This study describes the reproductive microhabitat of Melanophryniscus montevidensis and its use in two water bodies (WBs) in Barra de la Laguna de Rocha, Uruguay. Monthly field trips were performed between March 2012 and February 2013. Variables related to the WBs and vegetation, as well as parameters linked to the usage the amphibians make of the site (e.g: distance to the border of the pond, water depth and the vegetation use) were recorded. The behavior shown by the individuals during the breeding activity was recorded. This activity occurs in shallow temporary WBs with abundant hydrophilic vegetation. The individuals were found more frequently in areas near the edge of the pond, which has denser vegetation. The calling males were found closer to the border of the pond, and they showed better body condition than the non-calling males. In addition to calling activities, males used alternative tactics to find couples, such as active search of females, and aggressive behaviors, such as male displacing and physical combat. Such behaviors are common in anurans with explosive reproductive dynamics. The characterization of the reproductive microhabitats permits the proposal of strategies for the conservation of the species in Uruguay, given that the loss and fragmentation of habitats is one of the main causes considered for the decrease in their populations.

Paddy fields have traditionally been viewed as the key foraging habitats for the endangered crested ibis (Nipponia nippon). With the population of this species now increasing, its distribution has expanded to both lowland areas and outside the nature reserve. However, little is known about the current foraging habitat preferences of these birds, especially during winter. In this research, a total of 54 used sites and 50 unused sites were investigated during winter from December 2011 to January 2012. The results of logistic regression analysis indicate that soil softness, human disturbance, and distance to the nearest road were important factors. For the site plots of winter-flooded paddy fields, the birds prefer the paddy fields with higher coverage of vegetation, except softer foraging sites and lower human-related disturbance. In lowland areas, the size of winter-flooded paddy fields was not a limiting factor, due to the availability of other wetlands capable of providing abundant food. The micro-habitat characteristics were important indicators of foraging habitat quality rather than the size of winter-flooded paddy fields, and the food accessibility may play an important role in the process of foraging habitat use. We suggest the improvement of the foraging micro-habitat and environmental characteristics would be effective in ensuring the availability of food in the dispersed lowland areas. The local people still needed to be encouraged and compensated by their single-cropping cultivation, ploughed the paddy fields after harvesting and irrigated them with shallow water flooded in the original core areas of the nature reserve.

To evaluate the seed shadow generated by wild Japanese martens (Martes melampus), we combined data on their ranging behavior from the northern foot of Mt. Fuji, central Japan (seven males and three females) with data on gut passage time obtained from martens in Toyama Municipal Family Park Zoo (three males and one female). The movement distances varied, and mean distances for 0–1, 2–3, and 4–5 h intervals were 152.4, 734.7, and 1,162.4 m, respectively, with no significant sex difference. The mean gut passage time of ingested seeds was 7.4 h (range: 0.6–51.7 h), and two-thirds were defecated within 12 h. Seeds of fleshy fruits was frequently transported to 501–1,000 m, and 20% of ingested seeds were transported > 1,000 m from feeding sites. We found positive correlations between body size and home range of the animals in Japan and their seed dispersal distances. We conclude that Japanese martens are medium-range dispersers that can transport seeds from the source to open habitats conducive for germination and/or growth, partly due to scent marking behaviors.

The genomes of vertebrates are globally methylated, but a small portion of genomic regions are known to be hypomethylated. Although hypomethylated domains (HMDs) have been implicated in transcriptional regulation in various ways, how a HMD is determined in a particular genomic region remains elusive. To search for DNA motifs essential for the formation of HMDs, we performed the genome-wide comparative analysis of genome and DNA methylation patterns of the two medaka inbred lines, Hd-rRII1 and HNI-II, which are derived from northern and southern subpopulations of Japan and exhibit high levels of genetic variations (SNP, ∼ 3%). We successfully mapped > 70% of HMDs in both genomes and found that the majority of those mapped HMDs are conserved between the two lines (common HMDs). Unexpectedly, the average genetic variations are similar in the common HMD and other genome regions. However, we identified short well-conserved motifs that are specifically enriched in HMDs, suggesting that they may play roles in the establishment of HMDs in the medaka genome.

