Transgenerational acclimation to acidified seawater and gene expression patterns in a sea urchin.

Transgenerational responses of susceptible calcifying organisms to progressive ocean acidification are an important issue in reducing uncertainty of future predictions. In this study, a two-generation rearing experiment was conducted using mature Mesocentrotus nudus, a major edible sea urchin that occurs along the coasts of northern Japan. Morphological observations and comprehensive gene expression analysis (RNA-seq) of resulting larvae were performed to examine transgenerational acclimation to acidified seawater. Two generations of rearing experiments showed that larvae derived from parents acclimated to acidified seawater tended to have higher survival and show less reduction in body size when exposed to acidified seawater of the same pH, suggesting that a positive carry-over effect occurred. RNA-seq analysis showed that gene expression patterns of larvae originated from both acclimated and non-acclimated parents to acidified seawater tended to be different than control condition, and the gene expression pattern of larvae originated from acclimated parents was substantially different than that of larvae of non-acclimated and control parents.

Development of a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry for human urine (RaDPi-U).

Drug screening tests are mandatory in the search for drugs in forensic biological samples, and immunological methods and mass spectrometry (e.g., gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry) are commonly used for that purpose. However, these methods have some drawbacks, and developing new screening methods is required. In this study, we develop a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry (PESI-MS/MS), which is an ambient ionization mass spectrometry method, for human urine, named RaDPi-U. RaDPi-U is carried out in three steps: (1) mixing urine with internal standard (IS) solution and ethanol, followed by vortexing; (2) pipetting the mixture onto a sample plate for PESI; and (3) rapid-fire analysis by PESI-MS/MS. RaDPi-U targets 40 forensically important drugs, which include illegal drugs, hypnotics, and psychoactive substances. The analytical results were obtained within 3 min because of the above-mentioned simple workflow of RaDPi-U. The calibration curves of each analyte were constructed using the IS method, and they were quantitatively valid, resulting in good linearity (0.972-0.999) with a satisfactory lower limit of detection and lower limit of quantitation (0.01-7.1 ng/mL and 0.02-21 ng/mL, respectively). Further, both trueness and precisions were 28% or less, demonstrating the high reliability and repeatability of the method. Finally, we applied RaDPi-U to three postmortem urine specimens and successfully detected different drugs in each urine sample. The practicality of the method is proven, and RaDPi-U will be a strong tool as a rapid-fire drug screening method not only in forensic toxicology but also in clinical toxicology.

Single-polyp metabolomics for coral health assessment.

Coral reef ecosystems supported by environmentally sensitive reef-building corals face serious threats from human activities. Our understanding of these reef threats is hampered by the lack of sufficiently sensitive coral environmental impact assessment systems. In this study, we established a platform for metabolomic analysis at the single-coral-polyp level using state-of-the-art mass spectrometry (probe electrospray ionization/tandem mass spectrometry; PESI/MS/MS) capable of fine-scale analysis. We analyzed the impact of the organic UV filter, benzophenone (BP), which has a negative impact on corals. We also analyzed ammonium and nitrate samples, which affect the environmental sensitivity of coral-zooxanthella (Symbiodiniaceae) holobionts, to provide new insights into coral biology with a focus on metabolites. The method established in this study breaks new ground by combining PESI/MS/MS with a technique for coral polyps that can control the presence or absence of zooxanthellae in corals, enabling functions of zooxanthellae to be assessed on a polyp-by-polyp basis for the first time. This system will clarify biological mechanisms of corals and will become an important model system for environmental impact assessment using marine organisms.

Metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer.

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in material cycling in various aquatic ecosystems, our current understanding of the microbial community in groundwater ecosystems is remarkably insufficient. To assess the groundwater in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. 16S rRNA amplicon analysis showed that bacteria accounted for 94-98% of the microbial communities, which included archaea at all three sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes such as narG and napA than the other two sites. This site was thought to have undergone denitrification that was driven by high quantities of dissolved organic carbon (DOC). In contrast, site 2 was characterized by a high nitrate-nitrogen (NO3-N) content and a low amount of DOC, and this site yielded a moderate amount of denitrification genes. Site 1 showed markedly low amounts of all nitrogen metabolism genes. Overall, nitrogen metabolism in the Ryukyu limestone aquifer was found to change based on environmental factors.

