DOI: 10.5061/dryad.mkkwh70wt
title: Facultative chemosynthesis in a deep-sea anemone from hydrothermal vents in the Pescadero Basin, Gulf of California

description: 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 (&lt; 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.
description: <b><i>Specimen
 collections</i></b> All specimens and water
 samples were collected from active vent sites within the Pescadero Basin,
 Gulf of California (~ 3700 m depth), using the ROV
 <i>SuBastian</i> during the R/V <i>Falkor</i>
 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 <i>ROV</i> SuBastian.
 <b><i>DNA extraction</i></b>
 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. <b><i>Molecular
 analysis of the microbial community</i></b> A 1000-bp region of
 the gene coding for <i>napA</i> (periplasmic nitrate
 reductase) was amplified directly from <i>Ostiactis</i>
 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 <i>aprA </i>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 <i>de
 novo</i> 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 <i>de
 novo</i> 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 &lt; 0.05. Statistical analyses of beta diversity (e.g. ANOSIM)
 were performed with Primer E. <b><i>Molecular
 analysis of the anemone host
 </i></b><em><b>Ostiactis
 pearseae</b></em> 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<i>.<b> </b></i>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.<b><i> </i></b>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.<b><i>
 </i></b>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.
description: 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