The ability to discriminate among individuals plays a fundamental role in the establishment of social relationships in animals. We examined how Mongolian gerbils (Meriones unguiculatus) discriminate among individuals using odor. In the first experiment, the ability of male gerbils to discriminate among five odor sources from different individuals was investigated using a habituation-dishabituation paradigm. After male gerbils had been habituated to a scent from one individual, they were exposed to familiar and unfamiliar scents from different donors simultaneously. Where urine and ventral gland secretions were used, the subjects spent more time investigating novel odors than familiar ones, suggesting that they were able to discriminate individual differences in these odor sources. However, with the scents of feces and saliva, they could detect, but could not discriminate individual differences; with scent from inside the pinnae, they could not even detect. In the second experiment, we tested whether cross-habituation occurred between the scents of urine and ventral gland secretions. A male was exposed repeatedly to urine from one of two familiar donor males during four habituation trials, and was then exposed to the ventral gland secretions from two donors simultaneously. The subject males spent more time investigating scents of ventral gland secretions, but there was no difference in the investigation time between ventral gland scents from the two donors. These results suggest that male gerbils discriminate among individuals using odors from urine and ventral gland secretions and that cross-habituation may not occur between these scents during social-memory formation.

Field observations were conducted on egg attendance in Chirixalus eiffingeri from April to August 2003. Parental attendance during embryonic development was performed exclusively by males. The frequency of egg attendance was low (27%), but it had a distinct diel pattern in which males were observed to attend eggs more frequently at night than during the day. Attendance frequency significantly decreased with increasing developmental stage of the embryos, but it was not statistically significantly related to clutch size. Field observations confirmed that male frogs actively moisten egg clutches using their ventral surfaces, presumably to prevent desiccation of egg clutches. The non-significant relationship between hatching success and frequency of egg attendance suggests that embryonic survival of C. eiffingeri is more than a function of egg attendance, and ecological and environmental factors, such as climate and characteristics of microhabitats, may also influence the survivorship of the embryos.

The epidermis of an ascidian larva derived from animal-hemisphere cells is regionalized along the anterior-posterior (AP) axis through inductive signals emanating from vegetal-hemisphere cells in early stages of the development. Previously, we showed by blastomere isolation and ablation experiments that the contact between the animal and vegetal hemispheres until the 32-cell stage is necessary for the proper AP patterning of the epidermis in the tailbud-stage embryo. We here addressed the patterning mechanism of the posteriormost epidermis using a tail-tip epidermis marker, HrTT-1. Employing blastomere isolation and ablation experiments along with knockdown of a master regulator gene for posterior mesoderm, we have demonstrated that presence of the posterior vegetal cells after the 32-cell stage is necessary for the expression of HrTT-1. To explore the timing and nature of the influence of the posterior vegetal cells, we treated the embryos with FGF signaling inhibitors at various developmental stages and found that HrTT-1 expression was lost from embryos treated with the inhibitors from stages earlier than the late neurula stage, just prior to the onset of HrTT-1 expression but not after the initial tailbud stage, at which the expression of HrTT-1 had started. In embryos lacking HrTT-1 expression, the expression domain of Hrcad, which would otherwise be localized anterior to that of HrTT-1, expanded to the tail-tip. These results suggest that FGF signaling from the neurula to initial tailbud stages is necessary for the initiation but not maintenance of HrTT-1 expression in the tail-tip epidermis. The contact with posterior vegetal cells until and after the 32-cell stage may be required for FGF signaling to occur in the posterior tail, which in turn regionalizes the tail-tip epidermal territory.

The amino-acid sequences of the T-domain region of the Tbx4 gene, which is required for hindlimb development, are 100% identical in humans and mice. Cetaceans have lost most of their hindlimb structure, although hindlimb buds are present in very early cetacean embryos. To examine whether the Tbx4 gene has the same function in cetaceans as in other mammals, we analyzed Tbx4 sequences from cetaceans, dugong, artiodactyls and marine carnivores. A total of 39 primers were designed using human and dog Tbx4 nucleotide sequences. Exons 3, 4, 5, 6, 7, and 8 of the Tbx4 genes from cetaceans, artiodactyls, and marine carnivores were sequenced. Non-synonymous substitution sites were detected in the T-domain regions from some cetacean species, but were not detected in those from artiodactyls, the dugong, or the carnivores. The C-terminal regions contained a number of non-synonymous substitutions. Although some indels were present, they were in groups of three nucleotides and therefore did not cause frame shifts. The d_N/d_S values for the T-domain and C-terminal regions of the cetacean and artiodactylous Tbx4 genes were much lower than 1, indicating that the Tbx4 gene maintains it function in cetaceans, although full expression leading to hindlimb development is suppressed.

