Marine pollution: Let us not forget beach sand

Background: Assessing the chemical or bacterial contamination in marine waters and sediments is a very common approach to evaluate marine pollution and associated risks. However, toxicity and organic pollution of beach sands have not yet been considered, except in adjacent waters. In the present study, the toxicity and the chemical contamination of natural beach sands collected 20 m from the shoreline at two sites located on the Mediterranean Sea (Marseille and La Marana, Corsica) were studied. Results: Up to 16.93% (net percentage) abnormal or dead larvae was observed in elutriates prepared from the urban beach sand sample (Marseille); no significant toxicity was observed in the sample collected from the reference beach in La Marana. Results of Fourier transform infrared spectroscopy analyses revealed that no microplastics were present in either of the samples. Several polycyclic aromatic hydrocarbons [PAHs] in both samples and a larger number of individual PAHs in the urban sample than in the sample collected from the reference beach were detected. In addition, the antioxidant dioctyldiphenylamine was detected in both beach sand samples, whereby a higher concentration was found in La Marana than in Marseille. Calculated PAH concentrations in elutriates were generally higher than measured ones. Conclusions: The results of this preliminary study provide evidence of toxicity and the presence of organic trace contaminants in beach sands from France. According to our results, monitoring using a combination of biotests and chemical analyses is recommended, especially of sediments from beaches abandoned to urban and industrial areas.

Relations entre apports terrigènes et conchyliculture dans les Pertuis Charentais

This work presents interactions between quantitative and qualitative river freshwater inputs and the shellfish farming (oyster and mussel) in the Pertuis Charentais. The quantity of freshwater (i.e. salinity) seems to have a weak influence on the shellfish farming contrarily to its quality determined by particulate and dissolved matters contained in the water. In autumn and winter, large precipitations have a "globally positive" effect amending the coastal ecosystem. Associated dissolved nutriments and the organic matter largely determine the quality of the coming spring growth for bred shellfish, itself controlling in turn the annual yield efficiencies. However, in winter their effects are postponed because of strong mineral load, low luminosity and temperature, then limiting the primary production. The spring contributions, directly linked to territorial practices, agriculture and tourism are more variable in quantity and quality from one year to another. They often correspond to high-risk inflows since numerous substances from anthropogenic watersheds can be found diluted in the coastal zone as in the Pertuis Charentais. Their impacts on in situ estuarine ecosystems are still poorly known since these substances are mainly studied and estimated in laboratory in controlled conditions. Several studies showed anthropogenic contaminations (i.e. cadmium, pesticides) could have significant direct or indirect effects on shellfish farming. For instance, the "summer" mortalities between 1990 and 2000 in the South of the Marennes-Oléron bay (MOB), that induced environmental and physiological oyster disorders, could be linked to pesticide effects, measured during consecutive years on the oyster bed of Ronce Perquis in the South of the MOB. The weak results from the spring larval rearing of the IFREMER experimental hatchery in the South of the bay, and chromosomal abnormalities measured on the stocks of wild oysters of the Pertuis could confirm a high-risk spring environment for the shellfish farming. In summer terrestrial inputs are reduced by low precipitations, anthropogenic water removals (drinking water, irrigation) and by plant evapotranspiration. Consequently certain years, a significant salinity increase in water masses of the Pertuis Charentais is observed. However, based on long-term observations, the significant interannual variability noticed in freshwater contributions constitutes one of the most important facts of these last years. When contributions are weak (i.e. 1991 and 2011), the mean annual salinity is 34.5 in the MOB. To the contrary, other years (i.e. 1977, 1981, 1983 and 1988), the mean salinity reduced to 30.5 shows the significant freshwater contributions to the bay. Elsewhere, particularly in the mediterranean region, oyster breeding water conditions characterized by high salinity values show the freshwater does not seem to be necessary for biological functions of the Pacific oyster Crassostrea gigas. Indeed, the oyster embryonic life in particular is well adapted to high salinity values as long as trophic resources are substantial and temperatures remain high. These two factors firstly condition the embryonic survival before the water salinity. Besides, in the Pertuis Charentais, wind conditions and the geographical bloodstock position rather determine the success of the larvae capture than seawater physic-chemical conditions. Finally, a misunderstanding still remains on summer freshwater contributions to the oyster larvae food supply.

