Influence of OSHV-1 oyster mortality episode on dissolved inorganic fluxes: An ex situ experiment at the individual scale

Highlights

• This study is the first one to show consequences of oyster mortality episode in matter cycle using a lab approach.

• Dissolved inorganic fluxes at the water-shell interface of Pacific oyster significantly varied during mortality episode.

• OsHV-1 infection decreases oxygen consumption and ammonium excretion of oyster juveniles.

• Mineralisation of the flesh of dead oysters leads to an increase in NH₄ and PO₄ fluxes and a decrease in N/P ratio.

Abstract

Ostreid herpesvirus 1 (OsHV-1 μvar) infection has caused significant mortalities in juvenile oysters (Crassostrea gigas). In contrast to the practices of other animal production industries, sick and dead oysters are not separated from live ones and are left to decay in the surrounding environment, with unknown consequences on fluxes of dissolved materials. A laboratory approach was used in this study to test the influence of oyster mortality episode on dissolved inorganic fluxes at the oyster interface, dissociating (i) the effect of viral infection on metabolism of juvenile oysters and (ii) the effect of flesh decomposition on oxygen consumption and nutrient releases at the individual scale. Nine batches of juvenile oysters (Individual Total wet weight 1 g) were infected via injection of OsHV-1 enriched inoculums at different viral loads (10⁸ and 10⁹ OsHV-1 DNA copies per oyster) to explore infection thresholds. Oysters injected with filtered seawater were used as controls (C). Oysters were maintained under standard conditions to avoid stress linked to hypoxia, starvation, or ammonia excess. Before, after the injection and during the mortality episode, i.e. at days 1, 3, 7, 10 and 14, nine oysters per treatment were incubated in individual metabolic chambers to quantify oxygen, ammonium and phosphate fluxes at the seawater-oyster interface. Nine empty chambers served as a reference. Injections of the two viral loads of OsHV-1 induced similar mortality rates (38%), beginning at day 3 and lasting until day 14. The observed mortality kinetics were slower than those reported in previous experimental pathology studies, but comparable to those observed in the field (Thau lagoon, France). This study highlights that oxygen and nutrient fluxes significantly varied during mortality episode. Indeed (i) OsHV-1 infection firstly modifies oyster metabolism, with significant decreases in oxygen consumption and ammonium excretion, and (ii) dead oysters lead to a strong increase of ammonium (6 fold) and phosphate (41 fold) fluxes and a decrease in the N/P ratio due to mineralisation of their flesh. The latter may modify the structure of the planktonic community in the field during mortality episode. This study is a first step of the MORTAFLUX program. The second step was to in situ confirm this abnormal nutrient loading during a mortality episode and show its impact on bacterio-, phyto- and protozoo-plankton.

Introduction

Mortality episodes in cultured bivalves have been reported worldwide and have been associated with infection by a range of viral and bacterial pathogens (Barbosa Solomieu et al., 2015). Thus, the history of oyster culture consists of a succession of developmental phases using different species, followed by collapses caused by diseases as described in France (Buestel et al., 2009), where the indigenous species Ostrea edulis was replaced first with Crassostrea angulata in the 1920's, then C. gigas in the 1970s. First mortalities of the Pacific oyster, Crassostrea gigas in France, were reported in the early 1990s in hatcheries and nurseries, for larval and juvenile stages, and later in the field (Nicolas et al., 1992, Renault et al., 1994). These events have been primarily attributed to a herpes-like virus, based on histological and electron microscopy examinations (Renault et al., 1994, Renault et al., 2000a, Renault et al., 2000b) and PCR procedures (Le Deuff and Renault, 1999, Renault et al., 2000b). This herpes-like virus was later described and named ostreid herpesvirus 1 (OsHV-1) (Minson et al., 2000, Davison et al., 2005). Since 2008, a new variant, OsHV-1 μvar, has emerged (Segarra et al., 2010) and has induced abnormal juvenile mortality rates ranging from 80 to 100% along the French coast (Garcia et al., 2011). Mortality of oysters coincided with infections involving the ostreid herpes virus and also bacteria of the group Vibrio splendidus (Pernet et al., 2012, Petton et al., 2015b). These oyster mortality episodes have caused important economic losses in France since 2008 (Girard and Pérez Agúndez, 2014). The development of molecular tools has subsequently allowed the detection of these viruses in many countries in Europe, USA, Australia, New Zealand and in East Asia (Mineur et al., 2014 for review). In 2016, mortalities are still a key topic since they continue to occur each year in France at rates ranging from 35 to 70% (RESCO networks: http://wwz.ifremer.fr/observatoire_conchylicole). Thus mortality of Pacific oyster remain a huge problem, both from economic and scientific points of view in France and worldwide.

