Comparative electrophoretic patterns of lactase dehydrogenase and malate dehydrogenase in five Lake Victoria cichlid species

Biochemical techniques designed to compare species on the basis of protein differences were started by NUTTALL (1904) who used immunological methods to compare the serum of humans with that of other primates. Since then more refined techniques have led to better results at the protein level in taxonomy, The analyses of proteins are considered to be the simplest indirect approach to understanding the structure and function of the genetic material, deoxyribonucleic acid (DNA). Interest in these analyses arises because of the close relationship between protein structure and gene structure. Thus by comparing the properties of homologous proteins from different taxa one is in essence comparins their genes (GORMAN er al., 1971). It is now an established fact that genetic information coded in molecules of DNA is translated through a series of reactions in the structure of proteins which form the principal morphological units of the animal body at the molecular level of organization (SIBLEY, 1952). A convenient method of comparing molecular differences between species is to measure the electrophoretic mobility of proteins in a starch gel medium (ASPINWALL and TSUYUKI, 1968) or acrylamide gel (RAYMOND and WEINTRAUB, 1959; BOUCK and BALL, 1968). Proteins with enzymatic properties can be compared on the basis of catalytic activity in the presence or absence of inhibitors (KAPLAN et al., 1959); BAILEY et al., t 1970). A combination of gel electrophoresis and histochemical enzyme detection techniques (HUNTER and MARKERT, 1957) makes it possible to combine electrophoretic mobility anti catalytic activity comparison, Enzyme patterns exhibited in starch gel or acrylamide gel have been used to classify different species. BOUCK and BALL (1968)working with lactate dehydrogenase in species of Trout found that each Trout species had LDH pattern characterbtic of that species. ASPINIWALL and TSUYUKI (1968) used muscle protein electrophoretic patterns to identify hybrid fishes. TSUYUKI and ROBERTS (1963) and TSUYUKI et al. (1964-65) found that myogen protein patterns in fishes were species specific. The myogen patterns within one family were remarkably parallel with the existing morphometric classification and these patterns constituted a single criterion by which the fishes could be identified. The fish used in these investigations were collected from shallow waters (10 metres) of Lake Victoria in two areas, Jinja and Kisumu, using gillnets and beach-seines. The study included ten specimens of each of the following specIes: (l) Haplochromis michaeli (2) Haploehromis obems (3) Astatoreochromis ulluaudi (4) Tilapia zillii and (5) Tilapia nilotica.

A molecular genetic investigation of the population structure of Atlantic menhaden (Brevoortia tyrannus)

Atlantic menhaden (Brevoortia tyrannus), through landings, support one of the largest commercial fisheries in the United States. Recent consolidation of the once coast-wide reduction fishery to waters within and around Chesapeake Bay has raised concerns over the possibility of the loss of unique genetic variation resulting from concentrated fishing pressure. To address this question, we surveyed variation at the mitochondrial cytochrome c oxidase subunit I (COI) gene region and seven nuclear microsatellite loci to evaluate stock structure of Atlantic menhaden. Samples were collected from up to three cohorts of Atlantic menhaden at four geographic locations along the U.S. Atlantic coast in 2006 and 2007, and from the closely related Gulf menhaden (B. patronus) in the Gulf of Mexico. Genetic divergence between Atlantic menhaden and Gulf menhaden, based on the COI gene region sequences and microsatellite loci, was more characteristic of conspecific populations than separate species. Hierarchical analyses of molecular variance indicated a homogeneous distribution of genetic variation within Atlantic menhaden. No significant variation was found between young-of-the-year menhaden (YOY) collected early and late in the season within Chesapeake Bay, between young-of-the-year and yearling menhaden collected in the Chesapeake Bay during the same year, between YOY and yearling menhaden taken in Chesapeake Bay in successive years, or among combined YOY and yearling Atlantic menhaden collected in both years from the four geographic locations. The genetic connectivity between the regional collections indicates that the concentration of fishing pressure in and around Chesapeake Bay will not result in a significant loss of unique genetic variation.

