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.