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Observations on the inherent variability of measuring lysozyme activity in coho salmon (Oncorhynchus kisutch)
Comparative Biochemistry and Physiology, Part B 138 (2004) 207 – 211 www.elsevier.com/locate/cbpb
Observations on the inherent variability of measuring lysozyme activity in coho salmon (Oncorhynchus kisutch) S.K. Balfry a,*, G.K. Iwama b a
Faculty of Agricultural Sciences, University of British Columbia, 4160 Marine Drive, West Vancouver, British Columbia, Canada V7V 1N6 b Institute of Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 3Z1 Received 29 September 2003; received in revised form 8 December 2003; accepted 9 December 2003
Abstract Lysozyme activity is a common measurement of innate immunity. It has also been used to investigate genetic variation and an animal’s responses to factors such as stress, infections and variations in diet. This research demonstrates the inherent variation in lysozyme activity in unstimulated coho salmon (Oncorhynchus kisutch). The role of maternal contribution, early life stage development and fish mass are considered. Genetic variation within and between strains of coho was found to be significant at selected life stages. Our results indicate that strain differences in lysozyme activity are more accurately measured by comparing the genetic variation after the eyed stage, when maternal effects are reduced. A positive correlation between plasma/serum lysozyme activity and fish mass is reported here. In summary, this study shows the role of maternal, developmental stage and size in lysozyme activity in fish, and emphasizes the importance of considering such variables when measuring the variability of lysozyme activity in fish. D 2004 Elsevier Inc. All rights reserved. Keywords: Lysozyme; Genetic variation; Coho salmon; Maternal effects
1. Introduction Lysozyme is an important bacteriolytic agent found in a variety of freshwater and marine fish species (Lie et al., 1989). Leucocytes such as monocytes, macrophages and polymorphonuclear granulocytes are known to synthesize and secrete lysozyme in fish (Murray and Fletcher, 1976). Kidney tissue appears to have the greatest concentration of lysozyme activity, likely due to the high concentration of these leucocytes in the anterior hematopoietic portion of the kidney. Lysozyme has also been detected in many other fish tissues such as spleen, liver, skin, mucus, gills, muscle, ovary and eggs (Takahashi et al., 1986; Lie et al., 1989; Yousif et al., 1991; Takemura and Takano, 1995). Lysozyme isolated from fish has been found to be effective as a bacteriolytic agent against both Gram-positive and Gramnegative fish pathogens (Grinde, 1989; Yousif et al., 1994). Lysozyme is therefore an important factor in protecting fish against bacterial pathogens, due to its antibacterial properties and because it is located in areas that are in frequent contact with pathogens (i.e., kidney and skin mucus). * Corresponding author. Tel.: +1-604-666-6036; fax: +1-604-666-3497. E-mail address: [email protected] (S.K. Balfry). 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2003.12.010
Lysozyme activity is known to change according to the state of health, stress, sex, season, temperature and degree of sexual maturity (Fletcher and White, 1973; Fletcher et al., 1977; Mo¨ck and Peters, 1990). The genetic variation of lysozyme has also been established (Grinde et al., 1988; Røed et al., 1989; Lund et al., 1995; Balfry et al., 1997, 2001), and research into breeding selection programs are being developed that utilize lysozyme activity measurements as selection criteria (Fevolden et al., 1991, 1992, 2002; Fevolden and Røed, 1993; Røed et al., 2003). The present study demonstrates the inherent variability in lysozyme activity, with an examination of the importance of maternal contribution, early life stage development and fish size. The coho salmon (Oncorhynchus kisutch) strains used for this project were part of a large study designed to examine strain differences in innate disease resistance.
