Effect of environmental parameters on immune response of the Indian spiny lobster, Panulirus homarus (Linnaeus, 1758)

Fish & Shellfish Immunology 23 (2007) 928e936 www.elsevier.com/locate/fsi

Effect of environmental parameters on immune response of the Indian spiny lobster, Panulirus homarus (Linnaeus, 1758) Bindhu Verghese a,*,1, E.V. Radhakrishnan a, Abinash Padhi b b

a Central Marine Fisheries Research Institute, P.B. No. 1603, Cochin 682018, Kerala, India Department of Biological Science, 600 S. College Avenue, University of Tulsa, Tulsa, OK 74104, USA

Received 17 October 2006; revised 16 January 2007; accepted 26 January 2007 Available online 9 February 2007

Abstract The high export value of the Indian spiny lobster Panulirus homarus increasingly attracts the aquaculturists for farming and fattening. However, lack of knowledge on the effect of environmental parameters on the immune system of this animal could result in high mortality, which ultimately may cause major loss to the industry. Here, we report the effect of salinity (20, 25, 35, 40, and 45&), pH (5.0, 8.0, and 9.5), dissolved oxygen (DO) (1 and 5 mg L1), and ammonia-N concentration (0, 0.5, 1.5 and 3 mg L1) on the immune response of P. homarus measured in the haemolymph in terms of Total Haemocyte Count (THC), phenoloxidase (PO) activity, and NBT-reduction. Our data showed significant reduction (P < 0.05) in THC, and NBT-reduction at lower (20&) and higher (45&) salinities. However, PO activity showed significant disparity, showing an increasing trend from 20 to 45&. Significant reduction (P < 0.05) in THC and PO activity under acidic and alkaline conditions, under hypoxic condition (1 mg L1), and at the higher ammonia-N concentrations than their respective optimal conditions were observed. Thus, suggesting that extreme environmental parameters can induce modifications in the immune system of the spiny lobster P. homarus, which may enhance their susceptibility to opportunistic pathogens. The humoral parameters such as THC, PO activity, and NBT-reduction can be used as potential stress indicators for healthy management of spiny lobsters. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Panulirus homarus; Total haemocyte count; Phenoloxidase; Superoxide anion production; Environmental parameters

1. Introduction The Indian spiny lobster Panulirus homarus is a truly marine species inhabiting the inshore sea along the southern coast of India. Live lobsters of this species are exported to Southeast Asian countries. The high demand for live lobsters in the export market and the decline of wild catches has attracted aquaculturists to venture into lobster farming. Due to lack of hatchery technology, the success of lobster farming largely depends on the fattening of wild caught * Correspondence to: Department of Biological Science, 600 S. College Avenue, University of Tulsa, Tulsa, OK 74104, USA. Tel.: þ1 918 631 2765; fax: þ1 918 631 2762. E-mail address: [email protected] (B. Verghese). 1 Present address: Department of Biological Science, 600. S. College Ave. University of Tulsa, OK-74104, USA 1050-4648/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.01.021

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lobsters [1]. The wild caught commercial grade lobsters meant for export are maintained carefully in secondary holding centres whereas the undersized lobsters are stocked at high densities in fattening ponds. Although farming trials in indoor systems have shown culture potential for this species [1], the high stocking density coupled with poor water quality management can lead to devastating consequences on the successful maintenance of these lobsters. Knowledge of the immune response of lobsters with regard to water quality and stress could provide insight into developing proper management strategies. Environmental parameters such as temperature, salinity, oxygen, ammonia content, and pH have shown significant effects on the immune system of crustaceans [2e7]. Salinity is one of the major factors that may significantly affect the physiology of aquatic organism. For example, a reduction in salinity resulted in increased haemolymph total protein and haemocyanin plasma level in Homarus americanus [8]. Similarly, lower salinity caused significant increase in oxygen consumption in Penaeus japonicus [9]. Wide fluctuation in pH, dissolved oxygen, and ammonia also affect culture conditions [6]. Elevated amounts of environmental ammonia have been reported to affect growth and moulting of crustaceans [10]. Though lethal and sublethal effects of ammonia on metabolism and osmoregulation of shrimps have been well studied [11,12], recently the focus has been towards understanding the effect of ammonia on their immune system [3,13,14]. A decrease in dissolved oxygen (DO) concentration can also hinder the metabolic performance, growth, and moulting [15]. Decrease in DO concentration can also have a negative effect on crustacean immune system [5,6,16] and may decrease resistance to diseases. Although influence of environmental parameters on the immune system of fishes, molluscs, and shrimps have been reported (e.g. refs. [2,5,17,18]), the influence of such factors on the immune response of spiny lobsters are poorly understood. The impact of these environmental parameters can be directly assessed from the immune response of the organism. In decapods, cellular defenses rely on haemocytes with several functions such as coagulation, phagocytosis, encapsulation [5], and the production of melanin by the phenoloxidase (PO) activating system [9]. Previous studies reported that the PO activities are sensitive to salinity fluctuation; for example, in Litopenaeus schmitti, the PO activity is significantly reduced at lower salinity [4], and in case of Penaeus californiensis its activity increases with increase in salinity [7]. Similarly, Total Haemocyte Count (THC) and NBT-reduction in crustaceans are all affected by fluctuation in salinity, temperature, pH, ammonia content, and dissolved oxygen [6]. The impact of environmental parameters on the immune response of lobsters can be directly assessed from haemolymph by measuring PO activity, THC, and NBT-reduction. Here, we report the influence of salinity, pH, dissolved oxygen, and ammonia-N on total circulating haemocytes, PO activity and NBT-reduction in the spiny lobster P. homarus.

