Conceptual and methodological advances for holobiont research.

Abstract

Having worked my entire career in a marine science institute, I have witnessed firsthand how microbiology has transformed from being ‘out of sight/out of mind’ to being central to our understanding of marine ecosystem processes. As an early career researcher, I clearly remember joining a workshop of senior scientists aimed at establishing a large research program on marine biodiversity. Plucking up the courage to propose they include microorganisms in their biodiversity assessments (since most marine diversity and biomass is microbial), I was first humoured, before being completely ignored. Moving forward, there is now barely a single research project in the same marine institute that doesn't consider the importance of microbial life, their role in primary production, in biogeochemical cycling, their utility as sensitive bioindicators of anthropogenic disturbance, as aetiological agents of disease, as a source of novel secondary metabolites, even their role in reef recovery as microbial biofilms induce metamorphosis of organisms such as the corals which build reef ecosystems (Bourne and Webster, 2013). Gazing into my crystal ball of where to next for our field, there is little doubt we are on the precipice of a revolution in holobiont research (McFall-Ngai et al., 2013). The term holobiont was first introduced back in 1991 by Lynne Margulis to describe all of the components of a symbiotic system, i.e. the host and its associated microbiome and virome (Margulis, 1991). In the early stages of holobiont research, there was consensus that microbes were important for the health and fitness of their hosts, but with the exception of a few classical, low diversity model systems (e.g. the bobtail squid and Vibrio fischerii (McFall-Ngai, 2008)) there were few insights into ‘why’ these symbiotic microorganisms were so fundamentally essential. However, application of new technologies including the unfolding ‘omics’ era is powering a wealth of discoveries into microbial physiologies that reveal the exquisite interplay between marine host and symbiont biology and ecology (e.g. see Lin et al., 2015). While new discoveries unfold almost daily, one area where we have failed to gain much conceptual momentum is in the contribution of microorganisms to host tolerance and their ability to facilitate acclimatisation of marine species in fluctuating and changing environments. We currently live in an era of rapid environmental change. Marine environments are becoming increasingly threatened by localized impacts such as declining water quality and global pressures derived from human-induced climate change (Hoegh-Guldberg et al., 2007; De'ath et al., 2012). The consequences of environmentally induced symbiont disruption can be devastating, exemplified by the recent global coral bleaching event or any number of reported disease outbreaks that have decimated populations of marine invertebrates (Harvell et al., 2007). Numerous field and experimental studies have shown that microorganisms associated with marine species shift with environmental perturbation (Bourne et al., 2008; Vega Thurber et al., 2009; Fan et al., 2012). However, with the exception of the coral-Symbiodinium model (e.g. see Howells et al., 2016; Figure 1), we have limited understanding of whether these shifts in symbiosis influence host tolerance or their ability to acclimatise or adapt to the new environmental conditions. Environmentally driven shifts in the microbiome can have significant functional consequences for the holobiont upon which selection can act. Local acclimatization of the holobiont could occur by recombination and mutation of symbiont genomes or via changes in the abundance of microbial symbionts, acquisition of novel microbial strains or horizontal gene transfer (Bordenstein and Theis, 2015). In particular, these latter processes can occur very rapidly when environmental conditions change which may be critically important for the evolution of marine species. Many marine species employ vertical transmission, or at least a combination of vertical transmission and acquisition from the environment to maintain their symbiont populations between generations (Webster et al., 2010). Transgenerational acclimatization of the holobiont could therefore theoretically occur if specific microorganisms or microbial genes that provide a fitness advantage to the host under stressed conditions are maintained and passed to subsequent generations. Corals form the essential framework of reef ecosystems. At the core of a healthy Acropora coral (A) is a dynamic relationship with microorganisms, including a mutually beneficial symbiosis with photosynthetic dinoflagellates (Symbiodinium) (B). It is also perhaps timely to explore the potential to microbially augment the tolerance of marine species, enhance their resilience or facilitate their recovery post-disturbance. A number of valid ethical and feasibility questions immediately spring to mind: (i) what are the unintended ecological consequences of such microbial manipulation, particularly in open, highly connected marine systems? (ii) will research focussed on microbiome manipulation detract from efforts to reduce the source of environmental pressure, (iii) as guardians of our marine environments, do we really want ‘Frankenstein’ species in our natural ecosystems? However, I would argue that we are already employing ‘microbial manipulation’ in human systems: probiotics promote a healthy gut microbiome (Gareau et al., 2010), faecal transplants are used to facilitate recovery from Clostridium difficile infections (Rohlke and Stollman, 2012) and phage therapy is used to treat a whole suite of diseases ranging from gastrointestinal infections to methicillin-resistant Staphylococcus aureus (Abedon et al., 2011). In a similar way, salt, drought and cold tolerance of commercially important plant crops are now being enhanced via inoculation with non-native fungal endophytes (Redman et al., 2011). The concerns expressed above are all valid, but shouldn't we at least be exploring novel solutions for our declining marine environments, particularly if the organisms and ecosystems at risk are on the brink of collapse? Phage therapy has already been used in some marine species, primarily in closed aquaculture systems, to treat infections caused by pathogens such as Vibrio anguillarum (Silva et al., 2014) but also in open reef systems to treat localized coral disease caused by Vibrio coralliilyticus (Efrony et al., 2007; Cohen et al., 2013) and Thalassomonas loyana (Atad et al., 2012). Enter the futuristic gene-editing tool CRISPR/Cas9 which has revolutionized the field of molecular biology by enabling researchers to apply precise, targeted changes to the genomes of living cells. This technology will have transformational effects on the field of holobiont research. While the potential of the CRISPR/Cas9 system for genome editing was only recognized a few years ago (Cong et al., 2013; Mali et al., 2013), there has already been an explosion of studies using this system to modify important genes in humans, plants, bacteria, yeast and a range of other animal model systems (reviewed by Jiang and Marraffini, 2015). Mutation of the Cas9 enzyme allows binding to the site that matches the guide RNA (gRNA), though prevents cutting, thereby effectively blocking other proteins from transcribing DNA into RNA and providing an elegant system to turn genes on and off without altering the DNA sequence (Qi et al., 2013). This approach therefore offers the ability to probe the functions of genes and gene regulators with unprecedented specificity which will undoubtedly affect a paradigm shift in our understanding of the genomic basis of symbiotic interactions in holobiont systems. Further, coupling of modified Cas9 enzymes to epigenetic modifiers such as those that add methyl groups to DNA or acetyl groups to histone proteins, will transform not only our understanding of the molecular basis of tolerance, but also our ability to enhance the environmental tolerance of marine holobionts. Researchers can now study how precisely placed epigenetic modifications affect gene expression and DNA dynamics in both host and symbiont populations. Researchers are also taking advantage of this method for assisted evolution, by introducing single point mutations (deletions or insertions) into particular target genes via a single gRNA or inducing large deletions or genomic rearrangements using pairs of gRNA-directed Cas9 nucleases. So returning my gaze to the crystal ball of holobiont research, how long can it be before we start trialing these genome editing approaches to enhance the environmental tolerance of marine holobionts, particularly critical, but endangered ecosystem engineers such as corals and their photosynthetic dinoflagellates? The Acropora polyp image was kindly provided by Katarina Damjanovic. David Bourne, Andrew Negri, Heidi Luter, Mari Carmen Pineda and Blake Ramsby are thanked for helpful discussions on these topics. N.S.W is funded by an Australian Research Council Future Fellowship FT120100480.