We studied seasonal and diurnal variation of singing activity in a single-song repertoire species, the savannah sparrow (Passerculus sandwichensis), to explore the possible functions of the song. We observed a total of 47 territorial males (11 over three years, 28 over two years) on a daily basis. For all paired and unpaired males, and across all stages of the breeding season, we recorded total number of songs produced (singing persistence), the number of instances of aggression per 10-minute period in each focal male, and the number of songs per 3-minute period (singing intensity). Male savannah sparrows showed different singing activity in different behavioural contexts: 1) unpaired males sang more persistently, but less intensely than paired males; 2) paired males markedly reduced their singing persistence, but showed higher singing intensity, especially in the evening; 3) singing intensity of paired males peaked during the egg-laying period. The different patterns of singing activity in relation to time of day, nuptial status, and female breeding stage suggest that though the birds each possess only a single song type, the differential activities may play important roles in intra- and inter-sexual communication systems: it is argued that 1) intense singing by paired males in the evening plays a role in territory defense, while persistent singing by unpaired males in the morning plays a role in mate attraction; 2) especially high evening singing activity during the egg-laying period may relate to changed female behavior at the nest associated with the onset of incubation.

Balanus rostratus is a large cold-water acorn barnacle distributed around the northern coast of the Pacific Ocean. In Mutsu Bay, Aomori, Japan, B. rostratus, which adhere naturally to scallop shells, are cultured as food. However, current culture methods do not generate sufficient supplies to satisfy market demand. Knowledge of the physiology of B. rostratus reproduction is important for the development of more efficient aquaculture methods. Previous studies have suggested that fatty acids and their metabolites play an important role in barnacle reproduction and development; however, few studies have analyzed lipids, particularly during ovary maturation. Here we analyzed lipid content, lipid class, and fatty acid composition of B. rostratus ovary throughout the year. The clutch in the present study was observed once per year at the end of November. The lipid content increased as the ovary underwent maturation. The proportion of triacylglycerol increased with increasing lipid content. The proportions of myristic acid, arachidonic acid, EPA and DHA significantly decreased in December. By contrast, the proportion of these fatty acids in lipid extracted from larvae was high relative to lipid extracted from B. rostratus ovary in December. These findings suggest that these fatty acids are transferred from the ovary to the larvae. Our novel findings on lipid metabolism during ovary maturation in B. rostratus indicate the importance of lipids during reproduction. This information may be useful in establishing methods for the aquaculture of B. rostratus.

Historically, many local grey wolf (Canis lupus) populations have undergone substantial reductions in size or become extinct. Among these, the wolf population once living in Sicily, the largest island in the Mediterranean Sea, was completely eradicated by human activity in the early decades of the 20th century. To gain a better understanding of the genetic identity of the Sicilian wolf, we used techniques for the study of ancient DNA to analyze the mitochondrial (mt) variability of six specimens stored in Italian museums. We were able to amplify a diagnostic mtDNA fragment of the control region (CR) in four of the samples. Two of the samples shared the same haplotype, differing by two substitutions from the currently most diffused Italian wolf haplotype (W14) and one substitution from the only other Italian haplotype (W16). The third sample showed a previously unreported wolf-like haplotype, and the fourth a haplotype commonly found in dogs. All of the wolf haplotypes analyzed in this study belonged to the mitochondrial haplogroup that includes haplotypes detected in all the known European Pleistocene wolves and in several modern southern European populations. Unfortunately, this endemic island population, which exhibited unique mtDNA variability, was definitively lost before it was possible to understand its taxonomic uniqueness and conservational value.

