Zoological Science

The population genetic structure and phylogeography of masu salmon were investigated by using variation in the mitochondrial NADH dehydrogenase subunit 5 gene (ND5) and six polymorphic microsatellite loci among a total of 895 fish representing 18 populations collected from Japan (9), Russia (7), and Korea (2) from 2000 to 2008. An analysis of ND5 nucleotide sequences revealed 22 variable sites in about 560 bp in the 5′ half of the gene, which defined 20 haplotypes, including some associated with geographical regions. Haplotype and nucleotide diversities were greater in the populations in Japan and Korea than in those in Russia, indicating greater genetic diversity in the Japanese and Korean populations than in the Russian populations. All the microsatellite loci examined showed a high level of variation, but the expected heterozygosity indicated a similar level of genetic diversity among the populations of the three regions, contrary to the results for ND5. However, AMOVA and pairwise population F _ST estimates for both ND5 and the microsatellite markers indicated a similar pattern of moderate genetic differentiation among populations of the three regions, and large population groups on the coasts of the Sea of Japan, Sea of Okhotsk, and Pacific Ocean in the Far East. From a mismatch distribution analysis and neutrality test, the observed genetic structure appears to have been influenced primarily by bottlenecks during glacial periods and population expansions during interglacial periods in the late Pleistocene.

Consensus on the taxonomic system and phylogenetic relationships for the anuran genus Fejervarya has yet to be established. Morphological characters in this genus are generally unsuitable for species identification. To carry out molecular species identification and solve phylogenetic problems, we collected 67 Fejervarya specimens from 12 Asian countries and sequenced part of the mitochondrial (mt) Cytb gene. We also sequenced the mt 12S and 16S rRNA genes and seven nuclear genes (BDNF, CXCR4, NCX1, RAG-1, RAG-2, Rhod, and Tyr) for 25 Fejervarya taxa. These molecular markers appear to be adequate for the identification of species. We subjected the molecular data molecular to phylogenetic analyses. In the resulting trees, topotypic F. limnocharis and “F. multistriata” (from China) formed a clade. On the other hand, neither “F. limnocharis” from the Japan mainland nor “F. limnocharis” from eastern Taiwan formed a clade with the real F. limnocharis, and the genetic divergences were larger than the species threshold for frog taxa proposed in previous studies (> 3% for 16S). These results may suggest that “F. multistriata” is a junior synonym of F. limnocharis, or that only some of the populations now recognized as “F. multistriata” correspond to F. limnocharis. Our results also suggest that several cryptic species may be included among the widely distributed Fejervarya species. Finally, our datasets support paraphyly for the genus Fejervarya, although alternative phylogenetic topologies, including Fejervarya monophyly, were not rejected by KH and SH tests.

We used field surveys and statistical models to investigate habitat associations of the endangered gold-spotted pond frog (Rana chosenica). The characteristics of its habitat are of great importance for effective conservation for this declining species in western South Korea. We evaluated a priori models that incorporated biotic and abiotic variables at the pond and landscape scales. The best-ranked model predicts that gold-spotted pond frogs will be more abundant at sites with fewer introduced American bullfrogs (Rana [= Lithobates] catesbeianus) and greater coverage of shallow, vegetated water. Our study leads us to conclude that limiting the spread and abundance of bullfrogs has the potential to aid conservation of the gold-spotted pond frog in our region.

From 5 March to 25 July 2008, ichthyoplankton drifting into the Three Gorges Reservoir from the upper reaches of the Yangtze River were sampled daily to investigate the species composition, abundance, and seasonal variation in early-stage fishes in this area. Twenty-eight species belonging to five orders and 17 families or subfamilies were identified by analyzing fish eggs and larvae, and a total of 14.16 billion individuals were estimated drifting through the sampling section during the investigation. Among the ichthyoplankton sampled, species in Cultrinae, Cobitidae, Gobioninae and Gobiidae, along with the common carp (Cyprinus carpio Linnaeus), comprised 89.6% of the total amount. Six peaks of drift density were identified during the sampling period, and a significant correlation was found between drift density with water discharge. The dominant species were different in each drift peak, indicating different spawning times for the major species. The total amount of the four major Chinese carps that drifted through the sampling section was estimated as 0.88 billion, indicating an increase in the population sizes of these species in the upper reaches of the Yangtze River after construction of the Three Gorges Dam. Actually, these reaches have become the largest spawning area for the four major Chinese carps in the Yangtze River. The large total amount of eggs and larvae drifting through this section demonstrated that the upper reaches of the Yangtze River provided important spawning sites for many fish species, and that conservation of this area should be of great concern.

Heat shock protein promoters (hsp promoters) are powerful tools for investigating gene functions, as the expression of targeted genes can be controlled simply by heating. However, there have been no reports of the utilization of an endogeneous medaka (Oryzias latipes) hsp promoter to induce exogenous gene expression in medaka. We identified and cloned a functional medaka hsp promoter (olphsp70.1) and verified its ability to act as an inducible promoter both in vitro and in vivo. The hsp promoter efficiently induced exogenous gene expression in cultured cells, developing embryos, and also in adult fishes. When used to control the expression of Venus, a variant of yellow fluorescent protein, in transgenic medaka, the hsp promoter was functional in all tissues except for the gonads of adults. These results indicate that the medaka hsp promoter can be a powerful tool for inducing exogenous gene expression and investigating gene functions both in vitro and in vivo in medaka.

