MicroRNAs (miRNAs) comprise a group of small noncoding RNA molecules thought to have contributed to the evolution of vertebrate brain homogeneity and diversity. The miRNA miR-124 is well conserved between invertebrates and vertebrates and is expressed abundantly in the central nervous system (CNS). We identified miR-124 in the medaka, Oryzias latipes, and investigated its role in neural development. The five candidate genes for medaka precursor miR-124 are unlinked on four different chromosomes and differ in nucleotide length. Their sequences suggest that they can generate functional miRNAs through conventional miRNA biogenesis by folding into stem-loop structures. Whole-mount in situ hybridization and northern blotting revealed that mature miR-124 is specifically expressed in the CNS and the eyes starting at two days post-fertilization. We also examined the sequences and expression of medaka Polypyrimidine tract binding protein 1 (Ptbp1), a possible direct target of miR-124. The 3′UTR of medaka Ptbp1 contains predicted binding motifs (target sites) for miR-124. A GFP reporter assay for the target sites or the entire 3′UTR showed that exogenous miR-124 silences PTBP1 expression in vivo. Our study suggests that medaka miR-124 is involved in post-transcriptional regulation of target genes in neural development and that medaka miR-124 homologs may have spatiotemporal roles different from those in other vertebrates.

Community compositions in continental islands have been strongly affected by the connection and separation of land via land bridges during the cycle of glacial and interglacial periods. The biota of the Japanese archipelago is a result of colonization from the adjacent mainland of East Asia via land bridges at the north and south that existed during the Pliocene and in glacial periods during the Pleistocene. The carrion beetle Necrophila jakowlewi (Coleoptera: Silphidae) is discontinuously distributed in inland regions of the East Asian mainland, the Korean Peninsula, the central area of Honshu, Japan, and several adjacent islands. This species is thought to have migrated into Japan via the southern land bridge. We conducted phylogenetic and phylogeographic analyses using a partial sequence of the cytochrome oxidase subunit I gene and estimated the divergence times among populations to elucidate the colonization process of N. jakowlewi into Japan. Populations of N. jakowlewi were estimated to have diverged in the Japanese archipelago during the last glacial period, whereas the related species, N.japonica and N. brunnicollis, diverged during the interglacial or the last glacial period. In N. jakowlewi, most haplotypes were unique to separate regions, suggesting that the regional populations have been segregated from one another without gene flow by geographic isolation due to rising sea level after the last glacial period.

We measured the fecundity (number of eggs laid and number of eggs hatched) of the Parthenogenese and sexual forms of the weevil Scepticus insularis (Roelofs) with and without effects from mating, using one sexual and two parthenogenetic populations (one of the latter sympatric and the other allopatric with sexuals). When mated sexuals and unmated parthenogens were compared, the average number of eggs laid per female was largest for allopatric parthenogens (274 eggs), followed by sympatric parthenogens (223), and smallest for sexuals (169), with a significant difference only between allopatric parthenogens and sexuals (P < 0.05). The average number of eggs hatched per female was largest for allopatric parthenogens (136 hatchlings), followed by sexuals (119), and smallest for sympatric parthenogens (59), with a significant difference only between allopatric and sympatric parthenogens. In addition, mating significantly increased the reproductive output of sympatric parthenogens (59 vs. 188 hatchlings per female) by increasing egg productivity and hatching rate, but not that of allopatric parthenogens (136 vs. 133). The productivity of parthenogens of S. insularis is thus nearly equal to (in unmated sympatric parthenogens) or much higher (in other categories of parthenogens) than that of conspecific sexuals at the start of the larval stage, provided that the sex ratio of sexuals is 0.5 and all else being equal. Our results further showed that fecundity and other important aspects of the demography of parthenogenetic females may vary locally, or depending on whether they are sympatric or allopatric with sexuals.