Whole transcriptome analysis of demersal fish eggs reveals complex responses to ocean deoxygenation and acidification.

Ocean acidification and deoxygenation co-occur in marine environments, causing deterioration of marine ecosystems. However, effects of compound stresses on marine organisms and their physiological coping mechanisms are largely unknown. Here, we show how high pCO2 and low dissolved oxygen (DO) cause transcriptomic changes in eggs of a demersal fish (Sillago japonica), which are fully exposed to such stresses in natural environment. Overall gene expression was affected more strongly by low DO than by high pCO2. Enrichment analysis detected significant stress responses such as glycolytic processes in response to low DO. Increased expression of a group of glycolytic genes under low DO conditions is presumably because oxygen depletion disables the electron transfer pathway, complementing ATP production in the glycolytic pathway. Contrary to expectations, apparent mitigation of gene expression changes was dominant under combined stress conditions, and may represent an innate fish adaptive trait for severe environments.

Methane diffusion affects characteristics of benthic communities in and around microbial mat-covered sediments in the northeastern Japan sea.

We investigated relationships between features of benthic macrofaunal communities and geochemical parameters in and around microbial mat-covered sediments associated with a methane seepage on Sakata Knoll in the eastern Japan Sea. A depression on top of the knoll corresponds to a gas-hydrate-bearing area with seepage of methane-rich fluid, and microbial mats cover the seafloor sediments. Sediment cores were collected at three sites for this study: one within a microbial mat, a second a few meters outside of the microbial mat, and a third from a reference site outside the gas-hydrate-bearing areas. Morphological analysis showed that the site inside the microbial mat had higher macrofaunal density and biomass compared with the other sites. 18S rRNA gene analysis showed that annelids were dominant in the surface sediment inside the microbial mat with the possible occurrence of microbial anaerobic oxidation of methane (AOM), whereas in the surface sediments outside the microbial mat and at the reference site the predominant species belonged to phylum Cercozoa. Morphological analysis also showed that the surface sediment inside the microbial mat noticeably favored annelids, with dorvilleid Ophryotrocha sp. and ampharetid Neosabellides sp. identified as major constituents. Statistical analysis showed that sulfidic sediment conditions with concentrations of H2S up to 121 µM resulting from AOM likely resulted in the predominance of annelids with tolerance to sulfide. Both the 18S rRNA genes and macrofaunal characteristics showed that benthic biodiversity among the three sites was greatest outside the microbial mat. The site outside the microbial mat may represent geochemical transition conditions, including a lower rate of upward methane gas-flow compared with the site inside the microbial mat. The high biodiversity there might result from the presence of species specifically suited to the transition zone as well as species also found in photosynthesis-based communities of the background environment.

Separate and combined effects of elevated pCO2 and temperature on the branching reef corals Acropora digitifera and Montipora digitata.

Ocean acidification (OA) and warming (OW) are major global threats to coral reef ecosystems; however, studies on their combined effects (OA + OW) are scarce. Therefore, we evaluated the effects of OA, OW, and OA + OW in the branching reef corals Acropora digitifera and Montipora digitata, which have been found to respond differently to environmental changes. Our results indicate that OW has a greater impact on A. digitifera and M. digitata than OA and that the former species is more vulnerable to OW than the latter. OW was the main stressor for increased mortality and decreased calcification in the OA + OW group, and the effect of OA + OW was additive in both species. Our findings suggest that the relative abundance and cover of M. digitata are expected to increase whereas those of A. digitifera may decrease in the near future in Okinawa.

Preliminary study on the acute effects of hydrogen sulfide on Amphipoda (Lysianassoidea; Pseudorchomene sp. and Anonyx sp.) collected from deep-sea floors in the Sea of Japan.

