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Antibody-producing cells correlated to body weight in juvenile chinook salmon (Oncorhynchus tshawytscha) acclimated to optimal and elevated temperatures
Fish & Shellfish Immunology (2001) 11, 653–659 doi:10.1006/fsim.2001.0342 Available online at http://www.idealibrary.com on
Antibody-producing cells correlated to body weight in juvenile chinook salmon (Oncorhynchus tshawytscha) acclimated to optimal and elevated temperatures* LAURA N. M. HARRAHY1, CARL B. SCHRECK1
AND
ALEC G. MAULE2
1
Oregon Cooperative Fish and Wildlife Research Unit, †Department of Fisheries and Wildlife, and U.S. Geological Survey, Oregon State University, Corvallis, OR 97331, U.S.A. and 2U.S. Geological Survey, Biological Resources Division, Columbia River Research Laboratory, 5501A Cook-Underwood Road, Cook, WA 98605, U.S.A. (Received 11 September 2000, accepted after revision 2 February 2001) The immune response of juvenile chinook salmon (Oncorhynchus tshawytscha) ranging in weight from approximately 10 to 55 g was compared when the fish were acclimated to either 13 or 21 C. A haemolytic plaque assay was conducted to determine di#erences in the number of antibody-producing cells (APC) among fish of a similar age but di#erent body weights. Regression analyses revealed significant increases in the number of APC with increasing body weight when fish were acclimated to either water temperature. These results emphasise the importance of standardising fish weight in immunological studies of salmonids before exploring the possible e#ects of acclimation temperatures. 2001 Academic Press Key words:
antibody production, body weight, temperature, chinook salmon, immune response.
I. Introduction Development and maturation of the immune system in fish is complex and can have high intraspecific variability. The ability of young fish to resist disease can depend on age, ontogeny, body size and external temperature (Wishkovsky et al., 1987; Bly & Clem, 1992; Schreck, 1996; Tatner, 1996; Schroder et al., 1998). While several studies have demonstrated di#erences in the specific immune function of vertebrates varying in age or stages of physical development (Tatner & Manning, 1983a; Castillo et al., 1993; Ravichandran et al., 1994; Saad et al., 1994), others have indicated the importance of the influence of body size (Johnson et al., 1982a, b; Nakanishi, 1983, 1991; Bootland et al., 1990; Tatner, 1996) or organ tissue weight (Tatner & Manning, 1983b) when assessing immune function. This study explores the possibility that body size may a#ect the specific immune response in juvenile chinook salmon regardless of acclimation temperature. *Oregon Agricultural Experiment Station Technical Report No. 11640. †Supported co-operatively by Oregon State University, the U.S.G.S. and the Oregon Department of Fish and Wildlife. 1050–4648/01/080653+07 $35.00/0
653
2001 Academic Press
654
L. N. M. HARRAHY ET AL.
The haemolytic plaque assay has been widely employed to assess immune system function since its development by Jerne & Nordin (1963). The assay measures the ability of B lymphocytes in vitro to produce plasma cells that release specific antibody in response to an antigen. Modifications of the assay are useful for assessing adaptive immunity in fish (Smith et al., 1967; Cunningham & Szenberg, 1968; Chiller et al., 1969; Rijkers et al., 1980; Tripp et al., 1987; Anderson, 1990; Arkoosh & Kaattari, 1992). The goal of this study was to provide a cautionary warning to researchers participating in studies involving temperature and adaptive immunity in fish. We hypothesised that juvenile chinook salmon of similar age, but di#ering size, di#ered in their ability to generate specific antibody-producing cells (APC) when acclimated to optimal and elevated temperatures. We had the opportunity to test for a potential correlation of APC with body size by taking advantage of data collected as part of an experiment in which juvenile chinook salmon were acclimated to two environmentally realistic temperatures. Chinook salmon acclimated to 13 C, the optimum temperature for this species, were compared to those acclimated to 21 C, an elevated temperature not uncommon in the northwestern U.S.A. during the late summer months (Brett, 1952; Armour, 1991). II. Materials and Methods Yearling spring chinook salmon parr were maintained at Oregon State University’s Fish Performance and Genetics Laboratory in 0·9 m circular tanks, with 2 l min 1 flow-through well water. Forty fish per tank were assigned randomly to three tanks at 13 C (control) or to three tanks at 21 C (treatment). Treatment fish were slowly acclimated from 13 to 21 C by temperature increases of 2 C per day, over 4 days. Temperatures