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Impact of different environmental factors on the circulating immunoglobulin levels in the Nile tilapia, Oreochromis niloticus
Aquaculture 241 (2004) 491 – 500 www.elsevier.com/locate/aqua-online
Impact of different environmental factors on the circulating immunoglobulin levels in the Nile tilapia, Oreochromis niloticus Miriam Domingueza, Akihiro Takemurab,*, Makoto Tsuchiyaa, Shigeo Nakamurab a
Laboratory of Ecology and Systematic, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan b Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, 3422 Sesoko, Motobu, Okinawa 905-0227, Japan Received 2 February 2004; received in revised form 24 June 2004; accepted 25 June 2004
Abstract Immunoglobulin M (IgM) in teleost fishes is the only component of the specific humoral defense system that is affected by environmental factors. In the present study, we examined the effect of various environmental factors (water temperature, salinity, pH and suspended solids) on the plasma IgM in the Nile tilapia, Oreochromis niloticus. For all treatments, fish were acclimatized in particular environmental conditions for 2 or 4 weeks. For fish reared at 18.4, 23 and 28 8C, the circulating IgM concentration increased with increased water temperature for 2 weeks. Rearing the fish at 33 8C resulted in a decrease in IgM concentration, suggesting that the fish possess an appropriate thermal range for production of immune substances. Plasma level of IgM increased significantly with salinity at 12 and 24 parts per thousand (ppt). On the other hand, plasma IgM concentration did not change by exposing the fish to acidification (pH 4.0) and suspended solids (20, 200, and 2000 mg/l). These results suggest that the specific immune system of tilapia changes by certain factors in aquatic environment. D 2004 Elsevier B.V. All rights reserved. Keywords: ELISA; Environment; Immunoglobulin; Low pH; Salinity; Suspended solids; Tilapia; Water temperature
* Corresponding author. Tel.: +81 980 47 6215; fax: +81 980 47 4919. E-mail address: [email protected] (A. Takemura). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.06.027
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1. Introduction The specific defense system of teleost fishes is continuously affected by periodic or unexpected changes in different factors of the aquatic environment. Fishes under suitable environmental conditions have a functional immune system and, consequently, high disease resistance, growth rate and reproductive activity are expected. On the contrary, unsuitable environmental conditions may act as acute or chronic stressors against fish and cause the suppression of the specific defense system. In the promotion of aquaculture, therefore, it is important to evaluate the relationships between changes in various environmental conditions and the effects on the immune system of fishes. Among different factors in aquatic environments, the effects of water temperature and salinity on the humoral defense system have been well studied. Production of humoral defense substances fluctuates with water temperature (Rijkers et al., 1980; Klesius, 1990; Suzuki et al., 1997; Magnado´ttir et al., 1999) and salinity (Marc et al., 1995; Yada et al., 1999, 2001, 2002; Yada and Azuma, 2002). Additionally, aquatic pollution often results in unexpected changes in the aquatic environment and occasionally affects the immune system of fishes (Almeida et al., 2002). In general, it seems that each fish species has a specific tolerance range with respect to different environmental conditions. To date, only a few studies have been done on adaptive changes in the humoral defense system under various environmental conditions in tilapia (genus Oreochromis), which are native to Africa and have been introduced as economically important aquaculture finfish to many countries (Naylor et al., 2000). The aim of the present study is to evaluate whether environmental changes affect the circulating immunoglobulin M (IgM) which is the only component of the specific humoral defense system in teleost fishes and can be detected in the blood circulation (Geir and Heidrun, 2000) as well as body surface (Suzuki et al., 1997). Using the Nile tilapia, Oreochromis niloticus, we assessed impact of water temperature and salinity on the circulating IgM. In addition, we examined the effects of low pH and suspended solids which are also critical environmental factors in some aquatic habitats.
2. Materials and methods 2.1. Fish The Nile tilapia, O. niloticus, used in the present study were caught from a pond near Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa, Japan, using a cast net. Their body mass ranged from 18.2 to 21.7 g. They were kept in tanks (1-metric ton capacity) supplied with filtered and aerated freshwater at the Sesoko Station. All the fish were acclimatized in the tanks at least for 2 weeks before the onset of the following experiments. All the experiments were repeated several times. 2.2. Effect of water temperature To investigate the effect of water temperature on plasma IgM levels, the fish (25 each) were transferred to four aquaria (60-l capacity) with a filtration and aeration system.
