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.

Mechanism of denitrification in subsurface-dammed Ryukyu limestone aquifer, southern Okinawa Island, Japan.

Denitrification crucially regulates the attenuation of groundwater nitrate and is unlikely to occur in a fast-flowing aquifer such as the Ryukyu limestone aquifer in southern Okinawa Island, Japan. However, evidences of denitrification have been observed in several wells within this region. This study analyzed environmental isotopes (δ15NNO3 and ẟ18ONO3) to derive the rationale for denitrification at this site. Additionally, the presence of two subsurface dams in the study area may influence the processes involved in nitrate attenuation. Herein, we analyzed 150 groundwater samples collected spatially and seasonally to characterize the variations in the groundwater chemistry and stable isotopes during denitrification. The values of δ15NNO3 and δ18ONO3 displayed a progressive trend up to +59.7 ‰ and + 21 ‰, respectively, whereas the concentrations of NO3--N decreased to 0.1 mg L-1. In several wells, the enrichment factors of δ15NNO3 ranged from -6.6 to -2.1, indicating rapid denitrification, and the δ15NNO3 to δ18ONO3 ratios varied from 1.3:1 to 2:1, confirming the occurrence of denitrification. Denitrification intensively proceeds under conditions of depleted dissolved oxygen concentrations (<2 mg L-1), sluggish groundwater flow with longer residence times, high concentrations of dissolved organic carbon (>1.2 mg L-1), and low groundwater levels during the dry season with precipitation rates of <100 mm per month (Jun-Sep). SF6 analysis indicated the exclusive occurrence of denitrification in specific wells with groundwater residence times exceeding 30 years. These wells are located in close proximity to the major NE-SW fault system in the Komesu area, where the hydraulic gradient was below 0.005. Detailed geological and lithological investigations based on borehole data revealed that subsurface dams did not cause denitrification while the major NE-SW fault system uplifted the impermeable basement rock of the Shimajiri Group, creating a lithological gap at an equivalent depth that ultimately formed a sluggish groundwater area, promoting denitrification.

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.

Phosphate bound to calcareous sediments hampers skeletal development of juvenile coral.

To test the hypothesis that terrestrial runoff affects the functions of calcareous sediments in coral reefs and hampers the development of corals, we analysed calcareous sediments with different levels of bound phosphate, collected from reef areas of Okinawajima, Japan. We confirmed that phosphate bound to calcareous sediments was readily released into ambient seawater, resulting in much higher concentrations of phosphorous in seawater from heavily polluted areas (4.3-19.0 µM as compared with less than 0.096 µM in natural ambient seawater). Additionally, we examined the effect of phosphate released from calcareous sediments on the development of Acropora digitifera coral juveniles. We found that high phosphate concentrations in seawater clearly inhibit the skeletal formation of coral juveniles. Our results demonstrate that calcareous sediments in reef areas play a crucial role in mediating the impact of terrestrial runoff on corals by storing and releasing phosphate in seawater.

N-Acetyl-d-Glucosamine-Binding Lectin in Acropora tenuis Attracts Specific Symbiodiniaceae Cell Culture Strains.

Many corals establish symbiosis with Symbiodiniaceae cells from surrounding environments, but very few Symbiodiniaceae cells exist in the water column. Given that the N-acetyl-d-glucosamine-binding lectin ActL attracts Symbiodiniaceae cells, we hypothesized that corals must attract Symbiodiniaceae cells using ActL to acquire them. Anti-ActL antibody inhibited acquisition of Symbiodiniaceae cells, and rearing seawater for juvenile Acropora tenuis contained ActL, suggesting that juvenile A. tenuis discharge ActL to attract these cells. Among eight Symbiodiniaceae cultured strains, ActL attracted NBRC102920 (Symbiodinium tridacnidorum) most strongly followed by CS-161 (Symbiodinium tridacnidorum), CCMP2556 (Durusdinium trenchii), and CCMP1633 (Breviolum sp.); however, it did not attract GTP-A6-Sy (Symbiodinium natans), CCMP421 (Effrenium voratum), FKM0207 (Fugacium sp.), and CS-156 (Fugacium sp.). Juvenile polyps of A. tenuis acquired limited Symbiodiniaceae cell strains, and the number of acquired Symbiodiniaceae cells in a polyp also differed from each other. The number of Symbiodiniaceae cells acquired by juvenile polyps of A. tenuis was correlated with the ActL chemotactic activity. Thus, ActL could be used to attract select Symbiodiniaceae cells and help Symbiodiniaceae cell acquisition in juvenile polyps of A. tenuis, facilitating establishment of symbiosis between A. tenuis and Symbiodiniaceae cells.

