Acquisition of bacterial associates and their roles in early coral life-stages
Coral sexual reproduction occurs either through spawning where gametes are released into the water column, or through internally fertilizing gametes and brooding larvae inside the coral polyp (Fadlallah 1983). In spawning corals, the establishment of coral-bacteria symbioses is widely believed to happen horizontally (i.e., through acquisition of symbionts from the environment) during the coral pelagic larval phase, or even after settlement and metamorphosis (Apprill et al. 2009; Sharp et al. 2010). Conversely, in brooders, bacteria can be vertically transmitted (i.e., directly passed from the parent colony to the planula before its release). Bacterial communities exhibit dynamic changes between the different ontogenetic stages of coral development (Damjanovic et al. 2019; Epstein et al. 2019), likely reflecting a succession of microbial functions relevant for the holobiont (Bernasconi et al. 2019) and potentially following a winnowing process (Epstein et al. 2019).
A broad phylogenetic range of bacteria can be vertically or horizontally acquired. The most common associates of gametes, embryos, and larvae of brooders and spawners include the genera Roseobacter, Marinobacter, Alteromonas, Vibrio, Bradyrhizobia, and Endozoicomonas. Roseobacter- and Alteromonas-affiliated sequences are consistently found in very early developmental stages of several coral species (Apprill et al. 2009; Ceh et al. 2012, 2013; Sharp et al. 2012). These taxa are metabolically diverse and have been linked with the production of antibiotic compounds to counter coral pathogens (Piekarski et al. 2009). Furthermore, Alteromonas spp., Vibrio spp., and the diazotroph Bradyrhizobia might provide biologically available nitrogen to coral larvae (Ceh et al. 2013; Lema et al. 2014). The transmission of potentially beneficial bacteria from parent coral colonies to gametes or early ontogenetic stages might be a mechanism to ensure microbial inheritance across generations. In addition, bacterial behavior is likely to play a role in microbiome establishment. Indeed, many environmentally acquired bacterial symbionts use chemotaxis—the ability to direct their movement towards or away from specific chemicals—to locate their hosts (Raina et al. 2019). Chemotaxis may be a particularly prevalent mechanism employed on reefs because the coral surface is characterized by strong gradients of organic compounds that can act as cues for microorganisms (Ochsenkiihn et al. 2018). Several coral- associated bacterial families, such as Endozoicomonadaceae. Rhodobacteraceae, or Oceanospirillaceae, exhibit chemoattraction towards constituents of coral mucus (Tout et al. 2015). Chemotaxis and motility, however, can also enable coral pathogens, such as Vibrio shiloi and V. coralliilyticus, to locate and infect their hosts (Banin et al. 2001;
Meron et al. 2009) using chemical cues such as dimethylsulfoniopropionate (DMSP) present in the coral mucus (Garren et al. 2014).
Bacteria are also directly involved in the transition between pelagic and benthic life stages in the coral life cycle. Indeed, after a pelagic phase (typically ranging from a week to more than 100 days), coral larvae must attach to a suitable reef structure to metamorphose into colony-forming juveniles (Connolly and Baird 20Ю). Habitat-specific environmental cues, mainly produced by specific bacteria associated with crustose coralline algae (CCA), contribute to coral larval recruitment and metamorphosis (Harrington et al. 2004; Webster et al. 2004; Tebben et al. 2015). Clear shifts in the bacterial community structure of CCA occur following thermal stress, resulting in significant reduction of coral larvae recruitment (Webster et al. 2011). Similarly, antibiotic treatment of larval cultures reduced settlement rates, suggesting that the presence of certain bacteria is essential for settlement induction (Vermeij et al. 2009).
Understanding the dynamic interplay of coral-associated microbes in relation to the prevailing environment is deeply interconnected with the setup, maintenance, and inheritance of microbial symbiotic relationships. The underlying premise is that microbial associates can adapt quickly to the surrounding environment and contribute functions that support coral holobiont health and resilience (Reshef et al. 2006; Ziegler et al. 2017, 2019; Bang et al. 2018). In recent years, efforts have been channeled into coral "probiotics” applications. Their ultimate goal is to fast-track ecological adaptation to global climate change by designing physiologically augmented coral holobionts (Peixoto et al. 2017). The research field of coral probiotics includes the isolation and screening of native bacterial associates for functional genes beneficial to coral health, and subsequent physiological assays to determine holobiont performance after inoculation with putatively beneficial bacterial isolates (Rosado et al. 2018). Experimental inoculation with mixed consortia of native coral bacterial isolates harboring dinitrogen fixation (nifH), denitrification (nirK), and DMSP-degrading (dmdA) genes resulted in partial mitigation of coral bleaching compared to controls or corals challenged with the temperature-dependent pathogen Vibrio coralliilyticus (Rosado et al. 2018). Open questions to this line of research are the temporal stability of the observed beneficial effects, the underlying mechanistic nature, and the potential for application of coral probiotics at the reef scale.