2. Materials and methods 2.1. Fish Two different year classes of coho salmon (O. kisutch) strains were used for this study. In the first year of study,
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comparisons were made between the Kitimat and Quinsam River coho strains. These were comprised of nine and seven full sib families, respectively. In the second year, five coho strains were compared: Kitimat, Quinsam, Robertson, Chehalis and Capilano. Each strain was represented by 10 full sib families. In each year, the families were reared separately until approximately 3 g mean mass, at which time the fish were fin clipped to identify strain and equal numbers were combined into common rearing tanks. The fish were fed daily to satiation, maintained in dechlorinated freshwater (2 –18 jC) and exposed to natural photoperiod. 2.2. Lysozyme assay The lysoplate method (Osserman and Lawlor, 1966, with modifications outlined by Yousif et al., 1994) was used to determine lysozyme activity in serum, plasma, eggs, alevin, fry and kidney samples. Briefly, this assay involved preparing agar plates (lysoagar) containing 0.60 mg/ml Micrococcus lysodeikticus (Sigma), 0.02 M NaCl, 0.50% Agarose (Sigma) in phosphate buffer (PB, 0.06 M, pH 6.0). Wells (approximately 3 mm diameter) were punched into the lysoagar which was air dried. Hen egg white lysozyme (HEWL, Sigma) standards were used. The activity of the HEWL standard (under the assay conditions described) was measured using the turbidimetric method described by the supplier of the HEWL (Sigma), with modifications by Grinde (1989). Samples were placed (in triplicate) onto separate plates, along with the HEWL standards and incubated overnight in a moist chamber. The zones of clearance surrounding each well were then measured with calipers and compared to the HEWL standards using regression analysis. Lysozyme activity of serum and plasma has been found to be the same (Mo¨ck and Peters, 1990). We were therefore able to pool both results in order to test for correlations between lysozyme activity and fish mass. The lysozyme activity of kidney, gill, whole egg, alevin and fry samples was determined by first diluting (1:4 w/v PB) and homogenizing (Polytron homogenizer) the tissues. The tissue homogenates were then centrifuged and the resultant supernatants assayed for lysozyme activity. Comparisons were made between the lysozyme activity of the whole homogenate and the supernatant (no significant differences were found). 2.3. Sampling Changes in lysozyme activity in the early life history stages were determined in both year classes for all strains. In the first year, unfertilized eggs, eyed eggs, alevins and 1 g fry were sampled, while in year, only unfertilized eggs were sampled. Variation within and between strains was examined. In both years, the relationship between maternal lysozyme activity and progeny was determined
from comparisons between kidney (years one and two) and serum (year two) lysozyme activity in the females, sampled at the time of egg take, and her unfertilized eggs. The relationship between serum lysozyme and fish mass (strains combined for each year class) was established by sampling the fish at selected times over the course of a two-year rearing period. 2.4. Statistical analysis Prior to the statistical analyses, the lysozyme data were log-transformed. Measures of genetic variation within and between the strains were performed by analysis of variance tests and where only two strains were compared (year one). Student t-tests were used (Sokal and Rohlf, 1981). Pearson product – moment correlation tests were used to measure the strength of the association between the following variables: maternal kidney lysozyme activity and unfertilized egg lysozyme activity, maternal serum lysozyme activity and unfertilized egg activity, fish mass and serum/plasma lysozyme activity. Statistical significance for all tests was determined, where p < 0.05.
3. Results Significant positive correlations between the lysozyme activity in the maternal kidney and her unfertilized eggs (Fig. 1) were detected in the first year coho (r2 = 0.65, p < 0.01, n = 16), but not in the second year two coho (r2 = 0.09, p>0.05, n = 50). However, the second year coho did show a significant positive correlation between maternal serum lysozyme activity and unfertilized eggs (r2 = 0.39, p < 0.01, n = 50). Correlations between maternal kidney and serum lysozyme activity with unfertilized egg lysozyme activity were generally not significant ( p>0.05) when each strain was examined separately (data not presented). Strain differences in lysozyme activity at the various early life stages (Fig. 2) were significant at the unfertilized egg and 1 g fry stages. The Quinsam coho had higher activity as unfertilized eggs, but as the development progressed past the egg stage (i.e., alevin and 1 g fry), the Kitimat coho had higher lysozyme activity. There was a highly significant positive correlation (r2 = 0.63, p < 0.0001, n = 114) between serum/plasma lysozyme activity and fish mass (Fig. 3) in the second year coho, while no correlation was found in the first year coho (r2 = 0.06, p>0.05, n = 116). Similar sample sizes were used in the comparisons, but the second year two coho were sampled over a wider range of masses (46 – 805 g), than the first year coho (8.3 –57.3 g). Genetic variation in lysozyme activity was examined in both year classes of coho. Variation in lysozyme activity within (i.e., family variation) and between
