Current environmental status of the oyster farms on Lake Kamo in Japan; viral control of the harmful bloom of Heterocapsa circularisquama.

Lake Kamo is an enclosed, low-inflow estuary connected to the open sea that is famous for oyster farming in Japan. In the fall of 2009, this lake experienced its first bloom of the dinoflagellate Heterocapsa circularisquama, which selectively kills bivalve mollusks. This species has been detected exclusively in southwestern Japan. The completely unexpected outbreak of H. circularisquama in the northern region is believed to have been caused by the contamination of purchased seedlings with this species. The water quality and nutrient data collected by our group from July through October over the past 10 years revealed that the environment of Lake Kamo has not changed significantly. However, in the open water around Sado Island, where Lake Kamo is located, the water temperature has increased by 1.80 °C in the last 100 years, which is equivalent to 2-3-fold the world average. This has resulted in a rise in the sea level, which is expected to further deteriorate the water exchange between Lake Kamo and the open sea and low dissolved oxygen in the bottom layer of the Lake and the associated dissolution of nutrients from the bottom sediment. Therefore, seawater exchange has become insufficient and the lake has become nutrient rich, making it prone to the establishment of microorganisms, such as H. circularisquama, once they have been introduced. We developed a method to mitigate the damage caused by the bloom by spraying sediments containing the H. circularisquama RNA virus (HcRNAV), which infects H. circularisquama. After ∼10 years of performing various verification tests, including field trials, this method was used at the Lake in 2019. During the 2019 H. circularisquama growth season, a small amount of sediment containing HcRNAV was sprayed on the lake three times, which resulted in a decrease in H. circularisquama and an increase in HcRNAV, indicating that this method is effective in diminishing the bloom.

Development of sandwich enzyme-linked immunosorbent assays for chub mackerel Scomber japonicus gonadotropins and regulation of their secretion in female reproduction.

The pituitary gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play critical roles in regulating gonadal development and sexual maturation in vertebrates. We developed non-competitive enzyme-linked immunosorbent assays (ELISAs) to measure Fsh and Lh in chub mackerel Scomber japonicus, which is a commercially important scombrid species. Mouse monoclonal antibodies specific for Fsh and Lh, and a rabbit polyclonal antibody against both Gths were produced by immunization with hormones purified from chub mackerel pituitaries. These monoclonal and polyclonal antibodies were used as capture and detection antibodies in the developed sandwich ELISAs. The ELISAs were reproducible, sensitive, and specific for chub mackerel Fsh and Lh. Parallelism between the standard curve and serial dilutions of chub mackerel serum and pituitary extract was observed for both Fsh and Lh ELISAs. Comparison between vitellogenic and immature females revealed that Fsh is secreted during vitellogenesis and Lh is barely released during immaturity. After gonadotropin-releasing hormone analog (GnRHa) injection, vitellogenic females showed increases in serum Lh, whereas serum levels of Fsh did not vary. Moreover, the serum steroid profiles revealed that estradiol-17ß was continuously produced after GnRHa treatment, whereas 17,20ß-dihydroxy-4-pregnen-3-one secretion was transiently induced. These results indicate that, in vitellogenic females, GnRHa stimulates the release of Lh, but not Fsh, which results in acceleration of vitellogenesis and induction of oocyte maturation via steroid production.

Assessment of the risk posed by three antifouling biocides to Pacific oyster embryos and larvae in Hiroshima Bay, Japan.

The Pacific oyster (Crassostrea gigas) is an important species in oyster farming worldwide, including in Japan. Hiroshima Bay is one of the most important oyster farming areas in Japan. We investigated the occurrence of antifouling biocides used worldwide including diuron, Irgarol 1051 (Irgarol), and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT), which have been detected at sub-ppb levels in seawater in Japan, and estimated their no observed effect concentrations (NOECs). In recent years, the spat settlement of Pacific oysters has become poor, which presents a challenge for oyster aquaculture in Hiroshima Bay; hence, we conducted embryotoxicity and larva settlement tests using Pacific oysters. Compared to diuron and Irgarol, DCOIT exhibited a higher toxicity toward oyster embryos, and the minimum 24-h NOEC toxicity value for the oyster embryos was <3 ng/L. The highest concentrations of diuron, Irgarol, and DCOIT in the environmental seawater in the Seto Inland Sea were 27.6, 3.2, and 24 ng/L, respectively. Considering the NOECs, the environmental concentrations of these biocides suggest that the ecological risks posed by diuron and Irgarol are low, whereas those posed by DCOIT are high. However, the rate of detection of DCOIT was low because it degraded rapidly in the seawater before treatment for chemical analysis, except in the case of the treatment on the research vessel.

Partial revision of Japanese Pectinariidae (Annelida: Polychaeta), including redescriptions of poorly known species.

