Zoological Science

The Japanese eel has two characteristics advantageous for the study of the mechanisms controlling spermatogenesis. One is the possibility of artificial induction of the complete process of spermato-genesis from spermatogonial proliferation to spermiogenesis by exogenous gonadotropin injection, and the other is the possibility of inducing this process in an in vitro testicular organ culture or germ-Sertoli cell coculture system. Using the eel system, we analyzed the control mechanisms of spermatogenesis. In Japanese eel, the whole process of spermatogenesis is regulated by several sex steroid hormones. Spermatogonial stem cell renewal is promoted by estradiol-17β (the natural estrogen in vertebrates). Spermatogonial proliferation can be induced by 11-ketotestosterone, the main androgen in teleost. IGF-I is necessary for the action of 11-ketotestosterone in the initiation of spermatogenesis. The action of 11-ketotestosterone is mediated by other factors, such as activin B, produced by Sertoli cells. Although 11-ketotestosterone also induce meiosis and spermiogenesis, the control mechanisms of these processes are not clear. After spermiogenesis, immature spermatozoa undergo sperm maturation, thereby becoming capable of fertilization. Sperm maturation is regulated by 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), which is progestogen in teleosts. The 17α,20β-DP acts directly on spermatozoa to activate the carbonic anhydrase existed in the spermatozoa. This enzymatic activation causes an increase in the seminal plasma pH, enabling spermatozoa to motile.

When socially isolated from siblings, newly hatched chicks emit distress calls at a high frequency. As a step toward identifying brain mechanisms responsible for recognition of conspecifics, we analyzed sensory cues critical for suppressing distress calls in socially isolated quail chicks at post-hatch day 2 to 4. Each subject of wild-type chicks was reared with another wild-type companion chick for 1–2 nights before tests. As the first step, we found that distress call was completely suppressed when accompanied by another wild-type chick, irrespective of whether the subject had previously been familiarized with the chick or not. When paired with an albino chick, on the other hand, the subject emitted significantly more calls, suggesting that the wild-type plumage is important. As the next step, in search of the critical sensory modality, we found that visual display of a conspecific chick suppressed the distress call even without accompanying acoustical stimuli. On the other hand, the acoustical stimuli alone were without effects. Olfactory as well as tactile sensations also proved irrelevant. As the final step, we tried to identify the significant sub-modalities in the visual images of conspecifics. Degraded visual image of a moving chick (degraded in fine details) caused a significant but incomplete suppression, whereas an anesthetized chick was without effects. On the other hand, distress calls were completely suppressed even without behavioral correspondence or exact plumage coloration. All of these data suggest that visual image of a moving chick should be given at a high spatial resolution, and wild-type plumage pattern might provide important visual cues.

Fluorescence properties from the wings of male M. sulkowskyi and Papilio xuthus butterflies were investigated for the first time over a wide range of excitation and emission wavelengths using a three-dimensional (3-D) fluorescence method.

From the 3-D plots of the pale blue region of the wings of M. sulkowskyi, four contour peaks were observed. On the other hand, from the yellow region of the wings of Papilio xuthus, one peak was observed. The origins of these contour peaks are discussed based on a comparison with standard pigments. Of the four contour peaks seen for M. sulkowskyi, a specific peak (A), located at an excitation wavelength of about 325 nm and an emission wavelength of about 410 nm, was thought to be mainly due to a mixture or compound of three pteridine pigments (i.e., biopterin, pterin and isoxanthopterin). The single specific peak observed for Papilio xuthus, located at an excitation wavelength of about 400 nm and an emission wavelength of about 470 nm, was thought to be due to the Papiliochrom II pigment.

Based on these results, the possibility of using 3-D fluorescence analysis as a tool for classifying butterflies is briefly described.

Post-hatching development of the brain in the oegopsid squid, Todarodes pacificus was described using conventional histological and Cajal's silver impregnation methods. The oegopsid squids spend a specific paralarval period before attaining adult-like juveniles. In the just-hatched paralarvae, the brain lobes (lower and intermediate motor centers) are differentiating only in the ventral part of the brain (subesophageal mass, SBM), and development of the dorsal part of the brain (supraesophageal mass, SPM) shows a heterochronic delay. In the SPM, an arched bundle of axonal tracts (transverse arch, TA) crosses the region over the oral ingrowth. In the early paralarval period, the basal lobes and precommissural lobe (higher motor centers) begin to develop along the TA. A little later, a pair of longitudinal axonal tracts (supraesophageal ladder, SPRL) elongates anteriorly from the TA, and accessory lobes (centers for memory and learning) and superior buccal lobes begin to differentiate along the SPRL. In the mid paralarval period, the lobes of the olfactory center and the peduncle lobe develop well in each optic tract region. In the late paralarvae, all brain lobes become identifiable and the brain shows substantially the same organization as that in the adults. The dorsal-most region of the SPM largely increases in volume with striking growth of the accessory lobes. The SBM elongates in anterior and posterior directions and the rostral end (anterior SBM) separates from the middle SBM. The optic lobes become very large with neuropils arranged in layers. In the juveniles, the neuropils increase in relative volume to the perikaryal layers, and neuronal somata enlarge markedly in some lobes. The retarded development of higher motor centers during paralarval development suggests that the early paralarvae of T. pacificus are not active predators but suspension feeders.

