The biota of the southern part of the Ryukyu Archipelago (South Ryukyu) is considered to have been formed relatively recently by organisms that migrated from Taiwan Island and continental China. However, phylogeographic patterns of insect species in South Ryukyu remain largely unexplored. In this study, we elucidated the intraspecific phylogeny and divergence time of Vagitanus terminalis, a small cicada species distributed from continental China to Taiwan Island and South Ryukyu. We conducted phylogenetic analyses using two mitochondrial and two nuclear gene sequences, and genome-wide DNA sequences and SNPs obtained through MIG-seq. We found that V. terminalis populations from Sichuan in continental China and Taiwan Island were genetically closely related despite the large geographical distance between them. In contrast, in South Ryukyu substantial genetic differentiation was detected between Yaeyama Islands and Miyako Island. The Yaeyama Islands populations were estimated to have diverged from Miyako Island and Taiwan-continental China populations approximately 2.4 million years ago (confidence interval: 1.1–4.0), during Early Pleistocene when the formation of the Ryukyu Archipelago began. These results suggested that colonization to the two island areas in South Ryukyu occurred at different times and via different routes, although precise phylogenetic relationships and divergence times among these populations and Taiwan-continental China populations remain to be clarified through additional taxon sampling and sequence data. Overall, our findings revealed substantial genetic differentiation among non-endemic insect populations in South Ryukyu, including the presence of relict populations with an ancient origin.
Fissiparity, a form of asexual reproduction, involves the loss of significant body parts followed by regeneration. In echinoderms, this mode of reproduction has been documented in numerous species, particularly among holothurians, asteroids, and ophiuroids. Fissiparous brittle stars typically exhibit six-fold symmetry and restore this body plan by regenerating the missing half of the disk and three arms after fission. However, the detailed regeneration process and its dynamics in natural populations remain poorly understood. Here, we characterized post-fission regeneration in the brittle star Ophiactis savignyi by defining eight stages based on external morphology and histological features. Our observations revealed that, during early disk regeneration, the paired lateral arm primordia developed prior to the central primordium, suggesting that regenerating individuals may transiently pass through a five-armed configuration before re-establishing six-fold symmetry. We further quantified the frequency of each regeneration stage in a wild population and examined its relationships with season and body size. Early regeneration stages were less frequent from winter to spring and among large individuals, indicating that fissiparity is more active during warmer periods and in smaller individuals. In addition, swelling of interradial regions containing gonads was observed in some large individuals, suggesting a size-dependent life-history strategy in which asexual reproduction predominates in smaller individuals and sexual reproduction becomes more prominent with growth.
High-altitude environments expose poultry to low temperature, hypoxia, and strong UV radiation, but how combined environmental gradients shape plumage lightness remains insufficiently resolved. Using Qiangshan cloud chickens from Wenchuan, we analyzed 150 dorsal plumage samples collected along 900–2800 m. Plumage lightness (L-value) was measured by colorimetry, and eumelanin content was quantified by alkaline dissolution spectrophotometry. Five environmental variables (UV index, altitude, mean annual temperature, annual precipitation, and forest coverage) were integrated from field monitoring, meteorological records, and remote sensing data. We combined correlation analysis, hierarchical regression, principal component analysis (PCA), path analysis, and interaction surface modeling to partition direct, indirect, and synergistic effects. L-value was negatively associated with altitude and melanin content, and the melanin-L-value relationship remained stable across altitude strata. PCA summarized environmental variation into PC1 (UV index, altitude, temperature) and PC2 (precipitation, forest coverage), jointly explaining 88.4% of L-value variance. Path analysis showed that melanin exerted a masking effect on the PC1 pathway while partially mediating the PC2 pathway (mediation proportion 76.2%). A significant PC1 × PC2 interaction indicated non-additive control of plumage lightness under combined environmental extremes. These results support a mechanism in which high-altitude stress promotes melanin deposition and darker plumage as a photoprotective and thermoregulatory adaptation, and they highlight the importance of environmental interactions in shaping poultry phenotypic responses.
Insect wings often bear dense arrays of nanoscale protrusions known as nanopillars that confer functions such as water repellency, self-cleaning, and antibacterial activity. However, how these cuticular nanostructures develop remains poorly understood. Here, I investigate the development of wing nanopillars in the dragonfly Anax parthenope and compare it with that in the cicada Hyalessa maculaticollis. Using electron microscopy, I examined the formation of the wing surface from early cuticle deposition to post-ecdysial stages. In the dragonfly, the wing surface remained smooth throughout cuticle formation and nanopillars appeared only several hours after ecdysis, indicating that they likely arise through physicochemical self-organization of wax-based lipids secreted onto the wing surface. In contrast, nanopillars were already present on pharate wings of the cicada, indicating that they form before ecdysis, although the precise timing and mechanism remain unresolved. These findings show that superficially similar nanostructures on insect wings can differ in developmental timing and may arise through different developmental routes, highlighting the diversity of mechanisms underlying cuticular surface patterning.
