Background

Numerous deep-sea invertebrates have formed symbiotic associations with internal chemosynthetic bacteria in order to harness inorganic energy sources typically unavailable to most animals. Despite success in nearly all marine habitats and their well-known associations with photosynthetic symbionts, Cnidaria remain without a clear dependence on hydrothermal vents and chemosynthetic bacterial symbionts specifically.

Results

A new chemosynthetic symbiosis between the sea anemone Ostiactis pearseae (Daly & Gusmão, 2007) and intracellular bacteria was discovered at ~3700 m deep hydrothermal vents in the southern Pescadero Basin, Gulf of California. Unlike most sea anemones observed from chemically-reduced habitats, this species was observed in and amongst vigorously venting fluids, side-by-side with the chemosynthetic tubeworm Oasisia aff. alvinae. Individuals of O. pearseae displayed carbon, nitrogen, and sulfur tissue isotope values suggestive of a distinct nutritional strategy from conventional Actiniaria suspension feeding or prey capture (average d¹³C -29.1‰, d¹⁵N 1.6‰, and d³⁴S -1.1‰). Molecular and microscopic evidence confirmed the presence of intracellular SUP05-related bacteria housed in the tentacle epidermis of O. pearseae specimens collected from 5 hydrothermally-active structures within two vent fields ~2 km apart. SUP05 bacteria dominated the O. pearseae bacterial community (64-96% of the total bacterial community based on 16S rRNA sequencing), but were not recovered from other nearby anemones, and were generally rare in the surrounding water (< 7% of the total community). Further, the specific Ostiactis-associated SUP05 phylotypes were not detected in the environment, indicating a specific association. Two unusual candidate bacterial phyla (the OD1 and BD1-5 groups) also appeared to associate exclusively with O. pearseae and may play a role in sulfur cycling.

Conclusion

Ostiactis pearseae represents the first member of Cnidaria described to date to have a physical and nutritional alliance with chemosynthetic bacteria. The facultative nature of this symbiosis is consistent with the dynamic relationships formed by both the SUP05 bacterial group and Anthozoa. The advantages gained by appropriating metabolic and structural resources from each other presumably contribute to their striking abundance in the Pescadero Basin, at the deepest known hydrothermal vents in the Pacific Ocean.

Specimen collections

All specimens and water samples were collected from active vent sites within the Pescadero Basin, Gulf of California (~ 3700 m depth), using the ROV SuBastian during the R/V Falkor expedition FK103118 (October-November 2018), specifically from six sites at two vent fields within ~2 km of each other; the previously described Auka vent field (refs) and a newly discovered JaichMaa 'ja'ag vent field. Sea anemones were collected by ROV manipulator or suction sampler and preserved shipboard as described below in each analysis section. Targeted water samples (2 L) were collected via Niskin bottle mounted on ROV SuBastian.

DNA extraction

Specimens for molecular analysis (Table 1) were preserved immediately upon collection in ~90% ethanol and stored at 4°C. Total genomic DNA was extracted from tissues using the Qiagen DNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. 2L water samples were filtered onto a 0.22 µm Sterivex-GP polyethersulfone filter (Millipore-Sigma, St. Louis, MO, USA) and frozen at -80°C until DNA analysis. DNA extraction from Sterivex PES filters was also performed using the Qiagen DNeasy kit, according to the manufacturer’s instructions, with the exception of the first step where 2 ml of ATL lysis buffer was added to the Sterivex filter, via luer lock and syringe, and rotated at 56°C for 12 hours. This solution was recovered from the filter, also via luer lock and syringe, and processed as usual.

Molecular analysis of the microbial community

A 1000-bp region of the gene coding for napA (periplasmic nitrate reductase) was amplified directly from Ostiactis tissues using the primers V16F (5′-GCNCCNTG-YMGNTTYTGYGG-3′) and V17R (5′-RTGYTGRTTRAANCCCATNGTCCA-3; Flanagan et al. 1999), while a 408 bp fragment of the aprA gene (subunit of particulate methane monooxygenase enzyme) was generated using primers, aps1F (5-TGGCAGATCATGATYMAYGG-3) and aps4R (5-GCGCCAACYGGRCCRTA-3, described in Blazejak et al. 2006). A 1465-bp fragment of the 16S rRNA gene was amplified using the primers 27F and 1492R (ref). Annealing conditions of 50°C, 50°C and 54°C were used for napA, aprA, and 16SrRNA, respectively. Otherwise, all thermal protocols included the following steps: an initial 5 min denaturation at 94°C, then 1 min at 94°C,
1 min annealing step, and 1 min at 72°C, for 25 cycles, and a final 5 min extension at 72°C. Amplification products were sequenced directly using Sanger sequencing, via Laragen Inc.

