Microsatellite markers reveal population structure in gilthead sea bream Sparus auratus from the Atlantic Ocean and Mediterranean Sea

ABSTRACT:   This study was carried out to investigate the genetic variability and the population genetics of Sparus auratus. In fact, despite its importance in Mediterranean fisheries and aquaculture, very little it is known concerning its population structure. Samples of wild gilthead sea bream were collected in seven different localities along the Mediterranean and Atlantic coasts and were genetically characterized by means of microsatellite markers. Genotyping at four microsatellite loci revealed high polymorphism (7–38 alleles/locus) and expected heterozygosities, which ranged from 0.80 to 0.85. A slight but significant population structure was found ( F _ST = 0.010). In fact, at least three populations of gilthead sea bream within the Western Mediterranean Sea were identified (Sardinian Sea, Sardinian Channel and Central Tyrrhenian Sea), which are also genetically differentiated from those of the Atlantic Ocean and the Adriatic Sea.     

Genetic difference between Ezo-awabi <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> and Kuro-awabi <Emphasis Type="Italic">H. discus discus</Emphasis> populations: Microsatellite-based population analysis in Japanese abalone

ABSTRACT:   This study documents the genetic population relationships between Ezo-awabi Haliotis discus hannai and Kuro-awabi H. discus discus by means of the microsatellite technique with an emphasis on the extent of genetic difference between Ezo- and Kuro-awabi populations. Eight markers were employed to screen five populations each collected from Ezo- and Kuro-awabi habitats. All eight loci showed polymorphisms in all populations (number of alleles per locus, 11.1–14.1; averaged expected heterozygosity, 0.64–0.70). The number of loci that yielded significant genetic heterogeneities (allele frequency distribution and/or pairwise θ ) between Ezo- and Kuro-awabi populations was larger than that generated in comparisons between populations within Ezo- and Kuro-awabi habitats. According to the bootstrap neighbor-joining trees constructed on the basis of two genetic distance measures ( D _A and D _ST), the 10 populations fell into two clusters of populations (Ezo-awabi and Kuro-awabi habitat groups), and the results of hierarchical amova testing also supported the neighbor-joining clustering. The outcomes presented here suggest that the microsatellite markers used in this study are potentially an efficient means to differentiate Ezo- and Kuro-awabi populations.     

Evaluation of genetic diversity of eight grouper species <Emphasis Type="Italic">Epinephelus</Emphasis> spp. based on microsatellite variations

ABSTRACT:   Genetic diversity among eight species of grouper, Epinephelus bleekeri , E. coioides , E. malabaricus , E. ongus , E. akaara , E. maculatus , E. merra and E. fuscoguttatus , was studied using six microsatellite loci, Em-01 *, Em-03 *, Em-07 *, Em-08 *, Em-10 *, and CA-07 *, with the aim of exploring the feasibility of using microsatellite data for species identification. The results showed high levels of genetic differentiation among species ( F _ST  = 0.4403 and R _ST  = 0.4954). Species identification based on fixed allelic differences was possible between E. coioides , E. ongus , and E. fuscoguttatus at Em-01 * and between E. fuscoguttatus and E. ongus at Em-08 *. Private alleles were found in all species, except for E. ongus . Pairwise F _ST ranged 0.238–0.578 ( P  < 0.008 Bonferroni correction), and Nei's genetic distance ranged 0.433–2.710. Size homoplasy was observed at Em-03 * 157 allele, which was characterized by a T–C transition at the 119th nucleotide site of PCR products. The genetic assignment test unambiguously assigned each individual to the correct species. Thus, this test can be used for species identification of unknown individuals when the multilocus genotypes of the six microsatellite loci are available. The phylogenetic (neighbor-joining) tree, which was constructed based on the genetic distance matrix, separated the eight grouper species into two main groups.     

