Population structure and demographic history for year cohort dynamics of landlocked ayu Plecoglossus altivelis altivelis in dam reservoir of Japan.

Ayu Plecoglossus altivelis altivelis is a valuable osmeroid species for inland fishery in Japan. It is classified into two ecological forms of amphidromous migrating between rivers and sea and landlocked migrating between rivers and lakes or dam reservoirs. The number of dams and their reservoirs has remarkably increased in the twenty-first century under climate change, because of their respective roles in hydropower generation with negligible carbon emissions and in flood control. Dam reservoirs therefore become increasingly important as inland nursery grounds of ayu. In this study, we investigated the reproduction status of landlocked ayu migrating in the Haidzuka Dam reservoir and the Tabusa River in western Japan by molecular phylogenetic analysis based on population structure and demographic history for year cohort dynamics. A total of 849 individuals were collected monthly from October 2018 to September 2021 according to an annual life cycle of ayu. Nucleotide sequences of the partial mitochondrial DNA control region yielded 31 haplotypes, consisting of 4 shared haplotypes among the 2019, 2020 and 2021 cohorts and 27 unique haplotypes. The overall haplotype diversity and nucleotide diversity were calculated to be relatively low at 0.3503 ± 0.0206 and 0.0077 ± 0.0045, respectively, suggesting a founder event by dominant haplotypes. Star-shaped radiational haplotypes from dominant shared haplotypes on the median-joining network likely support a founder event. Although pairwise ФST values were determined to be very low among the year cohorts, only the 2019 cohort was found to have a significant difference from the 2020 and 2021 cohorts, for both of which Tajima's D values were also statistically significant. For the overall population, multimodal mismatch distribution and negative Tajima's D and Fu's Fs values in the neutrality test suggested population expansion or population subdivision. The native riverine population in the Tabusa River suffered habitat fragmentation and population bottleneck from dam construction, and therefore severe founder effect remained behind the artificially landlocked population with a low level of genetic diversity in the Haidzuka Dam reservoir.

Stock assessment of landlocked ayu Plecoglossus altivelis altivelis in Japan through length-based models.

Ayu Plecoglossus altivelis altivelis is a key commercially and culturally important freshwater osmeroid in Japan. Its native population is mostly an amphidromous form migrating between rivers and the sea, and not only native but also artificially landlocked forms are found in lakes and dam reservoirs. This study was undertaken to execute population feasibility and maximum sustainable yield (MSY) analysis of an artificially landlocked form of ayu during January 2018 to December 2020 in the Haidzuka reservoir and its connected Tabusa River, western Japan. FAO-ICLARM Stock Assessment Tools-II and empirical models were employed to estimate the growth function and population parameters. The estimated asymptotic length was 19.50 cm, and the growth coefficient was 0.73 year-1 with a growth performance index of 2.443. The length at first maturity and length at optimum yield were calculated as 10.77 cm and 12.63 cm, respectively, which were lower than the length at first capture (Lc = 13.15 cm), suggesting the mesh size of fishing gear favoring the sustainability of the reproductive potential of this population. The calculated total, natural, and fishing mortalities were 2.45, 1.19, and 1.26 year-1, respectively. The recruitment pattern was continuous round the year with two pulses where the peak was during July. The current level of exploitation (0.51) was slightly higher than the maximum exploitation rate (0.45), indicating a little overharvesting. The MSY of ayu in the Haidzuka reservoir was estimated to be 211 metric tons if the recommended Lc is maintained. Results of this study illustrate the first information on population characteristics of landlocked ayu and will help the development of suitable management policies for ayu fishery in lakes and dam reservoirs.

Stochastic optimal switching model for migrating population dynamics.

An optimal switching control formalism combined with the stochastic dynamic programming is, for the first time, applied to modelling life cycle of migrating population dynamics with non-overlapping generations. The migration behaviour between habitats is efficiently described as impulsive switching based on stochastic differential equations, which is a new standpoint for modelling the biological phenomenon. The population dynamics is assumed to occur so that the reproductive success is maximized under an expectation. Finding the optimal migration strategy ultimately reduces to solving an optimality equation of the quasi-variational type. We show an effective linkage between our optimality equation and the basic reproduction number. Our model is applied to numerical computation of optimal migration strategy and basic reproduction number of an amphidromous fish Plecoglossus altivelis altivelis in Japan as a target species.

