Effects of salinity and ph on immune parameters of the white shrimp Litopenaeus vannamei.

ABSTRACT The effect of salinity and pH on the immune response of the white shrimp Litopenaeus vannamei was studied over a period of 12-15 days. The results indicate that salinities between 5[per thousand] to 30[per thousand] and pH of approximately 7.0-9.5 affect hemocyte count, phenoloxidase activity, bacteriolytic activity and antibacterial activity. Phenoloxidase activity peaked at the 12th hour, whereas bacteriolytic activity and antibacterial activity were lowest. By the 6th day of exposure, every parameter returned to the values observed in controls except hemocyte count, which remained low with a decrease in salinity. From the 6th to the 15th day of the salinity test, the hemocyte count at every salinity were constant but significantly lower than control; significant changes in phenoloxidase activity, bacteriolytic activity and antibacterial activity were observed over the same period. During the first 3 days exposure to variable pH, hemocyte count and antibacterial activity decreased gradually as time elapsed at pH 7.0, 7.5, 9.0 and 9.5. Phenoloxidase activity peaked at the 12th hour, but the bacteriolytic activity fell. During the third to the 12th day of the pH test, every immune parameter was stable. The hemocyte count, bacteriolytic activity and antibacterial activity decreased, whereas the phenoloxidase activity increased in response to a change in pH.

KEY WORDS: salinity, pH, Litopenaeus vannamei, hemocyte count, phenoloxidase activity, bacteriolytic activity, antibacterial activity

INTRODUCTION

Disease has always been an important limiting factor in shrimp culture. In recent years, virus diseases have caused serious harm to shrimp culture (Lee et al. 1996, Lo & Kou 1998). The interaction of pathogeny, resistance and environmental factors all influence the etiology of shrimp disease (Lightner & Redman 1998). Many works have found that changes in environmental factors reduce immunologic activity and thus leading to enhanced sensitivity to pathogeny (Lightner & Redman 1998, Perazzolo et al. 2002, Li et al. 2002, Pan & Jiang 2002). This study examines the effects of salinity and pH on the immune capacity of the white shrimp Litopenaeus vannamei and discussed the environmental physiology mechanism of the shrimp immunologic regulation.

MATERIALS AND METHODS

Materials

Healthy L. vannamei adults used in this experiment were obtained from commercial farms in Yinghai, Qingdao, China. The average body length of the shrimp was 8.5 [+ or -] 0.5 cm. The shrimp were acclimated in tanks (30 cm x 40 cm x 50 cm) containing aerated water (salinity 30[per thousand], pH 8.0) with an air-lift at 24[degrees]C [+ or -] 0.5[degrees]C for 8-10 days prior to experimentation. Half of the water in each tank was renewed twice daily. During the acclimation period, the shrimp were fed with a formulated shrimp diet daily.

Methods

Experimental Design

Tests with two different conditions were conducted. In the salinity tests, seawater was diluted to the desired salinity of 5[per thousand], 10[per thousand], 15[per thousand], 20[per thousand] and 25[per thousand] with insolated tap water. In the pH tests, water was adjusted with 1 M HCl and 1M NaOH to a pH of 7.0, 7.5, 8.5, 9.0 and 9.5 and varied about [+ or -]0.1.

For each treatment, there were three replicate groups, and each group contained 42 shrimp respectively. Controls were maintained at a constant salinity of 30[per thousand] and pH of 8.0. The experiment conditions were identical to the acclimation period. Water was renewed twice daily with water of appropriate salinity/pH. No shrimp died during the experiment. Four shrimp were sampled randomLy from each group at 0 h, 3 h, 6 h, 9 h, 12 h, 1 d, 3 d, 6 d, 9 d, 12 d and 15 d.

Swatch Preparation

Hemolymph was withdrawn from the ventral sinus into a 1.0 mL syringe containing an equal volume of precooled anticoagulant solution (580 mM NaCl, 13 mM Ca[Cl.sub.2] * 6[H.sub.2]O, 0.54 mM [Na.sub.2]HP[O.sub.4] * 12[H.sub.2]O, 12.6 mM KCl, 28 mM Mg[Cl.sub.2] * 6[H.sub.2]O, 50 mM Tris, pH 7.3). Samples of the hemolymph for the hemocyte count were mixed and used immediately. Other samples were held in a refrigerator (4[degrees]C) for 24 h, and were centrifuged (3000 rpm) at 4[degrees]C for 15 min, and then the supernatant was separated for measurements of other immune parameters.

