In 1926, Tage Lakjer hypothesized a replacement of the infratemporal bar in diapsids by a ligament spanning between quadrate and the upper jaw. As a similar ligament is also present in turtles, he argued for a diapsid origin of this group. Based on recent advances in the homologization of the tendinous framework in the reptile jaw adductor chamber — reviewed in this paper — one could argue for independent origins of the cheek ligaments in sauropsids. The quadratomaxillar ligament of turtles could, with reservation, be homologized with the quadrate aponeurosis of other sauropsids, as well as to the superficial tendon of m. masseter in mammals. These structures have a strong morphogenetic influence to cranial anatomy. Given such an identity, the hypothesis of a structural replacement of the lower temporal arcade in lizards would be refuted. Moreover, such a homology could be correlated to the evolution of the middle ear and to the origin of the chewing mechanism in mammals, which contributed to the evolutionary success of that group. The homologization presented herein is critically discussed and is open for revision. Nevertheless, the value of tendinous structures for fundamental homologisations in the vertebrate head is highlighted.

Anuran skin contains a calcified dermal layer, referred to as the Eberth-Kastschenko (EK) layer, which is found between the stratum spongiosum and the stratum compactum. Although it is established that some anuran species possess the EK layer, little is known about this layer from the standpoint of evolutionary and developmental biology. We conducted a morphological analysis by staining the dorsal skin from many species with alizarin red S to investigate the calcified layer. This layer was observed in all of the anurans tested, as well as in fishes and one species of caecilian with dermal scales, but not in urodeles, amniotes, or a scaleless caecilian. All of the investigated species with dermal scales exhibited a calcified layer in their dermis, while the anurans showed the EK layer, but no scales. We also analyzed the expression of genes related to scale formation (sparc, mmp9, and mmp2) in the dorsal skin of X. tropicalis. These genes were highly expressed at the metamorphic climax stage, which preceded the deposition of calcium. Furthermore, we examined the gene expression profile of amelogenin, the major protein found in the enamel matrix of the developing tooth. In X. tropicalis, amelogenin was upregulated in the skin at the climax stage and was expressed in the adult dermis at a high level. These data provide the first experimental evidence of the expression of amelogenin in the skin. These findings will lead to a better understanding of the developmental formation of the EK layer and the function of amelogenin.

In this study, we investigated the biochemical composition and digestive enzyme activity during embryonic development in the marine crab, Charybdis japonica. Water, protein, and ash content increased, while levels of lipids and carbohydrates decreased significantly during embryonic development, and a marked correlation between water content and egg volume (r = 0.97; P < 0.01) was recorded. The utilization percentages of lipids and carbohydrates were 32.72% and 91.56%, respectively. The predominant essential amino acids (EAA) were lysine, leucine, arginine and valine, and the major nonessential amino acids (NEAA) were glutamic acid and aspartic acid. From the fertilized egg stage to the protozoea stage, total amino acid (TAA) concentration increased from 52.18% to 55.11% on a dry weight basis, but the ratio of EAA/TAA decreased from 52.57% to 48.90%. The quantitatively more important fatty acids were C16:0, C18:1n-9c, C16:1, C22:6n-3 (docosahexaenoic acid, DHA), and C22:2. Polyunsaturates (PUFA) and monounsaturated (MUFA) were consumed at similar rates (34.20% and 36.70%, respectively); both were consumed at higher rates than saturates (SFA) (26.56%). In particuler, n-3 fatty acids decreased significantly, with a high consumption rate of 43.74%. Activities of trypsin and pepsin increased during both the early and later embryonic stages, but decreased during the middle stages. Lipase activity increased gradually during embryonic development, except in the protozoea stage with a significant decrease, while activities of amylase and cellulase showed an ascending trend after an initial decline. The activity of all digestive enzymes increased, except for that of lipase, from the heartbeat stage to the protozoea stage.

The Tohoku salamander, Hynobius lichenatus Boulenger, 1883, is a lentic breeding species widespread throughout montane regions of northeastern Japan. To explore intraspecific genetic variation and infer evolutionary history of H. lichenatus, we performed mitochondrial DNA analysis (complete 1141 bp sequences of the mitochondrial cytochrome b gene) using 215 adult and larval individuals collected from 75 localities, encompassing known distributional range of the species. Hynobius lichenatus proved to be monophyletic, including three well-supported and geographically structured clades (Clade I from northern Kanto, Clade II from southern Tohoku, and Clade III from northern Tohoku). These clades, respectively, comprise several subclades, and show genetic distances as large as those seen between different species of Hynobius. Results of population statistic analyses indicate that all clades and most subclades have maintained high genetic diversity and demographic stability over long periods. Molecular dating indicates divergence in H. lichenatus concords with topographic evolution of northeastern Japan from late Miocene to early Pleistocene, suggesting that paleogeographic events in this region, such as orogenesis, sea level change, and volcanic activity, have been crucial for shaping genetic patterns and diversity in this species. Hynobius lichenatus greatly differs from many other animal species from northeastern Japan in its much older periods and the pattern of genetic differentiation, and is suggested as an old faunal element in this region.

