Eléments de connaissance sur la mortalité et la reproduction de la moule bleue (Mytilus edulis) sur la façade atlantique

Ce document s’inscrit dans le cadre de la problématique des mortalités de moules bleues sur les côtes françaises depuis l’hiver 2014. Il a été conçu comme une contribution aux projets Morbleu (projet de recherche) et Mytilobs (observatoire national sur les traits de vie de la moule). D’un point de vu historique, depuis le début du 20ème siècle, le principale « ennemi » de la moule reste la météo et en particulier, les tempêtes. Une seule crise de mortalité, majeure, est survenue en 1960-61 dans le pertuis Breton. Elle résulte d’une interaction entre le parasite mytilicola intestinalis et des conditions drastiques d’élevage sur bouchots dans la baie de l’Aiguillon. L’évaluation des mortalités de moules est difficile du fait des pratiques d’élevage et des pertes qui en résultent. Lorsque la crise survient en 2014, l’investigation scientifique s’appuie à la fois sur des déclarations (professionnels), des constats (DDTMs), des saisines (Repamo) et des mesures (réseau Mytilobs). L’intensité des mortalités, variable selon les sites et les années (2014 et 2015) est comparée à un référentiel de mortalités issu d’un réseau régional de suivi des mortalités de moules en paniers (Remoula, 2000-2010). En février 2014, les mortalités surviennent dans le pertuis Breton et s’étendent en quelques mois vers le sud (pertuis d’Antioche) et vers le Nord (Côte de Noirmoutier et Baie de Bourgneuf). Les simulations hydrodynamiques montrent la connectivité spatio-temporelle entre le site d’émergence des mortalités et les sites touchés quelques semaines plus tard. Alors qu’il existe plusieurs génomes de Mytililus edulis et des populations hybrides (entre Mytililus edulis et Mytilus galloprovincialis) sur les cotes de la France, seul le génome Mytililus edulis de la cote atlantique semble au départ concerné par ces mortalités. La quasi simultanéité d’occurrence d’épisodes de mortalités de moules en Rade de Brest (en hiver 2014), et sur les gisements profonds de la cote Est du Cotentin (dès le printemps 2014) reste aujourd’hui une énigme scientifique. Lorsque les mortalités surviennent en 2014 et lors de leur reprise en 2015, les moules sont en période de reproduction. Ce rapport synthétise également des résultats acquis sur la reproduction de Mytilus edulis sur la cote atlantique (Arcachon et Pertuis Charentais).

Evidences for an opportunistic and endophytic lifestyle of the Bursaphelenchus xylophilus -associated bacteria Serratia marcescens PWN146 isolated from wilting Pinus pinaster

Pine wilt disease (PWD) results from the interaction of three elements: the pathogenic nematode, Bursaphelenchus xylophilus; the insect-vector, Monochamus sp.; and the host tree, mostly Pinus species. Bacteria isolated from B. xylophilus may be a fourth element in this complex disease. However, the precise role of bacteria in this interaction is unclear as both plant-beneficial and as plant-pathogenic bacteria may be associated with PWD. Using whole genome sequencing and phenotypic characterization, we were able to investigate in more detail the genetic repertoire of Serratia marcescens PWN146, a bacterium associated with B. xylophilus. We show clear evidence that S. marcescens PWN146 is able to withstand and colonize the plant environment, without having any deleterious effects towards a susceptible host (Pinus thunbergii), B. xylophilus nor to the nematode model C. elegans. This bacterium is able to tolerate growth in presence of xenobiotic/organic compounds, and use phenylacetic acid as carbon source. Furthermore, we present a detailed list of S. marcescens PWN146 potentials to interfere with plant metabolism via hormonal pathways and/or nutritional acquisition, and to be competitive against other bacteria and/or fungi in terms of resource acquisition or production of antimicrobial compounds. Further investigation is required to understand the role of bacteria in PWD. We have now reinforced the theory that B. xylophilus-associated bacteria may have a plant origin.

The role of biological processes in affecting the dynamics and fate of microplastics in coastal marine systems

Microplastics (MP) are omnipresent contaminants in the marine environment. Ingestion of MP has been reported for a wide range of marine biota, but to what extent the uptake by organisms affects the dynamics and fate of MP in the marine system has received little attention. My thesis explored this topic by integrating laboratory tests and experiments, field quantitative surveys of MP distribution and dynamics, and the use of specialised analytical techniques such as Attenuated-Total-Reflectance- (ATR) and imaging- Fourier Transformed Infrared Spectroscopy (FTIR). I compared different methodologies to extract MP from wild invertebrate specimens, and selected the use of potassium hydroxide (KOH) as the most cost-effective approach. I used this approach to analyse the MP contamination in various invertebrate species with different ecological traits from European salt marshes. I found that 96% of the analysed specimens (330) did not contain any MP. As preliminary environmental analyses showed high levels of environmental MP contamination, I hypothesised that most MP do not accumulate into organisms but are rather fast egested. I subsequently used laboratory multi-trophic experiments and a long-term field experiment using the filter-feeding mussel Mytilus galloprovincialis and the detritus feeding polychaete Hediste diversicolor to test the aforementioned hypothesis. Overall, results showed that MP are ingested but rapidly egested by marine invertebrates, which may limit MP transfer via predator-prey interactions but at the same time enhance their transfer via detrital pathways in the sediments. These processes seem to be extremely variable over time, with potential unexplored environmental consequences. This rapid dynamics also limits the conclusions that can be derived from static observations of MP contents in marine organisms, not fully capturing the real levels of potential contaminations by marine species. This emphasises the need to consider such dynamics in future work to measure the uptake rates by organisms in natural systems.