A simple and reliable method for DNA extraction from bivalve mantle.

A simple and reliable method was developed for extracting genomic DNA from preserved mantle tissues of Pacific oyster Crassostrea gigas for reproducible PCR amplification. The method was based on destruction of the tissue using Proteinase K, Chelex 100 resin, detergents, and urea, followed by preferential capturing of genomic DNA with silica particles. Approximately 5 mg of mantle tissue provided a sufficient quality and quantity of DNA for several hundreds of PCR reactions amplifying the hypervariable mitochondrial DNA intergenic spacer, which is a useful genetic marker for population structure analysis of Pacific oyster. The method can be applied for DNA preparation from not only fresh and frozen but also ethanol-preserved mantle tissues, so this rapid and economical method can serve for investigating a large number of bivalve specimens collected in the field and next transported in ethanol at ambient temperature.

A novel mitochondrial intergenic spacer reflecting population structure of Pacific oyster.

Nucleotide sequence divergence in a novel major mitochondrial DNA intergenic spacer (IGS) of Pacific oyster Crassostrea gigas was analyzed for 29 cultured individuals within the Goseong population (Korea). A total of 7 variable sites were detected within the IGS, and the relative frequency of nucleotide alteration was determined to be 1.16%;. All alterations were due to a single nucleotide substitution, and 5 transitions and 2 transversions were observed. Among 29 specimens, only 8 haplotypes could be identified, and 6 of the haplotypes were unique to particular specimens. Pairwise genetic diversity of all 8 haplotypes was calculated to be 0.412+/-0.134 from multiple sequence substitutions based on the two-parameter model. The phylogenetic tree obtained for these haplotypes according to the neighbor-joining method illustrated a single cluster of linkages, which comprised 5 haplotypes associated with 23 specimens, while the other 3 haplotypes associated with 6 specimens were scattered. The results indicate that the IGS is higher polymorphic and thus more suitable as a genetic marker for population structure analysis of Pacific oyster than the mtDNA coding regions, such as cytochrome c oxidase I and 16S ribosomal RNA genes.

Molecular identification of hairtail species (Pisces: Trichiuridae) based on PCR-RFLP analysis of the mitochondrial 16S rRNA gene.

A rapid PCR-RFLP analysis was designed to identify 3 closely related species of hairtails: Trichiurus lepturus, T. japonicus, and Trichiurus sp. 2, basing on partial sequence data (600 bp) of the mitochondrial DNA encoding the 16S ribosomal RNA (16S rRNA) gene. Restriction digestion analysis of the unpurified PCR products of these 3 species, using EcoRI and VspI endonucleases, generated reproducible species-specific restriction patterns showing 2 fragments (250 bp and 350 bp) for T. lepturus in EcoRI digestion and 2 fragments (196 bp and 404 bp) for T. japonicus in VspI digestion, whereas no cleavage was observed for Trichiurus sp. 2 in both EcoRI and VspI digestions. The PCR-RFLP technique developed in this study proved to be a rapid, reliable and simple method that enables easy and accurate identification of these 3 closely related species of the genus Trichiurus.

A novel type of myofibril-bound serine protease from white croaker (Argyrosomus argentatus).

Myofibril-bound serine protease (MBSP) was purified from the myofibril fraction of white croaker (Argyrosomus argentatus) muscle and its enzymatic properties were compared with other fish MBSPs. White croaker MBSP was extracted by the heat treatment of myofibrils and then purified by a series of column chromatographies on Q-Sepharose, Sephacryl S-300, hydroxyapatite and Benzamidine Sepharose. The purified MBSP migrated as a single protein band at 67 kDa in SDS-PAGE under both reducing and non-reducing conditions. It was inhibited by Pefabloc SC, soybean trypsin inhibitor (STI), aprotinin and benzamidine, and was not affected by E-64, pepstatin A and EDTA. The enzyme was most active against Boc-Phe-Ser-Arg-MCA at pH 7.0 and 50 degrees C, and preferentially hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Asp-Pro-Arg-MCA. Unlike other marine fish MBSPs, white croaker MBSP considerably hydrolyzed Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA. Some enzymatic characteristics including the molecular structure and the substrate specificity for a lysine residue at the P(1) position are quite different not only from other fish MBSPs but also from soluble serine protease obtained from white croaker muscle (MSSP). White croaker MBSP could be therefore classified into a novel type of fish muscle MBSP.

