Zoological Science

We examined the importance of establishment and maintenance of territory on reproductive activity in the male dwarf gourami, Colisa lalia. After three males had been forced to fight for territory (five sets) for three weeks, social status was divided into three classes: the territorially dominant male, which guarded the territory under the floating nest; the second male which remained near the nest and occasionally attacked the dominant male; and the third male which was non-aggressive and remained at a distance from the other two males. Comparing testicular size by gonadosomatic indices (GSI) after three weeks of aggression, GSI of the dominant male (1.19 ± 0.07) was significantly larger than that of the second (0.81 ± 0.15) and the third (0.62 ± 0.08) males, as well as the initial control (not involved in any experiments: 0.85 ± 0.10, n = 5), indicating that the testes of the dominant males enlarge during territory defense. Histological observations of testes revealed that sperm production in the dominant males was more active compared to males of other classes, although spermatogenesis was confirmed in all males examined, suggesting that dominance accelerates sperm production. Social-status dependent development of testes suggests an absence of sperm competition due to the lack of sneaking by subordinate males. Since non-territorial males do not engage in alternative tactics (e.g., sneaking) leading to emission of semen, male C. lalia must obtain and defend territory if they are to increase their reproductive success.

The costs of parturition and lactation of female sika deer on Kinkazan Island (9.6 km² in size), northern Japan, which live at a high density (about 50 deer/km²), were evaluated by comparison of body weights of 481 females measured during a 15 year study (1993–2008). Weight data were chosen from only females that did not give birth in the preceding year. The mean body weight of females that did not give birth (“yelds”) was significantly lower (P < 0.001) than that of females who gave birth (“milks”); yelds' body weight was 93.1% and 83.5% that of milks in the preceding and parturition years, respectively. The yelds increased in body weight by the following March by 8.2% (P < 0.001), whereas milks did not. Among the milks, those whose fawns survived until the following May (“rearing milks”) lost body weight by 14.9% (P < 0.001). Milks who lost fawns within a week after birth (“early fawn-less milks”) did not lose body weight (P = 0.583), while those whose fawns died after the first autumn but died before May (“late fawn-less milks”) lost body weight by 19.9% (P < 0.001). These results indicate that sika deer females do not enter estrus unless they are heavy enough, and that both parturition and rearing are costly for sika deer mothers living in high-density conditions.

To investigate the molecular phylogeny and evolution of the family Canidae, nucleotide sequences of the zinc-finger-protein gene on the Y chromosome (ZFY, 924–1146 bp) and its homologous gene on the X chromosome (ZFX, 834–839 bp) for twelve canid species were determined. The phylogenetic relationships among species reconstructed by the paternal ZFY sequences closely agreed with those by mtDNA and autosomal DNA trees in previous reports, and strongly supported the phylogenetic affinity between the wolf-like canids clade and the South American canids clade. However, the branching order of some species differed between phylogenies of ZFY and ZFX genes: Cuon alpinus and Canis mesomelas were included in the wolf-like canid clades in the ZFY tree, whereas both species were clustered in a group of Chrysocyon brachyurus and Speothos venaticus in the ZFX tree. The topology difference between ZFY and ZFX trees may have resulted from the two-times higher substitution rate of the former than the latter, which was clarified in the present study. In addition, two types of transposable element sequence (SINE-I and SINE-II) were found to occur in the ZFY final intron of the twelve canid species examined. Because the SINE-I sequences were shared by all the species, they may have been inserted into the ZFY of the common ancestor before species radiation in Canidae. By contract, SINE-II found in only Canis aureus could have been inserted into ZFY independently after the speciation. The molecular diversity of SINE sequences of Canidae reflects evolutionary history of the species radiation.

To survey the diversity of anuran species in Bangladesh, we compared mitochondrial 16S rRNA gene sequences (approximately 1.4 kbp) from 107 Bangladesh frog specimens. The results of genetic divergence and phylogenetic analyses incorporating data from related species revealed the occurrence of at least eight cryptic species. Hoplobatrachus tigerinus from two districts diverged considerably, indicating the involvement of a cryptic species. Two Fejervarya sp. (large and medium types) and Hylarana cf. taipehensis formed lineages distinct from related species and are probably new species. Microhyla cf. ornata differed from M. ornata with respect to type locality area and involved two distinct species. In addition, we found that Hylarana sp. and Microhyla sp. did not match congeners examined to date in either morphology or 16S rRNA sequence. The occurrence of M. fissipes was tentatively suggested. Consequently, at least, 19 species were found from Bangladesh in this study. These findings revealed a rich anuran biodiversity in Bangladesh, which is unexpected considering the rather simple topographic features of the country.

Endosymbiotic algae of the genus Symbiodinium have been divided into nine clades (A–I) following genetic classification; some clades are known to have physiological properties that enable the coral hosts to adapt to different environmental conditions. To understand the relationships of coral-alga symbioses, we focused on Symbiodinium diversity in zooxanthellate corals living under the severe environmental conditions of the temperate region (30°–35°N) of Japan. We investigated Symbiodinium clades in 346 colonies belonging to 58 coral species from six locations. We then selected three coral species—Acropora hyacinthus, Acropora japonica, and Cyphastrea chalcidicum—to investigate whether Symbiodinium clades changed during winter or summer over the course of year (May 2009–Apr 2010) in Tanabe Bay, Japan. Three Symbiodinium clades (C, D, and F) were detected in corals in the temperate region. Notably, 56 coral species contained Symbiodinium clade C. Oulastrea crispata predominantly contained clade D, but traces of clade C were also detected in all samples. The temperate-specific species Alveopora japonica contained clades C and F simultaneously. Seasonal change of symbiont clades did not occur in the three coral species during the investigation period where SSTs range on 12.5–29.2°C. However, we found Acropora (2 spp.) and Cyphastrea (1 sp.) contained different subcladal types of clade C. These results reveal that most coral species harbored Symbiodinium clade C stably throughout the year, suggesting that Symbiodinium clade C shows low-temperature tolerance, and that two hypothetical possibilities; genetic differences of subcladal types generating physiological differences or wide physiological flexibility in the clade C.

