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Pearl oyster shell consists of two layers: a calcite prismatic layer (outer layer) and an aragonite nacreous layer (inner layer). Calcite and aragonite are CaCO₃ polymorphs, and their formations are controlled by shell‐forming tissue called mantle. Pearl sacs originating in the mantle form cultured pearls. Therefore, it has been widely accepted that pearl and shell are produced by the same process. However, this idea has been called into question by some recent mineralogical studies indicating microstructural and crystal‐polymorphic diversity in pearls. The pearl biomineralization process is still not fully understood in detail. Thus, in this study, we focused on the diversity of CaCO₃ polymorphism of non‐nacreous structures (NNSs) underlying the nacreous layer in pearl and regenerated shell, to reveal the biomineralization process of the Japanese pearl oyster (Pinctada fucata). Using Meigen's stain and scanning electron microscope‐energy dispersive X‐ray (SEM‐EDX), NNSs polymorphs in valuable and valueless pearls, in addition to regenerated shell, were compared. Aragonite was exclusively observed in the NNSs of valuable pearls, whereas calcite was dominant in those of valueless pearls. The same analysis of NNSs of regenerated shells was carried out. As in valueless pearls, almost all regenerated shell NNSs consisted of calcite, but one NNS was composed of aragonite. Accordingly, it seems that pearls are formed by the same biomineralization process as shell regeneration rather than shell formation.

The hermaphorodic clam Corbicula fluminea has the unusual reproduction mode of androgenesis. In fertilized eggs, all maternal chromosomes and centrosomes are simultaneously extruded as two polar bodies. To reveal the process of this unusual polar body formation, the process was observed by confocal microscopy. In particular, actin organization of oocytes labeled with anti-α- and γ-tubulin antibodies and rhodamine phalloidin were observed. The meiotic spindle was organized near the animal pole and was parallel to the oocyte surface at metaphase of the first meiosis. A thin actin layer was uniformly distributed across the oocyte membrane at metaphase. At anaphase of the first meiosis, two circular, actin-poor regions appeared where the meiotic half spindles docked. All chromosomes and centrosomes were located within the two bulges formed at anaphase in the actin-poor regions. No overlapping of microtubules was observed from either centrosome at anaphase. The centrosomes were located at the apical region of the bulges. Microtubules radiated for the bulge base. At late anaphase, around the actin-poor regions, actin accumulated and two actin rings became distinct. The diameter of these rings gradually decreased and cytokinesis occurred at the base of the bulges. Thus, all centrosomes and chromosomes were extruded together with the polar body. The present study suggests that centrosomes at the first meiosis could have the equal ability to approach and attach to the membrane and induce polar body formation.

The pearl culture industry using the Akoya pearl oyster Pinctada fucata in Japan has been suffering from low productivity due to the proportion of low-quality pearls, which are not round in shape and have blemishes on the surface, is fairly high with over 70% of pearls effected. Recent studies indicate that the immersion treatment of oysters in low salinity seawater (25 psu) in a tank for 8 days just after the implantation of nuclei has a positive effect on the production of high-quality pearls compared to the suspension of oysters in the sea (33 psu), so-called the “conventional method”, throughout two weeks of the post-operative care. However, a mechanism to increase the proportion of high-quality pearls in low salinity seawater is not fully known. In this study, we compared the periods from the implantation of nuclei to the completion of pearl-sac formation among implanted pearl oysters nursed in tanks containing low salinity (25 psu) or normal salinity (33 psu) seawater in tanks and in the sea (33 psu). After the immersion of the implanted oysters in 25 or 33 psu seawater in the tanks for 8 days, animals were suspended from a raft in an inland bay for the remainder of post-operative care period. The implanted oysters treated with the conventional method were all fastened from the raft throughout the post-operative care period. The oysters from the three treatments were 10% formalin-fixed at periodic intervals and thin slices of the nucleus-implanted part of body were prepared for the examination of the process of pearl-sac formation. The oysters suspended from the raft in the sea throughout the post-operative care period showed the shortest interval from the implantation to the pearl-sac formation, followed by those in normal salinity and low salinity treatments, respectively. This trend corresponds well to that in the proportion of high-quality pearls among the three treatments. The results of the present study suggest that the delay of pearl-sac formation is most likely one of the important factors in preventing deformities and blemishes of pearls in future culture production.

