Zoological Science

Complement receptor 3 (CR3), one of cell adhesion molecules, plays a crucial role in secretion of mammalian neutrophils. To study whether CR3 is also involved in neurosecretion of gastropod molluscs, we examined the CR3-like immunoreactivity in the central nervous system (CNS) and the superior and inferior tentacles of the terrestrial slug, Limax marginatus. In the CNS of L. marginatus, we did not detect the specific immunoreactivity at all. In contrast, the CR3-like immunoreactivity was observed in the cell bodies and processes of collar cells of the superior and inferior tentacles. In particular, granules contained in the cell bodies of collar cells exhibited the CR3-like immunoreactivity. Retrograde labeling of horseradish peroxidase applied on the sensory epithelia (SE) of superior and inferior tentacles showed that the collar cells of these tentacles project their processes to the SE. In the previous study, homogenate of the superior tentacles injected into the body cavity of slugs stimulated spermatogenesis and simultaneously inhibited egg-laying behavior, suggesting that the tentacular hormone acts as gonadotropic hormone. Taken together, CR3 expressed in the collar cells is considered to be involved in the secretion of tentacular hormone in L. marginatus.

When tail tips of the Japanese newt (Cynops pyrrhogaster pyrrhogaster) were amputated, large ionic currents were detected around the stump using a vibrating probe system. The initial wound currents flowed outwardly through the whole stump, and then localized within 24 hr toward the central region of the stump. Current densities reached a peak of about 450 μA • cm⁻² (about 900 times greater than before amputation) within one half of a day after amputation, and returned to levels similar to the intact tail tip by 4 days, i.e., the wound currents were transient. The swelling of the stump end (regenerate or blastema) occurred about 7 days after amputation. The major ion carrying the currents is thought to be Na⁺, because the currents were strongly inhibited by 0.5 mM ouabain added in the external medium or by Na⁺-deficient medium. These findings are consistent with those of newt limb stump.

Utilizing a basolateral membrane vesicle preparation containing Cl⁻-ATPase from Aplysia foregut, it was shown that orthovanodate inhibited Cl⁻-ATPase activity, ATP-dependent Cl⁻ transport and ATP-dependent membrane potential change. N-ethylmalemide (NEM) and p-chloromercurobenzoate (PCMBS) also inhibited the Cl⁻ pump biochemical and transport characteristics. However, bafilomycin, azide, DCCD or efrapeptin had no effect on the Cl⁻ pump characteristics suggesting that this Cl⁻ pump was a P-type ATPase.

The olfactory learning capability of the male cockroach Periplaneta americana was studied using an operant discriminatory conditioning paradigm. Cockroaches were highly motivated to search for a sucrose solution by giving them a diet that contained no sugar and by limiting their water supply, and they were individually trained to associate peppermint with sucrose solution and vanilla with NaCl solution. Their odor preference was tested before and after training by allowing them to choose between peppermint and vanilla sources. The time spent visiting each odor source served as a measure of odor preference. Cockroaches exhibited an innate preference for vanilla over peppermint but the preference for the latter increased after only one training session. The olfactory memory formed by a single training session remained for at least one week but significantly decayed from 2 to 24 hr after training. Memory formed by three training sessions was extremely robust, exhibiting no significant decay from one day to four weeks after training. The preference formed was easily altered by reversal training in which vanilla was associated with sucrose solution and peppermint with saline. This study shows that cockroaches have an excellent olfactory learning capability characterized by rapid acquisition, extremely long retention and easy re-writing of memory.

We purified the free-radical scavenger “water soluble protein (WSP)” from broad beans. Oxidative stress, as well as diminished activity of lysosomal proteolytic enzymes, are known to induce lipofuscin accumulation in a variety of cell types. In order to clarify the roles of oxidative stress and lysosomal proteolysis in lipofuscin accumulation, human lung fibroblasts were treated with WSP. Hydrocortisone (HC) which is known to inhibit superoxide generation, was compared with WSP. Cathepsin B activity in young cells showed a decrease during cell culture from 0.203±0.013 mU/μg protein (4 weeks) to 0.067±0.018 (6 weeks) with an unparalleled increase in lipofuscin (as autofluorescence intensity, AFI/μg protein) from 1.46±0.05 (4 weeks) to 1.55±0.04 (6 weeks). Similarly, the activity in old cells exhibited a decrease from 0.208±0.013 mU/μg protein (4 weeks) to 0.043±0.005 (6 weeks) with an unparalleled increase in lipofuscin from 2.11±0.06 AFI/μg protein (4 weeks) to 2.27±0.05 (6 weeks). Young cells treated with WSP (1.25 μg/ml) for 6 weeks showed increased cathepsin B activity (0.118±0.009 mU/μg protein) as compared to the control, but the activity in WSP treatment for 4 weeks decreased by 89%. Young cells treated with HC (5 μg/ml) were equal in cathepsin B activity to the control. On the other hand, old cells treated with WSP for 4 weeks exhibited the decreased activity (0.179±0.008 mU/μg protein). In contrast, HC treatment for 4 weeks increased the activity (36%). WSP treatment for 6 weeks was effective in reducing lipofuscin accumulation in young (25% decrease) and old (57%) cells as compared to the control. Similarly, HC treatments were more effective in reducing lipofuscin accumulation in young cells for 4weeks (31% decrease) and old cells (63%). The present results show that the free-radical scavenging in the cultured fibroblasts reduced the lipofuscin accumulation.

