Recent studies have suggested that, in some bird species, predator-elicited vocalizations of males may function as an advertisement to conspecific females, but the evidence remains scarce. Male Japanese bush warblers, Horornis diphone, are known for producing long, conspicuous songs (continuous songs), in response to both predators and conspecific females. In this work, I investigated the temporal and spatial associations of continuous songs with the presence of females. In seven surveys throughout a breeding season, male singing activity of continuous songs was low before females arrived at the breeding ground but increased thereafter. Singing activity was higher in territories where the presence of females was confirmed than in those where it was not. Over multiple days, I observed a single male from sunrise to sunset and observed that the locations where the male sang continuous songs were concentrated at the locations where the females were observed. These temporal and spatial associations of the continuous song with the presence of conspecific females support the hypothesis that the continuous song is an advertisement to females. Additional observations showed that females did not fly away or enter the bushes in response to the continuous songs as they did when they encountered danger, and that the male sang continuous songs even towards females that were not considered their mating partner. These findings are inconsistent with the hypothesis that the continuous song is an alarm to alert their mates of danger. This study shows the possibility that male Japanese bush warblers advertise their quality to potential mates by singing continuous songs under dangerous situations.

Due to limited spawning seasons, embryogenesis of corals has not fully been studied and the embryonic origin of gastrodermis remains uncertain in Acropora. We herein examined how embryonic endodermal cells develop into the gastrodermis and mesentery of polyps in Acropora tenuis. In juvenile polyps, the gastrodermis invaginates to form mesenteries, both of which were stained with rhodamine-phalloidin, an anti-myocyte-specific enhancer factor 2 (anti-AtMef2) antibody, and an anti-lipoxygenase homology domain-containing protein 1 (anti-AtLoxhd1) antibody. Rhoda-mine-phalloidin staining was traced back to the endodermal cells of 60–85 hpf ‘pear’-stage embryos through the larval stage. AtMef2 appeared in the blastomeres of a 12-hpf ‘prawnchip’-stage embryo that was a variant U-shaped blastula with a narrow blastocoel. AtMef2 temporarily disappeared from the nuclei of 28-hpf ‘donut’-stage embryos and reappeared in the endodermal cells of 40-hpf early ‘pear’-stage embryos, suggesting a transition from maternal to zygotic expression of Mef2. The blastopore closed without the invagination of blastomeres. The gastrocoel collapsed and the Mef2-positive endoderm was dissociated into single cells in the well-developed blastocoel filled with yolk cells. The mesoglea appeared in the yolk cell layer. AtLoxhd1 was traced back to the endodermal cells of ‘pear’-stage embryos. In 11-dpf larvae, Loxhd1-positive endodermal cells elongated in the vicinity of the mesoglea to adhere to each other and form the gastroderm epithelium in larvae. Therefore, in this coral, the inner wall of U-shaped early embryos is the cellular origin of the gastrodermis. Inner wall-derived endodermal cells move independently toward the mesoglea, where cell-cell adhesion occurs to establish the gastrodermis.

Melampus “sincaporensis,” Auriculastra “duplicata”, and Ellobium chinense are Red-Listed snail species of the family Ellobiidae that co-occur on salt marshes in mainland Japan. Here, we report the genetic diversity and population structures of the former two taxa in comparison with our previous data on E. chinense for the evaluation of connectivity and conservation values of their local populations. Analyses of 655-bp or 652-bp sequences of the mitochondrial cytochrome c oxidase subunit I gene showed the highest genetic diversity and panmictic structure for M. sp. cf. sincaporensis throughout its geographic range in Japan, whereas they showed the highest level of genetic subdivision for A. sp. cf. duplicata. Our laboratory observation of egg masses and planktotrophic larvae of A. “duplicata” and reference to previous ontogenetic data for the species of Melampus suggested differences in their fecundity and pelagic larval duration, which apparently have led to the contrasting levels of population differentiation in the study species. Particular need of conservation efforts was identified for the isolated population of A. “duplicata” in the Ise–Mikawa Bay area to avoid local extinction and shrinking of the species' geographic range. In addition, we present molecular and morphological evidence that individuals of A. “duplicata” from mangrove swamps in the northern part of Okinawa Island represent an independent, surviving lineage of a different species.