The influence of shell body color on pearls has been studied extensively, but the effect of shell interference color selection on pearls has not been fully understood yet. Because interference color is an important factor determining pearl quality, it is important to study whether the shell interference color of the donor and recipient oysters has any influence on the resulting pearls' interference color to cope with the market demands. We report that selective breeding of red- and blue-line Pinctada fucata martensii pearl oysters produce high-quality pearls based on shape, flaw, color, size, and luster assessment categories. Saibo from red- and blue-line donors was implanted with a rounded nucleus into the red- and blue-line recipient oysters from the same batch in four treatments which we refer to as DRMR (red-donor, red-recipient), DRMB (red-donor, blue-recipient), DBMR (blue-donor, red-recipient), and DBMB (blue-donor, blue-recipient) oysters. Pearl shape and flaws, body yellowness index (YI) and interference value color categories, luster value, total nacre and tablet thickness, and surface microstructure for these treatments are reported. More DRMR pearls are high grade (H) than DBMB, DBMR, and DRMB pearls. Using electronic pearl quality measuring devices, DRMR pearls have the lowest YI and highest interference color values, rendering them superior in color category. Whole nacre thickness of pearls estimated by the SEM on the pearl section reveal DRMR pearls have significantly (p .05), DRMR pearls are superior to those of other treatments in their shape, reduced number of flaws, color, and size. We consider that P. fucata martensii oysters with red shell nacre interference color improve pearl quality, and for the purposes of pearl culture should be selected for. These results are of commercial importance, especially where P. fucata is cultured for pearl production.

Several studies have examined early development in oysters, but few have reported the normal (expected) behavior of meiosis and early mitosis or of the organization of the spindle by the centrosomes. To establish this baseline, normal development of fertilized eggs in Crassostrea virginica from meiosis until the four—cell stage using confocal microscopy was documented. To visualize the centrosomes and microtubules, eggs were stained with antibodies for γ- and a-tubulin. In addition to establishing normal development, two additional features of early development were documented. First, centrosome behavior was documented. The centrosome associated with the female pronucleus disappeared after polar body formation. The single centrosome associated with the male pronucleus duplicated by late anaphase of meiosis II to eventually form the spindle for mitosis I, thus the two centrosomes in the first cleavage were derived from the sperm. Second, linkage between the cell and centrosome cycles was established. In the first and second mitosis, centrosome duplication began at early anaphase. At late anaphase, the centrosomes duplicated and were observed as two distinct units at each pole. Asynchrony was also observed at the four—cell stage between the large and small blastomeres, with chromatin and centrosome duplication showing more advanced stages in the large blastomere. This documentation will serve as a baseline for future studies on chromosome loss in polyploid oysters.

This study examines whole nacre thickness and monthly growth of pearls of Akoya pearl oyster, Pinctada fucata , which came from Japanese and hybrid strains and relate them to the water temperature of the aquaculture site in Ago Bay. Whole nacre thickness of the pearls was significantly different between Japanese and Hybrid strains (Student's t -test, p -value t -test, p -value t -test, p -value Statement of relevance The findings of this study will redound to the benefit of commercial aquaculture of pearl oyster in the world generally, and in Japan especially considering that cultured pearl is one of the important aquaculture products in Japan and some other Countries.

The Pacific oyster Crassostrea gigas is a commercially important bivalve distributed along the northwest Pacific coast. Here C. gigas in Japan was investigated using mtDNA and microsatellite markers to elucidate its genetic structure and phylogeny. On the basis of mtDNA all populations showed high genetic diversity with limited genetic differentiation among populations. The pattern of MtDNA diversity suggested that C. gigas had experienced population expansion about 112 Kya, prior to the last glacial maximum (LGM), which accorded well with other marine organisms. For microsatellites, a Bayesian-based assignment test demonstrated that C. gigas is nearly panmictic. However, on the basis of estimates of FST, Kumano populations differed significantly from other populations, a recent occurrence based on low RST. Irrespective of geographical distance, genetic similarity was observed in the main aquaculture regions with large-scale transportation of cultured spat. Unlike in the Yellow Sea, a genetic bottleneck was not detected in Japanese populations. These results imply, contrary to the prevailing view, that C. gigas in Japan was demographically stable during the LGM. Gene flow by larval dispersal seems to be regionally restricted to localities of congenital areas by ocean currents, while genetic homogenization by cultivated oysters might have occurred in aquaculture areas.

