Comprehensive study on assessment of sturgeons population genetic structure in the Caspian Sea

The population genetic structure of five Caspian Sea sturgeon species was investigated. Totally 1121 samples of caudal and unault's fin tissue of the sturgeons (Acipenser persicus, A. gueldenstaedtii, A. stellatus, A. nudiventris and Huso huso) were collected from the Volga River (Russia), Ural River (Kazakhstan), Kura River (Azerbaijan), Sepidrud River and the coastline of the south Caspian in the Iranian waters as well as from the sampling stations selected for the marine survey for sturgeon stock assessment in the Caspian Sea. All samples were 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 by Agarose gel (1%) electrophoresis and spectrophotometry. The population genetic structure of Ship and Persian sturgeon was studied using both PCR-RFLP (D-loop and ND5/6 gene) and microsatellite technique and that of H. huso, A. stellatus and A. persicus were studied using microsatellite technique. After amplification of genes using PCR, the RFLP technique was used to digest mtDNA using restriction enzyme. The PCR products were electrophoresed on 6% sequencing polyacrylamide gels followed by silver nitrate staining. Data for PCR-RFLP were analyzed using REAP program and those from microsatellite technique were analyzed using Gene Alex. Population genetic parameters including allele frequency, expected and observed heterozygosity, effective allele, Shannon's index were determined. Genetic identity and distance were calculated following Nei criteria and Hardy Weinberg equilibrium was tested based on X2 and analysis of molecular variance (AMOVA) using Reap and Gen Alex at 99% confidence limit. Phylogenetic relationship was determined and drawn using TFPGA program. The population genetic structure and genetic diversity of the 1121 sturgeon specimens were determined. Three independent populations were identified for Acipenser persicus (two populations in the south Caspian in the Iranian waters and one in the north Caspian). Three independent populations were identified for A. gueldenstaedtii (Volga, Ural and South Caspian populations) using the microsatellite technique. Population genetic structure using PCR-RFLP revealed no genetic differentiation among the A. gueldenstaedtii specimens studied from the different regions using ND5/6 gene, while two populations (Ural and south Caspian populations) were detected for this species with the same technique using D-loop genes. Four independent populations (Volga, Ural, Kura and Sepidrud populations) were reported for A. stellatus using the microsatellite technique and four more populations which most probably belong to the autumn and spring races of the above mentioned independent populations were identified for this species. The present study also identified two populations for H. huso; The North Caspian population (in Volga and Ural Rivers) and The South Caspian population (in Golestan and Guilan regions) which were significantly different from each other (P<0.01). The genetic population structure of A. nudiventris was studied using the microsatellite and PCR-RFLP techniques which revealed two populations for this species one in the Ural River and the other in the Sepidrud River (South Caspian). Comparison of the ND5/6 and D-loop genes studies in Russian sturgeon revealed that the D-loop gene is better than the ND5/6 genes in population's differentiation and is therefore strongly recommended for population genetic studies on sturgeons in the Caspian Sea. Genetic diversity studied using microsatellite technique was higher and more accurate as compared to that using RFLP. Nevertheless the RFLP technique was able to introduce molecular markers for the population’s species pacific identification. On developing suitable primers these studies can be speeded up and the cost of such studies can be cut down. However the drawback in using microsatellite technique for population genetic studies is that it cannot introduce a molecular marker for the identification of populations. The present study was able to introduce molecular markers to differentiate the ship sturgeon population in the south Caspian from that in the Ural River using the PCR-RFLP technique Based on the results obtained it is strongly recommended that all activities related to restocking and rehabilitation of sturgeon stocks in Iran be conducted on the basis of genetic principles. Also serious and immediate measures should be taken for the restoration and conservation of rare population of native species of Iran particularly in the Sepidrud region using genetic markers before they are become extinct