Spatial differentiation in the eastern Pacific yellowfin tuna revealed by microsatellite variation

ABSTRACT:   Five eastern Pacific yellowfin tuna samples (four northern: 10–25°N, 95–130°W, and one southern: 16–18°S, 95–97°W), collected from fishing vessels between 1994 and 2002 were analyzed for variation at seven microsatellite loci to evaluate their spatial genetic homogeneity. Single-locus homogeneity exact tests revealed significant genetic differentiation caused by three of seven loci. Pairwise sample comparisons of multilocus allele-frequency homogeneity tests and subpopulation division ( F _ST), revealed significant differentiation in comparisons between north equatorial samples and the south equatorial sample. amova analysis among collections grouped as northern and southern populations separated by the equator in the eastern Pacific confirmed the differentiation observed. These results may be considered as preliminary evidence of the presence of discrete populations in the eastern Pacific yellowfin tuna. The possibility that the spatial differentiation observed corresponds to temporal variation or non-random sampling cannot be discarded. The genetic differences encountered need to be corroborated by increasing sample sizes, including temporal replicates, and by the use of an alternative marker.     

Population structure and partial anadromy in Oncorhynchus mykiss from Kamchatka: relevance for conservation strategies around the Pacific Rim

Abstract –  Conservation of life-history diversity found in Oncorhynchus mykiss requires knowledge of the underlying population structure and genetic basis of this variability. We analysed variation at 10 microsatellite loci from seven rivers across Kamchatka to identify population structure and to test for divergence between life-history forms. We found lower heterozygosity in Kamchatkan populations compared with North American populations, but population structure was substantial (region-wide F _ST = 0.11) and followed an isolation-by-distance pattern similar to that reported for older North American populations. We found no evidence for genetic divergence between resident and anadromous individuals in the Sopochnaya River or between typically anadromous individuals and 'half-pounders' in the Utkholok River. A review of other studies of reproductive isolation, in combination with our results, suggests: (1) that pristine populations of steelhead should be expected to exhibit partial anadromy; and (2) that managing anadromous and resident individuals separately without demonstrating reproductive isolation is biologically unsound.     

Body shape variation within and among three species of darters (Perciformes: Percidae)

Abstract –  Using geometric morphometric (GM) techniques, we quantified intra and interspecific variation in female body shape using five collections each of Etheostoma caeruleum Storer, Etheostoma nigrum Rafinesque, and Etheostoma stigmaeum (Jordan). Nested multivariate analysis of variance showed significant variation in body shape among populations (Wilks' Λ = 0.0687, F _144,7585.1 = 19.35, P  < 0.0001), as well as among species (Wilks' Λ = 8.7 × 10⁻⁶, F _24,2 = 28.18, P  = 0.0348). Etheostoma caeruleum displayed greater body depth relative to both E. nigrum and E. stigmaeum , whereas E. nigrum displayed a compressed arrangement of mid-body landmarks relative to both E. stigmaeum and E. caeruleum . The broader implications of these findings highlight the value of geometric morphometrics as both an exploratory and analytical approach. Conclusions drawn from comparisons among moderately differentiated darter species in future studies of body size and shape are likely robust to intraspecific variation within species, and will permit more rigorous investigations into the ecomorphology of these benthic stream fishes.     

Monitoring of the genetic variability of the hatchery and recaptured fish in the stock enhancement program of the rare species barfin flounder <Emphasis Type="Italic">Verasper moseri</Emphasis>

ABSTRACT:   In relation to the stock enhancement program for barfin flounder, hatchery juveniles produced in 2001 were genotyped using microsatellite DNA markers (msDNA) and then released to natural waters. Subsequently, recaptured individuals, designated as 'tentative recaptured', were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, pairwise F _ST and genic differentiation tests were used to estimate the genetic divergence between the wild samples, the hatchery broodstock, and the tentative recaptured samples. Analysis showed significant differentiation among these three groups. Pedigree determination by msDNA was used to establish the origin of the tentative recaptured individuals, in order to elucidate whether they were hatchery produced, 'real recaptured', or wild specimens. Wild individuals were not found. The effective population size of the real recaptured stock was very low ( N _e = 16.6). Equal family survivability was observed between the released and real recaptured stocks, indicating that the genetic variability of the released stock was maintained in the natural environment. Future broodstock management, breeding designs, and family contribution equalization of the offspring to be released will be required in this rare species to avoid unintended genetic differentiation between the wild population and the hatchery broodstock.     

