Zoological Science

Enucleated erythrocytes are characteristic of adult mammals. In contrast, fish, amphibians, reptiles, birds, and fetal mammals possess nucleated erythrocytes in their circulation. Erythroid maturation is regulated by erythropoietin (EPO) and its receptor (EPOR), which are conserved among vertebrates. In mammals, EPOR on the erythroid progenitor membrane disappears after terminal differentiation. However, in western clawed frog, Xenopus tropicalis, mature erythrocytes maintain EPOR expression, suggesting that they have non-canonical functions of the EPO-EPOR axis rather than proliferation and differentiation. In this study, we investigated the non-canonical functions of EPOR in Xenopus mature erythrocytes. EPO stimulation of peripheral erythrocytes did not induce proliferation but induced phosphorylation of intracellular proteins, including signal transducer and activator of transcription 5 (STAT5). RNA-Seq analysis of EPO-stimulated peripheral erythrocytes identified 45 differentially expressed genes (DEGs), including cytokine inducible SH2 containing protein gene (cish) and suppressor of cytokine signaling 3 gene (socs3), negative regulators of the EPOR-Janus kinase (JAK)-STAT pathway. These phosphorylation studies and pathway analysis demonstrated the activation of the JAK-STAT pathway through EPO-EPOR signaling in erythrocytes. Through comparison with EPO-responsive genes in mouse erythroid progenitors obtained from a public database, we identified 31 novel EPO-responsive genes indicating non-canonical functions. Among these, we focused on ornithine decarboxylase 1 gene (odc1), which is the rate-limiting enzyme in polyamine synthesis and affects hematopoietic progenitor differentiation and the endothelial cell response to hypoxic stress. An EPO-supplemented culture of erythrocytes showed increased odc1 expression followed by a decrease in polyamine-rich erythrocytes, suggesting EPO-responsive polyamine excretion. These findings will advance our knowledge of the unknown regulatory systems under the EPO-EPOR axis and functional differences between vertebrates' nucleated and enucleated erythrocytes.

A thorough understanding of the development of complex plumages in birds necessitates the acquisition of genetic data pertaining to the mechanism underlying this phenomenon from various avian species. The oriental honey-buzzard (Pernis ptilorhynchus orientalis), a tropical summer migrant to Northeast Asia, including Japan, exemplifies this aspect owing to the diversity of its ventral coloration and intra-feather barring patterns. However, genetic polymorphism responsible for this diversity has not been identified yet. This study aimed to investigate the link between dark-plumed phenotypes of this subspecies and haplotypes of the melanocortin-1-receptor (MC1R) gene. A draft sequence of MC1R was constructed using next generation sequencing and subsequently amplified using designed polymerase chain reaction (PCR) primers. The genome sequences of 32 honey-buzzard individuals were determined using PCR, and 12 MC1R haplotype sequences were obtained. Among these haplotypes, we found that unique haplotypes with nine non-synonymous substitutions and four or five synonymous substitutions in the coding region had a perfect correlation with the dark-plumed phenotype. The lack of correlation between the genotype of ASIP coding region and plumage phenotype reiterated that the dark morph is attributable to specific MC1R haplotypes. The absence of a correlation between genetic polymorphisms of MC1R and the intra-feather barring patterns, as well as the diversity observed within lighter ground color classes (pale and intermediate), implies the involvement of alternative molecular mechanisms in the manifestation of the aforementioned phenotypes.

Praesagittifera naikaiensis is an acoel flatworm that inhabits the sandy beaches in the intertidal zone of the Seto Inland Sea. This species carries Tetraselmis sp., a green unicellular chlorophyte, as a symbiont in its body, and depends on algal photosynthetic products to survive. However, the eggs of P. naikaiensis contain no symbiotic algae, and juvenile P. naikaiensis acquire symbionts from the surrounding environment through horizontal transfer after hatching, thereby establishing new symbiotic relationships in each generation. Other acoel species, Symsagittifera spp., also inhabit the Seto Inland Sea shores and acquire symbiotic green algae via horizontal transfers. To characterize their symbionts, these acoels were collected from a wide area of the Seto Inland Sea and partial nucleotide sequences of the chloroplast ribulose diphosphate carboxylase large subunit (rbcL) of the symbiotic algae were determined and used for molecular phylogenetic analysis. Symbionts of both P. naikaiensis and Symsagittifera spp. belonged to the genus Tetraselmis but were phylogenetically distant, and both species established symbiotic relationships with different symbionts even when they were sympatric. To test whether each species selects specific algae in the environment for symbiosis, we established algal strains from P. naikaiensis and Symsagittifera sp. symbionts and conducted uptake experiments on aposymbiotic juveniles of P. naikaiensis. The results suggest that symbiotic algae from Symsagittifera could be taken up by P. naikaiensis juveniles, but were unable to establish a normal symbiotic relationship with the juveniles.