Despite the importance of both pearl body and interference colors in determining pearl quality, only body color has been studied extensively. We report novel information on relationships between pearl nacre microstructure and interference color saturation and hue in pearls produced by Pinctada fucata martensii. The interference color saturation and hue of 60 round pearls was subjectively graded by eye by an expert. Nacre optical characteristics and microstructure of pearls of 1) high (grade A) and low (grade B) interference color saturation, and 2) pink and green interference hues are reported. Using electronic pearl quality measuring devices, grade A pearls have higher interference color values (0.050 ± 0.061) than grade B pearls (0.008 ± 0.039), consistent with subjective grading by eye. SEMs of surface microstructure reveal the nacre tablet area of grade A pearls, 4.079 ± 2.173 μm2, to be generally greater than that of grade B pearls, 2.940 ± 1.213 μm2 (P

The objectives of this study were to 1) determine the influence of environmental factors (water temperature, food availability [chlorophyll-a], salinity) on nacre deposition rate and tablet thickness at different positions in shells of two strains of pearl oyster, Pinctada fucata ( Gould, 1850 )—one from Japan, and one a Hybrid between this Japanese strain and another from China; 2) compare nacre deposition rates and tablet thickness in different shell positions within and between these two pearl oyster strains; and 3) determine relationships between shell nacre deposition rate and tablet thickness in host oysters of these two strains with those of their pearls. Nacre and tablet thickness were measured at three positions along the mantle pallial line (anterior, middle, and posterior), and one position close to the hinge, on the left valve of each oyster. Water temperature and chlorophyll-a were positively correlated (p 0.75) and salinity was negatively correlated (p .05) in nacre tablet thickness was apparent between strains. Correlation analysis of nacre deposition rate between the shell and pearls showed that all shell positions were highly correlated with their respective pearls (p 0.75). Environmental factors influence shell nacre deposition and thickness, and because shell nacre deposition is related to pearl nacre, pearl growth also. This influence varies in different shell positions, with nacre deposition in the posterior position on the shell the fastest, and the middle position showing the most stable and thin nacre tablet. Japanese pearl oysters exhibit better nacre deposition than Hybrid oysters and would be more appropriate as host oysters in Ago Bay, Japan, should culture occur in environmental conditions comparable to those in this study.

Sex, maturity, and the incidence of males in a population of hermaphroditic Corbicula fluminea Muller from Shirakawa River, Kyoto, Japan, are reported. Two shell color types (yellow and green) of this species are recognized. High frequencies of females (63.3% yellow and 58.5% green) are represented among individuals in this population. Histological examination of gonads and measurements of shell length suggest this species first matures as a female before commencing spermatogenesis (i.e., it is a protogynous simultaneous hermaphrodite). Low frequencies of males (mostly less than 5%), the testes of which are filled with spermatozoa, are reported; no histological evidence for any change from males to hermaphrodites, or hermaphrodites to males, is found. Requirements for maintaining males, and for their successful reproduction in self-compatible hermaphrodite populations, are proposed as follows: (1) the protogynous nature of the hermaphrodite, (2) low self-fertilization rates of hermaphrodites, (3) more spermatozoa and greater survival of males than hermaphrodites, and (4) high population densities, ensuring males and hermaphrodites are in close proximity.

The vasa orthologs have been used as a specific germ line molecular marker in many animal species. In this study, the expression of the vasa ortholog (povlg1) in adult and juvenile pearl oysters (Pinctada fucata) was observed by in situ hybridization. The in situ hybridization with povlg1 made it possible to detect the immature germ cells, which could not be detected by hematoxylin and eosin staining. During the reproductive season (May to July), spermatogonia, spermatocytes, oogonia, and oocytes showed povlg1 expression in the gonads of mature adult pearl oysters. By contrast, in spent pearl oysters in the nonreproductive season (September and October), only small ovoid (8.9×5.2 mm) povlg1-positive cells were observed in the base of acini. In juvenile (1 mo old) pearl oysters, a clump of germ cells first formed from several cells that were distributed symmetrically and lateral to the visceral mass. These cells then migrated to the ventrolateral periphery of the visceral mass in 2-mo-old oysters....

Genetic characteristics of four wild (Mie, Fukui, Shimane and Nagasaki) and five cultured populations (selectively bred for 12 years with an origin of Ehime population) of the Japanese pearl oyster, Pinctada fucata martensii, were evaluated by using AFLP markers. Six primer pairs generated 1019 loci in total, among which 45.2–55.1 % was polymorphic among populations. Although there was no significant difference in gene diversity between wild (0.170–0.174) and cultured (0.158–0.173) populations, genetic relatedness in cultured populations (0.316–0.450) was about three times higher than that in wild populations (0.110–0.165). In addition, genetic differentiation was about twenty times larger in cultured populations (Nei’s distance: 0.0111) than in wild populations (Nei’s distance: 0.0005). These results mean that selective breeding can cause marked inbreeding as well as large genetic differentiation among cultured populations in a short period. On the other hand, it was suggested that genetic homogenization in the wild, probably due to a large-scale transport of cultured oysters, had progressed in the sea around Japan. In conclusion, it is necessary to prevent inbreeding by the reconsideration of the style of selective breeding in cultured populations, while the escape of gametes or spats of cultured strains in the wild should be avoided for the preservation of genetic characteristics in native populations of P. f. martensii.