Proximate composition of some north-eastern Pacific forage fish species

To understand the relative dietary value of forage fish as prey in the Bering Sea and Gulf of Alaska, whole organisms of 13 species were analysed for proximate composition (protein, oil, ash and moisture content). Eulachon ( Thaleichthys pacificus ) were high in oil (total lipid) (16.8% to 21.4%) and low in moisture (64.6% to 70.8%). Oil in capelin ( Mallotus villosus ) ranged from 2.1% to 14.0%. Juveniles of walleye pollock ( Theragra chalcogramma ), Atka mackerel ( Pleurogrammus monopterygius ), Pacific herring ( Clupea pallasii ), and prowfish ( Zaprora silenus ) had low oil contents (< 1.8%) and high moisture contents (> 80.3%). Rankings of median proximate values illustrate the similarities. Surf smelt ( Hypomesus pretiosus ), rainbow smelt ( Osmerus mordax ), pricklebacks ( Lumpenus spp.), Atka mackerel, Pacific sand lance ( Ammodytes hexapterus ) and Pacific sandfish ( Trichodon trichodon ) ranked high in median protein content (> 15.4%). Median ash content for all species ranged from 0.6% to 3.3%. Total wet mass caloric content (kcal g^–1) was calculated for the four main species and a linear model was developed for caloric content as a function of moisture. The linear models (caloric content = b₀ + b₁ × moisture) were Pacific sand lance and Pacific sandfish (b₀ = 7.82, b₁ = – 0.09); eulachon (b₀ = 7.97, b₁ = – 0.08); and capelin (b₀ = 9.70, b₁ = – 0.11).     

Pedigree analysis of recaptured fish in the stock enhancement program of spotted halibut Verasper variegatus

ABSTRACT:   Spotted halibut Verasper variegatus hatchery juveniles produced in 2002 were genotyped using three microsatellite DNA markers (msDNA) and then released into natural waters. Subsequently, recaptured individuals were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, from the genetic point of view, a pairwise F _ST test was implemented to estimate the genetic divergence between the wild captive broodstock, the hatchery offspring and the recaptured samples. The analysis showed significant differentiation between the broodstock and recaptured samples. Pedigree determination using msDNA was used to calculate the effective population size of the recaptured stock, which was found to be very low ( N _e ≈ 8). Equal family survivability was observed between the two recaptured stocks, but not between the released and recaptured stock. The number of identified families was higher and more equalized in the hatchery offspring compared to the recaptured samples, where the number of families declined. This fact was caused by an unequal family survivability just before or just after release. Separately, the number of contributing parents to the hatchery offspring was lower than the broodstock census number. Consequently, these two facts caused the genetic divergence of the recaptured stock from the broodstock.     

Population genetics of the European trout (Salmo trutta L.) migration system in the river Rhine: recolonisation by sea trout

Abstract –  Allozyme genetics (34 loci) is studied in up to 1010 European trout ( Salmo trutta ) from the Rhine, Meuse, Weser, Elbe and Danube river systems in Central Europe. Population samples from single collection sites, chiefly small streams ( G _CG = 0.2126), rather than the divergence of the trout from Atlantic and Danubian drainages ( G _SG = 0.0711), contributed to the overall gene diversity of G _ST = 0.2824. Sea trout ( n  = 164) and brown trout ( n  = 767) in Atlantic rivers adhere to the same biogeographical stock, but anadromous trout from the Rhine and the Elbe display more genetic cohesion than resident brown trout from the Rhine system alone. Strayers from the Elbe could have founded the recently re-established sea trout population of the Rhine, after a few decades of absence or precarious rarity. Migrants may even connect the Rhine and Elbe stocks by ongoing gene flow. A release–recapture study confirms that all trout in the Rhine belong to one partly migratory population network: Six of 2400 juvenile sea trout released into a tributary of the Rhine were later recorded as emigrants to the Rhine delta, against three of 1600 released brown trout. One migrant had entered the open North Sea, but the other dispersers were recorded in fresh waters of the Rhine delta (Ijsselmeer, Amstelmeer). Stocking presumably elevated both heterozygosity and fixation indices of brown trout, but this effect is subtle within the range of the Atlantic population group. Improved sea trout management in the Rhine, and modifications to brown trout stocking in the upper Danubian area are recommended.     