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maternal effects become significant only under adverse conditions (i.e., presence of pathogens, poor water quality), due to increased additive genetic variation. The maternal effects on lysozyme activity were not measured in this study, but significant positive correlations between maternal lysozyme activity (kidney and serum) and unfertilized egg lysozyme activity were detected. Strain differences in the lysozyme activity of unfertilized and eyed eggs generally reflected maternal lysozyme activity. However, after the eyed stage, the pattern of strain differences in lysozyme activity differed. There was a reverse in the activity level from Quinsam strain having higher levels prior to the eyed egg stage to higher activity levels in the Kitimat strain after the eyed egg stage. This change was likely due to a reduction in the maternal effects, associated with the adsorption of yolk material and the development of the lysozyme-producing leucocytes in the developing fish. Takemura (1993) reported a similar decrease in maternally derived IgM-like proteins (MLPs) in larval tilapia (Oreoochromis mossambicus) as the yolk was absorbed. MLPs were found in their lowest concentration when all the yolk was gone and the larvae were beginning to feed independently. After the first feeding, the MLP rapidly increased as the larvae produced their own MLPs. The coho salmon examined here showed no increase in lysozyme activity until after the eyed stage, which supports the suggestion that as with MLP in tilapia, the coho had exhausted the maternally derived lysozyme and were independently producing lysozyme. Genetic variation within and between strains of coho was found to be significant at selected life stages. However, in light of our findings here, we believe that strain differences in lysozyme activity are more accurately measured by Fig. 1. Correlations between maternal lysozyme activity and mean unfertilized egg lysozyme activity in coho salmon (Oncorhynchus kisutch) collected in (a) Year one—maternal kidney vs. egg lysozyme: r 2 = 0.65, p < 0.01, n = 16; (b) Year two—maternal kidney vs. egg lysozyme: r 2 = 0.09, p>0.05, n = 50; (c) Year two—maternal serum vs. egg lysozyme: r 2 = 0.39, p < 0.01, n = 50.
strains at various life stages was statistically significant (Table 1).
4. Discussion Maternal contributions to disease resistance have been described as significant non-additive, maternal effects on survival. The significance of such effects appears to be related to species and life history. For example, maternal effects on survival are highly significant for up to 1 year in the platy (Xiphophorus maculatus; Price and Bone, 1985), while in salmonids, the effects are most pronounced up to the eyed stage (Kanis et al., 1976). Withler et al. (1987) suggest that even in the earliest stages of development,
Fig. 2. Coho salmon (Oncorhynchus kisutch) strain comparison of lysozyme activity in selected stages of early development. *Refers to significant strain differences ( p < 0.05).
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should be noted that a significant negative correlation between plasma lysozyme activity and weight has also been reported in salmonids (Fevolden et al., 2002). These researchers, however, measured lysozyme activity from fish that had been subjected to a confinement stress (Fevolden et al., 1991, 2001), and therefore the results may not be comparable to the results reported here, which were obtained from fish that had not been subjected to a stressor. The relationship between fish mass, growth rate and growth hormones is unclear at this time and therefore we recommend that when comparing different groups of fish for differences in lysozyme activity, the fish be of similar masses. This study shows maternal, developmental stage and size effects on lysozyme activity in coho salmon, and emphasizes the importance of considering such variables when measuring the variability of lysozyme activity in fish. To minimize the confounding effects of environment on the results, groups of fish should be reared under identical, preferably communal conditions. The fish should be of similar size, as this research has shown significant effects of mass on lysozyme activity. In addition, the age of the fish should be considered because maternal effects on the activity of lysozyme appear to be significant at very early stages of development and therefore can have a confounding effect on estimates of this variable. Fig. 3. Correlations between fish mass and serum lysozyme activity in individual coho salmon (Oncorhynchus kisutch) collected in (a) Year one: r2 = 0.06, p>0.05, n = 116; (b) Year two: r2 = 0.63, p < 0.001, n = 114.
