Genetic divergences and hybridization within the Sebastes inermis complex.

The Sebastes inermis complex includes three sympatric species (Sebastes cheni, viz Sebastes inermis, and Sebastes ventricosus) with clear ecomorphological differences, albeit incomplete reproductive isolation. The presence of putative morphological hybrids (PMH) with plausibly higher fitness than the parent species indicates the need to confirm whether hybridization occurs within the complex. In this sense, we assessed the dynamics of genetic divergence and hybridization within the species complex using a panel of 10 microsatellite loci, and sequences of the mitochondrial control region (D-loop) and the intron-free rhodopsin (RH1) gene. The analyses revealed the presence of three distinct genetic clusters, large genetic distances using D-loop sequences, and distinctive mutations within the RH1 gene. These results are consistent with the descriptions of the three species. Two microsatellite loci had signatures of divergent selection, indicating that they are linked to genomic regions that are crucial for speciation. Furthermore, nonsynonymous mutations within the RH1 gene detected in S. cheni and "Kumano" (a PMH) suggest dissimilar adaptations related to visual perception in dim-light environments. The presence of individuals with admixed ancestry between two species confirmed hybridization. The presence of nonsynonymous mutations within the RH1 gene and the admixed ancestry of the "Kumano" morphotype highlight the potential role of hybridization in generating novelties within the species complex. We discuss possible outcomes of hybridization within the species complex, considering hybrid fitness and assortative mating. Overall, our findings indicate that the genetic divergence of each species is maintained in the presence of hybridization, as expected in a scenario of speciation-with-gene-flow.

Redescription and molecular characterization of Mothocya parvostis Bruce, 1986 (Crustacea: Isopoda: Cymothoidae) parasitic on Japanese halfbeak, Hyporhamphus sajori (Temminck & Schlegel, 1846) (Hemiramphidae) with Mothocya sajori Bruce, 1986 placed into synonymy.

Two species of Mothocya have previously been recorded from Hyporhamphus sajori: M. parvostis Bruce, 1986 and M. sajori Bruce, 1986. Mothocya parvostis is re-described based on the ovigerous female type and additional materials collected from the host from in and around the type locality. Morphological re-examination of fresh specimens and the type materials together with genetic data show that the M. sajori and M. parvostis are the same species, differing primarily in size, therefore we have placed Mothocya sajori Bruce, 1986 into a junior synonym of Mothocya parvostis Bruce, 1986. Mothocya parvostis is characterized by the following combinations of characters: 1) body slightly to moderately twisted to one side; 2) pereonite 7 posterior margin moderately to deeply recessed; 3) uropodal rami extending to pleotelson posterior margin; and 4) uropod rami bluntly rounded, exopod 1.5 times as long as peduncle. The differences of four morphological features for M. parvostis and M. sajori was quantified. Furthermore, a total of 635 isopods infesting H. sajori were collected from all over Japan to conduct quantitative morphological and molecular sequence analyses (mitochondrial cytochrome c oxidase subunit I and 16S rRNA). Although the four quantitative features did not overlap between the two species in type specimens, all quantitative morphological values of newly collected specimens in this study did not display a bimodal distribution. In addition, our molecular analyses found only a single clade for our newly collected specimens in neighbor-joining tree.

Molecular and Morphological Characterizations of the Fish Parasitic Isopod Mothocya parvostis (Crustacea: Cymothoidae) Parasitizing Optional Intermediate Hosts: Juveniles of the Cobaltcap Silverside Hypoatherina tsurugae and Yellowfin Seabream Acanthopagrus latus.

Mothocya parvostis (Isopoda: Cymothoidae) is a parasitic crustacean that infests the opercular cavities of fishes. Its main final host is the Japanese halfbeak, Hyporhamphus sajori. However, M. parvostis also infests the black sea bream, Acanthopagrus schelgelii, as an optional intermediate host. Understanding the use of optional intermediate hosts is important for understanding the life history of Cymothoidae, and further information should be obtained. In this study, we aim to investigate the life cycle of M. parvostis. We collected and examined 20 mancae and 144 juveniles of M. parvostis from 129 cobaltcap silversides, Hypoatherina tsurugae, and 494 yellowfin seabreams, Acanthopagrus latus. Molecular analysis of the cytochrome c oxidase subunit I gene and 16S rRNA genes revealed that cymothoid mancae and juveniles from the two fish species were identified to be M. parvostis. All M. parvostis on H. tsurugae and A. latus might be mancae or juveniles, with no adult parasites; thus, H. tsurugae and A. latus juveniles were optional intermediate hosts of M. parvostis. In the results of morphological description, M. parvostis juveniles infesting the final host H. sajori lacked swimming setae, while juveniles parasitizing the two optional intermediate hosts had them. Mothocya parvostis mancae infested juveniles of both species just after metamorphosis, grew with the host. As the fish grows further, the parasite detached from the fish. The parasitic status of M. parvostis in the three optional intermediate hosts indicated that M. parvostis likely reproduced from June to December, and different optional intermediate hosts were used depending on the time of year in Hiroshima Bay. Therefore, a parasitic strategy involving optional intermediate hosts might increase the infestation success of M. parvostis to H. sajori.