Age,growth and age at sexual maturity of fan ray <Emphasis Type="Italic">Platyrhina sinensis</Emphasis> (Batoidea: Platyrhinidae) in Ariake Bay,Japan

Abstract:   Age, growth and sexual maturity of the fan ray Platyrhina sinensis in Ariake Bay, Japan were determined from specimens collected from May 2002 to September 2006. Age determination was conducted by vertebral centrum analysis using soft X-radiography. Annual band pair deposition was determined by marginal increment and edge analyses. The von Bertalanffy growth model best described the overall pattern of growth for both males and females (males L _∝  = 455.2, k  = 0.56, t ₀ = −1.09; females L _∝  = 555.8, k  = 0.28, t ₀ = −1.77; L _∝ is the theoretical asymptotic total length in mm, k is the growth rate coefficient and t ₀ is the theoretical time at zero length). Parameter estimates suggest that females attain a larger asymptotic total length and grow more slowly than males. The observed maximum ages were 5 years for males and 12 years for females. Age at 50% sexual maturity was 2.1 years for males and 2.9 years for females. The results indicate that this species is relatively fast-growing, short-lived and early maturing compared with many batoid species.     

Management of gene diversity in the endemic killifish Aphanius iberus: revising Operational Conservation Units

Abstract –  Conservation of genetic diversity within species requires effective management techniques. To study the spatial structure of the killifish Aphanius iberus and to improve ongoing conservation practices, we used genetic approaches to assess the genetic structure of this endemic Iberian species. Twenty locations were analysed by allozyme electrophoresis and allelic variation was detected at 11 of 17 loci examined. Results of our study enabled us to redefine its conservation policy based on Operational Conservation Units (OCUs), which were proposed in 1996. The new OCUs recognise the genetic isolation of some marshland populations ( F _ST = 0.247) and incorporate reintroductions that occurred over the last 10 years. We also propose that the status of A. iberus in the International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species be changed from data deficient to endangered. Our work exemplifies how temporal genetic monitoring of populations can be a powerful tool to improve conservation programmes.     

Microsatellite variation reveals fine-scale genetic structure of masu salmon, Oncorhynchus masou, within the Atsuta River

Abstract –  To describe in detail the population genetic structure of masu salmon Oncorhynchus masou , seven anadromous populations in the Atsuta River were investigated by analysis of eight polymorphic microsatellite loci. Exact test and genetic differentiation ( F _ST) revealed that significant differentiation was observed at the within-river scale. Principal components analysis revealed that the spatial pattern of genetic composition was related to geographical location of each population. Furthermore, a significant correlation was observed between genetic differentiation and geographical distance, indicating that dispersal is less likely to occur between distant populations. The results of this study imply that masu salmon have potential to show clear genetic structure at the microgeographical scale (21 km) due to precise homing behaviour.     