Molecular mechanisms controlling physiological plasticity in marine mussels under the influence of natural and anthropogenic stress factors

Marine mussels are exceptionally well-adapted to live in transitional habitats where they are exposed to fluctuating environmental parameters and elevated levels of natural and anthropogenic stressors throughout their lifecycle. However, there is a dearth of information about the molecular mechanisms that assist in dealing with environmental changes. This project aims to investigate the molecular mechanisms governing acclimatory and stress responses of the Mediterranean mussel (Mytilus galloprovincialis) by addressing relevant life stages and environmental stressors of emerging concern. The experimental approach consisted of two phases to explore (i) the physiological processes at early life history and the consequences of plastic pollution and (ii) the adult physiology processes under natural habitats. As the first phase, I employed a plastic leachate (styrene monomer), and polystyrene microplastics to understand the modulation of cytoprotective mechanisms during the early embryo stages. Results revealed the onset of transcriptional impairments of genes involved in MXR-related transporters and other physiological processes induced by styrene and PS-MPs. In the second phase, as a preliminary analysis, microbiota profile of adult mussels at the tissue scale and its surrounding water was explored to understand microbiota structures that may reflect peculiar adaptations to the respective tissue functions. The broader experiment has been implemented to understand the variability of transcriptional profiles in the mussel digestive glands in the natural setting. All the genes employed in this study have shown possibilities to use as molecular biomarker responses throughout the year for monitoring the physiology of mussels living in a particular environment and, in turn, more properly detecting changes in the environment. As a whole, my studies provide insights into the interactions between environmental parameters, and intrinsic characters, and physiology of marine bivalves, and it could help to interpretation of responses correctly under stress conditions and climate change scenarios.

Characterization of microplastics ingested by marine benthos - a methodological and field-experimental study

Microplastics have become ubiquitous pollutants in the marine environment. Ingestion of microplastics by a wide range of marine organisms has been recorded both in laboratory and field studies. Despite growing concern for microplastics, few studies have evaluated their concentrations and distribution in wild populations. Further, there is a need to identify cost-effective standardized methodologies for microplastics extraction and analysis in organisms. In this thesis I present: (i) the results of a multi-scale field sampling to quantify and characterize microplastics occurrence and distribution in 4 benthic marine invertebrates from saltmarshes along the North Adriatic Italian coastal lagoons; (ii) a comparison of the effects and cost-effectiveness of two extraction protocols for microplastics isolation on microfibers and on wild collected organisms; (iii) the development of a novel field- based technique to quantify and characterize the microplastic uptake rates of wild and farmed populations of mussels (Mytilus galloprovincialis) through the analysis of their biodeposits. I found very low and patchy amounts of microplastics in the gastrointestinal tracts of sampled organisms. The omnivorous crab Carcinus aestuarii was the species with the highest amounts of microplastics, but there was a notable variation among individuals. There were no substantial differences between enzymatic and alkaline extraction methods. However, the alkaline extraction was quicker and cheaper. Biodeposit traps proved to be an effective method to estimate mussel ingestion rates. However their performance differed significantly among sites, suggesting that the method, as currently designed, is sensible to local environmental conditions. There were no differences in the ingestion rates of microplastics between farmed and wild mussels. The estimates of microplastic ingestion and the validated procedures for their extraction provide a strong basis for future work on microplastic pollution.

Distribuzione spaziale e temporale delle specie macrobentoniche aliene e native in ambienti portuali

La maggior parte degli insediamenti urbani si trova in prossimità delle coste e più del 50% della popolazione vive entro 100 km dal mare. Questo significa che l’ambiente marino è fortemente influenzato dalle attività antropiche e, per questo, è necessario creare un piano di gestione degli ambienti costieri, in quanto la resilienza dell’habitat marino dipende dall’uso sostenibile degli ecosistemi marini urbani. Lo scopo dello studio è quello di analizzare la distribuzione spaziale e temporale delle specie native e aliene, presenti sulle banchine del porto-canale di Ravenna. Sono stati analizzati i popolamenti intertidali di 6 siti lungo il canale Candiano dal 2016 al 2018 nei mesi di maggio. I risultati hanno illustrato un gradiente terra-mare a partire dai siti più interni lungo il porto-canale. Le specie più abbondanti identificate sono i due mitili Mytilus Galloprovincialis (specie nativa) e Xenostrobus securis (specie invasiva); queste specie si avvicendano nelle stazioni centrali del Candiano e si cedono il posto a vicenda: la specie nativa rimane nelle acque esterne e meno impattate mentre quella invasiva resta nei siti interni più soggetti a disturbi di vario tipo, come quello antropico. La terza specie più numerosa è Sphaeroma serratum, isopode nativo del Mediterraneo, presente da sempre nelle zone lagunari del ravennate; in questo studio è stato rinvenuto nelle stazioni più interne, che potrebbe sfruttare come rifugio da possibili competitori. I risultati hanno mostrato l’abbondanza e la diversità delle specie, indigene e non ,del porto di Ravenna: su un totale di 71 taxa diversi, le specie aliene rappresentavano il 17%; queste sono state rinvenute soprattutto nei siti di campionamento più interni, dove sono presenti condizioni più limitanti e sfavorevoli per le specie native. Inoltre, è stato osservato come la diversità specifica aumenti lungo il gradiente mare-terra, considerando le zone con maggiore diversità anche quelle con maggiore specie aliene.