In most animals, aging is an irreversible process; however the species Turritopsis sp. has been observed to undergo a rejuvenation process as many as 14 times. In the present study, we used multiplexed RNA libraries to obtain the transcriptome from four developmental stages (St) of Turritopsis sp., including (I) immature medusa, (II) dumpling, (III) dumpling with a short stolon, and (IV) polyp, which had recently rejuvenated. A total of 4.02 billion paired-end reads were assembled de novo, yielding 90,327 contigs. Our analyses revealed that significant blast hits were recovered for 74% of the assembled contigs, and 19% were successfully annotated with gene ontology (GO) terms. A BLAST search demonstrated that 32% of the contigs were most similar to Hydra vulgarissequences. Raw reads from each sample were mapped against the contigs to find St-specific genes. This represents the first comprehensive set of de novo transcriptome data for this species, which may provide clues toward a better understanding of cyclical rejuvenation in multicellular animals.

The objective of the present study was to analyze chondrogenesis and the ossification pattern of the limbs of Melanosuchus niger in order to contribute with possible discussions on homology and the fusion pattern of autopodial elements and phylogeny. In the Reserva Extrativista do Lago Cuniã, Rondônia, Brazil, six nests were marked and two eggs removed from each nest at 24-hour intervals until hatching. Embryos were cleared using KOH; bone tissue was stained with alizarin red S and cartilage with Alcian blue. Routine staining with HE was also performed. In the pectoral girdle, the scapula showed ossification centers before the coracoid process. In the pelvic girdle, the ilium and the ischium were condensed as a single cartilage, although ossification took place through two separate centers, forming distinct elements in the adult. The pubis developed from an independent cartilaginous center with free end, which reflects its function in breathing. In the initial stages, the stylopodium and the zeugopodium developed from the condensation of a Y-shaped cartilage in the limbs, and differentiation of the primary axis and digital arch were observed. The greatest changes were observed in the mesopodia. In their evolution, Crocodylia underwent a vast reduction in the number of autopodial elements as a consequence of fusions and ossification of some elements. This study shows that the chondrogenesis and ossification sequences are dissociated. Moreover, the differences between M. niger and other species show clear variation in the patterns for these events in Alligatoridae.

The pale grass blue butterfly has been used to assess the biological effects of the Fukushima nuclear accident. Zizeeria and Zizina are two closely related genera of grass blue butterflies that are widely distributed in tropical to temperate Asia, Australia, and Africa, making them suitable environmental indicators for these areas. However, the morphological features of the immature stages have been examined only in fragmentary fashion. Here, we reared Zizeeria maha argia, Zizeeria maha okinawana, Zizeeria karsandra karsandra, Zizina emelina emelina, Zizina otis labradus, and Zizina otis riukuensis using a standard rearing method that was developed for Zizeeria maha, and comparatively identified morphological traits to effectively classify the immature stages of species or subspecies. Morphological information on these and other subspecies including Zizeeria knysna knysna and Zizina otis antanossa from Africa was also collected from literature. The subspecies were all reared successfully. The subspecies all had dorsal nectary and tentacle organs with similar morphology. For the subspecies of Zizeeria maha, only minor morphological differences were noted. Similarly, the subspecies of Zizina otis shared many traits. Most importantly, Zizeeria and Zizina differed in the shape of the sensory hairs that accompany the dorsal nectary organ; Zizeeriahad pointed hairs, and Zizina had blunt or rounded hairs. However, Zizina emelina exhibited several intermediate features between these two genera. Overall, the morphological traits did not completely reflect the conventional systematic relationships. This comparative study describes the efficient rearing of the grass blue butterflies and provides a morphological basis for the use of these species as environmental indicators.

We investigated the phylogenetic relationships and divergence times within the genus Auritibicen(Cicadidae: Cicadinae: Cryptotympanini), analyzing five Japanese species (A. japonicus, A. bihamatus,A. kyushyuensis, A. esakii and A. flammatus) and three species from East Asian mainland and Taiwan (A. atrofasciatus, A. intermedius and A. chujoi) using mitochondrial cytochrome oxidase subunit I (COI) and nuclear elongation factor 1-alpha (EF-1a) gene sequences. Although the EF-1a gene tree did not resolve the relationships among these Auritibicen species, the trees based on COI gene and the combined data set showed that Japanese taxa comprised three distinct lineages: the individual species A. flammatus and A. bihamatus, and the A. japonicus group, comprising A. japonicus, A. esakii and A. kyushyuensis from Japan and A. intermedius from Korea. In A. kyushyuensis, which comprises three populations in Kyushu, western Honshu and Shikoku, the specimens from western Honshu and Shikoku were closely related to each other, but not to the specimen from Kyushu; instead, they were sister to the Korean A. intermedius. The incongruence between the gene tree and species tree necessitates further population genetic and morphological studies to confirm the classification and species status of the western Honshu and Shikoku populations of A. kyushyuensis, which were originally described as two independent species. Divergence time estimation suggested that the most recent common ancestor of Auritibicen species studied dated back to the late Pliocene and that the species of the A. japonicus group diverged during the mid Pleistocene. Thus, the Pleistocene climatic fluctuation may have promoted the divergence of the Auritibicen species.

