Prokaryotic communities of the French Polynesian sponge Dactylospongia metachromia display a site-specific and stable diversity during an aquaculture trial.

Dynamics of microbiomes through time are fundamental regarding survival and resilience of their hosts when facing environmental alterations. As for marine species with commercial applications, such as marine sponges, assessing the temporal change of prokaryotic communities allows us to better consider the adaptation of sponges to aquaculture designs. The present study aims to investigate the factors shaping the microbiome of the sponge Dactylospongia metachromia, in a context of aquaculture development in French Polynesia, Rangiroa, Tuamotu archipelago. A temporal approach targeting explants collected during farming trials revealed a relative high stability of the prokaryotic diversity, meanwhile a complementary biogeographical study confirmed a spatial specificity amongst samples at different longitudinal scales. Results from this additional spatial analysis confirmed that differences in prokaryotic communities might first be explained by environmental changes (mainly temperature and salinity), while no significant effect of the host phylogeny was observed. The core community of D. metachromia is thus characterized by a high spatiotemporal constancy, which is a good prospect for the sustainable exploitation of this species towards drug development. Indeed, a microbiome stability across locations and throughout the farming process, as evidenced by our results, should go against a negative influence of sponge translocation during in situ aquaculture.

Metallic trace element contamination of the giant clam Tridacna maxima in French Polynesia.

This study is a first assessment of Metallic Trace Elements (MTE) contamination of four lagoons in French Polynesia, chosen because their main anthropic uses are potential sources of MTE (military, agriculture, maritime). We used the giant clam Tridacna maxima as a biointegrator of fifteen MTE to assess the environmental footprint of human activities. The study of organotropism showed that not only the kidney but also the gonad and digestive system (GDS) are accumulator organs of interest for ETM biomonitoring. This result confirms that heterotrophic feeding is the pathway of ETM accumulation. Here, T. maxima appeared to be a super-accumulator of Ni (2700 ppm.dw), but also revealed at Tubuai (As, Fe, Mn) and Arutua (Cd, Zn) the environmental footprint of agricultural and maritime activities. The concentration of Cd recorded in Arutua (5 ppm.dw) both in KID and GDS, also underline the need for better control of effluents resulting from human activities.

Effects of elevated temperature and pCO2 on the respiration, biomineralization and photophysiology of the giant clam Tridacna maxima.

Many reef organisms, such as the giant clams, are confronted with global change effects. Abnormally high seawater temperatures can lead to mass bleaching events and subsequent mortality, while ocean acidification may impact biomineralization processes. Despite its strong ecological and socio-economic importance, its responses to these threats still need to be explored. We investigated physiological responses of 4-year-old Tridacna maxima to realistic levels of temperature (+1.5°C) and partial pressure of carbon dioxide (pCO2) (+800 µatm of CO2) predicted for 2100 in French Polynesian lagoons during the warmer season. During a 65-day crossed-factorial experiment, individuals were exposed to two temperatures (29.2°C, 30.7°C) and two pCO2 (430 µatm, 1212 µatm) conditions. The impact of each environmental parameter and their potential synergetic effect were evaluated based on respiration, biomineralization and photophysiology. Kinetics of thermal and/or acidification stress were evaluated by performing measurements at different times of exposure (29, 41, 53, 65 days). At 30.7°C, the holobiont O2 production, symbiont photosynthetic yield and density were negatively impacted. High pCO2 had a significant negative effect on shell growth rate, symbiont photosynthetic yield and density. No significant differences of the shell microstructure were observed between control and experimental conditions in the first 29 days; however, modifications (i.e. less-cohesive lamellae) appeared from 41 days in all temperature and pCO2 conditions. No significant synergetic effect was found. Present thermal conditions (29.2°C) appeared to be sufficiently stressful to induce a host acclimatization response. All these observations indicate that temperature and pCO2 are both forcing variables affecting T. maxima's physiology and jeopardize its survival under environmental conditions predicted for the end of this century.

