Takashi Asahida

Identification of consumed stone flounder, Kareius bicoloratus (Basilewsky), from the stomach contents of sand shrimp, Crangon affinis (De Haan) using mitochondrial DNA analysis

Abstract We have developed a method for the identification of consumed stone flounder, Kareius bicoloratus (Basilewsky) larvae and juveniles from the stomach contents of sand shrimp, Crangon affinis (De Haan). We succeeded in isolating stone flounder DNA from the stomach contents of sand shrimp, using a cell lysis buffer containing 8 M urea. DNA for PCR analysis was obtained from stomach contents even when it was isolated 5 hours after the termination of predation. However, the total DNA yield decreased as the time after predation progressed. Stone flounder was distinguished from other prey in the stomach contents of sand shrimp using PCR with universal primers for fish and restriction analysis of mtDNA. Successful amplification of stone flounder mtDNA was possible up until 4 h after the termination of predation, but no amplification was possible 5 h after the termination of predation. In addition, a part of the D-loop region of stone flounder mtDNA was cloned, sequenced, and used to design species-specific PCR primers that allow amplification for stone flounder. PCR products were not obtained from the total DNA of other possible prey of the sand shrimp: 11 fish species; 2 mysid species; one amphipod species; and 2 polychaete species. In this paper, we discuss the advantages and disadvantages of using the PCR method coupled with restriction analysis, and the species-specific PCR method to identify consumed larval and juvenile fish species in the guts of predators.

A ‘living fossil’ eel (Anguilliformes: Protanguillidae, fam. nov.) from an undersea cave in Palau

We report the discovery of an enigmatic, small eel-like fish from a 35 m-deep fringing-reef cave in the western Pacific Ocean Republic of Palau that exhibits an unusual suite of morphological characters. Many of these uniquely characterize the Recent members of the 19 families comprising the elopomorph order Anguilliformes, the true eels. Others are found among anguilliforms only in the Cretaceous fossils, and still others are primitive with respect to both Recent and fossil eels. Thus, morphological evidence explicitly places it as the most basal lineage (i.e. the sister group of extant anguilliforms). Phylogenetic analysis and divergence time estimation based on whole mitogenome sequences from various actinopterygians, including representatives of all eel families, demonstrate that this fish represents one of the most basal, independent lineages of the true eels, with a long evolutionary history comparable to that of the entire Anguilliformes (approx. 200 Myr). Such a long, independent evolutionary history dating back to the early Mesozoic and a retention of primitive morphological features (e.g. the presence of a premaxilla, metapterygoid, free symplectic, gill rakers, pseudobranch and distinct caudal fin rays) warrant recognition of this species as a ‘living fossil’ of the true eels, herein described as Protanguilla palau genus et species nov. in the new family Protanguillidae.

Species composition and dynamics of larval and juvenile fishes in the surf zone of Mauritius

The importance of the surf zone as a nursery ground for larval and juvenile fishes has been widely recognized, however the zone has yet to be studied in Mauritius. Recently, the coastal area of the island has been increasingly affected by human activities, especially by tourism. We collected fish samples with a hand pulled seine net during the period of August 2001 to March 2003 to clarify the fish fauna and the dynamics of fishes in the surf zone. Two sampling sites adjacent to river mouth areas and one sampling site adjacent to a mangrove area were selected for comparison of fish fauna in relation to environmental conditions. A total of 9,429 fish larvae and juveniles, representing at least 112 species from 48 families were collected. The abundant species were hardyhead silverside, Atherinomoruslacunosus, bluespot mullet, Valamugil seheli, and Ambassis spp., each contributing 16.2, 12.4, and 11.8% of the total number of individuals, respectively. Estuarine species dominated in the surf zone adjacent to the river mouth areas. Species composition and diversity changed seasonally. The number of fish increased during the rainy season. Species diversity increased at the turn of the seasons from the dry season to the rainy season. We conclude that species composition in each site was affected by environmental factors, such as the scale of the flux from the rivers, which is related to the precipitation. The results indicated that freshwater from the river is a trigger to aggregate larvae and juveniles in the surf zone.

Inhibiting roles of melanin-concentrating hormone for skin pigment dispersion in barfin flounder, Verasper moseri.

Barfin flounders change their surface color pattern to match their background. We have reported evidence of the association between hormones and body color changes in this fish. First, bolus intraperitoneal injection with melanin-concentrating hormone (MCH) immediately turned the skin color pale, while injection with melanocyte-stimulating hormone (MSH) did not change the skin color. Second, gene expression levels of MCH change in response to background color, while those of MSH do not. We also reported the expression of an MCH receptor gene (Mch-r2) in the skin of this fish. In this study, we aimed to further evaluate the roles of MCH in skin color change. First, long-term adaptation of adult barfin flounder to black or white background colors induced significantly different pigment migration patterns in both melanophores and xanthophores (P<0.05). However, continuous intraperitoneal injection with MCH did not influence chromatophore proliferation. Then, using in vitro experiments, we found that MCH aggregates both melanophores and xanthophores, and inhibits the pigment-dispersing activity of MSH in a similar manner. Finally, we identified transcripts of Mch-r2 in cells isolated from both melanophores and xanthophores. Taken together, the evidence suggests that MCH aggregates pigments via MCH-R2 in concert with the nervous system by overcoming the melanin-dispersing activities of MSH in barfin flounder.