The hard tissue of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the outer prismatic layer, bearing calcite, and the inner nacreous layer, bearing aragonite. An EDTA-insoluble fraction of the prismatic layer of P. fucata was extracted with urea. In-vitro crystallization experiments showed that this urea-soluble fraction contained the factor(s) that promoted the growth of calcite crystals. We purified a protein from this fraction and deduced the internal amino acid sequences EYDFDRPDPYDP and EYDFERPD. We performed 3′ RACE using primer DPPF1, encoding EYDFDRPDPYDP, and an oligo-dT adapter primer and amplified a fragment of approximately 300 bp. We screened cDNA libraries using the 300 bp fragment and obtained two clones that we named prismin 1 and 2. Both cDNAs encode proteins of 51 amino acids. Homology searches revealed 91% amino acid identity between prismin 1 and 2. The synthetic peptide DFDRPDPYDPYDRFD, corresponding to the carboxy terminal region of prismin 1, has calcite growing activity and calcium binding capability, showing that the carboxy-terminal region is a functional domain. Prismin 1 is expressed strongly in the outer edge and in the inner part of the mantle tissue. However, immunoblot analysis revealed that prismin protein exists only in the prismatic layer, not in the nacreous layer, despite the presence of the mRNA. Therefore, we conclude that prismin is a novel prismatic layer-specific calcite growth factor.

The streaked tenrec (Hemicentetes semispinosus) is equipped with a quill vibrating mechanism on the dorsal side of the caudal trunk that has evolved as an extraordinary sounding apparatus for communication. An arrangement of 15 or 16 light-brown quills was observed. Thickened cutaneous muscles were confirmed beneath quills. We named this structure the “quill vibrator disc” (QVD). The QVD was 16.8 mm long and 8.55 mm wide in a typical adult. Longitudinal musculature symmetrical about the sagittal plane was developed in the QVD. Myocytes were found immunohistochemically to contain mainly fast myosin but not slow myosin. These findings indicate that the QVD is a specialized apparatus in the cutaneous muscle that contributes to the vibration of quills and to the production of sound for communication.

Histochemical, lectin-histochemical, and immunohistochemical analyses were performed on parietal cells of the greater horseshoe bat, Rhinolophus ferrumequinum, to clarify the composition and distribution of oligosaccharide chains in the β-subunit of the protonic pump H⁺,K⁺-ATPase. PAS, Alcian Blue (pH 2.5) and Alcian Blue (pH 1.0) stainings detected only neutral glycoconjugates. Lectin-binding analyses included LTA, UEA-I, ConA, SBA, BSI-B4, AAA, DBA, PNA, and WGA. WGA-and PNA-bindings were also tested after β-elimination to detect O-linked glycans. Parietal cells were negative for binding to LTA and UEA-I, and to PNA and WGA after β-elimination, indicating the lack of (1,2) fucosylated residues and of N-linked glycans, respectively. Immunohistochemical tests with anti-α- and anti-β-H⁺,K⁺-ATPase were positive. Two alternative patterns of glycoconjugate distribution were found, i.e. a perinuclear and a diffuse one, indicating localization in the intracellular canaliculus and in the tubulovesicular system of the parietal cells, respectively. Both the subunits of the H⁺,K⁺-ATPase and the galactosyl/galactosaminyl residues were co-distributed in both the perinuclear and the diffuse patterns, suggesting that the residues are part of the protonic pump. Glycosyl/glycosaminyl and mannosyl groups were concentrated in the tubulovesicular system, and fucosylated residues were found almost exclusively in the intracellular canaliculi; thus they are probably not included in the oligosaccharide chains of β-H⁺,K⁺-ATPase. These findings indicate that the oligosaccharide chains linked to the β-H⁺,K⁺-ATPase subunit in R. ferrumequinum have distinct features compared to the other mammals studied and confirms the taxon specificity of the chains in the proton pump.

FMRFamide-gated Na⁺ channel (FaNaC) is a peptide-gated sodium channel in the epithelial Na⁺ channel/degenerin family. Although there are some data on the location of the putative peptide binding site, there is no structural information on the activation gating of FaNaC. Here, we addressed the function of a conserved aspartate residue in the second transmembrane domain of FaNaC. We used Aplysia kurodai FaNaC (AkFaNaC) and examined the function of the aspartate (D552) by site-directed mutagenesis and electrophysiological recording in Xenopus oocytes. We found that the macroscopic activation, desensitization, and potency of FMRFamide and its modification by external Ca²⁺ and Mg²⁺ are greatly affected by physicochemical properties of the amino acid at position 552. We conclude that D552 is situated in a key position that affects the gating properties of FaNaC.

Mus lepidoides of central Burma (Myanmar) was described 75 years ago but has since been dismissed as a regional variant of the Indian field mouse, M. booduga. DNA sequences of multiple mitochondrial and nuclear genes from recently collected specimens, combined with a fresh morphological reassessment, reaffirm the distinctiveness of M. lepidoides from M. booduga and from all other species of Mus. Mus lepidoides is so distinct in fact that it warrants placement in its own Species Group within subgenus Mus. Molecular and morphological assessments of phylogenetic affinities converge on the exciting possibility that M. lepidoides represents the previously elusive sibling taxon to the Mus musculus Species Group. If confirmed, this relationship would provide the previously missing connection between the main radiation of subgenus Mus in Southeast and South Asia, and the radiation of the M. musculus Species Group in western Asia and Europe. We speculate that a common ancestor of M. lepidoides and the M. musculus Species Group occupied a continuous but episodic tract of xeric habitat that linked central Burma with northern India at various times during the late Pliocene and Quaternary. Further molecular and cytogenetic studies on the phylogenetic position of M. lepidoides clearly represent a high priority in mouse research.