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The present study describes morphological development of the skeleton in the Persian sturgeon Acipenser persicus and discusses the hypothesis that expression of genes encoding vitamin K‐dependent proteins (VKDP) might be correlated with the mineralization of skeletal tissues during early development in sturgeons. Results showed that development of cartilage started just after hatching (mean ± S.D., 10·9 ± 0·7 mm in total length, LT) in the head and notochord, whereas the first signs of mineralization occurred in the dentary and in the dermopalatine and palatopterygoid elements of the upper jaw, coinciding with the onset of exogenous feeding (20·1 ± 1·5 mm LT). All branchial arch elements developed between 19·3 and 22·3 mm LT, whereas mineralization was only observed in tooth plates associated with the hypobranchial 1 and gill rakers at 20·8 ± 1·5 mm LT and 48·4 ± 6·4 mm LT, respectively. Quantitative real‐time PCR showed that transcripts of VKDP genes including bone Gla protein (bgp), matrix Gla protein (mgp) and Gla rich protein (grp) genes were significantly up‐regulated during the transition to exogenous feeding, supporting hypotheses about relevance of the above‐mentioned genes in chondrogenesis at early developmental stages. The strong mineralization of skeletal elements from 21·5 to 27·3 mm LT (20 days post hatch) was in accordance with the maximal levels of bgp, mgp and grp expression indicating a correlation between development of the skeleton and the expression of VKDP genes. These data are important for evaluating A. persicus larval quality, understanding the influence of rearing biotic and abiotic factors on skeletogenesis and recognizing the occurrence of skeletal deformities in this species. info:eu-repo/semantics/acceptedVersion

In order to introduce a new sturgeon hybrid to aquaculture industry, the possibility of hybrid production by crossing between Siberian and Persian sturgeons and its comparison with its pure parents using morphometric, molecular, cytogenetic and growth performance were conducted. For this purposes eggs and sperms of two pairs of Persian and Siberian sturgeon were collected and reciprocal crosses with 4 treatments were conducted. In treatment 1, Persian sturgeon as control (male and female of Persian sturgeon), treatment 2 (as type I hybrid) from crosses of male Persian sturgeon with eggs of female Siberian sturgeon in treatment 3 (type II hybrid) using crosses between male Siberian sturgeon and female Persian sturgeon and finally treatment 4 by crossing male and female of Siberian sturgeon were used. For each treatment 200 gr. of eggs were fertilized with one ml. of sperm and morphological, meristic parameters of parents and offspring were recorded. For growth comparison of fingerlings of control and hybrids in 3 different phases were measured. In phase (I), larvae rearing from 46 mg. up to 6600 mg. were conducted for a period of 10 weeks. Samples were collected once every two weeks and biological parameters were recorded. In rearing phase II for a period of 330 days, growth comparison were conducted for fish from 6.5 gr. to 750gr. and biological parameter were recorded once every month, where daily growth rate (gr./day), FCR, Specific growth coefficient (percentage per day), increase of body weight, food efficiency and protein efficiency ration were analyzed. In rearing phase (III), from 600 gr. to 2000 gr. for a period of 43 weeks, similar to phase (II) all biological parameters were analyzed, where 26 morphometric and meristic parameters of pure and hybrid parents and offspring were compared both for individual and ratio status of parameters. For genetic analysis and variation between control and hybrid specimens the microsatellite analysis were conducted. For this purposes, genomic DNA were extracted from fin tissues of brood stocks and fingerlings and 10 pairs of specific primers (Afug 56, Afug 63, Afug 241, Afug 229, OX 27, Afug 686, Afug 195, Afug 12, IS 68 and Afug 160) were used. Then PCR Products were electrophoresed on 6% polyacrylamide gel and then were stained with Nitrate silver and alleles were evaluated based on its length (base pair). Cytogenetic analysis and number of chromosome of control fish and hybrid were compared using white blood cell culture method and chromosome spread of parents as well as offspring were prepared by Gimsa staining and were visualized under light microscope. For sexual gonad development, laparoscopic method with light camera (Model M-CAM 1700) were used to differentiate 34 pieces of control and hybrid fish and gonad status of ovary and testis were analyzed. In present investigation the results indicate that, in phase (I) of larvae culture, a significant differences were observed after 10 weeks of feeding (p≤ 0.05) in hybrid and control groups. Maximum growth rate were observed in Siberian sturgeon larvae, followed by type I hybrid, type II hybrid and Persian sturgeon larvae. In phase (II) , growth rate of Persian sturgeon were lower than Siberian sturgeon. For period of 330 days of rearing periods, the control Siberian sturgeon had 1.4gr. weight increment per day, while Persian sturgeon had only1. gr./day. However