The search for a better understanding of these phenomena and to address this crisis have led scientists to focus on the causes and consequences of viral infection on oyster juvenile. Some studies have described viral entry (Jouaux et al., 2013) and distribution in organs and tissues (Segarra et al., 2016); others have identified changes in immune (Green et al., 2015, Green et al., 2016, He et al., 2015, Moreau et al., 2015,), physiological and biochemical responses (Corporeau et al., 2014, Tamayo et al., 2014) in Pacific oyster spat infected with OsHV1. These studies have provided a better understanding of the interactions between OsHV-1 and oysters at the organism scale. In parallel, some studies have envisaged solutions by highlighting the factors controlling these mortality episodes, such as size, genetics (Dégremont, 2013), energetic status (Pernet et al., 2010), husbandry practices (Paul-Pont et al., 2013, Pernet et al., 2014a, Whittington et al., 2015) and temperature (Pernet et al., 2012, Renault et al., 2014, Petton et al., 2015a). However, to date, no studies have investigated the consequences of these mortality events in the environment, and especially on matter cycle.

Unlike in most other animal production industries, sick and dead individuals are not separated from conspecifics in shellfish farms, a practice that can favour cross-contamination and spread of disease. Dead oysters are kept in the rearing environment until their flesh totally disappears. The consequences of this practice for (i) the transfer of pathogens and (ii) particulate and dissolved fluxes into the environment remain unknown.

Apart from a mortality context, shellfish farms are known to modify the ecosystem functions of coastal environments. Indeed, shellfish modify particulate and dissolved fluxes via their (i) respiration (Chapelle et al., 2000, Richard et al., 2006), excretion (Mazouni, 2004, Richard et al., 2007, Jansen et al., 2012, Lacoste and Gaertner-Mazouni, 2016), (ii) filtration (Dupuy et al., 2000, Trottet et al., 2008) and (iii) biodeposition (Callier et al., 2006, Callier et al., 2009, Robert et al., 2013). At high stocking densities in confined environments, shellfish can control seston biomass via filtration (Smaal et al., 2013, Filgueira et al., 2014a, Filgueira et al., 2014b), stimulate primary production via nitrogen excretion (Chapelle et al., 2000, Souchu et al., 2001, Mazouni, 2004) and modify the microbial plankton community structure (Froján et al., 2014, Mostajir et al., 2015). Many studies have focused on this topic in adult organisms (see Cranford et al., 2003, McKindsey et al., 2006, Forrest et al., 2009, Filgueira et al., 2015 for reviews), but few data are available on the juvenile stage, either under laboratory treatments (Dame, 1972, Goulletquer, 1999) or in situ (Meseck et al., 2012, van Broekhoven et al., 2014). Filtration, respiration and excretion rates depend on organism size (see Gosling, 2015a, Gosling, 2015b for reviews), so juveniles would not have the same influence on biogeochemical fluxes and planktonic communities as would be observed for adults.

In the context of viral infection, the shellfish/environment interactions could be modified. Diseases are known to decrease filtration (Newell, 1985, Flye-Sainte-Marie et al., 2007), excretion and respiration rates (Flye-Sainte-Marie et al., 2007) of marine bivalves. In cases of mortality, oyster flesh probably decomposes and mineralises, thereby causing an increase of nutrients, as observed for mussels (Lomstein et al., 2006) and jellyfish (West et al., 2009). Increases in nutrient loading may in turn induce changes in planktonic components, but this possibility remains to be investigated in cases of oyster mortality. The aim of the MORTAFLUX project is to determine the influence of mortality episode of juvenile oyster on fluxes in the benthic-pelagic coupling of the Thau lagoon that corresponds to the most important French oyster culture site on the Mediterranean Sea.

In this paper, we present the first part of this project. The aim was to evaluate the influence of OSHV-1 mortality episode of oyster juveniles on the dissolved inorganic fluxes at the individual level, i.e. at the water-oyster shell interface, dissociating (i) the effect of viral infection on metabolism of oyster and (ii) the effect of flesh decomposition on oxygen demand and ammonium and phosphate releases at the individual scale. This first study was carried out under laboratory conditions, with the aim of inducing OsHV-1 infection by intramuscular injection of viral inoculum, as performed previously (Schikorski et al., 2011b), maintaining oysters in standard conditions used in ecophysiology to avoid any stress linked to stocking conditions (hypoxia, starvation, ammonia excess). Finally, recorded data of this first study will be up scaled from individual to the rearing structure to be further compared with in situ data issued from the second study of Mortaflux that was carried out before within and after a mortality episode in the Thau lagoon (France).