Hermaphroditic freshwater clams in the genus Corbicula produce non-reductional spermatozoa. The DNA content of spermatozoa was almost identical with that of somatic cells in C. leana from Mie Prefecture, Japan. Hermaphroditic C. aff. fluminea from Saga Prefecture and C. fluminea from Taiwan also produce non-reductional spermatozoa. On the other hand, spermatozoa of the dioecious C. sandai had half the DNA found in somatic cells. Analysis of chromosome numbers suggests that C. leana (3n = 54 in somatic cells and 18 in meiotic cells) from Mie Prefecture and C. aff. fluminea (2n = 36 in gills and 18 bivalents in meiotic cells) from Saga Prefecture are triploids and diploids, respectively. C. leana, C. aff. fluminea, and C. fluminea may lack either first or second meiosis, resulting in non-reductional spermatozoa. We assume that gynogenetic reproduction occurs in both species; maternal chromosomes are also nonreductional, and spermatozoa activate development of the eggs, but do not contribute to the offspring.

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.

Current techniques for culturing the Akoya pearl oyster Pinctada fucata result in low yields of blemish-free, round pearls (high-quality pearls). We compared the effects of five factors on the proportion of high-quality pearls produced during culture using a generalized linear model (GLM). Two factors were physiological, shell-closing strength (SCS) and the whole wet weight of host pearl oysters, and three factors were procedural, post-operative care method, technician, and nucleus diameter. Our results suggest that post-operative care methods have the most significant effect on increasing the proportion of high-quality pearls. The proportion of high-quality pearls was five-fold higher in the group held in low salinity seawater than in the conventional treatment group. We propose a new post-operative care method in which oysters are immersed in low salinity seawater to increase the production of high-quality pearls.

Two shell color types, yellow (type I) and brown (type II), of hermaphrodite Corbicula fluminea clams from Ritto, Shiga Prefecture, Japan, are sympatric with both male and hermaphrodite Corbicula leana. In the present study, the mitochondrial DNA (mtDNA) cytochrome b and nuclear 28S rRNA genes of C. fluminea were sequenced to construct a haplotype network in order to investigate the genetic relationship with C. leana. Ninety C. fluminea samples revealed only two cytb haplotypes; the majority (97.8%) were CB7, while the remainder were CB1. In C. leana, only CB1 was detected in hermaphrodites, but both CB1 and CB7 were detected in males. Nuclear 28S rRNA haplotypes of C. fluminea type I individuals were divergent from those of hermaphrodite C. leana. However, C. fluminea type I clams shared haplotypes with male C. leana individuals, whereas C. fluminea type II individuals shared haplotypes with both hermaphrodite and male C. leana samples. These results suggest that it may be difficult to define a clear genetic border between these species.

In some bivalve species, paternal mitochondrial DNA (mtDNA) from sperm is transmitted to the offspring. This is called “doubly uniparental inheritance” (DUI). Under DUI, male offspring receive both paternal (M type) and maternal (F type) mtDNA. Females predominantly receive F type. Expression levels of M and F type mtDNA and mitochondrial RNA localization have not been studied extensively. In this study, we quantified M and F type mtDNA and their expression levels in male and female somatic tissues and gonads with real-time polymerase chain reaction (PCR) in the blue mussel, Mytilus galloprovincialis. M and F type expression patterns were studied with in situ hybridization, using probes specific to M and F type mtDNA in the cytochrome b region. We found that (i) F type mtDNA was expressed in somatic tissues and female gonads, while M type was not expressed in these tissues; (ii) M type expression in male gonads was limited, but strong expression was observed during early spermatogenesis; and (iii) F type expression ratios were significantly lower in female gonads than in somatic tissues and lower than both M and F type expression ratios in male gonads. We propose (i) different systems for M and F type tissue-specific transcriptional regulation; and (ii) different functions for F and M type mtDNA, with F type being functional in somatic tissues and female gonads and M type functioning only in spermatogenetic cells.

Doubly uniparental inheritance (DUI) of mitochondrial (mt) DNA has been reported in the blue mussel Mytilus galloprovincialis. In DUI, males inherit both paternal (M type) and maternal (F type) mtDNA. Here we investigated changes in M type mtDNA copy numbers and mitochondrial mass in testicular cells by real-time polymerase chain reaction and flow cytometry. The ratios of M type mtDNA copy numbers to nuclear DNA content were not different between haploid (1n), diploid (2n) and tetraploid (4n) spermatogenic cells. The mitochondrial mass decreased gradually during spermatogenesis. These results suggest that mtDNA and mitochondrial mass are maintained during spermatogenesis. We then traced M type mtDNA in larvae after fertilization. M type mtDNA was maintained up to 24 h after fertilization in the male-biased crosses, but decreased significantly in female-biased crosses (predicted by Mito Tracker staining pattern). These results are strikingly different from those reported for mammals and fish, where it is well known that the mitochondria and mtDNA are reduced during spermatogenesis and that sperm mitochondria and mtDNA are eliminated soon after fertilization. Thus, the M type mtDNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the DUI system in the blue mussel.