the hybrid type (I) had an average daily growth of 1.5gr/day and lower growth performance were observed in type II hybrid with daily growth of 1.5 gr./day. In phase (III), growth rates were varied at different age group. The daily growth rate of Siberian sturgeon was 4.5gr/day, while is control Persian sturgeon was 2.6 gr/day. The Siberian sturgeon within 47 weeks of rearing grown from 655 gr. to 2016 gr., however Persian sturgeon for the same period reached from Siberian sturgeon with male’s Persian sturgeon had higher type II hybrid showed opposite results for the same growing period and reached from 640 gr. to 1680 gr. In conclusion of growth performance can be stated that the hybrid from crosses between female Siberian sturgeon with male Persian sturgeon presented highest growth rate in comparison to control hybrid. This excellent achievement can be considered as a big step toward sturgeon aquaculture for meat and Caviar production. In analysis of morphometric characters 26 parameters were investigated which in 18 parameters showed significant differences (p≤ 0.05) which indicate clear distinguish able of hybrid performance in compare with parents where Siberian sturgeon has longer snout compare to Persian sturgeon and its size are intermediate in hybrids. In relative ratio of characters, out of 9 characters totally 7 characters showed significant differences (p≤ 0.05). In molecular analysis using microsatellite primers totally 400 DNA samples of hybrid as well as the control fish were investigated. Totally 784 allele were detected in which 363 alleles were belong to control fish and 421 alleles in hybrid. In this study, 19 alleles with different sizes were observed in hybrids rang from (120bp) in Aox27 primers (400bp) in AFUG229 primers. In comparing of all primers, the primer AFUG160 and AFUG195 with 2 alleles and primer AFUG686 with 7 alleles showed the minimum and maximum allele numbers.The banding patterns were monomorphic in 66 loci of control fish and 65 loci of hybrids, and in 77 loci of control fish and 73 loci of hybrids were diploid in 9 loci of control fish and 12 loci of hybrid fish were tetraploid, in one locus of control samples and 3 hybrid samples were pentaploid and finally within 10 pairs of primers 7 sets were disomic, 3 primers (Afug241, Afug686, Aox27) were tetraploid and 6 primers of (Afug63, Aox27, Afug688, Afug12, Is68, Afug160) showed excellent allele heritability which indicate the high efficiency of microsatellite techniques. In evaluation of the status of gonad development as well as determination of sexual stages laprascopic method indicated that out of 9 samples of Persian sturgeon (4 females and 5 male) were in stage of 2 & 3 gonad development. Out of 8 Siberian sturgeon (4 males+4 females) stage 2, 3, 4 were detected. In 7 gonad samples of hybrid (Female Persian and Male Siberian) totally 5 male and 2 female were observed at stage 3 and 4 gonad development. In analysis of 10 samples of type II hybrid (female Siberian × male Persian) 3 female, 6 males and 1 deformetic were observed. Cytological studies on 30 metaphase palate from 6 fish samples showed that the number of chromosome in hybrid are as same as parents 2n=250±10 where all control and hybrids similar to its parents showed 240 chromosome. In conclusion, It can stated that, this study successfully introduced a new sturgeon hybrid for sturgeon farming both for meat and caviar production. Considering the common name of sturgeon parents used for production this hybrid, we call this hybrid as (Siper) where Si instant for Siberian sturgeon and Per comes from Persian sturgeon. Iranian Fisheries Science Research Institute Published

Aquatic organisms present, not only simple sources of accumulated metal, but can interact with metals, altering their toxicity. Due to exposition of biosphere with metals, organisms have developed various defense mechanisms to protect themselves against adverse effects of these ions and their compounds. Metallothionein (MT) is one of that which represents a critical mechanism for detoxification of metals. The sterlet (Acipenser ruthenus) is a bottom feeding sturgeon species and because the fish are dependent on invertebrate species for food throughout their life cycle, the sterlet could be a good indicator of the quality of the state of water ecosystem. Addition of copper to water leads to the induction of MT. The present study analyzed MTgene primary structure that was excreted from the liver of sterlet exposed to sub-lethal copper concentrations (0.075 mgL^-1 ). At the beginning to identify the molecular structure of Metallothionein of sterlet, a cDNA encoding MT was purified from livers of sterlet, and the MTgene was amplified. The primary structure of sterlet metallothionein (S-MT) contained 20 cysteine residues, which is the same as MTs of teleost fishes. Although, the primary structure of S-MT contained 63 amino acids, which is longer than any MT identified in teleost fishes but similar to the structure of MT gene in lake sturgeon and white sturgeon. The complete nucleotide sequence of the S-MTgene has been detected. We have determined the structure of the fish copper-binding protein by DNA sequence analysis of the gene.

