Ayu or sweetfish, Plecoglossus altivelis, an amphidromous fish ranging in the northwestern Pacific, is economically important inland fisheries and aquaculture resources. Genetic characterization of wild Ayu and derived culture seeds with competent molecular genetic markers is still insufficient for their sustainable use. Microsatellite DNA markers with larger repeat motifs (e.g. tri- and tetra-nucleotide motifs) are convenient and accurate compared with those having mono- and di-nucleotide motifs, but the latter motifs characterized most Ayu microsatellite markers developed previously. Here, we isolated and characterized 17 polymorphic microsatellite DNA markers with tri- and tetra-nucleotide repeat motif using next-generation sequencing. Alleles per locus varied from 6 to 23. The observed and expected heterozygosities ranged from 0.542 to 1.000 and 0.709 to 0.951, respectively. Polymorphic information content (PIC) of 15 out of the 17 loci were high (≧ 0.700), suggesting them to be highly informative. Twelve of the 17 loci were used for preliminary assignment test among three collections, and successfully allocated the examined fish to the original populations. The novel polymorphic microsatellite markers developed herein will be useful to examine the genetic diversity and population structure of wild Ayu and the effect of seed transplantation on native populations, providing a tool for conservation and sustainable adaptive management of this species.

In marine aquaculture, fish populations constantly decrease throughout the cultivation period because of mortality and escape. Current production management systems provide limited opportunities to count the cultured fish, making it difficult to estimate accurately the fish population in the cage. To overcome this problem, an automatic fish identifying method based on particle tracking velocimetry (PTV) flow visualization technology is proposed in this paper. The proposed method utilizes an image processing unit that extracts individual fish from the acquired image and a motion analysis unit that calculates the motion vector for each individual. Thus, the accuracy of the extraction results in the image processing unit affects the system?s counting results. To validate the efficiency and robustness of the image extraction performed by the image processing unit, individuals were extracted from images using the open-source image deep learning semantic segmentation method (SegNet), which is able to distinguish between the background and foreground in the images via analysis at the pixel level. SegNet is able to improve the image discrimination performance by multiplying the learning paths, and the robustness of the detection results can be ensured by changing the layer structure according to the detection target. Accordingly, the use of SegNet was evaluated in terms of the number of layers and images in the training set. The results of this study indicate that the application of SegNet with PTV technology represents a promising method for the automatic identifying and behavioral tracking of fish in an aquaculture net cage.

Using nucleotide sequence variations found in the 5' half of the control region of the mitochondrial (mt) DNA, genetic diversity of chum (Oncorhynchus keta) and masu salmon (O. masou) was compared to get an insight into the specific difference, if any, in the genetic population structure of these two Pacific salmon species with different lifetime characteristics. Polymerase chain reaction (PCR)-based direct sequence analysis was successfully performed with the primers, tRNAthr-2 and tRNAphe-2, originally designed for chum salmon (Sato et al. 2001) and other sequencing primers for each species. Occasional occurrence of larger PCR products than regular-sized amplified fragments of about 1.0 kb were found in each population of masu salmon, whereas no size polymorphisms of PCR products were observed in chum salmon which constantly showed the regular PCR fragments. The occurrence of such irregular-sized fragments in masu salmon ranged from 3.3% to 12.5% in each population examined. Sequence analysis with primers of tRNApro-3, Okdl-L1, Omdl-H1 (designed for masu salmon), and tRNAphe-2 revealed that the irregular-sized PCR fragments were due to the occurrence of one to five repeats of an 80 bp unit in the 3' portion of the control region. Except for the observed sequence repeats, the structure of masu salmon control region and its flanking gene order were in keeping with the previous findings (Shedlock et al. 1992).

