Fish species identification in Canned Tuna by DNA Analysis (PCR-SSCP)

DNA in canned tuna is degraded into short fragments of a rew hundred base pairs. The polymerase chain reaction (PCR) was used to amplify short sequences of mitochondrial DNA, which were denatured and analysed by polyacrylamide gel electrophoresis (native PAGE) for detection of single strand conformation polymorphisms. Species specific patterns of DNA bands were obtained for a number of tuna and bonito species. DE: In Thunfischkonserven liegt die DNA in Form kurzkettiger Fragmente von wenigen Hundert Basenpaaren Länge vor. Mit Hilfe der Polymerase-Kettenreaktion (PCR) wurden kurze Sequenzen der mitochondrialen DNA vervielfältigt. Anschließend wurde die gebildete DNA in Einzelsträngen überführt, die durch eine native Polyacrylamidgel-Elektrophorese (PAGE) aufgetrennt wurde. Für eine Reihe von Thunfischen und Boniten ergaben die Einzelstränge artspezifische Bandenmuster, die auf unterschiedliche Konformationen der DNA-Stränge der einzelnen Fischarten zurückzuführen sind.

Molecular studies on the effect of germanium oxide on sponges, corals and ascidians

The stress response, at the molecular level, of the soft corals Dendronephthya klunzingeri and Heteroxenia sp., hard corals Acropora hyacinthus and A. valenciennesi, an ascidian Symplegma sp. and sponges Latruncula cortica and Callyspongia crassa to germanium oxide (GeO sub(2)) was evaluated. Evaluation was carried out using bioindicators. such as the level of expression of each of the heat shock proteins (HSPs) and the silicatein enzyme in response to the compound. However, the expression was measured by SDS Polyacrylamide Gel Electrophoresis (SDS PAGE) and western blotting. The harmful concentration of GeO sub(2) that produced noticeable molecular changes in the studied samples during the first 6-24 hours was 6 μg/ml. The two studied soft corals as well as the ascidian responded to the harmful concentration of germanium oxide by expressing the heat-shock protein 90 (hsp90), while the two hard corals responded by expressing hsp70, C. crassa by decreasing the level of silicatein enzyme and sponge L. cortica produced no change by any of the used biomarkers, The soft coral Heteroxenia sp. was found to be sensitive to mechanical stress during the experiment and it was more sensitive to 6 μg/ml of GeO sub(2) than the other soft coral D. klunzingeri. The two studied hard corals were sensitive to mechanical stress during the experiment, but A. hyacinth us showed higher sensitivity than A. valenciennesi. However, these 2 corals displayed reverse response to GeO sub(2). Primitive evidences were found in the SDS PAGE to distinguish the tissue of the soft coral from that of the hard coral on the molecular level; the soft coral showed two prominent protein bands (45 and 50 kDa) while the two prominent protein bands for hard corals were 31 and 116 kDa.

Structural and functional properties of the hemoglobin components from the Caspian Sea Acipenser persicus and Acipenser stellatus

Hemoglobin (Hb) variability is a commonly used index of phylogenetic differentiation and molecular adaptation in fish. In the current study, the structural and functional characteristics of Hbs from two Sturgeon species of the Southern Caspian Sea Basin were investigated. After extraction and separation of hemoglobin from whole blood , the polyacrylamide gel electrophoresis (SDSPAGE), native-PAGE and isoelectric focusing (IEF) were used to confirm Hb variability in these fishes. Ion-exchange on CM-cellulose chromatography was used for purification of the dominant Hbs from these fishes. The accuracy of the methods was confirmed by IEF and SDS-PAGE. Spectral studies using fluorescence spectrophotometery, circular dichroism spectropolarimetry (CD) analysis and UV–vis spectrophotometery. Oxygen affinities of these Hbs were compared using Hb-oxygen dissociation curves. Also, the dominant Hbs from these blood fishes were utilized for further experiments. The behavior of Hbs during the denaturation process by n-dodecyl trimethylammonium bromide (DTAB) is investigated by UV–vis spectrophotometer and circular dichroism spectropolarimetry. The thermal denaturation properties of the Hbs wereinvestigated by differential scanning calorimetry (DSC) and Hbs aggregation performed chemically in the presence of dithiotreitol (DTT) by UV–vis spectrophotometer and chemometric study. The results demonstrate a significant relationship between stability of fish hemoglobins and the ability of fish for entering to deeper depths. The UV–Vis absorption spectra identified species of hemoglobin and showed the concentration of oxyHb and metHb decreases and deoxyHb increases upon interaction with DTAB. Besides the UV–vis spectrophotometry, the interaction of DTAB with hemoglobins has been studied using circular dichroism spectropolarimetry analysis. This experiment was utilized to measure the unfolding mechanism and compared alpha-helix secondary structure under different conditions for Hbs. The results reveal that the Acipenser stellatus Hb in comparison with Acipenser persicus Hb has more stability and more structural compactness. Besides, the results confirm the hypothesis that there is a meaningful relation between average habitat depth, partial oxygen pressure, oxygen affinity, structural compactness of Hb, and its stability.