Target strength estimation of black porgy Acanthopagrus schlegeli using acoustic measurements and a scattering model

ABSTRACT:   Black porgy are hatched and released in marine ranching areas in Japan, Korea and Taiwan to maintain fisheries resources in coastal areas. If acoustic methods are used for field surveys, target strength ( TS ) information on the species is needed to convert acoustic data to biomass density. Target strength of black porgy ( Acanthopagrus schlegeli ) 15.5–32.9 cm fork length (nine individuals) was measured at 38 and 120 kHz (split beam) by ex situ measurements (cage and fishhook methods) of living fish, a tethered method with shock-frozen fish, and an acoustic scattering model, the Kirchhoff ray mode (KRM) model based on fish morphology. The ex situ TS -to-fork length ( FL , cm) relationships were TS _38kHz = 20log₁₀ FL  − 64.6 ( r  = 0.77) and TS _120kHz = 20log₁₀ FL  − 65.2 ( r  = 0.70). The values for the KRM model were TS _38kHz = 20log₁₀ FL  − 64.8 ( r  = 0.88) and TS _120kHz = 20log₁₀ FL  − 65.4 ( r  = 0.80). The agreement between the ex situ measurements and the KRM model for the mean TS was very good. In contrast, the results for the tethered method ( TS _38kHz = 20log₁₀ FL  − 69.3 [ r  = 0.42]) differed markedly from the other two methods, perhaps because of the effects of the shock-freezing procedure in air. The results of the ex situ measurements and scattering model provide basic information for studying TS and conducting acoustic surveys of black porgy, even though the tethered method must be modified.     

Genetic evaluation of interindividual relatedness for broodstock management of the rare species barfin flounder Verasper moseri using microsatellite DNA markers

ABSTRACT:   The barfin flounder has been recently declared to be rare, as the number of wild individuals in Japan has greatly reduced since 1975. The Japanese National Center for Stock Enhancement started the stocking program for this species on 1987. In the present study, microsatellite DNA loci were applied in order to determine the pedigree of the hatchery-produced juveniles for stock enhancement. Additionally, using six polymorphic microsatellite markers, the accuracy to assign dyads to categories of kinship was estimated using the relatedness estimator ( R _XY), similarity indexes (individual genetic identity [GI] and proportion of shared alleles [ P _S]), identification of kin groups using unweighted pair group method with arithmetic mean (UPGMA) clustering based on interindividual genetic distances (1- R _XY, 1- P _S, and 1-GI), a likelihood ratio approach, and partition methods to separate individuals into cohorts. The results were compared with the pedigree relationship previously obtained from parentage analysis. Estimation of the kinship relationship between individuals could be better inferred by means of UPGMA dendrogram clustering based on 1-GI or 1- P _S as interindividual genetic distances, or using computer software to perform partitioning of set of individuals.     

Common pearl oysters in China, Japan, and Australia are conspecific: evidence from ITS sequences and AFLP

ABSTRACT:   To elucidate the species status of Pinctada fucata in China, P. fucata martensii in Japan and P. imbricata in Australia, one population of each taxon was studied using internal transcribed spacer 1 and 2 (ITS1, and ITS2) and amplified fragment length polymorphism (AFLP) markers. ITS1 and ITS2 were 401–405 and 229–237 bp long, respectively. Twenty-nine ITS1 and 15 ITS2 unique genotypes were obtained from 44 and 34 individuals, respectively, with some genotypes shared by two or three populations. In AFLP analysis, each individual exhibited a distinct phenotype. No population had diagnostic markers. Mean genetic divergences within and among the three populations were very low and overlapped (between-population: 0.7–0.9% for ITS1, 0.9–1.3% for ITS2, and 53.3–55.6% for AFLP; within-population: 0.5–0.9% for ITS1, 0.8–1.2% for ITS2, and 50.4–53.6% for AFLP). Low levels of genetic differentiation were observed among the three populations while the Australian population is partially genetically isolated. Under an infinite allele model, genetic differentiation among populations was not significant based on a permutation test. Under an infinite site model, most F _ST values were not significant for ITS data although they were significant for AFLP data. Network analysis using ITS data indicated that individuals from the same population did not cluster together. Analysis of molecular variance ( amova ) demonstrated that > 94% variation was contributed by within-population variation. These findings suggest that the three taxa are conspecific and Pinctada fucata is the correct name.     