Measurement Method

Total hemocyte count (THC) was performed using a hemocytometer viewed under an Olympus phase contrast microscope.

Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome from L-dihydroxyphenylalanine (L-DOPA) as previously described (Ashida 1971) with some modifications. A 0.01 M solution of L-DOPA was prepared in 0.1 M-phosphate buffer (12.3 mL 0.2 M [Na.sub.2]HPO4, 87.7 mL 0.2 M Na[H.sub.2]PO4, pH 6.0). One hundred [micro]L serum and 100 [micro]L L-DOPA was to a spectroscopy cell (10.0 mm) containing 3 mL of phosphate buffer. After the mixture was agitated, the optical density at 490 nm was measured at 2 min intervals for 60 min. Phenoloxidase activity was determined from the increase in O.D. per minute at the environmental condition.

Bacteriolytic activity was measured using Micrococcus lysoleikticus (SIGMA) and antibacterial activity was measured with Vibrio parahaemolyticus and Vibro harveyi, according to the method described by Hultmark et al. 1980. Bacterial pellets of V. parahaemolyticus and V. harveyi were cultured on 2216E-beveled solid substrates for 18-20 h at 30[degrees]C, washed with 0.1 M phosphate buffer (pH 6.4) and the bacteria suspended in buffer at an [O.D..sub.570nm] of 0.3.

Each of the three bacterial suspensions (3 mL) were mixed with 50 [micro]L plasma in tubes in ice water (0[degrees]C) and the optical density at 570 nm ([A.sub.0]) measured. The tubes were then transferred to a water bath at 37[degrees]C for 30 min, then returned to ice water (0[degrees]C) for 10 min to stop the reaction and the optical density at 570 nm (A) was measured again.

The antibacterial activity was calculated as follows:

[U.sub.a.sup.2] = ([A.sub.0] - A)/A

The bacteriolytic activity was calculated as follows:

[U.sub.b] = ([A.sub.0] - A)/A

Statistical Analysis

The data were expressed as the mean [+ or -] SD of the replicated tanks (n = 12). One-way Analysis of Variance (ANOVA) and F test were used to test the significance of differences among treatments and various time intervals.

EXPERIMENTAL RESULTS

Effects of Salinity on Immune Parameters of L. vannamei

There was a progressive fall in hemocyte numbers in response to a decrease in salinity until day 6 of exposure. The hemocyte numbers of every treatment group became significantly different with the control from the 12th hour (F > [F.sub.0.05]). The hemocyte count remained stable from day 6 until the end of the experiment. Phenoloxidase activities progressively increase in response to a decrease in salinity. The phenoloxidase activities of every treatment group became markedly different with the control from the 6th hour (F > [F.sub.0.05]). The bacteriolytic activities and antibacterial activities progressively decreased with salinity decrease. The bacteriolytic activities of every treatment group became significantly different with the control from the 6th hour (F > [F.sub.0.05]). The antibacterial activity of salinity 25[per thousand] had no significant difference with the control during the experiment time (F < [F.sub.0.05]), whereas the antibacterial activity of salinity 20[per thousand] and 15[per thousand] became markedly different with control from the 6th hour and activity of salinity 10[per thousand] and 5[per thousand] became markedly different with control from the 3rd hour (F > [F.sub.0.05]). Recovery in phenoloxidase activity, bacteriolytic activity and antibacterial activity was seen with respect to control. Phenoloxidase activity and antibacterial activity had no significant difference with the control until day 6 (F < [F.sub.0.05]), and the bacteriolytic activity had no significant difference with the control until day 9 (F < [F.sub.0.05]). (Fig. 1).