Creating a sperm bank from sturgeon brood stocks in the south Caspian Sea

Sturgeon stocks are drastically decreasing in Caspian Sea during recent decades. This project has been proposed to collect the excess sperm of Caspian Sea Southern part sturgeon breeders, to cryopreserve and hold them for further use. During two spawning seasons (2006-2007), sperm quality and quantity analysis were conducted on 34 different male sturgeons of which 12 Persian sturgeon, 4 stellate sturgeon, 1 beluga and 1 ship sturgeon with better quality were chosen to cryopreserve. After dilution of fresh sperm with sturgeon sperm diluent (115 mM Tris, 23.4 mM sucrose and 15% DMSO) at the ratio of 1:1, the 0.5 ml straws were filled using special filling and sealing apparatus and frozen with a multi step freezing rate. To access the further quality and fertilization test, samples were thawed at 40 °C for 20 seconds. During the project performance, 1010 ml of Persian sturgeon sperm , 110 ml of ship sturgeon sperm , 90 ml of stellate and 80 ml of beluga sperm were cryopreserved. The reduction of frozen-thawed sperm motility in comparison with fresh samples showed 10-52.3 % decrease, but there was not any significant differences between samples after 7 months preservation in liquid Nitrogen (P < 0.005). River caught Persian sturgeon samples showed higher sperm density in comparison with nonriverine breeders (P < 0.05). Data showed that stellate sturgeon sperm showed better resistance to freezing condition in comparison with Persian sturgeon spermatozoa. Fertilization tests (4 hours after fertilization) showed 27.4% and 58.2% fertilization rate in frozen-thawed stellate and Persian sturgeon spermatozoa, respectively

The study of genetical differentiation of bisexual and parthenogenetic strains of Artemia urmiana by RAPD s method

The brine shrimp Artemia consists of a number of bisexual species and a large number of parthenogenetic forms, which collectively, inhabiting a wide range of hypersaline habitats. This research was carried out in order to study the genetic differentiation of Artemia urmiana and two parthenogenetic populations originated from surrounding lagoons of the Lake Urmia (Fesendoz, Saray village) and also to find the phylogenetic relationships of some Iranian Artemia (Qom, Arak, Golestan, and Shiraz) with a comparison to some foreign Artemia species (A. sinica, A. franciscana, A. parthenogenetica (Russia and Turkmenistan) using a molecular technique (PCRRAPD`s). Of a total number of 56 random primers (10 bp) survey, 29 and 26 oligonucleotides showed polymorphic patterns with highly reproducible bands in Artemia urmiana groups and Iranian Artemia groups.The highest percentage of polymorphic loci were in Artemia urmiana (P= 78.89 %) and in comparison with the other parthenogenetic forms, in parthenogenetic Artemia from Saray village (P= 59.8 %). The highest values of Shannon index were estimated in Artemia urmiana (I= 0.41), and in comparison with other parthenogenetic forms, in parthenogenetic Artemia from Saray village (I= 0.3). Analysis of molecular variance indicated that most variation were within populations (A. urmiana with parthenogenetic forms, FST= 0.35) and among populations (Iranian Artemia with foreign species, FST= 0.76). Genetic pattern was not in accordance to geographical distribution, which indicated the wide range of Artemia samples at different regions in Iran. UPGMA showed two distinct clusters in group I including A. urmiana with two parthenogenetic forms and four clusters in group II including Iranian Artemia with some foreign species. It is understood that two parthenogenetic populations (Fesendoz and Saray village) had close genetic relationship together with high value of heterozygosity in parthenogenetic Artemia from Saray village. Iranian Artemia (Qom, Arak, and Golestan) indicated close genetic relationships with A. parthenogenetica (Russia and Turkmenistan) with low values of heterozygosity. A sister group was generated including Shiraz and USA Artemia samples which attests a possible contamination of local water with A. franciscana. FST value revealed high genetic structuring at all studied groups. Besides, it is suggested that gene flow was not strong enough to prevent substantial genetic differentiation. Result could lead to a management programmes to use suitable Artemia strains with high genetic diversity, for culturing and establishment of gene bank of Artemia stocks in Lake Urmia

Determination of suitable size of Rutilus frisii kutum for releasing by evaluation of osmotic regulation ability

The study was done in Nutrition and Live Food Station was located in Bandar Anzali Ghaziyan. Juveniles weighted average 0.5, 1, 2.5, 5, 10, 15 and 20 g were randomly selected in three water conditions with a salinity of 11 ppt (Caspian sea water), water 7 ppt and freshwater (with three replicates per group) were included. At intervals of 0, 3, 6, 12, 24, 72, 168, 240 hours, blood samples were heparinized capillary tubes by caudal juveniles and ion concentrations Mg, Ca,Cl using the spectrophotometer and sodium and potassium ions with Flaym photometry (flame photometric), the osmotic pressure of blood plasma by osmometer and cortisol levels were measured by RIA method. To study the microstructure of gill and kidney tissue for each treatment , tissue samples by classical histological methods and stained with hematoxylin - eosin slides were prepared. The frequency and location of the enzyme Na^+, K^+ - ATPase and chloride cells with immunohistochemical localization was performed. Studies micrometric gill chloride cells and renal glomerular networks by software Image tool (version 2.0) was performed. Measurement of enzyme Na^+, K^+-ATPase, by Zaugg (1982) method was conducted. Data analyzed by one-way ANOVA (Oneway ANOVA) with Tukey's test was performed. Overall, the results of measuring ions and osmotic pressure on the tenth day of treatment, the osmotic potential juveniles 2.5, 5, 10, 20 gr in Caspian sea water and all groups except the 0.5 in water of 7 ppt confirmed. But in case of unfavorable conditions for the release in estuaries river and river, fish with weight 1 to 3 release directly to beach (where the salinity is 7 grams per liter) and fishes with weight from 10 to 20 gr to sea. Although suitable river conditions necessary condition for release of juveniles in riverine areas to adaptation juveniles occur gradually

