The effects of the delay and duration of wind self-generated during walking on the compensational recovery of escape direction were investigated in unilaterally cercus-ablated crickets, Gryllus bimaculatus. Artificial self-generated winds (self-stimulations; hereafter, SSts) from a nozzle set in front of a cricket placed on a styrofoam ball for stationary walking were used for training after unilateral cercus ablation. The delay and duration of artificial SSts were separately controlled. When the stimulus duration was fixed to 100 msec, the crickets trained with artificial SSts of 1000 msec delay showed a compensational recovery of the escape direction. However, no such compensational recovery was observed in crickets trained with artificial SSts of 1200, 1500, and 2000 msec delays. The relationship between the delay and duration of artificial SSts for compensational recovery was investigated. An artificial SSt with a longer delay required a longer-duration air current to cause a recovery of the escape direction. In contrast, an artificial SSt with a shorter delay was effective even when the duration was short. On the basis of the results obtained in the present study, we propose a hypothesis to explain the initial step for the compensation, that is, how the delay and duration of SSts are traded in terms of the compensational recovery of the escape direction.

We investigated the effects of extracellular proteinases of two Pseudomonas aeruginosa clinical isolates on the essential humoral immune response parameters in hemolymph of the insect model organism Galleria mellonella in vitro. Two culture media, rich LB and minimal M9, known to induce synthesis of different sets of proteinases secreted by P. aeruginosa were used. Changes in lysozyme, antibacterial and antifungal activities, as well as protein and peptide patterns in hemolymph treated with proteolytic fractions were evaluated. The effect of the proteolytic fractions on the apoLp-III level in hemolymph was determined by immunoblotting with antibodies against G. mellonella apolipophorin III (apoLp-III). We found that apoLp-III is hardly degraded by the proteinases of the proteolytic fractions of both clinical P. aeruginosa strains, in contrast to the high susceptibility of the protein to the proteinases of the entomopathogenic strain. The detected differences, together with the changes in the hemolymph protein and peptide patterns caused by the studied fractions, reflected the distinct composition of secreted proteinases of the entomopathogenic P. aeruginosa strain and the clinical strains tested. Our results also suggest the involvement of alkaline protease, the main proteinase of proteolytic fractions of P. aeruginosa grown in minimal medium, in the degradation of G. mellonella antimicrobial factors, such as lysozyme, antibacterial polypeptides, and proteins with antifungal activity. The diverse effects of the P. aeruginosa proteolytic fractions studied on the parameters of G. mellonella immune response indicate that this model insect may be useful in the analysis of the virulence factors of different P. aeruginosa strains.

Ascidian Ciona intestinalis tadpole larvae exhibit left-right asymmetry. The photoreceptors are situated on the right side of the sensory vesicle, and the tail curls along the left side of the trunk within the chorion. In tailbud embryos, the Ci-pitx gene is expressed in the left-side epidermis. It was previously reported that embryos generated from naked eggs, which lack the chorionic membrane and accessory cells (follicle cells attached to the outside of the chorion and test cells covering the inner surface of the chorion), show bilateral expression of Ci-pitx. This suggested that the chorion or accessory cells are needed for generation of asymmetry. Here, we show that a brief treatment with 60% artificial seawater (ASW) before, but not after, the neurula stage results in bilateral expression of Ci-pitx in the chorion of tailbud embryos, loss of follicle cells, and randomization of both the direction of tail curling and the locations of photoreceptors in larvae. This treatment also impaired the transient counterclockwise rotation within the chorion at the neurula stage. Nearly all test cells in the chorion died following 60% ASW treatment. These results suggest that dead test cells blocked the neural rotation and impaired left-right asymmetry. We also showed that tailbud embryos and larvae generated from defolliculated eggs produced by 80% ASW treatment, in which the test cells were alive, showed normal left-right asymmetry, suggesting that the follicle cells were not essential for asymmetric morphogenesis.

Two shell color types, yellow (type I) and brown (type II), of hermaphrodite Corbicula fluminea clams from Ritto, Shiga Prefecture, Japan, are sympatric with both male and hermaphrodite Corbicula leana. In the present study, the mitochondrial DNA (mtDNA) cytochrome b and nuclear 28S rRNA genes of C. fluminea were sequenced to construct a haplotype network in order to investigate the genetic relationship with C. leana. Ninety C. fluminea samples revealed only two cytb haplotypes; the majority (97.8%) were CB7, while the remainder were CB1. In C. leana, only CB1 was detected in hermaphrodites, but both CB1 and CB7 were detected in males. Nuclear 28S rRNA haplotypes of C. fluminea type I individuals were divergent from those of hermaphrodite C. leana. However, C. fluminea type I clams shared haplotypes with male C. leana individuals, whereas C. fluminea type II individuals shared haplotypes with both hermaphrodite and male C. leana samples. These results suggest that it may be difficult to define a clear genetic border between these species.

