Tissue-associated and vertically transmitted bacterial symbiont in the coral Pocillopora acuta

Abstract Coral microhabitats are colonized by a myriad of microorganisms, including diverse bacteria which are essential for host functioning and survival. However, the location, transmission, and functions of individual bacterial species living inside the coral tissues remain poorly studied. Here, we show that a previously undescribed bacterial symbiont of the coral Pocillopora acuta forms cell-associated microbial aggregates (CAMAs) within the mesenterial filaments. CAMAs were found in both adults and larval offspring, suggesting vertical transmission. In situ laser capture microdissection of CAMAs followed by 16S rRNA gene amplicon sequencing and shotgun metagenomics produced a near complete metagenome-assembled genome. We subsequently cultured the CAMA bacteria from Pocillopora acuta colonies, and sequenced and assembled their genomes. Phylogenetic analyses showed that the CAMA bacteria belong to an undescribed Endozoicomonadaceae genus and species, which we propose to name Candidatus Sororendozoicomonas aggregata gen. nov sp. nov. Metabolic pathway reconstruction from its genome sequence suggests this species can synthesize most amino acids, several B vitamins, and antioxidants, and participate in carbon cycling and prey digestion, which may be beneficial to its coral hosts. This study provides detailed insights into a new member of the widespread Endozoicomonadaceae family, thereby improving our understanding of coral holobiont functioning. Vertically transmitted, tissue-associated bacteria, such as Sororendozoicomonas aggregata may be key candidates for the development of microbiome manipulation approaches with long-term positive effects on the coral host.