To study the environmental impact of the assessment technologies for the development of shallow methane hydrate zones in the Sea of Japan, deep-sea amphipods (Pseudorchomene sp. and Anonyx sp.) were collected from a depth of approximately 1000 m and were tested for H2S toxicity. At 0.57 mg L-1 H2S, all specimens of Pseudorchomene sp. were dead after 96 h, whereas all individuals survived at 0.18 mg L-1. Moreover, Anonyx sp. had a survival rate of 17 % after 96 h at 0.24 mg L-1. A similar toxicity test was conducted with the coastal amphipod Merita sp., a detritivore, and all individuals died within 24 h at 0.15 mg L-1. These results suggested that compared with coastal detritivorous amphipods, deep-sea detritivorous amphipods, which also live near biomats with sediment H2S concentrations exceeding 10 mg L-1, showed a higher tolerance to H2S.

A new hexactinellid-sponge-associated zoantharian (Porifera, Hexasterophora) from the northwestern Pacific Ocean.

Symbiotic associations between zoantharians and sponges can be divided into two groups: those that associate with Demospongiae and those that associate with Hexactinellida. Parachurabanashinseimaruae Kise, gen. nov. et sp. nov., a new genus and a new species of Hexactinellida-associated zoantharian from Japanese waters, is described. It is characterized by a combination of the following: i) its host hexactinellid sponge, ii) very flat polyps, iii) cteniform endodermal marginal muscles, and iv) characteristic mutations in three mitochondrial regions (including a unique 26-bp deletion in 16S ribosomal DNA) and three nuclear regions. Parachurabanashinseimaruae Kise, gen. nov. et sp. nov. is the third genus in the family Parazoanthidae that is reported to be associated with Hexasterophora sponges. Although specimens have so far only been collected on Takuyo-Daigo Seamount off Minami-Torishima Island in Japan, unidentified zoantharians of similar description have been reported from the waters around Australia, indicating that the species might be widespread across the Pacific.

Microbial mat compositions and localization patterns explain the virulence of black band disease in corals.

Black band disease (BBD) in corals is characterized by a distinctive, band-like microbial mat, which spreads across the tissues and often kills infected colonies. The microbial mat is dominated by cyanobacteria but also commonly contains sulfide-oxidizing bacteria (SOB), sulfate-reducing bacteria (SRB), and other microbes. The migration rate in BBD varies across different environmental conditions, including temperature, light, and pH. However, whether variations in the migration rates reflect differences in the microbial consortium within the BBD mat remains unknown. Here, we show that the micro-scale surface structure, bacterial composition, and spatial distribution differed across BBD lesions with different migration rates. The migration rate was positively correlated with the relative abundance of potential SOBs belonging to Arcobacteraceae localized in the middle layer within the mat and negatively correlated with the relative abundance of other potential SOBs belonging to Rhodobacteraceae. Our study highlights the microbial composition in BBD as an important determinant of virulence.

Novel quantitative method for individual isotopomer of organic acids from 13C tracer experiments determines carbon flow in acetogenesis.

Anaerobic microbial acetogenesis is ubiquitous on Earth, and thus plays an important role in the global carbon cycle. The mechanism of carbon fixation in acetogens has attracted great interest from various studies for combatting climate change, and even for studying ancient metabolic pathways. Here, we developed a new, simple method for investigating carbon flows in the metabolic reaction of acetogen by conveniently and accurately determining the relative abundance of individual acetate- and/or formate-isotopomers formed in 13C labeling experiments. We measured the underivatized analyte by gas chromatography-mass spectrometry (GC-MS) coupled with a direct aqueous sample injection technique. The individual abundance of analyte isotopomers was calculated by the mass spectrum analysis using the least-squares approach. The validity of the method was demonstrated by determining known mixtures of unlabeled and 13C-labeled analytes. The developed method was applied to study the carbon fixation mechanism of the well-known acetogen Acetobacterium woodii grown on methanol and bicarbonate. We provided a quantitative reaction model for methanol metabolism of A. woodii, which indicated that methanol was not the sole carbon precursor of the acetate methyl group and that 20-22% of the methyl group was formed from CO2. In contrast, the carboxyl group of acetate appeared to form exclusively by CO2 fixation. Thus, our simple method, without laborious analytical procedures, has broad utility for the study of biochemical and chemical processes related to acetogenesis on Earth.

