We report on the behavior of a deep-sea tanaidacean, Gigantapseudes sp. (Apseudomorpha: Gigantapseudidae), recorded at the depths of 6446–6447 m by the manned submersible Shinkai 6500. From recordings of at least three individuals walking on the sea floor, we confirm that Gigantapseudes sp. is epibenthic, as previously inferred from leg shape. One individual was recorded entering a hole. All individuals in the videos kept pereopods 4 raised from the seafloor while walking, implying that those legs have a function other than for walking, such as mechano- or chemoreception, or posture control. Our in situ observations of behavior are the first for any deep-sea tanaidacean and illustrate the importance of recording high-resolution videos in the deep sea and archiving them for future use. Our identification of Gigantapseudes sp. from video footage provides the first record of this genus from Japanese waters and extends the northern limit of the known generic distribution.

Extracellular matrix (ECM) plays key roles in shaping fates of stem cells, not only by providing a suitable niche but also by mediating physical and biochemical cues. Despite intensive investigations on regeneration, the roles of ECM in fate determination of stem cells in animals with great regenerative potency, such as planarian, have remained unclear. Here, we developed a method for decellularizing and isolating extracellular matrix from planarians. Although the isolated scaffold appears translucent, it contains all the internal features resembling those of the structure of intact planarians, and we thus called it the “ECM-body”. Nuclear staining demonstrated that the ECM-body contains very few or no remaining cells. Histological sections displayed well-preserved morphological integrity of the specimen. Scanning electron microscopy showed a porous surface on the ECM-body, potentially suitable for housing cells. Furthermore, our preliminary experiment suggested that ECM-body can be utilized as a biomimetic scaffold for cell culture as it may support survival of injected neoblasts.

Acoel flatworms are simple bilaterians that lack digestive lumens and coelomic cavities. Although they are a significant taxon for evaluating the evolution of metazoans, suitable species for biological experiments are not available in Japan. We recently focused on Praesagittifera naikaiensis, which inhabits the sandy shores of intertidal zones in the Seto Inland Sea in Japan, as a candidate for a representative acoel species to be used in experiments. However, reports on its distribution range remain limited. Here, we surveyed the habitats of P. naikaiensis on 108 beaches along the Seto Inland Sea. Praesagittifera naikaiensis is reported here from 37 sites (six previously known and 31 newly discovered sites) spread over a wide area of the Seto Inland Sea, from Awaji Island in Hyogo Prefecture to Fukuoka Prefecture (364 km direct distance). Based on the mitochondrial cytochrome oxidase subunit I (COI) gene haplotypes, we evaluated the genetic diversity of 145 individuals collected from 33 sites. Out of 42 COI haplotypes, 13 haplotypes were shared by multiple individuals. The most frequent haplotype was observed in 67 individuals collected from 31 sites. Eight other haplotypes were detected at geographically distant locations (maximum of 299 km direct distance). Multiple haplotypes were found at 32 sites. These results demonstrate that sufficient genetic flow exists among P. naikaiensis populations throughout the Seto Inland Sea. Molecular phylogenetic analysis of the COI haplotypes of P. naikaiensis revealed that all specimens were grouped into one clade. The genetic homogeneity of the animals in this area favors their use as an experimental animal.

Genetic deviation between two deep-sea snailfishes, Careproctus pellucidus from the Pacific Ocean and Careproctus rastrinus from the Okhotsk Sea, of the Careproctus rastrinus species complex was analyzed, based on the nucleotide sequences of the mitochondrial cytochrome b (Cytb) gene. Our sampling revealed the occurrence of individuals with C. rastrinus-type Cytb genes off northeastern Japan, in the northwestern Pacific. Most of these individuals were collected from an area off Miyagi Prefecture, while few individuals were collected from areas to its north (off Iwate Prefecture) and south (off Fukushima and Ibaraki Prefectures). Phylogeographic analyses based on nucleotide sequences of the first intron region of the nuclear S7 ribosomal protein gene (S7) and 11 microsatellite loci indicated little genetic deviation between individuals with C. pellucidus-type and C. rastrinus-type Cytb genes in the area off Miyagi Prefecture. Significant genetic differences between the Pacific Ocean and the Okhotsk Sea populations may be attributed to unidirectional migration due to a greater current from the latter to the former. In addition, peritoneum and stomach colors of 10 and 17 specimens collected from the Pacific Ocean and the Okhotsk Sea, respectively, were evaluated quantitatively. The colors were significantly different for the two populations; however, they appeared to overlap. Although only one Pacific individual with a C. rastrinus-type Cytb gene was available for color evaluation, it was more similar to Pacific individuals than to Okhotsk Sea individuals. These results suggest inbreeding between C. pellucidus and C. rastrinus in the Pacific Ocean, off Miyagi Prefecture.

