Densitometric Analysis of Serum Transferrins in Two Air-Breathing Fish (Channidae:Channiformes)
Transferrin (TF) in vertebrates makes a reliable genetic marker system demonstrating extensive polymorphism and heterogeneity at the TF locus in fish species also. The present investigation was conducted on two Indian fish species viz., Channa punctatus and Channa gachua; to explore the quantitative expression of transferrins, where for the evaluation densitometric analysis of electrophoretically separated serum TF phenotypes was done. Distinct two peaks for heterozygous TF and single peak for homozygous TF was observed in densitographs. There was noticeable incidence of higher densitometric volumes of heterozygous TFs in comparison with the homozygous TFs in both the fish species. Statistically, considering the Percent Coefficient of Variation (%CoV) of the densitometric data, a small range of ~1.17-3.72% was calculated within each TF phenotype in both the populations whereas 17.79 and 24.33% of total divergence was accounted in C. punctatus and C. gachua, respectively. Further, the resulting significant value (p<0.05) after z-score analysis deduced that irrespective of variations TF phenotypes are expressed quantitatively as per the body requirement when the samples are randomly and normally distributed. This study demonstrates some species-specific electrophoretic and densitometric pattern of TFs and represents a promising and simple biochemical tool for discerning the closely related species.
February 23, 2010; Accepted: May 06, 2010;
Published: July 05, 2010
Transferrin (TF), a monomeric glycoprotein is structurally folded into two,
an N- and a C-lobe, each having one binding site for an iron atom. It occurs
in high concentrations (2-4 mg mL-1) in vertebrate sera playing a
central role as transport protein physiologically transporting iron from sites
of absorption and heme degradation to those of storage and utilization (Welch,
1990; Cnaani et al., 2002) thereby protecting
against iron intoxication. Studies on functional aspects of fish TF, despite
being limited, also reveal that it plays an important role in innate immune
system (Yano, 1996) and some evidence suggests a correlation
between TF levels in sera of fish species and certain clinical conditions (Winter
et al., 1980; Hirono and Aoki, 1995). Moreover,
TF polymorphism is reported to be one of the best appreciated characteristics
in most of the vertebrate species where transferrin locus is found to be polymorphic
presenting 2 to 13 alleles (Kirpichnikov, 1981).
The analysis of protein itself is the principle source of elucidating its functional
and biochemical properties. For determination of plasma protein concentrations
different methods like enzyme assay(Marjani et al.,
2007), spectrophotometric analysis (Prakash and Shetty,
2008) and nonspecific dye binding methods are commonly used (Harr,
2002). Since electrophoresis directly measures the protein present in a
plasma sample, the levels obtained by gel electrophoresis and densitometry are
a more accurate and recommended method for measurement of plasma proteins than
dye methods which suffer from variability when used in different species due
to differences in dye affinity (Harr, 2002). A system
of polyacrylamide gel electrophoresis (PAGE) permits rapid and direct comparison
of multiple samples of fish plasmas for population studies. The resulting electropherograms
are suitable either for enzyme screening or for densitometric scanning after
gels are stained for total proteins (Campenhout et al.,
2004; Gicking et al., 2004; Ahmad,
As far as studies representing TF polymorphism are concerned they show applications
in breeding and maintenance of stocks, determining gene frequencies, identifying
races and genetic structure of natural populations of several fish species (Lacy,
2000; Teixeira et al., 2002, 2008;
Nabi et al., 2003; Moller,
2005). Essentially, most of these studies have a focus on electrophoretic
surveys within and among different species whilst biochemical and molecular
aspects have also been explored (Stratil et al.,
1985; Welch, 1990; Nabi et
al., 2003, 2007; Jabeen
and Hasnain, 2008). Over the years, many experiments are designed to study
the relevance and to demonstrate the sensitivity and specificity of transferrin
heterogeneity among which densitometry is the most employed one (Gorogh
et al., 2005; Ahmad et al., 2007).
Notwithstanding the vastness of the resources and the diversity of fauna, the
information on the polymorphism of TFs of fish species inhabiting Indian subcontinent
is scarce. Sister species viz., Channa punctatus and Channa gachua
are an important freshwater, food fish of Southeast Asia. They belong to genus
Channa and are commonly called as snakehead or murrel. They are characterized
by their bimodal respiration and are capable of breathing atmospheric oxygen
with accessory air breathing organs. Regarding the clinical aspects of TF in
these fishes, former studies had shown that under laboratory conditions, C.
gachua survived longest during ulcerative sickness (Nabi
et al., 2000) whereas mixed inoculum of pseudomonads had failed to
cause severe symptoms in its sister species, C. punctatus (Pradhan
and Pal, 1990). From evolutionary perspective, quantitative and qualitative
status of several well-identified abundant proteins in lower vertebrates needs
The present investigation was envisaged as a contribution to the additional information on transferrin phenotypes of Channa punctatus and Channa gachua, the sister species belonging to the genus Channa. The current report deals with the densitometric analysis of electrophoretic patterns of serum TF phenotypes of both C. punctatus and C. gachua.
