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Genetic studies play a great role for determining the biology of bivalves, particularly those covering population genetics, phylogeny, breeding, stock management, and conservation. However, DNA sampling methods that require removal of bivalves from the water and/or opening of their shells often cause stress and damage to bivalves, which can be lethal. The invasiveness of DNA sampling has made it difficult to conduct genetic studies in threatened species, rare species, and/or breeding lineages. In the present study, we developed a non-invasive method for bivalve DNA sampling using the water-filled mantle cavity (WMC). Our method can extract DNA from a small WMC sample (about 100 µl), collected using a fine needle and syringe without opening the shell. We demonstrated that the WMC sample contains intact mitochondrial and nuclear DNA. DNA contamination from other organisms, such as adjacent bivalve individuals, did not affect the resulting PCR and DNA sequencing analyses. Finally, the individuals from whom WMC was collected remained alive for more than 2 months after the experiments. This non-invasive method will be of great assistance in investigating the genetics of bivalves.

Small planktonic crustaceans cause serious problems in invertebrate aquaculture tanks through predatory damage or competition for food resources with the aquaculture species. In sea cucumber aquaculture, they can cause the mass loss of culturing jeveniles for releasing, which is a major obstacle to stable seedling production. In this study, we developed a novel method for eradicating small planktonic crustaceans using a ‘cavitation’ shock wave. Cavitation can cause significant damage to the surfaces of metallic devices, such as propellers and pumps, via the shock waves that occur with the disruption of micro/nano-bubbles. Therefore, we subjected planktonic crustaceans, mainly copepods, to cavitation using a micro/nano-bubble generator. The cavitation treatment reduced the planktonic crustaceans in the aquaculture tanks by 63.3% compared with the control. Comparison of the body size distribution of crustaceans indicated that cavitation treatment kills crustaceans of all sizes equally. We also assessed the negative effects of the micro/nano-bubbles, which are a byproduct of cavitation treatment, on the aquaculture species. We exposed juvenile sea cucumbers (Apostichopus japonicus) and sea urchins (Strongylocentrotus intermedius) to micro/nano-bubbles, and then examined their survival rate four days later; all individuals were intact and uninjured. These results suggest that cavitation treatment is an effective method for controlling planktonic crustaceans without using chemicals.

Massive tsunami disturbances have potentially detrimental effects on genetic diversity and effective population size of coastal marine species, and evaluating these effects can be useful for devising conservation strategies for coastal marine environments. Local populations of the intertidal goby Chaenogobius annularis, which are distributed on scattered rocky beaches of the Japanese Archipelago, show demographic independence without overlapping generations, making this an ideal species with which to study the effects of tsunami disturbance on genetic diversity. Some of these populations were affected by the tsunami of the 2011 off the Pacific coast of Tohoku Earthquake. Here, we investigated the change in genetic diversity of a local population of this species, which was located close to the epicenter of the earthquake, across the cohorts before and after the tsunami and evaluated the impact of the tsunami disturbance. Genetic diversity was maintained after the tsunami, and no change in the effective population size was observed. Our results suggest that the tsunami disturbance has had no marked impact on the genetic diversity of C. annularis.

Various genome-scale data have been increasingly published in diverged species, but they can be reused for other purposes by re-analyzing in other ways. As a case study to utilize the published genome data, we developed microsatellite markers from the genome sequence data (assembled contigs and unassembled reads) of the marbled flounder Pseudopleuronectes yokohamae. No microsatellites were identified in the contig sequences, whereas the computer software found 781,773 sequences containing microsatellites with di- to hexa-nucleotide motif in the unassembled reads. For 86,732 unique sequences among them, a total of 331,368 primer pairs were designed. Screening based on PCR amplification, polymorphisms and accurate genotyping resulted in sixteen primer sets, which were later characterized using 45 samples collected in Onagawa Bay, Miyagi, Japan. The presence of null alleles was suggested at four loci in the studied population but no evidence of allelic dropout was found. The observed number of alleles and heterozygosity was 2-20 and 0-0.88889, respectively, indicating polymorphisms and usefulness for population genetic analyses of this species. In addition, a large number of the microsatellite primers developed in this study are potentially applicable also for kinship estimation, individual fingerprint and linkage map construction.