Quatrefoil light traps for free-swimming stages of cymothoid parasitic isopods and seasonal variation in their species compositions in the Seto Inland Sea, Japan.

Cymothoid parasitic isopods infest a wide range of fish of different taxa living in marine, brackish, and freshwater environments. Most research on the reproductive season of Cymothoidae has been done by collecting or monitoring host fish afflicted with cymothoid parasites. However, collecting ecological data on cymothoid species that infest non-commercial or endangered fishes is complex and challenging. We used a quatrefoil light trap to investigate the seasonal change in species composition of cymothoid free-swimming stages in the Seto Inland Sea, Japan. We also collected preliminary data for efficient light-trap sampling and showed its effectiveness in cymothoid-related research. From October 2020 to December 2021, 613 cymothoid free-swimming stages were sampled monthly. All obtained individuals were identified as Mothocya parvostis (596), Ceratothoa verrucosa (12), and Ceratothoa carinata (5) by DNA barcoding using cytochrome c oxidase subunit I and 16S rRNA gene sequences. Based on the number of M. parvostis mancae collected each month, M. parvostis was anticipated to reproduce from June to December, with two reproduction peaks each year, and C. verrucosa and C. carinata were expected to reproduce in June, July, and September, and September and October, respectively. In addition, free-swimming juveniles were captured, presumably after they had left their optional intermediate hosts. Furthermore, the most effective time to harvest cymothoids with light traps may be during high tide on the night of the new moon. This study serves as a methodological framework for future research on cymothoids using light traps.

Spawning time of black sea bream Acanthopagrus schlegelii, related to underwater photoperiodism in oyster farms.

The embryonic development times, spawning timing and hatching rates of the black sea bream Acanthopagrus schlegelii were examined to investigate the potential effect of seawater temperature, tides and photoperiod on the reproductive rhythm of this species in Hiroshima Bay, Japan. Low temperatures decreased hatching rates and extended the hatching time, and the minimum temperature threshold for hatching was 15°C. Back-calculated spawning times indicated that the peak of spawning occurred just before sunset and the reduction in diurnal light intensity around the oyster rafts acted as a trigger for spawning. In contrast, no correlation was found between spawning rhythms and tidal cycle. The results highlight the important role of oyster farms in the reproductive cycle and population dynamics of A. schlegelii in Hiroshima Bay, the main spawning ground for this species in Japan. The study findings provide insights for the sustainable management of this important sparid species.

The complete mitochondrial genome of Acanthopagrus pacificus (Perciformes, Sparidae) from Iriomotejima Island, Okinawa, Japan.

The Pacific seabream, Acanthopagrus pacificus, is a commercially valuable resource for fisheries around the West Pacific Ocean countries. Although the phylogenetic position of the A. pacificus within the genus Acanthopagrus changes by the differences in the target gene regions or fish sampling locations, the genetic information of the species is quite limited. In this study, the complete mitochondrial genome of A. pacificus from Iriomotejima Island, Okinawa, Japan was determined. The sequence is 16,640 bp in length and consists of 13 protein-coding genes, 2 rRNA genes (12S rRNA and 16S rRNA), 22 tRNA genes, and one control region. A reconstructed phylogenetic tree showed that A. pacificus has the furthest relationship to other Acanthopagrus species in the genus.

Oyster farms are the main spawning grounds of the black sea bream Acanthopagrus schlegelii in Hiroshima Bay, Japan.

Understanding the anthropogenic impact of oyster farms is essential for the management and conservation of marine fishes. In Japan, Hiroshima Bay is the region with the most intense oyster farming and thus suitable to study the impact of these farms. Here, we surveyed spherical planktonic eggs of the black sea bream Acanthopagrus schlegelii, one of the most abundant fish in the Bay. Our survey was performed at fourteen stations which included places with oyster farms and historical spawning grounds. We found the highest egg densities in four stations, one with historical spawning aggregations and three with major oyster farms. Besides, surveys at the innermost part of Hiroshima Bay, where two major rivers discharge, showed a low density of eggs indicating that black sea bream avoids spawning in low salinity areas. Our study suggests that oyster farms benefit spawners of black sea bream by providing more food sources than historical spawning grounds for efficient spawning. Yet, whether oyster farms represent a full advantage for the species remains unclear, particularly because they are known to host jellyfishes that prey on eggs and limit water flow that can influence the survival of fish eggs.

