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.
Fish_lab@netvision.net.il
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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Table 1. Means (±SD) and P values of Student T-tests for Measurements of
Biochemical and Immunological Parameters Taken from O. mossambicus and O.
aureus Before and After Stress1.
a. Normal Level:
Parameter
Glucose
(mg/100 ml)
NBT
(O.D., 540 nm)
Ceruloplasmin
(mg/ 100 ml)
Lysozyme
(IU/ml)
Cholesterol
(mg/100 ml)
Total protein2
(g/100 ml)
b. After Stress:
Glucose
(mg/ 100 ml)
NBT
(O.D., 540 nm)
Ceruloplasmin
(mg/ 100 ml)
Lysozyme
(IU/ml)
Cholesterol
(mg/100 ml)
Mean (±SD)
O. mossambicus
O. aureus
39.6 (±3.2)
37.7 (±7.5)
P value
0.40
0.13 (±0.03)
0.15 (±0.1)
0.68
55.2 (±23.7)
46.8 (±28.1)
0.42
80.0 (±36.6)
92.9 (±50.5)
0.63
177.6 (±25.9)
312.5 (±102.2)
0.0004
3.4 (±0.9)
5.5 (±1.2)
0.0001
130 (±20.0)
97.5 (±22.6)
0.001
0.13 (±0.02)
0.09 (±0.02)
0.0002
17.5 (±6)
32.7 (±15)
0.0035
90.6 (±33.9)
98.1 (±43.0)
0.63
173.8 (±29.3)
296.4 (±118.6)
0.003
1. Before stress N = 13. After stress N = 12.
2. Total protein was only measured before stress.
Table 2a. Means (±SD) and P values of Student T-tests for Measurements of
Biochemical Plasma Components Taken from O. mossambicus and O. aureus.
Mean (±SD)
Parameter
Total cholesterol1
O. mossambicus
164 (±12.7)
O. aureus
267 (±93.5)
P value
0.007
Magnesium1
4.09 (±0.29)
4.44 (±0.88)
0.346
Phosphorus1
12.9 (±1.6)
16.4 (±8.8)
0.246
Calcium1
18.21 (±3.4)
42.4 (±22.4)
0.008
Total Bilirubin1
0.15 (±0.02)
0.34 (±0.2)
0.018
Triglyciride1
241.4 (±79)
437.5 (±332)
0.01
Total protein2
3.0 (±0.3)
4.5 (±1.2)
0.004
Albumin2
1.4 (±0.2)
2.2 (±0.7)
0.006
Globulin2
1.6 (±0.2)
2.2 (±0.5)
0.003
Alkaline Phosphatase3
31 (±8.7)
35 (±11)
0.518
Aspartate Transferase3
46.5 (±25.5)
30.3 (±19.6)
0.236
Creatine Kinase3
953 (±643)
800 (±727)
0.625
Lactate Dehydrogenase3
1150 (±532)
464 (±123)
0.043
1) mg/100 ml.
2) g/100 ml.
3) U/liter.
Table 2b. Means (±SD) and P values of Student T-tests for Levels and Percentage of
Protein Fractions Determined by Electrophoresis of Serum Protein from Blood
Samples of O. mossambicus and O. aureus.
Parameter
Alpha1
O. mossambicus
1.4 (±0.3)
Mean (±SD)
O. aureus
P value
2.4 (±1.3)
0.042
Beta11
0.9 (±0.1)
0.9 (±0.2)
0.847
Beta21
0.3 (±0.1)
0.5 (±0.1)
0.006
IgM1
0.4 (±0.1)
0.7 (±0.3)
0.009
%Alpha2
40.1 (±2.8)
43.9 (±8.7)
0.218
%Beta12
26.1 (3.9)
18.8 (±5.4)
0.003
%Beta22
8.3 (±3.0)
10.2 (±4.5)
0.274
%IgM2
10.1 (±1.8)
14.1 (±4.3)
0.021
1) g/100 ml.
2) Percentage was calculated from total protein.
Figure 1. Four Representative Profiles of the Electrophoretic Distribution of Serum
Protein Fractions in O. mossambicus (A,B) and O. aureus (C,D). (Fractions were
determined to be (1) Albumin, (2) Alpha 1, (3) Alpha 2, (4) Beta 1, (5) Beta 2, (6)
Beta 3 (IgM). Alpha 2 could not be detected in O. aureus.)