Comparative Study of Biochemical and Non-specific Immunological Parameters in Two Tilapia Species (Oreochromis aureus and
O. mossambicus)
Yaniv Palti1*, Simon Tinman2, Avner Cnaani1, Yaakov Avidar3, Micha Ron1,
and Gideon Hulata1
1. Institute of Animal Science, Agricultural Research Organization, P.O. Box 6, Bet Dagan 50250, Israel.
2. The Central Fish Health Laboratory, Nir David 19150, Israel.
3. Biochemistry Department, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel.
Current Address: Dept. of Food Engineering and Biotechnology, The Technion, Haifa 32000, Israel.
ABSTRACT
Oreochromis aureus and O. mossambicus were compared in a preliminary study for differences in levels of blood biochemical and non-specific immunological parameters before and after exposure to acute stress, which was induced by air exposure for 10 minutes. Two experiments were conducted. In the first experiment comparisons were performed after two weeks of acclimation (“base-line” level), after stress, and for stress response. The latter was calculated for each fish by subtracting the level after stress exposure from the base line level. Significant differences (P < 0.01) were identified between species at the base-line level in total plasma cholesterol and total protein levels. Glucose concentration was significantly different after stress and in stress response. Significant differences between species were also identified in respiratory burst activity of phagocytes after stress and in ceruloplasmin activity after stress. No significant correlation was identified between body weight and each of the parameters tested, indicating that the differences detected in immunological parameters are not related to the notable size difference between the two groups used in this study. The significant differences in total cholesterol and protein were confirmed in the second experiment. Significant differences (P < 0.05) were also detected in levels of albumin, globulin, LDH, calcium, total bilirubin and triglycirides, which were measured by autoanalyzer, and in alpha, beta2, and IgM globulins, %beta1 and %IgM, which were separated and measured by agarose gel electrophoresis. Wide variation was detected within O. aureus in some of the parameters examined. A larger sample size should be used to learn if the differences are large enough to produce segregating O. aureus families for genetic analysis of those parameters. The differences identified suggest that hybrid families from the two species can be used to construct a segregating population for genetic analysis of immunological traits and stress response in tilapia.
INTRODUCTION
Fish diseases have become a major limiting factor in aquaculture. Current methods to control infectious diseases consist of hygiene, vaccination, drug therapy and eradication of infected populations. Improving infectious disease resistance by genetic means is an attractive alternative because of its prospects for prolonged protection. The significant genetic variation in disease resistance found in different fish species (reviewed by Chevassus and Dorson 1990; Fjalestad et al. 1993; Wiegertjes et al. 1996) suggests the possibility of such genetic improvement.
Strain and species differences in disease resistance were previously demonstrated in fish (Parsons et al. 1986; Dorson et al. 1991; Ibarra et al. 1991, 1994; LaPatra et al. 1993, Palti et al. 1999). Strain differences in disease resistance in coho salmon were found to be associated with components of the non-specific immune system (Whithler and Evelyn 1990; Balfry et al. 1994). Recently, strain differences in non-specific immunity were also found in tilapia (Balfry et al. 1997a).
Variation in disease resistance has traditionally been measured by the rate of survival after exposure to a pathogen. Such measurements can result in inaccurate estimation of genetic components of immunity in animals (Gavora and Spencer 1983). The innate (non-specific) immunity is thought to have a major role in disease resistance of fish (e.g. Roed et al. 1993; Balfry et al. 1997a,b). Several parameters of the innate immune response, such as respiratory burst activity, spontaneous haemolytic activity, lysozyme activity, complement concentration, and total IgM, were found to be associated with disease resistance in fish, and their heritability estimates were mostly moderate (reviewed by Wiegertjes et al. 1996). The strong link between stress and susceptibility to diseases in farm animals has long been acknowledged. Parameters of high and low stress response (e.g. cortisol and glucose levels in the blood) were also found to be associated with disease resistance in fish (Fevolden et al. 1991, 1992, 1993). Levels of blood plasma ions and enzymes with important metabolic functions can give indication to the general health of the fish (e.g. Williams and Wootten 1981; Asztalos and Nemcsok 1985; Heming and Paleczny 1987; Ellsaesser and Clem 1987; Waagbo et al. 1988).
Maita et al. (1998a) detected correlation between levels plasma lipids levels and resistance to pathogen infection in yellowtail and rainbow trout. Levels of plasma lipids in fish are affected by diet (Maita et al. 1998b) and by stress after exposure to low ambient dissolved oxygen (Maita et al. 1998c). Those findings suggest that plasma cholesterol can be an indicator for fish health and innate immunity.