Population structure and variance effective size of red snapper (Lutjanus campechanus) in the northern Gulf of Mexico*

We assayed allelic variation at 19 nuclear-encoded microsatellites among 1622 Gulf red snapper (Lutjanus campechanus) sampled from the 1995 and 1997 cohorts at each of three offshore localities in the northern Gulf of Mexico (Gulf). Localities represented western, central, and eastern subregions within the northern Gulf. Number of alleles per microsatellite per sample ranged from four to 23, and gene diversity ranged from 0.170 to 0.917. Tests of conformity to Hardy-Weinberg equilibrium expectations and of genotypic equilibrium between pairs of micro-satellites were generally nonsignificant following Bonferroni correction. Significant genic or genotypic heterogeneity (or both) among samples was detected at four microsatellites and over all microsatellites. Levels of divergence among samples were low (FST ≤0.001). Pairwise exact tests revealed that six of seven “significant” comparisons involved temporal rather than spatial heterogeneity. Contemporaneous or variance effective size (NeV) was estimated from the temporal variance in allele frequencies by using a maximum-likelihood method. Estimates of NeV ranged between 1098 and >75,000 and differed significantly among localities; the NeV estimate for the sample from the northcentral Gulf was >60 times as large as the estimates for the other two localities. The differences in variance effective size could ref lect differences in number of individuals successfully reproducing, differences in patterns and intensity of immigration, or both, and are consistent with the hypothesis, supported by life-history data, that different “demographic stocks” of red snapper are found in the northern Gulf. Estimates of NeV for red snapper in the northern Gulf were at least three orders of magnitude lower than current estimates of census size (N). The ratio of effective to census size (Ne/N) is far below that expected in an ideal population and may reflect high variance in individual reproductive success, high temporal and spatial variance in productivity among subregions or a combination of the two.

Limited genetic structure of Gulf Menhaden (Brevoortia patronus), as revealed by microsatellite markers developed for the genus Brevoortia (Clupeidae)

Long-term sustainable management of wild populations should be based on management actions that account for the genetic structure among populations. Knowledge of genetic structure and of the degree of demographic exchange between discreet [sic] populations allows managers to better define management units. However, adequate gene loci for population assessments are not always available. In this study, variable co-dominant DNA loci in the heavily exploited marine genus Brevoortia were developed with a microsatellite-enriched DNA library for the Gulf Menhaden (Brevoortia patronus). Microsatellite marker discovery was followed by genetic characterization of 4 endemic North American Brevoortia species, by using 14 novel loci as well as 5 previously described loci. Power analysis of these loci for use in species identification and genetic stock structure was used to assess their potential to improve the stock definition in the menhaden fishery of the Gulf of Mexico. These loci could be used to reliably identify menhaden species in the Gulf of Mexico with an estimated error rate of α=0.0001. Similarly, a power analysis completed on the basis of observed allele frequencies in Gulf Menhaden indicated that these markers can be used to detect very small levels of genetic divergence (Fst≈0.004) among simulated populations, with sample sizes as small as n=50 individuals. A cursory analysis of genetic structure among Gulf Menhaden sampled throughout the Gulf of Mexico indicated limited genetic structure among sampling locations, although the available sampling did not reach the target number (n=50) necessary to detect minimal values of significant structure.

The geographic basis for population structure in Fraser River chinook salmon (Oncorhynchus tshawytscha)