Intersexual selection results from several processes, such as differential allocation and differential access, in addition to mating skews by mate choice. These processes can contribute to the evolution, maintenance, and geographic differentiation of male ornamentation, although the importance of these processes in male ornamentation remains poorly understood. The Asian barn swallow Hirundo rustica gutturalis is a socially monogamous songbird that exhibits biparental care and has red throat patches twice as large as those of the nominate H. r. rustica. Our previous study showed that females paired to males with large throat patches had higher reproductive output in terms of multiple broods, although the underlying process resulting in selection for a large throat patch remained unclear. In the present study, we analyzed differential female access to males with large throat patches in H. r. gutturalis. We observed that males with large throat patches acquired older and fatter females, independent of male age class. In addition, females that mated to males with large throat patches returned to the study site more compared with others, indicating the high viability of these females, supporting differential access but not differential allocation. No other measures of male ornaments (i.e., tail length, white tail spots, or throat color value) were linked to female qualities. As these female qualities were associated with female reproductive output, males with large throat patches would obtain reproductive advantages, as found in our previous study. The current findings suggest the importance of differential access for the evolution of a large throat patch in this subspecies.

Hydrostatic pressure is the only one of a range of environmental parameters (water temperature, salinity, light availability, and so on) that increases in proportion with depth. Pressure tolerance is therefore essential to understand the foundation of populations and current diversity of faunal compositions at various depths. In the present study, we used a newly developed pressure chamber system to examine changes in larval activity of the salt-lake crustacean, Artemia franciscana, in response to a range of hydrostatic pressures. We showed that A. franciscana larvae were able to survive for a short period at pressures of ≤ 60 MPa (approximately equal to the pressure of 6000 m deep). At a pressure of > 20 MPa, larval motor ability was suppressed, but not lost. Meanwhile, at a pressure of > 40 MPa, some of the larval motor ability was lost without recovery after decompression. For all experiments, discordance of movement and timing between right and left appendages, was observed at pressures of > 20 MPa. Our results indicate that the limit of pressure for sustaining active behavior of A. franciscana larvae is ∼20 MPa, whereas the limit of pressure for survival is within the range 30–60 MPa. Thus, members of the genus Artemia possess the ability to resist a higher range of pressures than their natural habitat depth. Our findings demonstrated an example of an organism capable of invading deeper environment in terms of physical pressure tolerance, and indicate the need and importance of pressure study as an experimental method.

Reproductive strategies have evolved from a series of trade-offs between cost and timing of reproduction. We tested whether the reproductive effort of female Danube crested newts, Triturus dobrogicus, was environmentally or energetically constrained. We collected females migrating towards the water and kept them separately, with males. Deposited eggs were collected daily during the experiment. More eggs were deposited by older females and by females starting to reproduce earlier. Batches of eggs from females depositing more eggs had a lower hatching success, suggesting decreased viability. Oviposition lasted on average 22.7 days. Female newts showed no loss of weight during this period. At the end of the egg deposition period we injected a subset of females with hormones that triggered the deposition of additional eggs. This suggests that egg deposition in Danube crested newts is environmentally constrained, as females stopped oviposition despite having mature eggs in their ovaries.

To study the function of arginine vasotocin (AVT) in a specific courtship behavior (body undulation) in male Hynobius leechii, we injected various doses of AVT or an AVT V1a or V2 receptor antagonist into breeding and non-breeding males. After these injections, we placed the males alone or with breeding females in Petri dishes and measured the incidence and frequency of body undulation. Additionally, to test whether AVT modulates the olfactory response of males, we exposed breeding males that were injected with AVT to female odors and measured the same response. Both breeding and non-breeding males intraperitoneally injected with 50 or 100 µg of AVT exhibited body undulation. Additionally, breeding males intraperitoneally injected with 50 or 100 µg of AVT exhibited an increased frequency of body undulation when exposed to female odors. The intraperitoneal injection of 5, 25, or 50 µg of the AVT V1a or V2 receptor antagonist did not significantly decrease the incidence or frequency of body undulation of 100 µg AVT-injected breeding males. However, in a post-hoc experiment, the breeding males that were subcutaneously injected with 100 µg of the AVT V1a receptor antagonist exhibited a significant decrease in the frequency of body undulation, induced by exposure to females. Additionally, a central injection of 1 µg of AVT into the brain induced body undulation in breeding males. Our results show that AVT not only induces a specific courtship behavior in male H. leechii via AVT V1a receptors but also modulates the olfactory response.

Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

Recent molecular phylogenetic analyses have shown that the modern jawless vertebrates, hagfishes and lampreys, are more closely related to each other than to the other vertebrates, constituting a monophyletic group, the cyclostomes. In terms of their developmental morphology as well, it is possible to identify an embryonic pattern in hagfish embryos that is common to cyclostomes but not shared by jawed vertebrate embryos. On the basis of this pan-cyclostome embryonic pattern, we describe the developmental sequence of the chondrocranium and associated structures in the hagfish species Eptatretus burgeri and E. atami. Our aim was to establish homologies of the skeletal elements among cyclostomes by comparison of the developmental patterns with a lamprey, Lethenteron reissneri, to characterize further the cyclostome morphotype and its diversification in early vertebrate evolution. We show that the hagfish and lamprey chondrocrania can be compared perfectly at the level of modules corresponding to the craniofacial primordia constituting the cyclostome morphotype. In the adult anatomy, however, there are many instances in which homology cannot be established at the level of single skeletal elements, mainly because of the apparently highly apomorphic nature of the hagfish cranium. Even at the craniofacial modular level, the chondrocrania of cyclostomes and those of jawed vertebrates display very few primary homologies and are therefore very difficult to compare. We also discuss the problem of the homology of a neurocranial element, the trabecula.

We developed an individual identification method for goldfish based on morphological variation of the iris. Each goldfish has a few dark lines (eye marks) in the rostral and caudal portion of the iris, which are blood vessels underneath the silvery reflective layer. Through the blood vessels and the locally thin reflective layer, the pigment cell layer is partially visible as a dark line. The pattern of the blood vessels was found to be temporally stable and unique to each individual. Using this feature, we successfully identified 10 individual goldfish, each sampled three times within a 4-month time period. The eye mark identification method was confirmed for a further 20 goldfish by comparison with identification based on screening at polymorphic microsatellite loci. The eye mark method is 100% accurate and can be used for studies in which multiple observations of individually identified goldfish are needed over long time periods, to avoid invasive tagging or individual housing that may affect the behavior of the fish.

The relationship between sex steroid hormone profiles in plasma and gonadal function in hagfish is poorly understood. In the present study, plasma concentrations of estradiol, testosterone, and progesterone were examined with respect to gonadal development, sexual differences, and possible function of atretic follicles in the brown hagfish, Paramyxine atami, using a time-resolved fluoroimmunoassay. Plasma concentrations of these three hormones were low in juveniles of both sexes. In females, plasma estradiol showed a significant correlation with ovarian development, with the highest concentrations in late vitellogenic adults. Plasma testosterone and progesterone also increased significantly in non-vitellogenic adult females; however, plasma testosterone showed no significant differences among adult females at different ovarian developments, while plasma progesterone was significantly lower in late vitellogenic adults than it was in non-vitellogenic adults. Vitellogenic females that possessed atretic follicles showed significantly lower concentrations of all three hormones than females that only possessed normal follicles. In males, no significant differences were found in plasma estradiol or testosterone levels among groups of different developmental stages of the testis, while plasma progesterone showed a clear inverse relationship with testicular development. Thus, differences were found in plasma sex steroid hormone profiles between male and female P. atami. Moreover, plasma estradiol showed a significant correlation with ovarian development, which suggests that estradiol is involved in the regulation of ovarian development. The present study also revealed that steroid hormone production was strongly suppressed in females that possessed atretic follicles in their ovaries.

During the spawning process in starfish, oocytes are arrested at metaphase of meiosis I (MI) within the ovary, and reinitiate meiosis only after they have been released into the seawater. However, this arrest does not occur if the ovary is removed from the animal. As the pH of the coelomic fluid is buffered by CO₂/H /HCO₃^-, we investigated the involvement of gas concentrations in MI arrest. In vivo, the CO₂ level in the coelomic fluid was high (∼1.5% vs. 0.04% in air) and the O₂ level was low (0.1–1.0% vs. ∼20% in air). When these gas conditions were reproduced in isolated coelomic fluid or seawater, ovarian oocytes arrested at MI, just as in vivo. Isolated oocytes from the ovary required the similar high CO₂ and low O₂ level to remain arrested in MI and had an intracellular pH of ∼6.9. Intracellular pH increased to ∼7.3 when oocytes were transferred to seawater equilibrated with air, a condition that mimics that of spawning. We used ammonium acetate to clamp intracellular pH at different levels and found that MI arrest occurred when intracellular pH was ∼6.9. Our results support the idea that high CO₂ and low O₂ in the ovarian environment lead to low intracellular pH and MI arrest, while spawning into the seawater with low CO₂ and high O₂ results in high intracellular pH and release from MI arrest. The biological significance of MI arrest is that oocytes are spawned into seawater at the optimal physiological state of MI when the least polyspermy occurs.

