Food Web Structures and Mercury Exposure Pathway to Fish in Minamata Bay.

We analyzed total mercury content (THg) and carbon (δ13C) and nitrogen (δ15N) stable isotope ratios in fish, subtidal macrobenthos, and particulate organic matter (POM) as a proxy for pelagic phytoplankton and attached microalgae as a proxy for microphytobenthos to investigate the mercury exposure pathway in fish. For four seasons, samples of the above-mentioned organisms were collected on five occasions (July and October 2018 and January, April, and July 2019) in Minamata Bay. Isotope analysis showed that Minamata Bay food web structures were almost entirely fueled by microphytobenthos. The THg values of the fish and macrobenthos species were positively correlated with their δ13C. This indicates that their diets, which were highly fueled by microphytobenthos, led to high THg bioaccumulation in both macrobenthos and fish. The feeding habits of fishes differ depending on the species, and they prey on organisms of many taxa, including fish (mainly Japanese anchovy), crabs, shrimp, copepods, annelids, and algae. Fish species that preyed on benthic crustaceans had high THg. These results suggest that the main pathway of Hg bioaccumulation in fish from Minamata Bay is the benthic food chain, which is primarily linked to benthic crustaceans fueled by microphytobenthos.

Food sources are more important than biomagnification on mercury bioaccumulation in marine fishes.

Marine animals often accumulate various harmful substances through the foods they ingest. The bioaccumulation levels of these harmful substances are affected by the degrees of pollution in the food and of biomagnification; however, which of these sources is more important is not well-investigated for mercury (Hg) bioaccumulation. Here we addressed this issue in fishes that inhabit the waters around Minamata Bay, located off the west coast of Kyushu Island in Kumamoto Prefecture, Japan. The total Hg concentration (hereafter [THg]) and carbon and nitrogen stable isotope ratios (δ13C and δ15N) were analyzed in the muscle tissue of 10 fish species, of which more than five individuals were caught by gillnet. Except one species, each was separated into two trophic groups with respective lower and higher δ13C values ranging from -17‰ to -16‰ and -15‰ to -14‰, which suggested that the fishes depended more on either phytoplankton- and microphytobenthos-derived foods (i.e., pelagic and benthic trophic pathways), respectively. Linear mixed effects models showed that the Hg levels were significantly associated with both δ15N and the differences in the trophic groups. [THg] increased with δ15N (i.e., indicative of higher trophic levels), but the slopes did not differ between the two trophic groups. [THg] was significantly higher in the group with higher δ13C values than in those with lower δ13C values. The effect size from marginal R squared (R2) values showed that the variation in [THg] was strongly ascribed to the trophic group difference rather than δ15N. These results suggest that the substantial Hg bioaccumulation in the fishes of Minamata Bay is mainly an effect of ingesting the microphytobenthos-derived foods that contain Hg, and that the subsequent biomagnification is secondary.

The complete mitochondrial genome of Meretrix lusoria (Bivalvia: Veneroida: Veneridae) from Kumamoto, Japan.

We sequenced and assembled the complete mitochondrial genome sequence of the Meretrix lusoria, from Kumamoto, Japan. The length of mitogenome is 20,180 bp, including 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. The nucleotide composition of the mitogenome was 25.73% for A, 42.41% for T, 9.35% for C, and 22.49% for G. The AT and GC skewness of mitogenome sequence are -0.245 and 0.412, showing the T-skew and G-skew. The reconstructed phylogenetic relationships of 25 Bivalvia species based on 12 protein-coding genes were highly supported and the clade of all Meretrix clams included had a support value of 99%. Our results shall provide a better understanding in the evolutionary histories of the Veneroida and relative species.

Evolutionary history of the extant amphioxus lineage with shallow-branching diversification.