2. Materials and methods 2.1. Animal and experimental setup The lobsters were collected from Vizhinjam (8.41 N 77.0 E) located on the southwest coast of India and were transported to the Research Centre of Central Marine Fisheries Research Institute (CMFRI) at Calicut, 300 km north of Vizhinjam, in thermocol boxes containing cool saw dust and were acclimatised for two weeks at a temperature of 25  0.3  C, salinity 35&, pH 7.78  0.2, and dissolved oxygen 4.97  1.43 ml L1. The lobsters were maintained in individual fibreglass reinforced plastic tanks containing 100 L filtered seawater. All experimental lobsters were in the intermoult stage and weighed 104  43.88 g. The animals were fed with green mussel (Perna viridis) meat once daily at 10% of the bodyweight. Lobsters were gradually acclimatised to a range of salinities of 20, 25, 30, 40 and 45& either by reducing the salinity with freshwater or by adding saline water to increase the salinity both at a rate of 2% per hour and maintained for a week in respective salinities. The control animals were maintained at 35&. Lobsters for pH studies were acclimatised to a salinity of 35& and maintained for a week at pH 5.0, 8.0, and 9.5, where pH 8.0 was used as control. Seawater with lower and higher pH was made by adjusting the pH with 1 N HCl or 1 N NaOH, respectively. The animals for ammonia stress studies were maintained for a week in different concentrations of ammonia-N, 0.5 mg L1, 1.5 mg L1, and 3 mg L1 and a control at 0  0.05 mg L1. Different concentrations of ammonia-N were prepared by dissolving 38.2 g of NH4Cl in 1 L distilled water to make 10,000 mg L1 as stock solution. Water was replaced daily to maintain the concentration. For oxygen stress experiments, only two conditions were noted the hypoxic (1 mg L1) and the optimal level (5 mg L1) of oxygen. The oxygen levels were maintained by controlled aeration and continuous monitoring of the oxygen level. The control groups were maintained at the

