Journal of Microbiology and Biotechnology Research
Scholars Research Library
J. Microbiol. Biotech. Res., 2012, 2 (1):70-77
(http://scholarsresearchlibrary.com/archive.html)
ISSN : 2231 –3168
CODEN (USA) : JMBRB4
Serum protein electrophoretic characterization of C. anguillaris, H.
bidorsalis and their hybrids from the northeast Nigeria
M. Y. Diyaware1*, A. B. Haruna2, K. A. Abubakar3 and G. O. Omitogun4
1
Department of Fisheries, University of Maiduguri, Nigeria
Department of Fisheries, Federal University of Technology, Yola, Nigeria
3
Department of Biological Sciences, Federal University of Technology, Yola, Nigeria
4
Department of Animal Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria
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2
ABSTRACT
Electrophoresic characterization of Clarias anguillaris, Heterobranchus bidorsalis and their
hybrid were carried out using 12% Sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE). Twelve (12) nine-month fish samples, (three from each genetic mating) were used
for the experiment. The result showed no variation (p>0.05) among the number (11.67 = 12
each) of resolved serum protein bands of the two hybrids species. However, the putative parents
(Clarias anguillaris and Heterobranchus bidorsalis) showed significant (p<0.05) difference
among the number (11.33 = 11.00 and 10.00, respectively) of resolved serum protein bands. The
8th band was absent in Clarias anguillaris, while H. bidorsalis lacked 10th, 11th and 12th bands.
Clariabranchus lacks 10th band, while 12th band was not observed in Heteroclarias. The
UPGMA cluster analysis showed 96.10 % similarity coefficient for pure line C. anguillaris and
82.20% for H. bidorsalis. Generally, all the species had more than 80% similarity coefficient to
each other. This result serves as base line information for identification of African fish and their
hybrid incase of natural hybridization due to indiscriminate use.
Key words: Serum protein, Electrophoresis, Clarias anguillaris, Heterobranchus bidorsalis,
hybrids, Northeast Nigeria.
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INTRODUCTION
Electrophoresis has been used as a tool for examining biochemical variation in a population. It is
a technique where charged molecules migrate in a gel medium when an electric field is applied
providing a methods of size or charge fractionation of the molecules. It is used to isolate
individual components of protein or nucleic acids [1].
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Biochemical and molecular tools for identification of protein has been reported by [2], for
mitochondrial DNA [3]. Nuclear DNA Polymorphisms have been developed by [4] to study
genetic variation in different fish species.
Procedure for the identification of the species of fish from the separation of protein patterns
obtained by Sodium dodecylesulphate polyacrylamide gel electrophoresis (SDS-PAGE) was
described by [5]. According to then, the procedure also offers advantages over other methods
available for the identification of cooked fish. The first fertile hybrid generation ever known to
have been produced artificially between salmon (Salmo salar L.) and trout (Salmo trutta L.) was
examined electrophoretically by [6]. Analysis of protein polymorphisms was performed as a
means to identify different populations of carp [7, 8]. An electrophoretic taxonomic study on
serum ptoteins of Acanthobrama marmid, (Leucicus cephalus) and Chondrostoma regium have
also been studied [9]. Protein transferrins have been used to identify the genetic characterization
within the carp populations [7]. Electrophoretic separation was carried out on vertical
polyacrylamide gel by [10] to compare common carp (Cyprinus carpio L.). Likewise it was used
to evaluate the morphological and electrophoretic characteristics in the Southern Coast of the
Caspian Sea [11], of a hybrid between Eretmochelys imbricata and Caretta caretta
(Cheloniidae). The identification of two Salmonid smolts suspected to be hybrids between
Atlantic salmon (Salmo salar) and trout (S. trutta) using morphological evaluation was
confirmed by electrophoretic separation of serum proteins [12]. That of roach (Rutilus rutilus L.),
rudd (Scardinim erythrophthalmus L.), bream (Abramis brama L.), and their natural hybrids
have also been studied [13].