This study examines how testosterone levels and kisspeptin gene expression are affected in male Syrian hamsters following pinealectomy and photoperiod modification. By examining the rhythmic changes in these parameters under long (16L: 8D) and short (8L: 16D) photoperiods, we aim to elucidate the role of melatonin and photoperiod in regulating seasonal reproductive physiology. One hundred and ninety-two male Syrian hamsters were equally divided into four groups (n = 48/group). Two groups were pinealectomized (pinx) and the other two groups were sham-operated (control). Treatment and control groups were then exposed to either a long photoperiod (LP) of 16 hours light/8 hours dark or a short photoperiod (SP) of 8 hours light/16 hours dark for 30 days and then sampled at specific temporal intervals (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) at which hypothalamic tissues and serum samples were collected. Kiss1 mRNA expression and protein levels were measured using qPCR and Western blotting, while testosterone and melatonin levels were determined via ELISA. Under a long photoperiod, Kiss1 mRNA expression and protein levels exhibited significant diurnal variation, peaking during the light phase (P < 0.05). Pinealectomy disrupted these rhythms, leading to altered testosterone levels. Under a short photoperiod, a marked change in Kiss1 expression and testosterone was also observed, emphasizing photoperiod and melatonin's role in synchronizing reproductive rhythms. These findings highlight the central role of melatonin not just in modulating levels, but also in synchronizing the daily reproductive rhythms necessary for seasonal adaptation.
Small organisms such as water fleas and nematodes change their body stiffness in response to external stimuli and environmental alterations. Atomic force microscopy (AFM) is commonly used for stiffness measurement. However, the compression of the object by the cantilever is performed in the same direction as the observation, and this makes it difficult to observe the deformation of the object. Furthermore, the placement of the cantilever relative to the object is spatially quite limited and the driving distance is also very short: these limitations complicate measurements in organisms of various shapes and sizes. Here, we developed a new stiffness measurement technique using a cantilever attached to the tip of a micromanipulator, thereby overcoming these limitations of conventional AFM. During the compression of the object vertically downward, we observed the deformation from the side using a microscope tilted at 90°. The cantilever strain was monitored directly under the microscope rather than using the conventional optical reflection method. We used this approach to measure the stiffness of three small animal species, i.e., a tardigrade (Grevenius myrops), a nematode (Caenorhabditis elegans), and a water flea (Daphnia magna). We videotaped the compression of these organisms, and determined the strain distance of the object and cantilever from the images at different time points. A stress-strain curve was analyzed by plotting and Young's modulus was obtained as the curve's slope. Thus, Young's modulus data could be reliably obtained for varied organisms using the same device. This technology will facilitate measurements of stiffness of various small organisms.
Cerebratulus sardonyx sp. nov. is described from a single specimen collected at Kita-Koho Seamount on the Kyushu-Palau Ridge, a major submarine arc extending from southern Japan into the Philippine Sea. The specimen was obtained at a depth of 454 m using the remotely operated vehicle KM-ROV during cruise KM24-03C of the R/V Kaimei. An integrative taxonomic approach-combining external morphology of the living specimen, histology, and molecular phylogenetic analyses-was used to characterize the species. The new species is readily distinguished from all congeners by its vivid reddish-brown body crossed by numerous sharply defined opaque white transverse rings, together with a uniquely patterned head bearing orangish patches and alternating white and brown transverse bands. Phylogenetic analysis places the species within a clade referred to as Lineage J in a previous study, corresponding to the Cerebratulus clade. This discovery represents the first record of Cerebratulus Renier, 1804 from Japanese seamounts and the first described nemertean from the Kyushu-Palau Ridge.