We surveyed variation at 13 microsatellite loci in approximately 7400 chinook salmon sampled from 52 spawning sites in the Fraser River drainage during 1988–98 to examine the spatial and temporal basis of population structure in the watershed. Genetically discrete chinook salmon populations were associated with almost all spawning sites, although gene flow within some tributaries prevented or limited differentiation among spawning groups. The mean FST value over 52 samples and 13 loci surveyed was 0.039. Geographic structuring of populations was apparent: distinct groups were identified in the upper, middle, and lower Fraser River regions, and the north, south, and lower Thompson River regions. The geographically and temporally isolated Birkenhead River population of the lower Fraser region was sufficiently genetically distinctive to be treated as a separate region in a hierarchial analysis of gene diversity. Approximately 95% of genetic variation was contained within populations, and the remainder was accounted for by differentiation among regions (3.1%), among populations within regions (1.3%), and among years within populations (0.5%).Analysis of allelic diversity and private alleles did not support the suggestion that genetically distinctive populations of chinook salmon in the south Thompson were the result of postglacial hybridization of ocean-type and stream-type chinook in the Fraser River drainage. However, the relatively small amount of differentiation among Fraser River chinook salmon populations supports the suggestion that gene flow among genetically distinct groups of postglacial colonizing groups of chinook salmon has occurred, possibly prior to colonization of the Fraser River drainage.

Population structure of king mackerel (Scomberomorus cavalla) around peninsular Florida, as revealed by microsatellite DNA

A total of 1006 king mackerel (Scomberomorus cavalla) representing 20 discrete samples collected between 1996 and 1998 along the east (Atlantic) and west (Gulf) coasts of Florida and the Florida Keys were assayed for allelic variation at seven nuclear-encoded microsatellites. No significant deviations from Hardy-Weinberg equilibrium expectations were found for six of the microsatellites, and genotypes at all microsatellites were independent. Allele distributions at each microsatellite were independent of sex and age of individuals. Homogeneity tests of spatial distributions of alleles at the microsatellites revealed two weakly divergent “genetic” subpopulations or stocks of king mackerel in Florida waters—one along the Atlantic coast and one along the Gulf coast. Homogeneity tests of allele distributions when samples were pooled along seasonal (temporal) boundaries, consistent with the temporal boundaries used currently for stock assessment and allocation of the king mackerel resource, were nonsignificant. The degree of genetic divergence between the two “genetic” stocks was small: on average, only 0.19% of the total genetic variance across all samples assayed occurred between the two regions. Cluster analysis, assignment tests, and spatial autocorrelation analysis did not generate patterns that were consistent with either geographic or spatial-temporal boundaries. King mackerel sampled from the Florida Keys could not be assigned unequivocally to either “genetic” stock. The genetic data were not consistent with current spatial-temporal boundaries employed in stock assessment and allocation of the king mackerel resource. The genetic differences between king mackerel in the Atlantic versus those in the Gulf most likely stem from reduced gene flow (migration) between the Atlantic and Gulf in relation to gene flow (migration) along the Atlantic and Gulf coasts of peninsular Florida. This difference is consistent with findings for other marine fishes where data indicate that the southern Florida peninsula serves (or has served) as a biogeographic boundary.

Use of allozyme markers to determine the genetic structure of hatchery population of Thai pangas, Pangasius hypophthalmus of Jessore, Bangladesh

Genetic structure of hatchery population of Thai pangas (Pangasius hypophthalmus) of Jessore region, Bangladesh has been investigated from 1 January 2004 to 31 December 2004. Samples for this study were collected from five fish hatcheries viz. Asrom, Banchte Shekha, Chowdhury, Maola and Rezaul Haque. The enzymes were encoded by 15 gene loci: Adh-1*, Est-1*, G3pdh-2*, Gpi-1*, Gpi-2*, Idhp-1*, Idhp-2*, Ldh-1*, Ldh-2*, Mdh-1*, Mdh-2*, Pgm*, Sdh-1*, Sdh-2* and Sod*. Among them four (Est-1*, G3pdh-2*, Gpi-2*and Pgm*) were found to be polymorphic in different populations but only Gpi-2* was polymorphic in all the sampled populations. The mean proportion of polymorphic loci per population was the highest (26.7%) in Banchte Shekha hatchery while the mean proportion of heterozygous loci was 13.33% per individual in Banchte Shekha and Maola hatcheries. The UPGMA dendrogram of Nei's (1972) genetic distances indicated a relationship between the genetic distance and geographical difference. High genetic variability in stocks of Thai pangas was observed in the Banchte Shekha and Maola hatcheries and less variability was found in the other three hatcheries.