were monitored every 10 min in control and treatment tanks with electronic thermometers. All fish were maintained in their experimental tanks for a total of 3 weeks to allow su$cient time for acclimation. Although each of the selected fish were the same age and reared under identical conditions in the same stock trough, fish sizes were quite variable, ranging from approximately 10 to 55 g. All fish were fed a standardised ration of Oregon Moist Pellet twice daily. In order to standardise the amount of food required for energy consumption at each temperature, food quantities o#ered were set according to the feeding guide recommendations from the food manufacturer (BioDiet). Fish acclimated to 21 C received approximately 3·1% of their body weight each day, while fish acclimated to 13 C received 2·3%. These temperaturedependent feeding rations were also consistent with the recommendations by Brett & Groves (1979) in their description of physiological energetics. Finally, all fish were pre-conditioned to the presence of a dip net by swirling a net inside their tanks prior to each feeding. This procedure helped to reduce the amount of chasing during capture of the fish at sampling time. Sampling began on 4 November 1998. Control and treatment fish were sampled in triplicate over 3 days (one tank at each temperature sampled per day), in order to allow su$cient time for the sampling scheme. Head-kidney
ANTIBODY-PRODUCING CELLS IN CHINOOK SALMON
655
samples from fish acclimated to each temperature were collected at two di#erent sampling times each day. The fish were rapidly removed with a dip net and immediately killed by an anesthetic overdose of tricaine methanesulfanate (MS222) at 200 mg l 1 bu#ered with NaHCO3 . Each fish was then measured and weighed and the head-kidneys were removed under sterile conditions and placed into individual tubes containing 500 l of tissue culture medium (TCM). The TCM consisted of 10% foetal bovine serum, 1% L-glutamine and 0·1% gentamicin sulfate in RPMI 1640. A single cell suspension was made by gently aspirating the tissue and the medium with a sterile 1·0 cc syringe. Ten l of supernatant was transferred to a haemocytometer and leukocytes from each head kidney were counted. The number of leucocytes per head-kidney was standardised by adding or removing the proper amount of TCM for the number of cells counted in each sample. A haemolytic plaque assay was then conducted to determine the number of APC. The haemolytic plaque assay employed was as described by Tripp et al. (1987) and modified by Slater et al. (1995). Briefly, 50 l of picrylsulfonic acid-lipopolysaccharide (TNP-LPS) antigen was added to 50 l of head-kidney cells in culture in a 96 well plate. Duplicate in vitro samples were taken for each fish. The 96 well plate was secured in an incubation chamber containing blood gas and the cells were incubated at 17 C for 7 days. Following the incubation period, the cells were washed with 50 l of fresh TCM and mixed with 10 l of haptenated sheep red blood cells (SRBC) (SRBC coated in TNP antigen) and 10 l of complement (contained in rainbow trout serum). The cell mixtures were placed into Cunningham chambers (Cunningham & Szenberg, 1968) incubated at 17 C for 1·5 h, and plaques were then counted. Antibody production was determined by the number of APC per final pre-incubation leucocyte concentration in tissue culture. The APC data for both acclimation temperatures were tested for di#erences between the two sampling times each day with an analysis of variance (ANOVA), found to be insignificant (P>0·1), and pooled appropriately. Likewise, the number of APC were tested for di#erences between replicate sampling runs, also found to be insignificant (ANOVA, P>0·1), and the data were pooled. Fish weight and the number of APC from a total sample size of 28 fish were regressed for each acclimation temperature and for both temperatures combined. After determining that the linear model best fit the data, regression analyses were performed to examine possible correlations between fish weight and the number of APC. Finally, after determining that body size was not dependent on acclimation temperature (ANOVA, P>0·1), an analysis of covariance (ANCOVA) was run to test for temperature di#erences in the number of APC with fish weight included in the model as a covariate. III. Results and Discussion Significant, positive linear regressions were found for the number of APC and final weight in fish acclimated to 13 C (P=0·021) (Fig. 1a) and 21 C (P=0·0006) (Fig. 1b). No di#erences were found in the number of APC between fish acclimated to 13 and 21 C, and the number of APC in fish was dependent on the weight of the individual fish, regardless of acclimation temperature.