By immersing sea urchin embryos in seawater containing various concentrations of sucrose, we found that the level of osmotic pressure of blastocoel changed during gastrulation; the level was low around the onset of gastrulation and higher during secondary invagination. To learn how such changes in osmotic pressure related to invagination processes, embryos were shrunken or expanded by means of sucrose treatments, and the degree of invagination was monitored using Nile blue staining. The method elucidated that the cells invaginated during primary invagination occupied only top one third of archenteron at the end of secondary invagination, and that the cells constituting intestine and the posterior half of stomach were recruited into the archenteron after secondary invagination. When embryos were expanded, the degree of invagination was remarkably decreased, indicating that a higher level of expanding force hindered the fulfillment of invagination. On the other hand, shrinkage of blastocoel during secondary invagination increased the degree of invagination. Cell tracing experiments showed that the change in the degree of invagination estimated with Nile blue staining was accompanied with the change in the number of cells incorporated into the archenteron. When embryos were expanded, only the descendants of veg2 constituted the gut rudiment, while a considerable amount of the veg1 descendants were recruited into archenteron when embryos were shrunken. Obtained results suggest that change in the level of osmotic pressure of blastocoel would be necessary for the progress of gastrulation.

Concentrations of immunoreactive (ir-) inhibin in circulation, amniotic fluid and the ovary of embryos and newly-hatched ducks from Day 21 of incubation to one day of age were determined. The antiserum used was against bovine 31-kDa inhibin and was validated for RIA of inhibin using samples from female embryos. Plasma concentrations of FSH and LH were also determined by chicken RIA systems. Plasma concentrations of ir-inhibin were maintained at constant levels from day 21 to day 25, abruptly increased on day 26. After the increase, plasma concentrations of ir-inhibin quickly declined from day 27 to the day of hatch (day 28). Plasma concentrations of FSH were high on day 21, followed by an abrupt decline on day 22, gradually increased until day 24, and then decreased again on day 25. The maximal rise of plasma FSH was observed on day 27, and then rapidly decreased until the day after the hatch. No inverse relationship was observed between plasma concnetrations of ir-inhibin and FSH. Amniotic fluid concentrations of ir-inhibin were relatively low and remained constant between Day 21 and Day 25. Embryonic ovaries contained very low amounts of ir-inhibin. Ovarian inhibin levels were stabilized in early time points, temporally decreased on day 25, and abruptly increased on day 26. These results suggest that inhibin does not involved in the regulation of FSH secretion in the female duck embryo and the ovary of duck embryo is not the main source of circulating ir-inhibin.

We examined the effects of insulin (INS) and somatostatin 14 (SS-14) on glycemic regulation in larval and metamorphosing landlocked sea lamprey, Petromyzon marinus. Animals were injected intraperitoneally with either (Experiment 1) saline (0.6%), somatostatin-14 (SS-14; 50 or 500 ng/g body weight), insulin (INS; 10 or 100 ng/g body weight), or alloxan (20 or 200 μg/g body weight), or with (Experiment 2) normal rabbit serum, anti-SS-14, anti-lamprey SS-34 or anti-lamprey INS. Somatostatin-14 (500 ng/g) increased plasma glucose levels in larvae. Injection of anti-SS-14 and anti-lamprey SS-34 resulted in hypoglycemia compared to the controls. Insulin (100 ng/g) resulted in hypoglycemia in both larvae and stage 6 metamorphosing lamrpey. Acute insufficiency of lamprey INS in larvae treated with anti-lamprey INS elevated plasma glucose levels. Similarly, alloxan (200 μg/g, a cytotoxin of insulin-secreting cells) resulted in hyperglycemia in larvae. These data indicate that SS-14 is hyperglycemic in sea lamprey, whereas INS is hypoglycemic, and suggest that the glucoregulatory roles of SS-14 and INS emerged early during the evolution of vertebrates.

The swallowtail butterfly, Papilio xuthus L., exhibits seasonal diphenism, i.e., spring and summer morphs, the development of which is determined by photoperiod and temperature imposed during the larval stage. Larvae reared under long day conditions at 25°C develop into summer morphs without pupal diapause, while larvae reared under short day conditions at 20°C develop into spring morphs after diapause development at 4°C for more than three months. To investigate the neuroendocrine mechanism regulating seasonal morph development, the activity of the summer-morph-producing hormone (SMPH) was tried to assayed by using male short day pupae of P. xuthus that accomplished diapause development at 4°C. The wing patterns of male butterflies shifted toward summer morphs when short day pupae were treated with the SMPH at an adequate stage. SMPH activity was detected in 2% NaCl extracts of the brain-subesophageal ganglion (Br-SG) complexes of P. xuthus long day pupae. Chilled male short day pupae responded to P.xuthus SMPH in a dose-dependent manner and lost the responsiveness within 2 days of transfer to room temperature (about 25°C). This is a report to show the existence of SMPH in butterfly species having pupal diapause.