In ectotherms, body size differences between latitudes resulting from shorter activity periods at higher latitudes may disappear due to higher growth rates. Although such latitudinal variations have been examined for various taxa, only a few studies have examined such variations in Japanese vertebrates. Hynobius nigrescens is widely distributed in the northern part of Honshu, Japan, and although their larval period in the wild is shorter at high latitudes, there is no latitudinal variation in the body size of juveniles in the wild. Therefore, larvae may grow faster at high latitudes, and they can grow into juveniles with body sizes similar to those at low latitudes. To examine whether the growth rate of larvae is faster at high latitudes, we reared groups at 15°C and 20°C. We found that the larval period was shorter and the growth rate was greater at high latitudes. Next, we examined whether the body sizes of reared individuals differed between latitudes. We found that the body sizes of metamorphosed juveniles were smaller at high latitudes. Some ectotherms are known to feed more at high latitudes, leading to the disappearance of latitudinal differences in body size between latitudes. In this study, we provided the same amount of food regardless of latitude, which might have caused the differences in body size between latitudes. Our study suggested that the high growth rate at relatively high latitudes can compensate for the short activity period, leading to similar body sizes of juveniles among latitudes.

We report an entoproct epibiotic on the surface of a sea spider (Pycnogonida). The pycnogonid was identified as Nymphon sp. (Nymphonidae). The entoproct was colonial, with three zooids, and was identified both morphologically and by a molecular phylogeny as Barentsia sp. (Barentsiidae). The largest zooid had eight tentacles and was about 0.7 mm long, smaller than for most colonial entoproct species. We determined partial sequences for the 18S rRNA and 28S rRNA genes from the entoproct. In an 18S-based maximum likelihood tree (1507 characters), the entoproct was the sister taxon to Barentsia gracilis.

Most true predators are generalists, and it is difficult to examine their actual diets in the wild. In many cases, the prey that experience a large impact are also not well known. Amphibians are generalist predators that prey mainly on insects, and amphibian species active beginning in spring are considered to prey on food resources other than insects in this season due to the low number of insects. The Japanese red-bellied newt (Cynops pyrrhogaster) is a generalist predator widely distributed in Japan and is known to frequently prey on amphibian eggs. Here, we showed that amphibian eggs are a precious food resource for C. pyrrhogaster in spring. The total weights of stomach contents were compared among seasons and we found that the total weights of stomach contents in spring were the same as those in summer. Also, we examined whether C. pyrrhogaster preys on amphibian eggs instead of insects in spring. The composition of the taxonomic groups in their stomach contents was compared among the seasons, and the indicator groups in their stomach contents were identified in each season. We found that they preyed not only upon insects but also upon amphibian eggs in spring, while they preyed mainly upon insects in summer and autumn. Our results suggest that C. pyrrhogaster preys on amphibian eggs to compensate for the shortage of food in spring when the number of insects is low.

Although genome editing techniques have made significant progress, introducing exogenous genes into the genome through knock-in remains a challenge in many organisms. In silkworm Bombyx mori, TALEN-mediated knock-in methods have been established. However, difficulties in construction and limitations of the target sequence have hindered the application of these methods. In the present study, we verified several CRISPR/Cas9-mediated knock-in methods to expand the application of gene knock-in techniques and found that the short single-stranded oligodeoxynucleotide (ssODN)-mediated method is the most effective in silkworms. Using ssODN-mediated methods, we established knock-in silkworm strains that harbor an attP sequence, a 50 bp phiC31 integrase recognition site, at either the BmHr38 (Hormone receptor 38) or Bmdsx (doublesex) locus. Additionally, we found that the long ssODN (lsODN)-mediated method successfully introduced the GAL4 gene at the doublesex locus in embryos. The present study provides valuable information on CRISPR/Cas9-mediated knock-in methods in silkworms, expanding the utility of genome editing techniques in insects and paving the way for analyzing gene and genome function in silkworms.

