This study investigated defensive behavior by the microturbellarian Stenostomum sphagnetorum against the predatory ciliate Dileptus margaritifer. When S. sphagnetorum was attacked by D. margaritifer, the microturbellarian released a mass of mucous material from the site of attack and swam away, while D. margaritifer ingested the released material. If the attack was repeated, S. sphagnetorum was killed and eaten by the predator. Observations on the predator-prey interactions between these two organisms suggest that the discharge of mucous material by S. sphagnetorum is the main component of defensive behavior. The following findings support this hypothesis: 1) individuals of S. sphagnetorum artificially deprived of glandular secretions through lysozyme treatment were more vulnerable to D. margaritifer and were more sensitive to the predator's toxin than were untreated animals, while they were indistinguishable from intact S. sphagnetorum in external morphology and in the capacity to grow; 2) supernatant containing the D. margaritifer toxin induced in S. sphagnetorum the same behavioral reactions observed in actual encounters with D. margaritifer.

Giant scale insects (Drosicha: Coccoidea: Monophlebidae) were investigated for their symbiotic organs and bacterial endosymbionts. Two types of bacterial 16S rRNA gene sequences, flavobacterial and enterobacterial, were consistently detected in D. corpulenta and D. pinicola. The former sequences formed a compact clade in the Bacteroidetes, allied to the symbionts of cushion and armored scales. The latter sequences formed a robust clade in the γ-Proteobacteria, allied to enteric bacteria like Enterobacter aerogenes and Escherichia coli. Another type of 16S sequence derived from Wolbachia was also detected in D. pinicola. In-situ hybridization demonstrated that the flavobacterial and enterobacterial symbionts were localized in a pair of huge bacteriomes in the abdomen, the former in uninucleated peripheral bacteriocytes and the latter in syncytial central bacteriocytes. Electron microscopy confirmed the endocellular locations of the pleomorphic flavobacterial symbiont and the rod-shaped enterobacterial symbiont, and also revealed the location and fine structure of the Wolbachia symbiont in D. pinicola. Infection frequencies of the flavobacterial and enterobacterial symbionts were consistently 100% in populations of D. corpulenta and D. pinicola, while the Wolbachia symbiont exhibited 0% and 100% infection frequencies in D. corpulenta and D. pinicola, respectively. Neither the flavobacterial symbiont nor the enterobacterial symbiont exhibited AT-biased nucleotide composition or accelerated molecular evolution. The huge bacteriomes of Drosicha giant scales would provide a useful system for investigating biochemical, physiological, and genomic aspects of the host-symbiont and symbiont-symbiont interactions.

Molecular phylogenetic analyses of combined mitochondrial DNA sequences (2814 bp; cytochrome b gene, displacement loop region, and NADH dehydrogenase subunit 2 gene) identified nine groups among 49 individual Japanese martens, Martes melampus, collected from several areas in Japan. The grouping was not correlated with winter coat color, but was consistent with geography. In particular, the monophyly of 29 Tsushima martens, M. m. tsuensis, was supported by strong clade support and topological tests. Haplotype and nucleotide diversities were much lower for the Tsushima population than for any population on the Japanese main islands. In addition, analyses of heterozygosity in nuclear growth hormone receptor gene sequences (654 bp) showed genetic homogeneity for the Tsushima population. This evidence supports the view that the Tsushima marten's long history of isolation on small islands is responsible for its genetic distinctiveness and uniformity, validating the Tsushima population as an evolutionarily significant unit.

