Protein and Isozymes Electrophoresis for Identifying Some Transplanted Carp Species

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1 268 Bulgarian Journal of Agricultural Science, 12 (2006), National Centre for Agrarian Sciences Protein and Isozymes Electrophoresis for Identifying Some Transplanted Carp Species ELGHOBASHY PH. A 1.; A. A. RAMADAN 1 ; M. A. RASHD 2 and I. H. IBRAHEM 1 1 Genetics & Breeding Department., Central Laboratory for Aquaculture Research (CLAR) Agriculture Research Centre, Abbassa, Abou-Hammad, Sharkia, Egypt 2 Genetics Department, Fac. of Agric., Ain Shams Univ., Shubra El-Khima, Cairo, Egypt Abstract ELGHOBASHY PH. A., A. A. RAMADAN, M. A. RASHD and I. H. IBRAHEM, Protein and isozymes electrophoresis for identifying some transplanted carp species. Bulg. J. Agric. Sci.12: Genetics similarity and diversity of five cultured carp fishes collected from Abbassa area (CLAR) in Sharkia, Egypt were examined. SDS-PAGE for muscle protein and native-page for some isozymes, such as esterases and malate dehydrogenase were studied on ten individuals fish of each species. To discriminate isozymes variation among carp species, different synthetic Est substrates; α, β-n acetate and α-n propionate were tested. The results indicated that the soluble muscle protein patterns it may be conducted that each population had a unique banding pattern. Close relationship was noticed between bighead and black carp with similarity coefficient value of (94%). Silver carp had a different banding pattern and branched out as subgroup from each of bighead and black. The results of α, β naphthyl esterase showed perfect similarity within silver individuals. The average similarity values among the five carp species resulted from cluster analysis of α, β esterase isozymes data showed a close relationship between grass and silver carp (95%). High similarity in black, mirror carp, and grass carp while bighead and silver carp showed rear polymorphism with propionate esterase. Also the result showed that the variation in Mdh was limited and restricted on silver and mirror carp, while bighead, black and grass carp showed no variation within their populations. The final data of this work showed close relationship between bighead and black carp (85%). Moreover, quite close relationship was also noticed between each of black and silver carp followed by bighead and silver carp. Key words: electrophoresis, identification, protein, isozymes, carp species Introduction Cyprinids belong to the order Cypriniformes and the family Cyprinidae. This order includes over 1700 species and 220 genera of minnows and carp. Family Cyprinidae is the largest in the world, representing over 1400 species. The fish of this family are distributed throughout Africa, Asia, Europe, and North America and live

2 Protein and Isozymes Electrophoresis for Identifying Some almost exclusively in freshwater. Characteristics of Cyprinids include pharyngeal teeth, teeth used to grind food; a lack of an adipose fin; and the presence of barbels on many species. Cyprinids range greatly in size; from Danionella translucida at (1 cm) to the large Barbus tor, which reaches the length of 6 feet (1.8 m). The popular name of carp in Egypt is Mabrouk. Over the last decades in Egypt, carp fish production was steadily increased due to extension of fish hatcheries (Rothbard, 1981). All carps are less spawning naturally in nature, but artificially are more efficiency in hatcheries. Also, carps have relatively higher and faster growth rate but they have lower prices than tilapia or mullet because of their tiny bones in the flesh. Therefore, particular efforts have been made to overcome this problem using minced fish flesh as a basis of formulating products that makes it easier to be consumed (Ghazi et al., 1989; Shaltout, 1989). The carp species in Egypt are (Mirror) carp (Cyprinus carpio), Grass carp (Ctenopharyngodon idellus), Silver carp (Hypophthalmichthys molitrix), Bighead carp (Aristichthys nobilis) and Black carp (Mylopharyngodon piceus). Recently, isozymes polymorphism and protein banding patterns in addition to the molecular technique have been described. Polyacrylamide gel electrophoresis (PAGE) is an easy method for separating and visualizing proteins and isozymes to reveal patterns of ontogenetic and phylogenetic variations (Falk et al., 1996). The intraspecific variation in the protein patterns from different fish species, including some tilapias and carp, has been obtained mainly by the different staining intensities of bands. These variations have been attributed to one or more factors such as seasonal variation and sex differences or sexual maturity stages (Wu and Wang, 1992). Most studies of the proteins and isozymes electrophoresis of carp skeletal muscle myosin in SDS-PAGE revealed that the molecular weight determination of the light chain 3 (LC B3 B) was essentially affected by the electrophoretic techniques (Huriaux and Focant, 1978; Rashed et al., 1998). Five esterase isozymes in serum, liver and muscles of two stocks of grass carp, Ctenopharyngodon idellus was revealed. One polymorphic isozyme was found in liver and muscles, designated as Est-4. The two stocks showed significant differences of two alleles frequencies (Est- 1 and Est-2) (Bezrukov, 1987). According to Khan and Gadru (1988) the 11 to 16 electrophoretic patterns of protein for six cyprinid fish from Kashmir using PAGE were studied. The Mdh patterns tended to fluctuate at different pre and post hatching stages in carps (Padhi and Khud- Bukhsh, 1989). The protein electrophoresis was used also to examine the genetic variation and divergence among five species of Cyprinodontid fishes (Echelle et al., 1995) In addition Yang and Gui (1999) analyzed four isozyme systems, including esterase (EST), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD), by polyacrylamide gradient gel electrophoresis in liver, muscle and red blood cells of two artificial gynogenetic populations of silver carp in China, focused on the polymorphism of lactate and malate dehydrogenases in two lines of common carp by electrophoretic analysis of the enzymes extracted from blood erythrocytes was found in loci LDH-B1 and smdh-a1, 2, each