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oxygen saturation of 5 mg L1. In the hypoxic condition, the dissolved oxygen levels were maintained at 1 mg L1. All the stress tests were carried out in triplicates. 2.2. Total Haemocyte Count (THC) After exposure to each test condition for seven days except for oxygen stress experiments (24 h), haemolymph was drawn from each lobster from the ventral sinus of each individual. Haemolymph (0.1 ml) was drawn using a 2 ml syringe containing 0.9 ml of anticoagulant (sodium citrate 0.114 M, sodium chloride 0.10 M, pH 7.45). A drop of haemolymph in anticoagulant was placed in Neubauer haemocytometer and the THC was made using a phase contrast microscope. 2.3. Phenoloxidase (PO) assay Although in the spiny lobster (Genus: Panulirus) the proPO activating mechanism is similar to other crustaceans, unlike other crustaceans, most of the proPO-activity of spiny lobster has been detected in cell-free plasma [19]. In addition, Perazzolo et al. [20] reported a significant level of PO activity in the serum of Penaeus paulensis suggesting the use of serum for PO activity assay. Considering these evidence, in the present analyses we used serum for PO assay. PO activity was measured spectrophotometrically by recording the formation of dopachrome from L-dihydroxyphenylalanine (L-DOPA) [20]. The haemolymph was collected without anticoagulant and kept at 4  C for 5 min and the clot was broken using a micropestle and repeatedly centrifuged at 2000  g to remove serum. The serum was immediately used for PO analysis. Twenty ml serum was pre-incubated with 20 ml of trypsin (1 mg ml1) for 30 min at 37  C in 96 well microtitre plates. To each sample 20 ml of L-DOPA (3 mg ml1) was added and incubated for 5 min at 37  C. Distilled water (260 ml) was added to slow down the reaction. Activity of PO was detected spectrophotometrically using Bio-tek (EL 800) micro plate reader, measuring the formation of red pigment DOPA-chrome after 5, 10, 20, and 60 min absorbance at 490 nm. For control, L-DOPA alone was incubated with 0.45 M NaCl to monitor spontaneous oxidation. PO activity was expressed as IU mg protein1. The protein concentration of the sample was determined by Bradford method [21]. 2.4. Intracellular super oxide anion (O 2 ) production assay Respiratory burst activity of haemocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of super oxide anion production [22,23]. One hundred ml haemolymph was diluted with 400 ml of anticoagulant and was centrifuged at 300  g at 4  C for 10 min. The resultant haemocyte pellet was resuspended to 108 cells per ml in modified complete Hank’s balanced salt solution (MCHBBS) (10 mM CaCl2, 3 mM MgCl2, 5 mM MgSO4 and 24 mg ml1 HBSS). One hundred ml of haemocyte suspension was added to a flat bottom 96 well microlitre plates and cyto-centrifuged at 300  g at 4  C for 10 min. After removing the supernatant, 100 ml of trypsin (2 mg ml1) was added and allowed to react for 30 min at 37  C. MCHBBS was added to the remaining haemocyte suspension as a control. NBT (100 ml, 0.3% in MCHBBS) was added to the haemolymph and incubated for 30 min at 37  C. The staining reaction was terminated by removing the NBT solution and adding absolute methanol. After three washes with 70% methanol, the haemocytes were air dried and coated with a solution of 120 ml 2M KOH and 140 ml dimethyl sulfoxide were added to dissolve the cytoplasmic formazan [22,23]. The optical densities of the dissolved cytoplasmic formazan were measured at 630 nm with a Bio-tek (EL 800) microplate reader. The ratio of OD 630 nm of trypsin elicited haemocytes to OD 630 nm of control haemocytes was used as an index for comparing the effect of different treatments on phagocytic activity. 2.5. Analyses Each assay (THC, PO, and NBT-reduction) was repeated at least five times from at least ten individuals for each data point. One-way ANOVA followed by Post-Hoc multiple comparisons (except DO analysis) were carried out using the statistical program SPSS ver. 14.0. Significant levels for all analyses were set to P < 0.05.

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3. Results 3.1. Effect of salinity The effect of salinity on THC and PO activity is shown in Fig. 1A. THC was more or less consistent at salinities ranging from 30e40&. However, relative to THC at 30, 35 and 40&, THC at lower (20& and 25&) and higher salinities (45&) showed significant reduction (P < 0.05). Surprisingly, a significant difference in THC at 20 and 25& was also observed. While THC at 20 and 45& showed no significant difference, THC at 25 and 45& showed significant differences. These lines of evidence suggest that 20 and 45& are the extremes for the healthy maintenance of the lobster. The spiny lobster failed to survive below 20& and above 45&. The PO activity increased with increasing salinity (Fig. 1A). At lower salinities (20e25&), the PO activity was also significantly lower (P < 0.05) than the optimal salinity (35&). However, unlike THC, the PO activity at 45& was significantly higher than the PO activity measured at 20, 25, 30, 35, and 40&. In consistent with THC results, the PO activity at 20, and 25& showed significant difference. Similarly, the PO activity at 30, 35, and 40& also showed similar patterns as observed in THC (Fig. 1A). The intracellular O 2 production (NBT-reduction) ratio was also found to increase with increasing salinity (Fig. 1B). NBT-reduction values at lower salinities (20 and 25&) are significantly different from the higher salinities (40e45&). However, no significant differences were observed among the NBT-reduction values within the designated lower (i.e. between 20 and 25&) and higher (between 40 and 45&) salinities.