Genetic effect of domestication selective pressures on Pacific oyster at larval stage

Among bivalve species, the Pacific oyster, Crassostrea gigas, is the most economically important bivalve production over the world. Today, C. gigas is subject to an important production effort that leads to an intensive artificial selection. Larval stage is relatively unknown, specifically in a domestication context. Genetic consequence of artificial selection is still at a preliminary study. We aimed to tackle the consequence of inconscient domestication on the variance reproductive success focusing on larval stage, keystone of the life cycle. We studied two kinds of specific selective processes that common hatchery rearing practices exert : the effect of discarding the smallest larvae on genetic diversity and the artificial environment rearing effect via the temperature providing a contrast resembling wild versus hatchery conditions (20 and 26°C). In order to monitor the effect of the selection of fast growing larvae by sieving, growth variability and genetic diversity in a larval population descended from a factorial breeding was studied. We used a mixed-family approach to reduce potentially confounding environmental biais. The retrospective assignment of individuals to family groups has been performed using a three microsatellite markers set. Two different rearing were carried out in parallel. For three (replicates) 50-l tanks, the smallest larvae were progressively discarded by selective sieving, whereas for the three others no selective sieving was performed. The intensity of selective sieving was adjusted so as to discard 50% of the larvae over the whole rearing period in a progressive manner. As soon as the larvae reached the pediveliger stage, ready to settle larvae were sampled for genetic analysis. Regarding the artificial environment rearing effect via the temperature, we used a similar mixed-family approach. The progeny from a factorial breeding design was divided as follows: three (replicates) 50-l tanks were dedicaced to a rearing at 26°C versus 20°C for three others 50-l tanks. The whole size variability was preserved for this experiment. Individual growth measurements for larvae genetically identified have been performed at days 22 and 30 after fertilization for both conditions. In a same way, we collected individual measurements for genotyped juvenile oysters (80 days after fertilization). At a phenotypic scale, relative survival and settlement success for larvae with sieving were higher. Sieving appears as a time-saving process associated with a better relative survival ratio. But in the same time, our results confirm that a significant genetic variability exist for early developmental traits in the Pacific oyster. This is congruent with the results already obtained that investigated genetic variability and genetic correlations in early life-history traits of Crassostrea gigas. Discarding around 50% of the smallest larvae can lead to significant selection at the larval stage.

Impacts of Deepwater Horizon oil and associated dispersant on early development of the Eastern oyster Crassostrea virginica

The explosion of the Deepwater Horizon (DWH) oil platform resulted in large amounts of crude oil and dispersant Corexit 9500A® released into the Gulf of Mexico and coincided with the spawning season of the oyster, Crassostrea virginica. The effects of exposing gametes and embryos of C. virginica to dispersant alone (Corexit), mechanically (HEWAF) and chemically dispersed (CEWAF) DWH oil were evaluated. Fertilization success and the morphological development, growth, and survival of larvae were assessed. Gamete exposure reduced fertilization (HEWAF: EC201 h = 1650 μg tPAH50 L− 1; CEWAF: EC201 h = 19.4 μg tPAH50 L− 1; Corexit: EC201 h = 6.9 mg L− 1). CEWAF and Corexit showed a similar toxicity on early life stages at equivalent nominal concentrations. Oysters exposed from gametes to CEWAF and Corexit experienced more deleterious effects than oysters exposed from embryos. Results suggest the presence of oil and dispersant during oyster spawning season may interfere with larval development and subsequent recruitment.

Infection dynamics of Marteilia refringens in flat oyster Ostrea edulis and copepod Paracartia grani in a claire pond of Marennes-Oleron Bay

The protozoan parasite Marteilia refringens has been partly responsible for the severe decrease in the production of the European flat oyster Ostrea edulis Linnaeus in France since the 1970s. The calanoid copepod Paracartia grani Sars was recently found to be a host for M refringens in French shallow-water oyster ponds ('claires'). This study reconsidered M refringens transmission dynamics in the light of this finding, taking into account not only oyster infection dynamics and environmental factors but also data concerning the copepod host. P. grani population dynamics in the claire under study revealed that this species is the dominant planktonic copepod in this confined ecosystem. During winter, M refringens overwintered in O. edulis, with P. grani existing only as resting eggs in the sediment. The increase in temperature in spring controlled and synchronized both the release of M refringens sporangia in the oyster feces, and the hatching of the benthic resting eggs of the copepod. Infection of oysters by M refringens was limited to June, July and August, coinciding with (1) the highest temperature recorded in the claire, and (2) the highest abundance of P. grani. PCR detection of M refringens in P. grani during the summer period was linked to the release of parasite sporangia by the oyster. Our results are supported by previous results on the effective transmission of this parasite from the oyster to the copepod.