comparing the genetic variation after the eyed stage, when maternal effects are less pronounced. Measuring strain differences prior to the eyed stage appears to reflect maternal lysozyme activity. Maternal lysozyme activity within unfertilized eggs was likely influenced by many unknown, uncontrollable factors (i.e., environment, size, nutrition, disease and stress), so it was not possible to accurately demonstrate strain differences. Only those lysozyme measurements taken from fish after the eyed stage and reared under controlled conditions could be used with confidence to measure genetic variability. A positive correlation between plasma/serum lysozyme activity and fish mass is reported here for the first time. The wide range of masses (46 – 805 g) and large sample size (n = 114) obtained from sampling the year two coho, permitted significant results. Reports of positive correlation between growth rate and lysozyme activity have been suggested by research linking increased lysozyme activity with elevated plasma growth hormone levels (Marc et al., 1995; Yada et al., 2001). However, Fevolden et al. (2002) report that in Atlantic salmon (Salmo salar), there was a trend for a negative phenotypic correlation between serum lysozyme activity and specific growth rate and weight. It
Table 1 Examination of the genetic variation of mean lysozyme activity of various tissues, within and between strains of coho salmon (Oncorhynchus kisutch) collected in two different years Year class
Strain
Life stage
Within strain variation at each stage of development Year one Quinsam Unfertilized Eyed eggs Alevins Kitimat Unfertilized Eyed eggs Alevins Year two Quinsam Unfertilized Kitimat Unfertilized Robertson Unfertilized Capilano Unfertilized Chehalis Unfertilized Year class
Number of strains
p-value eggs
eggs
eggs eggs eggs eggs eggs
Life stage
Between strain variation at each stage of development Year one 2 Unfertilized eggs 2 Fry (1 g) Year two 5 Unfertilized eggs 5 Maternal kidney 5 Maternal serum
p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p-value p < 0.05 p < 0.05 p < 0.001 p < 0.05 p < 0.05
Year one, Quinsam and Kitimat River strains were represented by 9 and 7 full sib families, respectively. Year two, coho strains were each represented by 10 full sib families per strain. Variation determined from analysis of variance tests (n = 6 individuals per family). Statistical significance determined when p < 0.05.
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Acknowledgements This work was supported by the Canadian Bacterial Diseases Network to G.K.I. and a Science Council of B.C. GREAT Scholarship to S.K.B. We thank the personnel and managers of the Kitimat, Quinsam, Chehalis, Robertson, and Capilano River hatcheries, Canada, for their assistance in collecting broodstock and gametes. We are also grateful to Ms. Ellen Teng for her help in rearing the coho strains. Our thanks are due to Dr. L. Brown for her critical review of this manuscript.
References Balfry, S.K., Heath, D.D., Iwama, G.K., 1997. Genetic analysis of lysozyme activity and resistance to vibriosis in farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum). Aquac. Res. 28, 893 – 899. Balfry, S.K., Maule, A.G., Iwama, G.K., 2001. Coho salmon Oncorhynchus kisutch strain differences in disease resistance and non-specific immunity, following immersion challenges with Vibrio anguillarum. Dis. Aquat. Org. 47, 39 – 48. Fevolden, S.E., Røed, K.H., 1993. Cortisol and immune characteristics in rainbow trout (Oncorhynchus mykiss) selected for high and low tolerance to stress. J. Fish Biol. 43, 919 – 930. Fevolden, S.E., Refstie, T., Røed, K.H., 1991. Selection for high and low cortisol stress response in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Aquaculture 95, 53 – 65. Fevolden, S.E., Refstie, T., Røed, K.H., 1992. Disease resistance in rainbow (Oncorhynchus mykiss) selected for stress response. Aquaculture 104, 19 – 29. Fevolden, S.E., Røed, K.H., Fjalestad, K.T., 2002. Selection response of cortisol and lysozyme in rainbow trout and correlation to growth. Aquaculture 205, 61 – 75. Fletcher, T.C., White, A., 1973. Lysozyme activity in the plaice (Pleuroonectes platessa L.). Experientia 29, 1283 – 1285. Fletcher, T.C., White, A., Baldo, B.A., 1977. C-reactive protein-like precipitin and lysozyme in the lumpsucker Cyclopterus lumpus L. during the breeding season. Comp. Biochem. Physiol., B 57, 353 – 357. Grinde, B., 1989. Lysozyme from rainbow trout, Salmo gairdneri Richardson, as an antibacterial agent against fish pathogens. J. Fish Dis. 12, 95 – 104. Grinde, B., Lie, Ø., Poppe, T., Salte, R., 1988. Species and individual variation in lysozyme activity in fish of interest in aquaculture. Aquaculture 68, 299 – 304. Kanis, E., Refstie, T., Gjedrem, T., 1976. A genetic analysis of egg, alevin and fry mortality in salmon (Salmo salar), sea trout (Salmo trutta), and rainbow trout (Salmo gairdneri). Aquaculture 8, 259 – 268.