Influence of water temperature and food on the last stages of cultured pearl mineralization from the black-lip pearl oyster Pinctada margaritifera.

Environmental parameters, such as food level and water temperature, have been shown to be major factors influencing pearl oyster shell growth and molecular mechanisms involved in this biomineralization process. The present study investigates the effect of food level (i.e., microalgal concentration) and water temperature, in laboratory controlled conditions, on the last stages of pearl mineralization in order to assess their impact on pearl quality. To this end, grafted pearl oysters were fed at different levels of food and subjected to different water temperatures one month prior to harvest to evaluate the effect of these factors on 1) pearl and shell deposition rate, 2) expression of genes involved in biomineralization in pearl sacs, 3) nacre ultrastructure (tablet thickness and number of tablets deposited per day) and 4) pearl quality traits. Our results revealed that high water temperature stimulates both shell and pearl deposition rates. However, low water temperature led to thinner nacre tablets, a lower number of tablets deposited per day and impacted pearl quality with better luster and fewer defects. Conversely, the two tested food level had no significant effects on shell and pearl growth, pearl nacre ultrastructure or pearl quality. However, one gene, Aspein, was significantly downregulated in high food levels. These results will be helpful for the pearl industry. A wise strategy to increase pearl quality would be to rear pearl oysters at a high water temperature to increase pearl growth and consequently pearl size; and to harvest pearls after a period of low water temperature to enhance luster and to reduce the number of defects.

Consequences of an uncertain mass mortality regime triggered by climate variability on giant clam population management in the Pacific Ocean.

Despite actions to manage sustainably tropical Pacific Ocean reef fisheries, managers have faced failures and frustrations because of unpredicted mass mortality events triggered by climate variability. The consequences of these events on the long-term population dynamics of living resources need to be better understood for better management decisions. Here, we use a giant clam (Tridacna maxima) spatially explicit population model to compare the efficiency of several management strategies under various scenarios of natural mortality, including mass mortality due to climatic anomalies. The model was parameterized by in situ estimations of growth and mortality and fishing effort, and was validated by historical and new in situ surveys of giant clam stocks in two French Polynesia lagoons. Projections on the long run (100 years) suggested that the best management strategy was a decrease of fishing pressure through quota implementation, regardless of the mortality regime considered. In contrast, increasing the minimum legal size of catch and closing areas to fishing were less efficient. When high mortality occurred due to climate variability, the efficiency of all management scenarios decreased markedly. Simulating El Niño Southern Oscillation event by adding temporal autocorrelation in natural mortality rates increased the natural variability of stocks, and also decreased the efficiency of management. These results highlight the difficulties that managers in small Pacific islands can expect in the future in the face of global warming, climate anomalies and new mass mortalities.

Considering reefscape configuration and composition in biophysical models advance seascape genetics.

Previous seascape genetics studies have emphasized the role of ocean currents and geographic distances to explain the genetic structure of marine species, but the role of benthic habitat has been more rarely considered. Here, we compared the population genetic structure observed in West Pacific giant clam populations against model simulations that accounted habitat composition and configuration, geographical distance, and oceanic currents. Dispersal determined by geographical distance provided a modelled genetic structure in better agreement with the observations than dispersal by oceanic currents, possibly due to insufficient spatial resolution of available oceanographic and coastal circulation models. Considering both habitat composition and configuration significantly improved the match between simulated and observed genetic structures. This study emphasizes the importance of a reefscape genetics approach to population ecology, evolution and conservation in the sea.

Growth, Survival and Reproduction of the Giant Clam Tridacna maxima (Röding 1798, Bivalvia) in Two Contrasting Lagoons in French Polynesia.