Genetic diversity study of Caspian brown trout (Salmo trutta caspius Kessler, 1877) population in five rivers in the southern part of Caspian Sea, Iran

For study the genetic diversity of Caspian brown trout population in five rivers in the southern part of Caspian Sea in Iran 182 number generators in the fall and winter of 1390 were collected in Chalus, Sardab Rud, Cheshmeh Kileh, Kargan Rud and Astara rivers. Then about 3-5 g of soft and fresh tissue from the bottom fin fish removed and were fixed in ethanol 96°. Genomic DNA was extracted by using ammonium acetate, then quantity and quality of the extracted DNA were determined by using spectrophotometry and horizontal electrophoresis in 1% agarose gel. The polymerase chain reaction was performed by using 16 SSR primers and sequencing primers (D-Loop) and the quality of PCR products amplified by SSR method were performed by using horizontal electrophoresis in 2% agarose gel. Alleles and their sizes were determined by using vertical electrophoresis in 6% polyacrylamide gel and silver nitrate staining method. Gel images were recorded by gel documentarian, the bands were scored by using Photo- Capt software and statistical analysis was performed by using Gene Alex and Pop Gene software. Also the PCR sequencing products after quality assessment by usinghorizontal electrophoresis in 1.5% agarose gel were purified and sent to South Korea Bioneer Corporation for sequencing. Sequencing was performed by chain termination method and the statistical analysis was performed by using Bio- Edit, Mega, Arlequin and DNA SP software. The SSR method, 5 pairs of primers produced polymorphic bands and the average real and effective number of alleles were calculated 5.60±1.83 and 3.87±1.46 in the Cheshmeh Kileh river and 7.60±1.75 and 5.48±1.32 in the Karganrud river and the mean observed and expected heterozygosity were calculated 0.44 ±0.15 and 0.52 ±0.16 in the Cheshmeh Kileh river and 0.50 ±0.11 and 0.70±0.13 in the Karganrud river. Analysis of Molecular Variance results showed that significant differences in genetic diversity between and within populations and between and within individuals in the studied rivers (P<0.01). The sequencing method identified 35 different haplotype, the highest number of polymorphic position (251) and haplotype (14) were observed in the Chalus river. The highest mean observed number of alleles (2.24±0.48) was calculated in the Sardabrud river, the highest mean observed heterozygosity (1.00±0.03) was calculated in the Chalus river and the highest mean nucleotide diversity (0.13±0.07) was observed in the Sardabrud river and mean haplotype diversity was obtained (1) in three studied rivers. The overall results show that there are no same population of this fish in the studied rivers and Karganrud and Chalus rivers in the SSR and sequencing methods had the highest levels of genetic diversity.

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.

Studies on the electrophoretic patterns of fish muscle myogens

Electrophoretic patterns of the muscle myogens of prawns and some common Indian food fishes of marine and fresh water origin were obtained on polyacrylamide gel. It was observed that the characteristic band pattern which was species specific and was not altered by storage of fish in ice could be employed as a means of identification of fish species.

Manual for starch gel electrophoresis: A method for the detection of genetic variation

The procedure to conduct horizontal starch gel electrophoresis on enzymes is described in detail. Areas covered are (I) collection and storage of specimens, (2) preparation of tissues, (3) preparation of a starch gel, (4) application of enzyme extracts to a gel, (5) setting up a gel for electrophoresis, (6) slicing a gel, and (7) staining a gel. Recipes are also included for 47 enzyme stains and 3 selected gel buffers. (PDF file contains 26 pages.)

