Choosiness, or female receptivity to copulation, is a critical component in female preference for a mating partner, as it functions at the final step in the mating process by determining whether she accepts copulation with a given male. In spite of its importance in the evolutionary process of male traits via sexual selection, little is known about the genetic variation and inheritance pattern of female receptivity. Drosophila prolongata shows a unique courtship behavior, leg vibration, which increases female receptivity to copulation. In the present study, we analyzed variation in female receptivity and its inheritance pattern in isofemale strains of D. prolongata using leg vibration as an index. There was a significant difference in female receptivity among the strains examined. A high-receptivity phenotype was semi-dominantly expressed in F1 females of crosses between strains with low and high receptivity. Backcrossing F1 females to low-receptivity strains resulted in a lower level of receptivity, suggesting that the high-receptivity phenotype is controlled by multiple genes with epistatic interactions. These results indicate a genetic basis of female receptivity, shedding light on the evolutionary process of sexual selection in D. prolongata.

To determine whether the effects of low-dose-rate gamma (γ) irradiation are identifiable in the regeneration of murine hair follicles, we irradiated whole bodies of C57BL/10JHir mice in the first telogen phase of the hair cycle with ¹³⁷Cs γ-rays. The mice were examined for effects on hair follicles, including number, morphology, and pigmentation in the second anagen phase. Effects of γ-radiation on melanocyte stem cells were also investigated by the indirect immunolabeling of tyrosinase-related protein 2 (TRP2). Irradiated skin showed a decrease in hair follicle density and the induction of curved hair follicles along with the presence of white hairs and hypopigmented hair bulbs. There was a small, but not significant, change in the number of TRP2-positive melanocyte stem cells in the hair bulge region of the irradiated skin. These results suggest that low-dose rate γ-irradiation does not deplete melanocyte stem cells, but can damage stem cells and progenitors for both keratinocytes and melanocytes, thereby affecting the structure and pigmentation of regenerated hair follicles in the 2^nd anagen phase.

We surveyed natural population of the Drosophila ananassae species complex on Penang Island, Malaysia. Analyses of phenotypic traits, chromosome arrangements, molecular markers, and reproductive isolation suggest the existence of two species: D. ananassae and D. cf. parapallidosa. Molecular marker analysis indicates that D. cf. parapallidosa carries chromosome Y and 4 introgressions from D. ananassae. Thus, D. cf. parapallidosa seems to be a hybrid descendant that recently originated from a natural D. parapallidosa♀× D. ananassae♂ cross. Furthermore, D. cf. parapallidosa behaves differently from authentic D. parapallidosa with respect to its reproductive isolation from D. ananassae. Premating isolation is usually seen in only the D. ananassae♀× D. parapallidosa♂ cross, but we observed it in crosses of both directions between D. ananassae and D. cf. parapallidosa. In addition, hybrid males from the D. ananassae♀× D. parapallidosa♂ cross are usually sterile, but they were fertile when D. ananassae♀ were mated with D. cf. parapallidosa ♂. We attempted an artificial reconstruction of the hybrid species to simulate the evolutionary process(es) that produced D. cf. parapallidosa. This is a rare case of natural hybrid population in Drosophila and may be a useful system for elucidating speciation with gene flow.

Salamanders are expected to differentiate genetically among local populations due to their low dispersal ability, and are potentially susceptible to loss of genetic diversity if the population is isolated by habitat fragmentation. The salamander Hynobius tokyoensis is a lowland lentic breeder and endemic to a narrow area of central Japan. In this urban area, H. tokyoensis habitats are extensively fragmented and several populations are threatened with extinction, but information on genetic divergence and loss of genetic diversity is scarce. We performed mitochondrial (cyt b) and microsatellite (five loci) DNA analyses of 815 individuals from 46 populations in 12 regions across their entire distribution range. As a result, populations were clearly separated into northern and southern groups, and genetic differentiation among the 12 regions was also evident. Regional differentiation appears to be affected by a complex geographical history, but the genetic diversity of each population may have also been affected by recent habitat fragmentation. There were positive correlations between the mitochondrial and microsatellite DNA diversities. Some populations have lost genetic diversity in both mitochondrial and microsatellite DNAs; all such populations were at the peripheral edges of the species distribution range. Thus, even in attempts to restore genetic diversity in a small population by the transfer of outside individuals, efforts must be made to avoid genetic pollution.