To study the phylogeography of the ground beetle Carabus yamato in Japan, we compared 1,020-bp sequences of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene from 373 specimens from 37 localities with those of three parapatric species (C. albrechti, C. kimurai, and C. japonicus) that might share mitochondrial lineages with C. yamato through introgressive hybridization. We found 81 haplotypes from C. yamato. Of these, 17 haplotypes were considered to be of an introgressed lineage from C. albrechti, based on the phylogeny and geographic distribution. In addition, one haplotype of C. kimurai was likely an introgressant from C. yamato. Putative introgression events among the four species were restricted to these two directional cases. We analyzed the phylogeography of C. yamato using nested clade phylogeographical analysis and population genetic parameters. The mitochondrial lineages of C. yamato were estimated to have diverged no more than approximately 1.12 million years ago, implying that the estimated historical events occurred after the Early Pleistocene. Carabus yamato was inferred to have experienced a contraction of its distribution range, followed by recent range expansion. Populations in the western and eastern regions, segregated by Ise Bay and the Nobi and Okazaki Plains, diverged in the mitochondrial clades. The northern and most western populations possessed one clade only (except an introgressed lineage), whereas eastern and some southwestern populations possessed several diverged clades, which were considered to be ancestral; these populations may have been associated with refugia during glacial periods.

We studied genetic variation within the Japanese land snail Euhadra herklotsi, which occurs on Kyushu and the surrounding islands, using partial sequences of the mitochondrial COI gene and nuclear ITS2 genes. The phylogenetic analysis revealed the existence of two major clades: clade N in the north and clade S in the south. These clades were parapatric and overlapped in southern Kyushu. Genetic divergence was high in clade N, whereas it was much lower in clade S. In addition, isolation-by-distance within each clade was implied. Since no current geographical barriers separate these clades, the genetic structure of clade S might have been influenced by historical events, such as volcanic activity, and a resulting population bottleneck followed by range expansion. The genital characteristics of clade-S snails were distinct from those of clade-N snails, and snails in both clades were sympatric at one locality. The shells of clade-N snails were generally larger than those of clade-S snails, but the shell-size variation within each clade could not be explained simply by environmental variables. Our study suggests that E. herklotsi likely consists of two sibling species. The taxonomic status of the previously proposed subspecies of E. herklotsi and related species requires reassessment.

Antisymmetry in the direction of the mouth opening, to either the right (“lefty”) or left (“righty”), was documented in the scale-eating cichlid Perissodus microlepis. This study revealed the presence of lefty and righty mouth morphs in the herbivorous cichlid Neolamprologus moorii, although the degree of deviation was not large. Both species are biparental brooders and guard their young. We examined the inheritance pattern of the dimorphism (laterality) using parents and broods of P. microlepis and N. moorii collected in the wild. In P. microlepis, lefty-lefty pairs had a 2:1 frequency of lefty:righty young, lefty-righty pairs a similar number of each type of young, and righty-righty pairs only righty young. Similar inheritance patterns were observed in N. moorii. We propose two hypotheses to explain the inheritance pattern: Mendelian genetics with the lefty allele dominant over the righty and the dominant allele homozygous lethal, and cross-incompatibility that is predominant in lefty homozygotes.

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.

