1 1562-2916 Iranian Fisheries Research Organization 524 fish disease Genetic diversity of Mahisefid (Rutilus frisii kutum Kamensky 1901) in different rivers of the south Caspian Sea using PCR-RFLP Abdolhay H. A. Daud Siti Khalijah Rezvani Gilkolahi S. Pourkazemi M. Siraj Siti Shapor Javanmard A. 1 4 2012 11 2 235 251 10 06 2012   Mahisefid is the most popular fish in Iran with the highest economic value. The analysis of mitochondrial DNA has been extensively used as a marker for population genetic studies and is a powerful tool in studies of gene flow and evolutionary biology. Two hundred ninety four samples were collected from Sefid Rud River (100), Lamir River (98), Shir Rud River (48) and Tajan River (48) during spawning season. Out of 24 enzymes tested, four enzymes, namely TasI, HaeIII, HinfI and HincII were selected for this study. In the present study, the haplotype and nucleotide diversity of Mahisefid in four important rivers where fingerlings are produced, were carried out by using PCR-RFLP at mtDNA ND5/6 region. A total of 20 haplotypes were studied so that AAAA and BAAA haplotypes had the most frequency. The average haplotype frequency of AAAA and BAAA haplotypes were 29.93% and 27.55%, respectively. The maximum nucleotide diversity was 0.94%, the minimum was 0.80% and the average was 0.88%. Divergence between Lamir and Sefid Rud River and Shir Rud was 0.2% and between Lamir and Tajan, and Shir Rud River it was 0.1%. The average evolutionary distance was 0.015. The maximum evolutionary distance was 0.294 between ADAA and AAAB, ABAB and BDBA, BBAA and AABB. The average number of bases surveyed was 121.2 and the average number of fragments was 30.30. The study suggests that there was a low genetic variability in four populations of Mahisefid in the south of Caspian Sea. Mahisefid population can be divided into two main clusters, the first clade consists of Shir Rud River and Lamir River populations and the second clade consists of Tajan River and Sefid Rud River populations. The clustering of Mahisefid populations was not in accordance with their geographical areas or river systems.
525 Ecology Morphology and Phylogeny of Scrippsiella trochoidea (Dinophyceae) a potentially harmful bloom forming species isolated from the sediments of Iran’s south coast Attaran-Fariman G. Bolch C. J. S. 1 4 2012 11 2 252 270 10 06 2012 Phytoplankton cells and resting cysts of the species Scrippsiella trochoidea are regular and dominant components of the dinoflagellate flora of coastal marine waters and sediments around the world. This species is a common harmful bloom forming species in coastal waters. In this study, for the first time cyst of S. trochoidea were isolated from the sediments of southeast coast of Iran. Five strains from the germination of a single cyst belonged to S. trochoidea. In order to confirm identification of the species an excystment and encystment experiment, cyst and germinated cell morphology and plate pattern by light and electron microscopy (SEM) have been described. The nucleotide sequences of two highly diverse regions, the rDNA-ITS 1,2 and 5.8S-rDNA have been sequenced for all strains. Homologous sequences from GenBank with five Iranian strains were compared to find their phylogenetic relationship. Both NJ and MP phylogenetic and morphological analysis showed five strains of S. trochoidea from Iran were clustered with previously described S. trochoidea and Calciodinellum levantinum species, and its closest relationship was with Scrippsiella sp. strain with a 1.2-1.4% sequence divergence. Results indicate that molecular studies of rDNA if combined with morphological cyst and vegetative cells could be a valuable approach to identification and taxonomy of calciodinelloideae dinoflagellate. 526 Biology & physiology Microsatellite DNA diversity of Coilia mystus (Clupeiformes:Engraulidae) in three Chinese estuaries Fang Chen Qiqun Cheng 1 4 2012 11 2 271 282 10 06 2012  An analysis of eight microsatellite loci in 90 individuals was performed to define the genetic structure and variability of three estuarine populations of Coilia mystus: 30 individuals each  collected from ChangJiang River (Yangtze River) estuary-CJ, MinJiang River estuary-MJ, and ZhuJiang River (Pearl River) estuary-ZJ. The results showed polymorphic information contents (PIC) of 0.78, 0.77, and 0.64 in CJ, MJ, and ZJ, respectively. The mean observed number of alleles (Na) ranged from 7.38 to 11.88. The mean