Zoological Science

Certain behavioral acts of Pleurobranchaea japonica may be elicited in response to chemical stimulants applied to the rhinophores, tentacles or oral veil. These include feeding and escape. Upon treatment with chemical stimulants, the organs respond directly by extending or withdrawing. Beyond this, aversive responses involved withdrawal of the rhinophores, tentacles and oral veil plus contraction of the whole body. Feeding behavior was elicited by glycine, phenylalanine, proline, aspartic acid, alanine, asparagine, tryptophan and glutamine. An aversive response was induced by glutamate. Neural pathways responsible for these chemoreceptive reflexes have been identified. In addition to known nerves, a newly identified pair of nerves, the second pedal nerves arise from the pedal ganglion and send branches to the three organs. The nerves contain afferent pathways for chemoreception of the organs and efferent motor pathways for the movements of the organs. The rhinophore ganglion is responsible for the glutamate-induced contraction of the rhinophore. This withdrawal reflex of the rhinophore is under inhibitory control, exerted by the cerebral ganglion through the rhinophore nerve, and under an excitatory influence, emanating from the pedal ganglion through the second pedal nerve.

Previous theoretical studies and some empirical studies suggested that the factors which affect female availability should influence male reproductive behavior. We noticed the female post-emergence behavior as one such factor, and investigated how it might influence male reproductive strategy in the butterfly, Pieris rapae crucivora. In the field and the laboratory newly-emerged females stayed on the under-side of leaves 2.5 hr after emergence before they flew voluntarily into the open where they are more visible to males. Time spent before initial flight correlated negatively with ambient temperature, and initial flights increased significantly the possibility that the female would be detected by males. To evaluate whether these results could explain the time schedule of males' search for females in the field, female availability was estimated by shifting the observed schedule of female emergence according to the relationship between initial flight and effective cumulative temperature. The resultant female availability correlated well with temporal changes observed in the number of mate-seeking males. Thermal conditions may influence male reproductive behavior via female post-emergence behavior, as well as via male body temperature.

Three cell lines have been established from melanotic and amelanotic melanophores of wild and albino Rana nigromaculata and albino Rana porosa brevipoda after experiencing three culture crises. The population doubling rate increased with culture time. Cells from both wild and albino R. nigromaculata changed shape twice in ten years. They began as large pigmented or non-pigmented epithelial-like cells at the beginning of the cultures. The melanophores of albino R. nigromaculata unexpectedly became pigmented shortly after the beginning of the culture. Over time, they transiently aquired a small, non-pigmented, bipolar morphology and finally became large non-pigmented epithelial-like cells after about 8 to 10 years. On the contrary, cells from albino R. porosa brevipoda remained large, non-pigmented, epithelial-like cells over the entire course of the culture. Pigmented cells contained many mature melanosomes, while non-pigmented epithelial like cells contained premelanosomes with characteristic internal structures. Non-pigmented, fibro-blast-like cells contained numerous small vesicles in place of typical premelanosomes. The cells of established cell lines, including those from wild R. nigromaculata, were unpigmented and showed no tyrosinase activity in histochemical dopa reactions. The number of chromosomes deviated from the normal pattern in the three cell lines. Each of the three cell lines had a different modal number than the other two, and the chromosomal compositions of the cells within the same lines differed from one another. Even within the same cell lines, the mode changed as the culture progressed without any consistent trends.

Trichocyst discharge in Paramecium spp. is known to be mediated by rapid exocytosis. Applied stimuli induce fusion of the trichocyst membrane and plasma membrane within 30 ms. Both Ca²⁺ release from intracellular store(s) and Ca²⁺ influx from extracellular region have been suggested to be related to the trichocyst discharge.

We constructed a new system in which to record intracellular levels of Ca²⁺ ([Ca²⁺]_i) and microscopic images simultaneously without changing the optical path. With this system, we recorded [Ca²⁺]_i at 2 ms intervals and microscopic images of trichocyst discharge at video rate (33 ms intervals) simultaneously in Paramecium caudatum.