656
L. N. M. HARRAHY ET AL.
1000 (a) APC
800 600 400 200 0
20
40
60
40
60
40
60
Weight (g) 1000 (b) APC
800 600 400 200 0
20 Weight (g)
1000 (c) APC
800 600 400 200 0
20 Weight (g)
Fig. 1. Linear regressions of (a) antibody-producing cells (APC) per 106 leucocytes and fish weight of juvenile chinook salmon acclimated to 13 C and (b) 21 C (n=28). (c) A significant e#ect of fish weight on the number of APC is also found when data of fish acclimated to each temperature are combined (linear regression, P=0·0003) (n=56). Each point represents an individual fish.
The data from both acclimation temperatures were thus combined to reveal a significant, positive linear regression in APC and fish weight (P=0·0003) (Fig. 1c). These findings indicate that care should be taken to account for body size in immunological experiments involving antibody production in salmonids. These results are consistent with Nakanishi’s conclusion in his study of the marine teleost, Sebastiscus marmoratus, that larger fish tend to have a better immune response than smaller fish (Nakanishi, 1991). Although there were no di#erences in the regressions of fish weight and the production of specific antibody between acclimation temperatures, ANCOVA revealed significant di#erences in the number of APC when fish weight was included in the model as a covariate. In this case, fish acclimated to 21 C had a significantly higher number of APC than fish of the same weight acclimated
ANTIBODY-PRODUCING CELLS IN CHINOOK SALMON
657
to 13 C (P=0·034). Other studies have described how the immune response can be controlled by external temperature (Clem et al., 1984, 1991; Bly & Clem, 1992; Tatner, 1996). For example, the immune responses of channel catfish have been altered by both in vivo and in vitro temperature fluctuations. Lower temperatures tended to have an inhibitory e#ect on the production of specific antibody and warmer temperatures resulted in higher antibody responses (Clem et al., 1984; Bly & Clem, 1992). Our data support this outcome when the weight of the fish is accounted for in the analysis. Other researchers using the plaque assay for studies with salmonids (Tripp et al., 1987; Maule et al., 1989; Slater et al., 1995) may not have found an e#ect of body size on antibody production if size variation in the experimental fish was small. We are, at present, unable to explain why smaller fish of the same age tend to produce lymphocytes that are less responsive to the antigen in vitro. It has, however, been suggested that dominant fish, which generally tend to be larger, may stress smaller subordinate fish (Peters et al., 1991). Exhibition of such a stress response in small fish may lead to immunosuppression (Tripp et al., 1987; Maule et al., 1989). Though it is possible that the positive correlation of APC to body weight may have been driven by developmental stage, our results support Nakanishi’s and Tatner’s conclusions that body size, rather than age, is a better determinant of immunological maturity during ontogeny (Nakanishi, 1991; Tatner, 1996). Furthermore, they demonstrate that the influence of acclimation temperature on the adaptive immune system may be a#ected by body size. Our findings indicate that caution should be executed when selecting fish of similar age but varying body sizes for immunological studies in salmonids. We thank Rob Chitwood, manager of the Fish Performance and Genetics Laboratory at O.S.U. for his assistance with establishing the experimental conditions. We also thank Beth Siddens and Darren Lerner for aiding in sample collections and Wilfrido Contreras-Sanches for his guidance with data analysis.
References Anderson, D. P. (1990). Passive hemolytic plaque assay for detecting antibodyproducing cells in fish. In Techniques in Fish Immunology (J. S. Stolen, T. C. Fletcher, D. P. Anderson, B. S. Robertson & W. B. van Muiswinkle, eds) pp. 9–13. Fair Haven, NJ: SOS Publications. Arkoosh, M. R. & Kaattari, S. L. (1992). Induction of trout antibody-producing cells in microculture. In Techniques in Fish Immunology (J. S. Stolen, T. C. Fletcher, D. P. Anderson, S. L. Kaattari & A. F. Rowley, eds) pp. 67–77. Fair Haven, NJ: SOS Publications. Armour, C. L. (1991). Guidance for evaluating and recommending temperature regimes to protect fish. Instream Flow Information Paper. U.S. Fish and Wildlife Service, Fort Collins, CO. pp. 1–13. Bly, J. E. & Clem, L. W. (1992). Temperature and teleost immune functions. Fish & Shellfish Immunology 2, 159–171. Bootland, L. M., Dobos, P. & Stevenson, R. M. W. (1990). Fry age and size e#ects on immersion immunization of brook trout, Salvelinus fontinalis Mitchell, against infectious pancreatic necrosis virus. Journal of Fish Diseases 13, 113–125. Brett, J. R. (1952). Temperature tolerance in young Pacific salmon genus Oncorhynchus. Journal of the Fisheries Resources Board of Canada 6, 265–323.