observed and expected heterozygosities ranged from 0.09 to 0.21 and from 0.68 to 0.81, respectively. The ZJ population was least polymorphic. Highly significant deviations from Hardy-Weinberg equilibrium, mostly due to deficits of heterozygote, were found in these three populations. Pairwise FST and Reynolds’ distance indicated that the three estuarine populations were genetically distinct, in accordance with the principal component analysis (PCA) and the Bayesian model-based clustering algorithm analysis. The results showed that pairwise genetic differentiation among these three estuarine populations were relatively high, with possible divergence to subspecies level. This study provides microsatellite DNA evidence for assessing the genetic distinctness of C. mystus populations and will benefit fishery resource management and sustainable utilization of C. mystus.  527 Biology & physiology Anticancer effect of Dunaliella salina under stress and normal conditions against skin carcinoma cell line A431 in vitro Emtyazjoo Mo. Moghadasi Z. Rabbani M. Emtyazjoo Ma. Samadi S. Mossaffa N. 1 4 2012 11 2 283 293 10 06 2012 28 09 2013  Dunaliella salina, a green microalga, has been consumed as food and medicine for a long time.The anti-oxidant and anticancer effects are related to more production of β- carotene under stress conditions compared with normal circumstances.The scope of this study was survey of anticancer effect of the ethanol extract of Dunaliella salina algae under stress(EDSS) and normal (EDSN) conditions on death rate of skin carcinoma cell line A431 by MTT [3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide] method in vitro. Our results showed that the β-carotene amount in stress situation was 1.46 times more than in normal condition.The cytotoxic effect of EDSS exposed to 6.25, 12.5, 25, 50, 100 µg/ml in 6, 24, 48 h which was performed by MTT assay showed that there was a significant difference in the various time except in 6 h in both conditions.The results of compared death rate of A431cells exposed to various concentrations of EDSS and EDSN presented significant difference in 6, 24, and 48 h after exposure.  The LC50 of EDSS in 24 and 48 h were 15.4 and 0.4 µg/ml , respectively and 478.6 and 46.4 µg/ml for EDSN. 528 Biology & physiology Determination of developmental stages of embryo in the Sea Urchin, Echinometra mathaei Ghorani V. Mortazavi M. S. Mohammadi E. Sadripour E. Soltani M. Mahdavi Shahri N. Ghassemzadeh F. 1 4 2012 11 2 294 304 10 06 2012 Sea Urchin is one of the most useful tools in developmental biology studies because this organism has the simplest kind of developmental stages. We aimed to determine developmental stages and timetable of Echinometra mathaei embryo (the species of Persian Gulf). The spawning of E. mathaei was induced by 0.5M KCl injection (1ml) into the coelomic cavity. After fertilization, embryos were placed in beakers and were incubated at 29◦C and a salinity of 39 ppt until embryos reached the pluteus stage. The developmental stages of embryos and the timing of each stage including cleavage, morulae, blastula, gastrula, prism and pluteus larvae were studied under the microscope. Our results showed that after 30 hours from fertilization time, the embryos developed to pluteus larvae. E. mathaei had the shorter development time in comparison to the other Sea Urchin species. Therefore, it may be appropriate as a model organism in biological researches.  529 fish disease Histochemical study of the olfactory rosette of Cyprinus carpio (Linnaeus, 1758) Ghosh S. K. Chakrabarti P. 1 4 2012 11 2 305 314 10 06 2012   The distribution and localization of acid and neutral mucins in various cells lining the olfactory epithelium of Cyprinus carpio have been studied histochemically by employing the PAS-AB technique. Variations in the localization of protein in different cells lining the olfactory epithelium have been correlated with the functional significance of the region concerned. Intense localization of the silver stain in the surface of the olfactory epithelium as well as in the central core confirms the presence of different types of neurons. The localization and functional variations of alkaline phosphatase (ALPase) and adenosine-tri-phosphatase (ATPase) in the cells lining the olfactory epithelium of C. carpio have been discussed. 