Simultaneous application of Ca²⁺ chelator at 100 mM with secretagogue onto Paramecium cells resulted in only a slight increase in [Ca²⁺]_i (Δ[Ca²⁺]_i). Furthermore, no extrusion of trichocysts occurred. In contrast, application of secretagogue concomitant with Ca²⁺ chelator at 20 mM induced a Δ[Ca²⁺]_i composed of two phases. In this case, extrusion of trichocysts occurred. These observations directly indicated that Ca²⁺ influx from the extracellular medium in addition to Ca²⁺ release from intracellular store(s) contributes to Δ[Ca²⁺]_i during trichocyst discharge.

We compared chromosome morphologies for 11 species of Malagasy poison frogs, genus Mantella, and three outgroup taxa (genus Mantidactylus) using conventional and fluorescence staining techniques. All species studied had a karyotype of 2n=26, with five larger and eight smaller chromosome pairs. The 11th pair was acrocentic in Mantella nigricans which represents the first such observation in the genus. The nucleolus organizer region (NOR) was located at secondary constrictions on chromosome pair 2 in all Mantella studied and in Mantidactylus grandisonae (while located on other chromosomes in all other species of Mantidactylus studied so far). Heterochromatin distribution was highly variable among Mantella species; C-bands positively staining with DAPI and CMA₃ were observed. The possible structure of these bands, seemingly containing both A+T rich and C+G rich heterochromatin, is discussed. Phylogenetic reconstruction using chromosomal characters provided very little information. Evolution of the characters studied is probably either too fast (heterochromatin arrangement) or too slow (NOR location) to match the main cladogenetic events among Mantella species groups.

Tetrameric hemoglobins (Hbs) A and D were isolated from red blood cells of the Aldabra giant tortoises, Geochelone gigantea, by a hydrophobic interaction chromatography. After reduction and S-pyridylethylation, two sets of two types of α-chains (α-1 and α-2) and one β-chain were purified from the major Hb A and minor Hb D in molar ratios of about 1:1:2, respectively, by a reversed-phase column chromatography. The complete amino acid sequences of the three globin-chains from Hb A were determined: 141 amino acid residues for the two α-chains and 146 amino acid residues for the β-chain. Using computer analysis (amino acid maximum homology), the two α-chains shared a 96.5% sequence identity and had low sequence identities (37.8% for α-1 and 35.8% for α-2) with the β-chain of the same species, G. gigantea.

We constructed a phylogenetic tree of 28 primary globin structures from Reptilia (7 species of squamates, 4 species of turtles, 3 species of crocodiles and 1 species of sphenodontids), including the three globins of G. gigantea Hb A. The following results were obtained: (1) The two terrestrial species of Geochelone (G. gigantea and G. carbonaria) were closely related: 139 amino acid residues (95.2%) of the two β-globin chains were conserved; (2) Based on the divergence patterns of globin-chains, the sea turtle Caretta caretta was shown to be unusual relatedness form the groups of terrestrial and freshwater species in turtles. The molecular relationships appearing on the phylogenetic tree also support the traditional classification of reptiles and partly confirm previous molecular studies of reptilian hemoglobin evolution.

