*[ Received 25 June 2015 ; revised 21 December 2015

Transcription

1 Indian Journal of Geo Marine Sciences Vol. 46 (07), July 2017, pp Haematological parameters as predictive indicators of stress induced mortality in Pacific White Shrimp Penaeus vannamei (Boone, 1931) broodstock during transboundary shipment M. C. Remany* 1&2, Daly Cyriac 1, P. Krishna Kanth Varada Raju 1, Sruthi Prem. O. C 1, K. Karthick Kannan 3, Jaideep Kumar 3 & Y.C. Thampi Sam Raj 3 1 Dept. of Biotechnology, Sathyabama University, Jeppiar Nagar, Chennai , India 2 Rajiv Gandhi Centre for Aquaculture,MPEDA, Chennai , India 3 Rajiv Gandhi Centre for Aquaculture,MPEDA, Sattanathapuram , India *[ rgca_sbnpc@yahoo.com] Received 25 June 2015 ; revised 21 December 2015 The influence of stress due to different transit durations during transboundary shipment of Penaeus vannamei broodstock from the supplier source located at Florida, USA to the port of entry Chennai, India was evaluated in terms of haematological parameters, such as total protein, glucose and haemocyte count. The parameters studied were correlated with the percentage of transit mortality, recorded under different transit durations and with the water quality parameters in the shipment bags. Refractive index (RI) of haemolymph samples of brooders subjected to transit durations were monitored with the objective to use RI as a suitable proxy for haemolymph protein. A linear relation was observed between the RI and total protein content (r 2 = ). Among the parameters determined, total haemocyte count (THC) was significantly reduced (P<0.05) in broodstock subjected to longer transit duration, which had increased mean total ammonia level and decreased mean dissolved oxygen concentration coupled with low ph levels. The levels of total protein and glucose significantly high with increase in transit durations. Data obtained subjected to Tukey posthoc test indicated all determined haematological parameters were statistically significant (P<0.05) between control and each test groups. [Key words: Penaeus vannamei, broodstock, haematological parameters, transit stress, water quality] Introduction Haemolymph of crustaceans is a highly valuable tissue for ecotoxicological evaluation, environmental quality and assessment of environmental stress due to its ease of sampling and sensitive response to many disorder states 1. The haematological parameters assessed in the study were total protein, glucose, total haemocyte count (THC) and refractive index. Total protein can serve as significant source of metabolic energy for crustaceans 2,3 and therefore has been used to provide a rapid, non-destructive means of assessing an animal s physiological condition 4,5. Glucose is also a major component of circulating blood carbohydrates in crustaceans 6. Regardless of what form of carbohydrate is transported in blood, the metabolic oxidative processes are likely to be mediated via glucose at the cellular level 7,8. Furthermore, mobilization of glucose is generally accepted as means of providing extra metabolic energy resources 9. The third parameter that was analysed in the haemolymph was THC which reflects the well-being of the animal 10,11,12 and any change in haemocyte count (decreases under disease and environmental stress) and morphology is a sign of physiological or environmental stress 13. Haemocyte numbers also change during moulting, color variation and glycemia 14 which are controlled by the endocrine system. Though monitoring THC as a measure of stress for vannamei was investigated by several authors 15,16,17,18, studies on the relation between haemolymph parameters and shipment induced mortality is not yet documented. Further, the density of the haemolymph drawn from brooders subjected to different transit durations was also measured in terms of refractive index (RI) so as to draw a correlation between RI and the equation for the line of best fit. The equation derived can be used routinely to convert RIs of vannamei haemolymph to total protein and the observation made would provide a baseline data for the