Settlement, the transition from planktonic life to sedentary life, is a critical phase in the life cycle of corals, and is an important factor for population dynamics influencing new recruitment. However, previous studies on coral metamorphosis and settlement have overlooked actual processes from swimming to settlement on substrates. In this study, we discovered unique behavior of planula larvae of the coral Acropora that led to settlement on substrates covered by biofilms and we thus termed "pre-settling behavior". Pre-settling behavior consisted of three distinct steps in the sequence: (1) random crawling, (2) circular crawling, and (3) upright pausing. These steps were thought to correspond to the search for, evaluation of, and confirmation of appropriate settlement positions, respectively. Crawling speed and trajectories were analyzed, and their features are described. Since settlement occurs as adhesion of the aboral side to the substrate, it is conceivable that upright pausing is essential for metamorphosis to settled polyps as normal development. Frequent abortion of pre-settling behavior at any step to return to free swimming provides a view of repeated trials to seek settlement positions. Circular crawling in the counterclockwise direction was performed by forward movement with body curvature which determined the circle's radius, together with clockwise body spinning without forming helical trajectories. This movement suggests that changes of body curvature positions are synchronized with spinning to produce smooth, circular trajectories. The discovery of pre-settling behavior may contribute to understanding of settlement mechanisms and to coral seedlings production by elevating the settlement efficiency.
Ultrastructural studies of oogenesis in Heterotardigrada are limited. Here, we examined the ovarian organization of Actinarctus doryphorus using transmission electron microscopy and three-dimensional reconstruction. Four female specimens at different developmental stages, including a juvenile, were analyzed. The ovary is a single sac-like structure dorsally above the digestive tract, partially supported by longitudinal dorsal muscles. Early germ cells form anterior clusters interconnected by cytoplasmic bridges. Previtellogenic oocytes contain numerous mitochondria clustered around a prominent nuage, suggesting a conserved role in piRNA-mediated genome protection. Vitellogenic oocytes occupy the posterior half of the ovary, with the posterior-most cell developing as the egg and the remaining cells functioning as nurse cells. Nurse cells form a syncytium, and ER-rich accessory cells at the periphery likely contribute to yolk and material transfer. Large aggregations of lysosome-rich vesicles in peripheral ovarian cells suggest apoptotic degeneration of trophocytes and ER-rich cells after completing their function. These findings reveal that A. doryphorus exhibits polytrophic meroistic oogenesis with syncytial nurse cells, ER-rich accessory cells, and nuage-containing previtellogenic oocytes. This study provides the first comprehensive description of oogenesis in a heterotardigrade and highlights conserved mechanisms for cytoplasmic and genomic support during gametogenesis, suggesting avenues for exploring the evolutionary diversification of reproductive strategies in tardigrades.
With the globalization of human activities, invasions of non-native organisms into new habitats have increased. The masked palm civet (Paguma larvata) is an invasive alien species in Japan, with previous mitochondrial DNA studies indicating that Taiwan might be the source of the Japanese population. In addition, nuclear microsatellite data have shown a large genetic difference between the populations on Honshu and Shikoku islands. To further clarify the migration and expansion histories of this species, we analyzed the population genetics of P. larvata in Japan and Taiwan by using genome-wide SNP (single nucleotide polymorphism) data generated through ddRAD-seq. We found the previously unstudied population in western Honshu to be genetically closely related to that in central Honshu, with low genetic diversity due to a recent range expansion. The populations in Japan were genetically differentiated among Shikoku, western and central Honshu, and eastern Honshu. By admixture analysis, the Honshu and Shikoku populations showed affinity with one or two of them. Overall, the genetic diversity in Japan was lower than that in Taiwan, suggesting a founder effect during introduction from Taiwan. Within Japan, relatively higher variation in the eastern Honshu (especially Gunma Prefecture) populations indicated multiple introductions and/or different genetic lineages from those in Taiwan. Our study provides insights into the introduction history and migration routes of this alien species that can enhance management plans.
To understand the adaptation of body surface structures, the ultrastructure and optical properties of exumbrellar tissue were examined in cnidarian jellyfish, in which the epidermis is directly exposed to the external environment. The microscopic morphology of the exumbrellar epidermis varies from species to species. It can be flat or uneven, with/without cellular bulges, cilia, or an array of microvilli. Rigorous coupled wave analysis indicates that the light reflectance on the exumbrellar surface is expected to be very low owing to the very small differences in the refractive indices between exumbrella and seawater. The nanostructures modeled from the microvillar array of Spirocodon saltatrix reduced the reflection of light with large incident angles, which potentially affected the visibility of the contour of the umbrella. Therefore, the microvillar array may serve functions comparable to those of the nano-scale nipple array in some other metazoans. Moreover, an uneven pitch (distance between the apexes of neighboring microvilli) in the microvillar array potentially enhances the anti-reflection property. Although the difference in reflection reduction was only slight, this may be an example of unevenness in real biological structures that enhances the functional properties of the nanostructure.