3 270 Elghobashy A., A. A. Ramadan, M. A. Rashd and I. H. Ibrahem locus having two alleles (Ostaszewska and Martyniuk, 2000). The objectives of this research were assessment of population genetic variation between five carp species raised in Egypt namely, Bighead, Black, Mirror, Grass, and Silver carp using SDS-PAGE of watersoluble protein and PAGE of three isozyme systems; Esterase (Est) α and β-naphthyl acetate, α-naphthyl propionate and malate dehydrogenase (Mdh) and develop some biochemical and molecular genetic markers for identifying the carp in Egypt. Materials and Methods This investigation was carried out with cooperation between Genetics Department, Faculty of Agriculture, Ain Shams University, Shubra El-Kheima, Cairo, Egypt and the Central Laboratory for Aquaculture Research (CLAR), Abbassa, Abo-Hammad, Sharkia, Egypt. Carp Fishes Fingerlings (about 30 to 40 g) of five carp species namely Bighead (Aristichthys nobilis), Black carp (Mylopharyngodon piceus), Common (Mirror) carp (Cyprinus carpio), Grass carp (Ctenopharyngodon idellus ), Silver carp (Hypophthalmichthys molitrix) were collected from Abbassa location. Ten individuals from each species were sampled and dissected. All have the same number of chromosomes (2n=48) without mirror carp which has 2n=98 (Gregorian et al., 1995) while Wolfe (2001) found 2n = 104 duplicated chromosomes in common carp. Samples preparation Fish samples were placed directly in ultra low temperature freezer at -70 Pє PC until they were dissected. About 0.5 g of freshly skeletal muscle was taken and kept at 4 Pє PC until extraction. All samples were ground using liquid nitrogen and extracted in an appropriate volume of extraction buffer, then homogenized for 15 sec and centrifuged at rpm at 4 Pє PC for 10 min. Electrophoresis Soluble muscle protein and isozymes fractions were separated exclusively on 15 and 9% polyacrylamide gel respectively. Vertical slab (19.8 cm x 26.8 cm x 0.3 cm) gel electrophoretic apparatus (Manufactured by LABCONCO) was used (Payne 1976; Hussain et al., 1988 and Falk et al., 1996). PAGE of three isozyme systems; Esterase (Est) α and β- naphthyl acetate, α-naphthyl propionate and Malate dehydrogenase (Mdh) isozymes were used. Gels and Statistical analysis Electrophoresis was applied at Department of Genetics, Faculty of Agriculture, Ain Shams University. The protein and isozymes profiles were analysed and scanned using GelDoc2000 Instrument and Quantity-One Software Package supplemented by the manufacturer (Bio-Rad). The banding profiles were scored in binary manner (1.0) where 1 indicates band present while band absence was indicated by 0. The scored binary profiles were introduced to SPSS statistical software package (Ver. 8) to estimate both similarities and genetic distances for both within and among population relationships according to Bardakci and Skibinski (1994). For constructing a phylogeny tree, the data generated from protein and isozyme patterns were introduced also to SPSS package program (Ver. 8) according to binary values (1, 0). The output results in-

