Zoological Science

Due to its tectonically uplifted, mountainous landscape and copious rainfall, the Japanese archipelago possesses plentiful, persistent groundwater in alluvial plains. However, Phreatodytes, an endemic water beetle genus found in groundwater, is now critically endangered due to the devastation of subterranean water ecosystems. Phreatodytes beetles were collected from driven wells near the Ooi River estuary in central Japan. The beetles were distinguished from all described species by their thoracic and genital morphologies, and described as a new species, Phreatodytes haibaraensis. The unique habitat of this species is the hyporheic zone of a gravelly riverbed in an estuary, and the habitat represents the eastern limit of the known distribution range of the genus. Molecular phylogenetic analysis based on mitochondrial and nuclear gene sequences revealed that Phreatodytes is a member of the Noteridae and forms a clade with Notomicrus. This suggests that the genus colonized the hyporheic zone from surface waters, and that it adapted to subterranean interstitial life by becoming minute and slender, by losing its eyes, pigmentation, as well as its ability to fly and swim, and by developing a permanent underwater life cycle, and erect hairlike setae on the thorax and elytra. In addition to the beetle, several stygobiontic species, including mollusks and fishes, were found in pumped groundwater. These results suggest that the gravelly riverbed of a rapid river estuary in Central Japan harbors biodiverse, locally differentiated hyporheic organisms comprising a unique subterranean interstitial ecosystem.

We conducted a molecular phylogeographic analysis of Aegialites beetles sampled on the coasts of Hokkaido, northern Japan, using a partial sequence (553 bp) of the mitochondrial ND2 gene. The beetles are flightless and are adapted to the supralittoral zone of rocky shores. In all, 249 individuals from 17 populations were included in the molecular analysis. Haplotypes of the Aegialites beetles studied comprised two distinct lineages with different distributional ranges: one (lineage A) on the coasts of Hokkaido facing the Sea of Japan and the Sea of Okhotsk, and the other (lineage B) mostly on the Pacific coast and on the Shiretoko Peninsula in northeastern Hokkaido. These haplotype lineages clearly indicate two groups of populations, one comprising lineage A haplotypes (group A) and the other lineage B haplotypes (group B). Only one population, at Taisei facing the Sea of Japan on the Oshima Peninsula, contained a mixture of haplotypes from the two lineages. Genetic divergence among populations was larger in group B than in group A. Despite the unidimensional nature of the beetles' habitat, no correlation was found between the degree of genetic differentiation and geographic distance in either group of populations. Population genetic parameters suggested that the two lineages originated in the Pliocene, and that the populations in group A underwent a rapid increase in population size, whereas those in group B did not. We present a scenario for the allopatric divergence of the two lineages.

Zoanthids (Cnidaria, Hexacorallia) of the genus Epizoanthus are often found in association with other marine invertebrates, including gastropods and hermit crabs. However, little information exists on the specificity and nature of these associations due to a lack of investigation into Epizoanthus species diversity, and the taxonomy of Epizoanthus is therefore confused. In this study, analyses of morphological data (tentacle number, polyp size, etc) and molecular data (mitochondrial cytochrome oxidase subunit 1 = COI, 16S ribosomal DNA = 16S rDNA) were used to examine Epizoanthus specimens from Tosa Bay, Kochi, Japan. The Epizoanthus specimens were found on both live gastropods (Gemmula unedo) and hermit crabs (Paguristes palythophilus) inhabiting G. unedo and G. cosmoi shells. While morphological analyses did not show clear differences between examined specimens, both COI and mt 16S rDNA clearly divided the specimens into two groups, one associated only with hermit crabs (= Epizoanthus sp. C), and another associated only with living gastropods (= Epizoanthus sp. S). Unexpectedly, DNA sequences from both groups did not match with two previously reported Epizoanthus species from Japan (E. indicus, E. ramosus), indicating they both may be undescribed species. These results highlight the utility of DNA “barcoding” of unknown zoanthids, and will provide a foundation for re-examinations of Epizoanthus species diversity and specificity, which will be critical in understanding the evolution of these unique marine invertebrates.

The terrestrial slug Lehmannia valentiana was first recorded in Japan in the late 1950s and is now distributed throughout the country. Previous studies have revealed that in Osaka, southwestern Japan, L. valentiana reproduces from November to April. In the present study, in order to clarify the climatic adaptations of L. valentiana in Japan, we examined the life cycle of this slug in Sapporo, northern Japan. In the Sapporo population, the ratio of gonad weight to body weight reached a maximum in September. Most slugs had mature sperm from late August to April and large oocytes from September to April. Thus, the Sapporo population of L. valentiana commenced reproduction two months earlier than the Osaka population. We also examined the effect of various photoperiodic conditions on growth and reproductive maturation in both the Osaka and Sapporo populations. The effect of photoperiod on growth was different in the two populations. In both populations, however, reproductive maturation was induced by short days of photophase 14 h or less, and there was no obvious difference between the two populations, even though reproductive maturation in the wild commences in different seasons. This indicates the possibility that L. valentiana adapts to climatically different regions without changes in its critical daylength in photoperiodic response.