The raccoon dog (Nyctereutes procyonoides, Canidae, Carnivora) is highly adaptable to urban environments. Populations of carnivorans inhabiting urban areas sometimes differ ecologically and genetically from those in rural areas. However, there is little information on urban raccoon dogs. This study focused on raccoon dog populations in Tokyo, one of the most highly urbanized cities in the world. We examined the genotypes of 10 microsatellites for 101 fecal samples from raccoon dogs inhabiting the grounds of the Imperial Palace, a green space in central Tokyo. We successfully genotyped 58 samples originating from 31 individuals. We also analyzed muscle tissue samples from raccoon dogs from the grounds of the Imperial Palace, the Akasaka Imperial Grounds (a green space close to the Imperial Palace), and the surrounding urban area, and then investigated the genetic structure and diversity of these populations, and the genetic differentiation among them. The population on the grounds of the Imperial Palace was genetically differentiated from that in the Akasaka Imperial Grounds, suggesting that the roads and buildings act as barriers to gene flow. In addition, the population on the grounds of the Imperial Palace showed greater genetic difference from that in the surrounding area than that in the Akasaka Imperial Grounds. We speculate that the moats around the Imperial Palace restrict individual ranges within the palace grounds and limit migration and gene flow to other areas.

Dall's porpoise (Phocoenoides dalli) is a small toothed cetacean, widely inhabiting the North Pacific Ocean and adjacent seas, between about 30 and 62°N; however, only limited studies of its ecology have been made in nearshore areas. A cetacean sighting survey lasting 60 days was conducted during the 2012 summer cruise of the T/S Oshoro Maru (Hokkaido University, Japan) in the North Pacific Ocean and Bering Sea. Based on this data, the distribution of Dall's porpoises and the factors controlling it in the pelagic habitat were investigated. A total of 808 individual Dall's porpoises in 166 groups were sighted during a total of 469.6 hr and 4946.6 nm observations. The cruise consisted of three legs and the average porpoise group size was significantly larger in Leg 1. The sightings were concentrated at water depths of less than 1000 m and near eastern Aleutian passes. Sighting clusters were found on the 200 m isobath of the southeastern Bering Sea continental slope. There was a peak in sightings where the sea surface temperature (SST) was relatively cold, between 5 and 7°C. Although similar track routes were taken in Leg 1 and Leg 3, the number of sightings per unit effort was larger in Leg 1. This difference may have arisen from the significant rise in SST as the season progressed. Relatively large group size found in this study might relate with prey abundance along the Aleutian Islands.

Ghrelin was first isolated from human and rat as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In the present study, we determined the ghrelin cDNA sequence of the common marmoset (Callithrix jacchus), a small-bodied New World monkey, and investigated the distribution of ghrelin-producing cells in the gastrointestinal tract and localization profiles with somatostatin-producing cells. The marmoset ghrelin cDNA coding region was 354 base pairs, and showed high homology to that in human, rhesus monkey, and mouse. Marmoset ghrelin consists of 28 amino acids, and the N-terminal region is highly conserved as found in other mammalian species. Marmoset preproghrelin and mature ghrelin have 86.3% and 92.9% homology, respectively, to their human counterparts. Quantitative RT-PCR analysis showed that marmoset ghrelin mRNA is highly expressed in the stomach, but it is not detected in other tissues of the gastrointestinal tract. In addition, a large number of ghrelin mRNA-expressing cells and ghrelin-immunopositive cells were detected in the mucosal layer of the stomach, but not in the myenteric plexus. Moreover, all the ghrelin cells examined in the stomach were observed to be closed-type. Double staining showed that somatostatin-immunopositive cells were not co-localized with ghrelin-producing cells; however, a subset of somatostatin-immunopositive cells is directly adjacent to ghrelin-immunopositive cells. These findings suggest that the distribution of ghrelin cells in marmoset differs from that in rodents, and thus the marmoset may be a more useful model for the translational study of ghrelin in primates. In conclusion, we have clarified the expression and cell distribution of ghrelin in marmoset, which may represent a useful model in translational study.

The European honeybee (Apis mellifera L.) is used as a model organism in studies of the molecular and neural mechanisms underlying social behaviors and/or advanced brain functions. The entire honeybee genome has been sequenced, which has further advanced molecular biologic studies of the honeybee. Functions of genes of interest, however, remain largely to be elucidated in the honeybee due to the lack of effective reverse genetic methods. Moreover, genetically modified honeybees must be maintained under restricted laboratory conditions due to legal restrictions, further complicating the application of reverse genetics to this species. Here we applied CRISPR/Cas9 to the honeybee to develop an effective reverse genetic method. We targeted major royal jelly protein 1 (mrjp1) for genome editing, because this gene is predominantly expressed in adult workers and its mutation is not expected to affect normal development. By injecting sgRNA and Cas9 mRNA into 57 fertilized embryos collected within 3 h after oviposition, we successfully created six queens, one of which produced genome-edited male offspring. Of the 161 males produced, genotyping demonstrated that the genome was edited in 20 males. All of the processes necessary for producing these genome-edited queens and males were performed in the laboratory. Therefore, we developed essential techniques to create knockout honeybees by CRISPR/Cas9. Our findings also suggested that mrjp1 is dispensable for normal male development, at least till the pupal stage. This new technology could pave the way for future functional analyses of candidate genes involved in honeybee social behaviors.