An atlas of the developing brain up to hatching stage is established using conventional histo-logical methods in the oegopsid squid, Todarodes pacificus. The brain originates from placodal thickenings in the ectoderm at the end of epiboly. The neuroblasts composing the placodes ingress in a group and accumulate into ganglia under the proximal surface of the surface epithelium. Four pairs of the ganglia, pedal, palliovisceral, cerebral, and optic, form the brain primordium. These ganglia come into contact with one another, and eventually accumulate into a ring-like cluster (circumesophageal cluster) encircling the oral ingrowth and the inner yolk around the surface of the head. The circumesophageal cluster regionally differentiates into brain lobe anlagen through formation of neuropiles and nerve tracts. The neuropiles form a ladder-like structure with two longitudinal columns situated in the ventrolateral parts of the circumesophageal mass and some axonal tracts bridging the left and right columns (ladder-like framework). The brain is quite premature at the time of hatching, especially in the supraesophageal part. Though most brain nerves are already present, many brain lobes and commissures found in the adult brain are not yet differentiated. The present results show that the morphological processes of the brain formation are essentially common among the coleoid cephalopods. The similarity of the embryonic brain in Todarodes to the adult brain in Nautilus suggests that the coleoid brain has evolved on the basic plan as seen in the nautiloid brain.

Invertebrate insulin-related peptides play key roles in growth and metabolism. Genes encoding these peptides are specifically expressed in neurosecretory cells. Bombyxin genes of the silkmoth Bombyx mori encode insulin-related peptides and are expressed in four pairs of the neurosecretory cells in pars intercerebralis of brain. No regulatory element has been identified to confer the neurosecretory cell-specific expression of bombyxin gene. By promoter-deletion analysis and in vitro electroporation, we identified a transcription element essential for the cell-specific expression of bombyxin F1 gene, one of the bombyxin multifamily genes. The element was localized in the region from −170 to −159 bp upstream of the translation start site of the F1 gene. We named the element as BOSE, BOmbyxin gene-Specific Element. No protein that would bind to BOSE was found by searching the transcription factors database. In addition, an activator element responsible for increasing the expression level was identified in the region from −185 to −181 bp. The activator element is similar to the core element in gut-specific enhancer region of the mosquito Anopeles gambiae trypsin gene and the element essential for expression of a Bombyx chorion gene.

A high activity of ecto-ATP diphosphohydrolase (ATPDase, EC 3.6.1.5) is present on the surface of ovarian follicle cells of the starfish Asterina pectinifera. It is known that follicle cells play an important role in oocyte maturation by producing 1-methyladenine (1-MeAde) as the maturation-inducing substance. This study was undertaken to determine if ecto-ATPDase is associated with 1-MeAde production. 1-MeAde production is induced by a gonad-stimulating substance (GSS) secreted from nervous tissue. It has also been shown that G-proteins and adenylyl cyclase are involved in the action of GSS in 1-MeAde production. When isolated follicle cells were incubated with the ecto-ATPDase metabolites, adenosine and AMP, a slight decrease in intracellular cyclic AMP level was observed. Adenosine also inhibited adenylyl cyclase in the presence of GTP. Pertussis toxin reversed adenosine-induced inhibition, suggesting that adenosine acts via the Gi receptor. On the other hand, GSS could induce 1-MeAde production and an increase in cyclic AMP level regardless of the absence or presence of AMP or adenosine. GSS-dependent 1-MeAde production markedly decreased after washing of follicle cells with seawater containing low concentrations of Ca²⁺, but the activity of ecto-ATPDase remained constant. Neither NaF nor adenylimidodiphosphate, which are inhibitors of ecto-ATPDase, had any effect on GSS-induced 1-MeAde production. Thus, it is unlikely that ecto-ATPDase is directly involved in 1-MeAde production in starfish ovarian follicle cells. Since an increase in cyclic AMP level is indispensable for 1-MeAde production, it may be possible that adenosine and AMP produced by ecto-ATPDase play a role in prevention of precocity before oocyte maturation stage.

