DOI: 10.5061/dryad.5mkkwh7g9
title: <em>Vibrio pectenicida</em> strain FHCF-3 is a causative agent of sea star wasting disease

description: More than ten years following the onset of the sea star wasting disease
 (SSWD) epidemic, affecting over 20 asteroid species from Mexico to Alaska,
 the causative agent has been elusive. SSWD killed billions of the most
 susceptible species, sunflower sea stars (Pycnopodia helianthoides),
 initiating a trophic cascade involving unchecked urchin population growth
 and widespread loss of kelp forests. Identifying the causative agent
 underpins the development of recovery strategies. Here, we induced disease
 and subsequent mortality in exposure experiments using tissue extracts,
 coelomic fluid, and effluent water from wasting sunflower sea stars, with
 no mortality in controls. Deep sequencing of diseased sea star coelomic
 fluid samples from experiments and field outbreaks revealed a dominant
 proportion of reads assigned to the causative agent. Fulfilling Koch’s
 postulates, the pathogen, cultured from the coelomic fluid of a diseased
 sunflower sea star, caused disease and mortality in exposed sunflower sea
 stars, demonstrating that it is a causative agent of SSWD. This discovery
 will enable recovery efforts for sea stars and the ecosystems affected by
 their decline by facilitating culture-based experimental research and
 broad-scale screening for pathogen presence and abundance in the
 laboratory and field.
description: # Data for: *Vibrio pectenicida* strain FHCF-3 is a causative agent of sea
 star wasting disease Dryad
 DOI: [https://doi.org/10.5061/dryad.5mkkwh7g9](https://doi.org/10.5061/dryad.5mkkwh7g9) Title: Vibrio pectenicida strain FHCF-3 is a causative agent of sea star wasting disease Authors: Melanie B. Prentice1,2*, Grace Crandall3, Amy M. Chan1, Katherine M. Davis1,2†, Paul K. Hershberger4, Jan F. Finke1,2, Jason Hodin5, Andrew McCracken6, Colleen T. E. Kellogg2, Rute B. G. Clemente-Carvalho2, Carolyn Prentice2, Kevin X. Zhong1, Drew Harvell5,7, Curtis A. Suttle1,8,9,10, Alyssa-Lois M. Gehman2, 10* Affiliations: 1 Department of Earth, Ocean and Atmospheric Sciences, The University of British Columbia, Vancouver, BC, Canada. 2 Hakai Institute; Campbell River, BC, Canada. 3 School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA, USA. 4 U.S. Geological Survey, Western Fisheries Research Center, Marrowstone Marine Field Station; Nordland, WA, USA. 5 Friday Harbor Laboratories, University of Washington; Friday Harbor, WA, USA. 6 Department of Biology, University of Vermont; Burlington, VT, USA. 7 Department of Ecology and Evolutionary Biology, Cornell University; Ithaca, NY, USA. 8 Department of Microbiology & Immunology, The University of British Columbia; Vancouver, BC, Canada. 9 Department of Botany, The University of British Columbia, Vancouver, BC, Canada. 10 Institute for the Oceans and Fisheries, The University of British Columbia; Vancouver, BC, Canada. †Current address: Washington Department of Fish and Wildlife, Port Townsend, USA. *Corresponding authors. E-mail: [melanie.prentice@ubc.ca](mailto:melanie.prentice@ubc.ca), [alyssamina@gmail.com](mailto:alyssamina@gmail.com) ###### Principle Investigator Contact Information ``` Name: Alyssa-Lois M. Gehman Institution: The Hakai Institute & The University of British Columbia Email: alyssamina@gmail.com, alyssa.gehman@hakai.org ``` ###### Alternate Contact Information ``` Name: Melanie Prentice Institution: The University of British Columbia & The Hakai Institute Email: melanie.prentice@ubc.ca, melanie.prentice@hakai.org ``` ## Dataset Overview This dataset contains the data and code required to replicate analyses for metatranscriptomic and 16S rRNA gene amplicon datasets. We also provide R Code and associated data required to reproduce all figures associated with this manuscript. ### Dates of Data Collection ``` A. Metatranscriptomic dataset: 2022 B. 16S rRNA gene amplicon datasets: 2022-2023 ``` ### Data Spatial Scope Genetic datasets were generated from samples collected from controlled exposure experiments on wild sunflower sea stars (*Pycnopodia helianthoides*) collected in Washington, USA. Genetic