530 aquaculture The Antioxidant Vitamin (A, C, E) and the Lipid Peroxidation Levels in Some Tissues of Juvenile Rainbow trout (Oncorhynchus mykiss, W. 1792) at Different Oxygen Levels Tuna Keleştemur G. 1 4 2012 11 2 315 324 10 06 2012 In this study, the malondialdehyde (MDA) levels in the muscle, liver and kidney tissues and the antioxidant vitamin (A, C, E) levels in the muscle tissue of juvenile rainbow trout were investigated under different oxygen levels (3.5 mg O2/l, 4.5 mg O2/l and 7 mg O2/l) at the end of the 8 hour trial. Fish (initial weight and length, 30.19±2.2 g and 13.15±0.22 cm, respectively) were distributed into 9 fiberglass rectangular tanks. The highest MDA level of all tissues was obtained in 3.5 mg O2/l (p0.05). But the highest muscle vitamin C and E concentrations were obtained in 7 mg O2/l. (p<0.05), followed by 3.5 mg O2/l, while the lowest muscle vitamin C concentration was obtained in 4.5 mg O2/l. (p<0.05). 531 Toxicology Bioaccumulation of heavy metals Cu, Zn, and Hg in muscles and liver of the stellate sturgeon (Acipenser stellatus) in the Caspian Sea and their correlation with growth parameters Heidary S. Imanpour Namin J. Monsefrad F. 1 4 2012 11 2 325 337 10 06 2012 Bioaccumulation of Cu, Zn, Hg in muscles and liver tissues of stellate sturgeon (Acipenser stellatus) from the southern Caspian Sea was measured. Effects of condition factor (CF), age and hepatosomatic index (HSI) on metal accumulation were also determined. Some 40 stellate sturgeons were collected using gill nets in winter and spring 2007-2008 from the southern shores of the Caspian Sea. Total (TL) and fork (FL) length and weight of all specimens were measured. Cross sections of first ray of pectoral fins were used for age determination. Muscle and liver samples were taken and transported to the laboratory in ice packs and kept in -20 oC prior to analyses. In the laboratory samples were oven dried, digested with HClO3 and HNO3 and diluted with dionized water to 50cc to measure Cu, Zn, and Hg concentration with a flame spectrophotometer. Sequence of metals in Acipenser stellatus was Zn>Hg> Cu in muscle tissues and Zn> Cu> Hg in liver samples. Concentration of Zn was slightly in correlation with age (R= 0.3). A negative correlation was observed between Zn concentrations with condition factor (R= -0.3). Zn concentrations were negatively correlated in muscle tissues (R=-0.3). The results were compared to international standards proposed by MAAF, EEC and NHMRC. Our study showed that accumulation of heavy metals in sturgeon tissues was influenced by concentration of metals in sediment and the physiological state of fish. 532 Fish processing Protein and lipid changes of FPC produced from Caspian Sea Kilkas in VP and MAP during storage at different temperatures Khoshkhoo Zh. Motalebi A. A. Razavilar V. Khanipour A. A. 1 4 2012 11 2 338 346 10 06 2012 Fish Protein Concentrate (FPC) is a healthy, sustainable and high nutritive product that produced from fish and protein and other nutrients are more concentrated than in the fresh fish. The aim of this research is to study the sustainability of FPC produced from Kilka (combination of three Caspian Sea Kilka species, Clupeonella engrauliformis, C. grimmi and C. cultriventris which were not identified and processed separately) in VP (Vacuum Packaging) and MAP (Modified Atmosphere Packaging) at different temperatures during six months of storage. According to result of chemical analysis performed, protein content was evaluated 91.2%, lipid 0.5%, ash 3.6%, moisture 2.3%, TVN 10 mg/100g and peroxide 5 meq/kg in the produced FPC before packing. Amino acids and fatty acids were also determined. Lipid amount in FPC after 6 months at 35°C in VP changed from 0.50 to 0.45 and in MAP (combined of 60% CO2, 30% N2 and 10% O2), decreased from 0.50 to 0.36. It was also detected that increase in temperature leads to more decrease in lipid content but it was not significant (P>0.05). Protein content of FPC has changed from 91.2% to 73.6% during six months at 35°C in VP and 69.4% in MAP. But at 5°C, protein contents were changed from 91.2% to 88.4% and 81.2% in VP and MAP, respectively these changes were significant (P<0.05) but the decrease in MAP was again more than VP. 533 Genetics Genetic characterization of Vimba vimba persa (Pallas, 1814) in Southern parts of the Caspian Sea using microsatellite markers Mohamadian S. Rezavani Gilkolaei S. Rouhollahi S. Taghavi M. J. Nayerani M. Shirzad E. Taheri Mirghaed A. 1 4 2012 11 2 347 357 10 06 2012  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.   