The midgut epithelium of Acheta domesticus (Insecta, Orthoptera, Gryllidae), which is composed of columnar digestive cells and regenerative crypts, degenerates in two manners: necrotic and apoptotic. While necrosis was described in our previous paper, programmed cell death was the aim of the present studies. The first morphological signs of programmed cell death in midgut epithelium cells are alterations in the cytoplasm connected with shrinkage of the cells. Gradual modifications in a cell's structure cause it to be discharged into the midgut lumen, where it disintegrates. Autophagy is involved in the disintegration of organelles. The transitions of apoptotic cells are described at the ultrastructural level. Immunostaining methods were used in order to indicate the early stages of apoptosis when DNA fragmentation, which results from apoptotic signaling cascades, occurs.

We performed a phylogeographic analysis of the Japanese hare, Lepus brachyurus, using the mitochondrial cytochrome b gene (1140 bp). In total, 119 haplotypes were recovered from 197 samples isolated from 82 localities on three main islands of the Japanese archipelago: Honshu, Sikoku, Kyushu, Sado Island and the Oki Islands. Results showed two distinct clades at a genetic distance of 3.5%, equivalent to an estimated 1.2 million years. The two clades, encompassing seven subclades, showed an apparent geographic affinity to Kyushu, Shikoku and the nearby area of Honshu (southern group) by one clade, whereas the other clade covered the remaining area of Honshu (northern group). The landscape shape interpolation analysis exhibited a higher genetic diversity in the southern parts of central Honshu (northern group) and Shikoku and Kyushu regions (southern group), suggesting the existence of multiple geographical origins of population expansion in each clade. The Bayesian skyline plot analysis showed that lineage diversifications occurred about 0.35, 0.20 and 0.05 million years ago (Mya), which coincide closely with the glacial—interglacial cycles during the Pleistocene. Therefore, we suggest that the Japanese hare population once inhabited northern and southern refugia, and subsequently developed several populations through local demographic fluctuations. The present day demarcation in the northern and southern geographic groups is considered to be a temporal remnant of Pleistocene population dynamics and the geographic boundary between them could move or fade away in time.

Tektins are evolutionarily conserved filament-forming proteins localized in flagella and cilia, and have been reported to be involved in the stability and structural complexity of axonemal microtubules. Five mammalian Tektins (Tektin1–5) have been reported. Of these, Tektin2 (TEKT2) has been found to be required for normal flagellum structure and function. Tekt2-null sperm display flagellum bending and reduced motility, probably due to disruption of the dynein inner arm. However, the subcellular localization of TEKT2 in spermatozoa has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT2 in flagella of rat spermatozoa, we performed confocal laser scanning microscopy, extraction of flagella followed by immunoblot analysis, and immunogold electron microscopy. Extraction of sperm flagella by SDS-EDTA resulted in complete extraction of axonemal tubulins, while TEKT2 was only partially released from flagella, suggesting that TEKT2 might be present in the peri-axonemal component, not directly associated with axonemal tubulins. Confocal laser scanning microscopy and pre-embedding immunoelectron microscopy revealed that TEKT2 is associated with the surface of outer dense fibers (ODFs). TEKT2 may function as an ODF-affiliated molecule required for flagellum stability and sperm motility.

We sought to establish a new in vitro ovulation model using the whole ovaries of the medaka. Ovaries of the fish, which had been acclimated to the usual reproductive conditions (26°C, 14 h light/10 h dark) and which had then been kept at least one day at 30°C, were isolated 2 h before the expected in vivo ovulation time. When the ovaries were cultured in 90% medium 199 solution at 30°C or 36°C, oocytes were liberated with a gradual increase in the ovulation rate at 2 to 5 h of ovulation time. The maximum ovulation rate was ∼45%. Ovulated oocytes were fertilized and subsequently developed into adults. In vitro ovulation of medaka ovaries was inhibited by the addition of metalloproteinase inhibitors to the culture. In this in vitro ovulation model, the holes formed on the follicle layer upon follicle rupture at ovulation were sealed, strongly suggesting the importance of the germinal epithelium in the process. The present study indicates that our new in vitro ovulation model is useful for investigating the role of germinal epithelial cells in the ovulate process of the medaka fish.

Limnodrilus simplex sp. nov. (Oligochaeta: Naididae: Tubificinae) is described based on a single specimen from the mainstream of the Changjiang River near Anqing City, Anhui Province, China. The new species is assigned to Limnodrilus by the presence of long vasa deferentia, spindle-shaped atria with long ejaculatory ducts, large prostate glands, and thick cylindrical penial sheaths. It differs from its congeners in having simple-pointed chaetae and cuticularized penial sheaths without hoods. Limnodrilus simplex is closer to L. paramblysetus and L. amblysetus in possessing penial sheaths with relatively low length/maximum width ratio.

Here, we review the species of the Old World leafhopper genus Hishimonoides lshihara. Twelve species, including two new species, are recognized in the genus: H. arbudae Viraktamath et al. (India), H. aurifascialis Kuoh (China), H. bougainvilleae Viraktamath et al. (India, China), H. chinensis Anufriev (China), H. curvatus sp. nov. (China: Guangxi), H. dentimarginus Li and Zhang (China), H. miaolingensis Li and Zhang (China), H. orientalis Mahmood (Bangladesh, India), H. recurvatis Li (China), H. sellatiformis lshihara (China, Japan), H. spinosus Viraktamath et al. (India) and H. similis sp. nov. (China: Zhejiang, Gansu, Hunan). The type species and all known species from China are redescribed. One new synonym is revealed, Hishimonoides laterosporeus Li and Zhang, 2005, a junior synonym of Hishimonoides chinensis Anufriev, 1970. A key to the species in this genus is given, and their genital structures are illustrated.