The zebrafish (Danio rerio) has been used as a model for the study of endocrine disrupting chemicals. This study set out to determine the profiles of whole-body thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels during the development of zebrafish from embryo to adult. Enzyme-linked immunoassay was used to analyze whole-body T4 and T3 contents. The results showed that whole-body T4 and T3 levels remained stable during the pre-hatching period (0–3 d) and increased significantly during early development after hatching. The T3 level peaked at 0.28 ± 0.01 ng g^-1 body weight at 10 days post-fertilization (dpf), and T4 peaked at 0.58 ± 0.09 ng g^-1 body weight at 21 dpf. Both thyroid hormones subsequently declined during later development. This study establishes a baseline for thyroid hormones in zebrafish, which will be vital for the understanding of thyroid hormone functions and in future studies of thyroid toxicants in this species.

Animals possess systems for sensing environmental temperature using temperature-sensitive ion channels called transient receptor potential channels (TRPs). Various TRPs have been identified and characterized in mammals. However, those of ectotherms, such as reptiles, are less well studied. Here, we identify the V subfamily of TRP (TRPV) in two reptile species: Japanese grass lizard (Takydromus tachydromoides) and Japanese four-lined ratsnake (Elaphe quadrivirgata). Phylogenetic analysis of TRPVs indicated that ectothermic reptilian TRPVs are more similar to those of endothermic chicken and mammals, than to other ectotherms, such as frog and fish. Expression analysis of TRPV4 mRNA in the lizard showed that its expression in tissues and organs is specifically controlled in cold environments and hibernation. The mRNA was ubiquitously expressed in seven tissues/organs examined. Both cold-treatment and hibernation lowered TRPV4 expression, but in a tissue/organ-specific manner. Cold-treatment reduced TRPV4 expression in tongue and muscle, while in hibernation it was reduced more widely in brain, tongue, heart, lung, and muscle. Interestingly, however, levels of TRPV4 mRNA in the skin remained unaffected after entering hibernation and cold-treatment, implying that TRPV4 in the skin may act as an environmental temperature sensor throughout the reptilian life cycle, including hibernation. This is the first report, to our knowledge, to describe reptilian TRPV4 in relation to hibernation.

Amphibians can regenerate missing body parts, including limbs. The regulation of collagen has been considered to be important in limb regeneration. Collagen deposition is suppressed during limb regeneration, so we investigated collagen deposition and apical epithelial cap (AEC) formation during axolotl limb regeneration. The accessory limb model (ALM) has been developed as an alternative model for studying limb regeneration. Using this model, we investigated the relationship between nerves, epidermis, and collagen deposition. We found that Sp-9, an AEC marker gene, was upregulated by direct interaction between nerves and epidermis. However, collagen deposition hindered this interaction, and resulted in the failure of limb regeneration. During wound healing, an increase in deposition of collagen caused a decrease in the blastema induction rate in ALM. Wound healing and limb regeneration are alternate processes.

Few studies have conducted morphological analyses of crocodilians, and little information exists on differences between size-classes and sexes in Neotropical crocodilians. In this study, we measured nine morphological traits in 121 captive Morelet's crocodiles Crocodylus moreletii (81 females and 40 males). Our results revealed that individuals < 2 m total length do not exhibit sexual dimorphism in morphometric characteristics. However, for crocodiles over 2 m in length, males were significantly larger than females in terms of dorsal-cranial length, cranial width, snout width and snout-ventral length. In general, morphological traits demonstrated a strongly significant relationship with total length at the smaller size class of 150–200 cm length. However, in the highest size class of 250–300 cm length (large adult males), morphological traits were no longer significantly related with total length. Male crocodiles demonstrated allometric growth of cranial morphology with significantly greater increase in cranial width, snout width, and mid-snout width relative to total length at higher size classes. Morphological dimorphism and allometric growth may be associated with adaptive strategies for reproductive success.

A luteinizing hormone receptor (lhr) cDNA with high identity to other fish lhrs was fully cloned from the ovary of the Japanese eel (Anguilla japonica). The genes for two gonadotropin receptors (Gthr), follicle-stimulating hormone receptor (fshr) and lhr, were differentially expressed during oogenesis, which was artificially induced by salmon pituitary extract, a gonadotropin-rich source. Transcript abundance of fshr was significantly elevated at the early vitellogenic stage and peaked at the late vitellogenic stage, while lhr gene expression rapidly induced at the late vitellogenic stage and thereafter remained at a high level. The abundance of fshr and lhr transcripts was highest in the ovary in female eels. In addition to the ovary, forebrain was a major site for the fshr transcript, although the level did not change with reproductive status. Furthermore, it was examined how eel Gthrs were activated by two mammalian chrionic gonadtropin (CG), equine CG (eCG) and human CG (hCG), that have been used for study of fish reproduction as substitutes for homologous Gths. Both CGs specifically activated eel Lhr, but not Fshr, although the degree of effectiveness was different; thus the concentration of hCG (0.1 ng/ml) required for significant activation of Lhr was much lower than that of eCG (100 ng/ml). These data on gene expression and ligand-activation of Gthrs suggest that Fsh and Lh act differentially in the regulation of reproductive function in Japanese eel.