International Journal of Research in Biological Sciences

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1 Available online at International Journal of Research in Biological Sciences Universal Research Publications. All rights reserved ISSN Original Article Acid phosphatase as enzyme marker for health monitoring in Fenneropenaeus indicus under environmental perturbations and WSSV infection 1 Selven Subramanian 2 and * 1 Rosamma Philip 1 Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology, Kochi, India 2 Department of Zoology, Mar Athanasius College, Kothamangalam, Kerala. *Corresponding author: rosammap@gmail.com Tel: , Fax no: Received 25 June 2013; accepted 14 June 2013 Abstract The present study was conducted to identify suitable health indicators based on immune mechanisms of Fenneropenaeus indicus subjected to acute changes in salinity followed by challenge with white spot syndrome virus. The shrimps were maintained at 25 salinity for a period of seven days and the immune parameters like total haemolymph protein (THP), plasma protein (PP), serum protein (SP), total haemocyte count (THC), total free amino acids (TFAA), phenoloxidase (PO), intracellular anion production (NBT), alkaline phosphatase (ALP) and acid phosphatase (ACP) were analyzed. The salinity of the rearing water was then adjusted to two different salinities i.e., 25 to 5 and 25 to 35 followed by WSSV challenge. The analysis of haematological parameters showed that there are significant (p<0.05) differences between different groups of F. indicus. Correlation coefficients showed that all variables except total free amino acids exhibited a positive correlation with the survival rate. When multiple regression of survival rate on all immune parameters were considered, the amount of variability explained was 98% (R 2 =0.978). When significant regression coefficients among the immune parameters were taken into account, THC (p<0.001) and ACP (p<0.05) together are explaining 97% (R Square=0.972) of variability, indicating that these two are mainly responsible for the survival rate. Understanding of the immune defense profile in response to the environmental alterations may help to control the environmentally induced shrimp diseases by adopting proper prophylactic measures. The study was concluded that acid phosphatase and total haemocyte count are better indicators of F. indicus health under acute salinity stress post WSSV challenge. Of these acid phosphatase could be selected as enzyme marker for shrimp health monitoring as early warning systems of health hazards Universal Research Publications. All rights reserved Key words: Salinity, Immune response, WSSV, Fenneropenaeus indicus 1. Introduction White spot syndrome virus (WSSV) is one of the most dangerous pathogens of penaeid shrimp which causes massive production loss up to % in all shrimpgrowing countries ever since its first discovery in Asia in the early 1990s[1]. Application of traditional antibiotics used to prevent virus disease in vertebrate is not effective to cure virus disease of shrimp since no adaptive immunity exists in them. Shrimp diseases are always accompanied by changes in sea water quality [2 and 3]. Salinity is an important water quality parameter in shrimp culture systems and has been reported to influence the immune parameters of shrimp [4, 5,6,7,8 and9] To date no scientific evidence has been available on the suitability of haematological parameters as health indicators of shrimp species even though a number of studies have demonstrated the responses of immune parameters upon environmental alteration and microbial infection. Against this back ground, the present study was aimed at identifying suitable health markers in the immune parameters of F. indicus under acute salinity stress and WSSV challenge. 2 Materials and methods 2.1 Experimental animals and rearing conditions Adult Fenneropenaeus indicus of average wet weight ± 1.68g (Mean ± S.D.)were brought to the Laboratory within one hour of capture from a commercial shrimp farm located at Panangad, Kochi, India. Shrimps were reared in rectangular concrete tanks containing 25 clean sea water and allowed to acclimate for a period of seven days. Continuous aeration was provided using air pumps and the animals were maintained on a commercial shrimp diet 116