Marine organisms with a planktonic larval stage can passively disperse long distance and are thus expected to have a wider distribution range and lower geographic variation. However, recent molecular phylogenetic studies have revealed that they often display a clear geographic genetic structure or even form a geographically fragmented species complex. These genetic divergences can be facilitated by the presence of dispersal barriers such as oceanic currents and/or by the limitation of suitable habitats. Using comprehensive phylogenetic analyses, we evaluate how such dispersal barriers shape genetic divergence and speciation in the intertidal snail genus Monodonta. Our phylogenetic analysis revealed various patterns of cladogenesis in Monodonta in East Asia. Genetic segregation between the Japanese and Ryukyu Archipelagos are detected in M. labio and M. perplexa perplexa. However, the relationship of geographical border and lineages does not correspond to those two because they show different habitat preference. M. labio distributed in the Japanese mainland is separated by the boundary corresponding to the point from which oceanic currents split into different directions. In contrast, species inhabiting various environments such as M. confusa are not genetically separated in Japan. In the peripheral oceanic Ogasawara Islands, two Monodonta species form each endemic lineage, although these two underwent different colonization processes to the islands. These findings suggest that the genus Monodonta has been genetically diversified around Japan, probably due to its correlations with dispersal ability, oceanic current, and habitat preferences. These factors may be effective causes for diversification of marine gastropods with a planktonic stage.

Chlamys farreri , which is naturally distributed along the coasts of East Asia, is a commercially important mollusk in China, and its mariculture has been prevalent since the 1970s with the decrease of natural resources in the 1960s. To conserve its genetic resources and avoid genetic disturbance, the population structure of the natural population in Japan and genetic condition of the stock population in China were investigated using morphological, microsatellite, and mtDNA markers. Analysis of morphological variance in C . farreri revealed no distinct difference between the presumed 2 subspecies ( C . farreri akazara and C . farreri nipponensis ) or between the natural and cultured scallops. Population genetic analysis using 2 DNA markers showed that wild scallops in Japan could be divided into a northern group and a southern group with 2 subgroups. The magnitude of genetic divergence between these groups was below the subspecies level, suggesting that C . farreri comprises 1 species with geographic subpopulations. Analysis of the mtDNA markers indicated the genetic divergence between populations of Japan and China, while that of the microsatellite markers showed that 3 Chinese populations (Qingdao, Weihai, and Yantai) were genetically similar to the northern Japanese group, suggesting the possibility of introduction of foreign genomes from the Tohoku area. On the basis of the present results, the northern and southern groups in Japan and Dalian population in China should ideally be regarded as separate units for management and conserved as the source populations for breeding programs. In addition, the genetic relationship among natural and cultured populations in China and natural populations in Japan should be further investigated for a better genetic management of this species.

The Japanese sea cucumber Apostichopus japonicus has a pelagic-lecithotrophic larva. Here, we clarify larval dispersal among Japanese sea cucumber populations by describing the levels of distinctiveness and gene flow among red and green variant populations of A. japonicus across Toyama Bay using 11 polymorphic microsatellite markers. Samples of Japanese sea cucumber populations were collected from three localities (Noto, Shinminato, and Uozu) around Toyama Bay. Geographically separated populations of red and green variants were determined to be genetically distinct, supported by phylogenetic analysis and the distinctiveness range values of pairwise multilocus estimates of F ST. Contemporary migration analyses indicated that the majority of estimated migration events occurred within A. japonicus populations comprised of only either red or green variants. In a historical gene flow analysis, two best-fit models (n-island and stepping stone models) showed circulation for the gene migration in Toyama Bay among red variant populations. Our results provide useful information on the genetic structure of Japanese sea cucumber populations and will be helpful for genetic conservation and fisheries management of sea cucumber populations in Toyama Bay.

Elucidating the scale of gene flow among populations is an important challenge for understanding the ecological dynamics and local adaptation of marine organisms. We assessed whether gene flow is restricted even at a small spatial scale in the Japanese common intertidal goby Chaenogobius annularis, using highly polymorphic DNA markers, involving the mitochondrial DNA (mtDNA) control region and 15 microsatellite DNA (msDNA), because past ecological studies have suggested low dispersal ability for rocky intertidal fishes. We found significant heterogeneities between four neighboring local populations by both mtDNA and msDNA analyses. In addition, no genetic heterogeneity was detected by either method across generations within a population; it was considered that such genetic differentiation is retained across generations and that the gene flow of this species is restricted to within a radius of a few kilometers. This is the first report showing a clear genetic subdivision in rocky intertidal fish.