Contribution of bacteria to holobiont nutrient cycling
The ubiquitous coral symbionts Endozoicomonas harbor large numbers of genes involved in amino acid synthesis and carbohydrate cycling, suggesting its involvement in holobiont nutrient cycling (Neave et al. 2017), with different strains potentially exhibiting a different genetic and metabolic makeup (Neave et al. 2017; Pogoreutz et al. 2018). Taxonomy-based functional inference was used recently and suggested a role of Endozoicomonadaceae in processes related to nitrate reduction in giant clams (Rossbach et al. 2019). As such. Endozoicomonadaceae may provide otherwise inaccessible nitrogen sources, including ammonia, to the coral host.
Nitrogen (N) cycling is a critical component of holobiont health (Cardini et al. 2014; Radecker et al. 2015; Pogoreutz et al. 2017a). Most research on nitrogen cycling in the coral holobiont has focused on prokaryotic dinitrogen (N2) fixation (Shashar et al. 1994; Lesser et al. 2007; Radecker et al. 2014; Bednarz et al. 2017), while the assessment of other major nitrogen cycling pathways such as nitrification, denitrification, and ANAMMOX has only received marginal attention to date (Wafar et al. 1990; Tilstra et al. 2019), and is currently limited to describing the presence of functional genes in sequencing datasets (Wegley et al. 2007; Siboni et al. 2008; Neave et al. 2017). Prokaryotic N2 fixation commonly occurs in reef-building corals, helping supply the holobiont with “new” bioavailable nitrogen (Lesser et al. 2007; Cardini et al. 2015; Benavides et al. 2017). The biologically fixed nitrogen is then assimilated by both, coral (Benavides et al. 2016; Bednarz et al. 2017) and Symbiodiniaceae (Lesser et al. 2007; Cardini et al. 2015; Pogoreutz et al. 2017a). Nitrogen assimilation rates, however, appear to depend on environmental nitrogen availability, highlighting the importance of integrating environmental context into the study of coral holobiont function. The particular role of N2 fixation to coral heat stress (bleaching) remains to be determined. Elevated temperatures rapidly cause an increase in the relative abundance and activity of coral-associated N, fixers (Santos et al. 2014; Cardini et al. 2016). It was previously concluded that excess nitrogen supply has the potential to ameliorate the effects of heat stress caused by global warming in corals (Santos et al. 2014; Cardini et al. 2016). An increase in holobiont N, fixation, however, can shift the N:P ratio of dinoflagellate symbionts in Red Sea corals (Pogoreutz et al. 2017a), thereby destabilizing the coral-algae symbiosis, resulting in coral bleaching (Wiedenmann et al. 2012). Ultimately, whether increases in N2 fixation during heat stress have beneficial or detrimental effects on coral holobiont health may likely be determined by the environmental context (e.g., ambient nutrient regime), host nutritional state, or heterotrophic capacity (Bednarz et al. 2017; Pogoreutz et al. 2017b), and will require further mechanistic studies considering all major nitrogen cycling pathways.
In addition, symbiotic interactions between corals and bacteria might involve the cycling of essential compounds, for instance vitamins. Indeed, the cnidarian host may rely on bacterial symbionts for the provision of cobalamin, which is required for methionine synthesis by both corals and Symbiodiniaceae (Robbins et al. 2019). Concentrations of cobalamin in the coral gastrovascular cavity (coelenteron) are up to 35 times higher than in surrounding reef waters (Agostini et al. 2009), strongly suggesting that the dense bacterial communities harbored in the gastrovascular cavity are producing this essential molecule. In addition, some species from the genus Acropora are lacking an essential enzyme to synthesize the amino acid cysteine (Shinzato et al. 2011), and likely rely on their associated microbes for its provision.
Archaea associated with the coral holobiont
Corals associate with a diversity of archaea including representatives from the Crenarchaeota and Euryarchaeota. Crenarchaeota of the class Thermoprotei often dominate the archaeal community, while most abundant euryarchaeal members are affiliated to the Marine Group II and Thermoplasma (Kellogg 2004; Siboni et al.