Shell growth, reproduction, and natural mortality of the giant clam Tridacna maxima were characterized over a two-year-period in the lagoon of the high island of Tubuai (Austral Archipelago) and in the semi-closed lagoon of Tatakoto (Tuamotu Archipelago) in French Polynesia. We also recorded temperature, water level, tidal slope, tidal range, and mean wave height in both lagoons. Lower lagoon aperture and exposure to oceanic swells at Tatakoto than at Tubuai was responsible for lower lagoon water renewal, as well as higher variability in temperature and water level at Tatakoto across the studied period. These different environmental conditions had an impact on giant clams. Firstly, spawning events in the lagoon of Tatakoto, detected by gonad maturity indices in June and July 2014, were timed with high oceanic water inflow and a decrease in lagoon water temperature. Secondly, temperature explained differences in shell growth rates between seasons and lagoons, generating different growth curves for the two sites. Thirdly, local mortality rates were also found to likely be related to water renewal patterns. In conclusion, our study suggests that reef aperture and lagoon water renewal rates play an integral role in giant clam life history, with significant differences in rates of shell growth, mortality and fertility found between open versus semi-closed atoll lagoons in coral reef ecosystems.

Evidence of the bioaccumulation of ciguatoxins in giant clams (Tridacna maxima) exposed to Gambierdiscus spp. cells.

Ciguatera Fish Poisoning (CFP) is a foodborne disease classically related to the consumption of tropical coral reef fishes contaminated with ciguatoxins (CTXs), neurotoxins produced by dinoflagellates of the Gambierdiscus genus. Severe atypical ciguatera-like incidents involving giant clams, a marine resource highly consumed in the South Pacific, are also frequently reported in many Pacific Islands Countries and Territories. The present study was designed to assess the ability of giant clams to accumulate CTXs in their tissues and highlight the potential health risks associated with their consumption. Since giant clams are likely to be exposed to both free-swimming Gambierdiscus cells and dissolved CTXs in natural environment, ex situ contamination experiments were conducted as follows: giant clams were exposed to live or lyzed cells of TB92, a highly toxic strain of G. polynesiensis containing 5.83±0.85pg P-CTX-3C equiv.cell-1vs. HIT0, a weakly toxic strain of G. toxicus containing only (2.05±1.16)×10-3pg P-CTX-3C equiv.cell-1, administered over a 48h period at a concentration of 150cellsmL-1. The presence of CTXs in giant clams tissues was further assessed using the mouse neuroblastoma cell-based assay (CBA-N2a). Results showed that giant clams exposed to either lyzed or live cells of TB92 were able to bioaccumulate CTXs at concentrations well above the safety limit recommended for human consumption, i.e. 3.28±1.37 and 2.92±1.03ng P-CTX-3C equiv.g-1 flesh (wet weight), respectively, which represented approximately 3% of the total toxin load administered to the animals. In contrast, giant clams exposed to live or lyzed cells of HIT0 were found to be free of toxins, suggesting that in the nature, the risk of contamination of these bivalves is established only in the presence of highly toxic blooms of Gambierdiscus. Liquid chromatography-mass spectrometry (LC-MS/MS) analyses confirmed CBA-N2a results and also revealed that P-CTX-3B was the major CTX congener retained in the tissues of giant clams fed with TB92 cells. To the best of our knowledge, this study is the first to provide evidence of the bioaccumulation of Gambierdiscus CTXs in giant clams and confirms that these bivalve molluscs can actually constitute another pathway in ciguatera poisonings. While most monitoring programs currently focus on fish toxicity, these findings stress the importance of a concomitant surveillance of these marine invertebrates in applicable locations for an accurate assessment of ciguatera risk.

Effect of temperature, food availability, and estradiol injection on gametogenesis and gender in the pearl oyster Pinctada margaritifera.