Disappearance of the coloration pattern of the plumage and whole body hemorrhage is caused by an autosomal dominant black at hatch (Bh) mutation in the Japanese quail. In this study, we adopted genetically directed representational difference analysis (GDRDA) to obtain a genetic marker linked to Bh from our closed colony stock. A DNA pool of 10 wild-type (+/+) quails was subtracted from another DNA pool of 10 quails heterozygous for the Bh mutation (Bh/+). After analysis of 106 clones isolated by three series of GDRDA using three different restriction enzymes, one clone was shown to detect a restriction fragment length polymorphism (RFLP) between +/+ and Bh/+ quails. A genomic region flanking the polymorphic restriction site was cloned by inverse-PCR, and a PCR-RFLP maker linked to the Bh mutation was developed. The marker showed no recombination with the Bh mutation in 101 quails analyzed. This is the first report in which a genetic marker linked to a specific gene was successfully isolated by GDRDA using a closed colony population, and this strategy is expected to have wide applications in species whose genetic resources are not well established.

The alternatively spliced isoform of the nonmuscle myosin heavy chain IIB (MHC-IIB) with an insert of 21 amino acids near the actin-binding region, MHC-IIB(B2), is expressed specifically in the brain and spinal cord in Mammalia and Aves. We performed immunoblot analyses to elucidate the distribution of MHC-IIB(B2) in the brains of various animals. Nearly half of MHC-IIB existed as the B2 inserted isoform (MHC-IIB(B2)) in the cerebrum of the guinea-pig, rabbit and pig, while the non-B2 inserted isoform (MHCIIB(ΔB2)) was the dominant form in the cerebrum of the rat, mouse and hamster. In the guinea-pig, the amount of MHC-IIB(B2) expressed in the cerebrum was low compared to MHC-IIB(ΔB2) during the first postnatal week, but it increased to comparable levels during postnatal development. In the rat, the amount of MHC-IIB(B2) protein in the cerebrum remains low compared to its expression elsewhere in the brain throughout life. Our results regarding the distribution of MHC-IIB(B2) in the adult brain lead us to classify species into two types; one type expresses significantly less MHC-IIB(B2) in the cerebrum than in other portions of the brain, and the second type expresses it at comparable levels throughout the brain. Based on these results, we hypothesize that MHC-IIB(B2) modulates the role of MHC-IIB(ΔB2) in the regulation of synaptic structure and function in the mature brain, and that the requirements for such function by MHC-IIB(B2) have shifted gradually from the cerebellum to the cerebrum with evolutionary increases in brain size.

Temporal and spatial profiles of alkaline phosphatase (ALP) activity during the development of amphioxus have not been fully documented and thus the present study investigated this activity spectrophotometrically, electrophoretically and histochemically. The following results were observed: (1) spectrophotometrically, ALP activity increased markedly at the late gastrula stage and reached a plateau at 15 hr postfertilization; (2) the electrophoretic pattern of ALP isozymes changed dramatically during development; (3) ALP activity was initially localized in the posterior wall of the primitive gut and the anterior 5 to 6 somites at about the 15 hr larva stage, and then in the notochord and all the somites at about the 18 hr larva stage; (4) in 1-day larvae, ALP activity decreased in the posterior wall of the primitive gut and in the anterior 5 to 6 somites which had ALP activity at 15 hr, but it appeared in the newly formed somites, especially in the myosepta, the crevices cut in between adjoining somites; (5) in 2-day larvae, ALP activity was no longer visible in somites but became highly evident in most of the notochord except for its rostral region; and (6) when the lateral plate mesoderm pushed down ventrally on either side of the intestine and conjoined beneath the intestine, ALP activity was also detected in the conjoining lateral plate mesoderm. Apparently, two types of ALP exist in amphioxus larvae, the transient endodermal ALP and the constant mesodermal ALP, and the spatial and temporal correlation of ALP activity with the developing mesoderm, including the notochord, suggests that it plays a role in the differentiation of mesodermal structures during the development of amphioxus.