3.2. Effect of pH Marked differences in PO activity and in THC were also observed at different pH (Fig. 2A). In comparison to the control individuals at pH 8, the THC was significantly lower at pH 5 and pH 9.5 (Fig. 2A). The PO activity was also 10

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found to be significantly lower at pH 5 and pH 9.5 than the control pH. The results showed similar pattern in PO activity and THC (Fig. 2A). The relationship between NBT-reduction and pH is shown in Fig. 2B. The NBT-reduction at different pH did not show any significant differences. 3.3. Effect of dissolved oxygen (DO) Marked differences were observed between hypoxic (DO ¼ 1 mg L1) and control (DO ¼ 5 mg L1) condition in their respective PO activity and THC (Fig. 3A). The THC and PO activity significantly decreased when the DO was reduced to 1 mg L1 (Fig. 3A). The NBT-reduction also significantly decreased (P < 0.05) in the hypoxic condition. The intracellular O 2 production (NBT-reduction) under hypoxia and under optimal condition was 0.93  0.04 and 1.34  0.19, respectively. 3.4. Effect of ammonia The influence of different ammonia-N concentrations on the THC and PO activity are shown in Fig. 4A. The THC at 0.5 mg L1 ammonia-N showed significant reduction (P < 0.05) over the control. At 1.5 mg L1 and 3 mg L1ammonia-N concentrations, the THC showed a decline of 58.9% and 51.63%, respectively from the control.

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Fig. 3. Effect of dissolved oxygen (DO) concentrations on immune response of P. homarus. The DO concentrations at optimal (control) and hypoxic conditions are 5 and 1 mg L1, respectively. Data with different letters were significantly different (P < 0.05).

No significant difference in THC was observed at 1.5 mg L1 and 3 mg L1 ammonia-N concentrations. PO activity in the control was significantly higher (P < 0.05) than the 0.5, 1.5 mg L1 and 3 mg L1 ammonia-N concentrations. Despite the minor differences, both THC and PO activity at different concentrations showed similar trends (Fig. 4A). These lines of evidence indicate that both THC and PO are reliable indicators for stress measurement. The effect of different ammonia-N concentrations on the NBT-reduction is shown in Fig. 4B. The NBT-reduction at control was significantly higher than the NBT-reduction at 3 mg L1. Although NBT-reduction showed a decreasing 12