The population dynamics of lancelet Branchiostoma japonicum are reported for six sampling sites in the Seto Inland Sea, Japan, for November 2007–2016. Lancelet growth and life span varied spatially, being faster and shorter, respectively, at Stn 3 (off Marugame, western Bisan-seto) than at other sites; average body length at Stn 3 was 36.1 mm for 2-year-old lancelets, and 38.9–42.1 mm for 4-year-old lancelets at sites 1, 2, 4 and 6. Stepwise multiple regression analysis revealed adult growth rate to be significantly positively related to chlorophyll a concentration, and negatively correlated to lancelet density. Density of newly settled and adult lancelets varied spatially. Chlorophyll a concentration best predicted the density of newly settled lancelets, and sediment particle size best predicted that of adults, indicating that factors affecting lancelet density differ during their life history.

Intelectins (Itlns) are secretory lectins found in several chordate species that recognize carbohydrates on the bacterial cell surface depending on Ca²⁺ . In newly hatched larvae of Rana ornativentris (R. orn), Bufo japonicus formosus (B. jpn), and Cynops pyrrhogaster (C. pyr), an anti-Itln monoclonal antibody (mAb) labeled a subset of epidermal cells in whole-mount immunocytochemical assays. In western blot analyses, the mAb identified protein bands at approximately 33–37 kDa in the larval extracts and concentrated larval culture media. Using RT-PCR and RACE techniques, we isolated cDNAs from newly hatched larvae that encoded proteins of 343 (R. orn), 336 (B. jpn), and 337 (C. pyr) amino acids having 70%, 71%, and 60% identities with that of the Xenopus laevis embryonic epidermal lectin (XEEL), respectively. The proteins, designated REEL, BEEL, and CEEL, showed characteristics conserved among reported Itln proteins, and their amino acid sequences following the signal peptides were identical to those of the N-terminal peptides determined on Itln proteins in the respective larval extracts. Recombinant REEL (rREEL), rBEEL, and rCEEL proteins produced by HEK-293T cells were homo-oligomers of 34–37 kDa subunit peptides, which were similar to the Itlns found in the newly hatched larvae. The rEELs showed carbohydrate-binding specificities similar to that of XEEL and agglutinated Escherichia coli and Staphylococcus aureus cells depending on Ca²⁺ . These results suggest that REEL, BEEL, and CEEL are Itlns produced and secreted by epidermal cells of R. orn, B. jpn, and C. pyr larvae, respectively, and that Itlns have a conserved role as pathogen recognition molecules in the larval innate immune system.

The hynobiid salamander genus Hynobius is a basal taxon in the urodeles that plays a key role in understanding the evolution of sexual dimorphism in urodeles. We examined the age (juveniles vs. adults) and sexual differences in the external and skeletal morphology of a Japanese lentic breeding salamander, Hynobius setouchi. We found that juveniles had larger heads than those of adults, and most characters were larger in adult males than in females, except for trunk size. This was also observed in skeletal morphology, although some tail characters differed only in the skeleton. We suggest that intra- and interspecific comparisons of the external and skeletal characters of salamanders should consider age, sexual, and seasonal differences.

Crickets were arbitrarily fed a caffeine-containing diet during their lifetime or were orally administered caffeine in solution once during the conditioning session. These chronically or acutely treated crickets were used to test whether there were positive effects of caffeine on their learning performance. The effects of chronic exposure to caffeine on crickets' growth were also examined by measuring their body weight and counting the number of adults that emerged from larvae. Chronic treatment with relatively high concentrations of caffeine (5 mg/g and 0.5 mg/g in the diet) impaired the growth of crickets. Mortality was higher with 5 mg/g caffeine, and the survivors could not attain the final molt (eclosion). Body weight gain and eclosion rate were also negatively influenced in the group fed 0.5 mg/g caffeine. The olfactory learning paradigm was used to examine the effects of chronic caffeine treatment. Of the caffeine concentrations tested (0.5 µg/g to 0.5 mg/g), significant improvement in long-term memory (LTM) formation was observed only with 50 µg/g caffeine. Acute effects on olfactory learning were examined after oral administration of 0.1 nM to 10 µM caffeine solutions during the conditioning session. Administration of caffeine ≥ 1 nM consistently resulted in a significant improvement in LTM formation. These results suggest that chronic exposure to caffeine enhances learning performance in crickets at a specific dose (50 µg/g in the diet), although it inhibits their growth at higher doses (≥ 0.5 mg/g). In contrast, acute oral administration of caffeine in naive crickets may enhance their learning performance even at a very low (nanomolar) concentration.

