Akira Komaru

Cytological evidence of spontaneous androgenesis in the freshwater clam Corbicula leana Prime.

Abstract Cytological observations and DNA microfluorometry of the hermaphrodite freshwater triploid clam Corbicula leana revealed unusual androgenetic development as follows: (1) the maternal genome of zygotes was extruded as two polar bodies just after karyokinesis of the first meiosis, (2) only chromosomes derived from one male pronucleus constituted the metaphase of the first cleavage of zygotes, (3) DNA content of 7-day-old veliger larvae was identical to the somatic cells of the parent. This spontaneous androgenetic process in C. leana zygotes is the first case in the phylum Mollusca and may be related to the specialized mode of reproduction; i.e. hermaphroditism and self-fertilization.

Detection of induced triploid scallop, Chlamys nobilis, by DNA microfluorometry with DAPI staining

Abstract A simple method of detecting the ploidy levels of shellfish by DNA microfluorometry with DAPI staining was demonstrated. Triploid scallops, Chlamys nobilis, were induced by cytochalasin B treatment (0.5 mg/l) of fertilized eggs, commencing 15 min after insemination for 15 min duration. Ploidy levels were determined at 8–13 months of age by microfluorometry of cells from gill tissue and hemolymph stained with DAPI and excited with UV light. Fluorescence intensity per nucleus was measured using a microphotometer. The ploidy levels determined by microfluorometry were verified by both cytogenetic and flow cytometric assays.

HERMAPHRODITIC FRESHWATER CLAMS IN THE GENUS CORBICULA PRODUCE NON-REDUCTIONAL SPERMATOZOA WITH SOMATIC DNA CONTENT

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.

Effect of selection for shell coloration on growth rate and mortality in the Japanese pearl oyster, Pinctada fucata martensii

Abstract Effects of inbreeding on the growth rate and mortality are suggested on the basis of results of comparative growth experiments for a line of the Japanese pearl oyster, Pinctada fucata martensii , selected for white shell color in the prismatic layer. Growth in shell height, whole weight and mortality were measured using one inbred white cross and two hybrid white/brown crosses held in pearl nets under different densities. Analyses were conducted at the juvenile (3–12 months of age) and adult (12–20 months of age) stages. The growth rate was estimated by the differences between measurements at the start and after 4, 5, 6, 8 and 12 months in the juvenile experiment, and after 17 and 20 months in the adult experiment. Growth in whole weight and survival were significantly inferior in the inbred white crosses than in two hybrid crosses. With the exception of one case, there was no observation in which the performance of an inbred cross was significantly superior to that of the two hybrid crosses. There were significant effects of density on growth rate in weight, shell height and mortality.

Sperm mitochondrial DNA transmission to both male and female offspring in the blue mussel Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (M type) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, it has been reported that female mussels generally have only maternal mtDNA (F type). In this study, we examined the mode of mtDNA transmission in Mytilus galloprovincialis using M and F type‐specific primer sets. The ratio of M and F types were measured in each sample by SNaPshot. The M type was detected in the adductor muscle and female gonad of all females. In unfertilized eggs spawned by 84.6% of females (22/26), M type was also detected. The F type was more abundant than the M type in all females. Although the ratio of M type in females was very low, all females contained the M type. From these results, we propose a new possibility about DUI inheritance. The presence of M type in unfertilized eggs indicates that the M type of eggs may also contribute to M type inheritance.

Specific location of sperm mitochondria in mussel Mytilus galloprovincialis zygotes stained by MitoTracker

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.

Non-reductional Spermatozoa in Three Shell Color Types of the Freshwater Clam Corbicula fluminea in Taiwan

Abstract We found three distinct shell color types in samples of the freshwater clam Corbicula fluminea Müller collected at Hou Don, Keelung, Taiwan. DNA microfluorometric analysis revealed that these three types consisted of both diploids and triploids. DNA microfluorometry on sperm and somatic cells showed that both diploid and triploid produced non-reductional spermatozoa. These characteristics are similar to triploid C. leana Prime sampled in Japan. These findings suggest that Corbicula fluminea at different ploidy levels may be reproducing by androgenesis as already shown in C. leana from Japan.

Ultrastructure of spermatozoa from induced triploid Pacific oyster, Crassostrea gigas

Ultrastructure of spermatozoa produced by artificially induced triploid Pacific oysters, Crassostrea gigas, was studied by transmission and scanning electron microscopy. Spermatozoa from triploids (3n sperm) resembled sperm from diploids (2n sperm) and consisted of an acrosome, nucleus, four mitochondria, and flagellum. The head, acrosome, and flagellum of 3n sperm were significantly (P<0.05) larger than those of 2n sperm. The diameter of the mitochondria was not different between 3n and 2n sperm.

Different transcriptional ratios of male and female transmitted mitochondrial DNA and tissue‐specific expression patterns in the blue mussel, Mytilus galloprovincialis

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.

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.