2 (Higashimaru, Pvt.Ltd. Kochi). Water quality parameters viz. temperature, dissolved oxygen, NH 3, NO 2 and NO 3 were monitored daily following standard procedures [10] and maintained at optimal levels as per Table.1. Unused feed and faecal matter was siphoned out daily and 30 % water exchanged every alternate day. A biological filter was set up to maintain the appropriate levels of water quality parameters. After acclimating to 25 for seven days six (n=6) shrimps were sampled for baseline data. 2.2 Experimental setup and salinity adjustments. Shrimps of apparently uniform size were distributed in the experimental tanks containing 500L of seawater (n=35/tank). Shrimps in the intermoult stage only were used [11]. There were four treatment Groups (G-I, G-II, G- III and G-IV) and the experiment was conducted in triplicate i.e., 3 tanks per treatment. Salinity of all the tanks was adjusted to 25 prior to the experiment. After 12 hours of starvation, the salinity of G-I shrimps was lowered from 25 to 5 by diluting with fresh water. Whereas, the salinity of G-II was raised from 25 to 35 by adding sea water. The desired salinity was adjusted over a period of six hours. Shrimps of G-III and G-IV was maintained at 25 itself without any salinity change. All four groups were maintained on commercial diet. Ten minutes after the desired salinity level was reached six shrimps from each group (n=6) were sampled (post salinity day 0, PSD0). Water quality parameters were maintained at optimal levels as shown in Table WSSV challenge The shrimps of G-II, G-III and G-IV were then challenged with White Spot Syndrome Virus. Challenge was performed through oral administration i.e., by feeding white spot virus infected frozen tissue at the rate of 1g/shrimp. Group- I was maintained as the unchallenged control. Shrimps were sampled (n=6) after 24 h (post challenge day1, PCD 1), 48 h (post challenge day 2, PCD 2), 72 h (post challenge day 3, PCD 3) and 120 h of challenge (post challenge day 5, PCD 5). Before each sampling, the shrimps were starved for 12 hours to eliminate physiological variations caused by the ingested food [12]. Survival in each group was recorded daily for a period of 10 days with dead animals removed promptly. Mortality by WSSV infection was identified by checking the characteristic white spots on the carapace of infected shrimps and further confirmed by PCR detection [13]. 2.4 Extraction of haemolymph and estimation of immune parameters Haemolymph was withdrawn aseptically from rostral sinus using specially designed sterile capillary tubes of diameter 0.5mm, rinsed thoroughly with pre-cooled anticoagulant (0.02M sucrose, 0.01M tri-sodium citrate in 0.01M Tris- HCl, ph 7.6) according to Song and Hsieh [14]. The samples were transferred to sterile micro centrifuge tubes containing pre-cooled anticoagulant. Serum was obtained by keeping the haemolymph at room temperature without anticoagulant. This is allowed to clot and then centrifuged at 1700x g for 10min at 4ºC in a refrigerated centrifuge to get the serum. A fraction of haemolymph (0.1ml) was immediately centrifuged at 600x g for 15minutes to separate plasma and haemocytes. The haematological variables viz., total protein, plasma protein, serum protein as per Bradford [15] total free amino acids as per Yemm and Cocking [16] phenol oxidase as per Soderhall [17] NBT reduction as per Song and Hsieh 1994), [14] alkaline and acid phosphatases as per Gonzalez et al. [18] were estimated. An aliquot of haemolymph was placed in an Improved Neubauer Chamber and the haemocyte count (THC) was done using Light microscope. THC was expressed as number of cells per ml of haemolymph. 2.5 Statistical analysis A multiple comparison (Tukey) test was conducted to compare the significant differences among treatment groups using the software SPSS package. All data are presented as mean ± S.D. and the differences were regarded as statistically significant when p < Results 3.1 Effect of salinity changes on the haematological parameters of F. indicus The acute salinity stress has reduced the total haemolymph protein, plasma protein and serum protein concentration in shrimps held at 5. After virus challenge, THP, PLP and SRP of shrimps held at different salinity levels showed a considerable increase (p<0.05). Total free amino acid concentration in the haemolymph of shrimps has increased significantly (p < 0.05) after WSSV challenge except on post challenge day 2 (Table 2). Total haemocyte number showed significant decrease in shrimps held at 5 and 35 compared to 25 on PCD1 and then showed a gradual reduction (Fig.1). Fig. 1 Total haemocyte count of F. indicus subjected to acute salinity stress followed by WSSV challenge. Fig.2 Phenol oxidase activity of F. indicus subjected to acute salinity stress followed by WSSV challenge. 117