Comparison of Symbiodiniaceae diversities in different members of a Palythoa species complex (Cnidaria: Anthozoa: Zoantharia)-implications for ecological adaptations to different microhabitats.

In this study we compared genotypes of zoantharian host-associating algal symbionts among Palythoa species, which are among the dominant benthic reef organisms in the Ryukyu Archipelago, Japan, and evaluated Symbiodiniaceae diversities of closely related congeneric Palythoa species. We targeted a species complex of the zoantharian genus Palythoa (P. tuberculosa, P. sp. yoron, P. mutuki) living among different microhabitats in a narrow reef area of Tokunoshima Island. For phylogenetic analyses, we used two DNA marker regions; nuclear internal transcribed spacer (ITS) and plastid mini-circle non-coding region (psbAncr), both of which have previously been used to determine Symbiodiniaceae genotypes of zoantharian species. Our results showed that all Palythoa species hosted symbionts of the genus Cladocopium, with genotypic compositions of this genus showing some variations among the three different Palythoa species. Additionally, we found that the Cladocopium genotypic composition was statistically different among Palythoa species, and among P. tuberculosa specimens in different microhabitats. Our results suggest that ecological divergence among these three Palythoa species may be related to differing Symbiodiniaceae diversities that may in turn contribute to eco-physiological adaptation into different microhabitats on coral reefs.

Phosphate Enrichment Hampers Development of Juvenile Acropora digitifera Coral by Inhibiting Skeleton Formation.

Coral reef degradation due to various local stresses, such as nutrient enrichment and terrestrial run-off into coastal waters, is an increasing global concern. Inorganic phosphates have been considered to possibly inhibit skeleton formation in corals. Despite many studies available on the effects of nutrients on corals, a clear consensus on how nutrients exert deteriorative effects on corals has not been established satisfactorily. In this study, we examined the effects of phosphates and nitrates on in vitro aragonite CaCO3 formation by using biogenic polyamines and in vivo aragonite formation in the skeleton of juvenile Acropora digitifera corals. We showed that the phosphates at similar concentrations clearly inhibited both in vitro and in vivo CaCO3 formation. In contrast, nitrates inhibited neither in vitro aragonite CaCO3 formation nor in vivo aragonite formation in juvenile coral skeleton. Furthermore, our findings showed that inhibition of coral skeleton formation was due to absorption of phosphate on the skeleton, which inorganically inhibited normal development of juvenile coral skeleton.

Effects of moderate thermal anomalies on Acropora corals around Sesoko Island, Okinawa.

Over the past several decades, coral reef ecosystems have experienced recurring bleaching events. These events were predominantly caused by thermal anomalies, which vary widely in terms of severity and spatio-temporal distribution. Acropora corals, highly prominent contributors to the structural complexity of Pacific coral reefs, are sensitive to thermal stress. Response of Acropora corals to extremely high temperature has been well documented. However, studies on the effects of moderately high temperature on Acropora corals are limited. In the summer of 2016, a moderate coral bleaching event due to moderately high temperature was observed around Sesoko Island, Okinawa, Japan. The objective of this study was to examine thermal tolerance patterns of Acropora corals, across reefs with low to moderate thermal exposure (degree heating weeks ~2-5°C week). Field surveys on permanent plots were conducted from October 2015 to April 2017 to compare the population dynamics of adult Acropora corals 6 months before and after the bleaching events around Sesoko Island. Variability in thermal stress response was driven primarily by the degree of thermal stress. Wave action and turbidity may have mediated the thermal stress. Tabular and digitate coral morphologies were the most tolerant and susceptible to thermal stress, respectively. Growth inhibition after bleaching was more pronounced in the larger digitate and corymbose coral morphologies. This study indicates that Acropora populations around Sesoko Island can tolerate short-term, moderate thermal challenges.