4 Protein and Isozymes Electrophoresis for Identifying Some volved different unweighted pair-group method of analysis (UPGMA) and constructed dendrogram, according to Sneath and Sokal (1973). Results and Discussion Many electrophoretic studies have been conducted to identify the differences among carp species over the world. Genetic variants can be applied as markers to many different types of studies in natural and cultured fish populations. In order to resolve the expression of particular gene products, one must choose the appropriate tissue or such cellular fraction. In this regard, soluble muscle proteins and some isozyme systems were tested in five carp species with regard to the number and intensity of bands. I. SDS-PAGE for muscle-soluble protein Soluble muscle proteins of five carp species are shown in Figure 1. The densitometric analysis is summarized in Table 1. A total number of 19 bands were detected in bighead carp which represented two polymorphic bands at relative mobility ranges of 0.36 to 0.38 and M.W of 78 to 76 KDa, respectively. The homogeneity between individuals were large (95%) in this species. In black carp, the data represented also two polymorphic bands around relative mobilities of 0.42 to 0.47 and M.W of 48 to 40 KDa, respectively in samples 1 and 2. A maximum of 19 bands were scored. Similarly, high homogeneity (95%) were detected between individuals of black carp. As shown in Table 1, the muscle protein patterns of common (mirror) carp had total of 13 bands. Six polymorphic bands were noticed at relative mobilities (Rm) 0.04, 0.12, 0.18, 0.28, 0.35 to 0.76 with M.W of 141, 124, 112.2, 95, 78 to 25 KDa, respectively. In this specie; there was high polymorphism (14%) between their individuals. In grass carp, the SDS data showed total of 10 bands, three bands out of them were scored as polymorphic bands at relative mobility s of 0.09, 0.35 and 0.40 with M.W of 137, 81 and 72 KDa, respectively. The other seven bands were scored as monomorphic bands. In this specie there were some homogeneity (93%) between individuals and some polymorphism between anothers. Unlike above species, the protein patterns of silver carp represented highest similarity average (100%) between all individuals (Table 1). A total of 17 common bands were detected according to their relative mobility in all individuals and consequently very high homogeneity between silver carp individuals. As shown in Table 1, the similarity average values between individual of each of five species ranged of 0.95, 0.95, 0.86, 0.93 and 1.0 for bighead, black, mirror, grass, and silver carp, respectively. Phylogenetic relationships of soluble protein The similarity values among the five species of carp resulted from cluster analysis of protein data are shown in Table 2. Close relationship was noticed between bighead and black carp with similarity coefficient value of (0.94). Moreover, quite close relationship was also noticed between each of black and silver carp followed by bighead and silver carp with similarity value of 0.88 and 0.84, respectively. In addition, moderate relationship was reported between mirror carp and each of grass and silver carp as well as

5 272 Elghobashy A., A. A. Ramadan, M. A. Rashd and I. H. Ibrahem Fig. 1. SDS-PAGE profiles of soluble muscle proteins for five species of carp fishes

