Zoological Science

When female mice are mated, they form a memory of the pheromonal signal of the male with which they mated. Our research objective was to determine the neural mechanisms underlying learning and memory by employing a convenient model of pheromone-induced olfactory memory (pheromonal memory). Formation of pheromonal memory depends on the association between mating and exposure to pheromones. Synaptic plasticity involving this memory occurs in the accessory olfactory bulb (AOB), depending on vaginocervical stimulation at mating. The vaginocervical stimulation at mating reduces the dendrodendritic feedback inhibition of principal neurons (mitral/tufted (MT) cells) in the AOB and enhances their cell activity. The enhancement of activity induces on these plastic changes in dendrodendritic synapses, which in turn enhance GABA-mediated inhibition of MT cell activity. This “self-inhibition” of MT cells activity in response to pheromonal signals of the partner can disrupt its signals at the AOB thereby preventing the signals from reaching the central brain. The formation and maintenance of pheromonal memory is based on this inhibition mechanism.

To elucidate neural mechanisms underlying walking and jumping in insects, motor neurons supplying femoral muscles have been identified mainly in locusts and katydids, but not in crickets. In this study, the motor innervation patterns of the metathoracic flexor and extensor tibiae muscles in the cricket, Gryllus bimaculatus were investigated by differential back-fills and nerve recordings. Whereas the extensor tibiae muscle has an innervation pattern similar to that of other orthopterans, the flexor has an innervation unique to this species. The main body of the flexor muscle is divided into the proximal, middle and distal regions, which receive morphologically unique terminations from almost non-overlapping sets of motor neurons. The proximal region is innervated by about 12 moderate-sized excitatory motor neurons and two inhibitory neurons while the middle and distal regions are innervated by three and four large excitatory motor neurons, respectively. The most-distally located accessory flexor muscle, inserting on a common flexor apodeme with the main muscle, is innervated by at least four small excitatory (slow-type) and two common inhibitory motor neurons. The two excitatory and two inhibitory motor neurons that innervate the accessory flexor muscle also innervate the proximal bundles of the main flexor muscle. This suggests that the most proximal and distal parts of the flexor muscle participate synergistically in fine motor control while the rest participates in powerful drive of tibial flexion movement.

To examine whether α and β tubulin are glycoproteins, we used a pyridylamino labeling method and a monoclonal antibody, SG3-1, raised against NeuAcα2-3Gal structure. Alpha and β tubulin from both pig brain and HeLa cells were positive for the SG3-1 antibody by immunoblot assay. Sialidase treatment reduced the reactivity of the SG3-1 antibody to α and β tubulin molecules. N-linked oligosaccharide analysis also showed that α and β tubulin are glycosylated. Moreover, immunofluorescence analysis showed that the filamentous structure recognized by the SG3-1 antibody was overlapped with microtubules, especially in the vicinity of the nucleus. These results indicate that α and β tubulin are glycosylated with sialyloligosaccharides.

Morphological and biochemical investigations were made on the yolk formation in ovaries of the quail Coturnix japonica. Morphologically, two ways of nutrient uptake were observed in follicles. In small oocytes of white follicles, vitellogenin (VTG) was taken up through fluid-phase endocytosis which was assisted by follicular lining bodies. The lining bodies were produced in follicle cells. They adhered to the lateral cell membrane, moved along the membrane in the direction of the enclosed oocyte and were posted to the tips of the microvilli. These tips, now with lining bodies, were pinched off from the main cell body, engulfed by indented cell membranes of the oocyte, and transported to yolk spheres. In large oocytes of yellow follicles, VTG and very-low-density lipoproteins (VLDL) were taken up through receptor-mediated endocytosis. The VTG and VLDL particles diffused through the huge interspaces between follicle cells, and once in oocytes were transported to yolk spheres via coated vesicles. Immunohistochemistry showed that the VTG resides on or near the surface of the follicle cell membrane at the zona radiata whereas the cathepsin D resides at or near the oocytic cell membranes. Tubular and round vesicles in the cortical cytoplasm of oocytes were also stained with both antisera, suggesting that these vesicles are the sites where the VTG is enzymatically processed by cathepsin D. Upon analysis by SDS-PAGE, a profile similar to that of yolk-granule proteins was produced by incubating VTG with a quail cathepsin D of 40 kD.