The role of the isolation of the marginal seas during the Pleistocene in the genetic structure of black sea bream Acanthopagrus schlegelii (Bleeker, 1854) in the coastal waters of Japan.

The black sea bream Acanthopagrus schlegelii (Bleeker, 1854) is a commercially important species in Japanese waters. Assessing its population structure is essential to ensure its sustainability. In the Northwestern Pacific, historical glacial and interglacial periods during the Pleistocene have shaped the population structure of many coastal marine fishes. However, whether these events affected the population of black sea bream remains unknown. To test this hypothesis and to assess the population structure of black sea bream, we used 1,046 sequences of the mitochondrial control region from individuals collected throughout almost the entire Japanese coastal waters and combined them with 118 sequences from populations distributed in other marginal seas of the Northwestern Pacific Ocean. As in other coastal marine fish with similar distribution, we also found evidence that the glacial refugia on the marginal seas prompted the formation of three lineages in black sea bream. These lineages present signatures of population growth that coincided with the interglacial periods of the Pleistocene. While the origin of Lineages B and C remains unclear, the higher relative frequency of Lineage A in the southernmost location suggests its origin in the South China Sea. The non-significant pairwise ΦST and AMOVA of Japanese populations and the presence of these three lineages mixed in Japanese waters; strongly suggest that these lineages are homogenized in both the Sea of Japan and the Pacific Ocean. Our results indicate that the black sea bream should be managed as a single stock in Japan until the strength of connectivity in contemporary populations is further addressed using non-coding nuclear markers.

Infestation of the Parasitic Isopod Mothocya parvostis on Juveniles of the Black Sea Bream Acanthopagrus schlegelii as an Optional Intermediate Host in Hiroshima Bay.

In Hiroshima Bay, parasitic isopods of the genus Mothocya infest the black sea bream Acanthopagrus schlegelii (Bleeker, 1854) and the Japanese halfbeak Hyporhamphus sajori (Temminck and Schlegel, 1846), two fish species that are abundant and commercially important in the Seto Inland Sea of Japan. Immature and mature Mothocya individuals can infect both juveniles and adults of H. sajori, while immature Mothocya are known to parasitize juveniles of A. schlegelii; i.e., no Mothocya parasites are found in adult A. schlegelii. The identification of the immature Mothocya parasitizing juveniles of A. schlegelii remains uncertain, because Mothocya species are morphologically identifiable only based on adult females. Also, the biological/ecological relationship between the hosts and parasites has not been studied. Here, we identified the parasites on A. schlegelii as Mothocya parvostis Bruce, 1986 by molecular sequence analyses along with other parasites obtained from H. sajori, the latter being morphologically confirmed by comparison with paratype materials of M. parvostis as well as the similar congener Mothocya sajori Bruce, 1986. The growth rates of the infected A. schlegelii juveniles from June to September in the years 2013-2015 and 2018 were significantly lower than those of the uninfected ones, suggesting a negative effect of the infection on the hosts. Our data on the prevalence and duration of the infection, as well as the body size gain of the hosts and parasites, corroborate a hypothesis that M. parvostis would utilize A. schlegelii as an optional intermediate host before it reaches the final host, H. sajori.

Genus-level phylogeny of cephalopods using molecular markers: current status and problematic areas.

Comprising more than 800 extant species, the class Cephalopoda (octopuses, squid, cuttlefish, and nautiluses) is a fascinating group of marine conchiferan mollusks. Recently, the first cephalopod genome (of Octopus bimaculoides) was published, providing a genomic framework, which will enable more detailed investigations of cephalopod characteristics, including developmental, morphological, and behavioural traits. Meanwhile, a robust phylogeny of the members of the subclass Coleoidea (octopuses, squid, cuttlefishes) is crucial for comparative and evolutionary studies aiming to investigate the group's traits and innovations, but such a phylogeny has proven very challenging to obtain. Here, we present the results of phylogenetic inference at the genus level using mitochondrial and nuclear marker sequences available from public databases. Topologies are presented which show support for (1) the monophyly of the two main superorders, Octobrachia and Decabrachia, and (2) some of the interrelationships at the family level. We have mapped morphological characters onto the tree and conducted molecular dating analyses, obtaining congruent results with previous estimates of divergence in major lineages. Our study also identifies unresolved phylogenetic relationships within the cephalopod phylogeny and insufficient taxonomic sampling among squids excluding the Loliginidae in the Decabrachia and within the Order Cirromorphida in the Octobrachia. Genomic and transcriptomic resources should enable resolution of these issues in the relatively near future. We provide our alignment as an open access resource, to allow other researchers to reconstruct phylogenetic trees upon this work in the future.