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Lie, Ø., Evensen, Ø., Sørensen, A., Frøysadal, E., 1989. Study of lysozyme activity in some fish species. Dis. Aquat. Org. 6, 1 – 5. Lund, T., Gjedrem, T., Bentsen, H.B., Eide, D.M., Larsen, H.J.S., Røed, K.H., 1995. Genetic variation in immune parameters and associations to survival in Atlantic salmon. J. Fish Biol. 46, 748 – 758. Marc, A.M., Quentel, C., Severe, A., Le Bail, P.W., Boeuf, G., 1995. Changes in some endocrinological and non-specific immunological parameters during seawater exposure in brown trout. J. Fish Biol. 46, 1065 – 1081. Mo¨ck, A., Peters, G., 1990. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. J. Fish Biol. 37, 873 – 885. Murray, C.K., Fletcher, T.C., 1976. The immunohistochemical localization of lysozyme in plaice (Pleuronectes platessa L.) tissues. J. Fish Biol. 9, 329 – 334. Osserman, E.F., Lawlor, D.P., 1966. Serum and urinary lysozyme (Muramidase) in monocytic and monomyelocytic leukemia. J. Exp. Med. 124, 921 – 951. Price, D.J., Bone, L.M., 1985. Maternal effects and resistance to infection by Ichthyophthirius multifilis in Xiphophorus maculatus. In: Manning, M.J., Tatner, M.F. (Eds.), Fish Immunology. Academic Press, London, pp. 233 – 244. Røed, K.H., Larsen, H.J., Linder, D., Refstie, T., 1989. The genetic influence on natural immunity in rainbow trout. Anim. Genet. 20 (Suppl. 1), 54. Røed, K.H., Fevolden, S.-E., Fjalestad, K.T., 2003. Disease resistance and immune characteristics in rainbow trout (Oncorhynchus mykiss) selected for lysozyme activity. Aquaculture 209, 91 – 101. Sokal, R.R., Rohlf, F.J., 1981. Biometry, 2nd ed. Freeman, San Francisco. Takahashi, Y., Itami, T., Konegawa, K., 1986. Enzymatic properties of partially purified lysozyme from the skin of carp. Bull. Jpn. Soc. Sci. Fish. 52, 1209 – 1214. Takemura, A., 1993. Changes in an immunoglobulin M (IgM)-like protein during larval stages in tilapia, Oreochromis mossambicus. Aquaculture 115, 233 – 241. Takemura, A., Takano, K., 1995. Lysozyme in the ovary of tilapia (Oreoochromis mossambicus): its purification and some biological properties. Fish Physiol. Biochem. 14, 415 – 521. Withler, R.E., Clarke, W.C., Riddell, B.E., Kreiberg, H., 1987. Genetic variation in freshwater survival and growth of Chinook salmon (Oncoorhynchus tshawytscha). Aquaculture 64, 85 – 96. 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., B 129, 695 – 701. Yousif, A.N., Albright, L.J., Evelyn, T.P.T., 1991. Occurrence of lysozyme in the eggs of coho salmon Oncorhynchus kisutch. Dis. Aquat. Org. 10, 45 – 49. Yousif, A.N., Albright, L.J., Evelyn, T.P.T., 1994. In vitro evidence for the antibacterial role of lysozyme in salmonid eggs. Dis. Aquat. Org. 19, 15 – 19.
Observations on the inherent variability of measuring lysozyme activity in coho salmon (Oncorhynchus kisutch) S.K. Balfry a,*, G.K. Iwama b a
Faculty of Agricultural Sciences, University of British Columbia, 4160 Marine Drive, West Vancouver, British Columbia, Canada V7V 1N6 b Institute of Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 3Z1 Received 29 September 2003; received in revised form 8 December 2003; accepted 9 December 2003
Abstract Lysozyme activity is a common measurement of innate immunity. It has also been used to investigate genetic variation and an animal’s responses to factors such as stress, infections and variations in diet. This research demonstrates the inherent variation in lysozyme activity in unstimulated coho salmon (Oncorhynchus kisutch). The role of maternal contribution, early life stage development and fish mass are considered. Genetic variation within and between strains of coho was found to be significant at selected life stages. Our results indicate that strain differences in lysozyme activity are more accurately measured by comparing the genetic variation after the eyed stage, when maternal effects are reduced. A positive correlation between plasma/serum lysozyme activity and fish mass is reported here. In summary, this study shows the role of maternal, developmental stage and size in lysozyme activity in fish, and emphasizes the importance of considering such variables when measuring the variability of lysozyme activity in fish. D 2004 Elsevier Inc. All rights reserved. Keywords: Lysozyme; Genetic variation; Coho salmon; Maternal effects
1. Introduction Lysozyme is an important bacteriolytic agent found in a variety of freshwater and marine fish species (Lie et al., 1989). Leucocytes such as monocytes, macrophages and polymorphonuclear granulocytes are known to synthesize and secrete lysozyme in fish (Murray and Fletcher, 1976). Kidney tissue appears to have the greatest concentration of lysozyme activity, likely due to the high concentration of these leucocytes in the anterior hematopoietic portion of the kidney. Lysozyme has also been detected in many other fish tissues such as spleen, liver, skin, mucus, gills, muscle, ovary and eggs (Takahashi et al., 1986; Lie et al., 1989; Yousif et al., 1991; Takemura and Takano, 1995). Lysozyme isolated from fish has been found to be effective as a bacteriolytic agent against both Gram-positive and Gramnegative fish pathogens (Grinde, 1989; Yousif et al., 1994). Lysozyme is therefore an important factor in protecting fish against bacterial pathogens, due to its antibacterial properties and because it is located in areas that are in frequent contact with pathogens (i.e., kidney and skin mucus). * Corresponding author. Tel.: +1-604-666-6036; fax: +1-604-666-3497. E-mail address: [email protected] (S.K. Balfry). 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2003.12.010