2 INDIAN J. MAR. SCI., VOL. 46, NO. 07, JULY importers to determine the various physiological states (stress, nutrition, immunity, moulting, etc.) of the animal and also to grade the condition of the animal during its transport. Broodstock is a valuable live commodity purchased from the supplier at the rate of around 62 $ per unit (exclusive of custom clearance and documentation charges). The loss of a single brooder would mean a loss in seed (larval) production to the hatchery operator. Therefore, information on the influence of shipment induced stress on haemolymph parameters provides clue for both the hatchery operators and suppliers on the condition of the animal during transit. This would help both the suppliers and the hatchery operators to source alternate modes of flight cargoes which could reach the shipment port within the possible shortest time, thus curbing the loss of the valuable stock to the importers. Further, a basic information on haemolymph parameters such as cell count, haemolymph refractive index, etc. which can be recorded with common and cheap devices such as a light microscope, salino refractometer, etc. would help the hatchery operator to grade the condition of live brooders on its shipment. The water quality parameters which play a key role as the holding medium for the broodstock during shipment also provides baseline information on the broodstock condition. Materials and Methods Imported vannamei broodstock of size range g brought to the Aquatic Quarantine Facility were categorized into three groups, viz. A, B and C based upon the transit duration from the broodstock source. Brooders that were shipped to the quarantine facility within 34, 48 and 58 h from the supplier (Source) were assigned as Group A, Group B and Group C, respectively. Transit duration varied based upon the flight timings and availability of specific cargo flights from the port of shipment to the port of entry. Broodstock consignments were air lifted to India in oxygen packed shipment bags containing seawater of salinity 34.5 ppt, and at 18 0 C temperature (as provided in the packing list that accompanies the shipment), with activated charcoal. Bags were then again packed securely in thermocol boxes at the rate of 2 bags per box and airlifted. About 5 brooders were placed in one shipment bag containing 12 l of seawater (Fig. 1a & 1b). Immediately on arrival to the facility, the bags were received in designated broodstock receiving areas and only the shipment bags containing the shrimps were taken into the receiving tanks. The bags were opened and water quality parameters such as temperature, salinity, ph, Dissolved Oxygen (DO) and total ammonia were analysed. YSI multiparameter probe was used to record all the parameters, except total ammonia. The latter was determined according to standard methodology recommended by UNESCO 19 for water quality analysis. Fig. 1a - Broodstock consignment on arrival Fig. 1b - P. vannamei broodstock in shipment bag For the study of haematological parameters, the broodstock were carefully taken out from the bags and about 150 µl of haemolymph was withdrawn aseptically from the ventral sinus of the first abdominal segment into a 1 ml syringe. Of this, ~100 µl of the haemolymph was used fresh for the analysis of refractive index and the remaining 50 µl was collected in an eppendorf tube coated with equal volume of fixative 20 and used for total haemocyte count, protein and glucose analysis. Collection of haemolymph was done within the first ten minutes of the arrival in the intermoult (C stage) animals only to avoid any moult related variation. Similarly haemolymph samples were collected from equal number of sexes to check for any sex wise variation in the

3 1442 REMANY et al.: MORTALITY IN PACIFIC WHITE SHRIMP PENAEUS VANNAMEI (BOONE, 1931) parameters under study. Baseline haemolymph samples collected from the broodstock maintained in well acclimated condition at the Quarantine Centre was used as the control (0 h) for the study. Samples under each test group were collected in triplicates, each set comprising of 6 nos. of brooders with equal number of sexes. Haemolymph parameters such as total haemocyte count (THC), total protein, glucose and Refractive Index (RI) were assayed and data collected was correlated with the mortality during shipment. Refractive index of the haemolymph of both the control and test groups was checked so as to draw a relationship with the total protein content in haemolymph which would provide a nondestructive method to assess the physiological condition of the brooder. This technique would serve as a tool for the hatchery operator to monitor the condition of the brooders on import from other countries and also on transport to their hatchery for seed production. Determination of total haemocyte count (THC): The haemocytes collected in the fixative were counted in triplicates under phase contrast microscope (Zeiss) using an improved Neubauer haemocytometer (Fig. 2) and were reported as cells.ml -1. Fig. 2 - A view of the Haemocytes under 40X Total protein and glucose: About 60 µl of the collected haemolymph was immediately centrifuged for 2 min at 5000 rpm and the plasma was separated from the precipitated cells and stored at C for further analysis. The total protein and glucose content in the haemolymph