6 Protein and Isozymes Electrophoresis for Identifying Some Table 1 Common, polymorphic, total and similarity average of SDS-bands for five species of carps Bands Bighead Black Mirror Grass Silver Common Polymorphic Total Sim A Sim A = Similarity average between grass and silver carp with similarity value of 0.69, 0.58 and 0.52, respectively. These values are illustrated in the phonogram shown in Figure 5. It is concluded that bighead and black carp comprised one main group, while mirror and grass carp were comprised second main group. The species of silver carp branched out as subgroup from the first main group. From the results of muscle-soluble protein patterns it may be concluded that each species had a unique banding pattern. Both bighead and black carp were more similar to each other and quite unrelated to the other species (grass and mirror). While silver carp had a different banding pattern. The similarity coefficient can be considered as an indicator of homogeneity or resemblance within or among different species. In this study, high degree of polymorphism was observed, especially in the range of KDa for bighead, KDa for black carp, KDa for mirror carp and KDa for grass carp. In otherwise a highest similarity was reported for Silver carp individuals. Huriax and Focant (1978) obtained a comparable result by using SDS-PAGE to determine the molecular weight of the light chain of carp skeletal muscle myosin. While, Payusova and Tselikova (1993) found that muscle protein thermostability was higher in Vietnamese and Chinese silver carp. The genetic variation and divergence among five species of cyprinodontial fishes by protein electrophoreses was reported by Echell et al. (1995). Moreover Csizmadia et al. (1995) examined transferrrin (TF) protein in common carp using starch and Polyacrylamide gel electrophoresis to differentiate between sexes or species and discriminate between marine and freshwater fish. In the study of the genome duplications of the common carp, whole genome may have occurred in the early stages of vertebrate evolution. So, this duplication may explain the variation in chromosome number as well as the multiple gene copies and chromosome segments in fish species (Wolfe, 2001). II. Muscle isozymes variation Esterase (Est) and malate dehydrogenase (Mdh) isozymes were used in this study to interpret genetic variations and identification among different five species of carp fishes. Esterase isozymes (Est) Two kinds of substrates were used for Esterase visualize: (a) α and β-naphthyl acetate and (b) α-naphthyl propionate. Some of bands which have partial positive charges migrated to the negative pole

7 274 Elghobashy A., A. A. Ramadan, M. A. Rashd and I. H. Ibrahem while the others have partial negative charges migrated to the positive pole. (a) α- and β-naphthyl acetate Figure 2 represents the esterase isozyme patterns and the data analysis is summarized in Table 2. For bighead population with α and β-naphthyl acetate, three bands were detected. Individual s number 2 and 6 were different from the rest of individuals by the presence of band number 2. This population had two monomorphic bands at relative mobility of 0.38 and 0.64, respectively. Similarity average within this population was 0.87 (Table 2). In the black carp, according to the relative mobility of bands, four bands were detected. Individuals number 7, 9 and 10 were completely different from the others. This population had two monomorphic bands at relative mobility of (0.47 and 0.51). The average of similarity coefficient within this population was 0.77 (Table 2). This result indicated that the seven individuals of black carp had high similarity and others could be different from Black carp or supposed to environmental conditions. The muscle Est isozyme patterns of mirror carp shown in Figure 2 represent six monomorphic bands according to their relative mobility (Rm; 0.43, 0.60, 0.68, 0.82, 0.87 and 0.93) in all individuals. The samples number 2 and 4, which expressed extra band at relative mobility (Rm; 0.62). The average of similarity coefficient within this population was estimated to unity as shown in Table 2. From the result of esterase isozyme profiles of grass carp, maximum of four bands were detected according to their relative mobility. However, the total number of bands in each sample was 4 bands except carp No. 1, 9 and 10 which had three bands in each. This population had three monomorphic bands at relative mobility (Rm; 0.38, 0.42 and 0.64). The average of similarity coefficient was estimated to 0.88 within grass carp population as shown in Table 2. In silver carp population, the results of α, β naphthyl esterase showed perfect similarity between their individuals. This population had four monomorphic bands in all lanes at relative mobility s of 0.21, 0.34, 0.53 and 0.95; therefore similarity coefficient values were all equal to one as shown in Table 2. From the results of α, β naphthyl esterase patterns, two distinct zones were observed in applied fish species except silver pattern which recognized to three zones; A, B and C. Zone A (positive charge) had one band in bighead, two bands in black, grass carp and silver, three bands in mirror carp. While zone B had one band in black and silver carp, two bands in bighead and grass carp and three bands in mirror carp. Zone C was observed in silver carp which had one negatively charged band. This would indicate species differences with respect to Esterase s profiles. In addition the species differences can cause variation in the electrophoretic mobility. In general, certain pathological and extreme environmental conditions can cause variation in the quantity of a gene product but do not alter the electrophoretic mobility of the product (Buth, 1990). (b) α-naphthyl propionate Figures (3) represents the б-propionate esterase isozyme patterns of bighead, black, mirror, grass and silver carp and their final data are shown in Table 2. According to the relative mobility of bands, bighead, black and grass populations had

8 Protein and Isozymes Electrophoresis for Identifying Some Fig. 2. Native-PAGE profiles of soluble muscle Esterase α and β-naphthyl acetate for five species of carp fishes