Individual identification of goldfish from eye morphology: the eye mark method.

We developed an individual identification method for goldfish based on morphological variation of the iris. Each goldfish has a few dark lines (eye marks) in the rostral and caudal portion of the iris, which are blood vessels underneath the silvery reflective layer. Through the blood vessels and the locally thin reflective layer, the pigment cell layer is partially visible as a dark line. The pattern of the blood vessels was found to be temporally stable and unique to each individual. Using this feature, we successfully identified 10 individual goldfish, each sampled three times within a 4-month time period. The eye mark identification method was confirmed for a further 20 goldfish by comparison with identification based on screening at polymorphic microsatellite loci. The eye mark method is 100% accurate and can be used for studies in which multiple observations of individually identified goldfish are needed over long time periods, to avoid invasive tagging or individual housing that may affect the behavior of the fish.

Eleven novel polymorphic microsatellite Loci for oval squid Sepioteuthis lessoniana (Shiro-Ika type).

The oval squid Sepioteuthis lessoniana is one of the most economically important squid species in Japan; however, its population structure is poorly understood due to the lack of hypervariable markers. Such information is critical for managing sustainable fisheries, as well as for ensuring the existence of wild S. lessoniana stocks. Eleven candidate microsatellite loci were isolated from a small insert genomic DNA library. Polymorphisms in these 11 loci were screened in 24 wild individuals. The number of alleles per locus was found to range from 5 to 19 alleles, and the observed heterozygosity ranged from 0.292 to 0.958. No evidence for linkage disequilibrium was detected among all the loci. The genotypic proportions conformed to Hardy-Weinberg equilibrium, except at one locus. In conclusion, these polymorphic microsatellite loci may be used to develop a genetic framework to manage S. lessoniana in the future.

Comparisons of internal behavior after exposure to Flavobacterium psychrophilum between two ayu (Plecoglossus altivelis altivelis) strains showing different cumulative mortality to bacterial cold water disease.

Bacterial cold water disease (BCWD) in ayu (Plecoglossus altivelis altivelis) has a serious impact on aquaculture and fisheries. There is known to be a significant difference among ayu strains with regard to mortality caused by BCWD. In this study, the immune response of different ayu strains against Flavobacterium psychrophilum infection was observed. One strain was resistant to infection by F. psychrophilum, and the other was susceptible to infection by the same bacteria. The number of bacteria in the body was observed in each ayu strain, and the change in bacterial counts was similar. However, there was a significant difference in bacterial count in the spleen between the two strains on days 6, 9, 12 and 15 after exposure. To observe the immune response against F. psychrophilum, agglutination assay using serum was performed. An agglutination reaction in the resistant ayu strain was observed in 4 out of 6 ayu on day 6 after exposure, while no reactions in the susceptible ayu strain were observed in any sampled fish until day 12. However, some reactions in the susceptible ayu strain were observed in surviving ayu. These results indicate that there is a correlation between the presence of bacterial multiplication and agglutination reaction against F. psychrophilum.

Influence of host habitat on the occurrence of gill monogeneans Pseudodactylogyrus spp. on wild Japanese eels Anguilla japonica.

With technological improvements in otolith microchemical analysis, the flexible use of habitat from coastal marine to fresh waters has been discovered in Japanese eels Anguilla japonica. We examined the occurrence of 3 congeneric gill monogeneans-Pseudodactylogyrus anguillae, P. bini, and P. kamegaii-on wild Japanese eels, in relation to the host's flexibility. From April 2008 to October 2009, 114 eels were collected from a brackish-water cove and 2 rivers flowing into the cove in Ehime Prefecture, western Japan. Based on otolith microchemical analysis, the eels were discriminated according to the following 4 types of habitat use: freshwater residents (Type I), individuals utilizing low-salinity habitats (Type II), downstream habitat-shifters (Type III), and cove residents (Type IV). P. anguillae occurred mainly on Type I and II eels, while P. bini was primarily found on Type I eels. In contrast, P. kamegaii occurred mainly on Type III and IV eels. Thus, we conclude that species composition and infection levels of Pseudodactylogyrus spp. clearly differed with habitat-use patterns of Japanese eels. Also, since P. anguillae was scarcely found on either Type III or IV eels, this study suggests that previous identifications of monogeneans collected from European brackish-water localities as P. anguillae may require verification.