Eight species of Pectinariidae de Quatrefages, 1866 were recorded from Japan and adjacent waters. We studied four species of the family and redescribe the poorly known species from the Seto Inland Sea and Ariake Sound, Kyushu based on recently collected material. The species covered in this study are Amphictene japonica (Nilsson, 1928), Lagis bocki (Hessle, 1917), Pectinaria okudai (Imajima & Hartman, 1964) and Pectinaria hiuchiensis Kitamori, 1965.

Development of non-competitive enzyme-linked immunosorbent assays for mummichog Fundulus heteroclitus gonadotropins - examining seasonal variations in plasma FSH and LH levels in both sexes.

The mummichog Fundulus heteroclitus is an excellent experimental fish for reproductive physiology because of its adequate size, easiness for rearing, and controllable reproduction under laboratory conditions. Furthermore, it is the only species that the native GtHs and their subunits have been purified among small experimental fishes. In this study, homologous non-competitive enzyme-linked immunosorbent assays (ELISAs) for the mummichog FSH and LH were developed by raising monoclonal and polyclonal antibodies against the purified GtHs or their subunits, and the plasma hormone levels in various seasons were examined. The cross-reactivity of LH in the FSH ELISA and the cross-reactivity of FSH in the LH ELISA were low, 2.3% and 0.2% respectively, indicating high specificities of both GtH assays. The practical detection limits were 10 pg/well (0.125 ng/ml plasma) for the FSH ELISA and 8 pg/well (0.1 ng/ml plasma) for the LH ELISA. Plasma FSH levels in females indicated distinct correlations with ovarian stages: they were almost undetectable (<0.125 ng/ml) during the post-spawning immature phase (September), low values (0.3 ng/ml) during the cortical alveoli accumulation phase (December), considerably high (1.8 ng/ml) in the vitellogenic phase (February), and very high values (12 ng/ml) during the spawning season (June). The male FSH levels showed similar pattern of changes to that of females, also indicating distinct correlations with testicular activities. Plasma LH levels were considerably high during the spawning period in both sexes (3.3 ng/ml in females and 4.5 ng/ml in males). They were low or undetectable values in non-spawning seasons, and clear correlation with the gonadal stages was not observed. These results indicate the importance of FSH for various reproductive events in multiple spawning fishes, and are consistent with the general understanding that the LH is responsible for final gametes maturation in both sexes. Nonetheless, they further suggest that the role of LH for various reproductive events other than the final maturation may be limited.

Gene flow of Acanthaster planci (L.) in relation to ocean currents revealed by microsatellite analysis.

Population outbreaks of the coral-eating starfish, Acanthaster planci, are hypothesized to spread to many localities in the Indo-Pacific Ocean through dispersal of planktonic larvae. To elucidate the gene flow of A. planci across the Indo-Pacific in relation to ocean currents and to test the larval dispersal hypothesis, the genetic structure among 23 samples over the Indo-Pacific was analysed using seven highly polymorphic microsatellite loci. The F-statistics and genetic admixture analysis detected genetically distinct groups in accordance with ocean current systems, that is, the Southeast African group (Kenya and Mayotte), the Northwestern Pacific group (the Philippines and Japan), Palau, the North Central Pacific group (Majuro and Pohnpei), the Great Barrier Reef, Fiji, and French Polynesia, with a large genetic break between the Indian and Pacific Oceans. A pattern of significant isolation by distance was observed among all samples (P = 0.001, r = 0.88, n = 253, Mantel test), indicating restricted gene flow among the samples in accordance with geographical distances. The data also indicated strong gene flow within the Southeast African, Northwestern Pacific, and Great Barrier Reef groups. These results suggest that the western boundary currents have strong influence on gene flow of this species and may trigger secondary outbreaks.

Appearances and chronological changes of mummichog Fundulus heteroclitus FSH cells and LH cells during ontogeny, sexual differentiation, and gonadal development.

To examine relationships between gonadal stages and the initial appearance and subsequent development of gonadotrophs, hatched larvae of the mummichog Fundulus heteroclitus were reared until first maturity under suitable conditions for maturation (20 degrees C-16L). Evident FSH cells generally appeared 1-2 weeks after hatching (wah), around or slightly before the morphological sex differentiation which occurred at 2 wah. During this period, 3beta-hydroxysteroid dehydrogenase positive cells also appeared in the gonads. While FSH cells existed throughout the early phases of gonadal development such as cortical alveoli formation and basic spermatogenesis, LH cells appeared later (6-12 wah), after the commencement of the early gonadal development. Both FSH cells and LH cells were abundant at 36 wah when the fish had attained full maturity. These results indicate the possibility that FSH is responsible for gonadal differentiation by inducing steroidogenesis in the gonads, implying the importance of FSH on the early phases of gonadal development. These results also suggest cooperation of FSH and LH in later phases of gonadal development such as yolk globule accumulation and active spermatogenesis. The mode of changes in the abundances of the gonadotrophs according to the gonadal development was somewhat different from previously observed changes during the annual reproductive cycle in adult mummichog. Possible complementary roles of the two GTHs in vitellogenesis and spermatogenesis may be involved in the difference by providing flexibility to the controlling mechanism.