A new species of cavernicolous freshwater crab of the genus Sundathelphusa Bott, 1969 (Family Parathelphusidae), S. hades, is described from Mindanao in the Philippines. The new species has greatly reduced eyes, noticeably elongated ambulatory legs and lack of dark body pigmentation, all representing features associated with an obligate troglodyte. The species can be differentiated from the closest congener, S. cavernicola (Takeda, 1983) from Bohol in the Philippines by the form of its carapace and male first pleopod.

Histological comparisons of intestinal internal structures were made for the monophyletic lamprey group comprising parasitic Lethenteron japonicum, and nonparasitic L. kessleri and the northern form of L. reissneri, in order to verify the speciation hypothesis that the nonparasitic species have been derived from a congeneric parasitic species. In the larval stage of each species, the mucosal epithelial cells were regularly arranged around an inner layer of intestine, including the typhlosole. At the metamorphosed stage, L. japonicum possessed functional mucosal folds, reflecting an adaptive change for parasitic feeding after metamorphosis. The two nonparasitic species, in which feedings are absent after metamorphosis, also exhibited mucosal folds albeit in a degenerative condition, indicating the likely presence of functional or at least rudimentary mucosal folds in an ancestral parasitic species. This finding supports a previously advocated direction of speciation in lamprey satellite species, namely nonparasitic L. kessleri and the northern form of L. reissneri speciated from ancestral stocks of parasitic L. japonicum.

Previous phylogenetic studies using mitochondrial DNA sequences of the brown bear Ursus arctos have separately defined two major lineages in Europe, three in Alaska, and three in Hokkaido Island of Japan. To reconstruct phylogenetic relationships among worldwide populations of the species, nucleotide sequences of the mitochondrial DNA control region and cytochrome b were determined for some additional subpopulations of Asia (Gobi and Tibetan), and then all the data including previously reported sequences were compared. The resultant phylogenetic trees showed that the worldwide populations could be grouped into at least five lineages. One of the five lineages had a wide distributional range covering Eurasia, Alaska, and central Hokkaido. Moreover, it is likely that populations from eastern Hokkaido and eastern Alaska are the direct derivatives of a single lineage. These results suggest that brown bears may have widely colonized Eurasia and North America from their original areas somewhere in Eurasia more than once.

To assess the level of genetic variations of the Eurasian badger Meles meles in Japan, the entire sequences (1,140 base pairs) of the mitochondrial cytochrome b gene were phylogenetically examined. Most of substitutions between haplotypes were transitions resulting in synonymous mutations. A phylo-genetic tree reconstructed by sequence differences clearly showed that Japanese populations of Meles meles were differentiated from continental populations (from the Baikal area and eastern Europe) of M. meles. By contrast, genetic distances among Japanese populations were much smaller, and their geographic structures did not reflect geographic distances between sampling localities. The results indicate that polymorphisms of the ancestral populations still remain via loss of haplotypes by population size changes. In addition, M. meles could have occupied the present habitats in Japanese main islands (Honshu, Shikoku, and Kyushu) in a short period, possibly after the last glacial age.

Conventional and G-banded karyotypes of Crocidura attenuata Milne-Edwards, 1872 from Guangdong, southern China, are reported. The diploid chromosome number (2n) varied from 35 to 38 among specimens, while the fundamental arm number (FN) consistently was 54. Of the autosomes, 14 pairs including four meta- or submetacentric, three subtelocentric, and seven acrocentric pairs showed no variation in all specimens, whereas the remaining pairs showed Robertsonian polymorphism. The X and Y chromosomes were medium sized submetacentric and small acrocentric chromosomes, respectively. These karyotypes differ from that of C. attenuata from Taiwan, which has 40 chromosomes with 56 arms. Such differences are largely attributable to a non-Robertsonian rearrangement, where both 2n and FN values are different from each other. The largest metacentric pair observed in karyotypes from Guangdong may have resulted from the centromere-telomere translocation between an acrocentric and a subtelocentric pairs of karyotype homologous to that from Taiwan. Both morphometric difference and sequence divergence in mitochondrial cytochrome b gene between samples from Guangdong and Taiwan was relatively small. However, the nonRobertsonian rearrangement assumed between karyotypes of the shrews from Guangdong and Taiwan suggest that they are reproductively isolated from each other. The eastern continental and Taiwanese populations therefore may represent different species under the names, C. attenuata (sensu stricto), and C. tanakae Kuroda, 1938, respectively.