The effects of 2-bromopalmitate on the fatty acid composition in differentiating adipocytes of red sea bream (Pagrus major).

To determine whether external factors affect the adipogenic function of fish adipocytes, the effects of 2-bromopalmitate (a PPAR agonist) on the fatty acid composition in differentiating adipocytes of red sea bream were investigated in vitro. In the presence of 2-bromopalmitate, the red sea bream adipocytes were differentiated and the effects on the fatty acid composition and the adipogenic gene expression were analyzed. With the level of 2-bromopalmitate, the content of 16:1n-7, a delta-9 desaturation product, increased in association with the increase in a stearoyl CoA desaturase (SCD) gene expression level while the triglyceride accumulation was not affected. Subsequently, the effects on the bioconversion of the n-3 and n-6 fatty acids, which are main series of dietary essential fatty acids, were examined. In the presence of 300 microM of 18:3n-3 or 18:2n-6, red sea bream stromal-vascular cells accumulated the lipid in the cytoplasm within 3 days by the fatty acid uptake with the increase of corresponding fatty acid contents. Furthermore, in both the 18:3n-3 and 18:2n-6 stored cells, the products of delta-6 desaturation (18:4n-3 and 18:3n-6, respectively) and C(18-20) elongation (20:3n-3 and 20:2n-6, respectively) were detected. However, neither the delta-6 desatutration nor C(18-20) elongation of 18:3n-3 and 18:2n-6 were enhanced by 2-bromopalmitate treatment. In conclusion, the results indicate that the adipocyte function in fish, e.g. adipogenic gene expression and fatty acid composition, can be modified by external factors and a main effect of 2-bromopalmitate is the increase in the content of delta-9 desaturation product by stimulating the SCD gene expression.

Molecular characterization of peroxisome proliferator-activated receptors (PPARs) and their gene expression in the differentiating adipocytes of red sea bream Pagrus major.

To investigate the molecular mechanism of fish adipocyte differentiation, the three subtypes of PPAR genes (alpha, beta and gamma) were characterized in a marine teleost red sea bream (Pagrus major). The primary structures of red sea bream PPARs exhibited high degrees of similarities to their mammalian counterparts, and their gene expression was detected in various tissues including adipose tissue, heart and hepatopancreas. During the differentiation of primary cultured red sea bream adipocytes, three PPARs showed distinct expression patterns: The alpha subtype showed a transient increase and the beta gene expression tended to increase during adipocyte differentiation whereas the gene expression level of PPARgamma did not change. These results suggest that they play distinct roles in adipocyte differentiation in red sea bream. In the differentiating red sea bream adipocytes, mammalian PPAR agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2), ciglitazone and fenofibrate did not show clear effects on the adipogenic gene expression. However, 2-bromopalmitate increased the PPARgamma and related adipogenic gene expression levels, suggesting the gamma subtype plays a central role in red sea bream adipocyte differentiation and in addition, fatty acid metabolites can be used as modulators of adipocyte function. Thus our study highlighted the roles of PPARs in fish adipocyte differentiation and provided information on the molecular mechanisms of fish adipocyte development.

Molecular characterization of lipoprotein lipase, hepatic lipase and pancreatic lipase genes: effects of fasting and refeeding on their gene expression in red sea bream Pagrus major.

To investigate the nutritional regulation of lipid metabolism in fish, molecular characterization of lipases was conducted in red sea bream Pagrus major, and the effects of fasting and refeeding on their gene expression was examined. Together with data from a previous study, a total of four lipase genes were identified and characterized as lipoprotein lipase (LPL), hepatic lipase (HL) and pancreatic lipase (PL). These four lipase genes, termed LPL1, LPL2, HL and PL, share a high degree of similarity. LPL1 and LPL2 genes were expressed in various tissues including adipose tissue, gill, heart and hepatopancreas. HL gene was exclusively expressed in hepatopancreas. PL gene expression was detected in hepatopancreas and adipose tissue. Red sea bream LPL1 and LPL2 gene expression levels in hepatopancreas were increased during 48 h of fasting and decreased after refeeding, whereas no significant change in the expression levels of LPL1 and LPL2 was observed in adipose tissue, indicating that LPL1 and LPL2 gene expression is regulated in a tissue-specific manner in response to the nutritional state of fish. HL and PL gene expression was not affected by fasting and refeeding. The results of this study suggested that LPL, HL and PL gene expression is under different regulatory mechanisms in red sea bream with respect to the tissue-specificities and their nutritional regulation.

Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major.

Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.