Effect of tributyltin on veliger larvae of the Manila clam, Ruditapes philippinarum.

We investigated the effects of waterborne and maternal exposure to tributyltin (TBT) on veliger larvae of the Manila clam, Ruditapes philippinarum. In a waterborne exposure test, veliger larvae (D-larvae stage: 24h after fertilization) were exposed to TBT at measured concentrations of <0.01 (control), 0.055, 0.130, 0.340, and 0.600microg/l for 13d. The percentage of normal veliger larvae (the ratio of normal veliger larvae to all larvae) decreased significantly in all TBT treatment groups compared with that in the control group. In a maternal exposure test, 100 clams were exposed to TBT at measured concentrations of <0.01 (control), 0.061, and 0.310microg/l at 20-22 degrees C for 3 weeks, and the percentage of normal veliger larvae assessed for 13d. No maternal effects on veliger larvae from TBT were observed in TBT treatment groups as compared with the control group. These results demonstrate that waterborne TBT affects Manila clam veliger larvae, and indicates that TBT may have reduced Manila clam populations by preventing the development and survival of veliger larvae.

Complete mitochondrial genome sequences for Crown-of-thorns starfish Acanthaster planci and Acanthaster brevispinus.

BACKGROUND: The crown-of-thorns starfish, Acanthaster planci (L.), has been blamed for coral mortality in a large number of coral reef systems situated in the Indo-Pacific region. Because of its high fecundity and the long duration of the pelagic larval stage, the mechanism of outbreaks may be related to its meta-population dynamics, which should be examined by larval sampling and population genetic analysis. However, A. planci larvae have undistinguished morphological features compared with other asteroid larvae, hence it has been difficult to discriminate A. planci larvae in plankton samples without species-specific markers. Also, no tools are available to reveal the dispersal pathway of A. planci larvae. Therefore the development of highly polymorphic genetic markers has the potential to overcome these difficulties. To obtain genomic information for these purposes, the complete nucleotide sequences of the mitochondrial genome of A. planci and its putative sibling species, A. brevispinus were determined and their characteristics discussed. RESULTS: The complete mtDNA of A. planci and A. brevispinus are 16,234 bp and 16,254 bp in size, respectively. These values fall within the length variation range reported for other metazoan mitochondrial genomes. They contain 13 proteins, 2 rRNA, and 22 tRNA genes and the putative control region in the same order as the asteroid, Asterina pectinifera. The A + T contents of A. planci and A. brevispinus on their L strands that encode the majority of protein-coding genes are 56.3% and 56.4% respectively and are lower than that of A. pectinifera (61.2%). The percent similarity of nucleotide sequences between A. planci and A. brevispinus is found to be highest in the CO2 and CO3 regions (both 90.6%) and lowest in ND2 gene (84.2%) among the 13 protein-coding genes. In the deduced putative amino acid sequences, CO1 is highly conserved (99.2%), and ATP8 apparently evolves faster any of the other protein-coding gene (85.2%). CONCLUSION: The gene arrangement, base composition, codon usage and tRNA structure of A. planci are similar to those of A. brevispinus. However, there are significant variations between A. planci and A. brevispinus. Complete mtDNA sequences are useful for the study of phylogeny, larval detection and population genetics.

Effects of tributyltin maternal and/or waterborne exposure on the embryonic development of the Manila clam, Ruditapes philippinarum.

We examined the effect of tributyltin (TBT) on embryonic development of the Manila clam, Ruditapes philippinarum. In a maternal exposure test, 100 clams were exposed to TBT at measured concentrations of <0.01 (control), 0.061, 0.310, or 0.350 microg/l at 20-22 degrees C for 3 weeks, and the embryo developmental success (the ratio of normal D-larvae to all larvae) was measured. There was a significant negative correlation between embryo developmental success and TBT concentration in the female Manila clams (p < 0.001). These results indicated that TBT accumulated in the female clam decreased embryo developmental success. In a waterborne exposure test, fertilized eggs (4 h after fertilization) were exposed to TBT at measured concentrations of <0.01 (control), 0.062, 0.140, 0.320, or 0.640 microg/l for 23 h. Embryo developmental success was also significantly decreased in all TBT treatment groups compared with that in the control group. TBT accumulated in female adults and waterborne TBT clearly inhibit reproductive success of the clam.

Development of novel microsatellite DNA markers from the Pacific oyster Crassostrea gigas.

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2-20; observed heterozygosity, 0.119-0.925; unbiased expected heterozygosity, 0.139-0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.