Genetic differentiation in the intertidal goby Chaenogobius annularis was studied using allozyme markers. Samples were collected from six localities along the coasts of Japanese islands and the Korean Peninsula. Six out of 13 loci showed allelic variation in at least one population. Although the predominant alleles of 12 loci were the same among all populations, Mdh-1 showed clear differences among the populations located along the coasts of the Pacific Ocean and the Sea of Japan. These two possible geographic groups, the Pacific Ocean and the Sea of Japan groups, were characterized by diagnostic alleles of Mdh-1, namely Mdh-1 100 and Mdh-1 70.

To demonstrate inbreeding depression in various traits of Pacific abalone Haliotis discus hannai, 12 inbreeding full-sib families (brother-sister mating) and 13 outbreeding full-sib families were produced by factorial mating using the parents from two full-sib families. All 25 full-sib families were reared for 158 days after settlement, and 8 of the inbred and outbred full-sib families were reared until 3 y 4 mo of age. Inbreeding depression traits between inbred and outbred crosses were compared. No significant differences in fertilization rate, hatchability, and veliger survival rate were observed between the inbreeding and outbreeding crosses. Deformity rate in veliger larvae from the inbred crosses was significantly higher than that among the outbred crosses (t-test, P < 0.01). Significantly lower survival rates were observed in the inbreeding crosses at 109 days after settlement (t-test, P < 0.01) and between 1 y 4 mo-old and 3 y 4 mo-old crosses (t-test, P < 0.05). Mean increases in shell l...

‘Inbreeding depression’ may be an avoidable phenomenon for abalone culture. However, only a few studies have been carried out on inbreeding depression. In the present study, using six families produced in 1994, a factorial mating system including inbreeding and outbreeding was constructed in order to demonstrate inbreeding depression traits of the Pacific abalone. In total, 24 inbreeding and 21 outbreeding crosses were produced during three years (1999–2001) and these off-spring were reared for approximately one year. Significant differences in fertilization rate and growth were not observed between inbreeding and outbreeding corsses. However, the deformity rate of veliger larvae was always higher in inbreeding crosses than that of outbreeding crosses in all experiments. Moreover, a significantly high deformity rate was observed in some full-sib families of inbreeding. Alternatively, the survival rates of inbreeding crosses were much lower than for outbreeding crosses after about 4 months and one year in two rearing localities. These results indicate that inbreeding depression is observed in the traits of deformity rate and survival, but not in fertilization rate nor growth in the first generation of a full-sib family of the Pacific abalone.

Serotonin (5-hydroxytriptamine; 5-HT) is a major neurotransmitter that triggers oocyte maturation and sequential spawning in bivalve mollusks. A proteinous and heat-labile substance that proved to be a novel inhibitor of 5-HT-induced egg release from ovarian tissue was found in the cerebral and pedal ganglia (CPG) of the scallop Patinopecten yessoensis. The same inhibitory activity was also observed in the proteinous fraction from the supernatant of hemolymph. Histological observation demonstrated that the novel inhibitor prevented 5-HT from inducing oocyte maturation in the scallop ovary and that no prostaglandin F2alpha (PGF2alpha) inhibited 5-HT-induced oocyte maturation, although PGF2alpha strongly prohibited 5-HT-induced egg release through the gonoduct from ovarian tissue. The novel inhibitor from the scallop CPG also prohibited 5-HT-induced oocyte maturation of other bivalve species as well as scallops. The novel inhibitor, mediated through a receptor mechanism on oocyte membranes, blocked extracellular Ca2+ uptake into oocytes, which was observed in 5-HT-induced oocyte maturation. It is suggested that the novel inhibitor with a molecular mass of 60 kDa, named oocyte maturation arresting factor, which appears to be a universal substance for bivalve species, may be transported from the CPG to the ovary via hemolymph and may prohibit 5-HT-induced oocyte maturation due to the interference of extracellular Ca2+ influx into oocytes, eventually resulting in the inhibition of spawning. On the other hand, it seems that PGF2alpha inhibits 5-HT-induced transport of mature eggs through the gonoduct.