The genus Helice is comprised of four species, H. formosensis, H. latimera, H. tientsinensis, and H. tridens. A recent molecular analysis identified H. formosensis and H. tientsinensis as junior synonyms of H. latimera. We used three mitochondrial and two nuclear genes to further delineate species boundaries in Helice and to add to knowledge of the historical biogeography of the genus. The molecular data revealed deep divergences between the H. formosensis-latimera-tientsinensis complex and H. tridens. Clear genetic separations with incomplete lineage sorting and convincing morphological divergences were detected among H. formosensis, H. latimera, and H. tientsinensis, and a coalescence analysis revealed negligible gene flow among these species, except for weak unidirectional gene flow from H. formosensis to H. latimera. The estimated divergence time of 1.42–1.92 Ma between the H. formosensis-latimera-tientsinensis complex and H. tridens is consistent with the opening of the Tsushima/Korea Strait (1.71–1.52 Ma). The divergence time (∼22-730 ka) among H. formosensis, H. latimera, and H. tientsinensis indicates that the Taiwan Strait acted as a biogeographic barrier during major falls in sea level during the Pleistocene. Our findings indicate that H. formosensis, H. latimera, and H. tientsinensis are valid species, and that straits (Tokara Strait, Tsushima/Korea Strait, and Taiwan Strait), the Okinawa Trough, and currents (Kuroshio Current, Tsushima Current, and Taiwan Strait Warm Current) have acted as geographic barriers resulting in allopatric speciation among onshore marine animals in the northwestern Pacific.

We studied salinity tolerance and the effects of salinity on growth, development, and metamorphosis in Fejervarya limnocharis tadpoles living in brackish water. Specifically, we examined whether tadpoles exhibit adaptive plasticity in development when exposed to different salinities. Tadpoles collected on Green and Orchid Islands off Southeastern Taiwan were assigned to salinities of 0, 3, 5, 7, 9, 11, and 13 parts per thousand (ppt). The daily survival, weekly growth, and development of tadpoles were recorded until metamorphosis. More than 50% of tadpoles survived in 9 ppt for over a month, and a few individuals survived in 11 ppt for 20 days, suggesting that F. limnocharis tadpoles tolerate salinity better than the tadpoles of most species studied to date. Tadpoles at 9 ppt had lower survivorship, and retarded growth and development (from Gosner stage 26 to 35) compared to the other treatments. Tadpoles metamorphosed early at a smaller size as salinity increased, suggesting the existence of adaptive developmental plasticity in F. limnocharis in response to osmotic stress. Phenotypic plasticity in the age and size at metamorphosis in response to salinity may provide a means for tadpoles to adapt to the unpredictable salinity variation in coastal rock pools.

Triops granarius (Lucas) (Notostraca: Triopsidae) lives in paddy fields from the Kanto district to northern Kyushu, Japan. Changes in the size distribution of this species were examined in the paddy fields and then the effect of light on hatching was examined under quasi-natural and laboratory conditions. Adult tadpole shrimps were found about one week after irrigation and plowing in two paddy fields in Sakai, Japan. They developed rapidly and disappeared altogether about one month later. Under conditions of natural daylength and temperature, eggs laid in the soil did not hatch without being removed from the soil. Under constant light at 25°C, the lower the light intensity was, the longer the eggs took to hatch. Moreover, most eggs kept in constant darkness did not hatch, but many of them hatched within a short period after being transferred to constant light with an intensity of 0.3 W/m² or more. Because a 1-h light pulse was found to induce hatching, light is considered necessary for the resumption of embryonic development. These results suggest that eggs of T. granarius laid in the soil do not hatch without exposure to light; consequently, this species has a univoltine life cycle in the paddy fields. Histological observations revealed that under constant darkness, embryonic development was arrested at an early stage of organogenesis, in which the nauplius eye had not yet formed. We discuss the role of light in the regulation of embryogenesis in T. granarius.

Podocnemis expansa, known popularly as the giant Amazon river turtle, is widely exploited. This makes specimens available for various studies, and we used this opportunity to investigate aspects of the development of the plastron. We examined several stages of pre- and post- hatching development in embryos and hatchlings collected starting from day 18 of natural incubation. Embryos and hatchlings were cleared and double stained for cartilage and bone. The epiplastron, endoplastron, hyoplastron, hypoplastron, xiphiplastron, and mesoplastron bones form the complete plastron of this turtle. In stage 16, bone centers become visible in most of the bones of the plastron. Alizarin Red stain retention indicated that the sequence of bone ossification is as follows: first the hyoplastron and the hypoplastron, then the endoplastron followed by the xiphiplastron, and lastly the mesoplastron. The epiplastron bone shows an ossification center only in stage 20. All these elements have independent ossification centers and join together only later. The plastron closes completely seven months after hatching.