[14] also confirmed the morphometric characterization of the F1 progenies of the crosses between
Oreochromis niloticus and Sarotherodon galileus using SDS-PAGE which revealed that the
hybrid progenies inherited more of maternal biochemical properties than the male parents and
that females resolved proteins of a higher molecular weight than males which showed low-range
proteins. [15] compared three populations, each of Oreochromis aureus, O .mossambicus and O.
urolepis honorum and two each of red tilapia derived from hybridization of O. urolepis honorum
females and O. mossambicus males, for electrophoretic mobility of their enzymes at 27 enzymes
loci. They had constructed dichotomous keys based on relative electrophoretic mobility of
isoenzymes for the identification of the species.
Electrophoresis of serum proteins have been widely used in the classification of fish.
Discrimination of related taxonomy can be easily made according to their electrophoretic results
of serum proteins [16]. The serum proteins of five fish species of freshwater fish (Sarotheredon
galilaeus, Tilapia zillii, Orecohromis niloticus, Clarias lazera, and Barbus bynni) has been
studied electrophoretically [17]. They reporter 8 fractions of serum proteins in S. galilaeus, T.
zillii and O. niloticus while 7 and 5 fractions were found in C. lazera and B. bynni, respectively.
The importance of electrophoretic analysis in aquaculture to determine the purity of species of
fishes has been emphasized by several authors [18,19, 20].
There has been an increase in commercial production of Clariid catfishes and their hybrids in
Nigeria. Morphological features have used in many genetic and breeding studies to identify both
parental and hybrid stocks, the morphological characters tends to overlap each other due to
differences in environment or mixed up in the population during sample collection. African
catfish and there hybrids has high commercial value in Nigeria. Yet the electrophoretic
characterization (protein banding) of these species Clariid fish and their hybrids have not been
fully exploited except for report of [21] on the genetic diversity of commercial Clarias
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gariepinus population and that of [22] on interspecific and intergeneric hybrids of
Heterobranchus longiflis, Clarias gariepinus and Clarias anguillaris in the southern Nigeria.
There has been paucity of information on the electrophoretic characterization of the African
catfish from the northern Nigeria. This study investigates the protein profiles of the C.
anguillaris, H. bidorsalis, and their hybrids by SDS-PAGE from the semi-arid zone of Nigeria
with the view to providing base line information for further genetic studies.
MATERIALS AND METHODS
Experimental fish
The experimental fish was produce through intergeneric hybridization between C. anguillaris
obtained from fishermen in Lake Chad () and Heterobranchus bidorsalis from Lake Geriyo ().
Nine month old F1 C. anguillaris, H. bidorsalis, and their hybrids were collected from
polyethylene lined fish ponds of the Department of Fisheries Alau, University of Maiduguri
located at latitude 13o 86’N and 14o N and longitude 12oE and 13oE. The fish were conveyed to
the Fish biology laboratory of Department.
Blood sample collection and preparation
Blood samples (0.3-1.0ml) were collected from the caudal peduncle with means of 2ml
hypodermal syringe and needle of from each of the genetic mating in triplicates. The blood
samples were empties into centrifuge tube containing 1 ml of saline solution (0.9%). The
samples were allowed to clot and later centrifuged at 1000rpm for 10 minutes. The serum was
decanted into 5 ml plain heparin bottle and placed into ice pack. The samples were transported in
an ice pack to Biotechnology laboratory Department of Animal Science, Obafemi Awolowo
University (OAU), Ile-Ife, Nigeria for SDS-PAGE.