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Following acclimatization of the fish to basic experimental conditions for 2 weeks, water temperature in the three aquaria gradually increased to 23, 28 or 33 8C within 24 h and kept at respective temperature until samplings. One tank was kept at ambient temperatures which ranged between 16.9 and 20.3 8C (mean: 18.4 8C). Seven fish were taken from each aquarium at 0, 2 and 4 weeks after the onset of experiment and immediately anesthetized with 2-phenoxyethanol (Kanto Chemical, Tokyo, Japan). Blood samples from each fish were collected in heparinized capillary tubes (Terumo, Tokyo, Japan) from the caudal vasculature by cutting the tail. Plasma was obtained by centrifugation of the blood sample at 9600g, for 5 min, and stored at 30 8C until experimental analyses. 2.3. Effect of salinity Because preliminary experiments showed that all the fish died in full-strength salinity (35 parts per thousand, ppt), the highest salinity of the present experiment was set at 24 ppt. To investigate the effect of salinity on IgM levels, 25 fish each were transferred to three aquaria. Following acclimatization of the fish in the aquaria with freshwater under basic experimental conditions for 2 weeks, salinity of water in the aquarium was adjusted to 0, 12 and 24 ppt by diluting seawater with freshwater. Increase of salinities was done within 24 h. Water temperature of each aquarium was maintained at 28 8C and half of the water was changed every 2 days. Blood was collected from six individuals at 0, 2 and 4 weeks after salinity treatments were started, and plasma preparations were done as above. 2.4. Effect of low pH In preliminary experiments, O. niloticus were reared in different low pH conditions (pH 3.0, 4.0 and 5.0) for 2 weeks. Because rearing the fish at pH 3.0, but not pH 4.0, caused death in all the individuals, the lower limit for experimental pH was set at pH 4.0. Prior to initiation of the experiment, the fish (15 each) were transferred to two aquaria and were acclimatized in freshwater (pH 7.9) under basic experimental conditions for 2 weeks. Acidic condition (pH 4.0) was created within 24 h by adding 3 mol/l HCl to the aquaria and verified using a digital pH meter (HM-21P, Horiba, Tokyo, Japan). The control aquarium with 15 fish was kept at pH 7.9 during the experiment. The aquaria were aerated vigorously and 1/3 of water in each aquarium was changed every 2 days. Blood was collected from seven individuals at 0 and 2 weeks after the low-pH treatment started. Plasma preparations were done as indicated above. 2.5. Effect of suspended solids A sample of red soil was collected from the northern part of Okinawa Island. After vigorous washing and sieving (64 Am), the suspended solids were dried for 24 h at 60 8C with a drying oven (Isuzu, Tokyo, Japan). The fish (15 each) were transferred to four aquaria and were acclimatized in the aquaria under basic experimental conditions for 2 weeks. Within 6 h, dried sediment was added to each of the four aquaria at concentrations of 0, 20, 200 and 2000 mg/l. Suspension of red sediment was maintained by circulating
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water column. Blood was collected from six individuals at 0 and 2 weeks after sediment exposure treatment. Plasma preparations were done as before. 2.6. Measurement of plasma immunoglobulin M concentration Total IgM concentration in the plasma of tilapia was measured by enzyme-linked immunosorbent assay (ELISA) according to the method of Takemura (1993). Purified tilapia IgM, rabbit anti-tilapia IgM antibody (a-IgM) and a-IgM labeled with horseradish peroxidase (a-IgM HRP) was prepared in advance (Takemura, 1993). Each well of a 96-well microtiter plate (Becton Dickinson Labware, Franklin Lakes, NJ) was coated with 100 Al of a-IgM (6.8 Ag/ml) in 0.05 M sodium carbonate buffer, pH 9.6, and incubated for 2 h at 25 8C. Residual protein binding sites were blocked by adding 200 Al of 1 % gelatin (BioRad Laboratories, Richmond, CA) dissolved in 10 mM phosphate-buffered saline (PBS), pH 7.4, containing 0.05% Tween 20 (PBS–Tween) to the wells for 60 min at 25 8C. After washing the wells three times with PBS–Tween using a plate washer (Immunowash 1573, BioRad), 100 Al of plasma sample (1:10 000) or standards (serial dilution of purified tilapia IgM) was added to the well and then incubated overnight at 4 8C. All the dilutions were made with PBS–Tween. After washing the wells three times with PBS–Tween, 100 Al of a-IgM HRP (diluted 1:20 000 in PBS–Tween) was added to the wells and incubated for 2 h at 25 8C. After three successive washes with PBS–Tween, peroxidase activity was measured by adding 100 Al of 100 mM citrate buffer, pH 4.5, containing 0.01% o-phenylenediamine dihydrochloride (Sigma, St. Louis, MO) and 0.04% H2O2. Following 30-min incubation at 25 8C, the enzymatic reaction was stopped by adding 25 Al of 4 N H2SO4. The optical density of each sample was determined at 490 nm using a microplate reader 550 (BioRad). Computer software (Microplate Manager III, version 1.57) was used for conversion from optical density to IgM concentration. 2.7. Statistical analysis The plasma IgM concentration data were expressed as meanFS.E. Two-way analysis of variance (ANOVA) and Fisher’s protected least significance difference (PLSD) test were used to determine the statistical differences for the water temperature, salinity and suspended solids experiments. The Student’s t-test was also performed for analysis of IgM concentrations in the low-pH experiment.
3. Results and discussion 3.1. Water temperature Total IgM concentration in the blood circulation was compared among the fish that were kept at various water temperatures, 18.4 (control), 23, 28 and 33 8C. At 2 weeks after exposure, the plasma IgM concentration of the control fish (18.4 8C) was 168.64F29.73 Ag/ml. For the other temperature treatments, IgM concentration generally increased with
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water temperature after 2 weeks. However, only the fish kept at 28 and 33 8C had significantly higher IgM concentrations than the control fish. IgM concentration of the fish reared at 33 8C was not significantly different than the fish reared at 28 8C (Fig. 1a). Water temperature had similar effects on plasma IgM concentration at 4 weeks (Fig. 1b) for the fish kept at 28 8C. However, the concentration at 338C was significantly lower and did not differ from the control. The influence of water temperature on fluctuation of plasma IgM concentration has been previously studied in certain teleost fishes (Rijkers et al., 1980; Klesius, 1990; Suzuki et al., 1997; Magnado´ttir et al., 1999). Magnado´ttir et al. (1999) reported that Atlantic cod, Gadus morhua L., cultured for 12 months in water temperatures at 1, 7 and 14 8C had significant and interrelated increases in plasma IgM concentration with water temperature. Rijkers et al. (1980) reported that in common carp, Cyprinus carpio, both the humoral immune response (production of anti-sheep red blood cells) and cellular immune response (allograft survival time) changes with water temperature. In contrast, Klesius (1990) found no changes in plasma IgM concentration in channel catfish, Ictalurus punctatus, which were kept at 10 or 30 8C for 30 days. It is possible that the difference in results among various species is partially due to differences in thermal adaptation ability towards increased water temperatures tested. In the present study (using tilapia), the circulating IgM concentration increased with water temperature but decreased with time at the highest water temperature. This result suggests that O. niloticus has an optimal thermal range for immune function. Interestingly, there is a similar tendency for in vitro synthesis of vitellogenin, a female-specific protein (Kim and Takemura, 2003). When the hepatocytes of Oreochromis mossambicus were cultured with estradiol-17h at three different
Fig. 1. Plasma immunoglobulin levels of the Nile tilapia at 2 (a) and 4 weeks (b) after exposure to different water temperature (18.4 as a control, 23, 28 and 33 8C). Values are represented as meanFS.E. (n=7). Significant differences among aquaria are denoted by *pb0.05; ***pb0.001 compared with the control.
