Genetic characterization of Vimba vimba persa (Pallas, 1814) in southern parts of the Caspian Sea using microsatellite markers

Population genetic structure of Vimba vimba persa was investigated using microsatellite markers from 40 regions along the Iranian coastline of the Southern Caspian Sea (Anzali lagoon and Havigh River in Gilan province, BabolRoud River in Mazandaran province and GorganRoud River in Golestan province). Genomic DNA from 121 specimens was extracted from fin tissue by the Phenol-Chlorophorm method and PCR reaction was accomplished with 17 microsatellite primers, out of 17 microsatellite primers 13 loci were amplified, in which 10 of them were amplified with reasonable polymorphism and 3 were monomorphism. A total of 302 alleles were identified on average 7.55. Observed and expected heterozygosity averages were 0.80 and 0.77 respectively. Most cases significantly deviated from Hardy-Weinberg equilibrium (p≤0.01). The estimation of Fst (p≤0.01) revealed significant population structuring and an estimation of the four population of Vimba vimba persa was identified in the Caspian Sea in which restocking of these species should be considered

Investigation of genetic marker (Luciferase gene) production for detection and stigmatize Cyprinus carpio or Rutilus frisii kutum

In this study lusiferse gene extracted from Vibrio fischeri. V. fischeri strain was kindly provided by Iranian Research Organization for Science and Technology (IROST). Genomic DNA extraction was carried out by Phenol-chloroform. A DNA fragment encoding the luxA and LuxB was amplified by PCR using sense and antisense primers, specific restriction sites for BamH1 and Kpn1 were introduced into 5´ end of forward and reverse primer, respectively. The PCR product was purified from agarose gel and ligated into cleaved PT257R cloning vector. Following the confirmation of the cloned Luciferase transformed into E. coli. Recombinant clones were confirmed by specific PCR and restriction enzyme digestion analysis. The luxA and LuxB fragment was released subcloned in to the PcDNA3.1\hug and PcDNA3.1\neo expression vectors, respectively. recombinant plasmid was confirmed through restriction digestion using BamH1 and Kpn1 enzymes and subsequently, transformation procedure continued into NIH3T3 eukaryote cells by specific kit. Luminescence ability of recombinant clones was tested by NIH3T3 cells and dechanal (substrate) and Neomysin and hygromysin. The results showed that luminescence start after 2 hours and then increase after 6 hours. Inaddition, the protein identity was verified by western blot analysis, the protein bands 76 kD were detected which indicates protein expression of luxB, luxA

Genetic characterization of Vimba vimba persa in southern part of the Caspian Sea using microsatellite marker

Population genetic structure of Vimba vimba persa was investigated using microsatellite markers from 4 regions along the Iranian coastline of Southern the Caspian Sea (Anzali lagoon & havigh River in Gilan province, BabolRoud River in Maz&eran province & GorganRoud River in Golestan province). Genomic DNA from 121 specimens was extracted from fin tissue by phenol-Chlorophorm method & PCR reaction was accomplished with 17 microsatellite primers, out of 17 microsatellite primers 14 loci were amplified, in which 10 of them were amplified with reasonable polymorphism & 4 were monomorphism. Totally 302 alleles were identified on average 7.5 Observed & expected heterozygosity averages were 0.80 & 0.77 respectively. Most cases significantly deviated from Hardy-Weinberg equilibrium (p 0.01). The estimation of Fst (p 0.01) revealed significant population structuring & estimate four population of Vimba vimba persa is identified in the Caspian Sea. These studies were to apply & develop population genetic approaches to assist conservation, sustainable harvest & restocking of these populations

Global population structure of the great sturgeon (Huso huso) in the southern part of the Caspian Sea with emphasis on the Golestan Province

Report of the project entitled "determination of the global population structure of the great sturgeon (Huso huso) with emphasis on the Golestan Province" is in front of your attention. Outcome is the results of study on the samples collected from the total catch in the landing sites namely Bandar-e-Anzali, Babolsar, and Ashooradeh, in the three Northern provinces. 224 specimens were collected according to the sampling manual during the fishing seasons in 2005 and 2006. Three methods were applied in this study to find out the population structure of the great sturgeon in the southern Caspian Sea. These were as follows: Morpho-metric meristic Population dynamics Population genetics (microsatellite loci) Results indicate that the range of different age groups of the great sturgeon in the Golestan Province is larger than the other two provinces. Results show that the older age groups are found in the samples from the Golestan Province rather than the other two provinces. Results demonstrate that the sample size for the two methods (morpho-metric meristic and population dynamics) is not enough. In order to obtain precise results with less uncertainty one needs to collect more specimens. Therefore, according to the few number of the great sturgeon in the total sturgeon landing, it is recommended that researchers apply methods other than the two methods which are mentioned above in order to make sure that the results are most reliable. Population genetics methods including microsatellite loci are among the methods which could produce good results with minimum bias in this regard. Results indicate that there are two distinguishable populations of the great sturgeon (Huso huso) in the southern part of the Caspian Sea off the shores of the three Northern provinces. Results also show that these two populations are scattered in the two east end (Golestan Province) and west end (Guilan Province) of the southern Caspian Sea. The two populations have overlapped each other in the Mazandaran Province