Noninvasive genetic analysis is being used increasingly in field surveys. However, detecting large and middle-sized mammals, such as Carnivora species, using noninvasive samples, such as scat or hair, is time- and labor-intensive due to their low densities and elusive behaviors. As snow tracks are the most frequently encountered natural signs of terrestrial mammals in winter, we employed several methods to recover environmental DNA (eDNA) from snow tracks. We performed both DNA metabarcoding and Sanger sequence analyses, in combination with universal primers on the mitochondrial 12S rRNA gene for mammals and taxon-specific primers on the mitochondrial NADH dehydrogenase subunit 2 gene for Martes species (martens and sables in Mustelidae). Snow samples of four Martes melampus tracks, one Cervus nippon track, one Vulpes vulpes track, and the track of an unidentified Carnivora species were collected from a snowfall area in Kyoto, Japan, in February 2018. Regarding DNA metabarcoding analyses, the sequences of three Carnivora species (M. melampus, V. vulpes, and Canis lupus familiaris) and a deer (C. nippon) were obtained from their respective snow tracks. Using Sanger sequencing, eDNA on snow tracks was recovered at the species level except for M. melampus using universal primers, while eDNA of M. melampus was sequenced using Martes-specific primers. Snow track surveys in combination with eDNA techniques could dramatically improve the efficiency of monitoring and conservation of mammals.

The diversity of trunk muscle morphology of Salamandridae occupying different habitats (aquatic: Pachytriton labiatus; terrestrial: Tylototriton kweichowensis and Salamandra salamandra salamandra) was examined. Trunk muscles were dissected, and muscle weight ratios were quantified. The terrestrial species have larger abdominal trunk muscles than the aquatic species do. In contrast, the lateral hypaxial muscles are larger in the aquatic species. The M. rectus abdominis profundus is located subjacent to the M. rectus abdominis in the terrestrial species. In the aquatic species, the ventral muscle is composed of the M. rectus abdominis alone. The lateral hypaxial muscles in the aquatic species are suited to lateral bending during underwater locomotion in the denser aquatic medium. Larger abdominal muscles may be used in supporting body weight against gravity in the terrestrial species. The function of the M. rectus abdominis profundus may be to support the M. rectus abdominis in the terrestrial species. These findings suggest a divergent evolution of trunk muscle characteristics within the Salamandridae, which correlate with both habitats and modes of locomotion.

Anurans occupy a wide variety of habitats of diverse salinities, and their osmoregulatory ability is strongly regulated by hormones. In this study, we compared the adaptability and hormonal responses to osmotic stress between two kajika frogs, Buergeria japonica (B.j.) and B. buergeri, (B.b.), which inhabit coastal brackish waters (BW) in the Ryukyu Islands and freshwater (FW) in the Honshu, respectively. Both hematocrit and plasma Na⁺ concentration were significantly higher in B.j. than in B.b. when both were kept in FW. After transfer to one-third seawater (simulating the natural BW environment), which is slightly hypertonic to their body fluids, their body mass decreased and plasma Na concentration increased significantly in both species. After transfer, plasma Na⁺ concentration increased significantly in both species. We examined the gene expression of two major osmoregulatory hormones, arginine vasotocin (AVT) and atrial natriuretic peptide (ANP), after partial cloning of their cDNAs. ANP mRNA levels were more than 10-fold higher in B.j. than in B.b. in FW, but no significant difference was observed for AVT mRNA levels due to high variability, although the mean value of B.j. was twice that of B.b. Both AVT and ANP mRNA levels increased significantly after transfer to BW in B.b. but not in B.j., probably because of the high levels in FW. These results suggest that B.j. maintains high plasma Na⁺ concentration and anp gene expression to prepare for the future encounter of the high salinity. The unique preparatory mechanism may allow B.j. wide distribution in oceanic islands.

Serotonin (i.e., 5-hydroxytryptamine [5-HT]) plays a key role in stress responses in vertebrates. In mammals and teleosts, tryptophan hydroxylase (Tph), a rate-limiting enzyme in the biosynthesis of 5-HT, includes two paralogs: Tph 1 and Tph 2. The response of the Tphs to stress has been reported in mammals, but less is known about the responses of these enzymes to stress in fish. In the present study, we examined whether heat stress affects the mRNA expression of these Tphs in the brain of medaka fish (Oryzias latipes). We also determined the concentration of 5-HT in the brain, the mRNA expression of heat shock protein 90 alpha (Hsp90α) in the liver, plasma cortisol concentration, and blood glucose concentration in medaka. Whole-body exposure to repeated heat stress significantly decreased the mRNA expression of Tph1 and Tph2 in male and female medaka, whereas single heat stress did not affect the expression of either of the mRNAs. The 5-HT concentration also decreased significantly after repeated heat stress sessions in both sexes, but did not decrease after a single heat stress session. After single and repeated heat stress sessions, Hsp90α mRNA expression increased in both sexes; however, increments in the concentrations of plasma cortisol and blood glucose occurred in male, but not in female, medaka. These results suggest that both types of Tphs are involved in reducing 5-HT in the brain and are reliable indicators of chronic stress response in both sexes. However, stress responses in plasma cortisol and blood glucose concentrations differ between male and female medaka.