The present study was conducted to determine both the site at which cuticle materials are produced and the critical period for their production in the oviductal uterus of the Japanese quail, Coturnix japonica. An antiserum was produced against the 32-kDa band in electrophoretic profiles of cuticle materials obtained from eggshells decalcified with EDTA. Immunofluorescence and immunoelectron microscopic observations revealed that the 32-kDa protein was synthesized in luminal ciliated epithelial cells of the uterus until 21 h after the previous oviposition (the first phase) and then secreted during the 4 h before the next oviposition (the second phase). Scanning electron microscopic observations revealed that 10-µm-wide posts appear on the surface of the luminal epithelia during the first phase, and that they disappear during the second phase. During the second phase, air canals are formed in the eggshell by the retreat of the posts, and a cuticle layer forms on the eggshell. Our results indicate that the cuticle may function as a lubricant that facilitates egg rotation in the uterus.

Using mitochondrial 12S rRNA, 16S rRNA, and ND2 sequences, we investigated phylogenetic relationships among populations of two frog species endemic to China, both referred to the genus Feirana. A sister-group relationship between the two species was supported moderately in a maximum likelihood analysis and significantly in a Bayesian analysis, but not in a maximum parsimony analysis, of combined data for the three genes. Pending resolution of this incongruence, we provisionally maintain these species in the genus Feirana. Two major clades with a deep divergence are concordant with the species F. quadranus and “F.” taihangnica. In the present work, some populations from the Qinling Mountains and all those from the Funiu and the Zhongtiao-southern Taihang Mountains are referred to “F.” taihangnica rather than F. quadranus, whereas others are referred to F. quadranus. Consequently, the main body of the Qinling Mountains was identified as a large contact zone between these two species. On the basis of phylogenetic relationships and the distribution pattern of populations, we propose a hypothesis for the divergence of “F.” taihangnica: the ancestral species might have inhabited the westernmost Qinling Mountains and dispersed to the main Qinling Mountains, and then to the Zhongtiao-southern Taihang and Funiu Mountains. In contrast, two alternative hypotheses are suggested for F. quadranus: if the two species are confirmed as sister groups, F. quadranus might have dispersed from the westernmost Qinling to the Longmen, Qinling, Daba, and northern Wuling Mountains; alternatively, F. quadranus might have come from the northern Wuling Mountains and then dispersed to the Daba, Qinling, and Longmen mountains.

An immunological analysis using subtype-specific antisera of the major yolk protein lipovitellin (Lv) of the grey mullet (Mugil cephalus) confirmed the presence of the three corresponding Lv subtypes (LvA, LvB, and LvC) in vitellogenic ovaries of the marbled sole (Pleuronectes yokohamae). These three Lv subtypes were purified from sole ovaries by using various combinations of anion exchange, hydroxylapatite, immunoadsorbent, and gel-filtration chromatography. Purified LvA, LvB, and LvC had an apparent native mass of ∼482, ∼380, and ∼372 kDa, respectively, estimated by gel filtration. Analysis of their tertiary structures by SDS-PAGE indicated that LvA, LvB, and LvC were typical of teleost Lvs in having a heavy (H) chain (∼105, ∼102, and ∼107 kDa, respectively) and a light (L) chain (∼22, ∼19.5, and ∼25 kDa, respectively). The N-terminal amino acid (AA) sequences were obtained for the LvA H chain, the LvB H and L chains, and the LvC L chain and compared to the deduced AA sequences of their precursors, vitellogenins (Vgs), in several species. This comparison of LvA, LvB, and LvC with various teleost VgA, VgB, and VgC sequences, respectively, revealed high identities (60–100%). The purified Lv subtypes were subjected to double immunodiffusion using an antiserum against an unclassified Lv of the sole (Hashimoto et al., 1998); only the LvB subtype exhibited immunoreactivity with this antiserum. This result indicates that the previously developed immunoassay using this anti-Lv for the detection of sole Vg is effectively a VgB-specific assay.