Lysozyme activity is known to change according to the state of health, stress, sex, season, temperature and degree of sexual maturity (Fletcher and White, 1973; Fletcher et al., 1977; Mo¨ck and Peters, 1990). The genetic variation of lysozyme has also been established (Grinde et al., 1988; Røed et al., 1989; Lund et al., 1995; Balfry et al., 1997, 2001), and research into breeding selection programs are being developed that utilize lysozyme activity measurements as selection criteria (Fevolden et al., 1991, 1992, 2002; Fevolden and Røed, 1993; Røed et al., 2003). The present study demonstrates the inherent variability in lysozyme activity, with an examination of the importance of maternal contribution, early life stage development and fish size. The coho salmon (Oncorhynchus kisutch) strains used for this project were part of a large study designed to examine strain differences in innate disease resistance.
2. Materials and methods 2.1. Fish Two different year classes of coho salmon (O. kisutch) strains were used for this study. In the first year of study,
208
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comparisons were made between the Kitimat and Quinsam River coho strains. These were comprised of nine and seven full sib families, respectively. In the second year, five coho strains were compared: Kitimat, Quinsam, Robertson, Chehalis and Capilano. Each strain was represented by 10 full sib families. In each year, the families were reared separately until approximately 3 g mean mass, at which time the fish were fin clipped to identify strain and equal numbers were combined into common rearing tanks. The fish were fed daily to satiation, maintained in dechlorinated freshwater (2 –18 jC) and exposed to natural photoperiod. 2.2. Lysozyme assay The lysoplate method (Osserman and Lawlor, 1966, with modifications outlined by Yousif et al., 1994) was used to determine lysozyme activity in serum, plasma, eggs, alevin, fry and kidney samples. Briefly, this assay involved preparing agar plates (lysoagar) containing 0.60 mg/ml Micrococcus lysodeikticus (Sigma), 0.02 M NaCl, 0.50% Agarose (Sigma) in phosphate buffer (PB, 0.06 M, pH 6.0). Wells (approximately 3 mm diameter) were punched into the lysoagar which was air dried. Hen egg white lysozyme (HEWL, Sigma) standards were used. The activity of the HEWL standard (under the assay conditions described) was measured using the turbidimetric method described by the supplier of the HEWL (Sigma), with modifications by Grinde (1989). Samples were placed (in triplicate) onto separate plates, along with the HEWL standards and incubated overnight in a moist chamber. The zones of clearance surrounding each well were then measured with calipers and compared to the HEWL standards using regression analysis. Lysozyme activity of serum and plasma has been found to be the same (Mo¨ck and Peters, 1990). We were therefore able to pool both results in order to test for correlations between lysozyme activity and fish mass. The lysozyme activity of kidney, gill, whole egg, alevin and fry samples was determined by first diluting (1:4 w/v PB) and homogenizing (Polytron homogenizer) the tissues. The tissue homogenates were then centrifuged and the resultant supernatants assayed for lysozyme activity. Comparisons were made between the lysozyme activity of the whole homogenate and the supernatant (no significant differences were found). 2.3. Sampling Changes in lysozyme activity in the early life history stages were determined in both year classes for all strains. In the first year, unfertilized eggs, eyed eggs, alevins and 1 g fry were sampled, while in year, only unfertilized eggs were sampled. Variation within and between strains was examined. In both years, the relationship between maternal lysozyme activity and progeny was determined
from comparisons between kidney (years one and two) and serum (year two) lysozyme activity in the females, sampled at the time of egg take, and her unfertilized eggs. The relationship between serum lysozyme and fish mass (strains combined for each year class) was established by sampling the fish at selected times over the course of a two-year rearing period. 2.4. Statistical analysis Prior to the statistical analyses, the lysozyme data were log-transformed. Measures of genetic variation within and between the strains were performed by analysis of variance tests and where only two strains were compared (year one). Student t-tests were used (Sokal and Rohlf, 1981). Pearson product – moment correlation tests were used to measure the strength of the association between the following variables: maternal kidney lysozyme activity and unfertilized egg lysozyme activity, maternal serum lysozyme activity and unfertilized egg activity, fish mass and serum/plasma lysozyme activity. Statistical significance for all tests was determined, where p < 0.05.