Caffeine is a plant-derived alkaloid that is generally known as a central nervous system (CNS) stimulant. In order to examine the effects of caffeine on higher CNS functions in insects, we used an appetitive olfactory learning paradigm for the cricket Gryllus bimaculatus. Crickets can form significant long-term memories (LTMs) after repetitive training sessions, during which they associate a conditioned stimulus (CS: odor) with an unconditioned stimulus (US: reward). Administration of hemolymphal injections of caffeine established LTM after only single-trial conditioning over a wide range of caffeine dosages (1.6 µµg/kg to 39 mg/kg). We investigated the physiological mechanisms underlying this enhancement of olfactory learning performance pharmacologically, focusing on three major physiological roles of caffeine: 1) inhibition of phosphodiesterase (PDE), 2) agonism of ryanodine receptors, and 3) antagonism of adenosine receptors. Application of drugs relevant to these actions resulted in significant effects on LTM formation. These results suggest that externally applied caffeine enhances LTM formation in insect olfactory learning via multiple cellular mechanisms.

The Ryukyu wild boar (Sus scrofa riukiuanus) is an endemic, morphologically defined subspecies of the Eurasian wild boar (S. scrofa) found on five islands of the Ryukyu Archipelago (a group of small islands stretching from mainland Japan to Taiwan). Two hypothetical scenarios have been proposed regarding the origin of the current Ryukyu wild boar populations: 1) natural dispersal and 2) transportation and subsequent release by prehistoric humans. To test these two hypotheses, we compared the mitochondrial cytochrome b gene sequence (1140 base pairs) in 352 individual wild boar samples that included representatives of all five insular populations of the Ryukyu wild boar and populations of other conspecific subspecies in insular East and Southeast Asia and the Eurasian Continent. A total of 68 haplotypes were recognized, of which 12 were unique to the Ryukyu wild boar populations. The results of Bayesian phylogenetic analyses supported monophyly of the five Ryukyu populations (posterior probability value of 92), confirming the validity of the subspecies as a natural group. Coalescent analysis estimated the divergence times between the Ryukyu wild boar and the other conspecific subspecies as 144–465 thousand years ago (Kya), with a 95% HPD (highest posterior density) range of 51–837 Kya, and with no significant migration. Taking the broadly accepted date of initial human migration to the Ryukyus (no earlier than 50 Kya) into consideration, our results strongly suggest that the ancestral form of the Ryukyu wild boar first entered the Ryukyu Archipelago by natural dispersal prior to the arrival of prehistoric humans.

The signaler-eavesdropper interaction has been investigated for a wide range of organisms, and although many flies feed on calling frogs, this dynamic has been addressed only poorly in the austral Neotropics. We investigated this interaction in southern Brazil using pairs of suction traps (acoustic silent) broadcasting frog calls or an artificial white noise in ponds and streams. From 139 sessions, flies of the genera Corethrella (Corethrellidae), Forcipomyia (Ceratopogonidae) and Uranotaenia (Culicidae) were collected, including five Corethrella species, the most abundant of which was previously unknown and is formally described here. Additionally, we present the southernmost records of Corethrella lopesi, C. alticola and C. atricornis. Numbers of Forcipomyia midges and Uranotaenia mosquitoes did not differ between silent traps and traps broadcasting frog calls, and did not differ between white noise traps and adjacent silent traps. However, the number of female Corethrella was significantly higher in traps broadcasting calls of the pond-breeding frog P. aff. gracilis compared to adjacent silent traps; calls of this frog attracted the five Corethrella species and also collected significantly more female Corethrella than the white noise. By evaluating different taxa of flies and broadcasting different sounds, we demonstrated that Corethrella midges were attracted only to the acoustic cue of P. aff. gracilis calls, while Forcipomyia and Uranotaenia were captured in traps by chance. Our results suggest that female Corethrella feed on males of the common pond-breeding frog P. aff. gracilis in southern Brazil, and highlight the utility of frog call traps in revealing the diversity of Corethrella in the austral Neotropics.