We have developed an original in vitro bioassay using teleost scale, that has osteoclasts, osteoblasts, and bone matrix as each marker: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Using this scale in vitro bioassay, we examined the effects of seawater polluted with highly concentrated polycyclic aromatic hydrocarbons (PAHs) and nitro-polycyclic aromatic hydrocarbons (NPAHs) on osteoblastic and osteoclastic activities in the present study. Polluted seawater was collected from two sites (the Alexandria site on the Mediterranean Sea and the Suez Canal site on the Red Sea). Total levels of PAHs in the seawater from the Alexandria and Suez Canal sites were 1364.59 and 992.56 ng/l, respectively. We were able to detect NPAHs in both seawater samples. Total levels of NPAHs were detected in the seawater of the Alexandria site (12.749 ng/l) and the Suez Canal site (3.914 ng/l). Each sample of polluted seawater was added to culture medium at dilution rates of 50, 100, and 500, and incubated with the goldfish scales for 6 hrs. Thereafter, ALP and TRAP activities were measured. ALP activity was significantly suppressed by both polluted seawater samples diluted at least 500 times, but TRAP activity did not change. In addition, mRNA expressions of osteoblastic markers (ALP, osteocalcin, and the receptor activator of the NF-κB ligand) decreased significantly, as did the ALP enzyme activity. In fact, ALP activity decreased on treatment with PAHs and NPAHs. We conclude that seawater polluted with highly concentrated PAHs and NPAHs influences bone metabolism in teleosts.

The present study aimed to evaluate the osmoregulatory mechanism of Daisy's medaka, O. woworae,as well as demonstrate the major factors affecting the hypo-osmoregulatory characteristics of euryhaline and stenohaline medaka. The medaka phylogenetic tree indicates that Daisy's medaka belongs to the celebensis species group. The salinity tolerance of Daisy's medaka was assessed. Our findings revealed that 20‰ (hypertonic) saltwater (SW) was lethal to Daisy's medaka. However, 62.5% of individuals survived 10‰ (isotonic) SW with pre-acclimation to 5‰ SW for one week. This transfer regime, “Experimental (Exp.) 10‰ SW”, was used in the following experiments. After 10‰ SW-transfer, the plasma osmolality of Daisy's medaka significantly increased. The protein abundance and distribution of branchial Na , K -ATPase (NKA) and Na , K , 2Cl^- cotransporter 1 (NKCC1) were also examined after transfer to 10‰ SW for one week. Gill NKA activity increased significantly after transfer to 10‰ SW. Meanwhile, elevation of gill NKA αα-subunit protein-abundance was found in the 10‰ SW-acclimated fish. In gill cross-sections, more and larger NKA-immunoreactive (NKA-IR) cells were observed in the Exp. 10‰ SW medaka. The relative abundance of branchial NKCC1 protein increased significantly after transfer to 10‰ SW. NKCC1 was distributed in the basolateral membrane of NKA-IR cells of the Exp. 10‰ SW group. Furthermore, a higher abundance of NKCC1 protein was found in the gill homogenates of the euryhaline medaka, O. dancena, than in that of the stenohaline medaka, O. woworae.

Parthenogenetic oogenesis varies among and even within species. Based on cytological mechanisms, it can largely be divided into apomixis (ameiotic parthenogenesis) producing genetically identical progeny, and automixis (meiotic parthenogenesis) producing genetically non-identical progeny. Polyploidy is common in parthenogenetic species, although the association between parthenogenesis and polyploidy throughout evolution is poorly understood. Marmorkrebs, or the marbled crayfish, was first identified as a parthenogenetic decapod and was tentatively named as Procambarus fallax f. virginalis. Previous studies revealed that Marmorkrebs is triploid and produces genetically identical offspring, suggesting that apomixis occurs during parthenogenetic oogenesis. However, the behavior of chromosomes during the process of oogenesis is still not well characterized. In this study, we observed parthenogenetic oogenesis around the time of ovulation in P. fallax f. virginalis by histology and immunohistochemistry. During oogenesis, the chromosomes were separated into two groups and behaved independently from each other, and one complete division corresponding to mitosis (the second meiosis-like division) was observed. This suggests that parthenogenetic oogenesis in Marmorkrebs exhibits gonomery, a phenomenon commonly found in apomictic parthenogenesis in polyploid animals.