Genetic population structures of common scavenging species near hydrothermal vents in the Okinawa Trough.

Deep-sea mining of hydrothermal deposits off the coast of Japan is currently under consideration, and environmental baseline studies of the area are required to understand possible impacts. The aim of this study is to clarify population structures of dominant benthic megafaunal species near hydrothermal vent fields in the Okinawa Trough, using a population genetics approach. We examined dominant deep-sea scavenging species including eels, several amphipods, and a decapod and performed population genetic analyses based on the mitochondrial cytochrome c oxidase subunit I region. Several sites were sampled within Okinawa Trough to examine intra-population diversity while two other locations 1400-2400 km away were chosen for inter-population comparisons. For synaphobranchid eels Simenchelys parasitica and Synaphobranchus kaupii, our results showed significant intra-population diversity but no inter-population genetic differentiation, suggesting strong genetic connectivity and/or large population sizes. In addition, single nucleotide polymorphism analysis also confirmed strong genetic connectivity for Simenchelys parasitica. Among scavenging amphipods, we detected seven putative species using molecular phylogenetic analysis. We evaluated population structures of the most abundant species of amphipods and a decapod species (Nematocarcinus lanceopes). Our results provide basic information on the genetic population structures of benthic megafaunal species near hydrothermal vent fields, which can be used to select candidate species for future connectivity analysis with high-resolution genetic markers and aid understanding of the potential population impacts of environmental disturbances.

Evolutionary patterns of host switching, lifestyle mode, and the diversification history in symbiotic zoantharians.

Symbioses play important roles in forming the structural and distributional patterns of marine diversity. Understanding how interspecies interactions through symbioses contribute to biodiversity is an essential topic. Host switching has been considered as one of the main drivers of diversification in symbiotic systems. However, its process and patterns remain poorly investigated in the marine realm. Hexacoral species of the order Zoantharia (=zoantharians) are often epizoic on other marine invertebrates and generally use specific taxa as hosts. The present study investigates the patterns of host switching and the diversification history of zoantharians based on the most comprehensive molecular phylogenetic analyses to date, using sequences from three mitochondrial and three nuclear markers from representatives of 27 of 29 genera. Our results indicate that symbiotic zoantharians, in particular those within suborder Macrocnemina, diversified through repeated host switching. In addition, colonization of new host taxa appears to have driven morphological and ecological specialization in zoantharians. These findings have important implications for understanding the role of symbioses in the morphological and ecological evolution of marine invertebrates.

Short-term improvement of heat tolerance in naturally growing Acropora corals in Okinawa.

Mass bleaching and subsequent mortality of reef corals by heat stress has increased globally since the late 20th century, due to global warming. Some experimental studies have reported that corals may increase heat tolerance for short periods, but only a few such studies have monitored naturally-growing colonies. Therefore, we monitored the survival, growth, and bleaching status of Acropora corals in fixed plots by distinguishing individual colonies on a heat-sensitive reef flat in Okinawa, Japan. The level of heat stress, assessed by the modified version of degree heating week duration in July and August, when the seawater temperature was the highest, was minimally but significantly higher in 2017 than in 2016; however, the same colonies exhibited less bleaching and mortality in 2017 than in 2016. Another study conducted at the same site showed that the dominant unicellular endosymbiotic algal species did not change before and after the 2016 bleaching, indicating that shifting and switching of the Symbiodiniaceae community did not contribute to improved heat tolerance. Colonies that suffered from partial mortality in 2016 were completely bleached at higher rates in 2017 than those without partial mortality in 2016. The present results suggest that either genetic or epigenetic changes in coral hosts and/or algal symbionts, or the shifting or switching of microbes other than endosymbionts, may have improved coral holobiont heat tolerance.

Preliminary study to classify mechanisms of mitochondrial toxicity by in vitro metabolomics and bioinformatics.