The genetic diversity of Acipenser stellatus populations in the Caspian Sea was studied using microsatellite technique

A total of 361 caudal fin samples were collected from adult A. stellatus specimens caught in the north Caspian Sea, including specimens from Kazakhstan (Ural River), Russia (Volga River), Azerbaijan (Kura River), specimens caught in the south Caspian Sea including specimens from Fishery Zone 1 (from Astara to Anzali), Fishery Zone 2 (from Anzali to Ramsar), Fishery Zone 3 (from Nowshahr to Babolsar), Fishery Zone 4 (from Miyankaleh to Gomishan) as well as from specimens caught in Turkmenistan (all specimens were collected during the sturgeon stock assessment survey). About 2 g of fin tissue was removed from each caudal fin sample, stored in 96% ethyl alcohol and transferred to the genetic laboratory of the International Sturgeon Research Institute. Genomic DNA was extracted using phenol-chloroform method. The quality and quantity of DNA was assessed using 1% Agarose gel electrophoresis and Polymerase Chain Reaction (PCR) was conducted on the target DNA using 15 paired microsatellite primer. PCR products were electrophoresed on polyacrylamide gels (6%) that were stained using silver nitrate. Electrophoretic patterns and DNA bands were analyzed with BioCapt software. Allele count and frequency, genetic diversity, expected heterozygosity and observed heterozygosity allele number, and the effective allele number, genetic similarity and genetic distance, FST and RST were calculated. The Hardy Wienberg Equilibrium based on X2 and Analysis of Molecular Variance (AMOVA) at 10% confidence level was calculated using the Gene Alex software. Dendrogram for genetic distances and identities were calculated using TFPGA program for any level of the hierarchy. It is evident from the results obtained that the 15 paired primers studied, polymorphism was observed in 10 pairs in 12 loci, while one locus did not produce DNA bands. Mean allele number was 13.6. Mean observed and expected heterozygosity was 0.86 and 0.642, respectively. It was also seen that specimens from all regions were not in Hardy Wienberg Equilibrium in most of the loci (P≤0.001). Highest Fst (0.063) was observed when comparing specimens from Fishery Zone 2 and Fishery Zone 4 (Nm=3.7) and lowest FST (0.028) was observed when comparing specimens from the Volga River and those from the Ural River (8.7). Significant differences (P<0.01) were observed between RST recorded in the specimens studied. Highest genetic distance (0.604) and lowest genetic resemblance (0.547) were observed between specimens from Fishery zones 2 and 4. Lowest genetic distance (0.311) and highest genetic resemblance (0.733) was observed between specimens from Turkmenistan and specimens from Fishery zone 1. Based on the genetic dendrogeram tree derived by applying UPGMA algorithm, A. stellatus specimens from Fishery zone 2 or in other words specimens from the Sepidrud River belong to one cluster which divides into two clusters, one of which includes specimens from Fishery zones 1, 3 and 4 and specimens from Turkmenistan while the other cluster includes specimens from Ural, Volga and Kura Rivers. It is thus evident that the main population of this species belongs to the Sepidrud River. Results obtained from the present study show that at least eight different populations of A. stellatus are found in the north and south Caspian Sea, four of which are known populations including the Ural River population, the Volga River population, the Kura River population and the Sepidrud River populations. The four other populations identified belonging to Fishery zones 1, 3, and 4 and to Turkmenistan are most probably late or early spawners of the spring run and autumn run of each of the major rivers mentioned. Specific markers were also identified for each of the populations identified. The Ural River population can be identified using primers Spl-68, 54b and Spl-104, 163 170, 173, the Volga River population can be identified using primers LS-54b and Spl-104, 170, 173 113a and similarly the population from the Kura River can be identified using primers LS-34, 54b and Spl-163, 173 and that from the Sepidrud River can be identified using primers LS-19, 34, 54b and Spl-105, 113b. This study gives evidence of the presence of different populations of this species and calls for serious measures to be taken to protect the genetic stocks of these populations. Considering that the population of A. stellatus in Fishery zone 2 is an independent population of the Sepidrud River in the Gilan Province, the catch of these fishes in the region needs to be controlled and regulated in order to restore the declining stocks of this species.