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trend with the increase of ammonia-N concentration, no significant difference (P < 0.05) was observed among NBTreduction values of 0.5, 1.5, and 3 mg L1. 4. Discussion The present results demonstrate that fluctuation in environmental parameters such as salinity, pH, DO, and ammonia-N concentration significantly affect the immune function in P. homarus. For all stresses, THC, PO activity, and NBT-reduction followed similar trends for a given treatment. Fluctuation in salinity seems to have considerable impact on the THC and PO activity in many crustaceans including the freshwater prawn, Macrobrachium rosenbergii [2,3] and a number of euryhaline penaeids [4,6,7,24e26]. Concordantly, our data also supported that salinity is one of the important physico-chemical parameters that significantly affect the THC and PO activity of the stenohaline crustacean P. homarus. Although it is unequivocal that salinity is a pivotal physico-chemical parameter, it is still unclear to what extent each species from different habitats respond to the extreme conditions. For instance, THC seems to be lower for M. rosenbergii at 0& (optimal salinity for M. rosenbergii is 0 to 10&) and higher at 15& [2,3]. Similarly, most of the euryhaline penaeids have low THC at 12 to 13&, whereas THC increased at higher salinities 34e37& (optimal salinity for most of the penaeid shrimps ranged from 24e30&) [4,6,7,24e26]. In the stenohaline P. homarus, the sudden drop of THC at 45& clearly indicate a disparity in the pattern that was previously observed in other crustaceans despite showing an increase in THC until 40& (e.g., refs. [2e4,6,7,24e26]). We were curious to measure the THC at 50&; however, the animal could not survive in that high saline water. Thus, our data suggested that THC is significantly lower at extreme conditions, and it can be a good stress indicator. Including the present study, there has been several studies that showed a declining trend in THC with lower salinities (lower limit of respective organisms) (e.g., refs. [2e4,6,7,24e26]). However, the differential magnitude of THC at the lower or higher salinity tolerance limits of respective species could be directly associated with the energy requirements for osmotic regulation [6]. For example, euryhaline organism requires minimum energy to maintain homeostasis, whereas stenohalines require more energy under extreme (upper and lower bound) environmental conditions. One of the interesting findings in the present study is that both THC and PO activity showed an increasing trend until 40&, but showed contrasting pattern in THC and PO activity at 45& (Fig. 1A). The significant reduction in THC at salinity 45& could be associated with the significant decrease in granular cells as the proPO activating enzyme (PPAE) has to be released from the haemocyte granules, which has been suggested as the natural activation pathway in response to stress [19,27]. In addition, increase in PO activity could also be due to significant reduction in plasma inhibitors regulating the proPO system [18,27]. Significant reduction in THC at 20 and 45& salinity indicates the animal is under stress, and susceptibility to opportunistic pathogens is probably high at such extreme salinity level. The significant reduction in THC and PO activity at both higher and lower pH (Fig. 2A) suggested that these stenohaline lobsters could be under immunosupression at higher alkaline and acidic conditions, respectively. Although the underlying mechanisms for such a scenario in spiny lobsters are unclear, the possible explanation is that acidic or alkaline condition could hinder the growth, and moulting, as well as other physiological functions. As a consequence, it unswervingly affects the innate immune system, which directly reflects on the haemolymph. Deviation from the optimal pH has been reported to induce a modification in the haemocyte count and PO activity of M. rosenbergii [2]. Under hypoxia, significant reduction in PO activity and THC were also observed. Significant reduction in THC and PO activity at higher ammonia-N concentrations than the optimal concentration further indicate that THC and PO are the reliable stress indicators. Reduction in THC in lobsters exposed to high levels of ammonia is likely to decrease the phagocytic potential and other antimicrobial abilities of the organism (e.g., ref. [3]). Earlier studies reported that exposure of M. rosenbergii to higher concentrations of ammonia also reduce PO activity [3]. While the THC in P. stylirostris has also shown similar trends as observed in the present study, in contrast to the present study, PO activity in P. stylirostris showed an increasing trend [6]. Although the reason for such discrepancy in PO activity in P. homarus and P. stylirostris is unclear, it is hypothesized that behavioral differences, habitat preferences, intrinsic biological factors, and other physiological conditions of respective organisms could be the possible explanation. The effect of salinity, pH, DO, and ammonia-N concentration on the phagocytic activity of haemocytes was evaluated by quantifying super oxide anion production (O 2 ) using the NBT staining. Phagocytosis is an important cellular defense in crustacean which involves the engulfment of foreign substances/organisms and digesting them, which involves generation of reactive oxygen intermediates (ROIs) by the process called respiratory burst. The first ROI gen erated during this process is O 2 [28]. The O2 production depends on the number of haemocytes circulating in the

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haemolymph, thus drop in THC would result in the drop in O 2 production (NBT-reduction). The estimates of NBTreduction under each environmental parameter (salinity, pH, DO, and ammonia-N concentrations) were in concordance with their respective THC. Although it is not surprising to observe this concordant pattern as THC is directly associated with O 2 production [5], this relationship further validated our overall conclusion that these immune parameters are the reliable stress indicators for the Indian spiny lobster P. homarus. In conclusion, wide variations in environmental factors may induce alteration in the immune system in P. homarus that are often immunosuppressive, in terms of diminishing total haemocyte number and decrease of PO activity and phagocytic activity. Therefore, extreme environmental conditions can lead to detrimental effects like reduced disease resistance, growth impairment, poor reproductive performance, and lower survival. Hence, disease prevention due to stress or any other factor should begin from effective water quality management practices and maintenance of optimal environmental conditions. Acknowledgements The authors are grateful to the Director, Central Marine Fisheries Research Institute for the encouragement and for providing facilities to carry out this work. The authors are also thankful to Karen Dutoi for editing and comments for improving the manuscript. We thank R. Thangaraja and Binu Varghese for comments on the earlier version of the manuscript. We thank the two anonymous reviewers for their helpful comments on the earlier version of the manuscript. BV gratefully acknowledges Central Institute of Fisheries Education, Mumbai for granting the research fellowship. References [1] Radhakrishnan EV, Vijayakumaran M. Problems and prospects for lobster farming in India. In: Marine Fisheries Research and Management. Cochin: Central Marine Fisheries Research Institute; 2000. p. 753e64. 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