The black-lip pearl oyster Pinctada margaritifera is a protandrous hermaphrodite species. Its economic value has led to the development of controlled hatchery reproduction techniques, although many aspects remain to be optimized. In order to understand reproductive mechanisms and their controlling factors, two independent experiments were designed to test hypotheses of gametogenesis and sex ratio control by environmental and hormonal factors. In one, pearl oysters were exposed under controlled conditions at different combinations of temperature (24 and 28°C) and food level (10,000 and 40,000 cells mL(-1) ); whereas in the other, pearl oysters were conditioned under natural conditions into the lagoon and subjected to successive 17ß-estradiol injections (100 µg per injection). Gametogenesis and sex ratio were assessed by histology for each treatment. In parallel, mRNA expressions of nine marker genes of the sexual pathway (pmarg-foxl2, pmarg-c43476, pmarg-c45042, pmarg-c19309, pmarg-c54338, pmarg-vit6, pmarg-zglp1, pmarg-dmrt, and pmarg-fem1-like) were investigated. Maximum maturation was observed in the treatment combining the highest temperature (28°C) and the highest microalgae concentration (40,000 cells mL(-1) ), where the female sex tended to be maintained. Injection of 17ß-estradiol induced a significant increase of undetermined stage proportion 2 weeks after the final injection. These results suggest that gametogenesis and gender in adult pearl oysters can be controlled by environmental factors and estrogens. While there were no significant effects on relative gene expression, the 3-gene-pair expression ratio model of the sexual pathway of P. margaritifera, suggest a probable dominance of genetic sex determinism without excluding a mixed sex determination mode (genetic + environmental). J. Exp. Zool. 325A:13-24, 2016. © 2015 Wiley Periodicals, Inc.

Molecular signatures discriminating the male and the female sexual pathways in the pearl oyster Pinctada margaritifera.

The genomics of economically important marine bivalves is studied to provide better understanding of the molecular mechanisms underlying their different reproductive strategies. The recently available gonad transcriptome of the black-lip pearl oyster Pinctada margaritifera is a novel and powerful resource to study these mechanisms in marine mollusks displaying hermaphroditic features. In this study, RNAseq quantification data of the P. margaritifera gonad transcriptome were analyzed to identify candidate genes in histologically-characterized gonad samples to provide molecular signatures of the female and male sexual pathway in this pearl oyster. Based on the RNAseq data set, stringent expression analysis identified 1,937 contigs that were differentially expressed between the gonad histological categories. From the hierarchical clustering analysis, a new reproduction model is proposed, based on a dual histo-molecular analytical approach. Nine candidate genes were identified as markers of the sexual pathway: 7 for the female pathway and 2 for the male one. Their mRNA levels were assayed by real-time PCR on a new set of gonadic samples. A clustering method revealed four principal expression patterns based on the relative gene expression ratio. A multivariate regression tree realized on these new samples and validated on the previously analyzed RNAseq samples showed that the sexual pathway of P. margaritifera can be predicted by a 3-gene-pair expression ratio model of 4 different genes: pmarg-43476, pmarg-foxl2, pmarg-54338 and pmarg-fem1-like. This 3-gene-pair expression ratio model strongly suggests only the implication of pmarg-foxl2 and pmarg-fem1-like in the sex inversion of P. margaritifera. This work provides the first histo-molecular model of P. margaritifera reproduction and a gene expression signature of its sexual pathway discriminating the male and female pathways. These represent useful tools for understanding and studying sex inversion, sex differentiation and sex determinism in this species and other related species for aquaculture purposes such as genetic selection programs.

Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes.