Characterization and isolation of microsatellite in Persian sturgeon (Acipenser persicus, Borodine, 1897)

In order to have a sustainable management on Persian sturgeon (Acipenser persicus) as a highly commercial species in the South Caspian Sea, we need to identify its population structure and the level as well as its conservation status in their natural habitat. To develop a conservation program for this all Caspian Sea' sturgeon species it requires knowledge of its genetic diversity using reliable molecular marker to study population genetic structure. For these purposes, an enriched library was prepared based on a modified biotin-capture method. Approximately 1800 positive clones were screened for microsatellites in an Acipenser persicus genomic library. Of these 350 positively hybridizing clones were sequenced, and 81 clones were identified as having microsatellites with adequate flanking regions. We developed and tested 68 microsatellite primer pairs for Persian sturgeon. Out of 68 primer pairs developed, 11 pairs resulted in poor or no amplification, 13 were ambiguous, 6 were monomorphic, 20 were tetrasomic and 18 were octosomic in Persian sturgeon. While none of the markers showed disomic inheritance in Persian sturgeon and Russian sturgeon (A. gueldenstaedtii). Several of the markers appeared useful for studies stellate sturgeon (A. stellatus), ship sturgeon (A.nudiventris) and beluga (Huso huso). Nearly all the polymorphic pattern for ship, stellate and beluga displayed the simple banding patterns characteristic of disomic loci, while those for Russian sturgeon displayed banding patterns characteristic of tetraploid or higher polyploid levels. These markers may prove useful in a variety of future sturgeon population genetic studies in the Caspian Sea

An ecological investigating on controlling Mnemiopsis leidyi population activity 6: molecular test in identification of Mnemiopsis leidyi

In this study population genetic structure of Mnemiopsis leidyi were examined by 200 samples from the Caspian Sea. Samples were collected from Guilan province (Astara region), Mazandaran province (Amir Abad region), Golestan province (Gomishan region) and north part of the Caspian Sea. 50 samples were collected from each region. DNA was extracted by phenol - chlorophorm method and its concentration was 50 to 100 nanogram. PCR was performed using 18s rRNA gene. The PCR products of samples were digested by 12 restriction endonuclease enzymes. The digested products accompanied with standard marker (50 bp lader ). In order to measure fragment size samples were run on a 6% vertical polyacrylamide gel. The fragments were visualized by silver staining of the polyacrylamide gel. In this study, Dra I and Alu I enzymes showed different digestion pattern, as each of these enzymes had two genotypes. Other enzymes had similar digestion pattern. RAPD method used by 19 random primers. Ten of nineteen primers showed polymorphism patterns. Statistical analysis of data was performed by Popgene software. In this study, the maximum of genetic variation was in north of the Caspian Sea. Also, the maximum of genetic distance was between north area and Golestan coasts (0.089) and the minimum of genetic distance was between Mazandaran and Guilan coasts (0.001). The UOGMA dendogram showed two clusters. The samples of Mazandaran , Guilan and Golestan coasts placed in one cluster and samples of north area in other cluster The genetic diversity was significantly different between samples of north area and Golestan coasts (p≤0.05). As result, there is a significant genetic divergence between some of samples. Therefore, two genetic group of Mnemiopsis leidyi were identified in the Caspian Sea

Designing and establishment of ISO/IEC 17025 in 3aboratories of Caspian Sea ecological research center

The project was carried out between March of 2007 and November of 2009.Five laboratories of research center (Clinical pathology, Plankton, Molecular genetics, Pollutants, Analysis Instrument were selected for accreditation. The main stages for establishment of the system consisted of: 1-Conducting a gap analysis to compare the present state of the laboratories with ISO/IEC 17025 Training 2 General requirements for the competence of testing and calibration laboratories Validation of methods Estimation of uncertainty Internal audits 3- Performing of technical and management requirements 4-Submit of quality manual to Iran Accreditation System (IAS) in order to accredit In January of 2010 the laboratories were accredited by Iran Accreditation System (IAS). The main results were including: 1-Increase the accuracy of measurement 2-Improvement of the Repeatability and Reproducibility of the test methods 3-Traceability and standardization of test methods 4- Calibration of measurement instruments 6- Updating of test methods 7-Standardization of physical condition of the laboratories 8- Getting the certification from Iran Accreditation System (IAS)

