Functional characterization of the GABA transporter GAT-1 from the deep-sea mussel Bathymodiolus septemdierum.

Mammalian γ-aminobutyric acid (GABA) transporter subtype 1 (GAT-1) is a specific transporter for GABA, an inhibitory neurotransmitter in GABA-ergic neurons. GAT-1 belongs to the GAT group, in which five related transporters, GAT-2, GAT-3, GAT-4, CT1, and TAUT are known in mammals. By contrast, the deep-sea mussel, Bathymodiolus septemdierum has only two GAT group members, BsGAT-1 and BsTAUT, and their function in environmental adaptation is of interest to better understand the physiology of deep-sea organisms. Compared with BsTAUT, the function of BsGAT-1 is unknown. Here, we report the functional characterization of BsGAT-1. Analyses of BsGAT-1 expressed in Xenopus oocytes showed that it could transport GABA in a Na+- and Cl--dependent manner, with Km and Vmax values of 0.58 µM and 1.97 pmol/oocyte/h, respectively. BsGAT-1 activity was blocked by the GAT-1 selective inhibitors SKF89976A and ACHC. Competition assays indicated that BsGAT-1 has no affinity for taurine and thiotaurine. These characteristics were common with those of mammalian GAT-1, suggesting its conserved function in the nervous system. However, BsGAT-1 showed a certain affinity for hypotaurine, which is involved in sulfide detoxification in hydrothermal vent-specific animals. This result suggests an additional role for BsGAT-1 in sulfide detoxification, which may be specific to the deep-sea mussel. In a tissue distribution analysis, BsGAT-1 mRNA expression was observed in various tissues. The expression in the adductor and byssus retractor muscles, labial palp, and foot, which possibly contain ganglia, suggested a function in the neural system, while BsGAT-1 expression in other tissues might be related to sulfide detoxification.

Effect of sulfide, osmotic, and thermal stresses on taurine transporter mRNA levels in the gills of the hydrothermal vent-specific mussel Bathymodiolus septemdierum.

Hydrothermal vent environmental conditions are characterized by high sulfide concentrations, fluctuating osmolality, and irregular temperature elevations caused by vent effluents. These parameters represent potential stressors for organisms that inhabit the area around hydrothermal vents. Here, we aimed to obtain a better understanding of the adaptation mechanisms of marine species to hydrothermal vent environments. Specifically, we examined the effect of sulfide, osmolality, and thermal stress on the expression of taurine transporter (TAUT) mRNA in the gill of the deep-sea mussel Bathymodiolus septemdierum, which is a dominant species around hydrothermal vent sites. We analyzed TAUT mRNA levels by quantitative real-time polymerase chain reaction (PCR) in the gill of mussels exposed to sulfide (0.1 or 1mg/L Na2S·9H2O), hyper- (115% seawater) and hypo- (97.5%, 95.5%, and 85% seawater) osmotic conditions, and thermal stresses (12°C and 20°C) for 24 and 48h. The results showed that mussels exposed to relatively low levels of sulfide (0.1mg/L) and moderate heat stress (12°C) exhibited higher TAUT mRNA levels than the control. Although hyper- and hypo-osmotic stress did not significantly change TAUT mRNA levels, slight induction was observed in mussels exposed to low osmolality. Our results indicate that TAUT is involved in the coping mechanism of mussels to various hydrothermal vent stresses.

Dietary exposure experiments on the migration of chemical pollutants from microplastics to bivalves.

Plastics can contain two types of organic contaminants; absorbed from ambient water, and already contained as additives. To investigate the bioaccumulation of these substances, we conducted two types of exposure experiments using mussels and polyethylene microplastics with absorbed PCBs and containing four types of additives (BDE209, DBDPE, UV327 and UV234). After dietary exposure for 15 days, significantly higher concentrations of total PCBs, UV327 and UV234 were detected in the gonad of exposed groups than in the control groups, respectively. However, no significant differences in BDE209 or DBDPE levels were observed between the control and exposure groups. Although a higher transfer ratio was shown for PCB congeners with octanol-water partition coefficients (logKow) below 7, the ratio was lower for higher-hydrophobic PCBs with logKow above 7. This suggests that higher hydrophobic compounds (not only highly chlorinated PCBs, but also BDE209 and DBDPE) tend not to desorb or leach from plastics.

Divalent metal transporter-related protein restricts animals to marine habitats.

