To characterize the Osedax-associated bacterial diversity, we performed 16S rRNA gene amplicon sequencing of 37 specimens collected from two Pacific Ocean sites (Table 1; Fig. 1). Based on this analysis, the Campylobacterales was identified as the dominant bacterial group associated specifically with the Osedax trunk (67 ± 1 %; Figure S1). Arcobacter, Sulfurospirillum and Sulfurimonas were the primary Osedax-associated Campylobacterales genera recovered, and specific ribotypes were distinct from those known to associate with other animals from reducing habitats (only 82% 16S rRNA gene similarity; Fig. 2, Supp Materials). The only other common bacterial 16S rRNA gene barcode was from an uncultured member of the Kordiimonadales (Alphaproteobacteria; comprising 29 ± 7 % of the microbial community; Figure S1), was an uncultured member of the Kordiimonadales (Alphaprotoebacteria), closely related to those recovered previously from the external surface of Osedax (11) and sunken wood (58).
Fluorescence microscopy showed a close association of the Campylobacterales with the trunk epithelial surface of Osedax. The Campylobacterales were the only obvious bacteria present along the epidermis, based on overlap between the universal and specific bacterial probes (Figs. 3–4). They occurred along the full length of the trunk, and appeared very closely attached to the apical end of exposed epidermal labia, although some also appeared in epidermal cavities, as was seen via TEM (Figs. 4C-D, S2). Slight autofluorescence of a matrix in which the bacteria were embedded, made the determination of their specific position inconclusive via FISH microscopy. For the mucous tube, which is secreted by numerous glands on the trunk and is used by the worm to glide up and down, only non-Campylobacterales bacteria were identified via microscopy (Figure S3). No bacteria were observed on plume or root surfaces (Fig. 3A,G), despite significant surface area of both tissues.
To explore whether Osedax’s epibiont community changes as the whale carcass degrades, we collected the annelids from two whalefalls between 8 to 172 months (> 14 years) since carcass deposition; one intentionally deposited on the seafloor at 1018m depth in the Monterey Canyon (4) and a second discovered in an early stage of decomposition at 3239m depth on the Davidson seamount, both off the coast of California (Table 1; Fig. 1). Regardless of Osedax species, Arcobacteraceae were the dominant microbial group associated with the worm trunks at early time points (< 24 months, n = 16), constituting 59% of the average recovered Campylobacterales ribotypes, a significantly higher representation than at later time points (18–28%; ANOVA p < 0.02; Fig. 5). Osedax collected at early time points had relatively few aggregations of bacteria along the trunk that were visible by microscopy, while at later time points bacteria appeared to cover much more of the epithelial surface (Figs. 3–4, compared to Figure S4). A specific Arcobacteraceae ribotype occurred at the Davidson site, and was related to those associated with other eukaryote hosts (Fig. 2). Sulfurospirillum appeared to peak in abundance during the mid-stages of whalefall degradation (~ 50–60 months, n = 9), representing 41% of the recovered Campylobacterales ribotypes, compared to 12–16% at early and late stages (ANOVA p < 0.03; Fig. 5). While at later time points (> 140 months, n = 12) the dominant genus transitioned significantly to Sulfurimonas, averaging 71% of Campylobacterales ribotypes compared to early-mid time periods (25–40%; ANOVA p < 0.01; Fig. 5). All observed Sulfurospirillum and Sulfurimonas ribotypes were shared among Osedax species, regardless of Davidson or Monterey Canyon sites, suggesting that neither host species nor seafloor location, even at vastly different depths, plays a major role in assembling the Osedax Campylobacterales community (only 20–30% of the variation was influenced by either factor, according to one-way ANOSIM; Figure S5).
To better understand the physiological potential, and therefore ecological influences, of the Osedax epibionts, we performed metagenomic sequencing of a single specimen of Osedax frankpressi collected from a 3rd whalefall at 2891 m depth in Monterey Canyon. We identified four near complete genomes of the dominant Osedax epibionts (completeness scores of 93–100%; 0.6–6.8% contamination; Table 2), with nearly identical 16S rRNA gene sequences to those recovered via barcoding (99.6–100%; Fig. 5). These genomes belonged to Arcobacter (sensu lato, closely related to Arcobacter nitrofigilis, the type species of the genus; 50), Sulfurospirillum and Sulfurimonas, the 3 dominant Campylobacterales epibionts, the latter represented by 2 distinct genomes identifiable by a large difference in sequencing coverage depth (16 vs 268X; Table 2). The ‘high-coverage’ Sulfurimonas (268X), referred to further in the following metagenomic sections unless otherwise noted, was in far greater abundance than even the well-known primary Oceanospirillales endosymbiont (at 30X coverage). A single Kordiimonadales (Alphaproteobacteria) genome was also recovered from the external surface of Osedax, however, our microscopy analysis did not indicate integration into the Osedax epithelium, so we do not focus on it further (genome available at # PRJNA813420).