9 276 Elghobashy A., A. A. Ramadan, M. A. Rashd and I. H. Ibrahem Table 2 Collection data of similarity coefficient (Sc) within and between five species of carps derived from binary system of protein and isozymes bands Species Case Bighead Black Mirror Grass Silver Bighead total data 0.94 SDS data 0.95 total iso 0.92 α and β Est 0.87 prop Est 0.90 Mdh 1.00 Black total data SDS data total iso α and β Est prop Est Mdh Mirror total data SDS data total iso α and β Est prop Est Mdh Grass total data SDS data total iso α and β Est prop Est Mdh Silver total data SDS data total iso α and βest prop Est Mdh The bold No. indicate to (Sc) within population The light No. indicate to (Sc) between population.

10 Protein and Isozymes Electrophoresis for Identifying Some Fig. 3. Native-PAGE profiles of soluble muscle Est α-naphthyl propionate for five species of carp fishes

11 278 Elghobashy A., A. A. Ramadan, M. A. Rashd and I. H. Ibrahem total of two bands, while mirror and silver carp had four and three bands, respectively. However, with regard to, the muscle prop- Est isozyme profiles of the five species, all individuals of black, mirror and grass showed monomorphic bands while individuals of both bighead and silver carp showed one polymorphic band at Rm, 0.57 and 0.27, respectively. Therefore, the average of similarity values based on prop-esterase patterns for applied species was 0.90, 1.0, 1.0, 1.0 and 0.89 for bighead, black, mirror, grass and silver carp, respectively. In conclusion this system showed high similarity in black, mirror carp, and grass carp while bighead and silver carp showed rear polymorphism. Moreover two zones; A and B were observed in all profiles of species (Figure 3). Esterase is one of the most complicated systems that have been intensively studied in many organisms including fish. Consequently several esterase loci were recognized in different fish species (Kornfield et al., 1979; Van der Bank et al., 1989; Schmidt and Westhelde, 1994). The present results were comparable with that of Cai (1992) who found two types of Esterase bands in Grass carp, one type of Est bands was found with no detectable variation among organs except in liver. Ferreira (1986) used the elecrophoretic profile of esterase isozyme to analyze a stock of red mutant Tilapia. The electrophoretic analysis proved monomorphic esterase pattern. Also, Wu and Wang (1992) used starch and polyacrylamide gel electrophoresis to study the Est isozyme patterns in tissues from adult and embryonic Bighead carp. Est isozyme was present but showed no developmental change between adult and embryonic Bighead carp. Rashed et al. (1998) studied the polymorphisms among three populations of O. niloticus from different hatcheries in Egypt using starch gel electrophoresis of Esterase isozymes. High patterns were noted in liver and gonads for Est with б- naphthyl propionate. Yang and Gui (1999) examined Est isozyme in two artificial gynogenetic populations, which were obtained form different brood stocks of Silver carp. Some bands specific to the different gynogenetic populations were observed in Est isozyme zymograms in liver and muscle. It was suggested that these specific bands could be used as genetic markers for the artificial gynogentic populations. (2) Malate dehydrogenase (Mdh) Malate dehydrogenase catalyzes the interconversion of malate and oxaloacetate in the Kreb s cycle. Therefore, both mitochondrial and nuclear DNA produce Mdh subunits. Figure 4 represents the muscle Mdh isozyme patterns of applied carp species. According to the relative mobility (Rm) maximum 2, 4, 2, 2 and 3 bands were detected for each of bighead, black, mirror, grass and silver carp, respectively. All similarity coefficient values for every population were equal to unity as shown in Table 2. Data summarized in Table 2 showed that Mdh isozymes not appear any differences within population of all species while between species there are some variations. So the average of similarity between bighead with each of mirror and grass was equal to one (100%) whereas with black and silver carp was 0.51 and 0.74, respectively. Perfect similarity was found between mirror and grass carp. Also similarity average between silver and each of black, mirror and grass appeared restricted polymorphism due to total number of bands (Table 2). The results showed that

