Introduction
Microbial symbioses are the engine of coral reef ecosystems. Corals associate with endosymbiotic dinoflagellates of the familySymbiodiniaceae and a diverse microbiome (e.g. bacteria, archaea, viruses) which function as a unit and form a holobiont (Rohwer et al. 2002). The coral holobiont depends on key services such as nitrogen and sulfur cycling mediated by the associated microbiome (Wegley et al. 2007; Siboni et al. 2008; Raina et al. 2013; Rädecker et al. 2015). The coral surface mucous layer (SML) sustains a diverse and abundant community of these microbial partners (Koren and Rosenberg 2006; Sharon and Rosenberg 2008; Garren and Azam 2012; Ainsworth et al. 2015; Lima et al. 2020, 2022). The coral microbiome benefits from the high nitrogen content and organic matter in the SML (Wild et al. 2005; Rädecker et al. 2015) and provides protection against coral pathogens via production of antimicrobials (Ritchie 2006; Krediet et al. 2013). However, coral-associated microbial communities are sensitive to environmental changes, especially to increased temperature, which disrupt the beneficial services provided to the holobiont (Thurber et al. 2009; Vega Thurber et al. 2014; Raina et al. 2016; Zaneveld et al. 2016).
Coral reefs are at great risk of collapse as coral bleaching (i.e., loss of algal symbionts) and disease outbreaks have become more frequent in the last two decades, particularly correlated to rising seawater temperature, leading to major losses in coral cover worldwide (Willis et al. 2004; Maynard et al. 2015; Heron et al. 2016; Precht et al. 2016; Muller et al. 2018). These losses are pronounced on shallow water reefs of the Caribbean, where an overall decline in coral cover of up to 59 % has occurred since 1984 (Jackson et al. 2014). Corals live at their upper thermal limits, and therefore thermal thresholds may not be able to adjust to projected rises in seawater temperature in times of rapid environmental change (Berkelmans and Willis 1999; Fitt et al. 2001; Palumbi et al. 2014; Lough et al. 2018).
The mechanisms of resistance to environmental change in the coral holobiont are not completely understood, but the associated microbial community is a potential source of acquired heat-tolerance (Peixoto et al. 2017; Ziegler et al. 2017; Rosado et al. 2019; Doering et al. 2021; Santoro et al. 2021). There is also a potential evolutionary role of the microbiome as a source of genes (e.g. stress response genes) that can be used in thermal resilience and disease protection via mechanisms such as horizontal gene transfer (Webster and Reusch 2017). The use of metagenomics associated with physiological data in experimental settings is a recommended approach to further explore the role of the coral microbiome in heat-tolerance and stress response (Bourne et al. 2016). Another outstanding topic to be investigated is the relationship between stress and stability in the coral microbiome (Zaneveld et al. 2017).
According to the Anna Karenina Principle (AKP) for animal microbiomes, stress or disease will increase instability and result in low similarity among microbiomes exposed to the same disturbance (Zaneveld et al. 2017). In an analogy to a quote from Tostoy’s novel Anna Karenina , the AKP states that ‘all healthy microbiomes are similar; each dysbiotic microbiome is dysbiotic in its own way’. This increase in dissimilarity (i.e., microbial dispersion) among stressed microbiomes can be visualized in ordination methods (e.g., principal coordinate analysis) using a matrix of β-diversity distances between samples. When the AKP is shaping the microbial communities, healthy hosts present stable microbiomes that form tight clusters in ordination space, while stress leads to instability and higher β-diversity among microbiomes. The AKP seems to be shaping taxonomic composition in coral microbiomes under heat stress (Zaneveld et al. 2016; Ahmed et al. 2019), however, it needs to be further explored in the context of shotgun metagenomics for deeper coverage of microbial taxa and functional genes.
Here we investigate whether heat stress results in unstable coral SML microbiomes, as proposed by the AKP. We addressed these aims by exposing corals to a heat treatment and analyzing coral-algal physiological parameters and microbial taxa (genus level) and gene functions (stress response, nitrogen metabolism, and sulfur metabolism) associated with the coral SML using shotgun metagenomics.