A survey on relation of morphological, molecular and phylogenetic structure of zoanthids of the islands located in the Hormoz Strait (Hormoz, Qeshm, Larak, Hengam)

The order Zoantharia (Zoanthids) is one of the most neglected orders of cnidarians in the Persian Gulf. The present study aims to investigate the biodiversity of this order with morphological and molecular examination in the Persian Gulf. For this purpose, 123 colonies of zoanthids with variety of shape and colors have been collected from intertidal and shallow water zone of four islands, i. e. Hengam, Qeshm, Larak and Hormoz. After sampling, morphological characteristics of each specimen were recorded based on in situ photographs. Then DNA was extracted using the cetyl trimethyl ammonium bromide (CTAB) method. Both mitochondrial 16S ribosomal DNA (mt 16S rDNA) and cytochrome oxidase subunit I (COI) gene fragments were amplified and sequenced. The results of preliminary morphological identification integrated with two mitochondrial markers sequencing demonstrated the presence of five different species in this region; Zoanthus sansibaricus, Palythoa mutuki, Palythoa cf. mutuki, Palythoa tuberculosa and Neozoanthus persicus?. Although at first sight, morphological properties were not successful to delineate zoanthid species, they become reliable criteria to identify and delineate species in field studies after molecular identification.

The genetic structure and phylogenetics of pikeperch (Sander lucioperca) Anzali and Amirkolaye wetlands and perch (Perca fluviatilis) populations in Aras Dam and south-west of the Caspian Sea

