Zoological Science

Ciliated and microvillar olfactory receptor cells have been reported in many fish species, including teleosts and elasmobranchs. Morphological studies have suggested that microvillar cells are the only olfactory receptor cells in the elasmobranchs; however, there is no direct evidence for this hypothesis. Here we used a cat shark (Scyliorhinus torazame) to determine the cell type of the olfactory receptor cells in elasmobranchs. Retrograde labeling with a fluorescent dye, Dil, labeled only cells in the second layer from the surface of the olfactory epithelium, suggesting that ciliated cells located in the surface layer are not olfactory receptor cells. In addition, electron microscopic observation revealed that the labeled cells in the second layer have a thin dendritic knob extending from the cell body to the free surface of the epithelium. A part of the dendritic knob facing the mucous layer did not have ciliary structures. These results provide evidence that the aciliate cells are the only olfactory receptor cells in the cat shark olfactory organ.

Circadial spawning times of medusa of the bivalve-inhabiting hydrozoans Eugymnanthea japonica Kubota and Eutima japonica Uchida are confirmed to be morning (possibly sunrise) for the former species and night for the latter. Eugymnanthea, with small, short-lived, univoltine medusae, seems to have evolved from a form similar to Eutima japonica, with larger, longer-lived, multivoltine medusae; the morning spawning of medusae in Eugymnanthea may therefore be a newly evolved trait. Medusa release from polyps and spawning of medusae are not synchronous in Eugymnanthea japonica. This non-synchrony may represent an evolutionarily transitional state leading to the most advanced state, synchrony of these two reproductive events, as in certain other ephemeral hydrozoan medusae.

The jungle crow Corvus macrorhynchos Wagler, 1827, and the carrion crow Corvus corone L., 1758, are two closely related species with similar ecological requirements that occupy wide distribution ranges in the Palearctic. We studied patterns of their genetic variation by using sequences of the mitochondrial cytochrome b gene. Corvus macrorhynchos demonstrates a low level of variation and differentiation throughout its range, except for a highly diverged population of Cheju Island (Korea). The haplotype network shows two haplogroups. The island group comprises populations of Sakhalin, Hokkaido, Honshu, and Kyushu, while the haplotypes of Taiwan and Ryukyu Islands proved to be closer to the mainland group, which also includes populations from the Primorye, Khabarovsk, Amur, and Magadan regions in the Russian Far East. This pattern allowed us to develop a phylogeographic hypothesis regarding the two modes of settling of the island populations. Concerning C. corone, the presence of two distinct haplogroups was confirmed within the range of C. c. orientalis. Both haplogroups are found within the same populations in Kamchatka and North Sakhalin, which implies secondary contacts there. Populations of C. corone are found to be rather stable in the western parts of its range, while in the Far East populations experienced recent growth, as was observed for C. macrorhynchos in general. The two species appear to have passed through different evolutionary scenarios.

Large-scale systematic patterns of body size are a basic concern of evolutionary biology. Identifying body size variation along altitudinal gradients may help us to understand the evolution of life history of animals. In this study, we investigated altitudinal variation in body size, age and growth rate in Chinese endemic frog, Pelophylax pleuraden. Data sampled from five populations covering an altitudinal span of 1413 to 1935 m in Sichuan province revealed that body size from five populations did not co-vary with altitudes, not following Bergmann's rule. Average adult SVL differed significantly among populations in males, but not in females. For both sexes, average adult age differed significantly among populations. Post-metamorphic growth rate did not co-vary with altitude, and females grew faster than males in all populations. When controlling the effect of age, body size did not differ among populations in both sexes, suggesting that age did not affect variation in body size among populations. For females, there may be other factors, such as the allocation of energy between growth and reproduction, that eliminated the effect of age on body size. To our minds, the major reason of body size variation among populations in male frogs may be related to individual longevity. Our findings also suggest that factors other than age and growth rate may contribute to size differences among populations.

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E₂ (PGE₂) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE₂ (10^-7 and 10^-6 M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE₂ (10^-9 to 10^-6 M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE₂(10^-7 to 10^-6 M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE₂ treated-scales. Thus, PGE₂ acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE₂-injencted goldfish increased significantly. We conclude that, in teleosts, PGE₂ activates both osteoblasts and osteoclasts and participates in calcium metabolism.

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E2 (PGE2) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE2 (10^-7 and 10^-6 M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE2 (10^-9 to 10^-6 M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE2(10^-7 to 10^-6 M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE2 treated-scales. Thus, PGE2 acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE2-injencted goldfish increased significantly. We conclude that, in teleosts, PGE2 activates both osteoblasts and osteoclasts and participates in calcium metabolism.