534 aquaculture Control of euglenophyte bloom and fish production enhancement using duckweed and lime Mohammad Shadiqur Rahman Shahjahan Md. Mahfuzul Haque Md. Saleha Khan 1 4 2012 11 2 358 371 10 06 2012  Euglenophyte bloom is a common problem in most of the aquaculture ponds in Bangladesh. In the present study we conducted an experiment to control euglenophyte bloom for achieving better fish production using duckweed (Lemna minor) and lime. The experiment was carried out using four treatments, i.e., ponds were supplied with duckweed (T1), lime treatment (T2), both duckweed and lime (T3) and without supply of duckweed and lime (T4). Rohu, catla, mrigal, silver carp and silver barb were stocked and their gut contents were analyzed monthly. The ranges of water quality parameters were analyzed within the productive limit during the experimental period. The mean abundance of euglenophyte was significantly highest in T4 (17.62 ± 1.97 x 104 cells/L), followed by T2 (2.96 ± 0.20 x 104 cells/L), T1 (1.94 ± 0.35 x 104 cells/L) and T3 (1.53 ± 0.42 x 104 cells/L). Gut content analysis revealed that considerable amounts of euglenophyte were consumed by silver carp and silver barb, but not preferred by rohu, catla and mrigal. The gross yields of fish were 2133.37, 1967.76, 2816.52 and 1725.62 kg/ha/5 months in T1, T2, T3 and T4 respectively. The highest fish production in T3 and lowest fish production in T4 indicated the use of duckweed and lime is economically sustainable for controlling euglenophytes bloom, maintaining water quality and getting higher fish production.  535 Toxicology Accumulation Variations of selected heavy metals in Barbus xanthopterus in Karoon and Dez Rivers of Khuzestan, Iran Mohammadi M. Askary Sary A. Khodadadi M. 1 4 2012 11 2 372 382 10 06 2012  The concentrations of Cd, Pb, Ni and Hg were determined in gill, liver and muscle of Barbus xanthopterus in down streams of Karoon and Dez Rivers during summer 2009. Heavy metal concentrations varied significantly, depending on the types of the tissue in fish species. The levels of heavy metals such as Cd, Pb, Ni and Hg in the Barbus xanthopterus of Karoon River were higher than in Barbus xanthopterus of Dez River (P<0.05). In Karoon and Dez Rivers, the concentrations of Cd, Pb and Ni were in the sequence gill>liver>muscle. The concentration of Hg in Karoon River was in the sequence liver> gill> muscle but, in Dez River, it was in the sequence liver> muscle > gill. Among heavy metals (Cd, Pb, Ni and Hg), the accumulation of Pb was more than other heavy metals in fish (P<0.05). In both rivers, the accumulation of heavy metals in muscle of fish was higher than the world health organization (WHO) standard.   536 Biology & physiology Reproductive biology of Cobitis keyvani (Cobitidae) from the Talar River in the southern Caspian Sea basin Mousavi-Sabet H. Kamali A. Soltani M. Bani A. Esmaeili H. R. Khoshbavar Rostami H. Vatandoust S. Moradkhani Z. 1 4 2012 11 2 383 393 10 06 2012  Some aspects of the reproductive biology of Cobitis keyvani (Mousavi-Sabet, Yerli, Vatandoust, Ozeren and Moradkhani, 2012), an endemic Loach fish species from the Talar River in the southern Caspian Sea basin, in north of Iran was studied by regular monthly collections throughout a year. Fecundity, condition factor, gonado-somatic, modified gonado-somatic and Dobriyal indices were estimated. Regression analyses tested the dependence of fecundity on fish length, weight, gonad weight and age. Significant differences were observed between the total number of females and males, females being more abundant. The mature females and males were longer than 49 and 45 mm in total length, and +2 and +1 in age, respectively. The average egg’s diameter was 0.56 mm. The spawning of Cobitis keyvani from Talar River took place from May to late July, when the water temperature is between 18.7 to 24.0°C. At the beginning of the reproduction period, the average GSI values were 10.09%, ranging from 6 to 25% in ripe mature females. The absolute and relative fecundity were 2211 and 586 respectively. The absolute fecundity was significantly related to body weight and gonads weight. Based on the gonado-somatic and Dobriyal indices, it was concluded that the fish has a prolonged active reproductive period. Their long reproductive activity and high range of fecundity are types of adaptation by short-lived small fishes to environmental conditions.  