Tadashi Kitano

Stages of Embryonic Development of the Ice Goby (Shiro-uo), Leucopsarion petersii

Abstract A series of normal stages for the embryonic development of the ice goby (shiro-uo), Leucopsarion petersii, which belongs to the Perciformes, is described. Stages are based on morphological features, by utilizing the optical transparency of live embryos from the first cleavage to the hatching stage. Fertilized eggs were obtained by artificial insemination and normal embryogenesis was accomplished in a defined medium in plastic petri dishes at 19°C. Shiro-uo eggs were surrounded by a very thin and clear chorion and could be dechorionated with forceps very easily. Developmental stages were mostly comparable to those of other fish embryos described so far, but several differences were indicated, such as the third cleavage plane being horizontal, and that the length of the cleavage cycle increased gradually from the very early stages. Also, there were differences in the relative rates of organogenesis of the brain, eyes, otic vesicles, and somites when compared to the zebrafish and medaka.

Contrasting genetic structure of closely related giant water bugs: phylogeography of Appasus japonicus and Appasus major (Insecta: Heteroptera, Belostomatidae).

Appasus japonicus and A. major, two belostomatid species of the giant water bug found in parts of East Asia, have very similar morphological characteristics and ecological niches, and also overlapping habitats. However, the results of our previous published study utilizing molecular phylogenetic analyses of mitochondrial and nuclear DNA revealed extensive genetic differences, which indicated that the possibility of inter-specific hybridization was extremely unlikely. We collected A. japonicus and A. major from the Japanese Archipelago, Korean Peninsula, and Russian Far East, and conducted molecular analyses of mitochondrial DNA COI and 16S rRNA to compare phylogenetic relationships between these species. Three major clades were recognized within A. japonicus. Specimens from the Korean population constituted a monophyletic clade, and were a sister group of the western region of the Japanese Archipelago. The Eastern Japanese clade was clearly differentiated. Four major clades were recognized within A. major. Specimens from the Japanese and Korean populations revealed two distinct monophyletic clades. Significant differentiation was clearly observed between their genetic structures. Furthermore, the results of mismatch distribution and Bayesian skyline plot analyses suggested the possibility of a bottleneck effect or founder effect in two of the A. major clades. Collectively, these results demonstrated both similarities and differences in these two species even though their distribution widely overlaps in East Asia, with their morphological characteristics and ecological niches being very similar. These differences in genetic structures are considered to be due to their evolutionary history.

Noninvasive sampling of DNA from larval exuvia in diving beetles of the genera Cybister and Dytiscus

Nonlethal DNA sampling is a highly recommendable method in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from larvae of endangered diving beetles (Cybister brevis, C. lewisianus, C. limbatus, C. rugosus, Dytiscus sharpi sharpi and D. sharpi validus), we obtained the larval exuvia (molted skin) of these endangered diving beetles under laboratory conditions. A single exuvia 24 h after molting was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome‐c oxidase subunit I (COI), and the sequence of the COI gene could be determined directly. Sequences obtained from the exuvial samples were used to further find similarities within DDBJ/EMBL/GenBank. Genomic DNA from the samples was successfully isolated, and we identified the species. This process suggests that exuvia provides a good sample for extracting DNA from endangered diving beetle larvae without killing them.