AIM: Mitochondrial toxicity is one of the causes for drug-induced liver injury, and the classification of phenotypes or mitochondrial toxicity are highly required though there are no molecular-profiling approaches for classifying mitochondrial toxicity. Therefore, the aim of this study was to classify the mechanisms of mitochondrial toxicity by metabolic profiling in vitro and bioinformatics. MAIN METHODS: We applied an established gas chromatography tandem mass spectrometry-based metabolomics to human hepatoma grade 2 (HepG2) cells that were exposed to mitochondrial toxicants, whose mechanisms are different, such as rotenone (0.1 µM), carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 0.5 µM), nefazodone (20 µM), perhexiline (6.25 µM), or digitonin (positive cytotoxic substance, 4 µM). These concentrations were determined by the Mitochondrial ToxGlo Assay. Galactose medium was used for suppressing the Warburg effect in HepG2 cells, and the metabolome analysis successfully identified 125 metabolites in HepG2 cells. Multivariate, metabolic pathway and network analyses were performed by the R software. KEY FINDINGS: Metabolic profiling enabled the classifying the mitochondrial toxicity mechanisms of RCC inhibition and uncoupling. The metabolic profiles of respiratory chain complex (RCC) inhibitors (rotenone and nefazodone) and an uncoupler (CCCP) were fully differentiated from those of other compounds. The metabolic pathway analysis revealed that the RCC inhibitors and the uncoupler mainly disrupted TCA-cycle and related metabolic pathways. In addition, the correlation-based network analysis revealed that succinic acid, ß-alanine, and glutamic acid were potential metabolic indicators for RCC inhibition and uncoupling. SIGNIFICANCE: Our results provided new insights into classifying mechanisms of mitochondrial toxicity by in vitro metabolomics.

Exploring the trends of adaptation and evolution of sclerites with regards to habitat depth in sea pens.

Octocorals possess sclerites, small elements comprised of calcium carbonate (CaCO3) that are important diagnostic characters in octocoral taxonomy. Among octocorals, sea pens comprise a unique order (Pennatulacea) that live in a wide range of depths. Habitat depth is considered to be important in the diversification of octocoral species, but a lack of information on sea pens has limited studies on their adaptation and evolution across depth. Here, we aimed to reveal trends of adaptation and evolution of sclerite shapes in sea pens with regards to habitat depth via phylogenetic analyses and ancestral reconstruction analyses. Colony form of sea pens is suggested to have undergone convergent evolution and the loss of axis has occurred independently across the evolution of sea pens. Divergences of sea pen taxa and of sclerite forms are suggested to depend on habitat depths. In addition, their sclerite forms may be related to evolutionary history of the sclerite and the surrounding chemical environment as well as water temperature. Three-flanged sclerites may possess the tolerance towards the environment of the deep sea, while plate sclerites are suggested to be adapted towards shallower waters, and have evolved independently multiple times. The common ancestor form of sea pens was predicted to be deep-sea and similar to family Pseudumbellulidae in form, possessing sclerites intermediate in form to those of alcyonaceans and modern sea pens such as spindles, rods with spines, and three-flanged sclerites with serrated edges sclerites, as well as having an axis and bilateral traits.

Metabolomics and Data-Driven Bioinformatics Revealed Key Maternal Metabolites Related to Fetal Lethality via Di(2-ethylhexyl)phthalate Exposure in Pregnant Mice.

We performed serum metabolome analysis of di(2-ethylhexyl)phthalate (DEHP)-exposed and control pregnant mice. Pregnant mice (n = 5) were fed a DEHP-containing diet (0.1% or 0.2% DEHP) or a normal diet (control) from gestational days 0-18. After maternal exposure to 0.2% DEHP there were no surviving fetuses, indicating its strong fetal lethality. There were no significant differences in the numbers of fetuses and placentas between the 0.1% DEHP and control groups, although fetal viability differed significantly between them, suggesting that maternal exposure to 0.1% DEHP could inhibit fetal growth. Metabolomics successfully detected 169 metabolites in serum. Principal component analysis (PCA) demonstrated that the three groups were clearly separated on PCA score plots. The biological interpretation of PC1 was fetal lethality, whereas PC2 meant metabolic alteration of pregnant mice via DEHP exposure without fetal lethality. In particular, the first component was significantly correlated with fetal viability, demonstrating that maternal metabolome changes via DEHP exposure were strongly related to fetal lethality. Levels of some amino acids were significantly increased in the DEHP-exposed groups, whereas those of some fatty acids, nicotinic acid, and 1,5-anhydroglucitol were significantly decreased in the DEHP groups. DEHP-induced increases in glycine levels could cause fetal neurological disorders, and decreases in nicotinic acid could inhibit fetal growth. In addition, a machine-learning Random forest could determine 16 potential biomarkers of DEHP exposure, and data-driven network analysis revealed that nicotinic acid was the most influential hub metabolite in the metabolic network. These findings will be useful for understanding the effects of DEHP on the maternal metabolome in pregnancy and their relationship to fetal lethality.