Influence of one selected Tisochrysis lutea strain rich in lipids on Crassostrea gigas larval development and biochemical composition

Effects of a remarkably high overall lipid Tisochrysis lutea strain (T+) upon gross biochemical composition, fatty acid (FA), sterol and lipid class composition of Crassostrea gigas larvae were evaluated and compared with a normal strain of Tisochrysis lutea (T) and the diatom Chaetoceros neogracile (Cg). In a first experiment, the influence of different single diets (T, T+ and Cg) and a bispecific diet (TCg) was studied, whereas, effects of monospecific diets (T and T+) and bispecific diets (TCg and T+Cg) were evaluated in a second experiment. The strain T+ was very rich in triglycerides (TAG: 93–95% of total neutral lipids), saturated FA (45%), monounsaturated FA (31–33%) and total fatty acids (4.0–4.7 pg cell−1). Larval oyster survival and growth rate were positively correlated with 18:1n-7 and 20:1n-7, in storage lipids (SL), and negatively related to 14:0, 18:1n-9, 20:1n-9, 20:4n-6 and trans-22-dehydrocholesterol in membrane lipids (ML). Surprisingly, only the essential fatty acid 20:5n-3 in SL was correlated positively with larval survival. Correlations suggest that physiological disruption by overabundance of TAG, FFA and certain fatty acids in larvae fed T+ was largely responsible for the poor performance of these larvae. ‘High-lipid’ strains of microalgae, without regard to qualitative lipid composition, do not always improve bivalve larval performance.

Induction of apoptosis by UV in the flat oyster, Ostrea edulis

Apoptosis is a fundamental feature in the development of many organisms and tissue systems. It is also a mechanism of host defense against environmental stress factors or pathogens by contributing to the elimination of infected cells. Hemocytes play a key role in defense mechanisms in invertebrates and previous studies have shown that physical or chemical stress can increase apoptosis in hemocytes in mollusks. However this phenomenon has rarely been investigated in bivalves especially in the flat oyster Ostrea edulis. The apoptotic response of hemocytes from flat oysters, O. edulis, was investigated after exposure to UV and dexamethasone, two agents known to induce apoptosis in vertebrates. Flow cytometry and microscopy were combined to demonstrate that apoptosis occurs in flat oyster hemocytes. Investigated parameters like intracytoplasmic calcium activity, mitochondrial membrane potential and phosphatidyl-serine externalization were significantly modulated in cells exposed to UV whereas dexamethasone only induced an increase of DNA fragmentation. Morphological changes were also observed on UV-treated cells using fluorescence microscopy and transmission electron microscopy. Our results confirm the apoptotic effect of UV on hemocytes of O. edulis and suggest that apoptosis is an important mechanism developed by the flat oyster against stress factors.

Disruption of amylase genes by RNA interference affects reproduction in the Pacific oyster Crassostrea gigas

Feeding strategies and digestive capacities can have important implications for variation in energetic pathways associated with ecological and economically important traits, such as growth or reproduction in bivalve species. Here, we investigated the role of amylase in the digestive processes of Crassostrea gigas, using in vivo RNA interference. This approach also allowed us to investigate the relationship between energy intake by feeding and gametogenesis in oysters. Double-stranded (ds)RNA designed to target the two α-amylase genes A and B was injected in vivo into the visceral mass of oysters at two doses. These treatments caused significant reductions in mean mRNA levels of the amylase genes: −50.7% and −59% mRNA A, and −71.9% and −70.6% mRNA B in 15 and 75 µg dsRNA-injected oysters, respectively, relative to controls. Interestingly, reproductive knock-down phenotypes were observed for both sexes at 48 days post-injection, with a significant reduction of the gonad area (−22.5% relative to controls) and germ cell under-proliferation revealed by histology. In response to the higher dose of dsRNA, we also observed reductions in amylase activity (−53%) and absorption efficiency (−5%). Based on these data, dynamic energy budget modeling showed that the limitation of energy intake by feeding that was induced by injection of amylase dsRNA was insufficient to affect gonadic development at the level observed in the present study. This finding suggests that other driving mechanisms, such as endogenous hormonal modulation, might significantly change energy allocation to reproduction, and increase the maintenance rate in oysters in response to dsRNA injection.