Many species of Dytiscus diving beetles exhibit intrasexual dimorphism, e.g., the elytra is smooth in some females and grooved in others. However, the expression of the grooves and whether they are a product of heredity or the environment remain unknown. One Japanese species, Dytiscus sharpi sharpi Wehncke, 1875 , also shows female dimorphism, with grooved and smooth morphs, while D. sharpi validus Régimbart, 1899, only has a single morph (the grooved type). A hybrid of the two species should therefore provide a means of sorting out how the grooves are inherited. We found two independent wetlands of D. sharpi sharpi in Chiba Prefecture, Japan. One was a place where a high proportion of grooved females lived, and the others had high proportions of smooth females. After five to eight generations of beetles from two populations with different proportions of grooved females were reared under aquarium conditions constituting a common garden design, i.e., water temperature, water depth, and presence of a plant for oviposition, the differences remained. We mated smooth virgin females of D. sharpi sharpi with males of D. sharpi validus to obtain hybrid offspring. The elytral traits of the hybrid females produced only grooved forms. These results suggested that the female dimorphism is determined by genetics, and that the grooved morph was dominant over the smooth one, independent of environmental factors. In addition, the hybrid insects did not differ from the two subspecies insects in larval survivorship, pupation success, or sex ratio. They also showed neither morphological abnormality nor reduced survival.

Many species of Dytiscus diving beetles exhibit intrasexual dimorphism, e.g., the elytra is smooth in some females and grooved in others. However, the expression of the grooves and whether they are a product of heredity or the environment remain unknown. One Japanese species, Dytiscus sharpi sharpi Wehncke, 1875, also shows female dimorphism, with grooved and smooth morphs, while D. sharpi validus Régimbart, 1899, only has a single morph (the grooved type). A hybrid of the two species should therefore provide a means of sorting out how the grooves are inherited. We found two independent wetlands of D. sharpi sharpi in Chiba Prefecture, Japan. One was a place where a high proportion of grooved females lived, and the others had high proportions of smooth females. After five to eight generations of beetles from two populations with different proportions of grooved females were reared under aquarium conditions constituting a common garden design, i.e., water temperature, water depth, and presence of a plant for oviposition, the differences remained. We mated smooth virgin females of D. sharpi sharpi with males of D. sharpi validus to obtain hybrid offspring. The elytral traits of the hybrid females produced only grooved forms. These results suggested that the female dimorphism is determined by genetics, and that the grooved morph was dominant over the smooth one, independent of environmental factors. In addition, the hybrid insects did not differ from the two subspecies insects in larval survivorship, pupation success, or sex ratio. They also showed neither morphological abnormality nor reduced survival.

Scleractinian corals are of fundamental ecological significance in tropical and sub-tropical shallow water. This ecological success is attributed to their ability of formation of obligate endosymbioses with dinoflagellates of the genus Symbiodinium. Nevertheless, approximately one-third of reef-building coral species are critically endangered and the remainder are under threat from the effects of climate change and local impacts. Molecular and cellular mechanisms involved in stress responses and the establishment and collapse of the symbiosis are therefore an urgent subject of research. Metazoans possess large numbers of genes that participate in response to environmental stressors, and chemical defense genes included P450 and other oxidases, various conjugating enzymes, ATP-dependent efflux transporters, oxidative detoxification proteins, as well as transcription factors that regulate these genes. Here we searched those genes in recently decoded the coral Acropora digitifera genome. We found that this genome contains a set of chemical defense genes in numbers comparable with other cnidarians and metazoans and that there are some lineagespecific gene family expansions in the coral genome. These provide information for future research into molecular mechanisms involved in coral stress responses.

We reconstructed the demographic history of the plateau pika (Ochotona curzoniae) on the Qinghai-Tibetan plateau by using genetic variation data obtained from spatially distributed populations across much of the plateau. We obtained sequence data, including cob (1,140 bp) and D-loop sequences (732 bp), from 144 individuals at sites ranging from the high-altitude interior to the relatively low-altitude northeastern plateau, and identified 37 and 42 unique haplotypes, respectively. Phylogenetic analysis and haplotype networks based on the individual and the combined datasets of cob and D-loop sequences clustered all populations into four well-supported major groups, and the interaction between vicariance, dispersal, and habitat fragmentation resulted in the current geographical distribution and genetic diversity of O. curzoniae on the plateau. Analysis of molecular variance (AMOVA) and mismatch distribution suggested that the recent continuous uplifts of the plateau contributed to the radiation and diversification of the O. curzoniae populations occurring there.

To clarify the taxonomic status of the Burmoniscus ocellatus complex in Japan, we carried out morphological observations and phylogenetic analyses of specimens collected from Yonagunijima, Iriomotejima, Ishigakijima, and Miyakojima Islands of the southern Ryukyus and from Okinawajima Island of the central Ryukyus in southwestern Japan. Observations of the holotypes of Aphiloscia iriomotensis (Nunomura, 1986), Ap. ishigakiensis (Nunomura, 1986), and Ap. yonakuniensis (Nunomura, 1986), in addition to the specimens newly collected from the five islands, indicated that these specimens belong to the genus Burmoniscus. Analyses of five morphological characters of 268 specimens collected from the five islands showed that the body size of Okinawajima specimens is distinctly smaller than those of the specimens from the southern Ryukyus. The ranges of the five morphological characters tended to overlap among the specimens from Yonagunijima, Iriomotejima, Ishigakijima and Miyakojima Islands; these morphological characters were congruent with those of 6. ocellatus (Verhoeff, 1928). The phylogenetic analyses were based on three regions of mitochondrial DNA—COI, 12S rRNA, and 16S rRNA—and indicated that the specimens collected from the southern Ryukyus constitute a monophyletic group, which is clearly distinct from the clade composed of the Okinawajima specimens. These results strongly suggest that Ap. iriomotensis, Ap. ishigakiensis, and Ap. yonakuniensis are synonymous with B. ocellatus, a species widely distributed in the southern Ryukyus. On the other hand, the species from Okinawajima Island in the central Ryukyus is considered to be an undescribed Burmoniscus species, which is closely related to but clearly distinct from S. ocellatus.