12 Protein and Isozymes Electrophoresis for Identifying Some Fig. 4. Native-PAGE profiles of soluble muscle Malate dehydrogenase for five species of carp fishes variation in Mdh was limited and restricted to variation between species but no variation within population was noticed. Moreover, mitochondrial Mdh bands (slow migrated) were more intensive than fast bands. In general, many studies revealed that Mdh isozyme is a dimer and consists of two main forms; supernatant Mdh in cytoplasm (fast migration) and mitochondrial Mdh in the mitochondrial matrix. These two forms differ in electrophoretic mobility, kinetic behavior, amino acid composition and antigenic properties and they are controlled by separate genes (Banazak and Bradshow, 1975; Fisher et al., 1980). Cai (1992) also showed in Grass carp that the absolute activity of Mdh in muscle of the experimental group was only 5.7% higher than that of control group. There was no

13 280 Elghobashy A., A. A. Ramadan, M. A. Rashd and I. H. Ibrahem obvious variation in Mdh absolute activity of liver and kidney in either the experimental or control groups. Van der Bank et al. (1989) showed that the soluble forms of Mdh, which migrated anodally represented high activity in eye, heart, liver and white muscle. They reported that no heterozygotes were observed at any locus and the intermediate band was observed. Farias and Almeida-Val (1992) showed that all examined samples had six electrophoretic patterns, suggesting the existence of three gene Loci, namely s- Mdh-A, s-mdh-b1 and s-mdh-b2. According to Ostasezewska and Martyniuk (2000) wich studied two lines of Common carp, using electrophoretic analysis of the Mdh extracted from blood erythrocytes of the spawners of both lines, indicated that Loci S-Mdh-A1, 2, each locus having two alleles. Phylogenetic relationships of isozymes The total isozymes data within and between carp populations summarized in Table 2 showed that population of mirror carp had the highest value of similarity followed by silver, grass, bighead and black carp, respectively. The results indicated also that both bighead and grass carp were more affinity to each other followed by bighead with both mirror and black. The relationship between applied species of carp was illustrated in Figure 5. It is noticed close affinity (89%) in one main group between bighead and grass carp while black and silver carp represented second main group but mirror population was out group between the two groups as a taken part. Phylogenetic relationships of total data The total data resulted from protein and isozymes may give real interpreting. So similarity average values within and among the five species of carp resulted from cluster analysis of protein and isozymes shown in Table 2 showed close relationship between bighead and black carp (85%). Moreover, quite close relationship was also noticed between each of black and silver carp followed by bighead and silver carp with similarity value of 84% and 78%, respectively. In addition, moderate relationship was reported between mirror carp and each of grass and silver carp as well as between grass and silver carp with similarity value of 69%, 62% and 69%, respectively. Also both bighead and grass carp showed moderate relationship (65%). These values are illustrated in the phonogram shown in Figure 5. It is concluded that bighead and black carp comprised one main group and silver carp derived out as subgroup from it, while mirror and grass carp were comprised second main group. From the results of muscle-soluble protein patterns it may be concluded that each species had a unique banding pattern. Both bighead and black carp were more similar to each other and quite unrelated to the other species (grass and mirror). While silver carp had a different banding pattern. The similarity coefficient can be considered as an indicator of homogeneity or resemblance within or among different species. In this study, high degree of polymorphism was observed, especially in the range of KDa for bighead, KDa for black carp, KDa for mirror carp and KDa for grass carp. In otherwise a highest similarity was reported for silver carp individuals. However, in the absence of proper breeding plans, this has led to a gradual decline in the genetic quality of the seed. Consequently the negative effect of inbreeding started appearing gradually with the characteristic poor survival and slow

14 Protein and Isozymes Electrophoresis for Identifying Some Fig. 5. The phenogram relationships between five species of carp for total protein, total isozymes and total data using average linkage (between groups) growth, besides disease susceptibility of the hatchery produced seed (Ibrahim et al., 1982). Many studies indicated that the hybrids were produced by crossing the Chinese carp species such as hybrids between silver x grass carp, bighead x silver carp and between bighead x grass carp (Konradat, 1966; Bakos and Gorda, 1995). All this suggests that it is an appropriate time to act seriously about the genetic improvement of these carps. Unlike tilapia, carp doesn t make nests therefore carps need to special environmental for natural hatching. So all hatching processes conduct artificially and may give inquiring about the actual hybridization or crossing among these species. Overall, that still to more studies and research in this topic.

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