Amphioxus or lancelets have been regarded as a key animal in understanding the origin of vertebrates. However, the evolutionary history within this lineage remains unexplored. As the amphioxus lineage has likely been separated from other chordates for a very long time and displays a marked left-right asymmetry, its evolutionary history is potentially helpful in better understanding chordate and vertebrate origins. We studied the phylogenetic relationships within the extant amphioxus lineage based on mitochondrial genomes incorporating new Asymmetron and Epigonichthys populations, and based on previously reported nuclear transcriptomes. The resulting tree patterns are consistent, showing the Asymmetron clade diverging first, followed by the Epigonichthys and Branchiostoma clades splitting. Divergence time estimates based on nuclear transcriptomes with vertebrate calibrations support a shallow diversification of the extant amphioxus lineage in the Tertiary. These estimates fit well with the closure of seaways between oceans by continental drift, ocean currents, and present geographical distributions, and suggest a long cryptic history from the origin of amphioxus to its most recent diversification. Deduced character polarities based on phylogenetic analyses suggest that the common ancestor of the extant amphioxus existed in a tiny epibenthic state with larva-like appearance of extant amphioxus, likely with ciliate epidermis.

Experiments with claw models explain the function of the waving display of Ilyoplax pusilla (Brachyura: Dotillidae).

Males of the dotillid crab, Ilyoplax pusilla, perform waving displays during the reproductive season. Unlike many fiddler crabs (genus Uca), however, the function of the waving display is unclear for this species. Experiments using waving and static claw models over short (10 cm) and long (25 cm) distances were conducted to investigate responses by wandering females to artificial waving signals. In long-distance choice experiments, presuming undirected waving to unseen females as broadcast waving, females were equally likely to approach the waving and static claw sections during the non-reproductive season, but significantly more females (65 %) chose the waving claw section during the reproductive season. In short-distance choice experiments, presuming directed courtship waving toward a particular female, there was no significant difference between the waving and static claw models during the non-reproductive season, but significantly more females (88 %) chose the waving claw during the reproductive season. These results suggest that one function of the waving display of I. pusilla is mate attraction and that waving from a short distance is more effective.

Evolutionary origin of GnIH and NPFF in chordates: insights from novel amphioxus RFamide peptides.

Gonadotropin-inhibitory hormone (GnIH) is a newly identified hypothalamic neuropeptide that inhibits pituitary hormone secretion in vertebrates. GnIH has an LPXRFamide (X = L or Q) motif at the C-terminal in representative species of gnathostomes. On the other hand, neuropeptide FF (NPFF), a neuropeptide characterized as a pain-modulatory neuropeptide, in vertebrates has a PQRFamide motif similar to the C-terminal of GnIH, suggesting that GnIH and NPFF have diverged from a common ancestor. Because GnIH and NPFF belong to the RFamide peptide family in vertebrates, protochordate RFamide peptides may provide important insights into the evolutionary origin of GnIH and NPFF. In this study, we identified a novel gene encoding RFamide peptides and two genes of their putative receptors in the amphioxus Branchiostoma japonicum. Molecular phylogenetic analysis and synteny analysis indicated that these genes are closely related to the genes of GnIH and NPFF and their receptors of vertebrates. We further identified mature RFamide peptides and their receptors in protochordates. The identified amphioxus RFamide peptides inhibited forskolin induced cAMP signaling in the COS-7 cells with one of the identified amphioxus RFamide peptide receptors expressed. These results indicate that the identified protochordate RFamide peptide gene is a common ancestral form of GnIH and NPFF genes, suggesting that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. GnIH gene and NPFF gene may have diverged by whole-genome duplication in the course of vertebrate evolution.

Stable aquaculture of the Japanese lancelet Branchiostoma japonicum for 7 years.

Despite advances in the study on animal evolution in the last two decades, paucity of experimental data on cephalochordates comparable to those on the other chordates hinders an integrative understanding of chordate evolutionary history. To obtain lancelet data under well-controlled experiments, laboratory cultures of lancelets have been performed at several institutions. In a mass culture started in 2005, we have obtained up to three consecutive generations of Branchiostoma japonicum. Using sand substratum, survival rates of laboratory lancelets until maturation have improved to greater than 30%, much higher than compared to previously, and for adults the annual average survival rate was 82.3%. The high survival rate allows maintaining animals at least 6 years and potentially longer. Water temperatures lower than 23°C obviously reduced the frequency of spawning even after the onset of spawning period, and 1-2 days after changing the temperature at 25°C animals became spawned well. We also observed obvious sex reversal from male to female in individuals that had been cultured for 3 years or more. Our continuous culture has provided sufficient materials for vital experiments on early development and for studying metamorphosis, as well as for the conservation of wild populations. The subculture of successive laboratory generations will provide a valuable resource for genetic studies.