An analysis of genomic motifs involved in metabolism revealed differences in the capabilities among the Osedax epibionts. In general, there appeared to be a shift in metabolic strategy from heterotrophy to autotrophy along the temporal successional gradient, from Arcobacter (s.l.) and Sulfurospirillum at early-mid time points to Sulfurimonas at later time points (Fig. 6). All of the epibionts can metabolize hydrogen using a shared suite of hydrogenase enzymes (Groups 1, 2a and 4), although gene copy numbers vary. The epibionts differ, however, in their capacity to metabolize oxygen, carbon, nitrogen, and sulfur. For oxygen metabolism, Arcobacter and Sulfurospirillum possess cyoE and the cytochrome oxidase genes coxA/B - involved in processing heme and electron transport during aerobic respiration –which are absent in Sulfurimonas (Fig. 6). All Osedax-associated epibionts can reduce nitrate (via napA/B), however, only the Sulfurimonas epibionts possessed the nirS gene for nitrite reduction. In addition, the Arcobacter and Sulfurimonas possess genes involved in the reduction of nitric/nitrous oxide that are absent in the Sulfurospirillum. By contrast, the Sulfurimonas, which dominates the trunk surface at later stages of host decomposition, contained genes involved in carbon fixation through the reverse TCA cycle (aclA and aclB; Fig. 6) that are not found in the other epibionts. While all 3 genera can utilize sulfur compounds, the high-coverage Sulfurimonas epibiont has additional genes involved in sulfur metabolism, including two copies each of the sulfide:quinone oxidoreductase (sqr) and the soxZ gene, in addition to the full thiosulfate oxidation (sox) pathway. The sox pathway was notably absent from the low-coverage Sulfurimonas strain (Fig. 6).
The average amino acid identity (AAI) of the Osedax epibionts differed considerably from close relatives (N = 50; 55–78%), suggesting significant divergence time between the epibionts and free-living relatives with available genomes (Table S1). Despite having similar genome sizes to free-living deep-sea lineages, all 3 Osedax-associated Campylobacterales have decreased coding densities, and significantly more transposable elements which represented ~ 2–6% of their genomes, compared to < 1% for all but a few free-living relatives (Tables 3, S1). A pan-transposase analysis revealed that the epibionts shared numerous insertion sequence families among them; however, they did not share any with the primary symbiont (Table S2). Functional genes carried on transposons included those that encode for a Type I restriction modification system (hsdM superfamily), a leukotoxin export ATP-binding protein ltxB, and a toxin of the relE/parE family, which were shared by all Osedax Campylobacterales. An additional membrane fusion protein (MFP) of a Type 1 secretion system (T1SS) was also identified on a transposon in the high-coverage Sulfurimonas epibiont (Table S3). Lastly, all Osedax Campylobacterales shared a Mu-like bacteriophage, which was not present in free-living relatives, or in the primary Oceanospirillales endosymbiont. The phage appeared intact in the high-coverage Sulfurimonas, and is ~ 19 kb in size, with 15 open reading frames (ORFs), 13 of which code for proteins (7 are known viral proteins, 6 are hypothetical; Table S4), and two insertion sequences (attL and attR).
The Osedax-associated Arcobacter and Sulfurimonas also possessed additional genes that encode proteins involved in attachment and secretion system machinery (Table S3). For example, the Arcobacter contained 5 copies of the Type 5a secretion system (T5aSS), compared to 0–2 copies in close relatives. The Sulfurospirillum epibiont had a tight adherence (TAD) pilus, distinguishing it from close relatives. Similarly, the high-coverage Sulfurimonas epibiont had a Type 5a secretion system absent in close relatives, and a complete 14-gene Type 6 secretion system (T6SS) containing 15 copies of the tssD gene and 21 copies of the tssI gene — The tssD and tssI genes encode 2 of the 12 core T6SS subunits; the stacked hexameric rings (i.e., Hcp tube) that extend outward from the bacterial cell membrane and the distal cell-puncturing device (a trimer of VgrG), respectively.
Finally, the Osedax Campylobacterales genomes contained hundreds of genes encoding predicted secreted proteins, and within these, significantly more (31–59%) had eukaryotic-like protein domains (ELPs) compared to free-living relatives (2-30X; Tables 3, S1). These ELPs, which can be mobilized by the secretion systems described above, comprised 54 families, based on Pfam identification, plus 7 others of unknown function (Table S5). Several of the ELPs were shared among the 3 main Osedax Campylobacterales, including: ATP:guanido phosphotransferase (N-terminal domain), an integrase core domain, and a Helix-turn-helix (HTH)-like domain. The early colonizers Arcobacter and Sulfurospirillum also shared a homeobox-like domain, coding proteins in a large family of transcription factors that contain a highly conserved DNA-binding domain, and a second integrase core domain. The high- and low-coverage Sulfurimonas genomes, not unexpectedly based on their shared evolutionary history, shared many of their ELPs (~ 50%; Table S5).