Epiregulin-blocking antibody inhibits epiregulin-dependent EGFR signaling.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in many cellular functions including cell growth and migration. EGFR may be activated by EGF family ligands such as EGF and epiregulin (EREG). EREG is overexpressed in human colon and breast cancers, implying that EREG plays roles in tumorigenesis. Although EGF family members share a receptor, it is not well known whether their signaling pathways differ. In order to investigate EREG signaling, we established the anti-EREG antibody that inhibits EGFR downstream signaling stimulated by EREG but not by EGF. While the anti-EREG antibody has little effect on cell growth, it inhibits cell adhesion of EREG-expressing autocrine cancer cell lines. Our results suggest that anti-EREG antibodies represent valuable tools for elucidating EREG-specific signaling pathways, and may serve as therapeutic candidates for the treatment of cancers.

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES.

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro(103). A molecular dynamics simulation and mutational analyses revealed that Arg(40) in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.

Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies.

Growth factors are implicated in several processes essential for cancer progression. Specifically, epidermal growth factor (EGF) family members, including epiregulin (EREG), are important prognostic factors in many epithelial cancers, and treatments targeting these molecules have recently become available. Here, we constructed and expressed humanized anti-EREG antibodies by variable domain resurfacing based on the three-dimensional (3D) structure of the Fv fragment. However, the initial humanized antibody (HM0) had significantly decreased antigen-binding affinity. Molecular modeling results suggested that framework region (FR) residues latently important to antigen binding included residue 49 of the light chain variable region (VL). Back mutation of the VL49 residue (tyrosine to histidine) generated the humanized version HM1, which completely restored the binding affinity of its murine counterpart. Importantly, only one mutation in the framework may be necessary to recover the binding capability of a humanized antibody. Our data support that HM1 exerts potent antibody-dependent cellular cytotoxicity (ADCC). Hence, this antibody may have potential for further development as a candidate therapeutic agent and research tool.

Nasal allergy-like symptoms aggravated by ozone exposure in a concentration-dependent manner in guinea pigs.

Our previous study revealed that exposure to 0.4 ppm ozone (O(3)) enhanced nasal allergy-like reactions in guinea pigs. In the present study, we investigated the concentration-dependency of the effects of exposure to O(3) on the aggravation of nasal allergy-like reactions induced by repeated nasal administration of antigen. Guinea pigs were exposed to filtered air or 0.1-0.6 ppm O(3) for 5 weeks. After each weekly administration of ovalbumin (OVA), sneezes and nasal secretions were measured. The number of eosinophils infiltrating the nasal septum and the titers of OVA-specific antibody were measured 24h after the last administration. Ozone increased sneezing and nasal secretion induced by OVA, nasal responsiveness to physical stimuli, and the number of infiltrating eosinophils in a concentration-dependent manner. The titer of anti-OVA-IgG was increased in animals exposed to 0.6 ppm O(3). Thus, exposure to O(3) aggravated nasal allergy-like symptoms concentration dependently. The aggravation was caused by induction of nasal hyperresponsiveness, the infiltration of eosinophils, and the increase in the production of anti-OVA-IgG. The estimated maximum likelihood estimation concentrations (MLECs) and bench mark concentrations (BMCs) of O(3) for these indices were in the range of 0.09-0.18 and 0.02-0.06 ppm, respectively.