3 Table 1 Rearing conditions of Fenneropenaeus indicus Animal used Fenneropenaeus indicus Size of animal 15±1.68 g Stocking density (per tank) 35 Nos. Tank capacity 500L Feeding level 5-10% body weight Feeding frequency Twice daily Water temperature C ph NH ppm NO 3 below detectable NO ppm Dissolved Oxygen 6-7 mg/l Fig.3 Super oxide anion production in the haemolymph of F. indicus subjected to acute salinity stress followed by WSSV challenge. Table.2. Total haemolymph protein, plasma protein, serum protein and total free amino acid concentration of F. indicus subjected to acute salinity stress and then challenged with WSSV. Each value represents the mean ± SD. Values with different superscripts in the same rows vary significantly (p<0.05) among different salinity treatments. Control - unchallenged Parameters(mg/l) Duration Salinity( ) Control(25 ) THP Baseline ±5.36 PSD ±6.17 A 96.26±11.82 A ±14.74 A ±9.08 A PCD ±6.23 A ±11.93 A ±14.87 A ±11.88 A PCD ±5.69 A ±14.14 A ±9.05 A ±7.21 A PCD ±7.12 A ±22.19 A ±11.18 A ±15.49 A PCD ±6.23 A 87.71±13.87 A ±9.88 A 91.12±14.80 A PLP Baseline 92.26±3.14 PSD ± 5.76 B 81.28± 3.17 B 92.34± A 90.14±9.31 B PCD ±5.79 B ±10.45 B ±13.86 A 89.96±9.38 B PCD ±4.98 B 98.34±10.54 B 76.08±5.40 A ±6.15 B PCD ±5.39 B 80.88±10.54 B 62.82±6.67 A 93.09±13.23 B PCD ±5.68 B 65.36±10.34 B 59.96±5.90 A 77.82±12.64 B SRP Baseline 65.14±3.2 PSD ±4.24 B 57.28±7.18 A 64.82±11.54 A 60.25±7.39 AB PCD ±4.11 B 76.59±7.75 A 92.03±11.81 A 73.68±7.68 AB PCD ±5.22 B 67.56±7.24 A 58.43±4.15 A 69.21±4.24 AB PCD ±4.52 B 55.56±11.36 A 48.25±5.12 A 64.14±9.12 AB PCD ±4.29 B 44.91±7.10 A 46.05±4.53 A 53.62±8.71 AB TFAA Baseline 2.27±0.42 PSD0 2.19±0.44 A 3.18±0.43 B 2.24±0.18 B 2.71±0.81 B PCD1 2.26±0.34 A 3.06±0.18 B 2.65±0.63 B 2.99±0.82 B PCD2 2.32±0.42 A 1.62±0.48 B 1.77±0.27 B 1.53±0.33 B PCD3 2.41±0.36 A 3.51±0.83 B 3.92±0.51 B 3.75±0.69 B PCD5 2.34±0.34 A 3.90±0.96 B 3.25±0.71 B 2.63±0.61 B THP- total haemolymph protein, PLP- plasma protein, SRP- serum protein, TFAA- total free amino acid Fig.4 Alkaline phosphatase activity of F. indicus subjected to acute salinity stress followed by WSSV challenge. Fig.5 Acid phosphatase activity of F. indicus subjected to acute salinity stress followed by WSSV challenge. 118

4 Table 3 Correlation matrix between survival rate and immune parameters of F. indicus challenged with White spot syndrome virus under acute salinity stress Variables THP PLP SRP THC TFAA PO NBT ALP ACP SURVL THP PLP.851** SRP.848**.968** THC.600**.731**.861** TFAA PO.588**.727**.807**.852** NBT.540**.631**.581**.489** * ALP.663**.755**.728**.657** **.680** ACP.714**.755**.796**.766** **.748**.818** SURVL.635**.737**.871**.984** **.499**.636**.799** * p<0.05, ** p<0.01 THP- Total haemolymph protein, PLP-plasma protein, SRP-serum protein, THC- total haemocyte count, TFAA- total free amino acid PO- phenoloxidase, NBT- Nitroblue tetrazolium reduction, ALP- alkaline phosphatase, ACP- acid phosphatase, SURVL- survival Table 4 Multiple regression of survival rate and immune parameters of F. indicus challenged with White spot syndrome virus under acute salinity stress R Predictors- ACP, TFAA, PO, THP, NBT, ALP, THC, PLP, SRP Dependent Variable: SURVIVAL Variables THP PLP SRP THC PO NBT TFAA ALP ACP Significance *** * R Predictors- THC, ACP Dependent Variable: SURVIVAL p< 0.05, ** p< 0.01 *** p< THP- Total haemolymph protein, PLP-plasma protein, SRP-serum protein, THC- total haemocyte count, TFAA- total free amino acid PO- phenoloxidase, NBT- nitroblue tetrazolium reduction, ALP- alkaline phosphatase, ACP- acid phosphatase A profound increase (p < 0.05) in phenoloxidase activity of shrimps held at 5 and 25 salinity was found on PCD 1 of WSSV challenge and a gradual reduction (p < 0.05) in the phenoloxidase activity was observed from PCD 2 onwards. Super oxide anion production was higher (p < 0.05) in shrimps held at 5 and 35 after sudden salinity change and after WSSV challenge it was high in shrimps held at 35. Alkaline phosphatase activity and Acid phosphatase showed a significant increase on PCD1 (p < 0.05) and then a decline Shrimps held at 5 salinity showed early mortality on PCD3 onwards compared to other groups. Throughout the experiment the survival rate was least in shrimps