Alien Vs. Predator: Pathogens Open Niche Space for Opportunists, Unless Controlled by Predators

A peer-reviewed version of this preprint was published in PeerJ on 31 May 2017. View the peer-reviewed version (peerj.com/articles/3315), which is the preferred citable publication unless you speciﬁcally need to cite this preprint. Welsh RM, Rosales SM, Zaneveld JR, Payet JP, McMinds R, Hubbs SL, Vega Thurber RL. 2017. Alien vs. predator: bacterial challenge alters coral microbiomes unless controlled by Halobacteriovorax predators. PeerJ 5:e3315 https://doi.org/10.7717/peerj.3315 Alien vs. Predator: Pathogens open niche space for opportunists, unless controlled by predators Rory M Welsh, Rebecca Vega Thurber, Jesse R.R. Zaneveld, Jerome P Payet, Ryan McMinds, Stephanie M Rosales, Steve L Hubbs Coral microbiomes are known to play important roles in organismal health, response to environmental stress, and resistance to disease. Pathogens invading the coral microbiome encounter diverse assemblages of resident bacteria, ranging from defensive and metabolic symbionts to opportunistic bacteria that may turn harmful in compromised hosts. However, little is known about how these bacterial interactions influence the overall structure, stability, and function of the microbiome during the course of pathogen challenge. We sought to test how coral microbiome dynamics were affected by interactions between two of its members: Vibrio coralliilyticus, a known temperature- dependent coral pathogen, and Halobacteriovorax, a unique bacterial predator of Vibrio and other gram-negative bacteria. We challenged specimens of the important reef-building coral Montastraea cavernosa with Vibrio coralliilyticus pathogens in the presence or absence of Halobacteriovorax predators, and monitored microbial community dynamics with 16S rRNA gene time-series. In addition to its direct effects on corals, pathogen challenge reshaped coral microbiomes in ways that allowed for secondary blooms of opportunistic bacteria. As expected, Vibrio coralliilyticus addition increased the infiltration of Vibrio into coral tissues. This increase of Vibrios in coral tissue was accompanied by increased richness, and reduced stability (increased beta-diversity) of the rest of the microbiome, suggesting strong secondary effects of pathogen invasion on commensal and mutualistic coral bacteria. Moreover, after an initial increase in Vibrios, two opportunistic lineages (Rhodobacterales and Cytophagales) increased in coral tissues, suggesting that this pathogen opens niche space for opportunists. Based on the keystone role of predators in many ecosystems, we hypothesized that Halobacteriovorax predators might help protect corals by consuming gram-negative pathogens. In keeping with a protective role, Halobacteriovorax addition alone had only minor effects on the microbiome, and no infiltration of Halobacteriovorax into coral tissues was detected in amplicon libraries. Simultaneous challenge with both pathogen and predator eliminated detectable V. corallyticus infiltration into coral tissue samples, ameliorated changes to the rest of the coral microbiome, and prevented secondary blooms of opportunistic Rhodobacterales and Cytophagales. Thus, we show that primary infection by a coral pathogen is sufficient to PeerJ PrePrints | https://dx.doi.org/10.7287/peerj.preprints.1537v1 | CC-BY 4.0 Open Access | rec: 26 Nov 2015, publ: 26 Nov 2015 cause increases in opportunists, as seen in correlational studies. These data further provide a proof-of-principle demonstration that, under certain circumstances, host- associated bacterial predators can mitigate the ability of pathogens to infiltrate host tissue, and stabilize the microbiome against complex secondary changes that favor growth of opportunistic lineages. 1 Alien vs. Predator: Pathogens open niche space for opportunists, unless controlled by 2 predators 3 4 Rory M. Welsh1, Stephanie M. Rosales1, Jesse R. Zaneveld1, Jérôme P. Payet1, Ryan McMinds1, 5 Steven L. Hubbs1, and Rebecca Vega Thurber1 6 1Oregon State University, Dept. of Microbiology, 454 Nash Hall, Corvallis, OR, 97331, USA 7 8 Corresponding Author: 9 Rebecca Vega Thurber1 10 Oregon State University, Dept. of Microbiology, 454 Nash Hall, Corvallis, OR, 97331, USA 11 Email address: [email protected] 12 13 Abstract: 14 Coral microbiomes are known to play important roles in organismal health, response to 15 environmental stress, and resistance to disease. Pathogens invading the coral microbiome 16 encounter diverse assemblages of resident bacteria, ranging from defensive and metabolic 17 symbionts to opportunistic bacteria that may turn harmful in compromised hosts. However, little 18 is known about how these bacterial interactions influence the overall structure, stability, and 19 function of the microbiome during the course of pathogen challenge. We