Characterization of 38 kDa and 42 kDa chitinase isozymes from the liver of Japanese common squid Todarodes pacificus

ABSTRACT: Characterization was investigated on the 38 kDa and 42 kDa chitinase (EC3.2.1.14) isozymes from the liver of Japanese common squid Todarodes pacificus . Optimum pH toward colloidal chitin was observed at pH 3.0 for the 38 kDa chitinase, and pH 3.0 and 9.0 for the 42 kDa chitinase. K _m and k _cat of the 38 kDa and 42 kDa chitinases toward a longer substrate, glycol chitin, were 0.071 mg/mL and 1.22/s, and 0.074 mg/mL and 0.196/s, respectively. Alternatively, strong substrate inhibition of both chitinases were observed toward a short substrate, N -acetylchitopentaose (GlcNAc₅). Both chitinases decomposed not only chitin but also chitosan (D. A. 95%). The cleavage pattern and reaction rate were investigated using N -acetylchitooligosaccharides (GlcNAc_n, n  = 2–6). Both chitinases hydrolyzed GlcNAc_n ( n  = 4,5, and 6). The release of GlcNAc was not observed. The speed of the reaction was observed to be in the following order: GlcNAc₄ > GlcNAc₅ > GlcNAc₆ for the 38 kDa chitinase, and GlcNAc₆ > GlcNAc₅ > GlcNAc₄ for the 42 kDa chitinase. Both the chitinases released p -nitrophenol from p -nitrophenyl GlcNAc_n ( n  = 2, 3, and 4). N-terminal amino acid sequences of the 38 kDa and 42 kDa chitinases were YLLSXYFTNWSQYRPGAGKYFPQNI and EYRKVXYYTNWSQYREVPAKFFPEN, respectively.     

Culture-based fisheries in non-perennial reservoirs of Sri Lanka: influence of reservoir morphometry and stocking density on yield

Abstract  Culture-based fish yield in non-perennial reservoirs of Sri Lanka was related to reservoir morphometry and stocking density. The reservoirs were stocked mainly with fingerlings of one Chinese and three Indian major carp species, common carp, Cyprinus carpio L., and the genetically improved farmed tilapia strain of Nile tilapia, Oreochromis niloticus (L.), at four pre-determined species combinations and a range of stocking densities [ SD (fingerlings ha⁻¹)]. Twenty-three reservoirs were harvested successfully at the end of the culture period of 2002–2003. Basic limnological and morphometric parameters, including shoreline development ( D _L ) and shoreline area ratio ( R _LA ), were estimated for each of the 23 reservoirs. Bray–Curtis similarity and non-metric multidimensional scaling using mean values of limnological data revealed that reservoirs could be ordinated into two major clusters, one with intact sample distribution due to similar trophic characteristics and the other with scattered sample distribution. Reservoirs in the cluster with similar trophic characteristics showed significant correlation ( P  < 0.05) between R _LA and total fish yield ( Y ). A multiple regression equation, Y  = −693 + 4810  R _LA  + 0.484 SD , was generated to estimate fish harvest in relation to SD .     

An appraisal of factors constraining the success of fish stock enhancement programmes

Stocking and introduction of fish have been widely applied in Yunnan Province, China, for many years to mitigate for declines in natural stocks and commercial catches, and to restore or create new fisheries. The present paper assesses the results of activities concerned with stock enhancement programmes to evaluate the factors which have contributed towards the success of these projects. Factors such as natural lake conditions, stocking density, the age and size of fish stocked, and the mechanisms of stocking are discussed. Stocking density was evaluated using the equations Y _p = [ B ( P / B ) U _f]/ K _f or PY_p = (P × U_f)/K_f, which were based on the ecological carrying capacity of the recipient lake and other criteria. The stocking density in most lakes in Yunnan was high, but the recapture rates and total yield did not reach the predicted levels. This may have been an effect of the size of the fingerlings released (finger-lings >10 cm have a greater chance of survival). Key management measures for stocking and the ecological impacts of overstocking grass carp, Ctenopharyngodon idella (Valenciennes), are also described.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.