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temperatures (23, 28 and 33 8C), the temperature with the highest vitellogenin synthesis was 28 8C, not 23 or 33 8C. Because the optimal temperature for rearing of O. niloticus ranges from 25 to 29 8C, there may be a particular temperature range which is suitable for the production of the circulating proteins. Although the fish may be able to tolerate in the condition of higher temperatures such as 33 8C, there may be decreased ability to react to physiological stress. 3.2. Salinity At 2 weeks after initiation of the seawater treatments, a significant increase in plasma IgM concentration was noted in the fish reared at 24 ppt salinity. No significant differences were observed in the fish reared at 0 and 12 ppt salinity (Fig. 2a). After 4 weeks, the plasma IgM concentration increased significantly in the fish kept in salinities at 12 and 24 ppt (Fig. 2b). In this week, the plasma IgM concentration seemed to decrease from 12 to 24 ppt. However, there was no statistical difference between 12 and 24 ppt. These results suggest that IgM concentration in the plasma of O. niloticus changes after the fish were transferred to different salinities. In rainbow trout, Oncorhynchus mykiss, it has been reported that transfer of the fish from freshwater to 12 ppt or full-strength 29 ppt salinity (Yada et al., 2001) did not alter plasma IgM concentrations. A similar result was reported for O. mossambicus which were transferred from freshwater to 21 or 35 ppt salinity (Yada et al., 2002). In contrast with those results, the results of the present study showed clear changes in IgM concentration with increasing salinity. Because O. mossambicus exhibits higher salinity tolerance than
Fig. 2. Plasma immunoglobulin levels of the Nile tilapia at 2 (a) and 4 weeks (b) after exposure to different water salinity (0 as a control, 12 and 24 ppt). Values are represented as meanFS.E. (n=6). Significant differences among aquaria are denoted by **pb0.01 compared with the control.
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O. niloticus (Popma and Masser, 1999), the difference in the results between the two tilapia species may be partially due to tolerance to high salinity. Additionally, it is possible that fish size is related to high salinity tolerance, because O. mossambicus weighing 50 to 100 g were used in the experiment by Yada et al. (2002). This size was much higher than the fish size used in our experiment (from 18.2 to 21.7 g). A recent study showed that hypophysectomy of rainbow trout reared in freshwater caused a significant reduction in plasma IgM levels but not in lysozyme activity (Yada and Azuma, 2002). Yada et al. (1999) showed that hypophysial stimulation was needed to maintain circulation IgM levels in rainbow trout and that the replacement of prolactin or growth hormone restored plasma IgM to the control levels. In addition, IgM production in male and female rainbow trout decreased during the spawning season when sex steroid hormones normally increased (Suzuki et al., 1997). These results suggest a direct or indirect participation of several hormones in IgM fluctuation. We did not assess the role of the endocrine system for IgM production of O. niloticus. Further studies may be required in order to clarify the relationship of adaptation to environmental changes and exertion of endocrine system in this species. 3.3. Low pH Fig. 3 shows the effect of low pH on plasma concentration of IgM after 2 weeks exposure to low pH condition. There was no significant change in the plasma concentration of IgM between the control (pH 7.9) and the experimental group, suggesting that acid exposure does not affect IgM production after 2 weeks. Balm and Pottinger (1993) reported that when rainbow trout were exposed to acidic conditions (pH 4.0) for 14 days, they survived without activation of the pituitary– interrenal axis and displayed decreases in food consumption, hematocrit values and plasma proteins. Van et al. (1997) reported that in O. mossambicus, there were no significant differences in plasma sodium, chloride, cortisol and glucose between the control and the groups at pH 4.0 for 3, 17 and 37 days. This implies that ionic balance is maintained and that there is no activation of the pituitary–interrenal axis. These results suggest that adaptation to low pH is accompanied by few physiological changes. Therefore, it is
Fig. 3. Plasma immunoglobulin levels of the Nile tilapia at 2 weeks after exposure to water at low pH (7.9 as a control and 4.0). Values are represented as meanFS.E. (n=7).