Utilization and regulation of metals from seawater by marine organisms are important physiological processes. To better understand metal regulation, we searched the crown-of-thorns starfish genome for the divalent metal transporter (DMT) gene, a membrane protein responsible for uptake of divalent cations. We found two DMT-like sequences. One is an ortholog of vertebrate DMT, but the other is an unknown protein, which we named DMT-related protein (DMTRP). Functional analysis using a yeast expression system demonstrated that DMT transports various metals, like known DMTs, but DMTRP does not. In contrast, DMTRP reduced the intracellular concentration of some metals, especially zinc, suggesting its involvement in negative regulation of metal uptake. Phylogenetic distribution of the DMTRP gene in various metazoans, including sponges, protostomes, and deuterostomes, indicates that it originated early in metazoan evolution. However, the DMTRP gene is only retained in marine species, and its loss seems to have occurred independently in ecdysozoan and vertebrate lineages from which major freshwater and land animals appeared. DMTRP may be an evolutionary and ecological limitation, restricting organisms that possess it to marine habitats, whereas its loss may have allowed other organisms to invade freshwater and terrestrial habitats.

Genomics and Transcriptomics of the green mussel explain the durability of its byssus.

Mussels, which occupy important positions in marine ecosystems, attach tightly to underwater substrates using a proteinaceous holdfast known as the byssus, which is tough, durable, and resistant to enzymatic degradation. Although various byssal proteins have been identified, the mechanisms by which it achieves such durability are unknown. Here we report comprehensive identification of genes involved in byssus formation through whole-genome and foot-specific transcriptomic analyses of the green mussel, Perna viridis. Interestingly, proteins encoded by highly expressed genes include proteinase inhibitors and defense proteins, including lysozyme and lectins, in addition to structural proteins and protein modification enzymes that probably catalyze polymerization and insolubilization. This assemblage of structural and protective molecules constitutes a multi-pronged strategy to render the byssus highly resistant to environmental insults.

Size-dependent elimination of ingested microplastics in the Mediterranean mussel Mytilus galloprovincialis.

Filter feeding organisms have been reported to ingest microplastics (MP) in marine environments. However, information regarding how long the ingested MPs are retained in their digestive tracts remains limited. Here, we report the gut retention time (GRT90) and the long-term egestion time of three different sized polystyrene microspheres (1, 10, and 90 µm) in the Mediterranean mussel Mytilus galloprovincialis. We found significant differences in GRT90 with respect to MP size. With respect to the long-term egestion of MPs, most of the smaller MPs were excreted immediately, although some were detected intermittently until day 40. In comparison, larger MPs were slowly excreted in bulk, after which they were not detected. The results indicate that different sized MPs are retained differently in the digestive tract of mussels. The size-dependent effects of MPs should thus be considered when evaluating the effects of MPs in mussels.

Evolutionary history of the GABA transporter (GAT) group revealed by marine invertebrate GAT-1.

The GABA transporter (GAT) group is one of the major subgroups in the solute career 6 (SLC6) family of transmembrane proteins. The GAT group, which has been well studied in mammals, has 6 known members, i.e., a taurine transporter (TAUT), four GABA transporters (GAT-1, -2, -3, - 4), and a creatine transporter (CT1), which have important roles in maintaining physiological homeostasis. However, the GAT group has not been extensively investigated in invertebrates; only TAUT has been reported in marine invertebrates such as bivalves and krills, and GAT-1 has been reported in several insect species and nematodes. Thus, it is unknown how transporters in the GAT group arose during the course of animal evolution. In this study, we cloned GAT-1 cDNAs from the deep-sea mussel, Bathymodiolus septemdierum, and the Antarctic krill, Euphausia superba, whose TAUT cDNA has already been cloned. To understand the evolutionary history of the GAT group, we conducted phylogenetic and synteny analyses on the GAT group transporters of vertebrates and invertebrates. Our findings suggest that transporters of the GAT group evolved through the following processes. First, GAT-1 and CT1 arose by tandem duplication of an ancestral transporter gene before the divergence of Deuterostomia and Protostomia; next, the TAUT gene arose and GAT-3 was formed by the tandem duplication of the TAUT gene; and finally, GAT-2 and GAT-4 evolved from a GAT-3 gene by chromosomal duplication in the ancestral vertebrates. Based on synteny and phylogenetic evidence, the present naming of the GAT group members does not accurately reflect the evolutionary relationships.