Two types of Berardius are recognised by local whalers in Hokkaido, Japan. The first is the ordinary Baird’s beaked whale, B . bairdii , whereas the other is much smaller and entirely black. Previous molecular phylogenetic analyses revealed that the black type is one recognisable taxonomic unit within the Berardius clade but is distinct from the two known Berardius species. To determine the characteristics of the black type, we summarised external morphology and skull osteometric data obtained from four individuals, which included three individuals from Hokkaido and one additional individual from the United States National Museum of Natural History collection. The whales differed from all of their congeners by having the following unique characters: a substantially smaller body size of physically mature individuals, proportionately shorter beak, and darker body colour. Thus, we conclude that the whales are a third Berardius species.

James E. Seeb1, Richard L. Wilmot2, Shigehiko Urawa3, Syuiti Abe4, Lisa W. Seeb1, and Christian T. Smith1 1Alaska Department of Fish and Game, 333 Raspberry Road, Anchorage, Alaska 99518, USA 2U.S. Department of Commerce, NOAA, NMFS, Alaska Fisheries Science Center, Auke Bay Laboratory, 11305 Glacier Highway, Juneau, Alaska 99801, USA 3National Salmon Resources Center, 2-2 Nakanoshima, Toyohira-ku, Sapporo, Hokkaido 062-0922, Japan 4Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041-8611, Japan

Journal of Applied IchthyologyVolume 33, Issue 6 p. 1204-1207 SHORT COMMUNICATION Development and characterization of 13 polymorphic microsatellite DNA markers for pink salmon (Oncorhynchus gorbuscha) using next-generation sequencing approach H. Tsukagoshi, Corresponding Author H. Tsukagoshi tsuk@iwate-u.ac.jp orcid.org/0000-0003-2601-805X Sanriku Fisheries Research Center, Iwate University, Kamaishi, Iwate, Japan Correspondence Hideharu Tsukagoshi, Sanriku Fisheries Research Center, Iwate University, Kamaishi, Japan. Email: tsuk@iwate-u.ac.jpSearch for more papers by this authorS. Terui, S. Terui Sanriku Fisheries Research Center, Iwate University, Kamaishi, Iwate, JapanSearch for more papers by this authorS. Sato, S. Sato Hokkaido National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Sapporo, Hokkaido, JapanSearch for more papers by this authorS. Abe, S. Abe Sanriku Fisheries Research Center, Iwate University, Kamaishi, Iwate, JapanSearch for more papers by this author H. Tsukagoshi, Corresponding Author H. Tsukagoshi tsuk@iwate-u.ac.jp orcid.org/0000-0003-2601-805X Sanriku Fisheries Research Center, Iwate University, Kamaishi, Iwate, Japan Correspondence Hideharu Tsukagoshi, Sanriku Fisheries Research Center, Iwate University, Kamaishi, Japan. Email: tsuk@iwate-u.ac.jpSearch for more papers by this authorS. Terui, S. Terui Sanriku Fisheries Research Center, Iwate University, Kamaishi, Iwate, JapanSearch for more papers by this authorS. Sato, S. Sato Hokkaido National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Sapporo, Hokkaido, JapanSearch for more papers by this authorS. Abe, S. Abe Sanriku Fisheries Research Center, Iwate University, Kamaishi, Iwate, JapanSearch for more papers by this author First published: 25 July 2017 https://doi.org/10.1111/jai.13436Citations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume33, Issue6December 2017Pages 1204-1207 RelatedInformation

Because escape from a net cage and mortality are constant problems in fish farming, health control and management of facilities are important in aquaculture. In particular, the development of an accurate fish counting system has been strongly desired for the Pacific Bluefin tuna farming industry owing to the high market value of these fish. The current fish counting method, which involves human counting, results in poor accuracy; moreover, the method is cumbersome because the aquaculture net cage is so large that fish can only be counted when they move to another net cage. Therefore, we have developed an automated fish counting system by applying particle tracking velocimetry (PTV) analysis to a shoal of swimming fish inside a net cage. In essence, we treated the swimming fish as tracer particles and estimated the number of fish by analyzing the corresponding motion vectors. The proposed fish counting system comprises two main components: image processing and motion analysis, where the image-processing component abstracts the foreground and the motion analysis component traces the individual’s motion. In this study, we developed a Region Extraction and Centroid Computation (RECC) method and a Kalman filter and Chi-square (KC) test for the two main components. To evaluate the efficiency of our method, we constructed a closed system, placed an underwater video camera with a spherical curved lens at the bottom of the tank, and recorded a 360° view of a swimming school of Japanese rice fish (Oryzias latipes). Our study showed that almost all fish could be abstracted by the RECC method and the motion vectors could be calculated by the KC test. The recognition rate was approximately 90% when more than 180 individuals were observed within the frame of the video camera. These results suggest that the presented method has potential application as a fish counting system for industrial aquaculture.