低塩分海水養生による真珠の無キズ珠率向上効果を明らかにするため，挿核手術直後のアコヤガイを水槽（塩分 25 psu または 33 psu に調整）あるいは海上で 14 日間養生後，海上で養殖した場合の無キズ珠率を比較した。その結果，無キズ珠率は 25 psu の水槽で最も高く，次いで 33 psu の水槽，海上の順となった。水槽内での低塩分養生（25 psu）と海上養生による無キズ珠率を業者 10 名による実証実験で比較した結果でも，低塩分養生の無キズ珠率は有意に高く，低塩分海水養生は真珠の品質向上に有効と判断された。

Rhodeus atremius is an endemic bitterling fish from Japan comprising two endangered subspecies, R. a. atremius and R. a. suigensis. The latter subspecies, which had dramatically declined in last decades, was reported to contain two distinct mtDNA lineages. In order to estimate the optimized units for conservation management, the genetic structure of R. atremius was inferred by analyzing mtDNA and microsatellite markers (MS). Allelic richness and heterozygosity of MS in R. a. suigensis was less than half that in R. a. atremius. In R. a. suigensis, not only within-population genetic diversity but also among-population genetic divergence was low, with the exception of population Ah1, while the diversity was high and the divergence showed isolation by distance in R. a. atremius. In mtDNA and MS, R. a. suigensis concordantly formed a single lineage, while R. a. atremius encompassed four mtDNA lineages, two of which were completely admixed into one group on the basis of MS. In population Ah1 a striking introgression between the two subspecies was suggested by a Bayesian-based assignment test, with the presence of mtDNA haplotype of R. a. atremius. Contrary to the prevailing theory, R. a. suigensis corresponds to a single conservation unit, while three units seem appropriate for R. a. atremius. In addition, low genetic diversity of R. a. suigensis might have arisen mainly as a result of recent bottlenecks before population fragmentation, followed by current anthropogenic effects. Genetic introgression in population Ah1 was probably the result of human transplantation of R. a. atremius.

In bivalve, the distribution of primordial germ cells can be traced from early embryogenesis to the veliger larva by the expression of the vasa ortholog. However, the distribution of germ cells from metamorphosis to maturation in bivalves has not been examined extensively. In this study, we used in situ hybridization to observe expression of the Mytilus galloprovincialis vasa-like gene (Myvlg). The distribution of germ cells was clarified in immature mussels. We observed germ cells in adult mussels during the non-reproductive and reproductive seasons. Myvlg was specifically expressed in germ cells. Gametogenesis occurs in acini surrounded by connective tissue. Myvlg expression was detected in spermatogonia, spermatocytes, oogonia, and oocytes. In the non-reproductive season, gametes were not observed in the acini, but Myvlg was expressed in germinal stem cells along the acini. The expression intensity in the non-reproductive season, however, was much weaker than that in the reproductive season. Myvlg-positive cells proliferated during the non-reproductive season. In immature mussels, a pair of germ cell clumps was distributed laterally in the connective tissue between the nephric tubules and posterior byssal retractor muscle. Germ cells were also observed along pericardium. When immature mussels grew, a pair of germ cell clumps migrated anteriorly in the connective tissue along the outer epithelium at the dorsal region of the mantle base between the mantle and gill. The number of germ cells increased significantly as the mussels grew. This is the first report to observe the proliferation and migration of germ cells in immature mussels.

For pearl culture, the pearl oyster is forced open and a nucleus is implanted into the gonad with a mantle graft. The outer mantle epithelial cells of the implanted mantle graft elongate and surrounding the nucleus a pearl sac is formed. Shell matrix proteins secreted by the pearl sac play an important role in the regulation of pearl formation. Recently, seven shell matrix proteins were identified from the pearl oyster Pinctada fucata. However, there is a paucity of information on the function of these proteins and their gene expression patterns. Our study aims to elucidate the relationship between pearl type, quality, and gene expression patterns of six shell matrix proteins (msi60, n16, nacrein, msi31, prismalin-14, and aspein) in the pearl sac based on real-time PCR analysis. After culturing for about 2 months, the pearl sac tissues were collected from 22 individuals: 12 with high quality (HP), nine with low quality (LP), and one with organic (ORG) pearl formation. The surface of each of the 12 HP pearls was composed only of a nacreous layer; in contrast, that of the nine LP pearls was composed of nacreous and prismatic layers. The six target gene expressions were detected in all individuals. However, delta threshold cycle (ΔC(T)) for msi31 was significantly higher in the HP than in the LP individuals (Mann-Whitney's U test, p=0.02). This means that the relative expression level of msi31, which constitutes the framework of the prismatic layer, was higher in the LP than in the HP individuals.