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concluded that tilapia can acclimate to acidic water at pH 4.0, when the acidification rate is lowered gradually and additional stressors are avoided. Nagae et al. (2001) reported that the plasma IgM concentrations of carp, C. carpio, declined transiently 1 week after acidification (pH 4.5) and returned to the initial concentration at 2 and 4 weeks after acid exposure. Additionally, it was found that after acid exposure, phagocytic rates of peripheral blood leukocytes decreased significantly at 1 week and remained low even at 4 weeks after acid exposure (Nagae et al., 2001). We detected a significant elevation of plasma lysozyme activity at 2 weeks after acid exposure to pH 4.0 (Dominguez et al., unpublished data). Therefore, it is possible that acidification of aquatic condition affects the nonspecific immune activity of certain fish species. 3.4. Suspended solids Increasing delivery of sediment to streams has been recognized as one of the major environmental impacts of human development of land (Waters, 1995). Among many other things, high suspended sediment loads increase treatment costs for domestic and many industrial uses, damage fish food supplies and habitat and can injure fishes directly, depending on the duration and concentration (Newcombe and MacDonald, 1991; Waters, 1995; Newcombe and Jensen, 1996). Increases in sediment loads also can disrupt fish reproductive success by interfering with the viability of their eggs and fry (Waters, 1995). In tropical and subtropical islands, land area is occasionally occupied by red soil. Because red soil is discharged into rivers and carried to coastal areas following heavy rains in these islands, it is thought that red soil discharge results in damage to aquatic organisms and negatively alters the natural environment (Ota, 1994). Randal and Scott (1999) determined that roles of suspended sediment angularity and concentration were contributors to stress and mortality in juvenile coho salmon, Oncorhynchus kisutch, which were exposed to bextremely angularQ and broundQ silicate sediments (N40 g/l) for 96 h. It is possible that an acute effect of suspended solids occurs in the defense system of fishes. In the present study, we checked the effect of red suspended solids on plasma concentration of IgM in O. niloticus. Our results showed that plasma IgM concentration generally increased after exposure of the fish to different concentrations of suspended solids at 20, 200 and 2000 mg/l for 2 week (Fig. 4) but that the difference
Fig. 4. Plasma immunoglobulin levels of the Nile tilapia at 2 weeks after exposure to different concentrations of suspended solids (0 as a control, 20, 200 and 2000 mg/l). Values are represented as meanFS.E. (n=6).
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among the groups was not significantly different from the control fish which were not exposed. These results suggest that under our experimental conditions, the humoral defense system does not change after adaptation to high concentrations of suspended solids. However, it is notable that plasma cortisol level increased significantly within 6 h after exposure of the fish to 2000 mg/l suspended solids and, afterwards, decreased (Dominguez, data not shown). This suggests that high density of suspended solids causes acute but not chronic stress. For the present study, we used well-washed suspended solids. They yielded a pH of 8.0 which is similar to a bbasicQ condition. It seems that the effect of suspended solids is due to only their density because the red soil is normally acidic. Substances causing acidification may be washed out. This means that a composite effect of sedimentation on humoral defense system is not ruled out. It is believed that an increasing duration of exposure to concentration of suspended sediment potentially results in increasing harm to fish, and to other aquatic organisms (Newcombe and MacDonald, 1991; Anderson et al., 1996; Newcombe and Jensen, 1996). Such trend identification is of value in predicting the potential effects of sediments on aquatic organisms.
4. Conclusion This study provides significant evidence that both environmental temperature and salinity can impact the concentration of blood IgM of Nile tilapia. Second, it confirms and extends the hypothesis of Balm and Pottinger (1993) that tilapia exposed to low-pH water for 2 weeks can acclimate to such conditions, if the pH is gradually lowered. Thirdly, the effects of suspended solids on fish depend on characteristics and concentration of sediments as well as duration of sediment exposure. Acknowledgements The authors would like to thank the technical staffs of the Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus. We are also grateful to Dr. G. Curt Fiedler, University of Maryland University College, Asia Japan USAG-J, for his critical reading of the manuscript. References Almeida, J.A., Diniz, Y.S., Marques, S.F., Faine, L.A., Ribas, B.O., Burneiko, R.C., Novelli, E.L., 2002. The use of the oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination. Environ. Int. 27, 673 – 679. Anderson, P.G., Taylor, B.R., Balch, G.C., 1996. Quantifying the effects of sediment release on fish and their habitats. Can. Manuscr. Rep. Fish. Aquat. Sci. 2346, 110. Balm, P.H.M., Pottinger, T.G., 1993. Acclimation of rainbow trout (Oncorhynchus mykiss) to low environmental pH does not involve an activation of the pituitary–interrenal axis, but evokes adjustments in branchial structure. Can. J. Fish. Aquat. Sci. 50, 2532 – 2541.
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Geir, O.M., Heidrun, I.W., 2000. Serum protein and IgM profiles in connection with the smolting and vaccination of out-of-season Atlantic salmon (Salmo salar L.). Aquaculture 188, 189 – 201. Kim, B.H., Takemura, A., 2003. Culture conditions affect induction of vitellogenin synthesis by estradiol-17 h in primary cultures of tilapia hepatocytes. Comp. Biochem. Physiol. 135, 231 – 239. Klesius, P.H., 1990. Effect of size and temperature on the quantity of immunoglobulin in channel catfish, Ictalurus punctatus. Vet. Immunol. Immunopathol. 24, 187 – 195. Magnado´ttir, B., Jo´nsdo´ttir, H., Helgason, S., Bjfrnsson, B., Jbrgensen, T., Pilstrfm, L., 1999. Humoral immune parameters in Atlantic cod (Gadus morhua L.): I. The effects of environmental temperature. Comp. Biochem. Physiol. 122B, 173 – 180. Marc, A., Quentel, C., Severe, A., Le Bail, P., Boeuf, G., 1995. Changes in some endocrinological and nonspecific immunological parameters during seawater exposure in the brown trout. J. Fish Biol. 46, 1065 – 1081. Nagae, M., Ogawa, K., Kawahara, A., Yamaguchi, M., Nishimura, T., Ito, F., 2001. Effect of acidification stress on endocrine and immune functions in carp, Cyprinus carpio. Water, Air Soil Pollut. 