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Brett, J. R. & Groves, T. D. D. (1979). Physiological Energetics. In Fish Physiology, 8 (W. S. Hoar & D. J. Randall, eds) pp. 279–352. New York, San Francisco, London: Academic Press, Inc. Castillo, A., Sanchez, C., Dominguez, J., Kaattari, S. L. & Villena, A. J. (1993). Ontogeny of IgM and IgM-bearing cells in rainbow trout. Developmental and Comparative Immunology 17, 419–424. Chiller, J., Hodgins, H., Chambers, V. & Weiser, R. (1969). Immuno-competent cells in the spleen and anterior kidney. Journal of Immunology 102, 1193–1201. Clem, L. W., Faulmann, E., Miller, N., Ellsaesser, C. F. R., Lobb, C. J. & Cuchens, M. A. (1984). Temperature-mediated processes in teleost immunity: di#erential e#ects of in vitro and in vivo temperatures on mitogenic responses of channel catfish lymphocytes. Developmental and Comparative Immunology 8, 313–322. Clem, L. W., Miller, N. W. & Bly, J. E. (1991). Evolution of lymphocyte subpopulations: their interactions and temperature sensitivity. In The Phylogenesis of Immune Functions (N. Cohen & G. Warr, eds) pp. 191–213. Boca Raton, FL: CRC Press. Cunningham, A. J. & Szenberg, A. (1968). Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 599–601. Jerne, N. K. & Nordin, A. A. (1963). Plaque formation in agar by single antibodyproducing cells. Science 140, 405. Johnson, K. A., Flynn, J. K. & Amend, D. F. (1982a). Onset of immunity in salmonid fry vaccinated by direct immersion in Vibrio anguillarum and Yersinia ruckeri bacterins. Journal of Fish Diseases 5, 197–206. Johnson, K. A., Flynn, J. K. & Amend, D. F. (1982b). Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckeri and Vibrio anguillarum bacterins. Journal of Fish Diseases 5, 207–214. Maule, A. G., Tripp, R. A., Kaattari, S. L. & Schreck, C. B. (1989). Stress alters the immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha). Journal of Endocrinology 120, 135–142. Nakanishi, T. (1983). Studies on the role of the lymphoid organs in the immune responses of the marine teleost, Sebastiscus marmoratus. Ph.D. Thesis, University of Hokkaido, Japan. Nakanishi, T. (1991). Ontogeny of the immune system in Sebastiscus marmoratus: histogenesis of the lymphoid organs and e#ects of thymectomy. Environmental Biology of Fish 30, 135–145. Peters, G., Nubgen, A., Roobe, A. & Mock, A. (1991). Social stress induces structural and functional alterations of phagocytes in rainbow trout, Oncorhynchus mykiss. Fish & Shellfish Immunology 1, 17–32. Ravichandran, K. S., Osborne, B. A. & Goldsby, R. A. (1994). Quantitative analysis of the B cell repertoire by limiting dilution analysis and fluorescent in situ hybridization. Cell Immunology 154, 309–327. Rijkers, G. T., Frederix-Wolters, E. M. H. & van Muiskinkel, W. B. (1980). The haemolytic plaque assay in carp (Cyprinus carpio). Journal of Immunological Methods 33, 79–86. Saad, A. H., Mansour, M. H., Dorgham, V. & Badir, N. (1994). Age-related changes in the immune response of Bufo viridis. Developmental and Comparative Immunology Supplement 18, 597A. Schreck, C. B. (1996). Immunomodulation: Endogenous Factors. In The Fish Immune System: Organism, Pathogen, and Environment (G. Iwama & T. Nakanishi, eds) pp. 311–337. San Diego, CA: Academic Press, Inc. Schroder, M. B., Villena, A. J. & Jorgensen, T. O. (1998). Ontogeny of lymphoid organs and immunoglobulin producing cells in Atlantic cod (Gadus morhua L.). Developmental and Comparative Immunology 22, 507–517. Slater, C. H., Fitzpatrick, M. S. & Schreck, C. B. (1995). Androgens and immunocompetence in salmonids: specific binding in and reduced immunocompetence of salmonid lymphocytes exposed to natural and synthetic androgens. Aquaculture 136, 363–370.