MATERIALS AND METHODS
Samples of C. punctatus and C. gachua were obtained from local
fish markets of Aligarh during the month of May, 2009. Accessory air breathing
helped the fish to survive hypoxia during transportation in small water containers.
A total of 43 of C. punctatus and 32 of C. gachua were used for
serum collection. To minimize the seasonal and physiological variations (Samantaray
and Das, 1995), fishes were procured within a short time span of one month.
Blood samples were collected from live fish directly by cardiac puncture
using sterilized syringe according to the method mentioned by Nabi
et al. (2003). The sera from clotted blood was pipetted out and centrifuged
at 3,000 rpm for 10 min to sediment out contaminating blood corpuscles. Clear
sera were analyzed immediately or were stored at -20°C till further analysis.
Polyacrylamide Gel Electrophoresis (PAGE)
Serum proteins (1 μL of each serum sample) were separated according
to Laemmli (1970) protocol of polyacrylamide gel electrophoresis
(PAGE) with the only alteration that SDS was not added to any of the solutions.
After electrophoresis, the gels were stained with Coomassie Brilliant Blue (CBB)
R-250 and destained in 7% acetic acid.
The gel scans were used for software analyses where densitometric profiles
were generated using Scion Imaging software. The quantization of density of
all the TF phenotypes in a specific gel lane was done by UviDoc (ver 12.8) software
program. The densitometric volumes of each phenotype were taken as their relative
percentage values (arbitrary units) assuming them as relative transferrin concentration
in that band (Ahmad et al., 2007). For heterozygous
phenotypes (two-banded pattern), sum of band densitometric volumes of both TF
isoforms was taken as a whole.
Densitometric data was statistically treated (Daniel,
2005) where z-test was applied between the mean TF densitometric volumes
of the two populations and the differences were considered significant at p<0.05.
Percent Coefficient of Variance (% CoV) was also calculated to distinguish the
The results presented in Fig. 1 visibly showed that TF (bands marked in each gel lane), individually appears as one of the most negatively charged protein in a β-globulin region migrating faster than most of the others. Six phenotypes of TF were manifested in sera of C. punctatus whereas two were visualized in C. gachua. Out of the six TF phenotypes of C. punctatus; 13 homozygotes (2 AA, 7 BB, 4 CC) and 30 heterozygotes (13 BC, 9 AB, 8 AC) were found. While in C. gachua, there were 6 homozygous BB and 26 heterozygous AB. Moreover, not a single homozygous AA phenotype was observed in C. gachua.
The densitometric profiles of the respective gel lanes of TF phenotypes are shown in Fig. 1. The presence of corresponding TF isoforms in the form of two peaks for heterozygotes and one peak for homozygotes can be distinctly visualized in densitographs. In C. punctatus, heterozygous TF showed the mean range of 20.73-26.09% densitometric volumes which was distinctly higher than homozygous range of 13.68-16.68%. Similar case was observed in C. gachua where TF densitometric volume 17.17% of AB showed the marked increase over the homozygous BB value of 10.81 (Table 1).
Intra-specific differences as calculated %CoV are summarized in Fig.
2. In C. punctatus, the %CoV showed the variation between six TF
phenotypes where the significant values 1.17, 2.52, 1.58, 2.22 1.92, 1.41 (AA,
BB, CC, BC, AB, AC, respectively) were within the range of 1.17-2.52% accounting
for the total divergence of 17.79% within the population. Similarly, the range
of 3.23-3.72% in C. gachua between two recorded phenotypes showed the
total divergence of 24.33% (CoV) within the population where %CoV values were
3.23, 3.72 for BB and AB, respectively.
||Comparison of percent densitometric volumes of TF phenotypes
of Channa punctatus and C. gachua. (Each data point is the
arbitrary unit of average of individual loading)
||Electrophoresis patterns showing TF phenotypes of Channa
punctatus and C. gachua together with their densitometric tracings.
Each record represents a single scan without curve smoothing
||Calculated percent coefficient of variance (%CoV) within each
TF phenotype in both the populations of Channa punctatus and C.
Difference of mean densitometric volumes between the two populations (z) was
scored to be equal to 6.16. Results of two tailed P-value was 0.0001 indicating
the difference to be extremely significant.
Protein markers determined electrophoretically are of considerable importance in a number of aspects of animal breeding and livestock management where variations are generally correlated with the sexual differences, genetic diversity, temperature acclimatization or the seasonal effects. Selective breeding programmes have shown the beneficial effects of increased heterozygosity in polymorphs. The study of transferrins as the genetic marker has been facilitating because of the simplicity of inheritance of its locus and the ease of detection by electrophoresis. Although TF variants represent the polymorphism of a single locus, the data has been extremely useful in discerning the biochemical genetics of fish species also.