Paqr7 and paqr8 are genes encoding membrane progestin receptor αand β (mPRα and mPRβ, respectively), which are currently classified as members of the progestin and adipoQ receptor (PAQR) family. For mPRα, the first identified mPR gene, there are two paralogs in zebrafish, paqr7a and paqr7b. In order to elucidate the physiological functions of the mPR subtypes, we created gene knockout (KO) fish by editing seven paqr genes in zebrafish and analyzing their phenotypes. The null-mutant strains of paqr7a, paqr7b, and paqr8 presented no significant abnormalities in reproductive functions. Thus, we generated a triple-gene knockout (TKO) strain of these highly related genes. The TKO strain had reduced fecundity and a high percentage of abnormal embryos. The embryos exhibited various types of abnormal morphology. In histological sections, a reduction in the number of nucleoli in germinal vesicles was observed. Additionally, the distribution of the nucleolus was abnormal. The amount of 18S and 28S ribosomal RNAs in the oocytes significantly increased. These analyses indicate that paqr7a, paqr7b, and paqr8 are responsible for the production of the nucleolus, which is necessary for supplying the proper number of ribosomal RNAs into the cytoplasm of oocytes. Abnormal embryo development resulted from low-quality eggs in TKO zebrafish, suggesting that paqr7a, paqr7b, and paqr8 mutations affect oogenesis. These results indicate that Paqr7a, Paqr7b, and Paqr8 are required for the preparation of the nucleolus during oogenesis. Insufficient formation of the nucleolus resulted in a higher quantity of ribosomal RNA in the cytoplasm, causing abnormal embryo development.
Reconstructing ancestral reproductive systems is crucial for understanding the evolution of bilaterian body plans. Yet, despite extensive research on some organ systems of the last common bilaterian ancestor, the evolution and development of reproductive systems remain poorly understood. To address this gap, we investigated postembryonic sexual development in the xenacoelomorph Hofstenia atroviridis, a basal acoel species belonging to the infraorder Prosopharyngida. We reared H. atroviridis from eggs to adults and described the ontogeny of its reproductive organs using histology and immunohistochemistry with muscle and neural markers, along with observations of mating behaviors in each size class. Hofstenia atroviridis is a protandrous simultaneous hermaphrodite whose sexual maturity correlates with body size rather than age, as observed in Hofstenia miamia. The male copulatory system comprises a seminal vesicle, granular vesicle, penis, and a single copulatory stylet, surrounded by regionally specialized musculature and innervation. In contrast, the female system consists of paired, asaccate ovaries with large, follicle cell-bound oocytes, lacking a discrete gonopore or copulatory organ. Fertilization occurs internally through traumatic insemination, and eggs are likely released through the mouth. Our findings reveal that the male reproductive system of H. atroviridis exhibits unexpected complexity and structural differentiation despite the organism's overall simplicity. We propose that sexual selection acting on a hermaphroditic reproductive system may have driven the evolution of a multifunctional copulatory organ in the Urbilateria. This study establishes H. atroviridis as a promising and comparative model for exploring the evolutionary origins of animal reproductive systems.
The Zoological Collection at the Stazione Zoologica Anton Dohrn di Napoli has been accumulating since research activities began in 1872. Following the opening of the collection room, known as "Museo", after 1906, the former collections were gathered there and Reinhard Gast became the first curator. Between 1910 and 1914, he left behind an accession record of 1098 preparations. We have found that at least 624 of these preparations remain in the current collection. These preparations bear at least five distinct types of labels applied by successive curators over the last 110 years. One hundred preparations still retain Gast's original labels and containers, and clearly exhibit his characteristic showcase-style arrangements. They are often displayed vertically on glass plates with fine threads and placed in rectangular or cylindrical glass jars with black backgrounds, with some specimens arranged to evoke their natural habitat at the bottom of the sea. Gast also produced species reference cards describing the biological and ecological characteristics of each species. Taken together, these features suggest that Gast intended the collection not only as a taxonomic reference, but also as a visual and ecological representation of the marine fauna of the Gulf of Naples-an early form of a biodiversity information resource.
