with Dickson et al. (1982) and

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TABLE 1. Changes in biochemical constituents of V. parahaemolyticus-in-fected P. semisulcatus post-larvae. Values are means (±SD) of five replicates.Means in the same row with the same letter are not significantly (P > 0.05) dif-ferent.

Exposuretime (hours)

Total carbohydrates(µg/mg dry weight)

Total proteins(µg/mg dry weight)

Total lipids(µg/mg dry weight)

Control Infected Control Infected Control Infected

24 220.6±5.77a 192.5±6.82a 403.0±4.78b 490.6±4.90a 462.3±10.1a 447.6±9.84a

48 243.8±2.55a 146.2±3.23b 410.2±0.78b 520.8±6.41a 496.5±1.52a 417.8±6.67b

72 248.2±5.50a 121.2±1.45b 440.6±6.82b 630.5±3.90a 502.4±7.20a 386.5±5.59b

96 269.3±3.96a 96.50±5.50b 450.8±1.92b 720.6±5.65a 540.6±7.20a 280.3±4.48b

Radhakrishniah and Busappa (1986). According to Graney and Giesy(1986), the caloric concentrations showed a similar trend of decreaseunder stressful conditions in several crustaceans. Graney and Giesy(1986) reported that a reduction in caloric concentration by modifica-tions in its metabolism might occur under stressful conditions. Vibrioinfection results in carbohydrate depletion due to generalized distur-bance of carbohydrate metabolism (Jirvanichpaisal et al. 1994). Mobili-zation of carbohydrates from tissues as response to stressors in aquaticanimals has been observed by Giesy et al. (1998).

The lipid reserves showed a significant decrease after 48-hours expo-sure to V. parahaemolyticus. Changes in lipid content seem to be a com-mon stress-induced response in crustaceans (Torreblanca et al. 1992).Jirvanichpaisal et al. (1994) observed decreased lipid levels in tigershrimp, Penaeus monodon, infected with Vibrio harvei. Lipid deposi-tion in eggs represents a major energy source for the developing em-bryo, and hence, any interference with lipid metabolism in infected

Chinni and Yallapragada 51

TABLE 2. Changes in caloric concentration of V. parahaemolyticus-infectedP. semisulcatus post-larvae. Values are means (±SD) of five replicates. Meansin the same row with the same letter are not significantly (P > 0.05) different.

Exposure time (hours) Caloric concentration

Control Infected

24 7,011±120a 6,821±0.02a

48 7,516±0.02a 6,527±0.02a

72 7,700±120a 6,200±100b

96 8,012±300a 5,296±150b

TABLE 3. Changes in carbohydrate/protein and carbohydrate/lipid ratios ofV. parahaemolyticus-infected P. semisulcatus post-larvae. Values are means(±SD) of five replicates. Means in the same row with the same letter are not sig-nificantly (P > 0.05) different.

Exposure time (hours) Carbohydrate/protein ratio Carbohydrate/lipid ratio

Control Infected Control Infected

24 0.53±0.03a 0.45±0.02a 0.48±0.02b 0.43±0.01b

48 0.57±0.02a 0.31±0.02b 0.35±0.01b 0.33±0.03a

72 0.62±0.04a 0.23±0.01b 0.50±0.02b 0.33±0.03a

96 0.64±0.03a 0.14±0.01b 0.51±0.04b 0.30±0.02a

post-larvae or adult will directly influence the survival of next genera-tion (Giesy et al. 1988).

The observed increase in protein concentrations could be due to acti-vated protein synthesis during V. parahaemolyticus infection in post-larvae. Furthermore, the corresponding decrease in carbohydrates andlipids might be due to the diversion of these constituents to protein syn-thesis as a compensation mechanism under stress conditions in infectedpost-larvae of flower shrimp. In contrast to the present study, Claybrook(1993) reported that the structural proteins are used as energy sourcesunder stressful conditions in crustaceans.

In general, crustacean hemocytes contain high levels of carbohy-drates (Johnson and Davis 1972). According to the results carbohy-drate/protein and carbohydrate/lipid ratios appear to be decreased ininfected post-larvae suggesting a combined effect of structural damageand the infiltration of hemocytes in the epithelial tissue.

In the present investigation, the reduction of caloric concentration in-dicated utilization of more energy from carbohydrates and lipids understress conditions caused by V. parahaemolyticus. The abnormal re-sponses in carbohydrate, protein, and lipid metabolisms might be due totriggering of the compensatory mechanism to overcome the stresscaused by V. parahaemolyticus infection in flower shrimp post-larvae.Hence, measuring caloric concentrations can serve as a useful tool inevaluating physiological costs of combating stress conditions.

ACKNOWLEDGMENTS

The authors thank Kalavati C. and Aavindan N. for their help in iden-tification and isolation of pathogen and C. Vijayalakshmi for providinglaboratory facilities. The financial assistance of the Indian Council of Ag-ricultural Research, New Delhi is greatly appreciated.

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