The Coleoptera Cerambycidae (longicorn beetles) use wood under different states (living healthy, freshly snapped, completely rot, etc.) in a species-specific manner for their larval diet. Larvae of some Cerambycidae groups have mycetomes, accessory organs associated with the midgut that harbor fungal symbiont cells. The symbionts are thought to improve nutrient conditions; however, this has yet to be shown experimentally. To deduce the evolutionary history of this symbiosis, we investigated the characteristics of the mycetomes in the larvae of longicorn beetles collected in Japan. Lepturinae, Necydalinae, and Spondylidinae are the only groups that possess mycetomes, and these three groups' mycetomes and corresponding fungal cells exhibit different characteristics between the groups. However, the phylogenetic relationship of symbiont yeasts does not coincide with that of the corresponding longicorn beetle species, suggesting they have not co-speciated. The imperfect vertical transmission of symbiont yeasts from female to offspring is a mechanism that could accommodate the host-symbiont phylogenetic incongruence. Some Lepturinae species secondarily lost mycetomes. The loss is associated with their diet choice, suggesting that different conditions between feeding habits could have allowed species to discard this organ. We found that symbiont fungi encapsulated in the mycetomes are dispensable for larval growth if sufficient nutrients are given, suggesting that the role of symbiotic fungi could be compensated by the food larvae take. Aegosoma sinicum is a longicorn beetle classified to the subfamily Prioninae, which does not possess mycetomes. However, this species contains a restricted selection of yeast species in the larval gut, suggesting that the symbiosis between longicorn beetles and yeasts emerged before acquiring the mycetomes.

Sea urchins have a wide variety of symbionts on their body surfaces and inside their bodies. Copepods of the genus Clavisodalis (Taeniacanthidae) collected from the esophagus of sea urchins of the genera Diadema and Echinothrix in southern Japan were identified based on their morphological characteristics, and molecular analysis was conducted to determine whether genetic variation occurs in copepods from different localities and hosts. Morphological observations identified individuals from southern Japan as Clavisodalis sentifer Dojiri and Humes, 1982 , making this the first record of this species in the northern hemisphere and the first record of its genus in Japan. Morphological and molecular analysis suggested that the copepod specimens collected from multiple hosts across two genera would be the same species. Considering the typically observed high level of host specificity among taeniacanthid copepods, the utilization of hosts from two genera by C. sentifer is noteworthy.

The Japanese Archipelago hosts a rich butterfly fauna, and elucidating the genetic structures of multiple species is necessary to clarify their formation processes. This study aimed to reveal the genetic structure and distribution formation process of Parnassius citrinarius, which is widely distributed across the Japanese Archipelago from Hokkaido to Shikoku, through phylogeographic analysis based on the mitochondrial cytochrome c oxidase subunit I (COI) gene sequence. Thirty haplotypes were revealed from 311 individuals from 47 sites, indicating significant differences in the genetic structures between the eastern and western parts of the Japanese Archipelago. In Eastern Japan, multiple genetic clusters were found, with some sites harboring two clusters. The divergence times among populations in Eastern Japan were relatively recent, and no genetic differentiation was observed between regions, including between Hokkaido and Honshu, which are separated by a narrow strait. In contrast, in Western Japan, including Shikoku, unique genetic clusters were observed in each region. The phylogenetic relationships among populations were regionally clustered, and the divergence times were relatively ancient. The distribution and genetic structure of P. citrinarius in the Japanese Archipelago have been significantly influenced by temperature fluctuations and the presence of geographical barriers during the Pleistocene glacial-interglacial cycles, including the potential formation of refugia in Western Japan.