BACKGROUND: Black pearl farming is based on culture of the blacklip pearl oyster Pinctada margaritifera (Mollusca, lophotrochozoa), a protandrous hermaphrodite species. At first maturation, all individuals are males. The female sex appears progressively from two years old, which represents a limitation for broodstock conditioning for aquaculture production. In marine mollusks displaying hermaphroditic features, data on sexual determinism and differentiation, including the molecular sex determining cascade, are scarce. To increase genomic resources and identify the molecular mechanisms whereby gene expression may act in the sexual dimorphism of P. margaritifera, we performed gonad transcriptome analysis. RESULTS: The gonad transcriptome of P. margaritifera was sequenced from several gonadic samples of males and females at different development stages, using a Next-Generation-Sequencing method and RNAseq technology. After Illumina sequencing, assembly and annotation, we obtained 70,147 contigs of which 62.2% shared homologies with existing protein sequences, and 9% showed functional annotation with Gene Ontology terms. Differential expression analysis identified 1,993 differentially expressed contigs between the different categories of gonads. Clustering methods of samples revealed that the sex explained most of the variation in gonad gene expression. K-means clustering of differentially expressed contigs showed 815 and 574 contigs were more expressed in male and female gonads, respectively. The analysis of these contigs revealed the presence of known specific genes coding for proteins involved in sex determinism and/or differentiation, such as dmrt and fem-1 like for males, or foxl2 and vitellogenin for females. The specific gene expression profiles of pmarg-fem1-like, pmarg-dmrt and pmarg-foxl2 in different reproductive stages (undetermined, sexual inversion and regression) suggest that these three genes are potentially involved in the sperm-oocyte switch in P. margaritifera. CONCLUSIONS: The study provides a new transcriptomic tool to study reproduction in hermaphroditic marine mollusks. It identifies sex differentiation and potential sex determining genes in P. margaritifera, a protandrous hermaphrodite species.

Protective effect of Heliotropium foertherianum (Boraginaceae) folk remedy and its active compound, rosmarinic acid, against a Pacific ciguatoxin.

ETHNOPHARMACOLOGICAL RELEVANCE: Senescent leaves of Heliotropium foertherianum Diane & Hilger (Boraginaceae) are traditionally used in the Pacific region to treat Ciguatera Fish Poisoning. This plant contains rosmarinic acid that is known for its multiple biological activities. In the present study, H. foertherianum aqueous extract, rosmarinic acid and its derivatives were evaluated for their capacity to reduce the effect of ciguatoxins. MATERIALS AND METHODS: Aqueous extract of H. foertherianum leaves was prepared and studied for its effects against a Pacific ciguatoxin (P-CTX-1B) in the neuroblastoma cell assay and the receptor binding assay. Rosmarinic acid and six derivatives were also evaluated by means of these bioassays. For this purpose, we have developed an improved synthetic route for caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE). RESULTS: Both the aqueous extract of H. foertherianum leaves and rosmarinic acid showed inhibitory activities against a Pacific ciguatoxin in the above bioassays. Among all the molecules that were evaluated, rosmarinic acid was the most active compound. CONCLUSION: These results confirm further the potential of H. foertherianum in the treatment of Ciguatera Fish Poisoning.

Nutrient fluxes between water column and sediments: potential influence of the pearl oyster culture.

This study quantifies benthic nutrient fluxes and sedimentation rates in the Ahe Atoll lagoon (French Polynesia), in two stations located under pearl oyster frames, and two control stations away from the pearl culture facility. Dissolved inorganic nitrogen fluxes ranged between 2 and 35 µmol N m(-2) h(-1) and Soluble Reactive Phosphorus varied between -3 and 8.2 µmol P m(-2) h(-1). Particulate sedimentation rates beneath the oysters were approximately five times higher than in the control zone and the percentage of small particles (≤63µm) were about the twice. In contrast, sediment composition was similar under and outside the direct influence of oyster frames. In this ecosystem, where primary production is dependent on the available nitrogen, our study revealed that, while highly variable, benthic fluxes could sometimes contribute up to 28% of the nitrogen demand for primary production.

Physical and chemical control of the phytoplankton of Ahe lagoon, French Polynesia.

The environmental characteristics of Ahe deep lagoon (Tuamotu Archipelago, French Polynesia) were studied over 3 years with the aim of explaining the spatial and temporal variability of the natural food available for pearl oysters with a special focus on phytoplankton biomass and global photosynthesis/respiration ratio of the lagoon. Chlorophyll averaged 0.34±0.01 µg L(-1) and our findings did not confirm increased phytoplankton biomass in deep lagoonal waters. Phytoplankton production appears to be limited firstly by nitrogen and respiratory processes overpass photosynthetic processes at least in the north-eastern edge of the atoll. Grazing by pearl oysters in culture seems to decrease the POC concentration but not the phytoplankton biomass. Oysters graze mainly on non chlorophyllian particles.