was determined using the standard procedures 21,22. Bovine serum albumin and pure dextrose were used as the standards for protein and glucose, respectively. Transit mortality: The mortality of broodstock on arrival to the quarantine facility in each test groups was recorded for 9 consignments comprising of nos. of imported brooders and was used as the key index that can be extrapolated from the measured immunological (THC) and metabolic (total protein and glucose) parameters. Transit mortality was expressed in percentage. Correlation and regression analyses were performed to determine the relationships between transit mortality, metabolic (protein, glucose) and immune variables (THC). Water quality parameters which significantly varied between each test groups were also correlated with the determined metabolic and immune variables in addition to the transit mortality. Refractive index (RI): RI values of haemolymph were obtained by the use of a salino refractometer (ATAGO) with automatic temperature compensation. Range of refractive index measured by the refractometer was 1.00 to 1.07 with a precision to the nearest 0.01 and a dilution1:1 v/v with distilled water was performed when haemolymph value overrode the upper limit. Correlation was then drawn between refractive index and total protein and the equation for the line of best fit was obtained. Statistical analysis: Statistical analysis of the data was performed by using Graphpad prism 5.0. Data collected on THC, protein and glucose was subjected to one way ANOVA. Significant levels for all analyses were set at P<0.05. Differences between means for each group (individual means) were determined by a post-hoc Tukey test when the interaction between both independent variables was significant. Data are reported as mean ± standard error (SE). Results The water quality parameters recorded on arrival of consignments in relation to different transit durations are tabulated in table 1. Among all the recorded parameters ph, DO and total ammonia levels were significantly different (P<0.05) in all the test groups. ph and DO significantly reduced (P<0.05) with increase in transit duration. Mean ph value of the shipment water dropped from 8.14±0.029 at 34 h to 6.98±0.043 at 58 h through a value of 7.53±0.020 at 48 h. Post hoc analysis of the data showed a significant variation (P<0.05) in ph among all the test groups Similarly mean DO level also declined from 14.05±0.176 mg.l -1 at 34 h through

4 P ro te in (m g.m l - 1 ) INDIAN J. MAR. SCI., VOL. 46, NO. 07, JULY ±0.195 mg.l -1 at 48 h to 7.35±0.170 mg.l -1 at 58 h. The mean values of DO in all the test groups significantly differed (P<0.05) among the test groups A, B and C. Table 1.Water quality parameters (mean±se) monitored in shipment bags containing the broodstock on arrival (n=18) Parameters Test Group Group A (34 h) Group B (48 h) Group C (58 h) Temp ( C) 17.61±0.101 a 17.90±0.120 a 18.30±0.100 a Salinity (ppt) 33.00±0.161 a 33.22±0.102 a 33.66±0.110 a ph 8.14± ± ±0.043 DO (mg.l -1 ) 14.05± ± ±0.170 Total NH 3 (mg.n-l -1 ) 0.023± ± ±0.007 *Values without superscript are statistically different (P<0.05) between each test groups Table 2. Haematological parameters (mean±se) determined in control and stressed P. vannamei broodstock (n=18) Parameters Control Test Group 0 h Group A (34 h) Group B (48 h) Group C (58 h) Protein (mg.ml -1 ) ± ± ± ±0.850 Glucose (mg.ml -1 ) 0.220± ± ± ±0.007 THC (Cells.ml -1 ) ± ± ± ±0.170 RI 1.016± ± ± ±0.001 *Significant difference (P<0.05) between control and each test groups Total ammonia level in the shipment water significantly increased (P<0.05) with increase in transit duration. The mean value of total ammonia was recorded as 0.023±0.009 mg.n-l -1 for 34 h (Group A), 0.177±0.011 mg.n-l -1 for 48 h (Group B) and 0.288±0.007mg.N-l -1 for 58 h (Group C), respectively. Transit delay on arrival of broodstock from the source to the shipment port had a direct impact on all the measured parameters when compared with the control (Table 2). Total protein and glucose levels in the test groups ranged from to mg.ml -1 and 0.39 to 0.58 mg.ml -1, respectively. The levels of these metabolites were significantly higher (P<0.05) than the baseline concentrations which were only 57.58±1.8 and 0.22±0.03 mg ml -1, respectively, for protein and glucose. A pronounced increase in both the metabolic variables was recorded in brooders which were under transit stress for 58 h (Fig. 3). Significant difference (P<0.05) was also observed in protein and glucose between each test groups and the control. However, no significant variation in the two determined metabolites was observed between the sexes in any of the test groups or control. Total