IMPACT OF ENVIRONMENTAL CHANGES ON COASTAL PATHOGEN SYSTEMS – ENVICOPAS

ENVICOPAS relies on collection of field data, experimental works and modeling approaches.
The spatio-temporal dynamics of bacterial communities including populations of pathogenic Vibrio spp. and environmental factors influencing their distribution is investigated in France and Germany through a combination of wide (meta-barcoding and metagenomic) and specific tools like MALDI-TOF MS.
In addition, oyster microbiome (bacteria, viruses, protists) and their associations with known oyster pathogens will be characterized during natural infections processes in situ in French and German sites.

The ecological niche of pathogenic Vibrio spp. will be determined experimentally by defining their growth parameters in vitro under different environmental conditions.
Impact of experimental environmental disturbance on the oyster microbiome will be evaluated using the same approach as on samples from the field. Additionally, oyster survival will be investigated in the context of experimental exposure of oysters to human and oyster pathogens under different temperature conditions. These experimental works will also contribute to provide data required for the development of epidemiological models.

Compartmental epidemiological time-dependent models available for Vibrio aestuarianus and OsHV-1 but will be completed by evaluating the influence of the temperature. A similar approach will be developed for the first time for the parasite Bonamia ostreae. Based on ecological niche data, a modelling system will also be developed to understand and predict the occurrence of harmful Vibrio spp. and its bacteriophages.
In order to predict climate change consequences on the distribution of Vibrio spp., high-resolution hydrodynamic model for sites selected in Germany and France will be coupled with biological modules presented above.

Monitoring of pathogenic Vibrio spp. revealed the presence of major human pathogens in seawater collected in 2016. Interestingly, V. vulnificus and V. cholerae were isolated during the summer months at salinities above the optimal salinity required for their growth. Some new primers have been designed for metabarcoding approaches.
Monitoring of pathogens was completed on oysters collected in 2016 and revealed the presence of OsHV-1 in spat C. gigas during the mortality outbreak which occurred in May. Interestingly, differential dynamic was observed in Bonamia ostreae and Marteilia refringens prevalence in flat oysters. This biological material is now ready to be analysed by metabarcoding approach to evaluate the impact of field pathogen exposure on oyster microbiome.
Development works have been successfully performed to extract DNA and RNA from oyster samples and to amplify protists including main oyster pathogens and bacteria. These tools will be used to characterize protistome and bacteriome on samples collected from the field and oysters exposed to environmental disturbance in experimental conditions.
Spat C. gigas was infected with OsHV-1 after 3 weeks of acclimatization at different water temperatures and treatments. Oyster survival appeared not only significantly influenced by the temperature but also by water UV treatment. These results support our initial hypothesis concerning the involvement of microbiome in the development of the disease.
Experimental works carried out on C. gigas juveniles demonstrated the impact of temperature on the development and transmission of Vibrio aestuarianus. These data will allow completing the theorical model previously developed in the laboratory.
The bibliographic analysis of data available on Bonamia ostreae allowed designing a conceptual model of the transmission of the parasite and identifying key transmission parameters.

Water and oyster sampling and experimental works have almost been finalized.
The design and use of ftsZ primers for metabarcoding approaches will be of great interest to investigate Vibrios diversity
The protocol to extract DNA and RNA and to amplify protists and bacteria will be used to characterize protistome and bacteriome from oyster tissues and fluids. Potential association between microbiome and infection with known oyster pathogen will then been investigated. Metagenomics approach will also been tested in order to get viral sequences and to characterize the oyster virome.
Oysters experimentally infected with OsHV-1 or Vibrio aestuarianus afer an environmental disturbance will be tested by metabarcoding and metagenomic approaches in order to identify potential beneficial or detrimental associations between micro-organisms
Experimental assays carried out in 2016-2017 will also contribute to provide data required for the improvement of epidemiological models already available for both OsHV-1 and Vibrio aestuarianus.
Key transmission parameters identified through the conceptual model of Bonamia ostreae transmission will be investigated experimentally in 2018 including parasite shedding and survival.
The system of ordinary differential equations constituting the model of transmission of V. aestuarianus was shared with the German team of modelers and will be coupled with other biological modules and the hydrodynamic model In order to predict climate change consequences on the distribution and transmission of the bacteria.

Dupont, S. ; Petton, B.; Toulza, E.; Lokmer, A.; Montagnani, C.; Desdevises, Y. ; Pecqueur, D.; Salmeron, C.; Guillou, L. ; Desnues, C. ; La Scola, B.; de Lorgeril, J. ; Mitta, G. ; Gueguen, Y. ; Escoubas, J.-M., Oyster’s blood microbiota: Bacteria, Protists and Viruses, What else? International Conference on Holobionts, Paris April 19-21, 2017. Poster
Lupo Coralie, Faury Nicole, Ezanno Pauline, Dupuy Beatrice, Papin Mathias, Dutta Bhagat Lal, Morga Benjamin, Renault Tristan (2016). First insights of transmission dynamics of the ostreid herpesvirus 1 (OsHV-1 µVar) between Pacific oysters, Crassostrea gigas, using experimental infection data. AquaEpi I-2016 - 1st scientific conference in Aquatic Animal Epidemiology. September 20-22, 2016, Oslo, Norway.
Arzul Isabelle, Baillon Laury, Chollet Bruno, Dubreuil Christine, Garcia Céline, Heloury Purotu, Hussenot Mathieu, Serpin Delphine, Pouvreau Stéphane (2016). Differential dynamics of Bonamia ostreae and Marteilia refringens in a same population of flat oyster, Ostrea edulis, in rade de Brest, Brittany, France. World Aquaculture Society, Aquaculture 2016 : All in for Aquaculture. 2016 February 22-26, Las Vegas, Nevada, USA.
Jacquin Justine. Impact des changements environnementaux sur les Vibrio spp. pathogènes pour l’Homme dans les écosystèmes côtiers. 2017. Rapport M2R. 38p.

The Marine Strategy Framework directive aims to achieve good environmental status (GES) by 2020. Monitoring health and evaluating ecological status of marine ecosystems however require descriptors. Among them, marine microorganisms infecting wildlife and humans alike are interesting models. Indeed, these pathogens might be associated with public and animal health issues affecting notably economically and ecologically important marine species such as oyster. Massive mortality of oysters are more and more frequently reported and have been associated with viruses, bacteria (mainly Vibrio) as well as protozoans. These alarming phenomena result from direct and indirect factors that are only partly understood.
Oysters host communities of resident or transient microorganisms, i.e. the microbiota which can be a source and /or interact with some pathogens of the oysters and other species among which human. Thus investigating such pathosystems appears fundamental for economic and public health reasons. This task is however challenged by the complexity of biological interactions that should be considered, between oysters, pathogens of oysters and/or humans and the microbiota of the host.
The aim of ENVICOPAS not only resides in the description of how these microorganism communities coexist and interact, but also in the prediction of how environmental conditions are conductive to pathogen dispersion and disease development. The proposed project ENVICOPAS aims to investigate the dynamics of oyster and human pathogens under variable environmental physical drivers and connect these dynamics to changes observed in the oyster microbiota. Using a combined “in vitro/in silico/in situ” approaches, projective tools will be developed to advance prediction of dispersion of human pathogenic Vibrios in seawater, their potential accumulation by oysters and complex interactions linking pathogens of oysters and human to their microbiota under different environmental scenarios. The practical value of the structure and composition of microbial communities in oysters and their surrounding water as indicator for GES is beyond the expected results of this study.