Shell matrix proteins play an important role in the regulation of shell and pearl formation in the Japanese pearl oyster (Pinctada fucata). There is a paucity of information on the function of these proteins and their gene expression patterns. The purpose of this study was to compare the gene expression patterns of six shell matrix proteins (nacrein, msi60, msi31, n16, prismalin-14 and aspein) in the mantle center (MC) (which forms the nacreous layer) and mantle edge (ME) (which forms the prismatic layer), with those in the pearl sac (PS) (which forms the nacreous layer on the pearl surface). There was a significant correlation between the PS and MC (P

The aim of this study was to evaluate the suitability of shell-closing strength (SCS), a newly developed trait, as a physiological indicator in the Japanese pearl oyster Pinctada fucata. SCS is the load value necessary to open the shell of a pearl oyster to 10 mm using a shell opener. We developed a new instrument for the measurement of SCS consisting of a force gauge and a shell opener, and examined the relationship between SCS and physiological and nutritive indexes in pearl oysters. The results of the experiments showed that (1) oysters exhibiting good physiological conditions can be selected by using SCS as the indicator; oysters with higher SCS showed lower mortality, higher condition factor, and glycogen content in the adductor muscles than those with lower SCS; (2) SCS varies remarkably among genetic oyster lineages through the culturing period, indicative of the involvement of genetic factors in the determination of SCS; (3) the SCS of pearl oyster tended to increase with the body and the adductor muscle weights, although the protein and glycogen contents of the soft tissue exhibited typical changes during the period in which the water temperature is high (spring to summer). These results suggested that SCS is an efficient indicator of health of pearl oysters, and is useful for breeding and culture management of this species.

To clarify factors reducing the motility and fertility of cryopreserved spermatozoa of the Japanese pearl oyster Pinctada fucata martensii, the structure of spermatozoa before and after cryopreservation was observed by scanning electron microscopy. Testicular spermatozoa were diluted with cryopreservation diluent (10% methanol+18% fetal bovine serum+72% sea water), and dispensed into 0.25-mL straws. The straws were cooled at a rate of approximately −20 °C/min to −50°C, and subsequently immersed in liquid nitrogen. Percentage motility of spermatozoa before cryopreservation was 69.9±4.2%, and that of cryopreserved spermatozoa was 24.0±1.8%, respectively. In cryopreserved spermatozoa, the percentage that lacked or had a deformed flagellum was 56.6±3.9%, while in fresh spermatozoa this was 8.7±2.0%. In cryopreserved spermatozoa, the percentage of deformed acrosomes was 76.6±5.2%, while in fresh spermatozoa this was only 0.9±0.3%. Cryopreserved spermatozoa with a normal acrosome and flagellum were only 15.4±3.5% of those in fresh spermatozoa. These results indicate that lesion of the flagellum and deformation of the acrosome occurred through the cryopreservation procedure, and both types of damage lead to loss of the motility and fertility in thawed spermatozoa.

In many bivalve species, paternal and maternal mitochondrial DNA (mtDNA) from sperm and eggs is transmitted to the offspring. This phenomenon is known as doubly uniparental inheritance (DUI). In these species, sperm mtDNA (M type) is inherited by the male gonad of the offspring. Egg mtDNA (F type) is inherited by both male and female somatic cells and female gonadal cells. In Mytilidae, sperm mitochondria are distributed in the cytoplasm of differentiating male germ cells because they are transmitted to the male gonad. In the present study, we investigated maternal inheritance of mtDNA in the Pacific oyster, Crassostrea gigas. Sequence analysis of two mitochondrial non-coding regions revealed an identical sequence pattern in the gametes and adductor muscle samples taken from six males and five females. To observe whether sperm mitochondria were specifically located in the cytoplasm of differentiating germ cells, their distribution was recorded in C. gigas fertilized eggs by vital staining with MitoTracker Green. Although the 1D blastomere was identified in the cytoplasm of differentiating germ cells, sperm mitochondria were located at the 1D blastomere in only 32% of eggs during the 8-cell stage. Thus, in C. gigas, sperm mitochondria do not specifically locate in the germ cell region at the 1D blastomere. We suggest that the distribution of sperm mitochondria is not associated with germ cell formation in C. gigas. Furthermore, as evidenced by the mtDNA sequences of two non-coding regions, we conclude that mitochondrial DNA is maternally inherited in this species.