Presently, the comparison of the electrophoretic patterns of serum samples
of Channa punctatus and C. gachua enabled to discriminate clearly
between their TF polymorphs (Fig. 1). So obtained species-specific
electrophoretic patterns along with their densitometric profiles (Fig.
1) ascertain the polymorphic nature of transferrin locus in the studied
species also. At this point, the genetics of C. punctatus represents
six TF phenotypes AA, BB, CC, AB, BC and AC exhibiting a 3-allele system (Nabi
et al., 2003). On the contrary, C. gachua TF exhibits a 2-allele
polymorphism at the transferrin locus (Sahoo and Khuda-Bukhsh, 1989) representing
three phenotypes AA, AB and BB categorizing the TF in a co-dominant biallelic
The sera samples extracted from 43 C. punctatus and 32 C. gachua fish species were subjected to non-SDS polyacrylamide gel electrophoresis. The gel patterns thus obtained were then utilized for generating density profiles and elucidating densitometric data. In native PAGE, the quantitation of the relative amounts of TF isoforms in a sample was done assuming to be extracted in proportion to their relative composition in intact samples. Moreover, the staining of the TF isoforms was considered to be stoichiometric. Under the conditions applied, several quantitative intra- as well as inter-species differences was noticed in TF bands after densitometric analysis of the gel patterns. Most important characteristic of all was the high amount of transferrin in heterozygous condition (Table 1) in both the fish populations.
It has been considered that the synthesis of iron chelators-compounds by certain
pathogens is necessary virulence factor for removal of iron from transferrin
(Arnold et al., 1977). Studies on coho salmon
and steelhead trout stocks have shown that TF genotypes provide resistance to
different diseases (Winter et al., 1980). Moreover,
allelic variability has been shown to make the stocks less prone to disease,
especially in the case of the transferrin loci (Calcagnotto
and Toledo-Filho, 2000) where bactericidal properties of transferrins are
more strongly expressed in heterozygotes (Hegenauer and
Saltman, 1975). These data suggest that heterozygous fish are metabolically
more efficient than less active individuals.
Judging by the present results, it appears that the elevated level of heterozygous
TF might somehow be inferred to their apparent role in defense mechanism. This
could be considered in view of the fact that our earlier study on albumin of
C. gachua had revealed the relative susceptibility of homozygous albumin
phenotype to ulcerative diseases (Jabeen et al.,
2001). Also, egg whites of Phasianus colchicus containing conalbumin
of the heterozygous type show a stronger inhibitory effect on the growth of
Saccharomyces cerevisiae than egg whites with conalbumin of the common
homozygous type (Lucotte and Kaminski, 1976). According
to this hypothesis, importance of heterozygosity of transferrin genotypes remains
an important factor in disease resistance. Regardless of the specific mechanism,
these data show that genetic variation is an important biological resource to
be conserved in captive populations of fishes where disease is likely to be
of primary concern. Nevertheless, it would be interesting to account this criterion
in detail since many fish species have a high fecundity of protein markers for
population studies and therefore considerable opportunity exists for differential
Statistical analysis of the densitometric data also revealed some marked characteristics
of TF phenotypes in C. punctatus and C. gachua. Each phenotype
in both the populations demonstrated the divergence (%CoV) within a small range
of 1-4% (min-max) (Fig. 2). Also, the densitometric volumes
of TFs showed maximum of ~25% of the total variation within both the populations.
Irrespective of these variations, the statistical significance (p<0.05) of
the study considering inter-specific difference (z-score) helped in concluding
that data constitute two independent simple random samples each drawn from a
normally distributed population. This independent distribution can be acknowledged,
considering the fact, that channid species are reproductively and completely
isolated (Banerjee et al., 1988). C. punctatus
not being a polyploid (Ponniah and John, 1998) might show
a different selection mechanism than a polyploid, C. gachua where, like
polyploid salmonids, a positive selection of beneficial mutants (Ford,
2000) has taken place. On the whole, this helps in attributing, that quantitatively,
TF is approximately normally distributed in both the populations where the protein
level is expressed as per the body requirement.
Consequently, this is the first ever report presenting the densitometric analysis of TF phenotypes from sera samples of C. punctatus and C. gachua. The species-specific unique electrophoretic mobility and density along with the similarity in occurrence of higher density of heterozygous TF make transferrins of C. punctatus and C. gachua a magnificent biochemical tool for elaborate exploration and will be of immense significance in comparing its polymorphic nature in closely related species.
This study was supported by a grant to the corresponding author from the Council of Scientific and Industrial Research (CSIR-New Delhi). Facilities provide by Aligarh Muslim University, Aligarh are also gratefully acknowledged.
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