International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 4(10), 20-23, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 20 Electrophoretic Analysis in two groups of enzymes of Musca domestica L. (Diptera: Muscidae) Tripathi M.1*, Agrawal U.R. 2 and Tripathi J.Department of Zoology, Iswar Saran Degree College, University of Allahabad, Allahabad-211004, INDIA Department of Zoology, CMP Degree College, University of Allahabad, Allahabad-211002, INDIA Available online at: www.isca.in, www.isca.me Received 17th August 2015, revised 28th August 2015, accepted 4th October 2015 Abstract Indian population of Musca domestica was examined by PAGE for allozyme variation. One to five enzyme loci in the glucose metabolizing system (Group I) and six to fourteen enzyme loci in the non-glucose metabolizing system (group II) were assayed. The parameter estimated were the allele frequencies, proportion of polymorphic loci and average heterozygosity for Group I and Group II loci. The present finding is that the genetic variability measured by allozyme variation is much higher for group II than for group I enzymes in M.domestica population. The loci coding for the hydrolytic and other nonspecific enzymes are much more variable than the loci coding for the enzymes of the glycolytic pathway, Kreb’s cycle and other specific enzymes. Keywords: Glucose metabolizing enzyme, non glucose metabolizing enzyme, heterozygosity, Musca domestica, genetic variation. Introduction Glucose metabolizing enzymes (Group I) show less heterozygosity as compared to the enzymes which are involved in non glucose metabolizing systems (Group II)1-6. It was postulated that Group I enzymes are characterized by a singular physiological substrate which is generally generated and utilized intracellularly, while Group II enzymes have multiple physiological substrate which originate from the external environment. Heterozygosity and polymorphism i.e. the proportion of the loci that are genetically variable are the most important measures to compare the amount of genetic variation within different populationGenetic variability in Musca domestica populations using electrophoretic technique to detect enzyme variants have revealed that 40% - 60% of the loci examined are polymorphic8-17. As most enzymes used have broad substrate specificities, it would be interesting to compare the degree of genetic variability in enzymes known to be active in energy metabolism. The present study was carried out to compare the amount of polymorphism and the degree of heterozygosity among glucose and non glucose metabolizing enzymes in Musca domesticafrom Allahabad, India. Material and MethodsLaboratory colonies of Musca domestica L. (Diptera: Muscidae) were established in the Cytogenetics research laboratory of Department of Zoology, University of Allahabad, Allahabad, India. In the present study adult male flies of approximately same age were taken and assayed for enzyme activity at 10 gene enzyme systems viz., malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), acid phosphatase (ACPH), esterase (EST), octanol dehydrogenase (ODH), alkaline phosphatase (APH) and aldehyde oxidase (AO). Sample preparation and electrophoretic procedure were according to the method of Tripathi et al. 201014. The staining protocol of Ayala et al.18 and Tsukamoto19 were followed for the analysis of enzyme activity. Band mobilities were measured and expressed as R (x 100) as per the method of Tsukamoto and Horio20. Electrophoretic genotypes were determined by comparison of the relative mobilities of the bands. The genotype information was used to calculate frequencies of allele for each enzyme21. The genotype information, thus obtained was used to estimate genetic variability using polymorphic loci, mean observed (H) and expected (H) heterozygosity. Results and Discussion Ten enzyme systems revealed fourteen (14) loci in Musca domestica. Table-1 represents the allelic frequencies and Chi square values. Out of 14 loci, only four are monomorphic and the rest are polymorphic. The enzymes investigated in the present study were categorized into glucose metabolizing (Group I) and non glucose metabolizing enzymes (Group II) following the Classification of Gillespie and Kojima and Kojima et al . Thus MDH, IDH, G6PD, ME and LDH were categorized as glucose metabolizing enzymes (Group I) and ACPH, EST, ODH, AO and APH were grouped as Non glucose metabolizing enzymes (Group II). Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 20-23, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 21 Table-1 Allele frequencies and Chi square values at ten enzyme loci in Musca domesticaLocus (n) Allele frequencies Chi square values a B MDH 50 0.54 0.46 6.33 IDH 50 0.53 0.47 11.43 LDH 50 0.68 0.32 19.99 G6PD 50 1.00 - - ME 50 1.00 - - ACPH-1 50 0.58 0.42 17.37 ACPH-2 50 0.46 0.54 9.52 EST-1 50 0.59 0.41 2.30 EST-2 50 0.51 0.49 1.27 EST-3 50 0.46 0.54 6.33 ODH-1 50 1.00 - - ODH-2 50 0.60 0.40 8.68 APH 50 0.62 0.38 1.14 AO 501.00 - - (n)= number of individuals in each sample In Group I MDH, IDH and G6PD are polymorphic and the average heterozygosity is 0.148 for the five loci surveyed. In Group II among nine loci the activity at the loci APH and AO was confined to single locus, the activity of ACPH and ODH was confined to two loci while the activity of EST was confined to three loci. AO and ODH-1 are monomorphic and ACPH-1, ACPH-2, EST-1, EST-2, EST-3, ODH-2 and APH are polymorphic. Average heterozygosity is 0.253 for the nine loci surveyed (table-2). A comparison of proportion of the polymorphic loci of two groups revealed that the glucose metabolizing enzymes (Group I) were 60% polymorphic as compared to non glucose metabolizing enzymes (Group-II) which were 77.77% polymorphic. Thus the data shows that the loci in Group I are less polymorphic than the loci in Group II as also opined by Gillespie and Kojima. Similarly the average heterozygosity for glucose metabolizing enzymes is less than the non glucose metabolizing enzymes (table-2). It seems that the enzymes of Group II which include various non-specific hydrolytic enzymes are the most variable, whereas the enzymes of Group I, which are more specific in their mode of action and play a more significant role in cellular physiology, harbor less variation. In other Dipterans2,3,5,6,22 and other animals (echinoderms and fishes)23-28 similar pattern of result was observed between the two groups of enzymes. However, Frydenberg and Simmonson29 found that glucose metabolizing enzymes in eel pout population tend to be as polymorphic as non glucose metabolizing enzymes. They concluded that the hypothesis of Gillespie and Kojima may not be a general one for animal species. Band findings on Drosophila melanogaster populations supported the conclusions of Frydenberg and Simmonson29. But in Indian M. domestica populationgreater polymorphism and genetic variability is maintained in the enzymes of group II.Thus the hypothesis proposed by Gillespie and Kojima that glucose metabolizing enzymes tends to be less polymorphic than non glucose metabolizing enzymes, is supported by the data from Musca domestica. Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 20-23, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 22 Table-2 Average heterozygosities for Group I and Group II enzymes in Musca domesticaLocus Heterozygosity Group I Ho (Observed) HE (Expected) MDH .320 .497 IDH .260 .498 LDH .160 .435 G6PD - - ME - - Mean heterozygosity .148 .286 Group II ACPH-1 .200 .487 ACPH-2 .280 .497 EST-1 .380 .484 EST-2 .420 .499 EST-3 .320 .497 ODH-1 - - ODH-2 .280 .480 APH .400 .471 AO - - Mean heterozygosity .253 .379 Conclusion It seems that in M.domestica the glucose metabolizing enzymes (Group I) are more stringent against electrophoretic variation than that of non-glucose metabolizing enzymes (Group II). These results can be explained by the neutral theory of Kimura30. Since the glucose metabolizing enzymes have functional importance therefore functional constraint of these enzymes is much stronger. The enzymes of group II because of a nonspecific and less significant role in the physiology of organism, can tolerate far greater electrophoretic variations. 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