sought to test how coral 20 microbiome dynamics were affected by interactions between two of its members: Vibrio 21 coralliilyticus, a known temperature-dependent coral pathogen, and Halobacteriovorax, a unique 22 bacterial predator of Vibrio and other gram-negative bacteria. We challenged specimens of the 23 important reef-building coral Montastraea cavernosa with Vibrio coralliilyticus pathogens in the 24 presence or absence of Halobacteriovorax predators, and monitored microbial community 25 dynamics with 16S rRNA gene time-series. In addition to its direct effects on corals, pathogen 26 challenge reshaped coral microbiomes in ways that allowed for secondary blooms of 27 opportunistic bacteria. As expected, Vibrio coralliilyticus addition increased the infiltration of 28 Vibrio into coral tissues. This increase of Vibrios in coral tissue was accompanied by increased 29 richness, and reduced stability (increased beta-diversity) of the rest of the microbiome, 30 suggesting strong secondary effects of pathogen invasion on commensal and mutualistic coral 31 bacteria. Moreover, after an initial increase in Vibrios, two opportunistic lineages 32 (Rhodobacterales and Cytophagales) increased in coral tissues, suggesting that this pathogen 33 opens niche space for opportunists. Halobacteriovorax predators are commonly present at low- 34 abundance on coral surfaces. Based on the keystone role of predators in many ecosystems, we 35 hypothesized that Halobacteriovorax predators might help protect corals by consuming gram- 36 negative pathogens. In keeping with a protective role, Halobacteriovorax addition alone had only 37 minor effects on the microbiome, and no infiltration of Halobacteriovorax into coral tissues was 38 detected in amplicon libraries. Simultaneous challenge with both pathogen and predator 39 eliminated detectable V. corallyticus infiltration into coral tissue samples, ameliorated changes to 40 the rest of the coral microbiome, and prevented secondary blooms of opportunistic 41 Rhodobacterales and Cytophagales. Thus, we show that primary infection by a coral pathogen is 42 sufficient to cause increases in opportunists, as seen in correlational studies. These data further 43 provide a proof-of-principle demonstration that, under certain circumstances, host-associated 44 bacterial predators can mitigate the ability of pathogens to infiltrate host tissue, and stabilize the 45 microbiome against complex secondary changes that favor growth of opportunistic lineages. 46 47 48 Introduction 49 Coral reefs have experienced sharp declines in coral cover from environmental factors 50 (De’ath et al., 2012), temperature induced bleaching (Fitt & Warner, 1995), and disease (Bourne 51 et al., 2009; Burge et al., 2014) with some areas of the Caribbean experiencing as much as 80% 52 coral loss over the past several decades (Gardner et al., 2003). While many studies have 53 identified microbial consortia that increase in diseased corals (e.g. Gignoux-Wolfsohn et al. 54 2015), etiological agents are unknown for the majority of coral diseases (Mouchka, Hewson & 55 Harvell, 2010). Currently Vibrio coralliilyticus is the most well described model for interactions 56 between corals, the environment, and pathogenic bacteria (Ben-Haim et al., 2003). Several V. 57 coralliilyticus virulence factors are temperature-dependent and upregulated above 27 ºC (Kimes 58 et al., 2012), and it has been suggested that host tissue invasion can only occur above this 59 threshold (Vidal-Dupiol et al., 2011). Given the continuous rise in sea surface temperatures due 60 to global climate change (Hoegh-guldberg et al., 2007), and the projected increased variability of 61 temperature extremes, it is likely that the incidence of infections by V. coralliilyticus and other 62 temperature-dependent pathogens will increase (Maynard et al., 2015). Bacterial communities of 63 diseased corals are also known to have large numbers of opportunistic pathogens and secondary 64 colonizers (Gignoux-Wolfsohn & Vollmer, 2015). It has been hypothesized that the majority of 65 coral disease may be the result of normally-benign coral microbionts that become opportunistic 66 pathogens during physiological stress to the host (Lesser et al., 2007a). Thus, the linkages 67 between infection by a primary pathogen and secondary opportunistic infection remain an area of 68 active exploration. 69 Corals also form mutualistic and commensal partnerships with diverse microorganisms, 70 ranging from endosymbiotic photosynthetic dinoflagellates (Symbiodinium spp.), to consortia of 71 archaea, fungi, and bacteria. These multi-domain communities make coral meta-organisms 72 among the most diverse systems on the planet (Bayer et al., 2013). Although the role of 73 Symbiodinium in the coral holobiont

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