Five families of gynogenetic diploid Pacific oyster (Crassostrea gigas) were induced by inhibiting the second polar body in meiotic cell division of eggs fertilized with UV-irradiated sperm. Segregation patterns of eight microsatellite loci were investigated in the gynogenetic diploid offspring; the proportion of heterozygous progeny was used to estimate microsatellite–centromere (M–C) distances. Mendelian inheritance was confirmed for the eight loci by examining the genotypic segregation in the control crosses. Three of the eight microsatellite loci showed the existence of null alleles in four control crosses. All gynogenetic offspring only possessed the alleles of the mother, indicating 100% success level for the five families. The M–C recombination frequency estimates ranged from 0.62 to 0.77 (0.72 mean), comparable to those in the oyster based on allozyme markers and suggesting that meiotic gynogenesis does not appear to be a very efficient inbreeding method in the oyster. Recombination frequencies observed were often higher than the theoretical maximum of 0.67, indicating the existence of positive interference after a single chiasma formation in some chromosomes. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution toward assembly of genetic maps in C. gigas.

Inheritance of 9 microsatellite loci was examined in 3 families of gynogenetic Pacific abalone Haliotis discus hannai produced by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division. The proportion of heterozygous progeny was used to estimate marker-centromere (M-C) distances. All loci conformed to Mendelian segregation in the control crosses when null alleles were accounted for. The absence of paternal alleles confirmed the gynogenetic origin of the offspring and indicated 100% success for 3 families. Estimated recombinant frequencies ranged from 0.10 to 0.60, which is lower than those observed in other gynogenetic diploid animals. The mean recombination frequency was 0.22, corresponding to a fixation index of 0.78 in one generation. This is 3.12 times the increase in homozygosity expected after one generation of sib mating (0.25), suggesting meiotic gynogenesis may be an effective means of rapid inbreeding in the abalone. M-C map distances for the 9 loci varied between 5 and 30 cM under the assumption of complete interference. The information about M-C distances will be useful for future gene mapping in H. discus hannai.

In order to assess the utility of microsatellite DNA markers for detecting changes of genetic diversity in hatchery strains and for estimating their genetic relationships, we used six microsatellite markers to estimate the level of genetic diversity within three hatchery strains and two wild populations of Pacific abalone, and compared the degree of genetic differentiation between them. High polymorphism at the microsatellite loci was found within both hatchery and wild abalone populations. Compared to wild populations, all the hatchery strains showed less genetic variation as revealed in lower number of alleles and lower expected heterozygosity, indicating that bottleneck effects occurred when each strain was founded. Significant differentiation was found between the hatchery strains, and between the hatchery strains and wild populations (Fst range: 0.059–0.427; Rst range: 0.056–0.351), and no obvious difference was detected between the wild populations (Fst=0.004; Rst=0.007). According to the neighbor-joining tree topology constructed on the basis of genetic distances among individuals, almost all individuals from each hatchery strain were closely clustered, demonstrating the feasibility of microsatellite analysis for discrimination between hatchery strains. The results obtained in this study indicate that it is necessary to genetically characterize the abalone strains that are being released every year in order to monitor the effect on the genetic diversity of wild populations.

The present study was undertaken to determine scallop vitellogenin (Vtg) cDNA sequence, to identify Vtg synthesizing cell, and to analyze the regulation of Vtg mRNA expression. Clones containing partial cDNA sequence of Vtg were isolated from cDNA library of the scallop ovary by immunoscreening with the anti-scallop vitellin (Vn) serum. The deduced amino acid sequence of the clone containing the longest cDNA insert (1,689 bp) was identified as a member of the lipid transport protein family and exhibited about 20-35% identity with Vtgs of other oviparous animals. Northern blot analysis identified a single transcript longer than 10 kb in the ovary. Dot blot analysis of the ovary showed a high amount of Vtg mRNA during the growing stage and the level was retained until spawning stage. In situ hybridization demonstrated the expression of Vtg mRNA in the auxiliary cells closely associated with growing oocytes, suggesting that the synthesis of a major yolk protein in the scallop occurs through hetero-synthetic pathway without mediation through the blood flow but occurs de novo in the ovary. The content of Vtg mRNA in the ovarian tissue cultured in vitro with vitellogenesis promoting factor (VPF), which strongly promotes Vtg protein synthesis, from the cerebral plus pedal ganglion (CPG) showed no change. The transcription of Vtg mRNA appeared to be promoted by estradiol-17beta (E2) not by VPF although VPF may enhance the translation of Vtg mRNA.