Analyzing the effects of aquaculture and rebuild aquatics stocks on fisheries development in the Caspian Sea

Using SWOT analysis with traditional matrix and quantitative matrix methods, the strategies to benefit from strengths and opportunities to vanishing weaknesses and decrease of threats for strategic management of aquaculture in Iranian Northern provinces was surveyed. Utilizing researches held in fisheries research centers and existence of aquaculture experts in vicinity, activation of veterinary organization in production and post production, and entering new brood stocks with desirable growth are the main strategies for developing rainbow trout and Chinese carps culture in Iranian Northern provinces. In sturgeon fishes culture, independence of farms from fingerlings produced by governmental complex and diversification the cultured fishes using new species and hybrids can develop this part of aquaculture. Avoiding illegal fishing in sea and rivers, preserving the population of Caspian Sea fishes, entering new fish species which their resources are decreased extremely to breeding and fingerling production cycle, and using pike perch and bream while they migrate to Anzali Lagoon from Caspian Sea, instead of freshwater brood stocks will cause the output of sea enhancement and sea ranching activities to increase, and fisheries return coefficient of these fishes will rise. Using quantitative matrix and scoring method, it was defined that aquaculture in Iranian Northern provinces are extremely approved and acceptable and the score of strengths is more than weaknesses and opportunities more than threats. By using strategic management, these activities can be more developed

Investigation on probable viral agents associated with grass carp and silver carp mortality in Guilan province

Warm water aquaculture development along with import of eyed eggs, Broodstocks and ornamental fish also transferring of fingerlings and broodstocks from one province to another one has led to spreading of some unwanted diseases. According to the Gilan Fisheries Department report, mortality rate of silver carp(Hypophthalmichthys molitrix) in the summer of 2010 caused losing of silver carp production more than 1162 tons which was valued over than twenty-three milion rials that was increased to about 40 million rials in 1391. Also grass carp had a less mortality rate in some farms. Observed symptoms and severity of losses increased the probability of viral diseases such as spring viremia of carp, koi herpes virus and grass carp reovirus. In this research, totally 411 silver carp and grass carp with 5 to 2300 gram weight from 83 farms were sampled during 2011-2012. Clinical symptoms was different in various farms and subcutaneous hemorrhages on the flanks, red fin base or total fin and Red belly and operculum, mild exophthalmia, hemorrhaging in the abdominal cavity (yellow, transparent or red fluid) and petechial haemorrhages of skin and viscera and Ascites in abdominal cavity and some silver carps and grass carps were observed with palied liver and gill. In some cases swelling muscle was observed in Silver carp. Several fries with the severe emaciation were observed. Difference between clinical symptoms can indicate the presence of different causes of mortality in different farms. Various water colors from completely transparent to dark green, yellow or slightly brown observed in different pool, which shows the difference in overall culture management including water quality management. The minimum Water turbidity using secchi disk was 40 centimeters to one meter page in the fields. Source semi-deep wells or deep pools that were used as water source. Average water temperature, oxygen and pH in fileds 26/5-31 °C , 4/3-5/7 mg / L and 7/4-9/38. In order to virological surveys, sampling of internal organs, including kidneys, and spleen were done.and Homogeneous target tissues after spending 0/45 micron filter on EPC and BF2 cell line were inoculated to observe cell damage(CPE) in case of confirmation of antibody detection methods brilliant, RT-PCR and PCR done. In cell culture examination, a kind of cell damage (CPE-like changes) was observed after inoculating of 3 samples of Silver carp those were suspected to SVC, but in the second and third passages there were no sign of cell damage. It may be because of toxic effect of tissue filtrate on the cell monolayer. Concerning sample of Grass carp on two EPC and BF2 cells no evidence of cell damage was found. In indirect fluorescent antibody test, 72 hours after inoculation of tissues filtrates of of silver carp and grass carp samples on EPC, no positive reaction was observed. PCR and RT-PCR tests using specific primer pairs were done to test all of the silver carp and grass carp samples for SVC and KHV diseases Also for GCRV in grass carp samples. In addition, simultaneously commercial PCR kits applied for testing of SVC and KHV (IQ2000. Taiwan). The results of PCR and RT-PCR tests showed no evidence of robdovirus Carpio and grass carp reovirus RNA and no sign of koi herpesvirus DNA in tested samples. In conventional PCR and RT-PCR tests Using specific primer pairs none of positive bands related to SVCV(470 bp) and GCRV(292bp, 697bp and 320bp) were not confirmed in the tested samples. Also in electrophoresis of PCR products using IQ2000 kit revealed 471 bp and 640 bp negative bands in all samples and the positive bands were not observed. For confirmation of the results, three suspected samples were sent to Europe Union Reference Laboratory and none of mentioned diseases were confirmed. Simultaneously bacteriolocical examination applied for 26 using blood agar. In bacterial culture and related analyzes 6 isolates of Pseudomonas and 21isolates of Aeromonas hydrophila were detected from 26 farms while no virus was confirmed in the same samples. Based on the results of cell culture, PCR, RT-PCR and IFAT tests no one of SVCV, KHV and GCRV viruses were confirmed in tested samples. Therefore, the etiological reason of the mortalities in the tested farms was not viral diseases and should be prevent and control by biosecurity and health management in the farms

