We investigated Ophiodaphne formata, an exceptional sexually dimorphic ophiuroid, in situ using scuba, and in the laboratory. Solitary males, solitary females, and pairs of coupled individuals were found associated with the irregular echinoid sand dollar Astriclypeus manni. We observed that coupled females, solitary males, and solitary females always hold their aboral surface against the oral surface of the host. Coupled males and females with disk diameters > 0.63 mm and > 3.7 mm, respectively, are considered to be ripe for reproduction. In order to couple, a solitary male approaches the margin of a female's disk, quickly inverts, interdigitates its arms with the female's, and keeps its mouth pressed against that of the female. At first, solitary males and females attach to their host singly and, secondly, couple with each other in order to engage in reproductive behavior. Solitary and coupled individuals are capable of changing their positions on the host, and transferring from one host to another. Coupled individuals separate after a period of attachment on the host.

Conspecific avoidance may influence the spatial distribution of colonies in some ants. House-hunting ants (Temnothorax albipennis) avoid nesting in areas where non-nestmates have nested previously. However, no reports are available on conspecific avoidance during nest selection in other ants. In the present study, we investigated nest selection in another nomadic species, the Japanese queenless ant, Pristomyrmex punctatus. Two-choice tests revealed that, similar to house-hunting ants, P. punctatus preferred nests soiled by nestmates to clean nests. However, unlike house-hunting ants, P. punctatus also preferred nests soiled by non-nestmates to a clean nest. Given the choice between a nest soiled by nestmates and one soiled by strangers, P. punctatus, unlike house-haunting ants, showed no significant preference. Thus, conspecific avoidance in nest selection was not observed in P. punctatus. Interspecific differences in ecological factors may drive the evolution of different nest selection strategies.

Most amphibians belonging to Rhacophoridae have an arboreal life. A large amount of bioactive peptides have been identified from amphibian skin, but none from amphibians belonging to Rhacophoridae have been reported to date. A tachykinin-like peptide, tachykinin-Thel, was purified and characterized from skin secretions of the tree frog, Theloderma kwangsiensis. Its primary structure was determined as KPSPDRFYGLM-NH2 by Edman degradation and mass spectrometry analysis. cDNA sequence encoding the precursor of tachykinin-Thel was cloned from the skin cDNA library. Tachykinin-Thel induced the contraction of isolated ileum smooth muscle in a dose-dependent manner. The current work reported a bioactive peptide, tachykinin-Thel, from Rhacophoridae amphibians and confirmed the presence of tachykinin-like peptide in the skin of the tree frog, which may facilitate their arboreal life.

The Papilio xuthus (Lepidoptera: Papilionidae) pupa expresses novel soluble proteins that undergo reversible temperature-dependent coacervate-formation. We purified two coacervate-forming proteins, PX-1 and PX-4, from the wings of pharate adults. PX-1 and PX-4 form coacervates upon warming. Transmission electron microscopy analysis revealed that these proteins assemble ordered bead-like ultrastructures. We cloned and sequenced PX-1 and PX-4 cDNAs. The PX-1 and PX-4 amino acid sequences contain many hydrophobic residues and show homologies to insect cuticular proteins. Moreover, when recombinant PX-1 and PX-4 were overexpressed in Escherichia coli, both recombinant proteins exhibited temperature-dependent coacervation. Furthermore, analyses of truncated mutants of PX-1 suggest that both the Val/Pro-rich region and Gly/lle-rich regions of PX-1 are involved in such coacervation.

As amniotes diversified, mammals may have modified mechanisms of cellular pluripotency along with the acquisition of a placenta. What then defined pluripotent states in the ancestral amniotes? To study the evolutionary background of pluripotency in amniotes, we tested the effects of extracellular effectors on primary culture cells from avian and reptile embryos in serum-free medium. When treated with a combination of a MEK inhibitor and a GSK3 inhibitor (2i condition), chicken early embryos formed domed colonies (DCs), which were morphologically indistinguishable from the colonies formed by mouse and rat naïve embryonic stem cells. However, no DCs formed when cells from further-developed embryos were cultured in the 2i condition, indicating that there is a clear boundary of DC-forming ability at around the stage of primitive streak elongation. Quail embryos at the blastoderm and cleavage stages also formed DCs in the 2i condition, which is consistent with the notion that the appearance of DCs corresponds with the presence of pluripotent cells in embryos. Gecko blastoderms also formed DCs in the 2i condition, but gastrulas did not. ERK activation by bFGF caused an effect opposite to that of the 2i condition, namely, it dispersed colonies of cells even from early embryos in all species examined. These results suggest that the regulation of pluripotency by FGF/ERK signaling may date back at least to the common ancestor of mammals, birds, and reptiles. However, gene expression analysis indicated the possibility that mammalian pluripotency transcription factors function differently in non-mammalian amniotes.