Both male and female red sea urchins, Pseudocentrotus depressus, accumulate a large quantity of the major yolk protein (MYP) in the nutritive phagocytes of immature gonads before the initiation of game-togenesis. To examine the accumulation mechanism of this protein in the gonad, we cloned full-length cDNA encoding vitellogenin (Vg; the MYP precursor in the coelomic fluid), and investigated its expression in various tissues of immature adults. The nucleotide sequence of Vg contains an open reading frame of 4050 bp encoding 1349 amino acids. The deduced amino acid sequence near the N-terminal showed 25% homology to the vertebrate transferrin family. Vitellogenin mRNA was detected in the ovary, testis, stomach, intestine and rectum by Northern blot analysis, with the highest level of mRNA expression in the gonad. Weak expression was also detected in the esophagus and coelomocytes by RT-PCR. In situ hybridization demonstrated that nutritive phagocytes, which exclusively fill the lumina of the immature gonad, contained Vg mRNA. These results suggested that the MYP stored in the immature gonads is synthesized and accumulated mainly within the nutritive phagocytes.

Development of the gonads and embryo in the deep-sea sponge-associated shrimp, Spongicola japonica Kubo, was observed based on field samples collected from July 1993 to June 2000. Histological observation of gonads revealed that the gonadal maturities were divided into 4 and 5 stages in males and females, respectively. In females the smallest mature size was 5.5–6.0 mm in carapace length (CL), and the beginning of oocyte development was related to body size. The beginning of spermatogenesis was not related to body size. Grouped males started spermatogenesis at more than 3.0 mm CL, but the immature testis was recognized at 4.0–4.5 mm CL in solitary males. Embryonic development was classified into 12 stages based on the morphology of embryonic appendages. Ovarian maturity was almost always synchronized with embryonic stages in ovigerous females, and the females with fully developed embryos had fully ripened ovaries. The ovigerous females molted after hatching juveniles, and laid new eggs. Reproductive seasonality was not recognized in S. japonica. The induction of reproduction in this species may be controlled not by any physical environmental factors, but by other factors such as body size.

In Xenopus laevis, the spermatogenic cells derived from a primary spermatogonium divide synchronously and form a cyst surrounded by Sertoli cells. Therefore, it is easy to know how many times the spermatogenic cells divide from the primary spermatogonium by counting the cells in a cyst. In the present report, the distinctive feature of mitotic divisions during the spermatogonial stage in Xenopus laevis is described as having two phases. The first phase took place during five mitotic divisions. During this phase, the nucleus of primary spermatogonium, which was identified by its large size and lobulated shape with weak stainability, progressively decreased in size and became round with heterochromatin patches. In the second phase, the spermatogonia, that had completed the fifth mitotic division and morphologically differentiated into the secondary spermatogonia, could undergo up to three additional mitotic divisions without any morphological change. From the observation of the cysts that consisted of approximately 2⁵, 2⁶, 2⁷, or 2⁸ primary spermatocytes, it was further suggested that the secondary spermatogonia at the second phase could also enter a meiotic phase. All primary spermatocytes were believed to have the ability to undergo the meiotic phase-specific gene expression and two meiotic divisions because the round spermatids in the cyst consisting of approximately 2⁷, 2⁸, 2⁹, or 2¹⁰ cells expressed SP4 mRNA which was transcribed in the spermatogenic cells at mid-pachytene stage and thereafter. These observations suggested that after the fifth mitotic division, the spermatogenic cells differentiated into the secondary spermatogonia and acquired an ability for mitosis to meiosis conversion.

The influence of web design on prey capture efficiency was investigated in the orb-webs of Octonoba sybotides under different prey conditions (abundant and limited). The insects captured by artificial traps under limited prey conditions were smaller than those captured under abundant prey conditions. O. sybotides webs with spiral stabilimenta (SP webs) have a narrower mesh and a larger catching area than webs with linear stabilimenta (LN webs). Previous studies have shown that the form of the stabilimentum is changed in response to the internal energetic state of individual spiders. Food-deprived spiders tend to construct SP webs, and food-satiated spiders tend to construct LN webs. The total thread length of SP webs is significantly longer than that of LN webs; so food-deprived spiders seem to invest more energy in foraging than do satiated spiders. When prey was abundant, prey sizes and capture rates for SP webs and LN webs were similar. LN webs may have an advantage over SP webs in prey capture when prey is abundant, because SP webs appear to be more costly than LN webs. On the other hand, SP webs caught more prey than LN webs under limited-prey conditions, when the prey was smaller than under abundant-prey conditions. The average size of prey captured on SP webs was smaller than that on LN webs; SP webs seem to catch more prey than LN webs by catching smaller prey efficiently in prey-limited conditions. Since the food-deprived and food-satiated conditions of spiders in the field may correspond to the potential prey abundance in their habitat, O. sybotides seems to change its web dimensions in order to capture prey efficiently under different prey conditions.