In this preliminary study we compared the levels of parameters of the innate immune response and other blood plasma components in O. aureus and O. mossambicus to identify significant differences between the two species. Identification of differences between species in parameters of non-specific immunity and stress response is necessary for constructing hybrid families for genetic analysis of immunological traits in tilapia.
MATERIALS AND METHODS
Fish
Two experiments were performed. In the first experiment we used 26 three years old O. aureus and O. mossambicus from the purebred stocks kept at the Department of Aquaculture, Agricultural Research Organization (A.R.O.), Bet Dagan, Israel. The O. aureus strain originated from the Mehadrin stock (Hulata et al. 1993). The O. mossambicus strain originated from a stock introduced to Israel from Natal, South Africa, in 1975 (Hulata 1988). Size range was 75 – 330 g and 110 – 315 g for O. aureus and O. mossambicus, respectively. Average (±SD) was 163 g (±83) for O. aureus and 209g (±47) for O. mossambicus. The fish were tagged individually and reared communally in two tanks. For the second experiment we randomly sampled 20 adult O. aureus and O. mossambicus from the ARO rearing tanks (10 from each species). The fish were fed daily with a commercial pelleted tilapia feed, 30% protein (Zemach Mills, Israel).
Sampling Schedules
Each of the biochemical and immunological parameters recorded in the first experiment was measured in blood samples taken after two weeks of acclimation period at the “base line” level. Two weeks later, fish were exposed to a 10 minutes air exposure stress. Water temperature in the rearing tanks where the fish were kept during the experiment was 25ºC. Parameters were measured again from blood samples taken 4 hours after the stress exposure. Sample size was 13 fish from each species at the base line level. One fish was lost from each group between sampling, and therefore, 12 fish were used from each species after stress. Blood samples for the second experiment were only taken from fish kept at normal rearing conditions.
Assays to Measure Biochemical and Non-specific Immunological Responses:
Experiment I
Measurements were performed for glucose concentration, ceruloplasmin activity, lysozyme activity, total protein and total cholesterol, and respiratory burst activity of blood cells. The glucose concentration in fish blood is expected to increase four hours after stress exposure (Vijayan et al. 1997; Melamed et al. 1999). It was measured immediately after bleeding by a kit of Haemo-Glukotest 20-800 R (Reflolux S, Boehringer Manheim). Ceruloplasmin is an alpha globulin component of the blood plasma, involved in copper ion transport and oxygen reduction. Its activity and concentration in the plasma is measured by spectrophotometry. Lysozyme is an important enzyme in the blood that actively lyses bacteria. We used an assay based on the lysis of Micrococcus lysodeikitus for determining its activity. Lysozyme activity over time is measured by a spectrophotometric assay. Chicken egg white lysozyme was used as a standard control (Ellis 1990). Total protein and total cholesterol were measured according to established procedures (Doumas 1975; and Allain et al. 1974, respectively). Total protein was only measured at the base line level. Respiratory burst activity of phagocytes was measured by spectrophotometric assay of nitroblue tetrazolium (NBT) activity (Anderson and Siwicki 1995; Efthimiou 1996).
Experiment II
Measurements were performed at 30ºC using the Selective Autoanalyzers (Supra and Progress, Kone Inc., Finland) at the Kimron Veterinary Institute, Bet Dagan, Israel, according to established procedures. The components measured by the autoanalyzers are listed in Table 2a. Globulin levels were determined indirectly by subtracting the measurement of albumin from total protein. Protein fraction levels (Table 2b) were determined by agarose gel electrophoresis following the procedure described by Rehulka (1993). Purified IgM that was contributed by Prof. Ramy Avtalion, Bar Ilan University, was used as a standard to identify the IgM fraction
Statistical analysis
Student t-test analyses were performed to identify significant differences between O. aureus and O. mossambicus in each of the parameters at the base line level and after acute stress, and for stress response. The latter was calculated for each fish by subtracting the level after stress exposure from the base line level. F-tests were used to identify significant differences between variances of the two species for each of the parameters. Unequal variance t-test (Montgomery 1991) was used for parameters with significant variance differences. Correlations of body weight and different parameters were estimated to determine whether biochemical and immunological differences between the two species were caused by the notable size difference between the two groups. Correlations were estimated within species and also for the pooled data from both species.
RESULTS
Significant differences (P < 0.01) were identified between O. aureus and O. mossambicus in total plasma cholesterol and total protein at the base-line level (Table 1a), and in glucose concentration, NBT and ceruloplasmin activity after stress (Table 1b). Body weight was not correlated to the parameters tested (P > 0.1). The significant differences in total cholesterol and protein were confirmed in the second experiment (Table 2a). Significant differences (P < 0.05) were also identified in the second experiment in levels of albumin, globulin, LDH, calcium, total bilirubin and triglycirides (Table 2a). Electrophoresis revealed significant differences in levels of globulins alpha, beta2 and IgM, and also in %beta1, and %IgM (Table 2b).