The inheritance mode of six previously published and newly developed microsatellite markers was investigated in newly hatched Haliotis discus hannai larvae from four controlled crosses, and the feasibility of these markers for kinship estimation was also examined. Microsatellite DNA was successfully amplified from H. discus hannai larvae using the Chelex extraction method, and at least 15 microsatellite loci could be analyzed in a single trochophore larva. All six microsatellite loci were compatible with Mendelian inheritance. Neither evidence of sex-linked barriers to transmission nor evidence of major barriers to fertilization between gametes from the parents was shown. One of the six microsatellite loci showed the existence of null alleles in one family, indicating that the loci should be used in population studies with caution. Although the null allele heterozygotes were considered as homozygotes in the calculation of genetic distance, offspring from four full-sib families were unambiguously discriminated in the neighbor-joining dendrogram. The result demonstrates that the microsatellite markers might be capable of discriminating between related and unrelated abalone larvae in the situation where no pedigree information is available.

In order to evaluate genetic differentiation among three color variants (red, green and black) of Japanese sea cucumber, Stichopus japonicus, 11 isozyme loci were used as genetic markers for 448 individuals collected from four localities around Japan. Significant differences of allele frequencies were observed between all pairs of color types sympatrically. Average genetic distances at seven loci between the three sympatric color types were 0.0173 between the red and the other color types, and 0.0015 between the green and the black types. A dendrogram drawn from genetic distances among the three color types across all four localities showed two distinct clusters comprising all the red types and all the green and the black types, respectively. These results indicated that the red type showed definite genetic differentiation from the other color types.

Twelve polymorphic microsatellite markers were isolated from the kelp Ecklonia cava Kjellman (Phaeophyceae, Laminariales). The applicability of these markers was confirmed by genotyping of 24 individuals from a natural population in Japan. All 12 loci were polymorphic, with the number of alleles ranging from 2 to 11 per locus. The range of expected heterozygosities in the population was 0.187–0.867. These informative microsatellite markers will be useful in genetic studies of this species.

SUMMARY: To obtain useful information about the mechanism of the induction of gynogenesis in the Pacific abalone, nuclear behavior in normal and gynogenetic eggs was examined during meiosis and first cleavage. After its incorporation into the egg cytoplasm, the UV-irradiated sperm nucleus gradually dispersed and eventually developed into the male pronucleus, behaving like a normal sperm until contact with the female pronucleus. At mitotic prophase, the male pronucleus in the gynogenetic eggs did not form chromosomes like the female pronucleus, but became a dense chromatin body, which did not participate in karyokinesis. At completion of cytokinesis of the first cleavage, the UV-irradiated sperm nucleus was seen in the cytoplasm of one of the two blastomeres near the cleavage furrow. As a clear haploid mode was observed in the frequency distribution of chromosome numbers in gynogenetic larval cells, it is suggested that the paternal genome in the gynogenetic eggs does not contribute to the next generation.

Effects of ultraviolet (UV) irradiation on genetic inactivation and morphological structure of sperms were examined in the scallop, Patinopecten yessoensis . Haploid gynogenesis of the scallop was successfully induced by 50–60 s UV irradiation of 720 μW cm −2 s −1 . The fertilization rate apparently decreased with increasing irradiation time, and the development of the eggs fertilized with the genetically inactivated sperms terminated before reaching the D-shaped larvae stage. Scanning electron microscopy (SEM) showed clear destruction of the sperm acrosome and flagellum in the UV-irradiated sperms. As the duration of UV irradiation increased, the acrosome of sperms tended to suffer greater damage, until the sperms eventually lost their flagella. Abnormalities in these structures have appeared to account, at least in part, for the decline of the fertilization rate of eggs inseminated with UV-irradiated sperms.