haemocyte count in the haemolymph significantly reduced as the stress duration increased. THC of the broodstock which were subjected to 34 h shipment reduced to ± 0.14 x 10 6 cells ml -1 from ± 0.87 x 10 6 cells ml -1 in the control, whereas those shipped for 48 and 58 h showed further reduction in cell counts to ± 0.18 x 10 6 and 9.75 ± 0.17 x 10 6 cells ml -1, respectively. Variation of the cell counts were significantly high among each test groups and as well as with the control (P<0.05). Although not significant, a slight decrease in THC was observed in female broodstock, under each test groups T r a n s it D u r a tio n (h ) G lu c o s e (m g.m l - 1 ) G lu c o s e Fig. 3-Protein and glucose levels (mean±se) in P. vannamei broodstock subjected to control (0 h) and test transit durations P r o te in Refractive index and total protein content in the haemolymph were subjected to linear regression analysis and the equation obtained was y=103.4x The correlation obtained, when all the data were plotted together, was r 2 = No variation in refractive index was observed between the two sexes.

5 T H C (x ) c e lls /m l 1444 REMANY et al.: MORTALITY IN PACIFIC WHITE SHRIMP PENAEUS VANNAMEI (BOONE, 1931) Table.3. Correlation matrix (Pearson) on transit mortality, haematological and water quality parameters of P. vannamei subjected to with or without transit stress Parameters THC TP TG RI TM ph DO NH 3 THC 1 TP TG RI TM ph DO NH *Values given are statistically different (P<0.05) THC-Total Haemocyte Count, TP-Total Protein, TG-Total Glucose, RI-Refractive Index, TM-Transit Mortality Pearson correlation coefficients showed that transit mortality and all the other determined variables except THC exhibited positive correlation with the total ammonia level. THC showed a positive correlation with DO level in the shipment water whereas a negative correlation was observed with increase in total ammonia. Similarly drop in ph with increased transit duration showed positive correlation with THC. All the determined metabolic variables exhibited a greater degree of correlation with each other (Table 3) and also to the shipment water quality parameters mainly ph, total ammonia and DO. When multiple regression of transit mortality on THC, total protein, glucose and refractive index was considered, the amount of variability explained was 85.90% (R Square=0.859). The mean percentage of transit mortality observed in the 9 consignments under each test group was found to increase with the increase in stress due to increase in transit duration (Fig. 4) Transit D uration (h ) T ra n s it M o rta lity (% ) T H C (x ) c e lls /m l Transit M ortality (% ) Fig. 4. Relation between THC and Transit mortality (mean±se) in P. vannamei broodstock exposed to different transit durations and control (0 h) Statistically significant variation was observed between the control and the test group of 58 h. Similarly variation in transit mortality was significant among the test groups of 34 and 58 h. However, the difference in transit mortality was not significant between control Vs 34 h and 48 Vs 58 h (P>0.05). Discussion The shipment induced stress during the transboundary shipment of vannamei in different transit durations from USA to India had a profound influence on the protein, glucose and THC levels in haemolymph. These changes were reflected in the transit mortality of the stocks that were imported. Transit mortality which was used as the reference index in the present study was found to increase with the determined metabolic parameters and increased shipment durations. The two fold increase in glucose content observed in the present study is indicative of the increased glucose supply to the tissues, when the animal is in stressed condition on shipment of 58 h. This observation is comparable to other work analyzing stress influence 23. Hall and van Ham 24 also had reported a significant increase in glucose level in P. monodon in stressed condition. Furthermore, glucose mobilization is generally accepted as means of providing extra metabolic energy resources 9 to combat stress. In the present investigation, glucose level probably elevated with increase shipment duration to ward of stress. Such observation had also been documented in studies related to salinity stress 25. Total haemolymph protein measured under different transit durations increased 1.8 times that of the control group, under 58 h of shipment stress. This pattern of elevation of protein in haemolymph has been reported in stressed Homarus americanus 26. However, Racotta et al 23 has reported slight declining trend in protein level in stressed juvenile vannamei. Increased protein level under acute salinity stress condition was