Total plasma cholesterol levels were significantly higher in O. aureus at the base line level and after stress, however, there was no difference in cholesterol levels in response to stress. Glucose blood concentration was significantly higher in O. mossambicus after stress and also in stress response values. Total protein, albumin, globulin alpha and beta2, IgM and %IgM were also significantly higher in O. aureus. Percent beta1 was significantly higher in O. mossambicus. A notable difference between the two species was observed in the profile of electrophoretic distribution of protein fractions (Figure 1).
Variance within O. aureus was significantly greater than within O. mossambicus (P = 0.05) in the following parameters: NBT before stress, ceruloplasmin after stress, cholesterol, magnesium, phosphorus, calcium, total bilirubin, triglycirides, total protein in the second experiment, alpha protein and IgM. LDH variance was significantly greater in O. mossambicus.
DISCUSSION
Increase in glucose concentration is a secondary response to stress, and the level of increase is a measurement for stress response. The aquaculture environment exposes the fish to a regime of repeated acute stress, which has deleterious effects on growth, reproduction and the immune response (Pottinger and Carrick 1999). The results indicate stronger stress response in O. mosambicuss, suggesting that this species may be more sensitive for stress. Glucose blood concentration was the only parameter in which significant differences in stress response were detected.
Significant differences were also identified in NBT and in ceruloplasmin activity after stress, but not in stress response values. An increase in NBT values after stress was observed in O. aureus, but no change was observed in O. mosambicus. Respiratory burst activity (measured by NBT) is one of the most important bactericidal mechanisms in fish (Secombes and Fletcher 1992). Balfry et al. (1997a) observed significant strain differences in NBT between red and wild type O. niloticus. Our results provide additional evidence for genetic influence on this important component of the non-specific immunity in tilapia. The reduction in ceruloplasmin activity after stress was stronger in O. mosambicuss than it was in O. aureus. The oxygen reduction activity of ceruloplasmin is also involved in non-specific immunity, and the differences detected between the two species in its activity after stress may indicate genetic control on this trait.
Balfry et al. (1997a) also identified a significant difference between red and wild-type O. niloticus in lysozyme activity following Vibrio pararahaemelyticus challenge. Such difference was not identified between O. aureus and O. mossambicus in this study. It may be that a bacterial challenge can also trigger differences in lysozyme activity in O. aureus and O. mossambicus, but it is also possible that levels of this parameter of the immune response are similar in both species.
Total cholesterol level was found to be associated with disease resistance in fish (Maita et al. 1998a). Our findings indicate that there may be a genetic influence on plasma cholesterol level in tilapia. Such putative genetic factor(s) may also influence disease resistance. Higher levels of serum protein, globulin and IgM are thought to be associated with stronger innate response in fish (Wiegertjes et al. 1996). A disease challenge of fish from the two species can help in determining whether higher globulin and IgM levels in O. aureus are associated with improved disease resistance. The profile of electrophoretic protein fractions in O. mossambicus was similar to the carp profile described by Rehulka (1993), which enabled identification of the different protein fractions. The alpha 2 fraction in O. aureus was not detected by the eletrophoresis method used here, suggesting that it is very low and may be even absent in this species.
Biochemical differences were also identified in levels of LDH, calcium, total bilirubin and triglycirides. The immunological significance of those differences is currently unknown. It is also important to note that 22 parameters were tested in the second experiment. Therefore, it is expected that at least one of the differences identified is a false positive due to type I error of 5%, and the data should be treated as preliminary results.
Wide variation was detected within O. aureus in some of the parameters examined. A larger sample size should be used to learn if the differences are large enough to produce segregating families for genetic analysis of those parameters. Larger differences were identified between the two species in a broader range of immunological parameters and in stress response. It is therefore concluded that crosses between the two species should be more informative for genetic analysis of non-specific immunological parameters and stress response.
In this study we identified significant differences in non-specific immunity and stress response between two tilapia species. Further research is needed to determine if the immunological differences are associated with variation in disease resistance. The differences identified between O. aureus and O. mossambicus suggest that hybrid families from the two species may be used to construct a segregating population for genetic analysis of immunological traits and stress response.
ACKNOWLEDGEMENTS
This study was supported by research grant number US-2664-95 from BARD, the United States – Israel Binational Agricultural Research and Development Fund. The contribution of Y.P. to this study was supported by BARD postdoctoral grant number FU-268-97.
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