The eggshells of 56 chelonians were examined by electron microscopy and X-ray diffractometry. They were classified into six types in terms of the matrix structure of their calcareous layer; type I was composed of a thin calcareous layer with minerals in an amorphous structure; type II with shell units composed of mammillary cores calcified with aragonite crystals; type III with shell units composed of mammillary cores, plus a single palisade layer also calcified with aragonite crystals, and with each shell unit separated; type IV with shell units the same as type III, but tightly packed together; type V with shell units composed of mammillary cores plus two palisade layers; and type VI with a cuticle layer calcified with calcite crystals over the same structure as that of type V. X-ray diffraction analyses at the outer surface of eggshells showed a gradual change in crystal disposition from the random disposition of type II to the single direction-oriented disposition of type V. The shell height was approximately parallel to the development of the palisade-layer matrix. The limiting membrane of all eggshell types was perforated with canals and that of type I was partially missing. Type I had a parchment shell, types II and III had a pliable shell (some were rigid) and types IV to VI had rigid shells. The present study showed that the hardness of eggshells can be determined by the composition of the shell matrices, as shell matrices are the framework for mineralization.

Fishermen often anecdotally report an unexpected increase of fish caught in small tributary streams during floods, presumably due to refuge-seeking behavior from the main stem. From a population perspective, this implies the significance of refuge habitats and connectivity for population viability against natural disturbances. Despite the plausibility, however, surprisingly few studies have examined the tributary refuge hypothesis, mainly due to the difficulty in field survey during floods. Here, we made use of a large-scale controlled flood to assess whether fishes move into tributaries during flooding in the main stem. A planned water release from the Satsunai River Dam located on Hokkaido Island in Japan rapidly increased the main stem discharge by more than 20-fold. Before, during, and after flooding censuses in four tributaries provided evidence of the refuge-seeking behavior of fishes from the main stem. For example, more than 10 Dolly Varden char, a salmonid fish, were caught in a tributary during the flood, even though almost no individuals were captured before or after the flood. The fish responded immediately to the flooding, suggesting the need for studies during disturbances. In addition, the likelihood of refuge movements varied among tributaries, suggesting the importance of local environmental differences between tributary and the main stem habitats. This is the first study to experimentally confirm the tributary refuge hypothesis, and underscores the roles of habitat diversity and connectivity during disturbances, even though some habitats are not used during normal conditions.

The evolutionary phenomena associated with divergence in chemical signals between populations of the same species help to understand the process of speciation. Animals detect and react to semiochemicals and pheromones used in communication. Comparison between populations of the same species that are geographically isolated from one another allows us to determine the genetic or environmental factors responsible for chemical differentiation. Acanthodactylus boskianus from the east and west of Egypt were used as an example to compare the geographical diversity in chemical fingerprints of this species' femoral gland secretions and its phylogeography. Chemical analysis via GC—MS showed that the two geographically distinct populations' odor fingerprints are quantitatively different despite sharing the same components of the secretions. Phylogenetic analysis showed that the eastern and western Egyptian populations are genetically distinct and that chemical divergence of these lizards' odor profiles may be an example of signal evolution.

In Japan, rice paddies have acted as substitute habitats for pond-breeding frogs. However, frog populations are declining due to the loss of habitat and environmental changes in rice paddy areas. Frogs need both aquatic and terrestrial habitats to complete their life history; in rice paddy areas, levees that surround rice paddies provide terrestrial habitats for basking, foraging, and shelter from predators. Studying microhabitat use at levees is important to elucidating the ecological roles of levees and to properly managing them to support frog populations. In this study, we conducted surveys in lowland modernized rice paddy areas in Shiga Prefecture in which a common species, Hyla japonica, and an endangered species, Pelophylax porosa brevipoda, were found. We captured frogs at levees and recorded environmental factors related to levee vegetation, rice paddy conditions, and weather. We constructed generalized linear mixed models to examine the effects of environmental factors on juvenile and adult H. japonica and on small and large juveniles, females, and males of P. p. brevipoda. Our results showed distinct microhabitat uses at levees in different species, sexes, and body sizes. In general, abundance was high at levees with vegetation that provided shelter. The water depth in rice paddies negatively influenced juvenile H. japonica and large juvenile and small female P. p. brevipoda, and positively influenced small male P. p. brevipoda. The maintenance of a mosaic structure of levees was important not only to support frog populations but also to maintain frog diversity in the area.