Sexual differences are commonly described as male-biased sexual dimorphism, female-biased dimorphism, and monomorphism. Nevertheless, detailed patterns of sexual dimorphism have only been explored in a few rodent species. We used geometric morphometrics to investigate patterns of sexual dimorphism, static allometry, and modularity in the skull of the Ethiopian white-footed mouse (Stenocephalemys albipes). Procrustes analysis of variance showed nonsignificant sexual dimorphism for the size and shape of the cranium. Significant sexual differences were detected in the mandibular size but not in the mandibular shape. Discriminant analysis indicated nonsignificant differences between sexes in the Procrustes distance of the cranium and mandible. Additionally, scatter allometric plots demonstrated similar patterns of allometry in both sexes. Allometric variation showed an elongation of the rostrum, widening of the zygomatic bones, and narrowing of the braincase. We also observed an expanding of the ascending ramus and shortening of the alveolar part for the mandible. These may indicate the existence of modularity in the skull, which was supported by modularity analysis. Although these trends have been thought to be a growth-related phenomenon of the mammalian skull, functional requirements may also have an effect. In conclusion, our results indicate sexual monomorphism in the skull of S. albipes, which may have no effect on allometric variation and modularity patterns.

The chestnut tiger butterfly, Parantica sita, can undertake long-distance migrations. They flap their wings for power flight and hold the wings for gliding; such repertoires of wing movements may be the key to explaining their excellent flight abilities. Measuring flight muscle activity using the electromyogram (EMG) is the first step toward understanding the neuromuscular mechanism of active flight control. Free-flight EMG measurements have, however, not been reported in butterflies. This study developed a method to acquire two-channel EMGs from free-flying P. sita. Stable EMG recordings were acquired using a monopolar electrode by attaching a small pre-amplifier to the dorsal mesonotum. The common-mode noise between channels was resolved by inserting a reference electrode into the mesonotum midline. The EMGs of five flight muscles were measured during free-flight and their activation phases were analyzed. The EMGs of all five muscles demonstrated a burst of spikes per stroke cycle, in contrast to the few spikes per cycle in the EMGs of hawkmoths, which would reflect the differences in wing kinematics and flight abilities. Further analyses, combining the technique developed in this study with high-speed videography, will clarify the neuromuscular mechanisms underlying the flight ability of P. sita.

Planktonic larvae of sessile metazoans select substrates for settlement based on various factors. Phallusia philippinensis larvae (Ascidiacea: Phlebobranchia: Ascidiidae) showed a negative preference for nano-scale nipple arrays (dense arrays of papillae-like nanostructures approximately 100 nm in height). To clarify whether ascidian larvae discriminate between nano-structure sizes for substrate selection, three different sizes of periodic nano-folds were fabricated using two-beam interference exposure, and substrate selection assays were performed on the three types of nano-folds and flat surfaces made of the same material. The substrate selection assay with 500–2000 freshly hatched larvae was carried out in nine replicates. The ascidian larvae showed a positive preference for flat surfaces and a negative preference for substrates with a height of 120 nm and pitch of 600 nm. Manly's selection indices differed with the size of the periodic nano-folds, supporting the hypothesis that larvae directly or indirectly discriminate between nano-scale differences upon settlement. The present study is the first to show that differences in nanostructure size affect substrate selection during larval settlement of sessile animals. The evolutionary adaptive reasons for larvae to discriminate between nano-scale structures and select substrates for settlement are potentially important to effectively manage ascidian biofouling using non-toxic methods.