[FIGURE 1 OMITTED]

Effects of pH Change on Immune Parameters of L. vannamei

Fall in hemocyte numbers in response to a change in pH from the optimum of 8.0 and 8.5 was seen. The hemocyte numbers of pH 7.5, 9.0 and 9.5 became significantly different with control from the third day and the hemocyte numbers of pH 7.0 became significantly different with the control from the first day (F > [F.sub.0.05]). Hemocyte numbers of pH 7.0, 7.5, 9.0, 9.5 became stable and had no significant different between them from day 3 until the end of the experiment (F < [F.sub.0.05]). Increase in phenoloxidase activity in response to a change in pH from the optimum of 8.0 was seen. Phenoloxidase activity of treatment groups became significantly different with the control from the third hour (F > [F.sub.0.05]). Phenoloxidase activity of pH 7.0, 7.5, 9.0, 9.5 became stable and no significant difference between them from day 3 until the end of the experiment (F < [F.sub.0.05]), whereas activity of pH 8.5 recovered and no longer significantly different compared with the control from day 3 until the end of the experiment (F < [F.sub.0.05]). Significant decrease in bacteriolytic activities of pH 7.0, 7.5, 9.0, 9.5 was seen from the 6th hour (F > [F.sub.0.05]) in response to a change in pH from the optimum of 8.0 and 8.5 and they became stable from day 3 until the end. Bacteriolytic activity of pH 9.0 appears to recover and returning the values close to the control pHs of 8.0 and 8.5. Fall in antibacterial activities in response to a change in pH from the optimum of 8.0 and 8.5 until day 3 of exposure was seen. Antibacterial activities of pH 7.0, 7.5, 9.0, 9.5 became significantly different with control from day 1 (F > [F.sub.0.05]) and became stable and had no significant difference between them from day 3 (F < [F.sub.0.05]) (Fig. 2).

[FIGURE 2 OMITTED]

DISCUSSION

Effects of Salinity on Immune Parameters of L. vannamei

Salinity is a very important environmental factor affecting the survival, growth and physiologic function of shrimp. Pan and Jiang (2002) have reported that during a short-term (10h) salinity change 30[per thousand] to 15[per thousand], the bacteriolytic activity and antibacterial activity of 2 reared shrimp (Fenneropenaeus chinensis and Litopenaeus vannamei) is reduced gradually, whereas phenoloxidase activity increased (2002). Li et al. found that hemocyte count of the shrimp Marsupenaeus janpolicus was reduced when salinity changed from 25[per thousand] to 9[per thousand] or to 33[per thousand] between 4 and 8 days, but phenoloxidase activity increased (2002). These studies indicate that a sudden change in salinities can cause an initial marked decline in shrimp immunocompetence and followed by a gradual recovery. Our results show that L. vannamei immune regulation system is able to recover from certain change in salinity within 6 days, and the recovery is independent of the change in salinity.

Cheng and Chen (2000) reported that hemocyte numbers in Macroblachium rosenbergii increased when salinity changed from 0[per thousand] to 5[per thousand], 10[per thousand] or 15[per thousand] over 7 days. Perazzolo et al. (2002) described that when salinity decreased gradually from 34[per thousand] to 22[per thousand] or 13[per thousand] over 2 or 3 days, the hemocyte count of Farfantepenaeus paulensis declined gradually before stabling between 7 and 28 days. In our study, from the 6 th to the 15 th day of following change, hemocyte count at every salinity was stable and the lower the salinity, the lower the hemocyte count. However no significant difference in phenoloxidase activity, bacteriolytic activity and antibacterial activity in hemolymph was observed over the same time period. Many studies have demonstrated that under the environmental stress the hemocyte count of crustaceans declines, enzyme activity related to diseases resistance decreases and the sensitivity to pathogens increases (Truscott & White 1990, Vargas-Albores et al. 1998, Winton 1998, 2000, Le Moullac & Haffner 2000). We suggest that hemocyte count of shrimp declines under low salinity because of an increase in hemolymph volume. Therefore the total hemocyte count has only declined in relation to hemolymph volume. It is probably for this reason that the immune enzyme activity per unit protein in the hemolymph shows no marked change with decreasing salinity.

