DOI: 10.5061/dryad.vmcvdncx9
title: Data from: Persistent, internal associations between bacteria and blood-feeding marine annelids and crustaceans

description: Persistent bacterial presence is believed to play an important role in
 host adaptation to specific niches that would otherwise be unavailable,
 including the exclusive consumption of blood by invertebrate parasites.
 Nearly all blood-feeding animals examined so far host internal bacterial
 symbionts that aid in some essential aspect of their nutrition. Obligate
 blood-feeding (OBF) invertebrates exist in the oceans, yet symbiotic
 associations between them and beneficial bacteria have not yet been
 explored. This study describes the microbiome of 6
 phylogenetically-diverse species of marine obligate blood-feeders,
 including leeches (both fish and elasmobranch specialists; ex.
 Pterobdella, Ostreobdella, and Branchellion), isopods (ex. Elthusa and
 Nerocila), and a copepod (ex. Lernanthropus). Amplicon sequencing analysis
 revealed the blood-feeding invertebrate microbiomes to be low in
 diversity, compared to host fish skin surfaces, seawater, and
 non-blood-feeding relatives, and dominated by only a few bacterial genera,
 including Vibrio (100% prevalence and comprising 39-81% of the average
 total recovered 16S rRNA gene sequences per OBF taxa). Vibrio cells were
 localized to the digestive lumen in and amongst the blood meal for all
 taxa examined via fluorescence microscopy. For Elthusa and Branchellion,
 Vibrio cells also appeared intracellularly within possible hemocytes,
 suggesting an interaction with the immune system. Additionally, Vibrio
 cultivated from four of the obligate blood-feeding marine taxa matched the
 dominant amplicons recovered, and all but one was able to effectively lyse
 vertebrate blood cells. Bacteria from 2 additional phyla and 3 families
 were also regularly recovered, albeit in much lower abundances, including
 members of the Oceanospirillaceae, Flavobacteracea, Porticoccaceae, and
 unidentified members of the gamma- and betaproteobacteria, depending on
 the invertebrate host. For the leech Pterobdella, the
 Oceanospirillaceae were also detected in the esophageal diverticula. For
 two crustacean taxa, Elthusa and Lernanthropus, the microbial communities
 associated with brooded eggs were very similar to the adults, indicating
 possible direct transmission. Virtually nothing is known about the
 influence of internal bacteria on the success of marine blood-feeders, but
 this evidence suggests their regular presence in marine parasites from
 several prominent groups.
description: Total genomic DNA was extracted from specimens, that had been
 rinsed in ethanol and dried, using the Qiagen DNeasy kit (Qiagen,
 Valencia, CA, USA) according to the manufacturer’s instructions. Swab and
 filter extractions deviated from manufacturer’s instructions by doubling
 reagent volumes during the initial lysis and incubation step (i.e. 360 ml
 ATL buffer and 40 ml Proteinase K) to cover the entire swab or filter
 area. 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′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA-3′) and 806R
 (5′-GTCTCGTGGGCTCGGAGATGTGTATA-AGAGACAGGGACTACHVGGGTWTCTAAT-3′; Caporaso
 et al. 2011). Note: Eight swabs and one non-blood-feeding isopod were not
 amplifiable for the 16S rRNA gene. 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′-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCG
 GCAGCGTC-3′ and P7
 5′-CAAGCAGAAGACGGCATACGAGAT-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<strong><em>
 </em></strong>Fisher Scientific (Waltham, MA, USA) QuantIT
 PicoGreen dsDNA Assay Kit on a BioRad CFX96 Touch Real-Time PCR Detection
 System. Barcoded samples were combined in equimolar amounts into a single
 tube and purified with Qiagen PCR Purification Kit 28104 before submission
 to Laragen (Culver City, CA, USA) for 2 × 250 bp paired end analysis on
 the Illumina MiSeq platform with PhiX addition of 20%.
 Amplicon 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 with 99% similarity using the UCLUST open reference clustering
 protocol, and then, the most abundant sequence was chosen as a
 representative for each. Taxonomic identification for each representative
 sequence was assigned using the Silva-138 database, and checked via BLAST.
 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. Non-metric
 multidimensional scaling ordination (NMDS), analysis of similarity
 (ANOSIM), and similarity percentage analysis (SIMPER) analyses were
 performed in Primer-E, after square-root transforming the dataset and
 calculating Bray–Curtis similarities (Clarke & Warwick,
 2001). For at least one specimen from each OBF taxon, a
 16S rRNA gene clone library was generated using the general primers 27F
 and 1492R (Lane 1991) and the TOPO-TA kit (ThermoFisher, Waltham, MA,
 USA), according to the manufacturer’s instructions. In this way, longer
 sequences of the 16S rRNA gene were recovered for nearly all dominant
 bacterial ribotypes (via Laragen, Inc; Supplementary Figure
 S1).<strong> </strong>Longer sequences were assembled using
 Sequencher v4.10.1 (GeneCodes Corp., Ann Arbor, MI, USA) and trees were
 generated using Geneious Prime v2022.2.1 (Biomatters, Inc. San Diego, CA,
 USA).
description: # Persistent, internal associations between bacteria and blood-feeding
 marine annelids and crustaceans Summary of 16S rRNA gene amplicon
 sequencing, binned at the 99% level. Columns are individual specimens, and
 rows are bacterial taxa. The number in each cell represents the number of
 hits recovered for that particular taxon group. The second file is a fasta
 file of sequences generated by cloning or direct sequencing of isolated
 bacteria ## Description of the Data and file structure There is one
 metadata file and a separate file with 3 results sheets separated by
 animal group (Crustaceans, Leeches, and Adults vs eggs). ## Sharing/access
 Information Links to other publicly accessible locations of the data: None