The aims of this study were to compare the fertility of fresh and cryopreserved spermatozoa of the Japanese pearl oyster, Pinctada fucata martensii and to develop artificial fertilization methods using the cryopreserved spermatozoa. The optimal egg density for the fertilization test was found to be 1.0 × 10 5 eggs ml − 1 seawater. When 1.0 × 10 5 eggs ml − 1 seawater were mixed with various numbers of fresh or cryopreserved spermatozoa (from 0.041 to 63 × 10 7 spermatozoa), high fertilization rates (about 60%) were obtained following addition of more than 3.5 × 10 7 spermatozoa when using either fresh or cryopreserved spermatozoa. With reduced numbers of spermatozoa of less than 1.1 × 10 7 (cryopreserved) or 0.12 × 10 7 (fresh) spermatozoa, the fertilization rates gradually decreased to 40% or less. More than 10 times the number of cryopreserved spermatozoa was necessary to obtain similar fertilization rates to those arising from the use of fresh spermatozoa.

In Mytilus mussels, paternal mitochondrial DNA (mtDNA) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, sperm mtDNA (M type) is inherited only by males. Female mussels receive maternal mtDNA (F type). However, in our previous study, we showed female and unfertilized eggs have both F and M types. We hypothesized that the two M types both from sperm and unfertilized eggs were transmitted to offspring. To test the hypothesis, we examined the number of M type haplotypes in mature M. galloprovincialis. The M type in larvae was compared with those of the parents. Cross experiments were carried out to test the inheritance of M type. In six of 20 mature mussels, two M types were detected by sequence analysis and polymerase chain reaction-restriction fragment length polymorphism. In cross experiments of larval samples from five of 12 crosses, double peak wave was observed by single nucleotide polymorphisms analysis. In these larval samples, the higher peak wave was identical to the parental M type. Larvae received much more paternal M type than the maternal ones. We demonstrated that two M types from sperm and unfertilized eggs were transmitted to offspring in M. galloprovincialis.

Effects of NH3 concentration in sea water and pH of sea water on the motility of spermatozoa obtained from testes were examined in the Japanese pearl oyster Percent motility at 30 s after dilution increased with increasing NH3 concentration in sea water from 0.75–2.0 mM. When spermatozoa were diluted with sea water containing 0.75 mM NH3, which is widely used as the insemination fluid in the hatchery of this species, the percent motility increased with time elapsed after dilution, and peaked at 5 min. For spermatozoa diluted with sea water containing 2.0 mM NH3, the percent motility increased rapidly and peaked at 30 s. The pH of sea water increased with increasing NH3 concentration from 8.2 (0 mM NH3) to 9.9 (5.0 mM NH3). When spermatozoa were diluted with artificial sea water at various pH (buffered without NH3 at 6.0–10.0), only spermatozoa diluted with artificial sea water of pH 10.0 were motile, and the percent was considerably lower than those in ammonical sea water. These results indicate that sea water containing 2.0 mM NH3 is a suitable solution for evaluating sperm motility, and that NH3 and/or ammonium ions may activate sperm motility in this species.

The system termed doubly uniparental inheritance (DUI) of mitochondrial transmission to progeny has been reported in Mytilus. Under DUI, it has been thought that males have both paternally (M type) and maternally (F type) transmitted mitochondrial DNA (mtDNA), and females have only F type. However, the presence of M type in females has been reported. To clarify the ratio of M type to F type mtDNA in female and male tissues to further our understanding of mitochondrial transmission, we developed a procedure to measure the copy numbers of the two types of mtDNA in Mytilus galloprovincialis using a real-time polymerase chain reaction assay. The following results were obtained by this method. In females, the copy numbers of M type mtDNA detected in adductor muscle, gonad and eggs were approximately 10 000-fold lower than those of F type. In males, F type dominated in adductor muscle, as in the female tissue. However, copy numbers of M type mtDNA were approximately 1000-fold higher than those of F type in gonad and 100 000-fold higher than those of F type in sperm. We examined the quantity relationship between the two types of mtDNA and the transmission mechanism of mtDNA in M. galloprovincialis.