Genetic population evaluation of two closely related flatfish species, the rare barfin flounder and spotted halibut, along the Japanese coast

Barfin flounder and spotted halibut have been selected as target species for stock enhancement in Japan. Understanding the genetic condition of the wild stock is a principal requirement in any stock enhancement program. The genetic variability of barfin flounder and spotted halibut, and the population structure of spotted halibut were evaluated using microsatellite DNA markers (msDNA) and the control region of the mitocondrial DNA (mtDNA). Barfin flounder and spotted halibut showed high genetic variability at the msDNA level. Barfin flounder A was 16.7 and He was 0.860; spotted halibut An ranged from 7.7 to 10.2 and He ranged from 0.710 to 0.774. At the mtDNA level, high haplotype (h=0.922) and low nucleotide (π=0.002) diversities were observed for barfin flounder; however, low haplotype and nucleotide diversities (h=0.603–0.620 and π=0.001–0.002), and very low haplotype and nucleotide diversities (h=0.193 and π=0.0003) were observed for spotted halibut in the north and south locations, respectively. Slight genetic differentiation among spotted halibut sampling locations was observed from the msDNA. MtDNA analyses showed genetic differentiation between north and south locations, but not within them. The designation of north-specific and south-specific management units in the future stock enhancement activities of spotted halibut is recommended.

Use of microsatellite locus flanking regions for phylogenetic analysis? A preliminary study of Sebastes subgenera

The systematics of the species-rich genus of Sebastes rockfish has not been resolved, and is unlikely to be resolved using morphological criteria. Using an alternative approach based on DNA sequence variation, we sampled the genome for random, presumably neutral, nuclear DNA sequences, by sequencing microsatellite flanking regions of 15 Sebastes species (representing 15 of 22 extant subgenera) and Hozukius emblemarius. We aligned sequences of flanking regions of eight of the 13 loci that amplified, which presumably are homologous. In aggregate, the aligned sequences included 848 bp, 53 bp of which were polymorphic. The base changes among species included 27 transitions, 20 transversions, three multiple substitutions, and three deletions or insertions. The flanking regions at different loci had different base substitution rates. Nucleotide divergence among Sebastes species ranged from 0.0012 to 0.0216, whereas nucleotide divergence between Sebastes and Hozukius species ranged from 0.0095 to 0.0240.We used maximum parsimony, neighbor-joining, and maximum likelihood methodstoexamine relationships among the species. H. emblemarius formed a separate group, and fiveof the western Pacific Ocean species ofrockfish clustered together, suggesting that they may have shared an evolutionary history distinct from many North American species. The flanking regions of some microsatellite loci contain DNA sequence variation that distinguishes rockfish species and may prove useful for clarifying relationships among rockfish, or other closely related species.

Stock structure of Japanese flounder inferred from morphological and genetic analyses

Stock structure of Japanese flounder Paralichthys olivaceus has been inferred mainly from either morphological or genetic analyses. However, because the results of both analyses did not always agree with each other, an inclusive conclusion has never been obtained. In this study, the stock structure has been inferred from both morphological and genetic analyses using 722 wild Japanese flounder collected from nine locations along the Japanese coast. The dorsal and anal fin ray counts were larger in the southern than in the northern populations. In total, 1041 bp of mitochondrial NADH dehydrogenase subunit-2 (ND2) and 1830 bp of ND5 sequences were aligned. There are 578 variable sites in the concatenated sequence from the two genes, which defined a total of 490 haplotypes. Both results of morphological and genetic analyses indicated that the western Kyushu group, which included the Nagasaki and Kagoshima populations, was divided from the other seven populations. This is the first report to reveal the heterogeneity of the western Kyushu group based on statistical analysis.

Larval and juvenile development of Lestidiops sphyraenopsis Hubbs, 1916 (Aulopiformes: Paralepididae) in the western North Pacific, with a reexamination of the holotype of Stemonosudis molesta (Marshall, 1955)

The early life stages of Lestidiops sphyraenopsis (Paralepididae) are described on the basis of 14 specimens [7.8 mm in notochord length (NL)–88.6 mm in standard length (SL)] collected from the western North Pacific, and the holotype of Stemonosudis molesta is reexamined. Larval L. sphyraenopsis occurred in the Kuroshio waters, and juveniles were taken in the Kuroshio–Oyashio transition waters. Diagnostic characters of larval and juvenile L. sphyraenopsis are 96–101 myomeres; 27–31 anal fin rays; 4–9 peritoneal pigment sections in larvae (7.8 mm NL–27.3 mm SL); dorsal and anal pigment patches present; and anus located anterior to a vertical through dorsal fin origin. Stemonosudis molesta, known only from the holotype from the South Pacific, is similar to immature specimens of L. sphyraenopsis, but can be clearly distinguished from the latter by having higher vertebral counts (105 vs. 96–101) and by morphometric and pigment differences. Consequently, S. molesta is a valid species, and the distribution of L. sphyraenopsis is restricted to the North Pacific.