data is also generated for samples collected from wild populations of sunflower sea stars in British Columbia, Canada. ### Funding The Nature Conservancy of California (DH, A-LMG) The Tula Foundation (A-LMG, CAS) Natural Sciences and Engineering Research Council of Canada Discovery Grant RPGIN-2020-06515 (CAS) Canadian Foundation for Innovation and British Columbia Knowledge Development Fund Infrastructure award #25412 (CAS) The University of British Columbia The U.S. Geological Survey, Biological Threats Research Program, Ecosystems Mission Area (PKH) Quantitative and Evolutionary STEM Traineeship NRT-1735316 (AM) ### Ethics Approval Wild *P. helianthoides* used in experiments were collected under scientific collection permits issued by the Washington Department of Fish and Wildlife (WDFW) (permit no. HARVELL 21-1172R, 22-175, 23-087, 24-053). Captive-bred *P. helianthoides* were transported to the USGS Marrowstone Marine Field Station under WDFW shellfish transfer permits (permit no. 23-1249, 24-1249). Samples from wild *P. helianthoides* collected in British Columbia, Canada, were collected under scientific collection permit XMCFR 18 2023 issued by Fisheries and Oceans Canada. Although research with sea stars is not explicitly regulated within the United States, we followed the ethical principle of reduction in all experiments, using the minimum number of required individuals per experiment to retain statistical power. Facilities housing the animals involved were accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAA-LAC) and inspected regularly by the Institutional Animal Use and Care Committee (IACUC) at the U.S. Geological Survey, Western Fisheries Research Center. ## Sharing/Access information ### Sharing/Access Metatranscriptomic and 16S rRNA gene sequence datasets are archived in the National Center for Biotechnology Information (NCBI) Short Read Archive (BioProject Number PRJNA1195080). The whole genome of *V. pectenicida* strain FHCF-3 is available from the NCBI GenBank Repository (Accession Number JBLZMR000000000), with raw sequence reads archived in the NCBI Short Read Archive (BioProject Number PRJNA1232168). The complete 16S rRNA gene sequences of *V. pectenicida* strain FHCF-3 is deposited in the NCBI GenBank Repository (Accessions PQ700178, PQ763222-PQ763229). #### Fig2_ExtDFig1_2_Data&Code.zip ### Content Description Contains: 1. Fig2_ExtDFig1_2_datacode.Rproj file for launching the project in R 2. Fig2_ExtDFig1_2_data_share.Rmd file with script used to generate figures 3. Fig2_data_21Mar25.csv file containing the data required to generate the Fig. 2 figure Column B (experiment_number): name or number of the experiment Column C (tmt): treatment type - type of exposure Column D (years): year of experiment Column E (response): categories of response being plotted, where "mean_start_twist_day" is the average number of days to stars starting to twist their arms, "mean_start_drop_day" is the average number of days before stars dropped an arm, and "mean_mort_days" is the average number of days to mortality Column F (exposure_days): the average value for each response in days since exposure Column G (response_type): labels for each response for the legend Column H (sdresponse): standard deviation of the mean values column I (sd): the value for plotting (sdresponse) Column J (facet): a variable indicating which panel of the figure to plot the data in 4. Extended_Data_Fig1_data.csv file containing the data required to generate the Extended Data Fig. 1 figure Column B (col.loc): treatment group names, where "lab" is lab-raised sea stars and "wild" is sea stars collected from the wild Column C (count): sample size for each group Column D (response): categories of response being plotted, where "mean_start_twist_day" is the average number of days to stars starting to twist their arms, "mean_start_drop_day" is the average number of days before stars dropped an arm, and "mean_mort_days" is the average number of days to mortality Column E (exposure_days): the average value for each response in days since exposure Column F (response_type): labels for each response for the legend Column G (sdresponse): standard deviation