Effects of pH on Immune Parameters of L. vannamei

At present many scholars believe that the optimal pH of L. vannamei is 7.6-8.5 (Allan & Maguire 1992). When shrimp were exposed to pH lower than 7.0, locomotor activity is limited and molting and growth are affected. Chen and Lin (1995) have reported that Penaeus chinensis juveniles oxygen consumption decreased as pH increased in the range of 7.0-8.5. Lin et al. (2000) reported that aqueous pH outside of a definite range affects respiration in crustaceans. Cheng and Chen & Chen (2000) have found that a pH change from 7.6-4.8 or 9.3 in 7 days decreased the hemocyte number of Macrobrachium rosenbergii, and suggested that M. rosenbergii showed the strongest disease resistance at pH 7.5-7.7. Pan and Jiang (2002) have found that pH fluctuation from 8.5-7.0 and 9.5 over a short time (10h) decreased the bacteriolytic activity and antibacterial activity of two species reared shrimp, whereas phenoloxidase activity increased significantly. In this study, we found that every immune parameter at each exposure pH was stable, and immune capacity of the control shrimp (pH 8.0) was the highest from the 3rd to the 12th day of pH change. Except for pH 8.5, the immune capacity decreased following an increase or decrease in pH from 8.5. The effect of pH on the immunocompetence mainly attribute to the gills, which are the main organ that regulates the Cl-/HC[O.sub.3.sup.-] and [Na.sup.+]/[H.sup.+] balance to maintain pH in vivo (Allan & Maguire 1992, Prqueux 1995). Meanwhile this regulation induces the oxygen consumption rate increase, more energy consumed (Savant & Amte 1995) and lead to a decline of the immune capacity in different pH levels.

Physiologic Mechanisms of Shrimp Immunologic Regulation

Crustaceans have a nonspecific immune response, including phagocytosis, coagulation and encapsulation of hemocyte and active factors released to hemolymph such as prophenoloxidase (proPO) system, agglutinant, bacteriolytic enzyme, antibacterial peptide and proteinase inhibitor (Soderhall & Cereniusl 1992). These immunological responses cooperate and many processes show complicated cascade reactions. Among them, the proPO system plays a key role in immunologic recognition and defense in crustacea, and the immune response to outside factors can be triggered and augmented by information transfer between cells. Soderhall and Unestam (1979) found the mechanisms of the proPO system of Astacus astacus, triggered by invasive agents (bacterium, fungus, etc.) and spontaneously activated in a normal physical condition are completely different. Many studies have demonstrated that changes in environmental factors triggers the proPO system and increases phenoloxidase activity in the hemolymph (Le Moullac et al. 1998, 2000, Pan & Jiang, 2002, Li et al. 2002, Perazzolo et al. 2002). We assume that the proPO system has different activating mechanisms and therefore the immunologic responses are also different. Therefore, the immunocompetence of crustacea should not be evaluated only by the numeric value of phenoloxidase activity, but should also consult the inducement mechanism of the immunity change, and make a comprehensive estimation of the immune level of the organism.

CONCLUSION

According to the immunological adaptation capacity to salinity change of L. vannamei, it is inadvisable that the environmental salinity change should not be more than 5[per thousand], and earnest attention should be paid to the shrimp in the initial 6 days of salinity change. Moreover, some technical measures should be taken to maintain the stabilization of aquatic environment salinity to prevent diseases. According to the immunological adaptation of L. vannamei to pH, we suggest that the pH of the environmental media should be maintained in the range of 8.0-8.5, and the variable of pH change should not exceed 0.5.

ACKNOWLEDGMENT

The study was funded by the Sea-prosper Science and Technology Project of Shandong Province, China.

LITERATURE CITED

Allan, G. L. & G. B. Maguire. 1992. Effect of pH and salinity on survival, growth and osmoregulation in Penaeus monodon Fabricius. Aquaculture 107:33-47.

Ashida, M. 1971. Purification and characterization of pro-phenoloxidase from hemolymph of the silkworm Bombyx morl. Arch. Biochem. Biophys. 144:749-762.

Barton, B. A., C. B. Schreck & L. D. Barton. 1987. Effect of chronic cortisol administration and daily acute stress on growth, physiological conditions, and stress responses in juvenile rainbow trout. Dis. Aquat. Organ. 2:173-185.

Carlos, R., M. Evemor & G. Gabriela. 1999. The effect of dissolved oxygen and salinity on oxygen consumption, ammonia excretion and osmotic pressure of Penaeus setiferus juveniles. J. Exp. Mar. Biol. Ecol. 234:41-57.

Chen, J. C. & C. Y. Lin. 1995. Responses of oxygen consumption, ammonia-N excretion and urea-N excretion of Penaeus chinensis exposed to ambient ammonia at different salinity and pH levels. Aquaculture 136:243-255.