Ascidians within order Phlebobranchia can accumulate extremely high levels of vanadium (V) in their blood cells. Several V-related proteins, including V-binding proteins (vanabins), have been isolated from V-accumulating ascidians. Vanabins were first isolated from a V-rich ascidian, Ascidia sydneiensis samea, belonging to Phlebobranchia. In this study, we searched for vanabin gene homologs in all available public databases containing tunicate genomic data and the draft genome database of A. sydneiensis samea constructed in this study. We found one to six vanabin genes per genome among all available genomes of Phlebobranchia species. We also found a vanabin gene in the genome of an Aplousobranchia species. Thus, we identified vanabins in seven ascidian species, and aligned these according to the C-Xn-C pattern common to the vanabin core domain. Based on a molecular phylogenetic tree derived from 18S ribosomal DNA and natural resistance-associated macrophage protein sequences, we propose a model for the evolution of vanabin gene structure via the following pathway: acquisition of ancestral vanabin by the common ancestor of Phlebobranchia and Aplousobranchia, followed by vanabin gene duplication; nonsynonymous mutation, insertion, and positive selection to create amino acid variation among vanabins; and extension of the vanabin gene cluster. The selective force acting within this pathway may have been V toxicity under high-V conditions.
The larval trematode taxon Monilicaecum ventricosum Yamaguti, 1942 (Didymozoidae) was redescribed from its type host, the Pacific saury Cololabis saira (Brevoort, 1856), collected from the North Pacific off Eastern Hokkaido, Japan. Because didymozoid larvae and adults differ markedly in morphology, linking these two stages using morphological data alone has traditionally been difficult. Consequently, the monotypic genus Monilicaecum Yamaguti, 1942 is currently regarded as a collective group, similar to genera established solely on larval forms of other trematodes. However, recent advances in molecular studies have enabled researchers to link larval and adult stages, clarify life cycles, and undertake more robust taxonomic evaluations. In this study, newly collected larval specimens were examined morphologically, and DNA barcodes (28S, ITS, and cox1) were generated to confirm their identity as M. ventricosum through morphological comparisons with type materials. These sequences were compared with those available in public databases to determine the corresponding adult stages and definitive hosts. Although no identical adult sequences were detected, M. ventricosum showed a close genetic relationship with four didymozoid species parasitizing the Australian yellowfin tuna, Thunnus albacares (Bonnaterre, 1788), suggesting that its definitive host is likely a species of Thunnus South, 1845. These findings provide essential molecular data for M. ventricosum, contribute to the taxonomic stability of Didymozoidae, and offer a foundation for future studies aimed at elucidating species boundaries and life-cycle connections within this group.
Two types of Cladonema pacificum (Cnidaria, Hydrozoa) can be distinguished based on whether their medusae spawn after a dark-light transition ("light type") or a light-dark transition ("dark type"). To clarify how these two types are related, we characterized C. pacificum from different sites in the Tohoku region of Japan. Essentially, only dark-type medusae were captured in the Pacific Ocean side, whereas light- and dark-type medusae were found sympatrically at some sampling points in Mutsu Bay and in the Sea of Japan side. We observed that the spawning times of the sympatric light and dark types differed in the field, suggesting that the gametes of the two types rarely encounter each other. A comparison of the DNA sequences of mitochondrial COI and nuclear ITS1 of the analyzed individuals suggests that the two types belong to separate clades. In crossbreeding experiments, offspring of the light and dark types (F1 hybrids) developed into planula larvae and metamorphosed into polyps, which then produced medusae with normal morphology. The F1 hybrid medusae had characteristics that were intermediate between the two types in terms of their spawning in response to both dark-light and light-dark cues, but with less sensitivity than the parent medusae. When F1 hybrids were used as one or both parents, planula larvae failed to metamorphose into normal polyps. These results indicate that the light and dark types of C. pacificum exhibit both prezygotic and postzygotic isolation, accompanied by distinct DNA sequences; thus, they can be considered as undergoing speciation or as already distinct species.
Tendon injuries in humans often lead to significant functional impairments, necessitating effective regeneration methods. While mammalian tendon healing typically results in scar formation and functional loss, certain species such as axolotls exhibit remarkable regenerative abilities. This study investigates axolotl tendon regeneration following complete transection (tenotomy) and volumetric loss (tenectomy). Axolotl tendons, identified through histological and gene expression analyses, consist of collagens and exhibit specific tendon markers. Our results indicated complete regeneration of transected and partially removed tendons, with regenerating fibers becoming organized over time. Gene expression patterns of tendon markers further support the regeneration process. Denervation experiments suggested that nerves play a minimal role in axolotl tendon regeneration. Additionally, grafting GFP-labeled tendon tissue into wild-type animals revealed cellular dynamics during regeneration. Our results suggest that regenerated tendons consist of cells from the damaged tendon tissue and/or surrounding tissues. This dual cellular source suggests a complex mechanism for axolotl tendon regeneration. Overall, axolotls demonstrate superior tendon regeneration abilities, shedding light on potential mechanisms for mammalian tendon healing improvement. Understanding such mechanisms could lead to more effective tendon regeneration strategies in humans.