of the mean values Column H (sd): the value for plotting (sdresponse) 5. Extended_Data_Fig2_data.csv file containing the data required to generate the Extended Data Fig. 2 figure Column B (star_ID): individual star ID Column C (exp_number): experiment number Column D (armtwist): number of arms twisting at time of observation Column E (exp_date): number of days post-exposure (inoculation) that observation was made Column F (control_cat): treatment group, where "exposed" are inoculated with live inocula, and "control" are inoculated with heat-killed inocula Column G (inoc_type): type of inocula used for exposure, where "homogenate" is tissue homogenate, "CF" is coelomic fluid, "water" is water (which is not injection but immersion exposure), and "culture" is *Vibrio pectenicida* bacterial culture Column H (star_arm_count): total number of arms each individual has Column I (movement): whether or not movement was observed from the last observation, where "NA" is no data, "1" is movement, and "0" is no movement #### Fig3_Data&Code.zip ### Content Description Contains: 1. RScript_Fig3_Metatranscriptomics_PCoA.R R script for generating the figure 2. Data_Normalized_Count_Matrix.csv data file containing the data required to generate the figure Row headers (down column A): sample IDs Column headers (along row 1): sequence annotations (i.e., family, genus or species IDs of microbial taxa identified in the metatranscriptomic sequencing dataset) 3. Vectors.csv data file generated during the analysis; this file was filtered (by r2 value) to create a new file which subsets all vectors to those sig. for plotting Column A is species IDs Column D is the r2 value used to sort and select species vectors to annotate on the plot 4. Vectors_Sig_ForPlotting.csv file generated during the analysis; this file contained sig. vectors for plotting The subset of vectors from the Vectors.csv file to annotate on the plot (r2&gt;0.9) #### Fig4_Data&Code.zip ### Content Description Contains: 1. Fig4_Data&Code.Rmd R script for generating the figure 2. data_2022E2_CFSamples_Vpec_RelativeAbundance.csv data file containing the data required to generate the figure panel A Column A (Sample ID): the sample ID Column B (Time): the time point that the sample was collected during the experiment, where "pre-inoculation" samples were collected before sea stars were exposed to SSWD (i.e., before injection with inoculum) and "post-inoculation" samples were collected post-exposure (injection); "inoculum" is a sample of the inoculum used for the exposure Column C (Experiment Treatment): experimental treatment group, where "exposed" individuals were injected with coelomic fluid collected from a wasting sunflower sea star, and "control" individuals were injected with heat-killed coelomic fluid Column D (Prop.Vp.Reads): relative abundance of 16S rRNA amplicon sequencing reads annotated as *Vibrio pectenicida* in each individual's dataset 3. data_Library4Field_CFSamples_Vpec_RelativeAbundance.csv data file containing the data required to generate the figure panel B Column A (Sample.ID): the sample ID Column B (Site): the name of the site where sampling occurred Column C (October.Disease.Signs): disease signs observed at the site in October, where "naive" is no disease observed at the site, and both "field exposed" and "wasting" are diseases observed at the site Column D (Time): the month the sample was collected Column E (Disease.Signs): disease signs observed at the time of sample collection, where "naive" is no disease signs observed in the individual being sampled or any other individuals at the site, "field exposed" is no disease signs observed in the individual being sampled but disease signs observed in other individuals at the site, and "wasting" is disease signs observed in the individual being sampled Column F (Prop.Vp.Reads): relative abundance of 16S rRNA amplicon sequencing reads annotated as *Vibrio pectenicida* in each individual's dataset #### Script_16S_QIIME2_decontam.sh ### Content Description This script contains an overview of the code used to run the analysis of 16S rRNA gene amplicon datasets. 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