We demonstrated the disembarkation of the bacterial-feeding nematode Caenorhabditis japonica dauer larvae (DL) from adult Parastrachia japonensis female insects and observed the propagation of nematodes in artificial insect nests. Our results clarify the process of propagation in this nematode species and provide insights into the nematode-insect relationship. Quiescent C. japonica DL resumed their mobility only at > 99.9% relative humidity (RH) at 25°C in the presence or absence of the carrier insect. In artificial nests with > 99.9% RH, DL resumed their mobility and the number of DL on female insects decreased gradually after oviposition, although numerous DL remained on the insects. Very few DL were detected on mother insects after hatching. Nematode propagation was observed on the egg mass after hatching and on nymphal carcasses; the total number of nematodes in the nest increased dramatically after this point. These results indicate that humidity is an important factor for disembarkation of C. japonica DL and that C. japonica propagates in the nest of P. japonensis where it feeds on the remains of eggs and nymph carcasses, indicating that C. japonica and P. japonensis have a unique phoretic and necromenic association.

Mesohabitat selection in fluvial fishes was studied in a small tropical stream of the Malay Peninsula. A total of 681 individuals representing 24 species were sampled at 45 stations within heterogeneous stream (ca. 1 km in length), in which water depth, water velocity, substrate size, and riparian canopy cover were measured as environmental variables. A canonical correspondence analysis (CCA) yielded a diagram that shows a specific mesohabitat selection of the fish assemblage, in which the species were plotted widely on the CCA1-CCA2 biplot. Generalized linear model also revealed a significant pattern of the mesohabitat selection of several species. Water velocity and substrate size mainly separated on CCA1, indicating variation of pool (deep, slow-flow section) and riffle (shallow, fast-flow section) structures is a primary factor of mesohabitat selection in the fluvial fish assemblage. The mean body weight of species significantly correlated with CCA1; larger species tended to inhabit pools, while small ones occupied riffles. The riparian canopy cover separated on CCA2. The trophic level of species significantly correlated with CCA2; herbivorous species (low trophic level) selected open sites without riparian cover, whereas omnivorous/carnivorous (middle-high trophic level) species preferred highly covered sites. In conclusion, our results suggest that mesohabitat selection is closely related to the species feeding habit, which is consistent with the results of previous studies.

The Harderian gland (HG) is an orbital gland found in many terrestrial vertebrates that possess a nictitating membrane. Using reverse-transcription polymerase chain reaction (RT-PCR), we cloned five cDNAs encoding antimicrobial peptide (AMP)-homologs, catesbeianalectin, ranacyclin-CBa, ranatuerin-1CBa, ranatuerin-2CBa, and ranatuerin-2CBb, from the bullfrog HG total RNA. Of these, catesbeianalectin has not been thoroughly studied in terms of its biological activities. We examined antimicrobial activities of the synthetic replicate of catesbeianalectin and its putative unprocessed precursor, catesbeianalectin-GK. Both peptides showed slight but significant growth inhibitory activity against the Gram-negative bacterium Escherichia coli. Subsequently, we tested catesbeianalectin and catesbeianalectin-GK for mast cell degranulation activity as a criterion of the release of N-acetyl-β-D-glucosaminidase from the mouse-derived mastocytoma cell line P-815, followed by the standard MTT assay to assess cell survival and recovery after peptide treatment. We found that catesbeianalectin and catesbeianalectin-GK invariably exhibited mast cell degranulation activity without cytotoxic effects. Hemagglutination assay revealed the presence of lectin-like activity in both catesbeianalectin and catesbeianalectin-GK. Our findings strongly suggest that these multifunctional host defense peptides in the amphibian HG are involved in innate immunodefense of the eye of the host against pathogenic environmental microorganisms.

The complete mitochondrial genome (mitogenome) of Gomphocerus sibiricus, consisting of 15,590 bp, was determined and analyzed. It displays typical genome organization found in other Caelifera mitogenomes: 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one A T rich region. In this paper, we focused on the comparative analyses of mitogenomes from four Gomphocerinae taxa to find characteristics of nucleotide composition and overlapping and non-coding regions. In addition, we compared variable sites in the structure of 22 tRNAs and two rRNAs, and differences in some common conserved elements of A T rich regions. Furthermore, we analyzed phylogenetic relationships of 12 Caelifera species using maximum likelihood (ML) and Bayesian inference (BI) based on two different datasets from mitogenomes. Our results reveal that G. sibiricus has a close relationship with Gomphocerus licenti of the four Gomphocerinae taxa examined in these analyses.