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Smith, A. M., Potter, M. & Merchant, E. B. (1967). Antibody-forming cells in the pronephros of the teleost Lepomis macrochirus. Journal of Immunology 99, 876–882. Tatner, M. F. (1996). Natural changes in the immune system of fish. In The Fish Immune System: Organism, Pathogen, and Environment (G. Iwama & T. Nakanishi, eds) pp. 255–287. San Diego, CA: Academic Press, Inc. Tatner, M. F. & Manning, M. J. (1983a). The ontogeny of cellular immunity in the rainbow trout, Salmo gairdneri Richardson, in relation to the stage of development of the lymphoid organs. Developmental and Comparative Immunology 7, 69–75. Tatner, M. F. & Manning, M. J. (1983b). Growth of the lymphoid organs in rainbow trout, Salmo gairdneri Richardson from one to fifteen months of age. Journal of Zoology 199, 503–520. Tripp, R. A., Maule, A. G., Schreck, C. B. & Kaattari, S. L. (1987). Cortisol mediated suppression of salmonid lymphocyte responses in vitro. Developmental and Comparative Immunology 11, 565–576. Wishkovsky, A., Garber, N. & Avtalion, R. R. (1987). The e#ects of fish age on the mortality caused by selected fish pathogens. Journal of Fish Biology 31, 243–244.
Antibody-producing cells correlated to body weight in juvenile chinook salmon (Oncorhynchus tshawytscha) acclimated to optimal and elevated temperatures* LAURA N. M. HARRAHY1, CARL B. SCHRECK1
AND
ALEC G. MAULE2
1
Oregon Cooperative Fish and Wildlife Research Unit, †Department of Fisheries and Wildlife, and U.S. Geological Survey, Oregon State University, Corvallis, OR 97331, U.S.A. and 2U.S. Geological Survey, Biological Resources Division, Columbia River Research Laboratory, 5501A Cook-Underwood Road, Cook, WA 98605, U.S.A. (Received 11 September 2000, accepted after revision 2 February 2001) The immune response of juvenile chinook salmon (Oncorhynchus tshawytscha) ranging in weight from approximately 10 to 55 g was compared when the fish were acclimated to either 13 or 21 C. A haemolytic plaque assay was conducted to determine di#erences in the number of antibody-producing cells (APC) among fish of a similar age but di#erent body weights. Regression analyses revealed significant increases in the number of APC with increasing body weight when fish were acclimated to either water temperature. These results emphasise the importance of standardising fish weight in immunological studies of salmonids before exploring the possible e#ects of acclimation temperatures. 2001 Academic Press Key words:
antibody production, body weight, temperature, chinook salmon, immune response.
I. Introduction Development and maturation of the immune system in fish is complex and can have high intraspecific variability. The ability of young fish to resist disease can depend on age, ontogeny, body size and external temperature (Wishkovsky et al., 1987; Bly & Clem, 1992; Schreck, 1996; Tatner, 1996; Schroder et al., 1998). While several studies have demonstrated di#erences in the specific immune function of vertebrates varying in age or stages of physical development (Tatner & Manning, 1983a; Castillo et al., 1993; Ravichandran et al., 1994; Saad et al., 1994), others have indicated the importance of the influence of body size (Johnson et al., 1982a, b; Nakanishi, 1983, 1991; Bootland et al., 1990; Tatner, 1996) or organ tissue weight (Tatner & Manning, 1983b) when assessing immune function. This study explores the possibility that body size may a#ect the specific immune response in juvenile chinook salmon regardless of acclimation temperature. *Oregon Agricultural Experiment Station Technical Report No. 11640. †Supported co-operatively by Oregon State University, the U.S.G.S. and the Oregon Department of Fish and Wildlife. 1050–4648/01/080653+07 $35.00/0