Rapid PCR-RFLP method for discrimination of imported and domestic mackerel.

With the ever-decreasing domestic fishery catch of Japanese mackerel Scomber japonicus, alternative Atlantic mackerel Scomber scombrus has been increasingly imported and currently accounts for approximately 34% of mackerel consumption in Japan. As there is no morphologic difference between the species after removal of their skin, not only fresh and frozen fillets but also processed seafood of S. scombrus are frequently marketed with mislabeling as S. japonicus. In this study, a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed to discriminate imported mackerel S. scombrus and domestic mackerel S. japonicus. PCR amplification for the nuclear 5S ribosomal DNA nontranscribed spacer was performed using Scomber-specific primers. Direct digestions of the PCR products using either PvuII or HaeIII restriction enzymes generated species-specific profiles, indicating that both enzymes enable the accurate identification of S. scombrus and S. japonicus. This robust and reproducible method can serve as molecular-based routine food inspection program to enforce labeling regulations.

Sequence polymorphism in a novel noncoding region of Pacific oyster mitochondrial DNA.

Nucleotide sequence polymorphism in a 641-bp novel major noncoding region of mitochondrial DNA (mtDNA-NC) of the Pacific oyster Crassostrea gigas was analysed for 29 cultured individuals within the Goseong population. A total of 30 variable sites were detected, and the relative frequency of nucleotide alteration was determined to be 4.68. Alterations were mostly single nucleotide substitutions. Transition, transversion, both transition and transversion, and both transversion and nucleotide deletion were observed at 18, 9, 2 and 1 sites, respectively. Among 29 specimens, 22 haplotypes were identified, and pairwise genetic diversity of haplotypes was calculated to be 0.988 from multiple sequence substitutions using the two-parameter model. A phylogenetic tree, obtained for haplotypes by the neighbor-joining method, showed a single cluster of linkages. The cluster comprised 11 haplotypes associating with 14 specimens, while the other 11 haplotypes associating with 15 specimens were scattered. This mtDNA-NC presenting a high nucleotide sequence polymorphism is a potential mtDNA control region. It therefore can serve as a genetic marker for intraspecies phylogenetic analysis of the Pacific oyster and is more useful than the less polymorphic mtDNA coding genes.

Identification of gadoid species (Pisces, Gadidae) by PCR-RFLP analysis.

A rapid PCR-RFLP analysis was optimized to identify the presence of 3 closely related gadoid fish species: Alaska pollack Theragra chalcogramma, Pacific cod Gadus macrocephalus and Atlantic cod Gadus morhua in commercial seafood products. Gadoid universal primers were designed for PCR amplification of a 558-bp fragment encoding the mitochondrial cytochrome b gene. Without purification of the PCR products, double digestion with Eco32I and Eco105I restriction enzymes generated reproducible species-specific restriction patterns visualizing 3 fragments (106 bp, 161 bp and 291 bp) in Alaska pollack and 2 fragments (106 bp and 452 bp) in Pacific cod, whereas no cleavage was observed in Atlantic cod. This PCR-RFLP analysis is simple, rapid and reliable, and therefore can be routinely applied to discover fraudulent substitution among 3 economically important gadoid species in commercial seafood products.

PCR-RFLP analysis of nuclear nontranscribed spacer for mackerel species identification.