The Persian sturgeon (Acipenser persicus) is more abundant sturgeon species in the South Caspian Sea and consist the highest proportion of Iranian Caviar, meat as well as bringing maximum foreign currency income, however from systematic point of view and differentiation of this species from Russian sturgeon (Acipenser gueldenstadttii) a serious challenging issues remain, where some Russian scientist are believe that the Persian sturgeon is not as an valid species and consider it as a subspecies of Russian sturgeon. This research conducted with the objective of identification and introducing a molecular marker based on specific DNA for differentiation of two species of Persian sturgeon and Russian sturgeon via a proved molecular marker method. For this purposes 8 different molecular approaches such: Microsatellite, AFLP, RAPD, sequencing of Cytb, 16sDNA, ND5, Growth Hormone gene and finally Single Nucleotide Polymorphism (SNP) were investigated. Based on applied methodology, between 5 to 16 caudal fin tissues were sampled for each species from different region of the Caspian Sea, Sefiedrud River, Ural and Volga rivers. Following DNA extraction, its quality and quantity were determined and the PCR experiment has been conducted using 5-110 primers according to various methods and type of gene. The PCR products were electrophoresed on Polyacrilamid or agarose gels and followed by silver and Ethidium Bromide staining. In RAPD method, polymorphic DNA band was cut on the gel followed by purification and then the segments were cloned in vector in Top10 strain of E.coli, and then sequenced. Meanwhile for Growth Hormone gene in Persian and Russian sturgeon the MEGA 4, Gene runner software were used to design the appropriate primers for PCR amplification. The PCR products were cloned in PTZ57R/T vector and transformed in Top10 E.coli strain and sequenced finally. For all other genes, similar methods were applied for PCR amplification and its products were sequenced and statistical analysis as well as phylogenetical tree was performed. In Single Nucleotide Polymorphism (SNP) method, after genomic library construction, in total 14.4 billion nucleotides were sequenced and similarity/ differentiation analysis of two species were investigated using specific bioinformatic software. Results indicated that Microsatellite and AFLP methods showed high level of genetic variation both within and between species. The Cytb gene, when 4 sample sequences from each species were compared two species were differentiated, however when analysis repeated over 15 samples, the sequence comparison couldn't differentiate two above mentioned species. Full sequence comparison of 16sDNA and mtDNA-ND5 gene showed variation in some nucleotide in both species of Persian and Russian sturgeon but no significant. Results of sequences obtained from cloned segment with RAPD method and also specific primer design based on produced sequences could succeed to discover a variable DNA band that able to differentiate two species from each other. Results of the present study also showed that the growth hormone gene (GH) of Persian and Russian sturgeon consists of 645 nucleotide that translate to 214 Amino Acids. The sequence comparison indicated that the gene coding growth hormone in Persian and Russian sturgeon had the highest similarity with GH of Mammals (71%), Anguilaformes (63%) and less similarity with bony fish (37%). Phylogenetic analysis indicates that Persian and Russian sturgeon in compare to other organism are ancient species and this gene is originated from a common ancestor. At present study the most appropriate results obtained from Single Nucleotide Polymorphism (SNP) method by sequencing 14.4 billion nucleotide from genome of two species of Persian and Russian sturgeon from North and the South Caspian Sea could prove that the Persian sturgeon is a valid and independent specie. This excellent results is the biggest scientific achievement for differentiation of two highly commercial important sturgeon species in the Caspian Sea in last two decades.