Cheng, W. & J. C. Chen. 2000. Effects of pH, temperature and salinity on immune parameters of the freshwater prawn Macrobrachium rosenbergii. Fish Shellfish Immunol. 10:387-391.

Dalla Via, G. J. 1986. Salinity responses of the juvenile shrimp Penaeus japonicus. 1. Oxygen consumption and estimations of productivity. Aquaculture 55:297-306.

Dalla Via, G. J. 1987. Effects of salinity and temperature on oxygen consumption in a freshwater population of Palaemonetes antenarius. Comp. Biochem. Physiol. 88A:299-305.

Hultmark, D., H. Steiner & T. Rasmusn. 1980. Insect immunity: purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophera cecropia. Eur. J. Biochem. 106:7-16.

Kutty, M. N., G. Mumgapoopathy & T. S. Krishnan. 1971. Influence of salinity and temperature on the oxygen consumption in young juveniles of the Indian shrimp Penaeus indicus. Mar. Biol. 11:125-131.

Lee, K. K., S. R. Yu & F. R. Chen. 1996. Virulence of Vibrio alginolyticus isolated from diseased tiger prawn Penaeus monodon. Curr. Microbiol. 32:229-231.

Le Moullac, G., D. Saulnier & D. Ansqer. 1998. Effect of hypoxic stress on the immune response and the resistance to vibriosis of the shrimp Penaeus stylirostris. Fish Shellfish Immunol. 8:621-629.

Le Moullac, G. & P. Haffner. 2000. Environmental factors affecting immune responses in Crustacea. Aquaculture 191:121-131.

Li, C. W., Y. Q. Guan & Z. M. Yu. 2002. Effect of salinity variation on outbreak of white spot syndrome and immunocompetence in Penaeus japonicus. Marine Environmental Science 21(4):6-9.

Lightner, D. V. & R. M. Redman. 1998. Shrimp disease and current diagnostic methods. Aquaculture 164:201-220.

Lin, X. T., Q. M. Zhang & Z. N. Xu. 2000. Proceeding of studies on respiratory metabolism decapod crustacean. J Fish China 24:575-580.

Lo, C. F. & G. H. Kou. 1998. Virus-associated white spot syndrome of shrimp in Taiwan: A review. Fish Pathology 33:365-371.

Pan, L. Q. & L. X. Jiang. 2002. Effect of sudden changes in salinity and pH on the immune activity of two species of shrimp. Journal of Ocean University of Qingdao 32:903-910.

Perazzolo, L. M., R. Gargioni & P. Ogliari. 2002. Evaluation of some hemato-immunological parameters in the shrimp Farfantepenaeus paulensis submitted to environmental and physiological stress. Aquaculture 214:19-33.

Pequeux, A. 1995. Osmotic regulation in crustaceans-Review. (J). J. Crustacean Biol. 15(1):1-60.

Savant, K. B. & G. K. Amte. 1995. Influence of some environmental factors on respiratory responses in the tropical estuarine crab Llyoplax gangetica (Kemp). (J). J. Environ. Biol. 16(4):311-317.

Soderhall, K. & T. Unestam. 1979. Activation of crayfish serum prophenoloxidase: The specificity of cell wall glucan activation and activation by purified fungal glycoproteins. Can. J. Microbiol. 25:406-414.

Soerhall, K. & L. Cereniusl. 1992. Crustacean immunity ann rev. (J). Fish Dis 10:3-23.

Truscott, R. & K. N. White. 1990. The influence of metal and temperature stress on the immune system of crabs. Funct. Ecol. 4:455-461.

Vargas-Albores, F., P. Hinojosa-Baltazar & G. Portillo-Clark. 1998. Influence of temperature and salinity on the yellowleg shrimp, Penaeus californiensis Holmes, prophenoloxidase system. Aquaculture Research 29:549-553.

Van Weerd, J. K. & J. Komen. 1998. The effect of chronic stress on growth in fish: a critical appraisal. Comp. Biochem. Physiol. 120:107-112.

PAN LU-QING, * JIANG LING-XU AND MIAO JING-JING

The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China 266003, China

* Corresponding author. E-mail: [e-mail]