Investigating the Environmental Survival of Marteilia refringens, a Marine Protozoan Parasite of the Flat Oyster Ostrea edulis, Through an Environmental DNA and Microscopy-Based Approach

Nicolas Mérou,Bruno Chollet,Stéphane Pouvreau,Cyrielle Lecadet,Tom Billon,Isabelle Arzul

doi:10.3389/fmars.2022.811284

Abstract

Outside-host parasite survival is a key parameter to better understand disease mechanisms, especially for marine pathogens transmitted from one host to another through an environmental stage. For non-cultivable micro-parasites like Marteilia refringens, a protozoan parasite infecting the flat oyster Ostrea edulis, investigating this parameter requires innovative approaches. In the present study, we have developed an Environmental DNA (eDNA)-based method allowing detecting and quantifying up to 25 and 10 parasites DNA in seawater and sediment, respectively. This method was used in combination with light and transmission electron microscopy (TEM) to study experimentally parasite survival in seawater and flat oyster faeces after its release from naturally infected oysters. M. refringens DNA could be detected up to 20 days, in both seawater and oyster faeces with a more stable detection over time in the latter. Light and transmission microscopy confirm that parasites leaving flat oysters are sporangia. We also observed a membrane dissolution of the sporangia over time that could indicate the release of parasite spore. This study not only improves our understanding of M. refringens ecology but also highlights the interest to combine molecular and microscopical analysis to study non-cultivable micro-parasites life cycle outside their host.

Highlights

The native European oyster, Ostrea edulis (Linnaeus, 1758) is naturally found from the Atlantic Ocean and North Sea to the Mediterranean, Adriatic and Black Seas
Three years old flat oysters O. edulis originated from a hatchery in La Rochelle (France) and maintained in claire ponds known to be infected with M. refringens on Oleron Island (NouvelleAquitaine, France) (Audemard et al, 2001) from April 2018 to April 2019 were maintained individually during 1,5 week at room temperature in 2 L aquaria containing decanted seawater: every three days, faeces samples were collected from each oyster and examined under light microscopy using Malassez-cell haemocytometer to check the presence of M. refringens
Real-Time PCR limit of detection (LOD), limit of quantification (LOQ), and efficiency were determined following analysis of DNA extracted from 1:10 dilution series of purified M. refringens applied on 1 μm polycarbonate membranes or mixed with 0.25 g of sediment

Summary

Introduction

The native European oyster, Ostrea edulis (Linnaeus, 1758) is naturally found from the Atlantic Ocean and North Sea to the Mediterranean, Adriatic and Black Seas. This oyster species has been consumed since Roman times. O. edulis populations showed first signs of overfishing in the XVIIIth century. Outside-Host Marteilia refringens Survival of O. edulis in Europe is explained by the emergence of epizootic diseases, including marteiliois (Buestel et al, 2009; Pogoda et al, 2019). Because of its impact on natural and farmed flat oyster populations, detection of this infection requires mandatory notification to the World Organization for Animal Health (OIE, 2021)

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Conclusion