Two morphotypes, with a large and small body size, of a brown frog Rana t. tagoi occur sympatrically in the Kinki region, central Honshu of Japan. Previous mitochondrial (mt) DNA genealogical study recognized two main lineages (A and B) and several sublineages in R. tagoi, where the small type was placed in the group A-1b, and the large type in groups A-1a and B-2a. Using haplotype network and structure analysis of three nuclear genes, we examined the discrepancy between morphology and mitochondrial genealogy. The results showed that the small type is reproductively isolated from its co-occurring large type (A-1a or B-2a), and that unlimited gene flow occurred between parapatrically occurring two mtDNA lineages of large types (A-1a and B-2a). Discordant genetic relationships between mtDNA and nuclear DNA results may be caused by the past mitochondrial introgression, and possibly, the incomplete lineage sorting. These results also suggest a heterospecific relationship between the large (A-1a and B-2a) and small types (A-1b). The large type is identified as Rana t. tagoi as it is genetically very close to the topotypes of the nominal subspecies, while the small type remains unnamed.

The Yellow River Delta, a stopover site, has become one of the breeding sites of oriental white storks (Ciconia boyciana) in China, with 28 breeding pairs. To gain insight into the characteristics of foraging habitat use during the breeding season, we surveyed the foraging habitats in 2009 and 2010. In 2009, using a quadrat sampling technique, we surveyed 74 quadrats in two breeding phases. Fourteen variables were analyzed with DCA. Oriental white storks mainly foraged in the reed swamp of recovered wetlands during the early breeding phase (70.29%), and in the reed swamp of unrecovered area during the late phase (32.74%), and in the open water of recovered wetlands (29.95%). The variation in proportional habitat use was not significant in the two breeding phases. Differences of the foraging sites in the two phases were extremely significant in terms of plant density, distance from nest, and distance from highway. In 2010, the storks also mainly used the reed wetlands for foraging (87.00%). The top three parameters on the first axis with the highest scores were distance from nest, plant height, plant coverage; on the second axis, the top three variables with the highest scores were plant density, number of other waterbird species, and water depth. These results showed that there are differences in the foraging habitat use of oriental white storks in the early and the late breeding phases. It is favorable for the breeding of oriental white storks to recover the reed wetland and to preserve a certain area of open water habitats.

The African clawed frog, Xenopus laevis, is widely used in biological studies. Ovulation of Xenopus is normally induced by the injection of human chorionic gonadotropin (hCG) into the dorsal lymph sac of fully-grown female frogs. Previously, we reported a novel method for inducing Xenopus ovulation by adding a mixture of steroids into the surrounding water. In the present study, we demonstrate how to induce reproductive behavior in male frogs using the same methodology. The types and concentrations of steroids were evaluated, and the efficiency of the selected steroid for the induction of ejaculation was examined. New procedures were also examined for inducing mating by mixing both females and males activated by steroids. In male frogs, testosterone was effective for the induction of physiological changes, accumulation of melanin in the hands and induction of amplexus. Time course experiments revealed that eight hours were sufficient to induce male reproductive behavior and ovulation in females. Finally, we established an efficient means of inducing pairing in frogs that involved pre-treatment of frogs with salt solution followed by testosterone for males and a mixture of estradiol and progesterone for females. Although the numbers of oocytes obtained were relatively fewer than those resulting from hCG injection, the fertilization rate of eggs ovulated using the new treatment method was similar to that with eggs obtained by hCG-injection, and juveniles developed normally. In conclusion, we have developed a novel method to induce pairing in frogs without the need for injections.

The Japanese pine sawyer, Monochamus alternatus Hope, 1842, an important forest pest, mainly occurs in Far East. It is the main vector of pine wood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. We determined the complete mitochondrial genome coding region of M. alternatus using long PCR and conserved primer walking. Our results show that the entire mitogenome coding region is 14,649 bp long, with 78.22% A T content [deposited in GenBank (JX987292)]. Positions and arrangement of the 37 genes encoded by the coding region are identical to those of two other longhorn beetles (Psacothea hilaris and Anoplophora glabripennis) for which the complete gene content and arrangement are known. All protein-coding genes start with a typical initiation codon ATN in insects. All tRNAs show standard clover-leaf structure, except the tRNA^Ser (AGN), which lacks dihydrouridine (DHU) arm. The most unusual feature found is the use of TCT as tRNA^Ser (AGN) anticodon instead of GCT, which is used in most other arthropods. This provides further insights into the diversity and evolution of the Cerambycidae family of long-horned beetles.

