Dynamics of the Sydney rock oyster microbiota before and during a QX disease event

Highlights

• Sydney rock oyster microbiota are different between QX positive and QX negative oysters before and during a disease event

• Microbiota variations are driven by relative abundance changes of several key OTUs

• There may be an important link between Mycoplasma and Borrelia species and, the health state of Sydney rock oysters

Abstract

The Sydney rock oyster (SRO; Saccostrea glomerata) is the most intensively farmed oyster species in Australia however, Queensland unknown (QX) disease has resulted in substantial losses and impeded productivity. QX disease is caused by infection with the parasite Marteilia sydneyi, and like other diseases, outbreaks are driven by a series of complex environmental and host factors such as seasonality, seawater salinity and oyster genetics. A potential but understudied factor in QX disease is the SRO microbiota, which we sought to examine before and during a QX disease outbreak. Using 16S rRNA (V1 – V3 region) amplicon sequencing, we examined the microbiota of SROs deployed in an estuary where QX disease occurs, with sampling conducted fortnightly over 22 weeks. Marteilia sydneyi was detected in the SROs by PCR (QX-positive), 16 weeks after the first sampling event and sporonts were observed in the digestive gland two weeks later on. There were no apparent patterns observed between the microbiota of QX-positive SROs with and without digestive gland sporonts however, the microbiota of QX-positive SROs was significantly different from those sampled prior to detection of M. sydneyi and from those negative for M. sydneyi post detection. As a result, shifts in microbiota structure occurred before sporulation in the digestive gland and either before or shortly after pathogen colonisation. The microbiota shifts associated with QX-positive oysters were principally driven by a relative abundance increase of operational taxonomic units (OTUs) assigned to unclassified species of the Borrelia and Candidatus Hepatoplasma genera and a relative abundance decrease in an OTU assigned to an unclassified species of the Mycoplasma genus. Since Mycoplasma species are common microbiota features of SROs and other oysters, we propose that there may be an important ecological link between Mycoplasma species and the health state of SROs.

Introduction

The Sydney rock oyster (SRO; Saccostrea glomerata) is native to Australia, where it is the most intensively cultivated oyster species (O'Connor and Dove, 2009; Schrobback et al., 2014) however, the SRO industry has been significantly impacted by a disease called QX (Queensland Unknown Disease), which has caused annual losses of SRO stocks of up to 100% in some cultivation regions (Peters and Raftos, 2003). QX disease was first detected in the late 1960s in Moreton Bay in the north-eastern Australian state of Queensland (Wolf, 1972). Since the late 1970s, QX has extensively spread across Queensland (Adlard and Ernst, 1995) and southwards into several New South Wales estuaries (Nell, 2007; Raftos et al., 2014). The disease is caused by a spore-forming protozoan parasite called Marteilia sydneyi that initiates its infection in the oyster's palps and gills as a uninucleate stem cell, and then over several weeks, migrates through connective tissue and the haemolymph into the digestive gland (Wolf, 1979; Kleeman et al., 2002). Once in the digestive gland, the parasite undergoes sporulation, forming mature sporonts containing two tricellular spores (Wolf, 1979; Kleeman et al., 2002) and causing blockage in the digestive gland resulting in starvation and death (Wolf, 1979).

Notably, the presence of M. sydneyi within an SRO farming estuary does not necessarily result in a QX disease outbreak (Adlard and Wesche, 2005) indicating that other factors, beyond the presence of the pathogen, are important for infection or progression of disease. For example, infection is thought to require an intermediate host(s) (Raftos et al., 2014). As with other oyster diseases, QX disease is likely driven by a convergence of environmental (e.g. water chemistry, temperature), host-specific (e.g. immunity and stress level) and pathogen-specific factors (Green et al., 2011; Raftos et al., 2014; King et al., 2019b). QX disease is seasonally recurrent, generally occurring in summer or autumn (depending on the location) (Wolf, 1979; Adlard and Ernst, 1995; Nell, 2007; Rubio et al., 2013). Additionally, low seawater salinity is considered a major contributing factor (Lester, 1986; Rubio et al., 2013) possibly through its inhibition of phenoloxidase (PO) activity in SROs (Butt et al., 2006), an enzyme in invertebrates that initiates host immune defences (Söderhäll and Cerenius, 1998). Decreased PO activity in SROs is known to be associated with increased susceptibility to QX disease (Peters and Raftos, 2003; Butt and Raftos, 2007) although the full mechanism(s) by which PO is involved in QX disease resistance is unresolved.

Within the more studied Pacific oyster (Magallana gigas, formally Crassostrea gigas) system, the microbiota is emerging as a key factor in disease dynamics (Petton et al., 2015; King et al., 2019b). For example, Pacific oysters with common genetics but varying microbiota have different mortality outcomes when challenged with the viral pathogen OsHV-1 (Pathirana et al., 2019). This is possibly explained by the fact that OsHV-1 supresses Pacific oyster immunity allowing opportunistic pathogens such as Vibrio species to infect (de Lorgeril et al., 2018). If Pacific oysters contain lower levels of opportunistic pathogens in their microbiota then they are less likely to be exposed to bacterial infection post OsHV-1 infection (Petton et al., 2015; King et al., 2019c; Pathirana et al., 2019). Additionally, other studies have made links between the oyster microbiota and disease (Lokmer and Wegner, 2015; King et al., 2019d), including one study that demonstrated the progressive replacement of a benign Vibrio population in the Pacific oyster microbiota with a virulent population during a mortality outbreak (Lemire et al., 2015) and suggesting that non-virulent bacteria may facilitate the disease of virulent bacteria (Lemire et al., 2015). Given the importance of the oyster microbiota within the disease dynamics of other oyster species, we propose that shifts in the SRO microbiota might also play a role in QX disease.

Previously, a clone library-based approach demonstrated that the digestive gland microbiota of SROs containing sporulating M. sydneyi is significantly different from uninfected oysters, with QX infected oysters dominated by an OTU closely related to a member of the Rickettsiales (see Green and Barnes, 2010). As sporulation in the digestive gland occurs in the late stages of QX disease, it is not possible to know if this OTU emerged prior to infection or as a consequence of infection and, whether it has a role in facilitating infection or driving QX disease progression. Using 16S rRNA amplicon sequencing, we have recently shown that the SRO microbiota associated with the adductor muscle is dominated by OTUs assigned to unclassified species of the Candidatus Hepatoplasma, Endozoicomonas and Mycoplasma genera, and that the microbiota is significantly influenced by location and season (Nguyen et al., 2020). Additionally, we found that selective breeding of SROs for QX disease resistance influences the structure of the microbiota, but only in winter before the typical QX disease period (late summer or early autumn) with OTUs assigned to unclassified species of the Mycoplasma, Borrelia and Endozoicomonas genera over-represented in the QX resistant SRO microbiota and OTUs assigned to unclassified species of the Pseudoalteromonas, Vibrio, and Candidatus Hepatoplasma genera over-represented in QX sensitive SRO microbiota (Nguyen et al., 2020). During this previous work, the SROs were deployed in non-QX disease areas and only two time points (one time point each in the Austral summer and winter) were compared therefore, a more comprehensive investigation of the SRO microbiota in QX disease dynamics is warranted. Here we employed fortnightly sampling to examine temporal shifts in the SRO microbiota before and during a QX disease event.