For marine benthic animals, the selection of substrate by larvae is important for their survival, with early post-settlement mortality often being affected by the microenvironment where they settle. We tested the substrate preference of the larvae of the ascidian Phallusia philippinensis toward nine commercially available substrates. In the present assay, the larvae settled on one of four substrates for seven substrate combinations with different wettabilities; we counted the number of settled larvae on each of the four substrates, and Manly's selection indices were compared to determine the preference rank of each substrate. Larvae significantly preferred more hydrophobic and oleophilic substrates to hydrophilic and oleophobic ones. While it is uncertain how the larvae detect the properties of the substrate surface, they might be able to sense the physical force, such as stickiness and repellent force. Although a hydrophobic surface is not common in a natural marine environment, the use of hydrophobic materials (as flypaper-like tools) for ascidian larvae might help to prevent the settlement of non-indigenous ascidians in aquaculture facilities.

Camponotus japonicus uses basiconic antennal sensilla (s. basiconica) to sense a colony-specific blend of species-specific cuticular hydrocarbons (CHCs). The inner portion of the s. basiconica is filled with sensillar lymph and chemosensory proteins (CSPs) presumed to transport CHCs to olfactory neuron receptors. Although 12 CSPs have been found in C. japonicus antennae, we focused on CjapCSP1 and CjapCSP13. The molecular basis of CSP1 function was explored by observation of its structure in solution at pH 4.0 and 7.0 through circular dichroism (CD) and X-ray solution scattering. Although the secondary structure did not vary with pH change, the radius of gyration (Rg) was larger by 5.3% (0.74 Å increase) at pH 4.0 than at pH 7.0. The dissociation constant (K_d) for CjapCSP1 measured with a fluorescent probe, 1-N-phenylnaphthylamine, was larger at pH 4.0 than at pH 7.0, suggesting that acidic pH triggers ligand dissociation. In contrast to CjapCSP1, the Rg of CjapCSP13 was slightly smaller at pH 4.0 than at pH 7.0. Western blotting and immunohistochemistry with protein-specific antisera revealed that both CjapCSP1 and CjapCSP13 are detected in the antennae, but differ in their specific internal localization. Binding to four compounds, including the ant CHC (z)-9-tricosene, was examined. Although both CjapCSP1 and CjapCSP13 bound to (z)-9-tricosene, CjapCSP13 bound with higher affinity than CjapCSP1 and showed different binding properties. CjapCSP1 and CjapCSP13 are synthesized by the same cells of the antenna, but function differently in CHC distribution due to differences in their localization and binding characteristics.

Species of the predatory gastropod genus ReishiaKuroda and Habe, 1971 (Muricidae) inhabit intertidal rocky shores in East Asia. Due to their highly variable external shell morphology, the taxonomy of this genus at species-level is still in need of re-evaluation. Using DNA-based delimitation methods, we aimed to ascertain the number of species of Reishia along the coasts of China and adjacent Asian areas. Also, we looked for diagnostic traits using morphology-based statistical approaches. Our genetic data suggest that the studied individuals comprised two separate species of a Reishia complex in this region, in contrast to the previously proposed four or more taxa. This conclusion is further supported by statistical analyses of shell morphological characteristics. The morphospecies R. bronni (Dunker, 1860), R. jubilaea (Tan and Sigurdsson, 1990), and R. luteostoma (Holten, 1803) were assigned to a single taxon, indicating that they might be synonyms of the same species. The morphospecies R. clavigera (Küster, 1860) singly formed one group, suggesting that it is likely a valid name. The estimated divergence time of the two identified taxa indicates that speciation might have been associated with the sea level and temperature fluctuations during the Plio-Pleistocene period. Our study on Reishia species provides crucial information for further research on the ecology, evolutionary biology, and conservation of this genus.