Ryo Ishibashi

Androgenetic Reproduction in a Freshwater Diploid Clam Corbicula fluminea (Bivalvia: Corbiculidae)

Abstract 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.

Pearl Microstructure and Expression of Shell Matrix Protein Genes MSI31 and MSI60 in the Pearl Sac Epithelium of Pinctada fucata by In Situ Hybridization

Expression patterns of the shell matrix protein genes MSI31 and MSI60 in the pearl sac epithelium were examined by in situ hybridization 38 days after implantation, and related to pearl quality. A pearl sac that produced a nacreous pearl showed very weak expression of MSI31 and strong expression of MSI60. A pearl sac, which yielded a prismatic pearl, strongly expressed MSI31 and very weakly expressed MSI60. In a complex pearl, whose surface consisted of a mosaic of both nacreous and prismatic layers, the expression pattern of MSI31 and MSI60 similarly corresponded to the underlying surface structures of the pearl. A nacreous pearl whose pearl sac showed strong MSI31 expression had an entirely nacreous surface composed of a laminar structure with unusual tablet growth at the corresponding site. MSI31 and MSI60 are the major components of the shell matrix proteins of the nacreous and prismatic layers. Clearly, high expression of MSI31 does not always result in prismatic secretion. These observations cannot be explained solely on the basis of the expression patterns of MSI31 and MSI60. We propose that, in addition to the MSI genes that form the prismatic and nacreous layers, upstream from these genes there are regulatory master genes that determine whether a nacreous layer (aragonite) or a prismatic layer (calcite) is formed.

Sperm Sphere in Unionid Mussels (Bivalvia: Unionidae)

Abstract We observed spermatozoa of five freshwater Japanese unionid mussel species under a light microscope. Males discharged spermatozoa as spherical masses. Spermatozoa embedded their heads into a spherical, colorless body with their tails extended around the sphere. We termed this spherical mass of sperm as a “sperm sphere”. Just after release the sperm spheres rotated rapidly by synchronous movement of the sperm tails. By the next day, the sperm spheres increased in size and some spermatozoa had detached from the sperm spheres. Spermatozoa embedded in the sperm sphere were active at least forty-eight hours after being discharged. Single spermatozoa in a balanced salt solution were also active until forty-eight hr after extraction from the gonads, while they lost their motility within a few minutes in freshwater. Spermatozoa may maintain their motility for a long time in the form of a sperm sphere and the use of sperm spheres may allow the fertilization of eggs discharged into the gill chambers of females far away from the male.

Comparison of Expression Patterns of Shell Matrix Protein Genes in the Mantle Tissues between High- and Low-Quality Pearl-Producing Recipients of the Pearl Oyster, Pinctada fucata

The production of a cultured pearl is the result of a complex interplay between the donor and recipient oysters. However, there is a paucity of information on the relationship between donor and recipient oyster gene expression patterns and pearl quality. Shell matrix proteins affect not only the formation of the shell, but also that of the pearls. We compared the gene expression patterns of five shell matrix proteins (msi60, nacrein, msi31, prismalin-14, and aspein) in the mantle edge (ME), which forms the prismatic layer, and the mantle center (MC), which forms the nacreous layer, between high- (HP) and low quality pearl- (LP) producing recipient oysters. After culturing for about two months, ME and MC tissues were collected from nine recipient oysters: four with HP, five with LP. In the ME, the average threshold cycle (&Dgr;CT) for aspein was higher in HP than in LP (t-test, p = 0.03). Additionally, in the MC, the average &Dgr;CT for msi60 was lower in HP than in LP (p = 0.06). This means the relative expression level of msi60 in the mantle of HP was higher than that of LP, and expression level of aspein in the mantle of HP was lower than that of LP. Pearl quality was closely related to the expression patterns of shell matrix protein genes of recipient oysters.

Phylogeography of the Brackish Water Clam Corbicula japonica Around the Japanese Archipelago Inferred from Mitochondrial COII Gene Sequences

We investigated the phylogeography of the Asian brackish water clam, Corbicula japonica, to clarify its demographic history using partial mitochondrial COII gene sequences (990 bp) from 283 individuals collected from around the Japanese archipelago and adjacent areas. Phylogenetic analyses revealed the presence of two major groups within our samples: monophyletic Group I comprising Lineages A-E of C. japonica and paraphyletic Group II consisting of Corbicula sp. Lineages A-C were distributed in Japan and Sakhalin Island, and Lineages D, E, and Corbicula sp. were distributed in the Korean Peninsula. Nested clade analysis (NCA) revealed that Lineage A—the dominant lineage in Japan—consisted of Pacific and Japan Sea lineages, the latter comprising southern and northern Japan Sea groups. Genetic diversity indices of the southern group were higher than those of the northern group, suggesting historical range expansion in the Sea of Japan from southwest to northeast. Geographical distribution of these genetic groups appears to have been influenced by major ocean currents around the Japanese archipelago. Dominant haplotypes in the star-shaped haplotype network of Lineage A were distributed throughout the entire distribution range of each genetic group, implying rapid range expansion of this species. The results of mismatch distribution analysis and molecular clock estimation suggest that expansion of lineage A occurred during the late Middle or Late Pleistocene. In contrast, restricted or past gene flow suggested by NCA and the many unique haplotypes (110/123; 89.4%) present in Lineage A suggest that gene flow among extant populations is rather limited.