130, 893 – 898. Naylor, R.L., Goldberg, R.J., Primavera, J.H., Kautsky, N., Beveridge, M.C.M., Clay, J., Folke, D., Lubchenco, J., Mooney, H., Troell, M., 2000. Effect of aquaculture on world fish supplies. Nature 405, 1017 – 1024. Newcombe, C.P., MacDonald, D.D., 1991. Effects of suspended sediments on aquatic ecosystems. North Am. J. Fish. Manage. 11, 72 – 82. Newcombe, C.P., Jensen, J.O.T., 1996. Channel suspended sediment and fisheries: a synthesis for quantitative assessment of risk and impact. North Am. J. Fish. Manage. 16, 693 – 727. Ota, M., 1994. The Okinawa prefecture red soils erosion prevention ordinance. No. 36, 20 pp. Popma, T., Masser, M., 1999. Tilapia: Life History and Biology. Southern Regional Aquaculture Center Pub. SRAC. 283, 4. Randal, G.L., Scott, G.H., 1999. Acute effects of suspended sediments angulatory on juvenile coho salmon (Oncorhynchus kisutch). Can. J. Fish. Aquat. Sci. 56, 862 – 867. Rijkers, G.T., Frederix-Wolters, E.M.H., Van Muiswinkel, W.B., 1980. The immune system of cyprinid fish. Kinetics and temperature dependence of antibody-producing cells in carp (Cyprinus carpio). Immunology 41, 91 – 97. Suzuki, Y., Otaka, T., Sato, S., Hou, Y., Aida, K., 1997. Reproduction related immunoglobulin changes in rainbow trout. Fish Physiol. Biochem. 17, 415 – 421. Takemura, A., 1993. Changes in an immunoglobulin M (IgM)-like protein during larval stages in tilapia Oreochromis mossambicus. Aquaculture 115, 233 – 241. Van, G., Van, E., Balm, P., Nieveen, M., Van, T., 1997. Tilapia are able to withstand long-term exposure to low environmental pH, judged by their energy status, ionic balance and plasma cortisol. J. Fish Biol. 51, 795 – 806. Waters, T.F., 1995. Sediment in streams: sources, biological effects, and control. American Fisheries Society Monograph vol. 7. Bethesda, MD, 251 pp. Yada, T., Azuma, T., 2002. Hypophysectomy depresses immune functions in rainbow trout. Comp. Biochem. Physiol. 131C, 93 – 100. Yada, T., Nagae, M., Moriyama, S., Azuma, T., 1999. Effects of prolactin and growth hormone on plasma immunoglobulin M levels of hypophysectomized rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 115, 46 – 52. Yada, T., Azuma, T., Takagi, Y., 2001. Stimulation of non-specific immune functions in seawater-acclimated rainbow trout, Oncorhynchus mykiss with reference to the role of growth hormone. Comp. Biochem. Physiol. 129B, 695 – 701. Yada, T., Uchida, K., Kajimura, S., Azuma, T., Hirano, T., Grau, E.G., 2002. Immunomodulatory effects of prolactin and growth hormone in the tilapia, Oreochromis mossambicus. J. Endocrinol. 173, 483 – 493.
Impact of different environmental factors on the circulating immunoglobulin levels in the Nile tilapia, Oreochromis niloticus Miriam Domingueza, Akihiro Takemurab,*, Makoto Tsuchiyaa, Shigeo Nakamurab a
Laboratory of Ecology and Systematic, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan b Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, 3422 Sesoko, Motobu, Okinawa 905-0227, Japan Received 2 February 2004; received in revised form 24 June 2004; accepted 25 June 2004
Abstract Immunoglobulin M (IgM) in teleost fishes is the only component of the specific humoral defense system that is affected by environmental factors. In the present study, we examined the effect of various environmental factors (water temperature, salinity, pH and suspended solids) on the plasma IgM in the Nile tilapia, Oreochromis niloticus. For all treatments, fish were acclimatized in particular environmental conditions for 2 or 4 weeks. For fish reared at 18.4, 23 and 28 8C, the circulating IgM concentration increased with increased water temperature for 2 weeks. Rearing the fish at 33 8C resulted in a decrease in IgM concentration, suggesting that the fish possess an appropriate thermal range for production of immune substances. Plasma level of IgM increased significantly with salinity at 12 and 24 parts per thousand (ppt). On the other hand, plasma IgM concentration did not change by exposing the fish to acidification (pH 4.0) and suspended solids (20, 200, and 2000 mg/l). These results suggest that the specific immune system of tilapia changes by certain factors in aquatic environment. D 2004 Elsevier B.V. All rights reserved. Keywords: ELISA; Environment; Immunoglobulin; Low pH; Salinity; Suspended solids; Tilapia; Water temperature
* Corresponding author. Tel.: +81 980 47 6215; fax: +81 980 47 4919. E-mail address: [email protected] (A. Takemura). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.06.027
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1. Introduction The specific defense system of teleost fishes is continuously affected by periodic or unexpected changes in different factors of the aquatic environment. Fishes under suitable environmental conditions have a functional immune system and, consequently, high disease resistance, growth rate and reproductive activity are expected. On the contrary, unsuitable environmental conditions may act as acute or chronic stressors against fish and cause the suppression of the specific defense system. In the promotion of aquaculture, therefore, it is important to evaluate the relationships between changes in various environmental conditions and the effects on the immune system of fishes. Among different factors in aquatic environments, the effects of water temperature and salinity on the humoral defense system have been well studied. Production of humoral defense substances fluctuates with water temperature (Rijkers et al., 1980; Klesius, 1990; Suzuki et al., 1997; Magnado´ttir et al., 1999) and salinity (Marc et al., 1995; Yada et al., 1999, 2001, 2002; Yada and Azuma, 2002). Additionally, aquatic pollution often results in unexpected changes in the aquatic environment and occasionally affects the immune system of fishes (Almeida et al., 2002). In general, it seems that each fish species has a specific tolerance range with respect to different environmental conditions. To date, only a few studies have been done on adaptive changes in the humoral defense system under various environmental conditions in tilapia (genus Oreochromis), which are native to Africa and have been introduced as economically important aquaculture finfish to many countries (Naylor et al., 2000). The aim of the present study is to evaluate whether environmental changes affect the circulating immunoglobulin M (IgM) which is the only component of the specific humoral defense system in teleost fishes and can be detected in the blood circulation (Geir and Heidrun, 2000) as well as body surface (Suzuki et al., 1997). Using the Nile tilapia, Oreochromis niloticus, we assessed impact of water temperature and salinity on the circulating IgM. In addition, we examined the effects of low pH and suspended solids which are also critical environmental factors in some aquatic habitats.