We compared the reproductive phenology and tadpole thermal physiology of two populations of Buergeria japonica in Taiwan. The population in Jentse breeds in a geothermal hot spring whose water is a warm, over 35°C year-round, whereas the population in Chukou breeds in a stream whose water temperature is closely correlated with fluctuations in air temperature and averages 23°C. The B. japonica population in Jentse breeds year-round, but the Chukou population breeds only from March to October. Tadpoles from Jentse that reared at 32°C attained larger sizes than tadpoles from Chukou that reared at 32°C, whereas tadpoles from both sites attained similar body size when reared at 22°C. Chukou tadpoles reared at 40°C died within 1–2 d, whereas Jentse tadpoles survived for 9 d at 40°C. All tadpoles tolerated heat well, but Jentse tadpoles had higher critical thermal maxima (CTMax) than Chukou tadpoles. Tadpoles from Jentse showed no metabolic compensation but those from Chukou showed an “inverse” metabolic compensation. Results suggest that B. japonica that breed in the geothermal hot spring has a prolonged breeding season, high growth rates and the reduction or absence of intra- and inter-specific competition. Evolution of a high level of heat tolerance and, possibly, behavioral temperature selection, enable tadpoles in the hotsprings to offset the thermal stress imposed by warm water.

Using skeletochronology, we compared age structure in the breeding season in two populations (Katata, Shiga Pref. and Shizuhara, Kyoto Pref.) of a pond frog, Rana nigromaculata from central Japan and elucidated interpopulation variation in some life history traits. Newly metamorphosed froglets were larger in SVL, heavier in body mass, and fattier in Katata than in Shizuhara. In both populations, the age at first reproduction was 2 yrs for males and 3 yrs for females. Longevity was estimated to be at least 5 and 6 yrs at Katata and Shizuhara, respectively, for both sexes. Females were on an average significantly older than males in both populations. The modal age in breeding males was 2 yrs in Katata but 4 yrs in Shizuhara, and Katata males (mean=3.27 yr) tended to be younger than Shizuhara males (mean=3.42 yr), although the difference was not significant. Similarly, females did not differ in the mean age in the two populations (4.1 yrs in both populations). In SVL, females were significantly larger than males in both populations. In males, SVL was larger in Shizuhara than in Katata, but females did not differ in SVL. Shizuhara males grew faster than Katata males, but females did not differ in growth rate in both populations. Interpopulation differences in age and size properties of R. nigromaculata seem to be related to degree of interspecific competition and food consumption. We briefly discussed use of skeletochronology in relation to amphibian declining problems.

Individual ages of postmetamorphic R. rugosa from three populations of Kyoto, Japan (Gondoike, Iwakura, and Kiyotaki), were determined skeletochronologically. From recaptured individuals, one LAG was confirmed to be formed per year. In all three populations, age at sexual maturity was 1–2 yrs in males and 2–3 yrs in females, though some females matured at the age of 1 yr. Longevity was at least 4 yrs in males and 5 yrs in females. Sexual size dimorphism was conspicuous in adults, and females were invariably larger than males. Females had a larger asymptotic size than males but growth rate differed between some localities only in males. In all populations, both sexes showed high variations in SVL and the variation was even higher in juveniles than in adults. This high variation is explained by a prolonged breeding season and resultant mixture of metamorphs with and without overwintering larval stage within a population of this species.