Mass mortality in bivalves and the intricate case of the Pacific oyster, Crassostrea gigas

Massive mortality outbreaks in cultured bivalves have been reported worldwide and they have been associated with infection by a range of viral and bacterial pathogens. Due to their economic and social impact, these episodes constitute a particularly sensitive issue in Pacific oyster (Crassostrea gigas) production. Since 2008, mortality outbreaks affecting C. gigas have increased in terms of intensity and geographic distribution. Epidemiologic surveys have lead to the incrimination of pathogens, specifically OsHV-1 and bacteria of the Vibrio genus, in particular Vibrio aestuarianus. Pathogen diversity may partially account for the variability in the outcome of infections. Host factors (age, reproductive status…) including their genetic background that has an impact on host susceptibility towards infection, also play a role herein. Finally, environmental factors have significant effects on the pathogens themselves, on the host and on the host-pathogen interaction. Further knowledge on pathogen diversity, classification, and spread, may contribute towards a better understanding of this issue and potential ways to mitigate the impact of these outbreaks.

Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea. The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum. GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification.

Biofloc contribution to antioxidant defence status, lipid nutrition and reproductive performance of broodstock of the shrimp Litopenaeus stylirostris: Consequences for the quality of eggs and larvae

The aim of this study was to determine biofloc contributions to the antioxidant status and lipid nutrition of broodstock of Litopenaeus stylirostris in relationship with their reproductive performance and the health of larvae produced. Shrimp broodstock reared with Biofloc technology (BFT) compared to Clear water (CW) exhibited a higher health status with (i) a better final survival rate during the reproduction period (52.6% in CW against 79.8% in BFT); (ii) higher glutathione level (GSH) and total antioxidant status (TAS), reduced oxidized/reduced glutathione ratio and a higher spawning rate and frequency as well as higher gonado-somatic index and number of spawned eggs. Finally, larvae from broodstock from BFT exhibited higher survival rates at the Zoe 2 (+ 37%) and Post Larvae 1 (+ 51%) stages when compared with those from females from CW treatment. The improved reproductive performance of the broodstock and higher larvae survival rate resulting from BFT treatment may be linked to the dietary supplement obtained by the shrimp from natural productivity during BFT rearing. Indeed, our study confirms that biofloc particulates represent a potential source of dietary glutathione and a significant source of lipids, particularly essential phospholipids and n-3 highly unsaturated fatty acids (HUFA) for shrimps. Thus, broodstock from BFT treatment accumulated phospholipids, n-3 HUFA and arachidonic acid, which are necessary for vitellogenesis, embryogenesis and pre-feeding larval development. The predominant essential fatty acids, arachidonic acid (ARA), eicopentaeonic acid (EPA) and docosahexaenoic acid (DHA), had levels in the eggs that were, respectively, 2.5, 2.8 and 3 fold higher for BFT compared to the CW treatment. Statement of Relevance Today, the influence of biofloc technology on shrimp broodstock is not enough described and no information was available on the larvae quality. Moreover, two key pieces of new information emerge from the present study. Firstly, biofloc is a source of further dietary lipids that can act as energetic substrates, but also as a source of phospholipids and essential fatty acids necessary to sustain reproduction, embryonic and larval development. Second, improving the reproduction of the broodstock also leads to an improvement in the quality of the larvae. We think that our research is new and important to increase knowledge on biofloc topic. We believe the paper will contribute to the development of more efficient and therefore more sustainable systems.

Macro-geographical differences influenced by family-based expression on cultured pearl grade, shape and colour in the black-lip "pearl oyster" Pinctada margaritifera: a preliminary bi-local case study in French Polynesia

In French Polynesia, the aquaculture of P. margaritifera is carried out in numerous grow-out sites, located over three archipelagos (Gambier, Society and Tuamotu). To evaluate the impact of macro-geographical effects of these growing sites on pearl quality traits, five hatcheries produced families were used as homogeneous donor oysters in an experimental graft. The molluscs were then reared in two commercial locations: Tahaa island (Society) and Rangiroa atoll (Tuamotu). At harvest, eight pearl quality traits were recorded and compared: surface defects, lustre, grade, circles, shape categories, darkness level, body and secondary colour and visual colour categories. Overall inter-site comparison revealed that: 1) all traits were affected by grow-out location except for lustre and round shape, and 2) a higher mean rate of valuable pearls was produced in Rangiroa. Indeed, for pearl grade, Rangiroa showed twice as many A-B and less reject samples than Tahaa. This was related to the number of surface defects (grade component): in Rangiroa, twice as many pearls had no defects and less pearls had up to 10 defects. Concerning pearl shape, more circled and baroque pearls were found in Tahaa (+10%). For colour variation, 10% more pearls have an attractive green overtone in Rangiroa than in Tahaa, where more grey bodycolor were harvested. Lustre does not seem to be affected by these two culture site (except at a family scale). This is the first time P. margaritifera donor family have been shown to vary in the quality of pearls they produce depending on their grow-out location.