SUMMARY: Effects of ultraviolet (UV) irradiation on genetical inactivation and morphological features of sperm were examined in the Pacific oyster. Ultraviolet light (254 nm) was effective for inactivating sperm chromosomes. Irradiation for 60 s at a UV intensity of 72 erg/mm 2 per s was the optimum dose to achieve haploid gynogenesis. The rates of the fertilization and the development of D-shaped larvae decreased with increasing irradiation time, and the development of the eggs fertilized with the genetically inactivated sperms terminated before reaching the D-shaped stage. Electron microscopy showed clear destruction of the sperm acrosome and flagellum in the UV-irradiated sperms. As the duration of irradiation increased, the acrosome of sperms tended to suffer greater damage until the sperms eventually lost their flagella. Abnormalities in these structures have appeared to account, at least in part, for the decline of the fertilization rate of eggs inseminated with UV-irradiated sperms.

Correspondance: Molecular Ecology Resources Primer Development Consortium, E-mail: editorial.office@molecol.com; International audience; This article documents the addition of 512 microsatellite marker loci and nine pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Alcippe morrisonia morrisonia, Bashania fangiana, Bashania fargesii, Chaetodon vagabundus, Colletes floralis, Coluber constrictor flaviventris, Coptotermes gestroi, Crotophaga major, Cyprinella lutrensis, Danaus plexippus, Fagus grandifolia, Falco tinnunculus, Fletcherimyia fletcheri, Hydrilla verticillata, Laterallus jamaicensis coturniculus, Leavenworthia alabamica, Marmosops incanus, Miichthys miiuy, Nasua nasua, Noturus exilis, Odontesthes bonariensis, Quadrula fragosa, Pinctada maxima, Pseudaletia separata, Pseudoperonospora cubensis, Podocarpus elatus, Portunus trituberculatus, Rhagoletis cerasi, Rhinella schneideri, Sarracenia alata, Skeletonema marinoi, Sminthurus viridis, Syngnathus abaster, Uroteuthis (Photololigo) chinensis, Verticillium dahliae, Wasmannia auropunctata, and Zygochlamys patagonica. These loci were cross-tested on the following species: Chaetodon baronessa, Falco columbarius, Falco eleonorae, Falco naumanni, Falco peregrinus, Falco subbuteo, Didelphis aurita, Gracilinanus microtarsus, Marmosops paulensis, Monodelphis Americana, Odontesthes hatcheri, Podocarpus grayi, Podocarpus lawrencei, Podocarpus smithii, Portunus pelagicus, Syngnathus acus, Syngnathus typhle,Uroteuthis (Photololigo) edulis, Uroteuthis (Photololigo) duvauceli and Verticillium albo-atrum. This article also documents the addition of nine sequencing primer pairs and sixteen allele specific primers or probes for Oncorhynchus mykiss and Oncorhynchus tshawytscha; these primers and assays were cross-tested in both species.

Nei's (1972) genetic distance were estimated among 21 populations of the common freshwater shrimp Paratya compressa improvisa, living in the northern part of Japan. The estimates were based on examination of 18 enzyme loci by starch-gel electrophoresis. These shrimp populations were genetically classified into three geographic groups on the basis of predominating alleles typical for two loci Fdp-1 and Fdp-2. The "Kanto group" consisted of individuals having Fdp-1a and Fdp-2c, the "Japan Sea group" having Fdp-1b and Fdp-2a, and the "Pacific Ocean group" having Fdp-1b and Fdp-2c. Nei's genetic distance among the three different geographic groups, varied from 0.0572 to 0.2136. Average genetic distances were 0.1001 between Pacific Ocean and Japan Sea groups, 0.0851 between Pacific Ocean and Kanto groups, and 0.1990 between Japan Sea and Kanto groups. These values of genetic distance among the three groups are comparable with those among different subspecies.