Study on viral nervouse necrosis (isolation, characterisation and pathogenesis) in golden grey mullet in the Caspian Sea and study of pathogenecity and possibility of transmission to the other fish species (Sturgeon fishes, Rutilus frisii kutum and reared Rainbow trout and Carp)

Study on Viral Nervouse necrosis (isolation, characterisation and pathogenesis) in Golden grey mullet in the Caspian Sea and study of pathogenecity and possibility of transmission to the other fish species (Sturgeon fishes, Rutilus frisii kutum and reared Rainbow trout and Carp) Viral Nervous Necrosis (VNN) is a worldwide disease affecting several species of cultured marine fish. For the past two decades, betanodavirus infections that cause Viral Nervous Necrosis (VNN) have emerged as major constraints on the culture and sea ranching of marine fish in almost all parts of the world. More than forty species mainly of marine origin have been so far affected and this number is likely to rise in future following the introduction of new species and the increase of aquaculture trade. Unknown acute mortality occurred in wild golden grey mullet Lisa auratus and Liza saliens in Iranian waters of Caspian Sea in recent years. In order to isolation and confirmation of causative agents of golden grey mullet mortality in the Caspian Sea, a complementary research investigation project was designed in 2005 and approved immediately in Iranian Fisheries Research Organization (IFRO). Many diagnostic aspects such as Virology (Cell culture and Elctereone Microscopy), Hemathology, Bacteriology, Histopathology, Molecular biology (NestedRT-PCR), Heavy metals measuerment and Serology (IFAT and IHC) were employed in mentioned multidiciplinary project. About 322 moribund fish samples which revealed skin darkening, erratic swimming behavior such as spiral and belly-up at rest and high distention of swimming bladder. Suspected samples were collected from coastal capture sites in iranian north proviences in 2006 till 2009. Targets tissue such as brain and eye were removed in strile condition and then kept in -80oC frezzer for cell culture and Nested-RT-PCR. Other tissue samples from liver, kidney, intestine, stomach, gill, skin and muscle, gall bladder and gonads were taken and fixed in 10% buffer formalin and same parts fixed in glutaraldehyde 3% for histopathology, IHC and EM respectively. Cytopathic effect (CPE) was observed in those cell cultures just six days after inoculation with the dilutions of the tested 312 homogenate supernatants. CPE in monolayers of cells cultured (SSN-1 cell line) was characterised by thin or rounded, refractile, granular cells with vacuoles. Nine samples were positive in virology assay. Nested- RT-PCR was done on suspected tissue samples and supernatant of CPE positive samples and 21 tissue samples and all CPE positive samples were positive. IFAT was selected as a confirmatory method for identifying viral strains replicating on cell cultures and carried out with rabbit anti-betanodavirus serum on suspected tissue samples and some smears of CPE positive samples. Some bright points approved betanodavirus antigen and confirmed cell culture and Nested-RT-PCR findings. In fixed tissue samples widespread and massive vacuolation were observed in brain, spinal cord, retina and optical nerve. In order to confirmation of diagnostic findings , IHC was done with monoclonal antibody antibetanodavirus and some red-brown points were observed. Theses findings revealed expected viral antigens and confirmed previous results. Moreover, virus particles with 25-30 nm in diameter were visualized in infected brain and retina using positive staining in TEM. Also pathogenicity test was employed to confirm the obtained results. So Guppy fish Poecilia reticulata and sturgeon fry were used instead of the experimental host due to ease of handling and susceptibility. After 15 days post infection, guppy bathed in VNN-infected tissue culture with 104 TCID50 showed clinical signs similar to naturally infected Golden grey mullet, and the mortality rate reached up to 100% in 75 dpi. When target organs were examined by cell culture isolation, serology, and histopathology, all revealed the presence of virus in the Guppy. Suspected supernatant injected to sturgeon fry through intravitreous injection and widespread vacuolation were observed in brain and spinal cord buy IHC and Real time PCR were negative. In conclusion, with attntion to obtained results in this investigation such as ecological factors, clinical signs, histopathological, virological and bacteriological results, molecular analysis, (IHC, IFAT, PCR), TEM demonstration, serological and hematological findings, it could be confirmed that VNNV was the main causative agent for disease outbreak in Golden grey mullet in Southern coastline of Caspian Sea