事業規模での精子大量凍結法を開発するため，容量の異なるストロー（0.25, 1, 2 mL）を用いて凍結したアコヤガイ精子による受精率を比較したところ，有意差は認められなかった。さらに 10 mL の希釈精子を板状に凍結したところ，解凍後の運動率はストロー法と同等であった。運動率が低い精子を凍結・解凍し，通常の 5 分間の媒精時間で人工授精を行うと，受精率は低かったが，媒精時間を延長することにより受精率は上昇し，未凍結の精子と同等となった。本結果から，本種精子を大量凍結保存できる可能性が示された。

We propose a hypothesis of ploidy elevation in the androgenetic clam Corbicula fluminea, based on an abnormal process of fertilization in clams collected at the Tone River, Ibaraki Prefecture, Japan. Most eggs showed androgenesis, that is, extrusion of all maternal chromosomes as two polar bodies during the first meiotic division. Most eggs did not form a female pronucleus, but only a male pronucleus. However, some eggs proceeded to the second meiosis and formed both a female and a male pronucleus. The formation of the female pronucleus suggests the hypothesis that ploidy elevation in androgenetic clams may have occurred by aberrant meiosis due to an altered orientation of the meiotic spindle.

In Mytilidae, mitochondrial DNA (mtDNA) in the offspring is inherited from male and female parents. Sperm mitochondria are only incorporated into the testes. This phenomenon is called doubly uniparental inheritance (DUI). Sperm mitochondria should locate in the primordial germ cell during development to maintain DUI. However, the mechanism of sperm mitochondria localization is still unknown. To reveal the mechanism, we followed the location of sperm mitochondria in Mytilus galloprovincialis zygotes fertilized with sperm stained by MitoTracker. Just after fertilization, sperm mitochondria, which were found to enter eggs from various sites, remained at sperm entry point. Five sperm mitochondria located at the male pronucleus. After pronuclear expansion, sperm mitochondria migrated to the center of the egg together with the male pronucleus. At anaphase of cleavage-I, the distribution pattern of sperm mitochondria was divided into two patterns. In pattern A, sperm mitochondria located in the equatorial region of the eggs. In pattern B, sperm mitochondria migrated and divided into two groups with chromosomes. From observations of colchicine-treated eggs, we suggest that sperm mitochondria migration from fertilization to anaphase of cleavage-I depends on the microtubules. The difference between pattern A and pattern B may be caused by whether sperm mitochondria migrated or not by the microtubules at cleavage-I.

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 used an artificial semen emission test to examine the semen transporting role of parasperm (unflagellated sperm), which are produced along with eusperm (normal sperm) by an incomplete meiosis in the marine sculpin Hemilepidotus gilberti. After separation of contents of semen (eusperm, parasperm, and seminal plasma) by centrifugation, the distance traveled by semen discharged vertically from a syringe into seawater was compared among various test semen; eusperm semen (eusperm re-mixed with seminal plasma), parasperm semen (parasperm re-mixed with seminal plasma), normal semen (eusperm and parasperm re-mixed with seminal plasma), and natural semen (unmodified semen). Parasperm semen traveled more than 1.5 times as far as the eusperm semen. The lateral dispersion width of normal semen after emission was significantly narrower than that of eusperm semen. These findings indicate that parasperm can reduce the lateral dispersion and prolong the distance semen travels. The eusperm ratio in the lower portion of the emitted semen did not differ from that in the upper portion, indicating that eusperm evenly distribute within the ejaculate and reach the lower portion of semen. Since males cannot closely approach eggs that are deposited in the narrow space between the female's belly and the spawning substrate, restraint of lateral dispersion and prolongation of the distance semen traveled would increase the number of eusperm arriving at eggs, despite reduction of eusperm in the ejaculate.

In many species of animals, males normally produce parasperm (dimorphic sperm) along with eusperm (normal sperm) during spermatogenesis. In the present study, to clarify the role of parasperm of the non-copulatory sculpin Hemilepidotus gilberti, whose reproduction is characterized by polyandrous oviposition involving sneaking by neighboring territorial males, we observed the movements of parasperm. Parasperm could not move by themselves, but they were transported in solutions by passive movement due to collisions with actively swimming eusperm. In the viscous ovarian fluid (OF), which isolates eggs from seawater by covering them during spawning, parasperm did not exhibit any movement. However, they could be transported by eusperm movement in solutions with dissolved OF, partly because the viscosity of the fluid become lower. And then, in some solutions parasperm formed lumps. Lump formation of parasperm was also observed at the boundary surface of an egg mass where OF contacted seawater. Eusperm added experimentally to a solution in which parasperm were forming lumps were engulfed in the lumps and never escaped. Thus, lump formation of parasperm would be obstacles for the later arriving eusperm. Although lumps formed against both kin and non-kin eusperm, parasperm are thought to be available to overcome sperm competition which would occur during spawning that involves sneaking being almost concurrent with lump formation. The territorial male eusperm reach the eggs while his parasperm hinder other males' eusperm from reaching the eggs. Thus, we concluded that parasperm of H. gilberti play a role on protection of paternity by blocking rival eusperm physically from approaching eggs.