Coastal ecological impacts from pumice rafts.

An explosive volcanic eruption occurred in the Ogasawara Islands on 13-15 August 2021, bringing unprecedented amounts of floating pumice to the coast of Okinawa Island in the Ryukyu Archipelago, 1300 km west of the volcano, approximately 2 months later. The coast of Okinawa Island, especially along the northern part, is home to many typical subtropical seascapes, including coral reefs and mangrove forests, so the possible impact of the large amount of pumice is attracting attention. Here, we report early evidence of ecosystem changes as a result of large-scale pumice stranding on coastal beaches, in estuaries and mangrove forests and passage across fringing coral reefs. Massive pumice drifts are major obstacles to fishing activities and ship traffic, but short and long-term changes in coastal ecosystems can also occur. The phenomena observed on Okinawa Island can be a preview of coastal impacts for the Kyushu, Shikoku, Honshu Islands, where pumice has subsequently washed ashore.

Calcification rates of a massive and a branching coral species were unrelated to diversity of endosymbiotic dinoflagellates.

BACKGROUND: To explore the possibility that endosymbiotic dinoflagellates (Symbiodiniaceae) are associated with coral calcification rates, we investigated the diversity of symbiotic algae in coral colonies with different calcification rates within massive and branching corals (Porites australiensis and Acropora digitifera). METHODS AND RESULTS: Genotyping symbiotic algae from colonies with different calcification rates revealed that all the colonies of both species harbored mainly Cladocopium (previously clade C of Symbiodinium). The Cladocopium symbionts in P. australiensis were mainly composed of C15 and C15bn, and those in A. digitifera of C50a and C50c. We did not detect clear relationships between symbiont compositions and calcification rates within the two coral species. CONCLUSIONS: Our results suggest that different coral calcification rates within species may be attributed to genetic factors of coral hosts themselves and/or within symbiont genotypes.

Visualisation of Phosphate in Subcalicoblastic Extracellular Calcifying Medium and on a Skeleton of Coral by Using a Novel Probe, Fluorescein-4-Isothiocyanate-Labelled Alendronic Acid.

The overload of nutrients of anthropogenic origin, including phosphate, onto coastal waters has been reported to have detrimental effects on corals. However, to the best of our knowledge, the phosphate concentration threshold for inhibiting coral calcification is unclear owing to a lack of information on the molecular mechanisms involved in the inhibitory effect of phosphate. Therefore, in this study, we prepared a new phosphate analogue, fluorescein-4-isothiocyanate (FITC)-labelled alendronic acid (FITC-AA), from commercially available reagents and used it as a novel probe to demonstrate its transfer pathway from ambient seawater into Acropora digitifera. When the juveniles at 1 d post-settlement were treated with FITC-AA in a laboratory tank, this phosphate analogue was found in the subcalicoblastic extracellular calcifying medium (SCM) and was absorbed on the basal plate in the juveniles within a few minutes. When the juveniles bear zooxanthellae at 3 months post-settlement, FITC-AA was observed on the corallite walls within a few minutes after adding ambient seawater. We concluded that FITC-AA in ambient seawater was transferred via a paracellular pathway to SCM and then absorbed on the coral CaCO3 skeletons because FITC-AA with a high polarity group cannot permeate through cell membranes.