The genera AngustipesColosi, 1922 and LatipesColosi, 1922 were originally proposed as “groups” within the genus Vaginulus Ferrussac, 1822, and since their establishment they have been variously considered valid or invalid until they gained the ultimate status of genus. The descriptions of both genera are general and broadly inclusive, and this fact has complicated taxonomic recognition. Additionally, incomplete descriptions and difficult identification of characteristics in the name-bearing type specimens demonstrate the need to revisit the species and revise the two genera. Herein, we broaden the description of Latipes erinaceusColosi, 1922 with respect to the circulatory system, the radula, the jaw, the position of entry of the ligation duct in the bursa copulatrix in relation to the canal of the bursa, the origin of the muscle of the penial gland, along with the morphometric characteristics of the phallus, the penial gland, the pedal gland, and the bursa copulatrix. We also propose new differential diagnoses for the genera Angustipes and Latipes, limited to the essential characteristics that enable taxonomic recognition. Hence, we propose the assignment of the species L. erinaceus, Latipes rosilus (Thiele, 1927), Latipes ribeirensis (Thiele, 1927), and Latipes absumptus (Colosi, 1921) to the genus Angustipes, based on the presence of morphological characteristics attributable to this genus, such as the phallus being short and conical; the bursa copulatrix being sessile or short, and lacking a head; the ligation duct inserted near the canal of the bursa; as well as on the similarity in phallus morphology with Angustipes difficilisColosi, 1922, the type species of this genus.

The morphology and molecular phylogeny of Parabistichella multilineae sp. nov., which was discovered in soil from Gagye Beach, South Korea, were investigated. The new species is characterized as follows: body size about 200 × 47 µm in vivo, slenderly lanceolate and twisted; two ellipsoidal macronuclei and usually two globular micronuclei; two types of cortical granules; four or five frontoventral rows, of which rows 1 and 2 are conspicuously developed and rows 3 and 4 are fully extended; and caudal cirri are lacking. Phylogenetic analyses based on the SSU rDNA sequences showed that the new species clusters in a soft polytomy with P. variabilis and Uroleptoides magnigranulosus.

A new species of stygobiontic interstitial annelid, Diurodrilus kunii sp. nov., is described based on material collected from medium sand sediment (φ = 1.2–1.7) at groundwater level (40–100 cm in depth; 5–15 m inland from splash zone) in the intertidal beach slope on Ishikari Beach, facing the Sea of Japan, Hokkaido, Japan. The new species differs from six known congeners in the arrangement of the anterior-head ventral ciliophores, the degree of development in the primary and secondary toes, and the shape of the spermatozoa. We inferred the phylogenetic position of the new species among other congeners for which 18S rRNA, 28S rRNA, and COI gene sequences were available in public databases. This is the first representative of the genus from the Northwest Pacific.

From previous research, it is known that hermit crabs predominantly dwell in vacated gastropod shells. Several epibiotic taxa are known to live on gastropod shells inhabited by hermit crabs, including some species of the zoantharian genus Epizoanthus. Although many previous taxonomic studies have focused on hermit-crab-associated Epizoanthus species, and have resulted in the description of several species, gastropod-associated species have received comparatively much less attention. At least five Epizoanthus species associated with gastropods have been formally described, but some species have not been found or examined since their original description. In Japan, specimens on the gastropod mollusk Guildfordia triumphans have been found and examined in previous studies, but no formal taxonomic conclusions were made. In this study, we formally describe Epizoanthus rinbou sp. n. from southern Japan based on molecular phylogenetic analyses combined with morphological observations. Epizoanthus rinbou sp. n. is located within an Epizoanthus clade consisting of species associated with gastropods, hermit crabs, sea urchins, and barnacles, as well as non-associated Epizoanthus species. The present study highlights the utility of molecular phylogeny for understanding the diversity and relationships of gastropod-associated Epizoanthus species.