The Chungan ground lizard, Takydromus sylvaticus, long known only from five females collected in Fujian Province of China, was recently found in Anhui Province of the country. The newly collected specimens included the first male specimens (n=7), in which the tail bases were stouter than in conspecific females. Ventral scales in these males were invariably keeled, whereas in females those forming the four median rows were smooth. Females had more dorsal scales (47–48) than males (42–43) in the median longitudinal row between axilla and groin. Takydromus sylvaticus differed from the closely related T. dorsalis in having more dorsal scales in the median longitudinal row between axilla and groin (42–48 versus 32–40, respectively), more rows of large dorsal scales between the hind legs (10 versus 7–8, respectively), shorter hind legs, two distinct white longitudinal stripes on each side of the body, and no black stripe in the preocular and postocular regions. The number of transverse rows of ventral scales between collar and femoral pores in T. sylvaticus (26–28) was larger, but with a slight overlap, than that in T. dorsalis (22–26). Ecological observations on T. sylvaticus were also documented.

In insects, four types of motoneurons have long been known, including fast motoneurons, slow motoneurons, common inhibitory motoneurons, and DUM neurons. They innervate the same muscle and control its contraction together. Recent studies in Drosophila have suggested the existence of another type of motoneuron, the common excitatory motoneuron. Here, we found that shakB-GAL4 produced by Jacob et al. (2000) labels this type of motoneuron in Drosophila larvae. We found that Drosophila larvae have two common excitatory motoneurons in each abdominal segment, RP2 for dorsal muscles and MNSNb/d-Is for ventral muscles. They innervate most of the internal longitudinal or oblique muscles on the dorsal or ventral body wall with type-Is terminals and use glutamate as a transmitter. Electrophysiological recording indicated that stimulation of the RP2 axon evoked excitatory junctional potential in a dorsal muscle.

To overcome difficulties in getting research materials of cephalochordate lancelets, which has severely hampered experimental studies of this animal, we have attempted to establish a culture system in the laboratory. Adult animals collected from the wild were maintained in 2.5-L plastic containers filled with natural seawater without sand substratum. They were fed daily with unicellular algae. About 25% of the animals collected in 2003, 2004, and 2005 developed gonads in our culture system. Some of the sexually mature animals collected in the breeding seasons in 2005 and 2006 spawned spontaneously in the plastic containers of this system. Broods obtained in 2005 were maintained longer than a year in a glass tank without sand substratum. The progeny born in the laboratory showed great individual variation in growth but metamorphosed normally, and some of them started to develop gonads around 10 months after fertilization. Our mass culture methods for both adults and their progeny made daily observation possible and allowed the constant spawning of animals collected from the wild, at least in the summer season. Our culture method saves labor in maintenance and is easily set up without any specific demands except for running seawater, though still required to better survival rate and spawning control. Lancelet populations maintained in the laboratory can promote studies on these animals across disciplines and especially contribute to elucidation of the evolutionary history of chordates.

Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177–186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile¹⁸⁷-Ser¹⁸⁸-Ala¹⁸⁹ and flanked at its N-terminus by Leu¹⁷⁴-Gly¹⁷⁵-Arg¹⁷⁶. This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbo-nyl (Boc)-Leu-Gly-Arg–4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45°C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40°C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males.

Tropical evergreen forests of Indian subcontinent, especially of the Western Ghats, are known hot spots of amphibian diversity, where many new anuran species await to be identified. Here we describe from the Sharavathi River basin of central Western Ghats a new shrub-frog taxon related to the anuran family Rhacophoridae. The new frog possesses the characteristic features of rhacophorids (dilated digit tips with differentiated pads circumscribed by a complete groove, intercalary cartilages on digits, T-shaped terminal phalanges and granular belly, the adaptive characters for arboreal life forms), but also a suite of unique features that distinguish it from all known congeners in the region. Morphogenetic analysis based on morphological characteristics and diversity in the mitochondrial 12S and 16S rRNA genes revealed it to be a new Philautus species that we named Philautus neelanethrus sp. nov. The phylogenetic analysis suggests the new frog to represent a relatively early Philautus species lineage recorded from the region. The distribution pattern of the species suggests its importance as a bioindicator of habitat health. In general, this relatively widespread species was found distributed only in non-overlapping small stretches, which indirectly indicates the fragmentation of the evergreen to moist deciduous forests that characterize the Western Ghats. Thus the discovery of the new rhacophorid species described here not only further reinforces the significance of the Western Ghats as a major hotspot of amphibian biodiversity, but also brings into focus the deterioration of forest habitats in the region and the need for prioritization of their conservation.