Individual variations in skull and lower carnassial morphology within a wild population of raccoon dog were examined using geometric morphometric techniques. We compared individual morphological variations by using relative warp analysis, and then tested morphological integration between the skull and carnassial by using partial least square (PLS) analysis. The most marked variation in skull shape was the dorsoventral flexion; i.e., deformation from klinorhynchy to airorhynchy. Two remarkable variations were observed, including tilting between the trigonid (or carnassial blade) and the talonid in the lower carnassial, and the relative sizes of the trigonid and the talonid. This observed variation in skull shape was similar to previous reports of variations among dog breeds that correlate with a polymorphism of the Runx2 gene. This polymorphism has also been reported to correlate with snout length, which is strongly related to carnivorous or omnivorous dietary adaptations, across the entire order Carnivora. Our results in the lower carnassial were also similar to previously reported patterns observed for carnivorous or omnivorous dietary adaptations among Carnivora. However, in our PLS analysis between skull and carnassial shapes, we only found a significant correlation in a lower dimension, suggesting a lower degree of integration. These results indicate that shape variations, which could be sources of natural selection in the skull and carnassial, were present in a wild population, suggesting high evolvability of these variations in the raccoon dog and the order Carnivora in general.

In the present study, we analyzed the ontogenetic scaling of humeri in the green turtle (Chelonia mydas) and loggerhead turtle (Caretta caretta). Green turtles have relatively thicker humeri than loggerhead turtles, indicating that the humerus of the green turtle can resist greater loads. Our results are consistent with isometry, or slightly negative allometry, of diameter in relation to length of the humerus in both species. Geometric similarity or isometry of the humerus in relation to body mass is supported by estimates of the cross-sectional properties of green turtles. Sea turtles are adapted for aquatic life, but also perform terrestrial locomotion. Thus, during terrestrial locomotion, which requires support against gravity, the observed scaling relationships indicate that there may be greater stress and fracture risk on the humeri of larger green turtles than on the humeri of smaller turtles. In aquatic habitats, in which limbs are mainly used for propulsion, the stress and fracture risk for green turtle humeri are estimated to increase with greater speed. This scaling pattern may be related to the possibility that smaller turtles swim at a relatively faster speed per body length.

Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblastic and osteoclastic activities were measured. Osteoblastic activity significantly increased under 2- to 4-G loading by both centrifugation and vibration. In contrast, we found that osteoclastic activity significantly decreased under 2- and 3-G loading in response to both centrifugation and vibration. Under 4-G loading, osteoclastic activity also decreased on centrifugation, but significantly increased under 4-G loading by vibration, concomitant with markedly increased osteoblastic activity. Expression of the receptor activator of the NF-κ;B ligand (RANKL), an activation factor of osteoclasts expressed in osteoblasts, increased significantly under 4-G loading by vibration but was unchanged by centrifugal loading. A protein sequence similar to osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, was found in medaka using our sequence analysis. The ratio of RANKL/OPG-like mRNAs in the vibration-loaded scales was significantly higher than that in the control scales, although there was no difference between centrifugal loaded scales and the control scales. Accordingly, medaka scales provide a useful model by which to analyze bone metabolism in response to physical strain.

Large amounts of neutral lipids (NLs) are stored as lipid droplets in the ooplasm of fish oocytes, providing an essential energy resource for developing embryos and larvae. However, little is known about the origin of such lipids or about mechanisms underlying their uptake and accumulation in oocytes. We have proposed a model for this lipidation of teleost oocytes, as follows: very low density lipoprotein (Vldl) is metabolized by lipoprotein lipase (Lpl) outside and/or inside of the oocyte and the resulting fatty acids (FAs) are then utilized for de novo biosynthesis of NLs. As a first step toward verification of this model, cDNAs for genes encoding two types of Lpl, lpl and lpl2, were cloned from the ovary of cutthroat trout, Oncorhynchus clarki. Examination of Lpl polypeptide sequences deduced from the cDNAs revealed features similar to LPLs/Lpls in other species, including several conserved structural and functional domains. Both types of lpl mRNA were highly expressed in lipid storage tissues (e.g., adipose tissue, muscle, and ovary) and were predominantly expressed in the granulosa cells of ovarian follicles. Ovarian lpl1 mRNA levels showed a remarkable peak in April (early oocyte lipid droplet stage) and then decreased to low values sustained until November (mid-vitellogenesis), after which time a small peak in lpl1 gene expression was observed in December (late vitellogenesis). The mRNA levels of lpl2 also were elevated in April and were highest in June (late lipid droplet stage), but did not show other pronounced changes. These results suggest that, in the cutthroat trout, Vldl is metabolized by the action of Lpls in the granulosa cell layer to generate free FAs for uptake and biosynthesis of neutral lipids by growing oocytes.