Survey on health status in aquaculture sturgeons centers (Mazandaran, Guilan And Golestan Provinces)

Study of survey health management and diseases in hatcheries and fish farms can help us to knowledge and application control methods such as: prevention, treatment and increase high levels of production in hatchery and farms, finally. This survey carried out from 2005 to 2008 for 4 years in sturgeon hatcheries and farms of Golestan province. Sturgeon fishes include Huso Huso, Ship sturgeon, Acipenser persicus collected and for virology, bacteriology, fungius and hematology examined. Also, physicochemical parameters measured and recorded in different stages of culture. Results of this study showed that all of samples in virology was negative and did not observe any doubetful causes. In bacteriology CFU was variation from 3/9 ×105 to 6/9×10. The most parasites that detected in this survey was Cocolanus espherolanus, Sceria binopsulus semiarmatus and Amphilina fuliacea that separates from Acipenser Percicus, especially. The results about hematology parameters some important hematological indices of ship sturgeon include: The total RBC for female and mail specimens measured as 5.3±1.5 ×10^5, 4.8±0.5×10^5 per mm^3 respectively. The amount of haematocrit and hemoglobin for female and mail determined: 34.3±2.8, 35±1.4 percent and 10.3±0.9, 8.9±0.8 gr/dl .The MCV: 216.3± 96.2, 736.5± 102.5, MCH: 720.2±309.5, 186±0.7 and MCHC: 30±0.8, 25.5±3.4 percent respectively.The total WBC were (female, male): 21320±1054, 20580±777 per mm^3 and neutrophil: 16.4±2.5, 17±1.4 percent and lymphocyte: 74.4±2.4, 73.5± 0.7 percent and eosinophil: 6±1.4, 6.4±0.5 percent, monocyte: 2.8±0.8, 3.5±0.7 percent. There was not any significant differences (p>0.05) between mentioned parameters in male and female (students t-test). Also evaluation of hematological parameters in bluga ( Huso huso) include: total RBC were (male , female) 5±0.3 ×105 , 4.9±0.6 ×105 per mm^3 ,respectively and hematocrit: 33.2±6.7 , 35.4±3.4 percent and hemoglobin: 11.2±1.5 , 12.2±1gr/dl and MCV: 669.9±172.2, 723.9±982.4 and MCH: 226.2±42.5, 249.5±35.4 and MCHC: 34.1±2.4, 34.6±3.6 percent respectively. The totals WBC were (male, female): 24800±707.1, 23042±1375.4 per mm^3 and neutrophil: 18.5±0.7, 21.4±1.1 percent and lymphocyte: 73.5±1.4, 68.4±1.1 percent and eosinophil: 5±2.8, 7±1.2 percent and monocyte: 3.5±3.5, 3.2±0.8 percent. According to statistically study the count of lymphocyte had significant difference between male and female fish and this count in male was higher than female. (p≥0.05)