653
2001 Academic Press
654
L. N. M. HARRAHY ET AL.
The haemolytic plaque assay has been widely employed to assess immune system function since its development by Jerne & Nordin (1963). The assay measures the ability of B lymphocytes in vitro to produce plasma cells that release specific antibody in response to an antigen. Modifications of the assay are useful for assessing adaptive immunity in fish (Smith et al., 1967; Cunningham & Szenberg, 1968; Chiller et al., 1969; Rijkers et al., 1980; Tripp et al., 1987; Anderson, 1990; Arkoosh & Kaattari, 1992). The goal of this study was to provide a cautionary warning to researchers participating in studies involving temperature and adaptive immunity in fish. We hypothesised that juvenile chinook salmon of similar age, but di#ering size, di#ered in their ability to generate specific antibody-producing cells (APC) when acclimated to optimal and elevated temperatures. We had the opportunity to test for a potential correlation of APC with body size by taking advantage of data collected as part of an experiment in which juvenile chinook salmon were acclimated to two environmentally realistic temperatures. Chinook salmon acclimated to 13 C, the optimum temperature for this species, were compared to those acclimated to 21 C, an elevated temperature not uncommon in the northwestern U.S.A. during the late summer months (Brett, 1952; Armour, 1991). II. Materials and Methods Yearling spring chinook salmon parr were maintained at Oregon State University’s Fish Performance and Genetics Laboratory in 0·9 m circular tanks, with 2 l min 1 flow-through well water. Forty fish per tank were assigned randomly to three tanks at 13 C (control) or to three tanks at 21 C (treatment). Treatment fish were slowly acclimated from 13 to 21 C by temperature increases of 2 C per day, over 4 days. Temperatures were monitored every 10 min in control and treatment tanks with electronic thermometers. All fish were maintained in their experimental tanks for a total of 3 weeks to allow su$cient time for acclimation. Although each of the selected fish were the same age and reared under identical conditions in the same stock trough, fish sizes were quite variable, ranging from approximately 10 to 55 g. All fish were fed a standardised ration of Oregon Moist Pellet twice daily. In order to standardise the amount of food required for energy consumption at each temperature, food quantities o#ered were set according to the feeding guide recommendations from the food manufacturer (BioDiet). Fish acclimated to 21 C received approximately 3·1% of their body weight each day, while fish acclimated to 13 C received 2·3%. These temperaturedependent feeding rations were also consistent with the recommendations by Brett & Groves (1979) in their description of physiological energetics. Finally, all fish were pre-conditioned to the presence of a dip net by swirling a net inside their tanks prior to each feeding. This procedure helped to reduce the amount of chasing during capture of the fish at sampling time. Sampling began on 4 November 1998. Control and treatment fish were sampled in triplicate over 3 days (one tank at each temperature sampled per day), in order to allow su$cient time for the sampling scheme. Head-kidney
ANTIBODY-PRODUCING CELLS IN CHINOOK SALMON
655
samples from fish acclimated to each temperature were collected at two di#erent sampling times each day. The fish were rapidly removed with a dip net and immediately killed by an anesthetic overdose of tricaine methanesulfanate (MS222) at 200 mg l 1 bu#ered with NaHCO3 . Each fish was then measured and weighed and the head-kidneys were removed under sterile conditions and placed into individual tubes containing 500 l of tissue culture medium (TCM). The TCM consisted of 10% foetal bovine serum, 1% L-glutamine and 0·1% gentamicin sulfate in RPMI 1640. A single cell suspension was made by gently aspirating the tissue and the medium with a sterile 1·0 cc syringe. Ten l of supernatant was transferred to a haemocytometer and leukocytes from each head kidney were counted. The number of leucocytes per head-kidney was standardised by adding or removing the proper amount of TCM for the number of cells counted in each sample. A haemolytic plaque assay was then conducted to determine the number of APC. The haemolytic plaque assay employed was as described by Tripp et al. (1987) and modified by Slater et al. (1995). Briefly, 50 l of picrylsulfonic acid-lipopolysaccharide (TNP-LPS) antigen was added to 50 l of head-kidney cells in culture in a 96 well plate. Duplicate in vitro samples were taken for each fish. The 96 well plate was secured in an incubation chamber containing blood gas and the cells were incubated at 17 C for 7 days. Following the incubation period, the cells were washed with 50 l of fresh TCM and mixed with 10 l of haptenated sheep red blood cells (SRBC) (SRBC coated in TNP antigen) and 10 l of complement (contained in rainbow trout serum). The cell mixtures were placed into Cunningham chambers (Cunningham & Szenberg, 1968) incubated at 17 C for 1·5 h, and plaques were then counted. Antibody production was determined by the number of APC per final pre-incubation leucocyte concentration in tissue culture. The APC data for both acclimation temperatures were tested for di#erences between the two sampling times each day with an analysis of variance (ANOVA), found to be insignificant (P>0·1), and pooled appropriately. Likewise, the number of APC were tested for di#erences between replicate sampling runs, also found to be insignificant (ANOVA, P>0·1), and the data were pooled. Fish weight and the number of APC from a total sample size of 28 fish were regressed for each acclimation temperature and for both temperatures combined. After determining that the linear model best fit the data, regression analyses were performed to examine possible correlations between fish weight and the number of APC. Finally, after determining that body size was not dependent on acclimation temperature (ANOVA, P>0·1), an analysis of covariance (ANCOVA) was run to test for temperature di#erences in the number of APC with fish weight included in the model as a covariate. III. Results and Discussion Significant, positive linear regressions were found for the number of APC and final weight in fish acclimated to 13 C (P=0·021) (Fig. 1a) and 21 C (P=0·0006) (Fig. 1b). No di#erences were found in the number of APC between fish acclimated to 13 and 21 C, and the number of APC in fish was dependent on the weight of the individual fish, regardless of acclimation temperature.