Hemichordates occupy a critical phylogenetic position among deuterostomes because they exhibit echinoderm-like larval morphology and chordate-like adult morphology. Analyses of the expression and function of hemichordate developmental genes will therefore provide insight into the evolution of deuterostome body plans. The distal-less/dlx gene encodes a homeodomain transcription factor and plays roles in the development of appendages and the brain in a variety of animals. Here we have characterized a distal-less gene (Pf-dlx) of the hemichordate Ptychodera flava. During embryogenesis, Pf-dlx is expressed in the whole aboral ectoderm of the blastula and gastrula. Later, its expression appears in several cells in the boundary region between the oral and aboral ectoderm. The tornaria larvae express Pf-dlx in some specific cells of the ciliary band. The results are discussed in terms of an ancestral function of the distal-less/dlx gene in the formation of the nervous system.

Present study tested a hypothesis that the seasonal reproductive pattern would be similar between two sympatric congeneric species of frogs, Buergeria japonica and B. robusta, by examining, fat body, liver and gonadal cycle. In total, 132 and 148 adult frogs, respectively, were collected from Taichung, Central Taiwan during July 1996 and July 1997. Both species were classified as prolonged breeders, breeding from March to August. This breeding season differs from those in conspecific populations from other localities. In both species, adult females attained larger snout-vent length (SVL), body mass (BM), head length (HL), and head width (HW) than males. The smaller size of male in these species is inconsistent with a previous energetic constraint hypothesis that smaller males are caused by the costs of advertising, maintaining territories, and lower food intake in prolonged breeding species. Neither B. japonica nor B. robusta showed a significant positive correlation between female ovary mass and female SVL, indicating that fecundity selection does not directly affect female body size. Also, timing of reproduction in three other anurans from the present study site is also discussed.

Present study showed the influence of photoperiod alterations on adrenal gland weight, adrenal lipids and plasma corticosterone profiles (adrenocortical function), and pineal and gonadal weights of two Indian tropical male and female birds belonging to nocturnal (Spotted owlet, Athene brama) and diurnal (Jungle bush quail, Perdicula asiatica) habitats. Exposure of both sexes of nocturnal birds to long photoperiod (LP, 16L:8D, light between 6:00 to 22:00hr) and continuous illumination (CL) during reproductive recrudescence and active phases, respectively, increased adrenal weight, adrenal lipids, such as phospholipids, free and esterified cholesterol, as well as plasma corticosterone levels. Concomitantly, the LP treatment elevated the gonado-somatic index while decreased the pineal gland weight, however, the CL treatment was ineffective to alter gondal and pineal activity. Exposure of both sexes of nocturnal birds to short photoperiod (SP, 8L:16D, light between 9:00 to 17:00hr) and continuous darkness (CD) during reproductive recrudescence and/or active phases inhibited adrenocortical function and gonadal activity while elevated the pineal gland weight. Exposure of both sexes of diurnal birds to SP and CD during the reproductive active phase produced similar changes on these variables like that of the nocturnal birds while the CL treatmet was ineffective. These results indicated that adrenocortical function and gonadal weights are photoperiod-dependent in both sexes of birds, irrespective of their habitats and can be positively correlated. Pineal gland activity showed an inverse pattern of changes with adrenocortical function and gonadal activity when exposed to different photoperiod regimes, irrespective of the habitat or sex of birds. Present study together with previous reports further indicate that pineal gland may mediate photic signals. Direct evidence is, however, required to support this hypothesis.

Present study showed effects of adrenal gland modulation on adrenocortical, pineal and gonadal activity in two Indian tropical male and female birds belonging to nocturnal (Spotted owlet, Athene brama) and diurnal (Jungle bush quail, Perdicula asiatica) habitats. Administration of adrenocorticotropin (ACTH) to both sexes of nocturnal or diurnal bird during regressive or active and inactive phases of the reproductive cycle elevated adrenal and gonadal weights when compared to vehicle control groups. Pineal mass showed no change for the ACTH treatment in the regressive phase while it decreased significantly in the inactive phase. Administration of ACTH elevated the adrenocortical function during the inactive phase in both species, which becomes evident from the increase in adrenal lipids and plasma corticosterone level. Conversely, ACTH treatment during peak adrenal activity (regressive phase in owlet and active phase in quail) decreased the adrenal free cholesterol (FC) while increased adrenal phospholipids (PL) and esterified cholesterol (EC) levels in both species. However, plasma corticosterone levels after the ACTH treatment did not show any change. Administration of corticosterone and dexamethasone to both sexes of nocturnal birds during the regressive phase decreased the gonadal weights while the adrenal gland mass showed no change. Dexamethasone treatment alone reduced the pineal gland mass in females. In diurnal birds, corticosteroids produced no change in adrenal, gonadal and pineal gland weights. However, corticosteroids elevated PL and EC in both species while adrenal FC was unaltered in the nocturnal bird but decreased in the diurnal bird. Administration of adrenal gland inhibitor, metyrapone, to both sexes of the diurnal bird reduced adrenal and gonadal weights and adrenal lipids while it elevated the pineal gland mass. However, in nocturnal birds, the treatment altered only adrenocortical function.