Population diversity studies of pharaoh cuttlefish (Sepia pharaonis) in the Persian Gulf and the Gulf of Oman

The pharaoh Cuttlefish (Sepia Pharaonis) is the most abundant species in the persian gulf and oman sea. The stock patterns of this species was studied conserning biological, morphological and electrophoretical aspects. In addition to measuring the biological patterns; 21 quantitative and qualitative factors were measured or counted. There tissues namely muscle, eye and liver were also used for further polyacrylamide electrophoresis analysis (SDS-Page method). The densitograms of protein bands of each tissue were prepared by Gel-Scanner and also the amount of bands' areas were estimated. The results of LSD test showed that tentacle length (TL), Tentacle club length (TCL) and TCL were indicative factors which they showed significant difference between male TL and female specimen of Bushehr and Balouchestan regions. Regarding to length and weight frequencies data the results indicated that males are always bigger than females and also, the cuttlefishes of the gulf of oman are bigger than persian gulf's samples. There were found a significant sample correlation (95%) between different quantitative parameters and the most correlation (0.963) was found to be between TL and TCL; whereas the less correlation (0.384) was observed between GL and LA3. However the gill length factor illustrated the less correlation with the other factors. The results of Cluster analysis for both sexes showed that the cuttlefishes of both studied regions belongs to seperate stocks. The electrophoretic experiments of proteins showed that the protein bands of the sample tissue of muscle and eye from both sampling areas revealed significant differences; whereas the tissue of liver wasn't recognized as an indicative tissue for population studies. Taking into consideration the findings of the present study including: (1) difference in spawning season, (2) results of dendrograms, (3) observed Significant differences in one-way analysis of variance (ANOVA) for morphometric measurements, (4) differences in body length and weight, (5) ecological variations of the persian gulf and oman sea, (6) as well as the results of electrophoresis of proteins have indicatend that: "The Pharaoh cuttlefishes of Bushehr and Balouchestan waters belong to two seperate stocks. Also, it is believed that each region has propably two different populations and more studies are needed to approve this result.

Rapid identification of European (Anguilla anguilla) and North American eel (Anguilla rostrata) by polymerase chain reaction.

A rapid and cost effective DNA test is described to identify European eel (Anguilla anguilla) and North American eel (Anguilla rostrata). By means of polymerase chain reaction (PCR) technique parts of the mitochondrial cytochrome b gene are amplified with species specific primers which are designed to produce PCR fragments of different characteristic sizes for European and American eel. The size differences can easily be made visible by agarose gel electrophoresis

Anwendung von Fischzellkulturen in der Meeresforschung

A large number of chemical pollutants can be found in the marine environment. So it is necessary to obtain informations about the toxic effects of this contaminant mixtures in general and especially on single cell level. We used an organic extract of a marine sediment from the North Sea to investigate its cyto- and genotoxicity with an in vitro system, the comet assay or single cell gel electrophoresis (SCGE). The comet assay can be applied for estimating genotoxic effects of chemicals on single cell level. First results confirm the sensitivity of this assay and its applicability in assessing genotoxic load in environmental samples. A permant cell line, the EPC (Ephithelioma papulosum cyprini) was used for the experiments. It was possible to demonstrate the suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells.

Genetic variation of rougheye rockfish (Sebastes aleutianus) and shortraker rockfish (S. borealis) inferred from allozymes

Rougheye rockfish (Sebastes aleutianus) and shortraker rockfish (Sebastes borealis) were collected from the Washington coast, the Gulf of Alaska, the southern Bering Sea, and the eastern Kamchatka coast of Russia (areas encompassing most of their geographic distribution) for population genetic analyses. Using starch gel electrophoresis, we analyzed 1027 rougheye rockfish and 615 shortraker rockfish for variation at 29 proteincoding loci. No genetic heterogeneity was found among shortraker rockfish throughout the sampled regions, although shortraker in the Aleutian Islands region, captured at deeper depths, were found to be significantly smaller in size than the shortraker caught in shallower waters from Southeast Alaska. Genetic analysis of the rougheye rockfish revealed two evolutionary lineages that exist in sympatry with little or no gene f low between them. When analyzed as two distinct species, neither lineage exhibited heterogeneity among regions. Sebastes aleutianus seems to inhabit waters throughout the Gulf of Alaska and more southern waters, whereas S. sp. cf. aleutianus inhabits waters throughout the Gulf of Alaska, Aleutian Islands, and Asia. The distribution of the two rougheye rockfish lineages may be related to depth where they are sympatric. The paler color morph, S. aleutianus, is found more abundantly in shallower waters and the darker color morph, Sebastes sp. cf. aleutianus, inhabits deeper waters. Sebastes sp. cf. aleutianus, also exhibited a significantly higher prevalence of two parasites, N. robusta and T. trituba, than did Sebastes aleutianus, in the 2001 samples, indicating a possible difference in habitat and (or) resource use between the two lineages.