Two shell color types of the exotic bivalve Corbicula fluminea were collected in Kyoto city, Japan. DNA microfluorometry revealed that both types were diploids with non-reductional spermatozoa. Maternal chromosomes were found to be extruded as two polar bodies at the first meiosis, and the second meiosis could not be observed. Only the male pronucleus was present in the egg cytoplasm and became metaphase chromosomes at the first mitosis. The present study indicates that the diploid C. fluminea in Japan has the same mode of androgenetic reproduction as the triploid C. leana.

We examined the effect of low temperatures on annexin V expression in newt testis. When newts were transferred to a low temperature (12°C), up-regulation of annexin V protein was observed in secondary spermatogonia. In primary spermatocytes, high levels of annexin V expression were observed at both 12°C and 22°C, but at 12°C the protein was localized in part of the cytoplasm of primary spermatocytes. These results indicate that in newt testis annexin V is a cold-sensitive protein, suggesting the possibility that annexin V might have a cold stress-related function in newt germ cells.

The relationship between gonadal development (histological evidence for spermiogenesis and/or spermatogenesis), sexual behavior (nest-building) and mRNA levels of gonadotropins (βFSH and βLH) and growth hormone (GH) in the male pituitary was investigated. Amplification of βFSH cDNA showed a significantly higher mRNA level in mature males (whether sexually active or not) than in juveniles. However, following PCR amplification of βLH cDNA, a significantly higher mRNA level was found in the sexually active group compared to the sexually inactive group. These results suggest that FSH may participate in spermatogenesis, whereas LH is more involved in spermiogenesis. The GH mRNA level increased slightly during the maturation process but no significant differences were found between the groups studied.

In teleosts, it is well known that plasma calcium levels increase as a result of treatment with estrogen for at least during 2 weeks and that calcitonin secretion is induced by estrogen. The present study examined the influence of bisphenol A on calcium homeostasis in goldfish and compared the above known estrogenic action. In goldfish kept in water containing bisphenol A (10⁻⁶ M), the plasma calcium concentration increased significantly (P<0.001) at 4 days but decreased significantly (P<0.05) at 8 days. By the treatment of bisphenol A, calcitonin secretion was not induced until 4 days. At 8 days, however, plasma calcitonin, as well as calcium, decreased significantly (P<0.05), although vitellogenin was detected in the plasma. Therefore, bisphenol A influences plasma calcium levels, but its action is different from that of estrogen, which indicates that bisphenol A affects the calcium homeostasis and might bring about abnormal conditions in teleosts.

The order Psittaciformes (parrots) has unique morphological features in the head that are evolutionarily novel. To better understand the unique evolution of the head in parrots, the developmental pattern of the skull of the budgerigar (Melopsittacus undulatus) was initially described on the basis of transparent skeletal specimens. Although the fundamental pattern of the skull development of birds is conserved in parrots, some differences were observed between parrots and other groups of birds. In parrots, the vacuity in the interorbital septum did not emerge throughout ontogeny, in contrast to other lineages of birds, for example Galliformes and Coliiformes. This feature seems to be concerned with the attachment of the unique jaw muscle of parrots, M. ethmomandibularis, to the interorbital septum. In spite of a prokinetic skull, the cranio-facial hinge of parrots was brought about by secondary transformation of dermal bones unlike that of birds with a standard prokinetic skull (e.g. Corvus) in which the nasal-frontal suture directly becomes a hinge of bending. To further understand the evolution of “pseudoprokinesis” in parrots, the construction of a robust avian phylogeny is desired. The parrot-specific suborbital arch and cranio-facial hinge are not seen until birds leave the nest and can feed themselves. In conclusion, these structures are considered to be essential for eating hard and/or large meals.

Two hundred and fifteen individuals of the tiny abyssal scallop (Delectopecten vitreus macrocheiricolus) were collected at a depth of around 3,000 m on the bottom of the Japan Sea during a cruise of the Yokosuka/Shinkai 6500 YK 01-06. The scallop population included specimens of 4 sizes, i.e., very small, small, middle, and large, with mantle sizes of 1–2mm, 3–5mm, 6–9mm, and 10–12mm, respectively. Histological observations revealed that the large-size group was at the stage just after oviposition or spermiation, or in the regressed stage of gametogenesis. The small- and middle-size groups were in the growing stage of gametogenesis. These results suggest that only the large-size individuals were responsible for reproduction and that the small- and middle-size individuals would participate in subsequent reproductions. This paper will discuss the reproductive strategy of this scallop living at such great depth.

The morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae are described in detail. This ascidian was collected from the stony shore in the cove near Shimoda Marine Research Center, University of Tsukuba (Shimoda, Shizuoka Prefecture, Japan). The ascidian colony was easily distinguished from colonies of other botryllids because it was very thin and bright pink in color. The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus. This ascidian was prolific, with 1-5 embryos on each side of a zooid, and the embryos of this ascidian developed in the peribranchial cavity without any brooding organs as in Botryllus scalaris. We observed the processes and features of the allorecognition reaction in colony specificity and found that allorejection occurred after fusion of the vascular system between two incompatible colonies. This manner of allorejection is also shown in B. scalaris and Botryllus delicatus; however, the reaction speed of allorejection is faster than that of B. delicatus and similar to that of B. scalaris. These results indicate that this ascidian might be closely related to B. scalaris.

Three new and two known species of the Drosophila (Sophophora) obscura species-group are reported from Yunnan Province, southern China. The sinobscura species-subgroup is newly established by D. sinobscura, D. hubeiensis and D. luguensis sp. nov. Geographic distribution of the obscura group in and around China is discussed, and a key to 10 Chinese species of the obscura group is provided.

Population genetic structure of the spotted seal, Phoca largha, along coastal regions of Hokkaido was investigated, using mitochondrial DNA (mtDNA) sequences. A 571-bp fragment of the mtDNA control region and adjacent threonine and proline transfer RNA genes was sequenced from 66 seals. We categorized all individuals into three groups considering sampling area and season: twenty-four seals from the Sea of Okhotsk in winter, 11 seals from the Sea of Okhotsk coast in fall, and 31 seals from the Sea of Japan coast in winter. From the 66 animals, 57 haplotypes were identified. Compared with the harbor seal sequence, all spotted seals examined shared two deletions in the control region, which distinguished between the two species. Forty-nine haplotypes were represented by a single individual, and haplotypes shared by more than two animals were generally restricted to same sampling-groups. Phylogenetic trees did not indicate clear geographic differences among the three groups. An Analysis of molecular variances (AMOVA) did not showed any significant population genetic structure in Hokkaido spotted seals (Φ_st = −0.003). Our results showed a high level of diversity but no genetic structure, and did not deny the possibility that seals in the Okhotsk breeding concentration mainly stayed in the fall Okhotsk and also inhabited in the winter Sea of Japan.

This study investigated geographic variation in the Asian lesser white-toothed shrew, Crocidura shantungensis Miller, 1901, among a number of localities in East Asia, including the Korean Peninsula, Tsushima Island, Cheju Island, and Taiwan, using univariate and multivariate analyses of cranial morphometric characters. Sexual differences were found in Taiwanese shrews, but were not evident in those from Korea or Tsushima. The samples from Taiwan had a larger overall cranial size than those from Korea and Tsushima, and were well separated from the latter two samples in the discriminant analyses. The overall size of the Cheju shrews was also greater, but lay within the variation of those from Taiwan. Crocidura dsinezumi quelpartis Kuroda, 1934, described from Cheju Island, is actually a junior synonym of C. shantungensis, and is not related to C. dsinezumi.

The Japanese silver crucian carp, Carassius langsdorfii, is a naturally polyploid fish that reproduces by gynogenesis, resulting in a geographically clustered clonal population. To determine the amount of clonal diversity in this species, we used genetic markers to compare fish from several Japanese locations. Samples were collected from Lake Kasumigaura (Ibaraki Pref.), Lake Imba (Chiba Pref.), Lake Fukushimagata (Niigata Pref.), Lake Biwa (Shiga Pref.), Lake Koyama (Tottori Pref.), and the Niyodo and Monobe Rivers (Kochi Pref.). The genetic markers used were the microsatellite loci GF1*, GF17*, and GF29*, as well as isozyme (creatinekinase CK*) and mtDNA (D-loop region) RFLP. In the seven locations sampled, 61 clonal lines were identified by combining the genotypes of the three microsatellite loci. Populations at all locations consisted of six to 28 clonal lines. Fourteen common clonal lines were detected in two or more locations, and 13 of these were observed in Lake Biwa. We suggest that these clonal lines have been widely distributed in Japanese freshwaters through the intentional stocking of fish species such as “gengorobuna” (C. cuvieri) and “ayu” (Plecoglossus altivelis altivelis) obtained from Lake Biwa and its adjacent waters. The CK* genotypes and mtDNA haplotypes of fish from Lake Kasumigaura differed from those of other locations.