Laboratory culture of the Oriental lancelet Branchiostoma belcheri.

To overcome difficulties in getting research materials of cephalochordate lancelets, which has severely hampered experimental studies of this animal, we have attempted to establish a culture system in the laboratory. Adult animals collected from the wild were maintained in 2.5-L plastic containers filled with natural seawater without sand substratum. They were fed daily with unicellular algae. About 25% of the animals collected in 2003, 2004, and 2005 developed gonads in our culture system. Some of the sexually mature animals collected in the breeding seasons in 2005 and 2006 spawned spontaneously in the plastic containers of this system. Broods obtained in 2005 were maintained longer than a year in a glass tank without sand substratum. The progeny born in the laboratory showed great individual variation in growth but metamorphosed normally, and some of them started to develop gonads around 10 months after fertilization. Our mass culture methods for both adults and their progeny made daily observation possible and allowed the constant spawning of animals collected from the wild, at least in the summer season. Our culture method saves labor in maintenance and is easily set up without any specific demands except for running seawater, though still required to better survival rate and spawning control. Lancelet populations maintained in the laboratory can promote studies on these animals across disciplines and especially contribute to elucidation of the evolutionary history of chordates.

Larval development of the oriental lancelet, Branchiostoma belcheri, in laboratory mass culture.

We are successfully maintaining a laboratory colony of the lancelet Branchiostoma belcheri bred in the laboratory. Based on living individuals in this mass culture, morphological characteristics from the seven-day larval to benthic juvenile stages have been studied. Most striking was that later larval development of B. belcheri showed great individual variation even in a rather stable culture environment. Metamorphosis first occurred on 60 days post fertilization (dpf) and was continuously observed throughout the present study up to 100 dpf. Morphological traits such as the number of primary gill slits and body size at the start of metamorphosis are apparently affected by culture condition. Body size measured in the largest individuals showed nearly linear growth at 0.087 mm/day. The variability found in larval development calls for caution when developmental stages and chronological ages are compared between populations. However, the developmental flexibility of this animal also raises the possibility that growth and sexual maturation could be controlled artificially in captivity.

Biology of the amphioxus, Branchiostoma belcheri in the Ariake Sea, Japan I. Population structure and growth.

We investigated the population structure and growth of the amphioxus Branchiostoma belcheri for four years in the southern Ariake Sea, Japan. We counted 62-66 myotomes and 251-310 dorsal fin-ray chambers, and these results support that this species is an intermediate form of B. belcheri and its subspecies B. belcheri tsingtauense. The ratio of females to males was 1:1.12. Males were more numerous than females among small individuals (< 40 mm body length), but we found no significant differences among large animals (50 mm body length). Spawning occurred from mid June to early July. Groups of newly settled young appeared from January to June of their second year. We observed a large fluctuation between years in the numbers of newly settled young. The estimated size of one-year-old individuals was 19.4 mm in body length; within the next 12 months, they reached 32.1 mm. Three- and four-year-old individuals measured 38.6 mm and 45.8 mm, respectively. Few grew beyond 60 mm; the largest specimen collected was a 64 mm male.

Biology of the amphioxus, Branchiostoma belcheri in the Ariake Sea, Japan II. Reproduction.

We investigated the reproduction of the amphioxus Branchiostoma belcheri in the southern Ariake Sea, Japan, from 1999 to 2002. Gonads were very small or almost absent from September to December and began to develop in January. Changes in the gonad length index indicated that spawning began in mid June, after the gonads had attained maximum size. Although most gametes were extruded during the first spawning, some remained in the gonads. Shrunken gonads were much smaller but contained gametes, and the second spawning occurred around 10 July. It was unclear whether all adults spawned twice, but a large proportion of individuals did. After the first spawning, both males and females lost 30% of their body weight. Most one-year-old individuals did not spawn, and the minimum size at maturity was ca. 20 mm. We found no termination of reproduction among very large individuals. The average number of gonads was significantly larger on the right side of the body (26.2 in males and 26.1 in females) than on the left side (24.0 in males and 23.5 in females). We found two hermaphrodites in a total of 11,184 specimens examined. Each had four or three ovaries among a total of 45 or 54 gonads.