2008). Interestingly, SML-associated archaeal sequences are most similar to obligate and facultative anaerobic and uncultivated archaea from anoxic environments, suggesting anaerobic microniches within the SML (Kellogg 2004). In terms of absolute abundance, archaeal cell numbers can comprise up to half of the prokaryotic community with an average of >107cells/cm2 on the surface of Porites astreoides colonies (Wegley et al. 2004).
The diversity of ammonia-oxidizing archaea (AOA) has also been evaluated in coral tissues by amplifying the amoA gene encoding the alpha-subunit of the ammonia monooxygenase (Beman et al. 2007; Siboni et al. 2008). In addition, AOA are suggested to be less host-specific and more geographically dependent (Siboni et al. 2012). The presence of amo genes in coral-associated archaea was also supported by an integrated genomic approach of Thaumarchaeota genomes assembled from Porites lutea metagenomes (Robbins et al. 2019). This study revealed the presence of other relevant key metabolic pathways, including the reductive tricarboxylic acid cycle, cobalamin synthesis, and taurine dioxygenase in the Thaumarchaeota genomes, suggesting these symbionts may contribute to the host’s demand for essential vitamins and carbon metabolism (Robbins et al. 2019).
Protists and fungi associated with the coral holobiont
Two photosynthetic alveolates, Chromera velia (Moore et al. 2008) and Vitrella brassicaformis (Obornik et al. 2012), which are the closest free-living relatives of the large parasitic phylum Apicomplexa, are protists commonly associated with corals worldwide (JanouSkovec et al. 2013). A recent transcriptomic study revealed that the coral host response to C. velia inoculation was similar to that of a parasite or pathogen infection in vertebrates (Mohamed et al. 2018). This suggests that C. velia, despite its photoautotrophic capabilities, is not involved in mutualistic interactions with corals, but rather parasitic or commensal (Mohamed et al. 2018). In addition to these two alveolates, the presence of apicomplexans in coral tissues has been reported for the past 30 years (Upton and Peters 1986; Toller et al. 2002; Slapeta and Linares 2013; Clerissi et al. 2018) and it was recently revealed that a single apicomplexan lineage is ubiquitously associated with corals, and might be the second most abundant microeukaryote group (after Symbiodiniaceae) associated with coral tissues (JanouSkovec et al. 2012; Kwong et al. 2019). Although the nature of the association between these “corallicolids” and the coral host remains unknown, their genomes lack all genes encoding for photosystem proteins, but retained the four ancestral genes involved in chlorophyll biosynthesis (Kwong et al. 2019).
Deeper in the skeleton, endolithic protist algae can form dense bands visible to the unaided eye below the tissues of many coral species and are often dominated by the filamentous green algae Ostreobium spp. (Siphonales, Chlorophyta) (Kornmann and Sahling 1980). Recent molecular studies have revealed the astonishing genetic diversity of this group, with up to 80 taxonomic units at the near-species level (Marcelino and Verbruggen 2016; Marcelino et al. 2017, 2018; Verbruggen et al. 2017). These filamentous algae colonize the skeleton of coral juveniles early in their development (Masse et al. 2018) and can interact with the coral tissue through transfers of photosynthates (Schlichter et al. 1995; Fine and Loya 2002; Pernice et al.
2019). High-throughput amplicon sequencing has also revealed the presence of other, less abundant, boring green microalgae closely related to Phaeophila, Bryopsis, Chlorodesmis, Cladophora, Pseudulvella, and red algae from the Bangiales order in coral skeletons (Marcelino and Verbruggen 2016).
Fungi are prevalent in corals and have been well studied in the coral skeleton where they penetrate the calcium carbonate microstructures and ultimately interact with Ostreobium cells (Le Campion-Alsumard et al. 1995). Along with endolithic algae, fungi are present in the newly deposited coral skeleton (Bentis et al. 2000; Golubic et al. 2005), where they exhibit rapid growth to match skeletal accretion (Le Campion-Alsumard et al. 1995). Fungi were the most abundant microorganisms in the Porites astreoides metagenome, contributing to 38% of the microbial sequences (Wegley et al. 2007). These fungi belonged mainly to the phylum Ascomycota, but also members of Basidiomycota and Chytridiomycota were detected. Based on their genomic potential, these organisms might play a role in nitrogen recycling through the reduction of nitrate and nitrite to ammonia and subsequent ammonia assimilation (Wegley et al. 2007).