2. Materials and methods 2.1. Fish The Nile tilapia, O. niloticus, used in the present study were caught from a pond near Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa, Japan, using a cast net. Their body mass ranged from 18.2 to 21.7 g. They were kept in tanks (1-metric ton capacity) supplied with filtered and aerated freshwater at the Sesoko Station. All the fish were acclimatized in the tanks at least for 2 weeks before the onset of the following experiments. All the experiments were repeated several times. 2.2. Effect of water temperature To investigate the effect of water temperature on plasma IgM levels, the fish (25 each) were transferred to four aquaria (60-l capacity) with a filtration and aeration system.
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Following acclimatization of the fish to basic experimental conditions for 2 weeks, water temperature in the three aquaria gradually increased to 23, 28 or 33 8C within 24 h and kept at respective temperature until samplings. One tank was kept at ambient temperatures which ranged between 16.9 and 20.3 8C (mean: 18.4 8C). Seven fish were taken from each aquarium at 0, 2 and 4 weeks after the onset of experiment and immediately anesthetized with 2-phenoxyethanol (Kanto Chemical, Tokyo, Japan). Blood samples from each fish were collected in heparinized capillary tubes (Terumo, Tokyo, Japan) from the caudal vasculature by cutting the tail. Plasma was obtained by centrifugation of the blood sample at 9600g, for 5 min, and stored at 30 8C until experimental analyses. 2.3. Effect of salinity Because preliminary experiments showed that all the fish died in full-strength salinity (35 parts per thousand, ppt), the highest salinity of the present experiment was set at 24 ppt. To investigate the effect of salinity on IgM levels, 25 fish each were transferred to three aquaria. Following acclimatization of the fish in the aquaria with freshwater under basic experimental conditions for 2 weeks, salinity of water in the aquarium was adjusted to 0, 12 and 24 ppt by diluting seawater with freshwater. Increase of salinities was done within 24 h. Water temperature of each aquarium was maintained at 28 8C and half of the water was changed every 2 days. Blood was collected from six individuals at 0, 2 and 4 weeks after salinity treatments were started, and plasma preparations were done as above. 2.4. Effect of low pH In preliminary experiments, O. niloticus were reared in different low pH conditions (pH 3.0, 4.0 and 5.0) for 2 weeks. Because rearing the fish at pH 3.0, but not pH 4.0, caused death in all the individuals, the lower limit for experimental pH was set at pH 4.0. Prior to initiation of the experiment, the fish (15 each) were transferred to two aquaria and were acclimatized in freshwater (pH 7.9) under basic experimental conditions for 2 weeks. Acidic condition (pH 4.0) was created within 24 h by adding 3 mol/l HCl to the aquaria and verified using a digital pH meter (HM-21P, Horiba, Tokyo, Japan). The control aquarium with 15 fish was kept at pH 7.9 during the experiment. The aquaria were aerated vigorously and 1/3 of water in each aquarium was changed every 2 days. Blood was collected from seven individuals at 0 and 2 weeks after the low-pH treatment started. Plasma preparations were done as indicated above. 2.5. Effect of suspended solids A sample of red soil was collected from the northern part of Okinawa Island. After vigorous washing and sieving (64 Am), the suspended solids were dried for 24 h at 60 8C with a drying oven (Isuzu, Tokyo, Japan). The fish (15 each) were transferred to four aquaria and were acclimatized in the aquaria under basic experimental conditions for 2 weeks. Within 6 h, dried sediment was added to each of the four aquaria at concentrations of 0, 20, 200 and 2000 mg/l. Suspension of red sediment was maintained by circulating
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water column. Blood was collected from six individuals at 0 and 2 weeks after sediment exposure treatment. Plasma preparations were done as before. 2.6. Measurement of plasma immunoglobulin M concentration Total IgM concentration in the plasma of tilapia was measured by enzyme-linked immunosorbent assay (ELISA) according to the method of Takemura (1993). Purified tilapia IgM, rabbit anti-tilapia IgM antibody (a-IgM) and a-IgM labeled with horseradish peroxidase (a-IgM HRP) was prepared in advance (Takemura, 1993). Each well of a 96-well microtiter plate (Becton Dickinson Labware, Franklin Lakes, NJ) was coated with 100 Al of a-IgM (6.8 Ag/ml) in 0.05 M sodium carbonate buffer, pH 9.6, and incubated for 2 h at 25 8C. Residual protein binding sites were blocked by adding 200 Al of 1 % gelatin (BioRad Laboratories, Richmond, CA) dissolved in 10 mM phosphate-buffered saline (PBS), pH 7.4, containing 0.05% Tween 20 (PBS–Tween) to the wells for 60 min at 25 8C. After washing the wells three times with PBS–Tween using a plate washer (Immunowash 1573, BioRad), 100 Al of plasma sample (1:10 000) or standards (serial dilution of purified tilapia IgM) was added to the well and then incubated overnight at 4 8C. All the dilutions were made with PBS–Tween. After washing the wells three times with PBS–Tween, 100 Al of a-IgM HRP (diluted 1:20 000 in PBS–Tween) was added to the wells and incubated for 2 h at 25 8C. After three successive washes with PBS–Tween, peroxidase activity was measured by adding 100 Al of 100 mM citrate buffer, pH 4.5, containing 0.01% o-phenylenediamine dihydrochloride (Sigma, St. Louis, MO) and 0.04% H2O2. Following 30-min incubation at 25 8C, the enzymatic reaction was stopped by adding 25 Al of 4 N H2SO4. The optical density of each sample was determined at 490 nm using a microplate reader 550 (BioRad). Computer software (Microplate Manager III, version 1.57) was used for conversion from optical density to IgM concentration. 2.7. Statistical analysis The plasma IgM concentration data were expressed as meanFS.E. Two-way analysis of variance (ANOVA) and Fisher’s protected least significance difference (PLSD) test were used to determine the statistical differences for the water temperature, salinity and suspended solids experiments. The Student’s t-test was also performed for analysis of IgM concentrations in the low-pH experiment.
