Data from: Persistent, internal associations between bacteria and blood-feeding marine annelids and crustaceans
Data files
Jan 18, 2025 version files 93.72 KB
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README.md
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Vampire_metadata.xlsx
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Vampires_Final_amplicon_data_v2.xlsx
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Vibrio_seqs_4GB_Nov2022.TXT
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Abstract
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.
README: 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
Methods
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 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 < 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). 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).