Postembryonic development of a larval tadpole into a juvenile frog involves the coordinated action of thyroid hormone (TH) across a diversity of tissues. Changes in the frog transcriptome represent a highly sensitive endpoint in the detection of developmental progression, and for the identification of environmental chemical contaminants that possess endocrine disruptive properties. Unfortunately, in contrast with their vital role as sentinels of environmental change, few gene expression tools currently exist for the majority of native North American frog species. We have isolated seven expressed gene sequences from the Northern green frog (Rana clamitans melanota) that encode proteins associated with TH-mediated postembryonic development and global stress response, and established a quantitative real-time polymerase chain reaction (qPCR) assay. We also obtained three additional species-specific gene sequences that functioned in the normalization of the expression data. Alterations in mRNA abundance profiles were identified in up to eight tissues during R. clamitans postembryonic development, and following exogenous administration of TH to premetamorphic tadpoles. Our results characterize tissue distribution and sensitivity to TH of select mRNA of a common North American frog species and support the potential use of this qPCR assay in identification of the presence of chemical agents in aquatic environments that modulate TH action.

The epithelial sodium channel (ENaC) has four subunits, namely α (alpha), β (beta), γ (gamma) and δ (delta). The functional ENaC is formed by the combination of either αβγ or δβγ subunits. The aim of the present study is to determine the combination of ENaC subunits predominant on the apical side of the frog skin, and the effect of ADH on sodium transport though these two ENaCs subunit combinations. The ventral abdominal skin of the frog, Rana hexadactyla was mounted in an Ussing-type chamber. The voltage-clamp method was performed to measure the ionic transport across the frog skin with normal Ringer solution (NR) on both sides. Evans blue (300 µM) and amiloride (100 µM) were added to the NR on the apical side and ADH (40 nM) was added on the serosal side. Statistical significance was analyzed by Student's paired t-test and repeated-measures ANOVA, P < 0.05 was considered significant. This study suggests that the ENaC of the frog skin consist of both αβγ and δβγ subunit combinations on the apical side. Though both types of subunit combination are present, the αβγ type was found to be more common than δβγ. ADH increases the sodium transport across the frog skin. The effect of ADH on sodium transport is achieved through the combination of δ-subunits, not through the combination of a-subunits in the skin of Pana hexadactyla.

A fish population of the carp family Cyprinidae with atypical phenotypic characteristics was observed in one of the main catchments of the Pollino National Park, a valuable, protected area in southern Italy. In this area, the Italian roach Rutilus rubilio (Bonaparte, 1837), a native endemic fish of Tyrrhenean regions, has been introduced in sympatric conditions with Squalius squalus (Bonaparte, 1837) and Telestes muticellus (Bonaparte, 1837). A molecular investigation was carried out to assess the genetic identity of the population with a view to conservation. Direct sequencing of a cytochrome b gene fragment was performed based on 30 individuals of cyprinid fish with atypical phenotype, in addition to 30 S. squalus, 10 T. muticellus, and 30 R. rubilio pure individuals collected in different Italian regions, which served as reference samples. Multiple sequence alignments demonstrated that 50% of atypical-cyprinid haplotypes were maternally inherited from either S. squalus or R. rubilio. No contribution by T. muticellus was determined. Our results indicate an intergeneric hybridization event between S. squalus and R. rubilio, as a consequence of trans-introduction activities of alien species.

A new species of the previously monospecific marine tardigrade family Neostygarctidae is described. Neostygarctus lovedeluxe n. sp. was found from a submarine cave in Miyako Islands, Japan. This is the first record of Neostygarctidae from the Pacific. The new species is easily distinguished from the previously known N. acanthophorus by its number of dorsal spines, as N. lovedeluxe has two spines each on the three dorsal body plates in contrast to one in N. acanthophorus. Furthermore the morphology of the two clawed juvenile is reported for the first time in Neostygarctidae, providing new insights into the common sequence in some ontogenic traits.