Nucleotide sequence analysis in the first half of the mitochondrial control region was used to estimate genetic variation and population structure of marbled sole Pleuronectes yokohamae and cresthead flounder Pleuronectes schrenki, obtained from 10 localities in Japan. Overall estimate of pairwise population FST and NJ tree suggested genetic distinction between P. yokohamae and P. schrenki. SAMOVA also demonstrated two distinct genetic groups of the samples, MS (marbled sole, Honshu and southernmost Hokkaido) and CF(cresthead flounder, Hokkaido), which was pertinent to the natural range of P. yokohamae and P. schrenki, respectively. However, the haplotypes common to P. yokohamae and P. schrenki were found in all four samples of the CF group and two of six samples of the MS group. Haplotype and nucleotide diversities were high within MS and CF groups, and the mismatch distribution analysis showed a bimodal profile in both groups, suggesting secondary contact between allopatrically evolved MS and CF groups. Thus, the observed haplotype sharing between two species might have been the consequence of interspecific hybridization after their divergence. In addition, FST estimates and Mantel test suggested that the population structure of P. yokohamae might have been influenced by low or restricted gene flow with isolation by distance, whereas no such population structuring was implied for P. schrenki. The observed difference in population structuring between two species was surmised by the difference of migration character.

A total of 18 novel microsatellite markers in masu salmon, an important fisheries resource around the Far East, were isolated using a next-generation sequencing approach. The number of alleles and expected heterozygosity per locus ranged 5–15 and 0.55847–0.92944, respectively. Four loci also were polymorphic in three other salmonid species after cross-species amplification. These newly isolated loci will provide important information for the sustainable adaptive management of masu salmon.

The red-crowned crane in Japan was once considered extinct due to hunting and habitat destruction in late nineteenth century; however, in 1926, a small group of cranes was rediscovered in the Kushiro Mire in eastern Hokkaido. Since then, various conservation efforts, including artificial feeding during winter, hunting prohibition, and habitat conservation, have increased the population size to >1400 by 2012. Despite such a successful population recovery, the genetic characteristics of the population have not been fully explored. To ensure the long-term persistence and evolutionary potential of cranes, accurate knowledge of the spatial distribution of genetic variation and its underlying causes are necessary. We assessed their genetic structure using 12 polymorphic microsatellite loci and inferred the mechanisms shaping the observed structure. Among the three regional groups in Hokkaido, we found generally low pairwise F ST values and no significant differences in genetic diversity, probably because of the population expansion in the recent past. In contrast, spatial autocorrelation analysis revealed a significant positive kinship at the short distance (0–15 km) and negative kinship at the long distance (155–205 km), showing a pattern of isolation by distance. The presence of isolation by distance on a small spatial scale despite the species’ strong flight ability is probably explained by the recolonization process and restricted dispersal due to natal philopatry in a non-equilibrium condition. Cranes in Hokkaido do not appear to be a panmictic (random mating) population; however they can be considered a single population without genetic discontinuity (i.e. a single management unit). Our findings confirm the importance of considering natal philopatry when developing management strategies such as dispersing cranes into unoccupied areas.