3. Results Significant positive correlations between the lysozyme activity in the maternal kidney and her unfertilized eggs (Fig. 1) were detected in the first year coho (r2 = 0.65, p < 0.01, n = 16), but not in the second year two coho (r2 = 0.09, p>0.05, n = 50). However, the second year coho did show a significant positive correlation between maternal serum lysozyme activity and unfertilized eggs (r2 = 0.39, p < 0.01, n = 50). Correlations between maternal kidney and serum lysozyme activity with unfertilized egg lysozyme activity were generally not significant ( p>0.05) when each strain was examined separately (data not presented). Strain differences in lysozyme activity at the various early life stages (Fig. 2) were significant at the unfertilized egg and 1 g fry stages. The Quinsam coho had higher activity as unfertilized eggs, but as the development progressed past the egg stage (i.e., alevin and 1 g fry), the Kitimat coho had higher lysozyme activity. There was a highly significant positive correlation (r2 = 0.63, p < 0.0001, n = 114) between serum/plasma lysozyme activity and fish mass (Fig. 3) in the second year coho, while no correlation was found in the first year coho (r2 = 0.06, p>0.05, n = 116). Similar sample sizes were used in the comparisons, but the second year two coho were sampled over a wider range of masses (46 – 805 g), than the first year coho (8.3 –57.3 g). Genetic variation in lysozyme activity was examined in both year classes of coho. Variation in lysozyme activity within (i.e., family variation) and between
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maternal effects become significant only under adverse conditions (i.e., presence of pathogens, poor water quality), due to increased additive genetic variation. The maternal effects on lysozyme activity were not measured in this study, but significant positive correlations between maternal lysozyme activity (kidney and serum) and unfertilized egg lysozyme activity were detected. Strain differences in the lysozyme activity of unfertilized and eyed eggs generally reflected maternal lysozyme activity. However, after the eyed stage, the pattern of strain differences in lysozyme activity differed. There was a reverse in the activity level from Quinsam strain having higher levels prior to the eyed egg stage to higher activity levels in the Kitimat strain after the eyed egg stage. This change was likely due to a reduction in the maternal effects, associated with the adsorption of yolk material and the development of the lysozyme-producing leucocytes in the developing fish. Takemura (1993) reported a similar decrease in maternally derived IgM-like proteins (MLPs) in larval tilapia (Oreoochromis mossambicus) as the yolk was absorbed. MLPs were found in their lowest concentration when all the yolk was gone and the larvae were beginning to feed independently. After the first feeding, the MLP rapidly increased as the larvae produced their own MLPs. The coho salmon examined here showed no increase in lysozyme activity until after the eyed stage, which supports the suggestion that as with MLP in tilapia, the coho had exhausted the maternally derived lysozyme and were independently producing lysozyme. Genetic variation within and between strains of coho was found to be significant at selected life stages. However, in light of our findings here, we believe that strain differences in lysozyme activity are more accurately measured by Fig. 1. Correlations between maternal lysozyme activity and mean unfertilized egg lysozyme activity in coho salmon (Oncorhynchus kisutch) collected in (a) Year one—maternal kidney vs. egg lysozyme: r 2 = 0.65, p < 0.01, n = 16; (b) Year two—maternal kidney vs. egg lysozyme: r 2 = 0.09, p>0.05, n = 50; (c) Year two—maternal serum vs. egg lysozyme: r 2 = 0.39, p < 0.01, n = 50.
strains at various life stages was statistically significant (Table 1).