Bucrates lanista, the most southerly distributed species in the genus Bucrates Burmeister, was originally described from Brazil based on a female collected in the state of Rio Grande do Sul, but the species has not been recorded since 1918. In this work, we report that B. lanista inhabits the Pantanal Wetland in the state of Mato Grosso do Sul and, for the first time, describe the male. Individuals of B. lanista are gregarious and present a brown/green color dimorphism; this behavior and color variation are also observed in species of closely related genera. Individuals from the Pantanal vary slightly from those of Rio Grande do Sul. The karyotype was determined to be 2n♂ = 21 = 20 X0 and 2n♀ = 22 = 20 XX. The X chromosome is metacentric and the largest of the complement, and all of the autosomes are submetacentrics. All chromosomes solely present telomeric (TTAGG)n repeats at their ends, and some chromosomes present positive and negative DAPI bands.

Capitella teleta Blake et al., 2009 is an opportunistic capitellid originally described from Massachusetts (USA), but also reported from the Mediterranean, NW Atlantic, and North Pacific, including Japan. This putatively wide distribution had not been tested with DNA sequence data; intraspecific variation in morphological characters diagnostic for the species had not been assessed with specimens from non-type localities, and the species status of the Japanese population(s) was uncertain. We examined the morphology and mitochondrial COI (cytochrome c oxidase subunit I) gene sequences of Capitella specimens from two localities (Ainan and Gamo) in Japan. Specimens from Ainan and Gamo differed from C. teleta from Massachusetts in methyl-green staining pattern, shape of the genital spines, and shape of the capillary chaetae; we concluded that these characters vary intraspecifically. Species delimitation analyses of COI sequences suggested that worms from Ainan and Massachusetts represent C. teleta; these populations share a COI haplotype. The specimens from Gamo may represent a distinct species and comprise a sister group to C. teleta s. str.; we refer to the Gamo population as Capitella aff. teleta. The average Kimura 2-parameter (K2P) distance between C. teleta s. str. and C. aff. teleta was 3.7%. The COI data indicate that C. teleta actually occurs in both the NW Atlantic and NW Pacific. Given the short planktonic larval duration of C. teleta, this broad distribution may have resulted from anthropogenic dispersal.

Deep-sea hydrothermal vent fields are among the most extreme habitats on Earth. Major research interests in these ecosystems have focused on the anomalous macrofauna, which are nourished by chemoautotrophic bacterial endosymbionts. In contrast, the meiofauna is largely overlooked in this chemosynthetic environment. The present study describes a new species, Thomontocypris shimanagai sp. nov. (Crustacea: Ostracoda), which was collected from the surface of colonies of neoverrucid barnacles and paralvinellid worms on the chimneys at the Myojin-sho submarine caldera. This is the first discovery of an ostracode from deep-sea hydrothermal vent environments in the western Pacific region. In addition to the species description, we discuss three aspects: 1) adaptation, 2) endemism, and 3) dispersal strategy of the hydrothermal vent ostracodes. Regarding these aspects, we conclude the following: 1) the new species may feed on sloughed-off tissues, mucus secretions, or fecal pellets of sessile organisms, rather than depend on chemoautotrophic bacteria as symbionts for energy; 2) as has been pointed out by other studies, Thomontocypris does not likely represent a vent-specific genus; however, this new species is considered to be endemic at the species level, as it has not been found outside of the type locality; and 3) this new species may have migrated from adjacent deep-sea chemosynthesis-based habitats, such as hydrothermal vents, with wood falls potentially having acted as stepping stones.

The Mugilogobius group consists of brackish water gobionellines widely distributed in the Indo-West Pacific region. Complete mitochondrial genome and morphological evidence was collected to estimate their phylogenetic relationship and taxonomic status. A total of 11 genera were sampled, including Brachygobius, Calamiana, Hemigobius, Mugilogobius, Pandaka, Pseudogobiopsis, Pseudogobius, Redigobius, Rhinogobius, Stigmatogobius, and Wuhanlinigobius, five of which were sequenced for the first time. A morphological phylogenetic tree was also reconstructed based on 35 characters. The molecular phylogenetic trees reveal that the Mugilogobius group contains four major clades. The present study also reveals that the adult male mouth size and forked sensory papillae row d can be considered as synapomorphies, and that the head pores on inter-orbital, anterior oculoscapular, and preopercular regions can be regarded as derived features among the Mugilogobius group. Furthermore, the absence of posterior oculoscapular pores may provide a clue for understanding the evolutionary history of the Mugilogobius group.