Here, we characterized CAMAs present in P. acuta colonies from Orpheus Island in the central GBR.Pocillopora acuta is an asexual brooder, producing fully formed, genetically identical larvae, and a sexual broadcast spawner, releasing gametes into the water column for external fertilization [11].In a previous study, the microbiome of three Orpheus Island colonies was assessed at three different life stages, finding that Endozoicomonas is highly abundant in larvae (5%-35%), recruits (10%-40%), and adults (40%-80%) [6].Detection of CAMAs in adults and larvae indicated possible vertical transmission [6].In a separate study, monitoring of 12 colonies from the same site throughout an entire year showed high abundances of Endozoicomonas (5%-70%), highlighting the stability of the association despite environmental f luctuations [12].Using samples from these two studies (Fig. S2A and B), we verified by f luorescence in situ hybridization (FISH) that both adults (X7 colony, three branches from the one colony [12]) and asexually produced larvae (OI2 and OI3 colonies, three larvae from each of the two colonies [6]) possessed CAMAs (Fig. 1A-E).In X7 adult polyps, CAMAs localized to the mesenterial filaments (Fig. 1A-C).In OI2 and OI3 larvae, CAMAs were found in the gastrodermis (Fig. 1D and E).
To taxonomically identify the bacteria present in the CAMAs, we excised CAMAs using laser capture microdissection (LCM), followed by 16S rRNA gene amplicon sequencing, in three replicate branches of the X7 colony and two replicates of three pooled larvae from each of the OI2 and OI3 colonies (Fig. S3, Table S1).Two ASVs assigned to Kistimonas (Endozoicomonadaceae ASVs 01 and 02) made up most of the reads in OI2 larvae and X7 branches  S2.
(Fig. 1F, Table S2).Even though the samples were collected at the same site, they originated from different host colonies and were collected several months apart, highlighting the temporal stability of this association among individuals and through ontogeny.
Kistimonas is part of the Endozoicomonadaceae family along with Endozoicomonas.Because of taxonomic discrepancies discussed below, we refer to these two ASVs as Endozoicomonadaceae ASVs 01 and 02.The two ASVs were also present in OI3 larvae at a lower relative abundance.In addition, three Endozoicomonas ASVs were found in X7 and were identical to Endozoicomonas present in P. acuta from Feather Reef [3].FISH using an Endozoicomonadaceae probe provided additional support for this taxonomy in both adult and larval CAMAs (Fig. S4).
Although Kistimonas was not reported in the original studies [6,12], a reanalysis of both previous studies reassigned three Endozoicomonas ASVs to Kistimonas (Fig. S5, Table S3), two of which were 100% identical to Endozoicomonadaceae ASVs 01 and 02 found in our excised CAMA samples.Virtually no Endozoicomonas strains (only one ASV at 0.02%-0.04%relative abundance) were detected in whole larvae or recruits in our reanalysis of the earlier datasets (Fig. S5), suggesting that Endozoicomonas are acquired horizontally in Orpheus Island P. acuta.Conversely, Endozoicomonadaceae ASVs 01 and 02 were present at all life stages (i.e.larvae, recruits, and adults of the same colonies, Fig. S5) and formed CAMAs, supporting their vertical transmission during asexual reproduction.Whether whole CAMAs or individual bacteria are transmitted from parent colony to asexually produced larvae remains unknown.The possibility of vertical transmission in sexually produced offspring (i.e. through the production of gametes) remains to be investigated.Strong evidence for vertical transmission of bacteria in corals has only been reported twice before [13,14].
Genome annotation (Figs 2B and S8, Table S6) revealed Pac_P3-11-1 encodes several genes indicative of host-symbiont interactions, including Types II and III secretion systems, 35 eukaryoticlike proteins (Table S7), and three secondary metabolites with putative antimicrobial activity (Table S8).This gene repertoire is similar to that of other coral-associated Endozoicomonadaceae [3,4,[8][9][10] and may regulate host colonization, aggregation, and vertical transmission.Additionally, we recovered pathways for the biosynthesis of all amino acids (except phenylalanine, tyrosine, and arginine), as well as ribof lavin (vitamin B 2 ), pantothenate (B 5 ), pyridoxine (B 6 ), biotin (B 7 ), and folate (B 9 ), which could assist with coral metabolism [3,4,9,10,16].Pac_P3-11-1 also shows potential for the scavenging of reactive oxygen species, which are believed to drive coral bleaching when present in excess [17], as it possesses pathways for the synthesis of the antioxidants heme, lipoic acid, and glutathione, and the dsyB gene, essential for the synthesis of dimethylsulfoniopropionate [18].Finally, we uncovered high potential for carbon cycling, with a full pathway for the degradation of D-galactose into UDP-glucose, a cellobiose phosphotransferase system (celABC) and glucosidase (celF) that can import extracellular cellobiose and degrade it into UDPglucose, and an endo-chitinase that can degrade chitin into Nacetylglucosamine.Cellobiose is a major cell wall component of some algae, including Symbiodiniaceae, and chitin is a major cell wall component of zooplankton and crustaceans, all potential food sources for corals.Endo-chitinases were recently found to be widespread in Endozoicomonadaceae and are potentially exported [19].Both sets of enzymes may therefore assist the coral host with prey digestion and provide carbon-and nitrogen-rich oligo-sugars to the holobiont.
In conclusion, we described and cultured the first coral tissue-associated, vertically transmitted member of the Endozoicomonadaceae family.Sororendozoicomonas was not found in other GBR P. acuta populations that harbor Endozoicomonas [3], suggesting geographic location may impact Endozoicomonadaceae associations.Additionally, Endozoicomonas CAMAs were previously found in pocilloporid tentacles [3,4], contrasting with Sororendozoicomonas' location in the mesenterial filaments, where it could aid in digestion and nutrient acquisition from host heterotrophic feeding.Other potentially beneficial functions, such as vitamin synthesis or antioxidant abilities, may also increase host fitness.This study provides additional insights into the wide array of functions, benefits, and lifestyles of Endozoicomonadaceae within coral holobionts and reinforces the ecological importance of this bacterial family.

Figure 2 .
Figure 2. Taxonomy and functional potential of S. aggregata, CAMA member of Orpheus Island P. acuta.(A) Maximum likelihood phylogeny of Endozoicomonadaceae based on 120 marker genes and 28 Endozoicomonadaceae genomes in addition to the two genomes recovered in this study.Zooshikella was chosen as the outgroup.Bootstrap support values based on 1000 replications are provided.Additional data on the reference genomes are available in TableS5.(B) Overview of the metabolic potential from the genome sequence of Pac_P3-11-1 recovered from P. acuta CAMAs.The pathways represented here are at least 80% complete (Fig.S7).Dashed arrows represent hypotheticals.T2SS: Type II secretion system; T3SS: Type III secretion system; DMSP: dimethylsulfoniopropionate; LPS: lipopolysaccharide; NAD: nicotinamide adenine dinucleotide; GlcNAc: N-acetylglucosamine.