The Neptune whelk, Neptunea arthritica, is a sublittoral snail from Pacific waters that is a food resource and supports a commercially important fishery in northern Japan. This species showed a severe decline during the 1970s and 1980s, possibly because of overfishing, imposex caused by tributyltin (TBT) pollution and parasite infection. In the present study, we investigated genetic variation among the populations of N. arthritica from eight localities in northern Japan, including Hokkaido and Aomori, using a mitochondrial DNA (mtDNA) marker, a partial sequence of the cytochrome c oxidase subunit I (COI) gene. We also addressed the evolutionary history of N. arthritica and human impact on the population genetic profiles of this species. The parsimony network showed 14 COI haplotypes separated into two groups (Groups A and B), with an intermediate haplotype connecting both groups. Among eight populations, six were fixed for only one or two haplotypes, and no geographic-genetic correlation was found; they were probably affected by random drift. These results contrasted with those from previous microsatellite analysis, which indicated that geographic structure was the result of restricted gene flow between populations. Our results suggested that N. arthritica diverged into Groups A and B during the Pliocene; however, recent TBT pollution and size-selective fishing pressure have reduced genetic diversity and concealed the natural population structure. The present study also suggested that human impact may cause longstanding and possibly irreversible modification of ecosystems, particularly for species forming discrete and relatively small local populations, such as N. arthritica. Thus, the combined use of mtDNA and microsatellite genetic data provides a powerful tool to investigate the health of biodiversity in molluscs.

Genetic relationships between homing anadromous and matured non-anadromous forms of masu salmon (Oncorhynchus masou) from one river of Hokkaido, Japan, and two rivers of Sakhalin, Russia, collected in 2001 to 2008 were examined using nucleotide sequence variation in the mitochondrial (mt) DNA NADH dehydrogenase subunit 5 gene (ND5) region and allelic polymorphisms at six microsatellite (ms) DNA loci. All the non-anadromous samples examined were genetic males identifi ed with GH-Y pseudogene. Signifi cant difference between anadromous and non-anadromous samples within the Shari and Sukhopletka Rivers was suggested with FST estimates and neighbor-joining trees using msDNA but not mtDNA marker. Analyses of mtDNA and msDNA also suggested that the genetic divergence among allopatric samples in Hokkaido and Sakhalin was larger than that between two life-history forms in the same river. The present fi ndings imply a possible genetic differentiation between the two life-history forms of masu salmon in the same river and distinct genetic structuring in the Far East.

Natural hybrids between the boreal species Hexagrammos octogrammus and two temperate species Hexagrammos agrammus and Hexagrammos otakii were observed frequently in southern Hokkaido, Japan. Previous studies revealed that H. octogrammus is a maternal ancestor of both hybrids; the hybrids are all fertile females and they frequently breed with paternal species. Although such rampant hybridization occurs, species boundaries have been maintained in the hybrid zone. Possible explanations for the absence of introgressions, despite the frequent backcrossing, might include clonal reproduction: parthenogenesis, gynogenesis and hybridogenesis. The natural hybrids produced haploid eggs that contained only the H. octogrammus genome (maternal ancestor) with discarded paternal genome and generated F1 -hybrid type offspring by fertilization with the haploid sperm of H. agrammus or H. otakii (paternal ancestor). This reproductive mode was found in an artificial backcross hybrid between the natural hybrid and a male of the paternal ancestor. These findings indicate that the natural hybrids adopt hybridogenesis with high possibility and produce successive generations through hybridogenesis by backcrossing with the paternal ancestor. These hybrids of Hexagrammos represent the first hybridogenetic system found from marine fishes that widely inhabit the North Pacific Ocean. In contrast with other hybridogenetic systems, these Hexagrammos hybrids coexist with all three ancestral species in the hybrid zone. The coexistence mechanism is also discussed.