537 Biology & physiology A Comparative study of synthetic carp GnRH and carp pituitary homogenate effects on in vitro steroidogenesis of oocytes in common carp (Cyprinus carpio) Salamat N. Erfani Majd N. Hashemitabar H. Mesbah M. AhangarPour A. 1 4 2012 11 2 394 404 10 06 2012 Vitellogenic stage carp ovaries were collected aseptically and the cells of ovarian follicles were dispersed by trypsin treatment and cultivated as monolayer. Control cultures were grown in medium M199 alone. Media for experimental cultures were supplemented either with 100 µl ml-1 of CPH and CPS-carp GnRH (cultivated pituitary cell secretion affected by 10-6, 10-8, 10-10 M carp GnRH). The direct effect of these factors on 17-α-Hydroxy progesterone (17α -OHP) and 17β-Estradiol (E2) secretion of isolated follicular cells in cell culture was assayed by appropriate Radioimmunoassay (RIA). According to the results the E2 was a main hormone secreted by cultivated carp granulosa and theca cells. Adding 100 µl ml-1of 10-6 M CPS-carp GnRH had no significant effect on cultivated granulosa and theca cells endocrine activity (P> 0.05). On the other hand, carp granulosa and theca cells treated with the lower contents of CPS-carp GnRH (10-8, 10 -10 M) showed a 4.02 and 3.74-fold increase in E2 concentration and 4.27 and 3.64-fold increase in 17α -OHP level, which was significantly higher than CPH (P< 0.05). Also, there was a significant increase of steroid secretion in group stimulated with 100 µl ml-1 of CPS and CPH, but there were no significant differences between them (P> 0.05). 538 Biochemistry Effects of propolis on some blood parameters and enzymes in carp exposed to arsenic Selamoglu Talas Z. Pinar Dundar S. Fuat Gulhan M. Orun I. Kakoolaki S. 1 4 2012 11 2 405 414 10 06 2012  The purpose of this study was to investigate the therapeutic effects of natural products like propolis on biochemical and hematologic parameters in carp (Cyprinus carpio, Linnaeus 1758) exposed to arsenic. In this study fish were exposed to 0.01 mg/L arsenic and 10 mg/L propolis for seven days. Our results indicated that triglyceride, urea, total cholesterol, cobalt, ALT (alanine amino transferase), AST (aspartate amino transferase), LDH (lactate dehydrogenase) values increased when exposed to arsenic (P<0.05) but reduced by combination of arsenic and propolis (P<0.05). Granulocyte, erythrocyte, hemoglobin, hematocrit values were decreased by use of arsenic in comparison to control group. These parameters increased in arsenic+propolis group (P<0.05). In addition levels of leucocyte, agranulocyte, MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration) increased in the arsenic group (P<0.05) and decreased in arsenic+propolis group (P<0.05). We can say that propolis can improve biochemical and hematologic functions of common carp blood, after being exposed to arsenic.  539 Biochemistry Identification of bester hybrids (female Huso huso Linnaeus, 1758 and male sterlet Acipenser ruthenus Linnaeus, 1758) using AFLP molecular technique Yarmohammadi M. Shabani A. Pourkazemi M. Baradaran Noveiri S. 1 4 2012 11 2 415 423 10 06 2012   In this study Amplified Fragment Length Polymorphism (AFLP) was applied to species identification of bester hybrids. Hybrids identification was performed by comparison of electrophoresis profiles with parental species. The simultaneous occurrence of diagnostic bands fixed in the parental species, genetic distance and identification and cluster analyses (UPGMA) allow a correct identification. We used 8 primer combinations (Eco+3, Mse+4) and a total of 250 bands (size range 40-1000 bp) were generated. Primer combinations of (E-AAT, M-CGAT) and (E-AAG, M-CGAT) produced diagnostic bands in hybrids and parental species. Moreover, the results of genetic identification showed that Bester hybrids are more similar to beluga (Huso huso) (0.68) in comparison with sterlet (Acipenser ruthenus) (0.45).  The results suggested that this technique could be suitable for precise identification of species and inter-generic hybrids like bester.  540 Fish processing Short Communication- Isolation of some human pathogens from fresh and smoked shad Tavakoli HR. Soltani M. Bahonar A. 1 4 2012 11 2 424 429 10 06 2012 541 Biochemistry Short Communication: Haematological and immunological responses of sea bass (Dicentrarchus labrax) to a short-term exposure to increased water levels of nitrate Vectesi D. Kotzamanis I. Henry M. A. Vatsos I. N. 1 4 2012 11 2 430 439 10 06 2012