6 INDIAN J. MAR. SCI., VOL. 46, NO. 07, JULY reported in adult P. monodon by Joseph and Philip 25. The observation of the present study clearly reveals the metabolic adjustments in the vannamei brooder, to combat shipment induced stress where the animal is confined to very less space for swimming activity. This corroborates with the findings of Zhang et al 27 in vannamei on protein mobilization from haemolymph and its subsequent reduction in response to swimming fatigue. Immediate variation in proteins could be a consequence of cellular release 28 and it has been suggested as a passive mechanism to maintain osmolality in crustaceans. Bower 29 has also documented the role of haemolymphatic protein as environmental stress indicator. Refractive index (RI) of the haemolymph collected from control and each of the test groups showed a direct correlation with the total protein content measured calorimetrically. RI significantly increased (P<0.05) with shipment stress, and protein and glucose content of the haemolymph. The findings of this study implies the utility of refractive index of haemolymph to evaluate protein content from the conversion factor obtained from the study without sacrification of the brooders and thus predict the degree of stress and mortality. As the shrimp immune system response is largely based on proteins, RI values serve as possible indicators of the overall physiological condition of the animal and hence can be used for grading the condition of live brooders from a small quantity (~100 µl) of haemolymph. Statistical analysis showed that there was a significant (P<0.05) reduction in THC in the transit stressed brooders when compared to the control. Low circulating level of haemocytes was negatively correlated to the transit mortality, total protein, glucose and RI. significant differences recorded in THC level between the control and test groups indicate that THC is very suitable to evaluate stress conditions resulting during transit. A significant reduction in THC was observed in brooders shipped under longer transit duration, which was also recorded with high percentage of transit mortality. Song et al 30 reported reduced level of THC in TSV infected vannamei. Reduction in THC was also interpreted as a major factor that decreased the immuno competence in crustaceans 31. A decrease in total haemocyte count (THC) was also reported in crustaceans exposed to stress conditions 32. Analysis of the water quality in the shipment bags indicated that the key parameters which had a significant influence on the haematological variables and transit mortality were total ammonia, dissolved oxygen and ph. Similar observations were reported by Sharma et al 33 in Indian white shrimp subjected to low ph where low THC values were reported. In the present study, increased level of total ammonia coupled with drop in ph and DO level contributed to stress which was reflected in the values of measured haematological parameters and transit mortality. Longer transit durations may have deleterious effect on water quality in the shipment bags which would in turn reduce the immunological activity of the shrimps making them prone to more stressed condition. These results suggest that the haemolymph parameters such as THC, total protein and glucose can potentially be used as reliable biomarkers of stress in brooders and thus helps to predict mortality in the stock. Conclusion The findings of the study documented in this paper is of direct relevance to the shrimp industry with the current trend of increased momentum of vannamei culture in the country. Refractive index used as a proxy in the present study for estimating total protein content can be very easily estimated in haemolymph using a simple refractometer, which is readily available with hatcheries and farms. This helps the hatchery operators to predict the broodstock condition on arrival to the hatcheries and take appropriate decision when to use them for induced spawning or seed production. Acknowledgement Authors are grateful to Ms. Leena Nair, IAS, Chairman, MPEDA and President, RGCA, for her continuous support and encouragement for the Project. References 1. Lewbart G A, Noga E J, Hancock A L and Bullis R A, Crustaceans, in: Invertebrate Medicine, edited by G.A. Lewbart, (Blackwell Publishing, IW) 2006, pp Claybrook D L, Nitrogen metabolism, in: The biology of Crustacea, edited by L. H. Mantel, (Academic Press, New York) 1983, pp Mugnier, C and Justou, C., Combined effect of external ammonia and moult stage on the blue shrimp Litopenaeus stylirostris physiological response, J. Exp. Mar. Biol. Ecol., 309 (2004)

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