The crucian carp Carassius carassius (Linnaeus, 1758) is a species with restricted and decreasing distribution in Europe. Six males and six females of the species from the Baltic Sea basin in Poland were examined to show sequentially CMA(3)/AgNO3 staining pattern, DAPI staining, and, for the first time in literature, molecular cytogenetic analysis using double-colour fluorescence in situ hybridisation (FISH) with 28S and 5S rDNA probes. The karyotype consisted of 20 m, 36 sm and 44 sta chromosomes, NF=156. The AgNO3 stained NORs were most frequently located terminally in the short arms of two sm and two sta elements, and CMA(3)-positive sites were also observed suggesting abundant GC-rich repetitive DNA in the regions. Other CMA(3)-positive sites in the short arms of six to ten sm and sta chromosomes were detected. The results based on 28S rDNA FISH confirmed the location of rDNA sites. DAPI-negative staining of NORs suggested the scarcity of AT-rich DNA in the regions. FISH with 5S rDNA probe revealed 8-14 loci (ten and 12 in respectively 49 and 29% of metaphases). They were located in two sm and eight to ten sta chromosomes and six of them were larger than others. Simultaneously, mapping of the two rDNA families on the chromosomes of C. carassius revealed that both 28S and 5S rDNA probes were located in different chromosomes. Molecular cytogenetic data of C. carassius presented here for the first time give an important insight into the structure of chromosomes of this polyploid and declining species and may be useful in its systematics.

The ploidy status of Acipenser mikadoi was examined using nuclear DNA contents, karyotypes and fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. In flow-cytometrically sorted specimens with 8.29.1 pg DNA content per somatic cell, i.e. genetic diploid, the best informative metaphase with 268 chromosomes had 80 biarmed meta- or submetacentric (M or SM) chromosomes, 48 monoarmed telocentric (T) chromosomes and 140 microchromosomes. In genetic triploid specimens with 12.613.0 pg DNA content, the best informative metaphase with 402 chromosomes showed 120 biarmed M or SM, 72 monoarmed T chromosomes and 210 microchromosomes. The rDNA FISH detected a maximum 18 and 27 signals in the diploid and triploid A. miakdoi, respectively. The obtained findings thus corroborated a clear parallel between nuclear DNA contents and karyological or FISH profiles in the genetic diploid and triploid specimens, suggesting 1.5 times chromosome complements of diploid counterparts or three sets of homologues in the triploid sturgeons. Moreover, the estimated genome size and the observed molecular cytogenetic features in the diploid A. mikadoi strongly suggest that this species is a member of a functional tetraploid group recently proposed in the literature.

When eggs from the Chinese tetraploid loach that had 100 chromosomes were fertilized with UV-irradiated sperm, we obtained viable gynogenetic progeny without any additional treatment for the duplication of maternal chromosomes, which survived beyond first feeding towards adult stage of development. Gynogenetic progeny were determined to be diploid since they possessed 50 chromosomes, along with two chromosomes bearing nucleolar organizing regions (NORs), detected by silver nitrate staining (Ag-NORs), chromomycin-A3 (CMA3)-positive sites and fluorescence in situ hybridization (FISH) signals for rDNA loci. In contrast, when gynogens were induced using eggs from diploid loach fertilized by UV-irradiated sperm, but without chromosome doubling, we found that all resultant progeny were non-viable haploid gynogens with 25 chromosomes, along with one NOR-bearing chromosome detected by Ag-NORs, CMA3 and FISH. These observations demonstrate the true genetic tetraploid nature of the Chinese loach possessing 100 chromosomes, and the potential use of this tetraploid as a source of functional diploid gametes for further ploidy manipulation experiments.

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Most males and females of intergeneric hybrid (BM) between female brook trout (Bt) Salvelinus fontinalis and male masu salmon (Ms) Oncorhynchus masou had undeveloped gonads, with abnormal germ cell development shown by histological examination. To understand the cause of this hybrid sterility, expression profiles of testicular proteins in the BM and parental species were examined with 2-DE coupled with MALDI-TOF/TOF MS. Compared with the parental species, more than 60% of differentially expressed protein spots were down-regulated in BM. A total of 16 up-regulated and 48 down-regulated proteins were identified in BM. Up-regulated were transferrin and other somatic cell-predominant proteins, whereas down-regulated were some germ cell-specific proteins such as DEAD box RNA helicase Vasa. Other pronouncedly down-regulated proteins included tubulins and heat shock proteins that are supposed to have roles in spermatogenesis. The present findings suggest direct association of the observed perturbation in protein expression with the failure of spermatogenesis and the sterility in the examined salmonid hybrids.