These results indicated that adrenal gland modulation possibly evoked a feedback response from the gland depending on the phase of reproductive (or adrenal) cycle and through this it altered pineal and gonadal activity differentially. Modulation of adrenal activity by ACTH produced a profound reduction in the pineal gland mass during the inactive phase of the reproductive cycle in both sexes of nocturnal and diurnal bird. Changes induced in the adrenocortical function also reflected to some extent on gonadal activity in both regressive/active and inactive phases of reproductive cycle in both species.

The embryonic and larval development of a freshwater goby, Micropercops swinhonis (Odontobutidae; Gobioidei), from Korea are described. One female spawned several times from mid-April to early July. About 100 to 500 eggs per batch were laid on the under surface of the nest guarded by a male. Eggs hatched 12 days after spawning. Larvae, about 3.7 mm in total length (TL) just after hatching, passed the exogenous feeding larval stage, and reached the juvenile stage of about 16 mm TL in about one month. The developmental process of M. swinhonis is quite different from that of the fluvial Odontobutis of the same family, which skips the exogenous feeding larval stage, but is rather similar to that of amphidromous Gobiidae. On the other hand, the left and right pelvic fin buds of M. swinhonis appear distant from each other as in Odontobutis, rather than Gobiidae.

We examined the nucleotide sequences of 500 bp variable portion from the 5′ end of mitochondrial (mt) DNA control region in about 500 individuals from 12 populations that were captured in 11 rivers, six in Hokkaido and five in Honshu, Japan. Comparison of the sequences showed 10 variable sites, defining a total of 12 haplotypes in the examined individuals. All the 12 haplotypes occurred in seven Hokkaido populations, whereas only six haplotypes were found in the five Honshu populations. Among these haplotypes, two were common in all the Hokkaido and Honshu populations. The AMOVA analysis inferred a genetic differentiation among three geographic regions, i.e. Hokkaido, Pacific Ocean coast in Honshu, and Japan Sea coast in Honshu. Haplotype diversity was higher in the populations of Hokkaido than those of Honshu, indicating a greater genetic variation in the Hokkaido than the Honshu populations. The estimates of pairwise population F_ST suggested that the regional differentiation was mostly ascribed to the divergence between populations in Hokkaido and the Pacific coast in Honshu.

To investigate genetic diversity among populations of the Japanese giant flying squirrel Petaurista leucogenys, the mitochondrial DNA control region sequences (1,052–1,054 bases) were determined in 37 specimens from 17 localities on the Honshu, Shikoku, and Kyushu Islands of Japan. Of the 37 animals examined, 24 haplotypes were identified. All haplotypes from Kyushu consisted of 1,052 bases, whereas those from Honshu and Shikoku consisted of 1,054 bases including two insertions, except for three haplotypes (which had 1,052 or 1,053 bases). Phylogenetic relationships reconstructed using neighbor-joining and maximum parsimony methods indicated that P. leucogenys is essentially separated into three major lineages: Group A consisting of a single haplotype from Kyushu, Group B consisting of some haplotypes from Kyushu and one haplotype from Honshu, and Group C consisting mostly of haplotypes from Honshu and Shikoku. Animals with the Kyushu haplotypes were split into two lineages (Groups A and B), suggesting that Group A diverged at an earlier point from the other groups. Genetic distances in Group C were not related to geographic distances between sampling localities, indicating that ancestral populations of this group recently expanded their distribution in a short time, possibly after the last glacial stage.

Scincella boettgeri and S. formosensis are two small lygosomine skinks endemic to the southern Ryukyus and Taiwan, respectively. Taxonomic separation of these two species depends on only a few external characters that are seemingly more or less variable within each species. To investigate the geographic pattern of their morphological variations, multivariate analyses were performed for 23 meristic and 16 morphometric characters in 680 specimens of S. boettgeri from 12 islands of the Miyako and Yaeyama Groups and 193 specimens of S. formosensis from ten localities in Taiwan. The canonical discriminant analysis (CDA) for samples representing local populations using meristic data completely separated the southern Ryukyu and Taiwanese assemblages, supporting their assignments to different taxa. On the other hand, the analyses using morphometric data remained their variations partially overlapping each other. Of the samples of S. boettgeri examined, the Haterumajima sample markedly varied from the others, whereas the Kaohsiung sample was somewhat divergent among the S. formosensis samples. Neighbor-joining phenograms derived from Mahalanobis distances among the samples showed branching patterns apparently inconsistent with the topographical (for S. formosensis) or putative geohistorical relationships of their localities (for S. boettgeri). This suggests that in these species external quantitative characters often rapidly vary under the influence of localized environments rather than of the passage of time after geographical isolations.