Urodele amphibians are thought to have poorer immune responses than evolutionary more ancestral vertebrate classes, such as bony fish. We investigated skin graft rejection and transplantation immunity in Urodele amphibians, Japanese newts, and Asiatic salamanders, and compared these findings to those from transplants in several species of frogs. The skin grafts used in this study were either allogeneic or xenogeneic. The mean survival time of the first set of allografts at 20°C was approximately 60 days for chronic responses in Urodela and 20 days for acute responses in Anura. As the graft survival times of urodeles were significantly longer than those of anurans, even when urodeles were repeatedly grafted from identical donors, there appear to be substantial differences in transplantation immunity between Urodela and Anura. These slow responses in Urodela may not be accompanied by the expansion of cytotoxic T cells, as observed in fish and anuran species, which are known to have functional major histocompatibility complex (MHC)-class I systems. In our study, approximately five histo-incompatible immunogenic components were found to be involved in chronic responses in newts. Similar chronic responses were also observed in xenograft rejection in newts. In contrast, xenografts were rejected in frogs due to an accelerated acute response, possibly involving natural killer cells. Our findings that some anti-allogeneic components appear to be shared with xenogeneic components indicate that the diversification of the acquired immune system is poorly developed in Urodela.

The present study aims at assessing allometric relationships in the Sigmodontinae rodents (Calomys tener, Akodon cf. montensis, Necromys lasiurus, Oligoryzomys flavescens, and Oligoryzomys nigripes), and morphological variation among different habitats in human-dominated environments in Southeastern Brazil. We captured rodents using pitfall traps placed in Eucalyptus plantations, abandoned pastures, and remnants of secondary native vegetation, and took the following measurements: body mass, total length, body length, left hind foot length, and left ear length. Males were usually larger than females, except in N. lasiurus. There was no intraspecific difference in body condition among habitats, suggesting that Eucalyptus may not have a deleterious effect upon its residents. However, A. cf montensis from Eucalyptus plantations had longer feet than those from other vegetation associations, suggesting a possible adaptive response to the lower cover in the plantation environment, and its consequent higher predation risk, or alternatively that only individuals with greater dispersal ability are found in Eucalyptus plantations. Future studies should investigate a possible co-evolutionary predator-prey relationship, including rapid evolution by Sigmodontinae rodents in anthropogenic landscapes.

To characterize the neural elements involved in the higher-order control of spontaneous walking in insects, we recorded extracellular spike activity in the protocerebrum of freely walking crickets (Gryllus bimaculatus). Locomotor behavior was simultaneously recorded using a newly developed motion tracking system. We focused on spike units that altered their firing patterns during walking. According to their activity patterns with reference to walking bouts, these locomotor-related spike units were classified into the following four types: continuously activated unit during walking (type 1); continuously inhibited unit during walking (type 2); transiently activated unit at the onset of walking (type 3); and transiently activated unit at the termination of walking (type 4). The type 1 unit was the most dominant group (25 out of 33 units), whereas only a few units each were recorded for types 2–4. Some of the locomotor-related units tended to change firing pattern before the onset or termination of walking bouts. Spike activity in some type 1 units was found to be closely correlated with walking speed. When firing timing was compared between unit pairs, their temporal relationships (synchronization/desynchronization) altered, depending on the behavioral state (standing/walking). Mechanical stimuli applied to the body surface elicited excitatory responses in the majority of the units. Histological observations revealed that the recorded sites were concentrated near or within the mushroom body and central complex in the protocerebrum.

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. Recently, GSS was purified from radial nerves of the starfish Asterina pectinifera and identified as a relaxin-like peptide. This study examines the mechanism of GSS secretion from radial nerves. When radial nerves isolated from A. pectinifera were incubated in artificial seawater containing ionomycin as a calcium ionophore, GSS release increased in a dose-dependent manner; 50% activity of GSS release was obtained with approximately 10 µM ionomycin. Another calcium ionophore, A23187, also stimulated GSS release from radial nerves. In contrast, membrane permeable cyclic AMP and cyclic GMP analogs failed to induce GSS release. These results suggest that GSS secretion is induced by intracellular Ca² as a second messenger.