Sample preparation for SDS-PAGE analysis and gel staining
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using
Bio-Rad Mini Protein II cell (10ml capacity) in the Biotechnology Laboratory of Obafemi
Awolowo University, Ile-Ife, Nigeria. The method of analysis was a discontinuous buffer
system, which means that the buffer in the reservoir was of different pH and ionic strength from
the buffer used to cast the gel. The gel was prepared using 10% (10g of SDS/90ml in distilled
water) acrylamide/Bis acrylamide solution. 7.5% of β-mercaptoethanol (SIGMA) in sample
buffer was used for the preparation of the blood samples. The mixture of blood serum protein
and the sample buffer was added at a ratio 1:1 and heated at 95oC for 5 min in a water bath. The
mixture was then mixed carefully with a micropipette in order not to introduce air into the
mixture and loaded carefully into the wells on the gel. The separation of proteins was carried out
in the Bio-Rad Mini Protein II cell at 150V for an hour with the aid of Bio-Rad Electrophoresis
Power Supply (Model 200/2.0). After electrophoresis has been carried out, the gels was carefully
removed and placed in a staining/fixing solution composed of 3% Coommassie blue stain in 1:3
glacial acetic: methanol solution. The staining was done on a R100TH Rotatest shaker
(Luckham) for 0.5-1 hours. Thereafter, the staining solution was removed and the de-staining
solution added and allowed to de-stain for 3 hours on the shaker. The gel was scanned with a
table scanner for interpretation. The electrophoresis was repeated three (3) times to ensure
constancy of protein pattern. Relative mobility was determined using the following [23] formula:
Relative mobility (Rm) = Migration distance of a protein band / Migration distance of the fastest
migrating band
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M. Y. Diyaware et al
J. Microbiol. Biotech. Res., 2012, 2 (1):70-77
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Scoring of bands
Each gel was scored visually by placing it in a light box which allowed electrophoretic protein
bands to be seen clearly. Gels were scored visually: presence (1) or absence (0) of protein bands
classification was used. Rule (mm) was used to measure out the reference maker (RM).
Data analysis
Data collected from the SDS-PAGE were analyzed by means of PAST software package
(Version 2.0) to generate dendogram [24] of [25] UPGMA cluster analysis for standard genetic
distances based on band frequencies scores for various genetic mating. This was done to
determine the genetic similarity (Similarity Coefficient) between the various mating
combinations. The numbers of scored bands from all cross combinations (treatments) were also
subjected to one-way analysis of variance (ANOVA). Differences between the mean were
determined using LSD (p=0.05) with means of statistix 8.0 for windows.
RESULTS AND DISCUSSION
The mean number of resolved serum proteins in C. anguillaris, H. bidorsalis, and their pure line
progenies are shown in Table 1. A total of 13 protein bands were resolved along the entire gel.
The pure line parents; C. anguillaris and H. bidorsalis had a mean total serum protein band of
11.33 and 10.00 respectively. The number of serum bands among the two putative parents varied
significantly (p<0.05). Both hybrids have similar mean number of serum protein.
The 8th band were absent in the pure line C. anguillaris, while the 9th band was not observed in
hybrid (♀C. anguillaris x ♂ H. bidorsalis). However, pure Hb x Hb did not show 10th, 11th and
12th bands. Only 12th band was absent in reciprocal hybrid hybrids (♀H. bidorsalis x ♂C.
anguillaris). All the genetic crosses had the 1st to 7th, 8th and the 13th bands.
A dendrogram generated by UPGMA cluster analysis [24] of [25] standard genetic distances
based on (band) frequencies scored in figure 1 show the similarity coefficient among the various
mating combinations is (Fig. 1). There was high level of genetic similarity among offspring of
various mating combinations. The pure line progenies of C. anguillaris and reciprocal hybrids
(♀H. bidorsalis x ♂C. anguillaris) has about 96.10% generic similarity coefficient; similarly, H.
bidorsalis and their reciprocal hybrids have 96% similarity. The pure line C. anguillaris
offspring has about 96.1% intra-species similarity coefficient, while H. bidorsalis had 82 %
similarities coefficient. However, pure line C. anguillaris has showed 90% similarity.
Table 1: Mean number of protein bands resolved in the in the inter-generic hybrid C. anguillaris x H.
bidorsalis and their hybrids
Cross combinations
♀Clarias anguillaris x ♂Clarias anguillaris
♀Clarias anguillaris x ♂ Heterobrancus bidorsalis
♀Heterobrancus bidorsalis x ♂Heterobrancus bidorsalis
♀ Heterobrancus bidorsalis x ♂Clarias anguillaris
Mean ± SE Resolved serum bands
11.33a ± 0.33
11.67 a ± 0.33
10.00b ± 1.52
11.67 a ± 0.33
The total number of serum protein bands resolved (13) along the entire gel in this study varied
with 16 protein bands reported by [22], for Heterobranchus longifilis, Clarias gariepinus,
Clarias anguillaris and their hybrids and to 7 protein bands reported by [17] for Clarias lazera.