Some anguillid eels migrate thousands of kilometers from their spawning grounds, dispersing across vast geographic areas to fresh and brackish water habitats, where they settle and grow. Japanese eels (Anguilla japonica) and giant mottled eels (A. marmorata) are both found in Japan, although their distributions differ, and their exact distributions are poorly known. We assumed that topographic distribution patterns of Japanese and giant mottled eels must differ among and within rivers along the northwest coast of Kyushu, Japan. Environmental DNA (eDNA) analysis was conducted at 87 sites in 23 rivers. Japanese eel eDNA was detected in 19 rivers (82.6%) and that of giant mottled eels was detected in eight (34.8%). We detected giant mottled eel eDNA in five rivers where they were previously unknown. eDNA for Japanese eels was detected at six of nine sites in the north (66.7%), 13 of 23 sites in Omura (56.5%), and 37 of 55 sites in the south (67.3%). In contrast, giant mottled eel eDNA was detected at one of nine sites in the north (11.1%), no sites in Omura, and 15 of 55 sites in the south (27.3%). There was no correlation between eDNA concentrations of the two species at 10 sites in the five rivers where eDNA of both species was detected. These findings suggest differences in the distribution of the two eel species and the northern distributional limit of giant mottled eels in the area facing the East China Sea.

In holometabolous insects, the larval body is almost completely decomposed and reconstructed into the adult body during the pupal-pharate adult stages. Therefore, the total energetic cost of this process is a key thermodynamic quantity necessary for evaluating the benefit of their life history. Here, we measured whole-body thermal dissipation of single pupae of the fruit fly, Drosophila melanogaster, during the period from puparium formation to adult eclosion as a function of age, using a high-precision isothermal calorimeter at T = 298 K. The mass-specific energy consumption during the period from the onset of larval-pupal apolysis to adult eclosion was determined to be 2.3 kJ/g for an individual of mass (adult) = 1.0 mg, while it was observed to follow Kleiber's law for individuals smaller than mass (adult) = 1.0 mg. During the pupal-pharate adult period, in addition to the U-shaped variation, several characteristic thermal dissipations related to various events, including somatic muscle contractions, ecdyses, pulsatile hormone secretion in a pharate adult, and vaporization of the exuvial fluid, were observed. The periodic bursts in the pharate adult stage grew exponentially, suggesting that the positive feedback in the metabolic system synchronized with the progression of development, making the energy consumption in this stage more efficient. The present study showed that high-precision calorimetry is a powerful and credible method for measuring not only the total energy spent during development but also the energy spent during every specific developmental event in an organism.

The circadian system comprises multiple clocks, including central and peripheral clocks. The central clock generally governs peripheral clocks to synchronize circadian rhythms throughout the animal body. However, whether the peripheral clock influences the central clock is unclear. This issue can be addressed through a system comprising a peripheral clock (compound eye clock [CE clock]) and central clock (the optic lobe [OL] clock) in the cricket Gryllus bimaculatus. We previously found that the compound eye regulates the free-running period (τ) and the stability of locomotor rhythms driven by the OL clock, as measured by the daily deviation of τ at 30°C. However, the role of the CE clock in this regulation remains unexplored. In this study, we investigated the importance of the CE clock in this regulation using RNA interference (RNAi) of the period (per) gene localized to the compound eye (per ^CE-RNAi). The per ^CE-RNAi abolished the compound eye rhythms of the electroretinogram (ERG) amplitude and clock gene expression but the locomotor rhythm driven by the OL clock was maintained. The locomotor rhythm of the tested crickets showed a significantly longer τ and greater daily variation of τ than those of control crickets treated with dsDsRed2. The variation of τ was comparable with that of crickets with the optic nerve severed. The τ was considerably longer but was comparable with that of crickets with the optic nerve severed. These results suggest that the CE clock regulates the OL clock to maintain and stabilize τ.

Western sand lance, Ammodytes japonicus, is known to have an estivation period, in which they cease feeding and stay in the sand from early summer to late autumn, followed by gonadal maturation. During the feeding period prior to estivation, they swim in daytime and spend the night in the sand. Before they start swimming, they show a typical behavior of head-exposing from the sand, which is likely to be related to foraging and predation avoidance. Our previous study revealed that melatonin regulates such diel behavior of this species. To elucidate the mechanisms of behavioral regulation throughout the life cycle of this sand lance, the present study examined the changes in behavior and melatonin secretion toward the estivation period. Both head-exposing and swimming behaviors were frequently observed at the transition period toward estivation. On the other hand, occurrence of these behaviors was suppressed just before entering estivation. Subsequently, it was found that plasma melatonin concentration was about three times higher at night than in daytime in the non-estivation period, while it was retained at high levels throughout the day in the estivation period. These results indicate that diurnal swimming behavior of sand lance from the feeding to estivation periods is associated with the daily cycle of melatonin secretion.