Nathalie Cochennec-Laureau

Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization

BackgroundThe shell of the pearl-producing bivalve Pinctada margaritifera is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in P. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell.ResultsWe report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 P. margaritifera biomineralization-related mantle protein sequences. Components of the current chitin-silk fibroin gel-acidic macromolecule model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered in P. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts, pmarg-aspein and pmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the P. margaritifera shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the Pinctada genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time.ConclusionsThis EST study made on the calcifying tissue of P. margaritifera is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of P. margaritifera shell, but also that the mantle transcripts encode proteins present in P. margaritifera shell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved in biomineralization. Data generated in this study supply the most comprehensive list of biomineralization-related sequences presently available among protostomian species, and represent a major breakthrough in the field of molluskan biomineralization.

Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell

Mollusca evolutionary success can be attributed partly to their efficiency to sustain and protect their soft body with an external biomineralized structure, the shell. Current knowledge of the protein set responsible for the formation of the shell microstructural polymorphism and unique properties remains largely patchy. In Pinctada margaritifera and Pinctada maxima, we identified 80 shell matrix proteins, among which 66 are entirely unique. This is the only description of the whole “biomineralization toolkit” of the matrices that, at least in part, is thought to regulate the formation of the prismatic and nacreous shell layers in the pearl oysters. We unambiguously demonstrate that prisms and nacre are assembled from very different protein repertoires. This suggests that these layers do not derive from each other.

Changes in circulating and tissue-infiltrating hemocyte parameters of European flat oysters, Ostrea edulis, naturally infected with Bonamia ostreae

We assayed European flat oyster, Ostrea edulis, hemocyte parameters, circulating and tissue-infiltrating hemocyte densities, circulating hemocyte type distribution and lysosomal enzyme contents, to possibly relate these hematological parameters to Bonamia ostreae infection. Circulating hemocyte densities were not statistically different between infected and uninfected oysters. In contrast, the number of tissue-infiltrating hemocytes increased with infection intensity suggesting a recruitment process at the site of infection and a possibility for cells to migrate from circulatory system to connective tissues. Lysosomal enzymes were localized mainly in granulocytes both infected and uninfected, and mean of alpha-naphtyl butyrate esterase activity decreased with increasing B. ostreae infection level. The main response observed was a change in hemocyte type distribution between uninfected and infected oysters and greater tissue-infiltrating hemocytes with increased infections. These results suggest that the decrease of circulating granulocytes, and, consequently of some cell enzyme activities may be related with B. ostreae infection.

Pmarg-Pearlin is a Matrix Protein Involved in Nacre Framework Formation in the Pearl Oyster Pinctada margaritifera

The shell of pearl oysters is organized in multiple layers of CaCO3 crystallites packed together in an organic matrix. Relationships between the components of the organic matrix and mechanisms of nacre formation currently constitute the main focus of research into biomineralization. In this study, we characterized the pearlin protein from the oyster Pinctada margaritifera (Pmarg); this shares structural features with other members of a matrix protein family, N14/N16/pearlin. Pmarg pearlin exhibits calcium‐ and chitin‐binding properties. Pmarg pearlin transcripts are distinctively localized in the mineralizing tissue responsible for nacre formation. More specifically, we demonstrate that Pmarg pearlin is localized within the interlamellar matrix of nacre aragonite tablets. Our results support recent models for multidomain matrix protein involvement in nacreous layer formation. We provide evidence here for the existence of a conserved family of nacre‐associated proteins in Pteriidae, and reassess the evolutionarily conserved set of biomineralization genes related to nacre formation in this taxa.

Improving the efficacy of sewage treatment decreases norovirus contamination in oysters

As human population increases worldwide, water quality will become increasingly problematic, and food consumed raw may be of higher risk. This is already evident for oysters grown in coastal areas - despite regulations based on bacterial indicators, oysters are still implicated in food-borne outbreaks worldwide. The pathogens most frequently detected are human noroviruses, which are shed at high concentrations in human excreta and are very resistant to environmental conditions. Sewage treatment plants usually apply a variety of steps such as activated sludge treatment, chlorine or UV disinfection to eliminate contaminants, these processes have variable efficacy. This study demonstrates the impact of replacing an old lagoon-based sewage treatment plant with a new membrane bioreactor sewage treatment plant on human norovirus levels in treated sewage and oysters. While comparable norovirus concentrations were detected in the influent samples, a clear difference was observed in effluent quality, as norovirus was only detected in one sample after treatment in the new membrane bioreactor system, confirming the efficiency of such technology. As a direct impact, oysters located close to the membrane bioreactor sewage outfall were less frequently contaminated by norovirus, and showed lower concentrations compared to the first period of the study when they were exposed to sewage effluent from the lagoon outfall. Shellfish located upstream showed comparable contamination levels suggesting that there are also other sources of norovirus contamination in the estuary. Considering the health benefits of shellfish consumption, improving wastewater quality will make an important contribution to enhancing the safety of shellfish and international food security.

Reduced phosphorus loads from the Loire and Vilaine Rivers were accompanied by increasing eutrophication in Vilaine Bay (South Brittany, France)

The evolution of eutrophication parameters (i.e., nutrients and phytoplankton biomass) during recent decades was examined in coastal waters of the Vilaine Bay (VB, France) in relation to changes in the Loire and Vilaine rivers. Dynamic linear models were used to study long-term trends and seasonality of dissolved inorganic nutrient and chlorophyll a concentrations (Chl a) in rivers and coastal waters. For the period 1997–2013, the reduction in dissolved riverine inorganic phosphorus (DIP) concentrations led to the decrease in their Chl a levels. However, while dissolved inorganic nitrogen (DIN) concentrations decreased only slightly in the Vilaine, they increased in the Loire, specifically in summer. Simultaneously, phytoplankton in the VB underwent profound changes with increase in biomass and change in the timing of the annual peak from spring to summer. The increase in phytoplankton biomass in the VB, manifested particularly by increased summer diatom abundances, was due to enhanced summer DIN loads from the Loire, sustained by internal regeneration of DIP and dissolved silicate (DSi) from sediments. The long-term trajectories of this case study evidence that significant reduction of P inputs without simultaneous N abatement was not yet sufficient to control eutrophication all along the Loire–Vilaine–VB continuum. Upstream rivers reveal indices of recoveries following the significant diminution of P, while eutrophication continues to increase downstream, especially when N is the limiting factor. More N input reduction, paying particular attention to diffuse N sources, is required to control eutrophication in receiving VB coastal waters. Internal benthic DIP and DSi recycling appears to have contributed to the worsening of summer VB water quality, augmenting the effects of anthropogenic DIN inputs. For this coastal ecosystem, nutrient management strategies should consider the role played by internal nutrient loads to tackle eutrophication processes.