656
L. N. M. HARRAHY ET AL.
1000 (a) APC
800 600 400 200 0
20
40
60
40
60
40
60
Weight (g) 1000 (b) APC
800 600 400 200 0
20 Weight (g)
1000 (c) APC
800 600 400 200 0
20 Weight (g)
Fig. 1. Linear regressions of (a) antibody-producing cells (APC) per 106 leucocytes and fish weight of juvenile chinook salmon acclimated to 13 C and (b) 21 C (n=28). (c) A significant e#ect of fish weight on the number of APC is also found when data of fish acclimated to each temperature are combined (linear regression, P=0·0003) (n=56). Each point represents an individual fish.
The data from both acclimation temperatures were thus combined to reveal a significant, positive linear regression in APC and fish weight (P=0·0003) (Fig. 1c). These findings indicate that care should be taken to account for body size in immunological experiments involving antibody production in salmonids. These results are consistent with Nakanishi’s conclusion in his study of the marine teleost, Sebastiscus marmoratus, that larger fish tend to have a better immune response than smaller fish (Nakanishi, 1991). Although there were no di#erences in the regressions of fish weight and the production of specific antibody between acclimation temperatures, ANCOVA revealed significant di#erences in the number of APC when fish weight was included in the model as a covariate. In this case, fish acclimated to 21 C had a significantly higher number of APC than fish of the same weight acclimated
ANTIBODY-PRODUCING CELLS IN CHINOOK SALMON
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to 13 C (P=0·034). Other studies have described how the immune response can be controlled by external temperature (Clem et al., 1984, 1991; Bly & Clem, 1992; Tatner, 1996). For example, the immune responses of channel catfish have been altered by both in vivo and in vitro temperature fluctuations. Lower temperatures tended to have an inhibitory e#ect on the production of specific antibody and warmer temperatures resulted in higher antibody responses (Clem et al., 1984; Bly & Clem, 1992). Our data support this outcome when the weight of the fish is accounted for in the analysis. Other researchers using the plaque assay for studies with salmonids (Tripp et al., 1987; Maule et al., 1989; Slater et al., 1995) may not have found an e#ect of body size on antibody production if size variation in the experimental fish was small. We are, at present, unable to explain why smaller fish of the same age tend to produce lymphocytes that are less responsive to the antigen in vitro. It has, however, been suggested that dominant fish, which generally tend to be larger, may stress smaller subordinate fish (Peters et al., 1991). Exhibition of such a stress response in small fish may lead to immunosuppression (Tripp et al., 1987; Maule et al., 1989). Though it is possible that the positive correlation of APC to body weight may have been driven by developmental stage, our results support Nakanishi’s and Tatner’s conclusions that body size, rather than age, is a better determinant of immunological maturity during ontogeny (Nakanishi, 1991; Tatner, 1996). Furthermore, they demonstrate that the influence of acclimation temperature on the adaptive immune system may be a#ected by body size. Our findings indicate that caution should be executed when selecting fish of similar age but varying body sizes for immunological studies in salmonids. We thank Rob Chitwood, manager of the Fish Performance and Genetics Laboratory at O.S.U. for his assistance with establishing the experimental conditions. We also thank Beth Siddens and Darren Lerner for aiding in sample collections and Wilfrido Contreras-Sanches for his guidance with data analysis.
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