The genetic structure of pikeperch (Sander lucioperca) and perch (Perca fluviatilis) populations was studied using microsatellite technique. A total of 207 specimens of adult pikeperch were collected from Aras dam (57 specimens), Anzali wetland (50 specimens), Talesh (50 specimens) and Chaboksar (50 specimens) coasts. Also a total of 158 specimens of adult perch were collected from Anzali (Abkenar (50 specimens)and Hendekhale(48 specimens)) and Amirkolaye(60 specimens) wetlands. About 2 g of each specimen's dorsal fin was removed, stored in 96% ethyl alcohol and transferred to the genetic laboratory of the International Sturgeon Research Institute. Genomic DNA was extracted using ammonium-acetate method. The quality and quantity of DNA was assessed using 1% agarose gel electrophoresis. Polymerase Chain Reaction (PCR) was conducted on the target DNA using 15 pairs of microsatellite primers. PCR products were electrophoresed on poly acryl amide gels (6%) that were stained that were stained using silver nitrate. DNA bands were analyzed with BioCapt software. Allele count and frequency, genetic diversity, expected and observed heterozygosity , allele number and the effective allele number, genetic similarity and genetic distance, Fst, Rst, Hardy Weinberg Equilibrium based on X2 and Analysis of Molecular Variance (AMOVA) at 10% confidence level was calculated using the Gene Alex software. Dendogram for genetic distances and identities were calculated using TFPGA program for any level of hierarchy. The results for P. fluviatilis showed that from 15 pair of primers that were examined 6 polymorphic and 7 monomorphic loci were produced, while 2 loci didn't produce any DNA bands. Mean allele number was 4.1±1.1 and mean observed and expected heterozygosity was 0.56±0.12 and 0.58±0.14 respectively. It was also seen that specimens from all regions were not in Hardy Weinberg Equilibrium in some of loci (P<0.001). Highest Fst (0.095) with Nm=2.37 was observed between Hendekhale and Amirkolaye and the lowest Fst (0.004) with Nm=59.31 was observed between Abkenar and Hendekhale. According to AMOVA Significant difference (P<0.05) was observed between recorded Rst in the studied regions in Anzali and Amirkolaye lagoons. In another words there are two distinct populations of this species in Anzali and Amirkolaye lagoons. The highest genetic distance (0.181) and lowest genetic resemblance (0.834) were observed between specimens from Hendekhale and Amirkolaye and the lowest genetic distance (0.099) and highest genetic 176 resemblance (0.981) were observed between specimens from Abkenar and Hendekhale. Based on the genetic dendogram tree derived by applying UPGMA algorithm, specimens from Anzali and Amirkolaye wetlands have the same ancestor. On the other hand there is no noticeable genetic distance between the specimens of these two regions. Also the results for S. lucioperca showed that from 15 pair of primers that were examined 6 polymorphic and 7 monomorphic loci were produced, while 2 loci didn't produce any DNA bands. Mean allele number was 3.0±0.6 and mean observed and expected heterozygosity was 0.52±0.21 and 0.50±0.14 respectively. It was also seen that specimens from all regions were not in Hardy Weinberg Equilibrium in some of loci (P<0.001). Highest Fst (0.093) with Nm=2.43 was observed between Aras dam and Anzali wetland and the lowest Fst (0.022) with Nm=11.27 was observed between Talesh and Chaboksar coasts. Significant differences (P<0.05) were observed between recorded Rst in the studied regions exept for Talesh and Chaboksar Coasts. In another words there are three distinct populations of this species in Caspian sea, Anzali wetland and Aras dam. Highest genetic distance (0.110) and lowest genetic resemblance (0.896) were observed between specimens from Aras dam and Anzali wetland and the lowest genetic distance (0.034) and highest genetic resemblance (0.966) were observed between specimens from Talesh and Chaboksar coasts. Based on the genetic dendogram tree derived by applying UPGMA algorithm, specimens from Talesh and Chaboksar coasts have the lowest genetic distance. On the other hand the main population of this species belongs to Anzali wetland. Phylogenetic relationship of these two species was inferred using mitochondrial cytochrome b gene sequencing. For this purpose 2 specimens of P. fluviatilis from Anzali wetland, 2 specimens of S. lucioperca from Aras dam and 2 specimens of S. lucioperca from Anzali wetland were sequenced and submitted in Gene Bank. These sequences were aligned with Clustal W. The phylogenic relationships were assessed with Mega 4. The results of evolutionary history studies of these species using Neighbor-Joining and Maximum Parsimony methods showed that the evolutionary origin of pikeperch in Aras Dam and Anzali wetland is common. On the other hand these two species had common ancestor in about 4 million years ago. Also different sequences of any region specimens are supposed as different haplotypes. 177 As a conclusion the results of this study showed that microsatellite and mtDNA sequencing methods respectively are effective in genetic structure and phylogenic studies of P. fluviatilis and S. lucioperca.

Population genetic structure of Neogobius caspius (Eichwald ,1831) in the South Caspian Sea using PCR-RFLP Marker

Neogobius caspius is a small benthic fish that is native to the Caspian Sea. The importance of this fish is because of it is role as a main food resource of the sturgeon fish. The genetic diversity of N. caspius population in the Caspian Sea was studied using PCR- RFLP technique. A total of 135 samples of N. caspius were collected from coastal line in the north Caspian sea, including specimens from coasts of Anzali , Torkman Port and Chalus. Genomic DNA was extracted by phenol-chloroform method and then was amplified using a pair primer of cytochrom b gene, 2 tRNA gene and the control region sequences by a thermal cycler. D2 (5'-CCGGAGTATGTAGGGCATTCTCAC-3'), CY1 (5'-YYTAACCRRGACYAATGACTTGA-3') 12 restriction enzyme were used to digest the target gene region including: Alul HincII —Tas1 —Rsa1 -MboI -DraI -BSeNI(BSRI) Alw261(BsmAI). Bsul 51 Hin11 Bsh12851- BsuRI(HaeIII) digested PCR products were observed by silver staining method followed by Polyacrylamide gel electrophoresis (PAGE). The results were shown the same pattern among the species. There was no polymorphism and no differentiation in population in the Neogobius caspius fish and all individuals have shown homogenous genotype.