Genetic analysis of juvenile coho salmon (Oncorhynchus kisutch) off Oregon and Washington reveals few Columbia River wild fish

Little is known about the ocean distributions of wild juvenile coho salmon off the Oregon-Washington coast. In this study we report tag recoveries and genetic mixed-stock estimates of juvenile fish caught in coastal waters near the Columbia River plume. To support the genetic estimates, we report an allozyme-frequency baseline for 89 wild and hatchery-reared coho salmon spawning populations, extending from northern California to southern British Columbia. The products of 59 allozyme-encoding loci were examined with starch-gel electrophoresis. Of these, 56 loci were polymorphic, and 29 loci had P0.95 levels of polymorphism. Average heterozygosities within populations ranged from 0.021 to 0.046 and averaged 0.033. Multidimensional scaling of chord genetic distances between samples resolved nine regional groups that were sufficiently distinct for genetic mixed-stock analysis. About 2.9% of the total gene diversity was due to differences among populations within these regions, and 2.6% was due to differences among the nine regions. This allele-frequency data base was used to estimate the stock proportions of 730 juvenile coho salmon in offshore samples collected from central Oregon to northern Washington in June and September-October 1998−2000. Genetic mixed-stock analysis, together with recoveries of tagged or fin-clipped fish, indicates that about one half of the juveniles came from Columbia River hatcheries. Only 22% of the ocean-caught juveniles were wild fish, originating largely from coastal Oregon and Washington rivers (about 20%). Unlike previous studies of tagged juveniles, both tag recoveries and genetic estimates indicate the presence of fish from British Columbia and Puget Sound in southern waters. The most salient feature of genetic mixed stock estimates was the paucity of wild juveniles from natural populations in the Columbia River Basin. This result reflects the large decrease in the abundances of these populations in the last few decades.

Electrophoretic and immunotaxonomic studies of three species of marine gastropods from Portonovo coast with reference to population management

Electrophoretic and serological studies of foot muscle protein of three species of Cerithiacea (Telescopium telescopium, Cerithidea fluviatilis and C. obtusum) were carried out to understand their relationships. Living specimens were collected from mud flats and mangrove swamps off Portonovo. Polyacrylamide electrophoresis of proteins from foot muscle extract of T. telescopium, C. fluviatilis and C. obtusum showed that the former had a different densitometric profile as well as more number of protein bands; but the later two species showed a closer related pattern as well as lesser number of protein bands. At the same time these two species are distinguished from each other in their total number of bands and Rf values. Immunological studies using micro-Ouchterlony double diffusion tests which absorbed antiserum indicated that C. fluviatilis and C. obtusum were more closely related as revealed by an identity reactions than T. telesopium as shown by non-identity reactions. Results are discussed in relation to ecological and morphological adaptations

Suitability of sodium lactate as cryoprotectant and its effect on gel forming ability and protein denaturation of croaker fish surimi during frozen storage

The effect of sodium lactate is compared with sucrose + sorbitol + sodium tri-poly phosphate as cryoprotectant on gel forming ability & protein denaturation of croaker surimi during frozen storage at -20±2°C for 90 days was evaluated. The quality of Croaker surimi with 6% (w/v) sodium lactate was examined in terms of biochemical parameters of muscle protein, thaw drip, gel strength and calcium ATPase activity :.omparing with those of surimi added with sucrose/sorbitol & without additive as control. Both the cryoprotectants minimized the negative effects of frozen storage on physico-chemical traits of myofibrillar proteins which was evident from the biochemical and sensory parameters. The residual Ca2+ ATPase activity and gel strength of surimi with sodium lactate were higher than those of control throughout 90 days of storage. Ca2+ A TPase activity and gel strength found a high positive correlation. From the results, it was found that sodium lactate was equally effective in preservation of croaker muscle protein native structure during frozen storage as the sucrose/ sorbitol and also less sweet without any risk of maillard browning.