The V4-V5 region of the 16S rRNA gene was amplified using bacterial primers with Illumina (San Diego, CA, USA) adapters on the 5′ end 515F (5′-TCGTCGGC-AGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHV-GGGTWTCTAAT-3′) (Caporaso et al. 2011). The PCR reaction mix was set up in duplicate for each sample with Q5 Hot Start High-Fidelity 2x Master Mix (New England Biolabs, Ipswich, MA, USA) and annealing conditions of 54°C for 25 cycles. Duplicate PCR samples were then pooled and 2.5 µL of each product was barcoded with Illumina NexteraXT index 2 Primers that include unique 8-bp barcodes (P5 5’-AATGATACGGCGACCACCGAG-ATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-3’ and P7 5’-CAAGCAGAA-GACGGCATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGG-3’). Secondary amplification with barcoded primers used conditions of 66°C annealing temperature and 10 cycles. Products were purified using Millipore-Sigma (St. Louis, MO, USA) MultiScreen Plate MSNU03010 with a vacuum manifold and quantified using Thermo-Fisher Scientific (Waltham, MA, USA) QuantIT PicoGreen dsDNA Assay Kit P11496 on the BioRad CFX96 Touch Real-Time PCR Detection System. Barcoded samples were combined in equimolar amounts into single tube and purified with Qiagen PCR Purification Kit 28104 before submission to Laragen (Culver City, CA, USA) for 2 x 250bp paired end analysis on the Illumina MiSeq platform with PhiX addition of 15-20%.

MiSeq 16S rRNA sequence data was processed in Quantitative Insights Into Microbial Ecology (v1.8.0). Raw sequence pairs were joined and quality-trimmed using the default parameters in QIIME. Sequences were clustered into de novo operational taxonomic units (OTUs) with 99% similarity using UCLUST open reference clustering protocol, and then, the most abundant sequence was chosen as representative for each de novo OTU. Taxonomic identification for each representative sequence was assigned using the Silva-119 database, clustered at 99% similarity. A threshold filter was used to remove any OTU that occurred below 0.01% in the combined samples dataset. Analyses are based on Bray-Curtis distances of fourth-root transformed data, which minimizes errors in the ordination due to PCR bias, while not sacrificing genuine differences between samples. Quantification and statistical analyses are described in the Results sections and figure legends. Comparisons were performed using ANOVA and statistical significance was declared at P < 0.05. Statistical analyses of beta diversity (e.g. ANOSIM) were performed with Primer E.

Molecular analysis of the anemone host Ostiactis pearseae

Phylogenetic relationships were determined via sequencing of three mitochondrial markers, the 12S rRNA, 16S rRNA, and cytochrome oxidase III genes, and the partial nuclear 18S rRNA gene. An 862-bp product of the 12S rRNA gene was amplified via primers ANTMT12SF (5’-AGCCAC-ACTTTCACTGAAACAAGG-3’) and ANTMT12SR (5’-GTTCCCYYWCYCTYA-CYATGTTACGAC-3’) according to Chen and Yu 2000. A 473-bp product of the 16S rRNA gene was amplified via primers ANEM16SA (5’-CACTGACCGTGATAATG-TAGCGT-3’) and ANEM16SB (5’-CCCCATGGTAGCTTTTATTCG-3’) according to Geller and Walton 2001. Finally, a 721-bp product of the cytochrome oxidase III (COIII) gene was amplified via primers COIIIF (5’-CATTTAGTTGATCCTAGGCCTTGACC-3’) and COIIIR (5’-CAAACCACATCTA-CAAAATGCCAATATC-3’) according to Geller and Walton 2001. Finally, a 502-bp product of the 18S rRNA gene was amplified via primers 18S-3F (5’- GTTCGATTC-CGGAGAGGGA-3’) and 18S-5R (5’-CTTGGCAAATGCTITCGC-3’) according to Giribet et al. 1996. Annealing conditions of 55°C, 51.5°C,  51°C and 54°C were used for 12SrRNA, 16SrRNA, COIII, and 18S rRNA, respectively. Otherwise, all thermal protocols included the following steps: an initial 5 min denaturation at 94°C, then 1 min at 94°C,
1 min annealing step, and 1 min at 72°C, for 30 cycles, and a final 5 min extension at 72°C.

There are 5 files:

The Ostiactis anemone gene sequences (12S, 16S, 18S, and COIII)

The Ostiactis anemone gene sequences (12S, 16S, 18S, and COIII) as an alignment

The SUPO5 bacteria genes (16S, napA, aprA)

The total bacterial community barcode results for 8 Ostiactis, 2 unidentified Kadosactinidae, 4 unidentified zoanthids, 4 seawater samples, and 11 hydrothermal vent worms (Oasisia and Riftia), for comparison

A 2-minute video of sampling O. pearseae via the ROV SuBastian, in and amongst the hydrothermal vent tubeworm, Oasisia