Scomber mackerel have been marketed in fresh and frozen forms and as processed seafood worldwide, and three species of Japanese mackerel S. japonicus, Pacific mackerel S. australasicus, and Atlantic mackerel S. scombrus have constituted a significant part of absolute Scombrid consumption in Japan. The present study was undertaken to develop a rapid and reliable method not only for differentiation of Scomber mackerel from related Scombrid fish by PCR amplification using Scomber genus-specific primers but also for identification of three Scomber mackerel species by PCR-RFLP analysis. Alignment of nucleotide sequences of the nuclear 5S ribosomal RNA gene (5S rDNA) among Scombrid fish allowed the selection of oligonucleotide primers specific for the Scomber genus. These primers enabled amplification of the nontranscribed spacer (NTS) of the 5S rDNA from S. japonicus, S. australasicus, and S. scombrus, whereas no amplification was demonstrated from other Scombrid fish. RFLP analysis of the PCR products with ScaI endonuclease generated unique restriction patterns for each Scomber species. This simple, robust, and reproducible PCR-RFLP technique using Scomber genus-specific primers can serve as a routine food inspection program to enforce labeling regulations of marketed Scombrid fish.

Genetic relationship between cultured populations of Pacific oyster revealed by RAPD analysis.

We developed random amplified polymorphic DNA (RAPD) analysis for the assessment of the genetic relationship between cultured populations of the Pacific oyster Crassostrea gigas Thunberg in Hiroshima and Goseong, the largest oyster farming areas in Japan and Korea, respectively. Of 25 arbitrary primers comprising decamer nucleotides of random sequences, polymerase chain reaction amplifications with 5 different primers gave reproducible electrophoretic patterns. A total of 49 RAPD markers were clearly identified for the Hiroshima and Goseong populations, and 46 markers were polymorphic presenting mean polymorphism rates of the respective populations at 92.29% and 93.32%. Pairwise genetic distances of each 20 individuals from these populations served to produce a UPGMA dendrogram. The dendrogram comprised two main clusters, one of which was a nested cluster including all individuals of the Hiroshima population along with 12 individuals of the Goseong population, and the other cluster included the remaining individuals of the Goseong population. Results indicate that RAPD markers are useful for the assessment of the genetic relationships between populations of the Pacific oyster and further that a significant portion of oysters imported from Korea could be genetically related to the Hiroshima population.

Relationship between gyrA mutations and quinolone resistance in Flavobacterium psychrophilum isolates.

Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of F. psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27 F. psychrophilum strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [Escherichia coli numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F. psychrophilum.

Genotyping of Flavobacterium psychrophilum using PCR-RFLP analysis.

Genetic variability among 242 strains of Flavobacterium psychrophilum was characterized using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. Universal Primers GYR-1 and GYR-1R, which were designed to amplify the gyrase subunit B gene (gyrB), yielded a 1178 bp PCR product encoding gyrB and a 290 bp PCR product of anonymous DNA from all F. psychrophilum strains tested. In the RFLP analysis of the anonymous 290 bp DNA marker, the restriction enzyme HinfI generated 2 cleavage patterns (Genotypes A and B). Genotype A was found only in isolates from ayu (n = 109), while Genotype B was found in isolates from coho salmon (n = 11), ayu (n = 35), rainbow trout (n = 43) and other fishes (n = 44). In the second experiment, Primers PSY-G1F and PSY-G1R specific for F. psychrophilum, were used to amplify gyrB. The specific primer pair amplified the expected size (1017 bp) PCR product from all F. psychrophilum strains. In the RFLP analysis of the gyrB, the restriction enzyme RsaI produced 2 genotypes, R and S. Genotype R was found in isolates from coho salmon (n = 6), ayu (n = 27), rainbow trout (n = 39) and other fishes (n = 4). Genotype S was found in isolates from coho salmon (n = 5), ayu (n = 117), rainbow trout (n = 4) and other fishes (n = 40).

Development of novel microsatellite DNA markers from the Pacific oyster Crassostrea gigas.

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2-20; observed heterozygosity, 0.119-0.925; unbiased expected heterozygosity, 0.139-0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.

Flexprep: scale-flexible rapid plasmid preparation for analysis of recombinant clones.

A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones, this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation. It can process up to 20 mL of E. coli cells carrying 3-10 kbp plasmid vectors in < 10 min. Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.