To clarify the taxonomic status of the Burmoniscus ocellatus complex in Japan, we carried out morphological observations and phylogenetic analyses of specimens collected from Yonagunijima, Iriomotejima, Ishigakijima, and Miyakojima Islands of the southern Ryukyus and from Okinawajima Island of the central Ryukyus in southwestern Japan. Observations of the holotypes of Aphiloscia iriomotensis ( Nunomura, 1986 ), Ap. ishigakiensis ( Nunomura, 1986 ), and Ap. yonakuniensis ( Nunomura, 1986 ), in addition to the specimens newly collected from the five islands, indicated that these specimens belong to the genus Burmoniscus. Analyses of five morphological characters of 268 specimens collected from the five islands showed that the body size of Okinawajima specimens is distinctly smaller than those of the specimens from the southern Ryukyus. The ranges of the five morphological characters tended to overlap among the specimens from Yonagunijima, Iriomotejima, Ishigakijima and Miyakojima Islands; these morphological characters were congruent with those of 6. ocellatus (Verhoeff, 1928). The phylogenetic analyses were based on three regions of mitochondrial DNA—COI, 12S rRNA, and 16S rRNA—and indicated that the specimens collected from the southern Ryukyus constitute a monophyletic group, which is clearly distinct from the clade composed of the Okinawajima specimens. These results strongly suggest that Ap. iriomotensis, Ap. ishigakiensis, and Ap. yonakuniensis are synonymous with B. ocellatus, a species widely distributed in the southern Ryukyus. On the other hand, the species from Okinawajima Island in the central Ryukyus is considered to be an undescribed Burmoniscus species, which is closely related to but clearly distinct from S. ocellatus.

The eel goby, genus Taenioides (Gobiidae: Amblyopinae), inhabits muddy bottoms of estuaries or shallow areas of seas in the Indo-Pacific Ocean. Among congeners, T. cirratus (Blyth, 1860) has been thought to be distributed in Japan, but taxonomic confusions remain as to which scientific names are applicable to Japanese Taenioides species, or more fundamentally, how many Taenioides species are distributed in Japan, due in part to the rarity of this group in museum collections and the morphological similarity among species. To clarify the species diversity of the genus Taenioides in Japan, we conducted phylogenetic analysis on the basis of partial mitochondrial DNA sequences and morphological observation of more than 100 specimens. As a result, four distinct species were distinguished from each other, on the basis of both genetic divergences (2.9–5.7%, 16S rRNA gene) and morphological differences (i.e., degree of development of dermal folds on the head, numbers of barbels and vertebrae). Although the identifications of four species need additional verifications, they were identified as T. anguillaris, T. snyderi, T. gracilis and T. cf. kentalleni, and the species name T. cirratus does not seem to be appropriate to any of four detected species. Museum collections indicate that the two species, which are distributed in the main islands of Japan, were collected frequently and treated as a single species. The other two were each collected only from a single locality of Okinawa Island in this study, of which one seems to be uncommon worldwide as well.

The eel goby, genus Taenioides (Gobiidae: Amblyopinae), inhabits muddy bottoms of estuaries or shallow areas of seas in the Indo-Pacific Ocean. Among congeners, T. cirratus ( Blyth, 1860 ) has been thought to be distributed in Japan, but taxonomic confusions remain as to which scientific names are applicable to Japanese Taenioides species, or more fundamentally, how many Taenioides species are distributed in Japan, due in part to the rarity of this group in museum collections and the morphological similarity among species. To clarify the species diversity of the genus Taenioides in Japan, we conducted phylogenetic analysis on the basis of partial mitochondrial DNA sequences and morphological observation of more than 100 specimens. As a result, four distinct species were distinguished from each other, on the basis of both genetic divergences (2.9–5.7%, 16S rRNA gene) and morphological differences (i.e., degree of development of dermal folds on the head, numbers of barbels and vertebrae). Although the identifications of four species need additional verifications, they were identified as T. anguillaris, T. snyderi, T. gracilis and T. cf. kentalleni, and the species name T. cirratus does not seem to be appropriate to any of four detected species. Museum collections indicate that the two species, which are distributed in the main islands of Japan, were collected frequently and treated as a single species. The other two were each collected only from a single locality of Okinawa Island in this study, of which one seems to be uncommon worldwide as well.