Evaluation of the impact of polyethylene microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae

Microplastics are present in marine habitats worldwide and may be ingested by low trophic organisms such as fish larvae, with uncertain physiological consequences. The present study aims at assessing the impact of polyethylene (PE 10-45µM) microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae. Fish were fed an inert diet including 0, 104 and 105 fluorescent microbeads per gram from 7 until 43 days post-hatching (dph). Microbeads were detected in the gastrointestinal tract in all fish fed diet incorporating PE. Our data revealed an efficient elimination of PE beads from the gut since no fluorescent was observed in the larvae after 48h depuration. While the mortality rate increased significantly with the amount of microbeads scored per larvae at 14 and 20 dph, only ingestion of the highest concentration slightly impacted mortality rates. Larval growth and inflammatory response through Interleukine-1-beta (IL-1) gene expression were not found to be affected while cytochrome-P450-1A1 (cyp1a1) expression level was significantly positively correlated with the number of microbeads scored per larva at 20 dph. Overall, these results suggest that ingestion of PE microbeads had limited impact on sea bass larvae possibly due to their high potential of egestion

Age-dependence of cultured pearl grade and colour in the black-lipped pearl oyster Pinctada margaritifera

Pinctada margaritifera is an economically important marine bivalve species for cultured pearl production in French Polynesian aquaculture. In order to evaluate the influence of donor oyster age on pearl quality traits, experiments were conducted over 6 years using both grafts and surgreffe operations. At harvest, 6 pearl quality traits were recorded and compared: surface defects, luster, grade, darkness level, and visual color. Analyzing the quality traits of pearls harvested in the initial graft process and those of pearls obtained from surgreffe experiments allowed a comparison of the influence of pearl sac cells originating from the initial mantle graft, which aged together with their recipient oysters. The results demonstrated a significant decrease between these successive grafts in luster, grade (A-B-C,) darkness level, and green color – traits that are of major importance in the pearl market. The duplicated graft experiment allowed the comparison of donor oyster families at 2 and 5 years old, where a mantle graft was inserted into recipient oysters aged 2.5 years old. The results showed the same tendencies to a lesser extent, with 1) an improved pearl grade, predominantly through a most important rate of 0 surface defect category, and 2) a green / grey ratio in favor of the younger donor. A comparison between the graft-surgreffe and the duplicated graft experiments also highlighted: 1) the indirect role played by the younger recipient oysters, which must be optimized for optimal pearl quality realization, and 2) the complex interplay between donor and recipient oysters.

Energy and Antioxidant Responses of Pacific Oyster Exposed to Trace Levels of Pesticides

Here, we assess the physiological effects induced by environmental concentrations of pesticides in Pacific oyster Crassostrea gigas. Oysters were exposed for 14 d to trace levels of metconazole (0.2 and 2 mu g/L), isoproturon (0.1 and 1 mu g/L), or both in a mixture (0.2 and 0.1 mu g/L, respectively). Exposure to trace levels of pesticides had no effect on the filtration rate, growth, and energy reserves of oysters. However, oysters exposed to metconazole and isoproturon showed an overactivation of the sensing-kinase AMP-activated protein kinase alpha (AMPK alpha), a key enzyme involved in energy metabolism and more particularly glycolysis. In the meantime, these exposed oysters showed a decrease in hexokinase and pyruvate kinase activities, whereas 2-DE proteomic revealed that fructose-1,6-bisphosphatase (F-1,6-BP), a key enzyme of gluconeogenesis, was upregulated. Activities of antioxidant enzymes were higher in oysters exposed to the highest pesticide concentrations. Both pesticides enhanced the superoxide dismutase activity of oysters. Isoproturon enhanced catalase activity, and metconazole enhanced peroxiredoxin activity. Overall, our results show that environmental concentrations of metconazole or isoproturon induced subtle changes in the energy and antioxidant metabolisms of oysters.