We compared the anatomy of the holotype of the palaeonemertean Cephalothrix simula (Iwata, 1952) with that of the holotypes of Cephalothrix hongkongiensis Sundberg, Gibson and Olsson, 2003 and Cephalothrix fasciculus (Iwata, 1952), as well as additional specimens from Fukue (type locality of C. simula) and Hiroshima, Japan. While there was no major morphological discordance between these specimens, we found discrepancies between the actual morphology and some statements in the original description of C. simula with respect to supposedly species-specific characters. Our observation indicates that these three species cannot be discriminated by the anatomical characters so far used to distinguish congeners. For objectivity of scientific names, topogenetypes of the mitochondrial cytochrome c oxidase subunit I (COI) sequences are designated for C. simula, C. hongkongiensis, and C. fasciculus. Analysis of COI sequence showed that the Hiroshima population can be identified as C. simula, which has been found in previous studies from Trieste, Italy, and also from both the Mediterranean and Atlantic coasts of the Iberian Peninsula, indicating an artificial introduction via (1) ballast water, (2) ship-fouling communities, or (3) the commercially cultured oyster Crassostrea gigas (Thunberg, 1793) brought from Japan to France in 1970s. Cephalothrix simula is known to be toxic, as it contains large amounts of tetrodotoxin (TTX). We report here that the grass puffer Takifugu niphobles (Jordan and Snyder, 1901)—also known to contain TTX— consumes C. simula. We suggest that the puffer may be able to accumulate TTX by eating C. simula.

A new species of the genus Sarsarietellus from Korean waters, S. orientalis n. sp. is described based on both sexes. This is very closely related to S. suluensis Ohtsuka, Nishida and Machida, 2005, but is readily distinguished by the following characteristics: in the female (1) narrow head; (2) nearly symmetrical posterior corners of last pedigerous somite, not extending beyond the genital double-somite; (3) antennary endopod slightly longer than the exopod; (4) nearly equal length of three outer spines and the terminal spine of the exopod of the fifth leg, except for the proximal third outer spine; and in the male the left fifth leg with two whip-like setae on the tip of the second exopodal segment not being rotated outside. Sarsarietellus orientalis is the fourth species of the genus Sarsarietellus, while the male of Sarsarietellus species is the first reported. The habitat colonization of arietellids is reconsidered.

The mitochondrial cytochrome c-oxidase subunit I (cox1) can serve as a fast and accurate marker for the identification of animal species, and for the discovery of new species across the tree of life. Distinguishing species using this universal molecular marker, a technique known as DNA barcoding, relies on the identifying the gap between intra- and interspecific divergence. One of the difficulties could be wide-ranging, cosmopolitan species that show large amounts of morphological variation. The barn owl Tyto alba is a case in point. It occurs worldwide and varies morphologically, leading to the recognition of many subspecies or, more recently, species. We analysed data from the cox1 gene for 31 individuals of seven subspecies, and compared this with 214 sequences from 29 other owl species. Phylogenetic analysis of the T. alba samples gives very strong support for an Old World alba-clade (three subspecies) and a New World furcata-clade (four subspecies) that are genetically equidistant. The amount of intraspecific variation within each of these clades ranges from 0.66–0.99%, but variation among these clades ranges from 5.33–6.20%. Combined these data suggest that barn owl of the Old World is indeed best considered a separate species different from that of the New World. For combined dataset, sample size of owl species (n between 1 and 21 sequences) increased with geographic range size but we did not find significant relationships between interspecific divergence and sample size or between interspecific divergence and geographic range. For 21/24 species of owls with sample sizes of n ≥4 the maximum interspecific divergences was ≤ 3.00%. However, similar to those found in barn owls, the largest amount of divergence (3.23–4.09%) was present in two other wide-ranging species (Strix nebulosa and Aegolius funereus) raising the possibility of multiple species in other wide-ranging owls as well.