maintained at 5. Comparatively higher survival has been shown by shrimps held at 25. Animals in the control group (unchallenged) showed 100% survival (Fig.6). in the activity was noted. ACP activity was higher in shrimps held at 35 compared to those held at other salinity levels (Fig. 2-5). Correlation coefficients showed that all variables except total free amino acids exhibited a positive correlation with the survival rate (Table.3). When multiple regression of survival rate on all immune parameters were considered, the amount of variability explained was 98% (R 2 =0.978). When significant regression coefficients among the immune parameters were taken into account, it was found that THC (p<0.001) and ACP (p<0.05) together are explaining the 97% (R 2 =0.972) Fig.6 of variability, indicating that these two are mainly stress followed by WSSV challenge. responsible for the survival rate (Table 4). 3.2 Effect of salinity on WSSV infectivity and mortality in F. indicus Post challenge survival rates showed significant variation (p < 0.05) in all treatment groups. Maximum survival was observed for shrimps maintained at 25 followed by 35 and least in shrimps held at Survival of F. indicus subjected to acute salinity 4 Discussion The present study documented the variations in the immune mechanisms of Fenneropenaeus indicus on challenge with WSSV under acute salinity change from 25 to 5 and 25 to 35. There were measurable variations (p<0.05) in the haematological parameters of shrimps held at 5, 25 and 35 salinity and challenged with WSSV.

5 In the present study F. indicus was more susceptible to WSSV at 5 compared to those at 25 and 35 indicating the stress at hyposmotic condition leading to immune suppression. This increase in susceptibility was well correlated with the reduction in haematological parameters. F. indicus was found to be more susceptible to WSSV when the animals were exposed to 5 and 35 from 25 in 96 h. In addition, F. indicus was more susceptible at 5 compared to 35. Wang and Chen [19] observed that L. vannamei was more susceptible to V. alginolyticus when the animals were transferred to 5 and 15 from 25 in 24 h. Liu et al. [6] reported that acute salinity change from 22 to 14 resulted in rapid proliferation of WSSV in Fenneropenaeus chinensis, after 10 h of acute salinity change and the shrimps were more susceptible to WSSV following salinity changes. Since acute salinity changes may result in physiological and biochemical adaptive changes in shrimps having to expend corresponding amounts of energy in osmoregulation, they become more susceptible to pathogens. The protein components viz., THP, PLP and SRP in the haemolymph of F. indicus was found to decrease after salinity stress and maximum reduction was found in shrimps held at 5 salinity. However after WSSV challenge, there was a considerable increase in these parameters in the haemolymph. Increase in haemolymph proteins owing to the increase in viral titre has been previously reported in Manduca sexta [20] and in F. indicus [21 and 22]. The plasma protein plays a vital role in the immunity of crustaceans; it not only correlates with the infection of pathogen [23 and 24] but also with environmental stress [25]. In the present study, the total free amino acid levels of F. indicus were found to increase after salinity and WSSV stress indicating the metabolic adjustments to prevent the osmotic stress at suboptimal salinities. It appears that a low circulating haemocyte number in crustaceans is strongly correlated with a greater sensitivity to pathogens as well as a low THC can be an indication of higher susceptibility to the infectious disease. In our study there was significant difference in the total haemocyte count of shrimps examined during the experiment at same sampling time in different salinity levels. THC was highest in shrimps maintained at 25 and was lowest in shrimps at 5 compared to those at 35. The THC of P. monodon infected with WSSV also decreased significantly [26]. There are many factors which contribute to lower THC such as haemocyte lysis [27] cell recruitment towards infected tissues and nodule formation [28] or interference with haematopoiesis. Following salinity stress, a reduction in PO activity could be observed in the present study. It has been previously reported that prophenoloxidase activity increased directly with salinity for the yellow leg shrimp Farfantepenaeus californiensis reared in salinity levels