This variation could be due to the differences in the species and the environment where the
experimental fish species were collected.
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9
4
7
6
5
2
1
3
10
11
12
8
M. Y. Diyaware et al
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1
2
3
4
5
6
7
8
9
10
11
12
1
0.98
0.96
Similarity Coefficient
0.94
0.92
0.9
0.88
0.86
0.84
0.82
0.8
0
Fig: 2. Dendrogram generated by UPGMA cluster analysis of Nei’s standard genetic distances based on
scored electrophoretic protein band frequencies
The mean number of serum protein bands observed in pure line C. anguillaris during this study
differed from the report of [22] for similar fish species, but corresponds to that of pure line
Clarias gariepinus progenies. The number of protein bands for pure line H. longifilis reported by
[22] was also higher than that of pure line H. bidorsalis reported in this study. However, the
protein band resolved in this study for the reciprocal hybrids is similar to those reported by [22]
for hybrid between Heterobranchus longifilis (♂) x Clarias gariepinus (♀).
The consistent appearance of some similar bands (1-7, 8, and13, corresponding to globulin,
transferring and albumin sub-fractions of serum protein). The high similarity coefficient (above
80%) observed in all the genetic crosses in this study confirm that Clarias anguillaris and H.
bidorsalis are closely related which permitted successful hybridization between the two species.
The close relationships between the genera of Heterobranchus and Clarias have been
emphasized by [25] using morphological and osteological features. According to [26], the
species from the subgenus Clarias are more closely related to Heterobranchus than to some
other genus of Clariidae [22] also confirmed inter-specific and inter-generic relationships
between the Heterobranchus and two subspecies of Clarias (Heterobranchus longifilis, Clarias
gariepinus, and Clarias anguillaris.
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CaCa
CaCa
CaCa
CaHb
CaHb
CaHb
HbHb
HbHb
HbHb
HbCa
RM
0.22
0.4
0.5
0.6
0.71
0.74
0.79
0.81
0.84
0.88
0.91
0.95
1.00
+
Figure 1: A representative of Coomassie-blue stained SDS-PAGE gel showing banding patterns of Clarias
anguillaris, Heterobranchus bidorsalis, and their hybrids.
1-3 = Female Clarias anguillaris x Male Clarias anguillaris
4-6 = Female Clarias anguillaris x male Heterobranchus bidorsalis
7-9 = Female Heterobranchus bidorsalis x Heterobranchus bidorsalis
10-12 = Female Heterobranchus bidorsalis x Clarias anguillaris
Genetic distances indicated a greater similarity between geographically isolated (hatchery)
populations than between hatchery and wild populations of C. gariepinus [28, 21].
The higher number (11.67 = 12) of resolved serum proteins in both hybrids could have been
inherited from the pure-line parents. The absence of 8th band in C. anguillaris, 10th band in
Clariabranchus, 10th, 11th in pure H. bidorsalis, and 12th in the reciprocal hybrids
(Heteroclarias) could possibly be used as a marker for differentiating this species from the other
Clariids in the Northeast region of Nigeria. The absence of 12th band in the reciprocal hybrid
(Heteroclarias) differentiated it from the normal hybrid (Clariabranchus).
The low number of differentiating protein bands observed in this study may not be sufficient
enough to be used as diagnostic tool for identifying them in this region, hence more samples
from the populations in the Northeast are needed.
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The two hybrids evidently inherited more of their maternal protein bands as revealed by SDSPAGE. Similar result was also reported by [22] and [12] for inter-specific and inter-generic
hybridization of African catfish and crosses Oreochromis niloticus x Sarotherodon galileus
respectively.
Acknowledgement
The authors wishes to thank Mr. Akin Babatunde of Biotechnology Laboratory of the
Department of Animal Science, Obafemi Awolowo, University of Ile-Ife for the assistance in
running the electrophoreses analysis. The first author also thanks the efforts of Mr. Wasiu A.
Olaniyi and Mal Nuradeen Shifaluwo for their tireless during this study.
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