3. Results and discussion 3.1. Water temperature Total IgM concentration in the blood circulation was compared among the fish that were kept at various water temperatures, 18.4 (control), 23, 28 and 33 8C. At 2 weeks after exposure, the plasma IgM concentration of the control fish (18.4 8C) was 168.64F29.73 Ag/ml. For the other temperature treatments, IgM concentration generally increased with
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water temperature after 2 weeks. However, only the fish kept at 28 and 33 8C had significantly higher IgM concentrations than the control fish. IgM concentration of the fish reared at 33 8C was not significantly different than the fish reared at 28 8C (Fig. 1a). Water temperature had similar effects on plasma IgM concentration at 4 weeks (Fig. 1b) for the fish kept at 28 8C. However, the concentration at 338C was significantly lower and did not differ from the control. The influence of water temperature on fluctuation of plasma IgM concentration has been previously studied in certain teleost fishes (Rijkers et al., 1980; Klesius, 1990; Suzuki et al., 1997; Magnado´ttir et al., 1999). Magnado´ttir et al. (1999) reported that Atlantic cod, Gadus morhua L., cultured for 12 months in water temperatures at 1, 7 and 14 8C had significant and interrelated increases in plasma IgM concentration with water temperature. Rijkers et al. (1980) reported that in common carp, Cyprinus carpio, both the humoral immune response (production of anti-sheep red blood cells) and cellular immune response (allograft survival time) changes with water temperature. In contrast, Klesius (1990) found no changes in plasma IgM concentration in channel catfish, Ictalurus punctatus, which were kept at 10 or 30 8C for 30 days. It is possible that the difference in results among various species is partially due to differences in thermal adaptation ability towards increased water temperatures tested. In the present study (using tilapia), the circulating IgM concentration increased with water temperature but decreased with time at the highest water temperature. This result suggests that O. niloticus has an optimal thermal range for immune function. Interestingly, there is a similar tendency for in vitro synthesis of vitellogenin, a female-specific protein (Kim and Takemura, 2003). When the hepatocytes of Oreochromis mossambicus were cultured with estradiol-17h at three different
Fig. 1. Plasma immunoglobulin levels of the Nile tilapia at 2 (a) and 4 weeks (b) after exposure to different water temperature (18.4 as a control, 23, 28 and 33 8C). Values are represented as meanFS.E. (n=7). Significant differences among aquaria are denoted by *pb0.05; ***pb0.001 compared with the control.
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temperatures (23, 28 and 33 8C), the temperature with the highest vitellogenin synthesis was 28 8C, not 23 or 33 8C. Because the optimal temperature for rearing of O. niloticus ranges from 25 to 29 8C, there may be a particular temperature range which is suitable for the production of the circulating proteins. Although the fish may be able to tolerate in the condition of higher temperatures such as 33 8C, there may be decreased ability to react to physiological stress. 3.2. Salinity At 2 weeks after initiation of the seawater treatments, a significant increase in plasma IgM concentration was noted in the fish reared at 24 ppt salinity. No significant differences were observed in the fish reared at 0 and 12 ppt salinity (Fig. 2a). After 4 weeks, the plasma IgM concentration increased significantly in the fish kept in salinities at 12 and 24 ppt (Fig. 2b). In this week, the plasma IgM concentration seemed to decrease from 12 to 24 ppt. However, there was no statistical difference between 12 and 24 ppt. These results suggest that IgM concentration in the plasma of O. niloticus changes after the fish were transferred to different salinities. In rainbow trout, Oncorhynchus mykiss, it has been reported that transfer of the fish from freshwater to 12 ppt or full-strength 29 ppt salinity (Yada et al., 2001) did not alter plasma IgM concentrations. A similar result was reported for O. mossambicus which were transferred from freshwater to 21 or 35 ppt salinity (Yada et al., 2002). In contrast with those results, the results of the present study showed clear changes in IgM concentration with increasing salinity. Because O. mossambicus exhibits higher salinity tolerance than
Fig. 2. Plasma immunoglobulin levels of the Nile tilapia at 2 (a) and 4 weeks (b) after exposure to different water salinity (0 as a control, 12 and 24 ppt). Values are represented as meanFS.E. (n=6). Significant differences among aquaria are denoted by **pb0.01 compared with the control.
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O. niloticus (Popma and Masser, 1999), the difference in the results between the two tilapia species may be partially due to tolerance to high salinity. Additionally, it is possible that fish size is related to high salinity tolerance, because O. mossambicus weighing 50 to 100 g were used in the experiment by Yada et al. (2002). This size was much higher than the fish size used in our experiment (from 18.2 to 21.7 g). A recent study showed that hypophysectomy of rainbow trout reared in freshwater caused a significant reduction in plasma IgM levels but not in lysozyme activity (Yada and Azuma, 2002). Yada et al. (1999) showed that hypophysial stimulation was needed to maintain circulation IgM levels in rainbow trout and that the replacement of prolactin or growth hormone restored plasma IgM to the control levels. In addition, IgM production in male and female rainbow trout decreased during the spawning season when sex steroid hormones normally increased (Suzuki et al., 1997). These results suggest a direct or indirect participation of several hormones in IgM fluctuation. We did not assess the role of the endocrine system for IgM production of O. niloticus. Further studies may be required in order to clarify the relationship of adaptation to environmental changes and exertion of endocrine system in this species. 3.3. Low pH Fig. 3 shows the effect of low pH on plasma concentration of IgM after 2 weeks exposure to low pH condition. There was no significant change in the plasma concentration of IgM between the control (pH 7.9) and the experimental group, suggesting that acid exposure does not affect IgM production after 2 weeks. Balm and Pottinger (1993) reported that when rainbow trout were exposed to acidic conditions (pH 4.0) for 14 days, they survived without activation of the pituitary– interrenal axis and displayed decreases in food consumption, hematocrit values and plasma proteins. Van et al. (1997) reported that in O. mossambicus, there were no significant differences in plasma sodium, chloride, cortisol and glucose between the control and the groups at pH 4.0 for 3, 17 and 37 days. This implies that ionic balance is maintained and that there is no activation of the pituitary–interrenal axis. These results suggest that adaptation to low pH is accompanied by few physiological changes. Therefore, it is
Fig. 3. Plasma immunoglobulin levels of the Nile tilapia at 2 weeks after exposure to water at low pH (7.9 as a control and 4.0). Values are represented as meanFS.E. (n=7).