of 28, 32, 36, 40 and 44 [29], white shrimp L. vannamei reared in salinity levels of 5, 15, 25 and 35 [30], and tiger shrimp P. monodon reared in salinity levels of 5, 15 and 25 [31]. Cheng et al. [32] have reported that phenol oxidase activity of M. rosenbergii was significantly higher for animals reared at 5 and 10 than those reared in fresh water and 15. The apparent reduction in phenol oxidase activity in shrimps held at hypoosmotic condition indicates that the prophenol oxidase cascade has been affected by osmotic stress and the animal s immune vigour is suppressed under hypoosmotic condition. Then, PO activity increased on PCD 1 in shrimps at all salinities after WSSV challenge when compared to control (25 ). Significant difference in the phenol oxidase activity could also be observed at same exposure time in different salinities, the lowest being at 35 followed by 5 and 25. Salinity stress has induced an increase in super oxide anion production in shrimps held at 5 and 35. Intracellular superoxide anion production in shrimps maintained at 5 was maximum at PCD 1 and gradually reduced towards the end of the experiment. Similar pattern was shown by animals at 25 and 35. Cheng et al. [33] reported that H.diversicolar supertexta when transferred to 20, 25 and 35 decreased the release of superoxide anion as compared to the abalone reared in 30. They could not distinguish whether the decrease in superoxide anion resulted from decreased NADPH oxidase activity or from increased activity of superoxide dismutase (SOD) responsible for scavenging superoxide anions. The present study shows that there was significant increase in the superoxide anion production in shrimps maintained at 35 compared to those held at 5 and 25 salinities. The current study reports a reduction in the activity of alkaline and acid phosphatase in shrimps subjected to acute salinity stress. Lovett et al. [34] reported the presence of an alkaline phosphatase at ph 9.1 in the posterior gills of C. sapidus and its alterations with respect to environmental salinity. The specific activity of this alkaline phosphatase was greater in C. sapidus acclimated to 35 than crabs acclimated to 10. The changes in the activity of alkaline phosphatase in response to salinity suggested the role of this enzyme in modulating the osmoregulatory response of C. sapidus. WSSV challenge, however, enhanced the activity of these enzymes. Many researchers had showed that phosphatases play an important role in the immune system as a key compound of lysosomal enzymes [19]. It has been known that phosphatases play an important role in acute energy crisis in aquatic organisms and involve in cytolysis and differentiation processes. In addition, phosphatase is the most important element of lysosomal enzymes, in crustacean cells; they perform the double function of digestion and defense [35]. Liu et al. [36] reported that ACP was a sign of lysosomal activity to digest the invading organisms in shrimps and ACP and ALP activity could reflect directly the nonspecific immunological state in them. Cheng and Rodrick [37] observed that alkaline phosphatase was an important component of lysosomal enzymes that originate from haemocytes to destroy extracellular invaders. In the present study there was significant difference in the alkaline phosphatase activity of shrimps held at different salinities at the same sampling time. ALP activity of shrimps held at 5 salinity was significantly higher compared to those at 25 and 35 whereas ACP was maximum at 35 and lowest at

6 In the present study correlation coefficients showed that all variables except total free amino acids exhibited a positive correlation with the survival rate. When multiple regression of survival rate on all immune parameters were considered, the amount of variability explained was 98% (R 2 =0.978). When significant regression coefficients among the immune parameters were taken into account, it was found that THC (p<0.001) and ACP (p<0.05) together are explaining the 97% (R 2 =0.972) of variability, indicating that these two are mainly responsible for the survival rate. 5 Conclusions The present study gives information on the immune mechanisms of F. indicus under salinity stress and WSSV infection. The shrimp defense profile in response to environmental alterations and pathogenic invasion helped to assign biomarkers as early warning systems of health hazards. 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