4. Discussion Maternal contributions to disease resistance have been described as significant non-additive, maternal effects on survival. The significance of such effects appears to be related to species and life history. For example, maternal effects on survival are highly significant for up to 1 year in the platy (Xiphophorus maculatus; Price and Bone, 1985), while in salmonids, the effects are most pronounced up to the eyed stage (Kanis et al., 1976). Withler et al. (1987) suggest that even in the earliest stages of development,
Fig. 2. Coho salmon (Oncorhynchus kisutch) strain comparison of lysozyme activity in selected stages of early development. *Refers to significant strain differences ( p < 0.05).
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should be noted that a significant negative correlation between plasma lysozyme activity and weight has also been reported in salmonids (Fevolden et al., 2002). These researchers, however, measured lysozyme activity from fish that had been subjected to a confinement stress (Fevolden et al., 1991, 2001), and therefore the results may not be comparable to the results reported here, which were obtained from fish that had not been subjected to a stressor. The relationship between fish mass, growth rate and growth hormones is unclear at this time and therefore we recommend that when comparing different groups of fish for differences in lysozyme activity, the fish be of similar masses. This study shows maternal, developmental stage and size effects on lysozyme activity in coho salmon, and emphasizes the importance of considering such variables when measuring the variability of lysozyme activity in fish. To minimize the confounding effects of environment on the results, groups of fish should be reared under identical, preferably communal conditions. The fish should be of similar size, as this research has shown significant effects of mass on lysozyme activity. In addition, the age of the fish should be considered because maternal effects on the activity of lysozyme appear to be significant at very early stages of development and therefore can have a confounding effect on estimates of this variable. Fig. 3. Correlations between fish mass and serum lysozyme activity in individual coho salmon (Oncorhynchus kisutch) collected in (a) Year one: r2 = 0.06, p>0.05, n = 116; (b) Year two: r2 = 0.63, p < 0.001, n = 114.
comparing the genetic variation after the eyed stage, when maternal effects are less pronounced. Measuring strain differences prior to the eyed stage appears to reflect maternal lysozyme activity. Maternal lysozyme activity within unfertilized eggs was likely influenced by many unknown, uncontrollable factors (i.e., environment, size, nutrition, disease and stress), so it was not possible to accurately demonstrate strain differences. Only those lysozyme measurements taken from fish after the eyed stage and reared under controlled conditions could be used with confidence to measure genetic variability. A positive correlation between plasma/serum lysozyme activity and fish mass is reported here for the first time. The wide range of masses (46 – 805 g) and large sample size (n = 114) obtained from sampling the year two coho, permitted significant results. Reports of positive correlation between growth rate and lysozyme activity have been suggested by research linking increased lysozyme activity with elevated plasma growth hormone levels (Marc et al., 1995; Yada et al., 2001). However, Fevolden et al. (2002) report that in Atlantic salmon (Salmo salar), there was a trend for a negative phenotypic correlation between serum lysozyme activity and specific growth rate and weight. It
Table 1 Examination of the genetic variation of mean lysozyme activity of various tissues, within and between strains of coho salmon (Oncorhynchus kisutch) collected in two different years Year class
Strain
Life stage
Within strain variation at each stage of development Year one Quinsam Unfertilized Eyed eggs Alevins Kitimat Unfertilized Eyed eggs Alevins Year two Quinsam Unfertilized Kitimat Unfertilized Robertson Unfertilized Capilano Unfertilized Chehalis Unfertilized Year class
Number of strains
p-value eggs
eggs
eggs eggs eggs eggs eggs
Life stage
Between strain variation at each stage of development Year one 2 Unfertilized eggs 2 Fry (1 g) Year two 5 Unfertilized eggs 5 Maternal kidney 5 Maternal serum
p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p-value p < 0.05 p < 0.05 p < 0.001 p < 0.05 p < 0.05
Year one, Quinsam and Kitimat River strains were represented by 9 and 7 full sib families, respectively. Year two, coho strains were each represented by 10 full sib families per strain. Variation determined from analysis of variance tests (n = 6 individuals per family). Statistical significance determined when p < 0.05.
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Acknowledgements This work was supported by the Canadian Bacterial Diseases Network to G.K.I. and a Science Council of B.C. GREAT Scholarship to S.K.B. We thank the personnel and managers of the Kitimat, Quinsam, Chehalis, Robertson, and Capilano River hatcheries, Canada, for their assistance in collecting broodstock and gametes. We are also grateful to Ms. Ellen Teng for her help in rearing the coho strains. Our thanks are due to Dr. L. Brown for her critical review of this manuscript.
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