Milnesium tardigradum trispinosa Rahm, 1931 is characterized by its three spines at the posterior end of the animal, but has never been reported since its original description. Among mounted specimens of Milnesium sp. from Japan and M. tardigradum s. s. from France, several cases with these ‘three spines’ were observed. In these samples, the character was formed by an artifact of the fixation process. Images of these specimens show such a striking similarity to Milnesium tardigradum trispinosa that this taxon must be considered as having been erroneously established as a result of misinterpretation of an artifact and no longer valid.

The bivalve Tellimya fujitaniana (Yokoyama, 1927) (Galeommatoidea, Heterodonta) was described based on a fossil shell. Until now, the biology of living animals has not been reported. In this study, we found T. fujitaniana in a commensal relationship with the heart urchin Echinocardium cordatum (Pennant, 1777) (Spatangoida, Echinoidea) on the intertidal mud flats of the Seto Inland Sea, Japan. We investigated the morphology, host associations, and reproductive biology of this bivalve species. The elongate-ovate shell is covered by a reddish-brown ferruginous deposit. The mantle is exposed anteriorly to form a temporal siphon, while posteriorly one pair of short tentacles is exposed. Small individuals (shell length, SL, ≤ 2.1 mm) were attached to the host's body surface; middle-sized individuals (SL 3.0–3.2 mm) were attached to or stayed close to larger T. fujitanianathat were living freely in the host burrow. Nearly all the large individuals (SL ≥ 4.8 mm) lived freely in the host burrow, behind the urchin. This suggests that the host utilization pattern of T. fujitanianachanges with development. Specimens with SL ≥ 4.8 mm had mature gonads, mostly occupied by ova, and some individuals were brooding eggs or veliger larvae in the gills. This species was previously assigned to Fronsella. However, the morphology and ecology of this bivalve are very similar to those of Tellimya ferruginosa (the type species of the genus Tellimya) in the northeastern Atlantic Ocean. Thus, we have reassigned this species to genus Tellimya. We also confirmed that T. fujitaniana and T. ferruginosa can be genetically distinguished using the mitochondrial COI gene.

The bivalve superfamily Galeommatoidea is characterized by its symbiotic associations with other marine invertebrates. However, for many galeommatoideans, the host species remains unknown. Platomysia (Galeommatoidea) is a monotypic genus including a single species P. rugata, which is distinguished from other galeommatoideans in having distinct and evenly spaced commarginal ribs on its shell surface. This species was described based on a single right valve shell collected in Nanao Bay, Japan Sea, by Habe in 1951 and has been known only from Japanese waters. However, the biology of living animals has never been reported. We found that this species lives in the burrows of the sipunculan worm Sipunculus nudus in mud flats in the Seto Inland Sea, Japan. We investigated its host association and described its shell morphology and anatomy. In addition, we performed a phylogenetic analysis using two nuclear (18S and 28S ribosomal RNA) genes to determine its phylogenetic position in Galeommatoidea. The result suggests that this species belongs to the clade of commensal bivalves together with Pseudopythina, Byssobornia, and Pergrinamor. Platomysia rugata and other two groups of sipunculan-associated galeommatoideans were not monophyletic, suggesting that association with sipunculans occurred at least three times in the galeommatoid evolution.

In the present study, we report the identification of a sea anemone, Antennapeachia setouchi, collected in the Seto Inland Sea, which represents a new genus and new species. This new species has unusual tentacle and mesenterial arrangements that have not been observed in other species of Haloclavidae. There are 12 regular marginal tentacles and two ‘antenna tentacles,’ with the latter always rising upward and located on the oral disk near the mouth; the species is also characterized by its peculiar mesenterial pairs, consisting of a macrocneme and a microcneme. Furthermore, this species shows an interesting behavior: it can inflate its body like a balloon, lift above the seafloor, and drift with the sea current. The presence of a single, strong siphonoglyph, physa-like aboral end, and the lack of sphincter muscle classify this sea anemone within Haloclavidae. It resembles Peachia species, but cannot be classified in this genus as the new species has two pairs of mesenteries, consisting of a macrocneme and a microcneme, and irregular antenna tentacles. Therefore, we propose a new genus Antennapeachia to accommodate this species.