In order to estimate genetic differences and variability among and within cultured stocks of the masu salmon complex, restricition-fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) were performed for three masu salmon (Oncorhynchus masou masou) stocks and one amago salmon (O. m. macrostomus) stock. Based on our RFLP analysis using six 6-base recognized restriction endonucleases (BamHI, BglII, EcoRV, Hincll, HindIII, and XbaI), we concluded that: 1) the total size of mtDNA was about 17.3±0.5Kbp, 2) thirteen mtDNA haplo-types were observed among 59 individuals of the 4 stocks, 3) major haplotypes were different from each other and stock-specific haplotypes were observed in three out of four stocks, 4) the nucleotide diversity within the non-selected stocks ranged from 0.00170 to 0.00253, while no variation was observed in selected Amago-parr stock. The results suggest that the Amago-parr stock consists of only one maternal line while the other stocks consist of several maternal lines.

In order to estimate the degree of genetic divergence between two Paratya subspecies, Paratya compressa compressa and P. c. improvisa, which are common freshwater shrimp in Japan, isozymic analysis was carried out by starch gel electorophoresis. Eighteen loci coding for 15 enzymes were scored in both subspecies. Of the 18 loci, 7 divergent loci with no common allele were observed between the two subspecies. Nei's genetic distance between the subspecies was 0.597. Average heterozygosity was 0.044 in P. c. compressa but 0.069 in P. c. improvisa, indicating a difference in genetic variability between the two subspecies. These results indicate that the two subspecies are genetically divergent. The number of divergent loci and genetic similarities pointed to useful in-dices for genetic divergence between closely related species in the process of speciation.

The complete mitochondrial genome of Zhikong scallop Chlamys farreri is 21,695 bp in length and contains 12 protein-coding genes (the atp8 gene is absent, as in most bivalves), 2 ribosomal RNA genes, and 22 transfer RNA genes. The heavy strand has an overall A+T content of 58.7%. GC and AT skews for the mt genome of C. farreri are 0.337 and -0.184, respectively, indicating the nucleotide bias against C and A. The mitochondrial gene order of C. farreri differs drastically from the scallops Argopecten irradians, Mimachlamys nobilis and Placopecten magellanicus, which belong to the same family Pectinidae. 6623 bp non-coding nucleotides exist intergenically in the mitogenome of C. farreri, with a large continuous sequence (4763 bp) between tRNA ( Val ) and tRNA ( Asn ). Two repeat families are found in the large continuous sequence, which seems to be a common feature of scallops. Phylogenetic analysis based on 12 concatenated amino acid sequences of protein-coding genes supports the monophyly of Pectinidae and paraphyletic Pteriomorphia with respect to Heteroconchia.

Relationship between fitness advantage of isozymic genotype and a state of genetic constitution in a stock was examined in cultured stocks of rainbow trout in Japan. A number of stocks exhibited a larger average standard length in heterozygouse individuals under a state of “heterozygote excess” stocks at the Idh-3, Pgm-2 and Sod, significantly. However, an additive effect of advantage of heterozygotes at the three loci could not be observed. It sug-gests a fitness advantage of individuals heterozygous at each locus in cultured stocks of rainbow trout. On the other hand, a few stocks exhibited a larger standard length in homo-zygous individuals under a state of “homozygote excess” at the three loci, suggesting a fitness advantage of homozygous individuals. The mechanism of such phenomena could be explained by the relationship between the state of genetic constitution and quantity of dereterious genes in such a stack. An appearance of a larger average standard length in heterozygotes than in homozygotes could be explained by the resulting of a low fitness in homozygote individuals including many deleterious genes in the stocks. An appearance of larger average standard length in homozygotes than in heterozygotes could be explained by the elimination of deleterious genes during the process of inbreeding

The aim of this study was to quantitatively analyze a pattern of proliferation of gonial cells and to demonstrate neural involvement in spermatogonial proliferation of the scallop by the in vitro experiment. Immunocytochemistry for incorporated BrdU was used to identify mitotically active gonial cells. The pattern of proliferation of gonial cells was divided into two phases: phase I; oogonia and spermatogonia slowly proliferate through the growing stage: phase II; oogonia develop into oocytes and spermatogonia start to proliferate rapidly from the mature stage through the spawning stage. The neurons detected with anti-mammalian (m)GnRH antibody were distributed sparsely in the pedal ganglion and predominantly in the cerebral ganglion of both sexes at the growing stage. The extracts from the cerebral and pedal ganglion (CPG) of both sexes collected at the growing stage promoted proliferation of spermatogonia in the in vitro culture of the testicular tissue as well as mGnRH. However, CPG extract had no effect on oogonial proliferation. The increased mitotic activity induced by CPG and mGnRH was abolished by the addition of mGnRH antagonists and anti-mGnRH antibody, suggesting that the spermatogonial proliferation is regulated by GnRH-like peptide in CPG of the scallop. The same mitotic activity as CPG extract and mGnRH was observed in the hemocyte lysate, but not in the serum. These findings suggest that the spermatogonial proliferation at phase II in the scallop may be under the neuroendocrine control by GnRH neuron in CPG.