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concluded that tilapia can acclimate to acidic water at pH 4.0, when the acidification rate is lowered gradually and additional stressors are avoided. Nagae et al. (2001) reported that the plasma IgM concentrations of carp, C. carpio, declined transiently 1 week after acidification (pH 4.5) and returned to the initial concentration at 2 and 4 weeks after acid exposure. Additionally, it was found that after acid exposure, phagocytic rates of peripheral blood leukocytes decreased significantly at 1 week and remained low even at 4 weeks after acid exposure (Nagae et al., 2001). We detected a significant elevation of plasma lysozyme activity at 2 weeks after acid exposure to pH 4.0 (Dominguez et al., unpublished data). Therefore, it is possible that acidification of aquatic condition affects the nonspecific immune activity of certain fish species. 3.4. Suspended solids Increasing delivery of sediment to streams has been recognized as one of the major environmental impacts of human development of land (Waters, 1995). Among many other things, high suspended sediment loads increase treatment costs for domestic and many industrial uses, damage fish food supplies and habitat and can injure fishes directly, depending on the duration and concentration (Newcombe and MacDonald, 1991; Waters, 1995; Newcombe and Jensen, 1996). Increases in sediment loads also can disrupt fish reproductive success by interfering with the viability of their eggs and fry (Waters, 1995). In tropical and subtropical islands, land area is occasionally occupied by red soil. Because red soil is discharged into rivers and carried to coastal areas following heavy rains in these islands, it is thought that red soil discharge results in damage to aquatic organisms and negatively alters the natural environment (Ota, 1994). Randal and Scott (1999) determined that roles of suspended sediment angularity and concentration were contributors to stress and mortality in juvenile coho salmon, Oncorhynchus kisutch, which were exposed to bextremely angularQ and broundQ silicate sediments (N40 g/l) for 96 h. It is possible that an acute effect of suspended solids occurs in the defense system of fishes. In the present study, we checked the effect of red suspended solids on plasma concentration of IgM in O. niloticus. Our results showed that plasma IgM concentration generally increased after exposure of the fish to different concentrations of suspended solids at 20, 200 and 2000 mg/l for 2 week (Fig. 4) but that the difference
Fig. 4. Plasma immunoglobulin levels of the Nile tilapia at 2 weeks after exposure to different concentrations of suspended solids (0 as a control, 20, 200 and 2000 mg/l). Values are represented as meanFS.E. (n=6).
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among the groups was not significantly different from the control fish which were not exposed. These results suggest that under our experimental conditions, the humoral defense system does not change after adaptation to high concentrations of suspended solids. However, it is notable that plasma cortisol level increased significantly within 6 h after exposure of the fish to 2000 mg/l suspended solids and, afterwards, decreased (Dominguez, data not shown). This suggests that high density of suspended solids causes acute but not chronic stress. For the present study, we used well-washed suspended solids. They yielded a pH of 8.0 which is similar to a bbasicQ condition. It seems that the effect of suspended solids is due to only their density because the red soil is normally acidic. Substances causing acidification may be washed out. This means that a composite effect of sedimentation on humoral defense system is not ruled out. It is believed that an increasing duration of exposure to concentration of suspended sediment potentially results in increasing harm to fish, and to other aquatic organisms (Newcombe and MacDonald, 1991; Anderson et al., 1996; Newcombe and Jensen, 1996). Such trend identification is of value in predicting the potential effects of sediments on aquatic organisms.
4. Conclusion This study provides significant evidence that both environmental temperature and salinity can impact the concentration of blood IgM of Nile tilapia. Second, it confirms and extends the hypothesis of Balm and Pottinger (1993) that tilapia exposed to low-pH water for 2 weeks can acclimate to such conditions, if the pH is gradually lowered. Thirdly, the effects of suspended solids on fish depend on characteristics and concentration of sediments as well as duration of sediment exposure. Acknowledgements The authors would like to thank the technical staffs of the Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus. We are also grateful to Dr. G. Curt Fiedler, University of Maryland University College, Asia Japan USAG-J, for his critical reading of the manuscript. References Almeida, J.A., Diniz, Y.S., Marques, S.F., Faine, L.A., Ribas, B.O., Burneiko, R.C., Novelli, E.L., 2002. The use of the oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination. Environ. Int. 27, 673 – 679. Anderson, P.G., Taylor, B.R., Balch, G.C., 1996. Quantifying the effects of sediment release on fish and their habitats. Can. Manuscr. Rep. Fish. Aquat. Sci. 2346, 110. Balm, P.H.M., Pottinger, T.G., 1993. Acclimation of rainbow trout (Oncorhynchus mykiss) to low environmental pH does not involve an activation of the pituitary–interrenal axis, but evokes adjustments in branchial structure. Can. J. Fish. Aquat. Sci. 50, 2532 – 2541.
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