Analytical Division, Faculty of Chemistry, Mazandaran University, Babolsar, Iran E-mail : samadi@umz.ac.ir Manuscript received online 14 April 2014, revised 02 May, accepted 10 May 2014 A few sample of zeolites were synthesized using two different techniques, first by conventional heating method (hydrothermal) and another by exposing the hydrogel to microwave radiation assisted by hydrothermal heating (Microwave treatment). Microwave heating decreased the nucleation and crystallization time. These zeolites powder were prepared at temperature of 450 K and at presence of organic template and without it. Templates were organic species added to synthesis media to aid/guide in the polymerization of the anionic building blocks that form the framework. The crystalline zeolites samples obtained were characterized by X-ray diffraction (XRD), FT-IR spectroscopy and scanning electron microscopy (SEM) techniques. It was established that phase purity, morphology, and the size of crystals of crystalline products were affected by molar ratios of the substrate, such as (H2O/Na2O) and present of organic template. Amorphous silica powder (Zeosil) was the preferred as silica source. The size of crystals in the microwave treatment samples are 5–10 µm without template and 10–15 µm with template and for the conventional heating heated samples are 20–25 µm without template and 50–60 m with template.

The population genetic structure of the Persian sturgeon (Acipenser persicus) in the 2, 3, 4 fisheries regions and Sefidrud River was investigated based on the DNA sequencing method during 2010 – 2013 sturgeon stock assessment in the south Caspian Sea . DNA samples were extracted using ammonium acetate, the quantity of DNA was measured at 260 and 280 nm using spectrophotometry by Nanodrop (ND 1000 model), and the quality was checked by 1% agarose gel electrophoresis. Two sets of mitochondrial gene (D-loop and cytochrom b) after synthesis were used for polymerase chain reaction (PCR). A Neighbor-Joining (NJ) tree was constructed for all haplotypes according to Kimura 2-parameter model using Mega Version 4.0.1, number of haplotypes, haplotype diversity (Hd) and nucleotide diversity and their corresponding variances, genetic divergence overall and between paired populations (Fst) by 10,000 permutations and exact test, the gamma distribution shape parameter for the rate heterogeneity among sites and nucleotide sequence, the historical demographic pattern of A.persicus using neutrality tests and mismatch distribution analysis (D test of Tajima and Fs test of Fu), also the concordance of the observed with the expected distribution under the sudden population model using the Harpending, s raggedness index (Hri) were analysed. All calculations were conducted using ARLEQUIN version 3.11 and DnaSP 4.0. The aligned mtDNA sequences of D-loop and cytochrom b genes were consisted of 500 and 700 base pairs (bp) respectively. 13 and 4 haplotypes were defined, the average haplotype diversity were 0.961 and 0.419, average nucleotide diversity were 0.038 and 0.002, The gamma distribution shape parameter were 0.19 and 0.20 indicating moderate mutation rate heterogeneity among sites in A.persicus. The lowest value of Fst for D-loop gene was calculated between Sefidrud and four fisheries region (-0.002) and the Fst values observed for cytochrom b gene was 0.04 with Nm=5.37 and not statistically significant. The exact test of population differentiation (non-differentiation exact P values) showed significant differences between Sefidrud and other areas (P ≤0.05) for D-loop gene and for cytochrom b gene was nonsignificant (P ≥0.05). The mismatch analysis produced a unimodal distribution of pairwise differences for both genes which was consistent with the sudden population expansion model. Tajima’s D and Fu’s Fs statistics were significantly negative (D= -0.84 and - 0.99, P>0.01; Fs= -0.220 and -0.079, >0.01). ARLEQUIN calculated the value of t as 13.65 and the time since population expansion was estimated to be approximately 1501 years before present based on the mutation rates for the control region and this value for cytochrom b gene t= 0.98 which population expansion time was 7.84 years before present. The results of this study based on D-loop gene showed that population of A.persicus in the Sefidrud River is differ from other studied areas. Therefore fisheries managements of this unique and valuable stock for restocking and conservation of gene pools is strongly recommended. Iranian Fisheries Science Research Institute Published