The genetic relationship among the three color variants (Red, Green, and Black) of the Japanese sea cucumber, S. japonicus, was investigated using 11 microsatellite markers. Genetic differentiation testing among the three sympatric color types showed the strong heterogeneity of Red (p < 0.001), while no significant difference was observed between Green and Black (p = 0.301 to 0.961). UPGMA trees constructed from 10 sample lots from 5 localities showed two distinct clusters, one from the Red types and the other from the Green and Black types. In addition, the sympatric Green and Black formed one subcluster with strong bootstrap support at each locality. These results indicate the separate species status of Red and the other color types, and also support the population identity of sympatric Green and Black.

Androgenesis, development from paternal but not maternal chromosomes, can be induced in some organisms including fish, but has not been induced previously in mollusk. In this study we investigated the induction of haploid androgenesis in the Pacific oyster by ultraviolet irradiation and observed nuclear behavior in the androgenetic eggs. Irradiation for 90 seconds at a UV intensity of 72 erg/mm2 per second (6480 erg/mm2) was the optimal dose to achieve haploid androgenesis. The fertilization and development rates of D-shaped larvae decreased with increasing exposure time, and the development of the genetically inactivated eggs terminated before reaching the D-shaped stage. Cytologic observations showed that UV irradiation did not affect germinal vesicle breakdown or chromosomal condensation but caused various nuclear behavioral patterns during meiosis and first mitosis: 21.7% of eggs extruded all maternal chromosomes as 2 or 3 polar bodies, and 59.1% of eggs formed one female pronucleus. The maternally derived nucleus did not participate, or partially participated, in the first karyokinesis. The cytologic evidence demonstrates that the male genome is directing development in haploids produced by UV irradiation.

Twenty microsatellite markers were first developed from the Japanese sea cucumber Stichopus japonicus using an enrichment protocol. Of the 20 microsatellite loci, 19 loci were polymorphic in the population examined. At these polymorphic loci, the number of alleles per locus varied from 2 to 15, and the observed heterozygosities ranged from 0.03 to 0.97, which is considerably higher than those previously found for allozymes. The high variability of the microsatellite markers identified in this study will make them excellent tools for genetic analyses of S. japonicus.

A complicated issue of very variable shell morphology in dogwhelks has led to a detailed discussion and set of complex analyses based on the methods of biochemical genetics, molecular genetics, and morphometrics. In this investigation, 29 allozyme loci and five morphometric shell characters were analysed for six samples of Nucella heyseana (Dunker, 1882) from Sakhalin Island, Primorye (Russia) and Onagawa Bay (Japan). An unweighted pair group method with arithmetic mean dendrogram was constructed using genetic distances for the six populations of N. heyseana sampled, and when combined with an earlier study of N. freycinetti Deshayes, 1841, indicated that two separate species are present. However, the population genetic analysis in combination with multivariate analysis of variance, discriminant and factor analyses for morphometric traits lead us to conclude that all six samples taken from Russian and Japanese waters belong to a single species. The taxon, known in Japanese and Korean literature as N. freycinetti, is actually N. heyseana and N. freycinetti is the appropriate name for another species discovered earlier. For N. heyseana Nei’s minimal unbiased genetic distances were examined at three hierarchical levels: (1) within Onagawa Bay (Pacific coast of Honshu), Dm = 0.0059 ± 0.0056; (2) within the group of populations in Peter the Great Bay (Japan Sea, Russia), Dm = 0.0083 ± 0.0067 (Kartavtsev et al., 2000), and (3) within the whole area investigated in the north-western Pacific, Dm = 0.1550 ± 0.0209. Allele frequency heterogeneity and hierarchical variability showed that each bay contains a genetically distinct population of this species.