Dataset: Mesocosm microbial analyses results

Name: Microbial taxa (amplicon sequence variant or ASV) statistical analyses for two seagrass genotypes from wasting disease mesocosm experiments at Bodega Marine Laboratory in July-Sept of 2015
Published: 2022-10-27
Keywords: climate change, host-parasite, microbiome, seagrass, wasting disease, oceans

Cite This Dataset

DOI:10.26008/1912/bco-dmo.883070.1

Spatial Extent: N:38.31753 E:-123.06572 S:38.31753 W:-123.06572

Temporal Extent: 2015-07-01 - 2015-09-14

Project:

CAREER: Linking genetic diversity, population density, and disease prevalence in seagrass and oyster ecosystems (Seagrass and Oyster Ecosystems)

Principal Investigator:

A. Randall Hughes (Northeastern University)

Scientist:

Katherine DuBois (University of California-Davis, UC Davis)

Melissa Kardish (University of California-Davis, UC Davis)

Forest Schenck (Northeastern University)

John J. Stachowicz (University of California-Davis, UC Davis)

BCO-DMO Data Manager:

Amber D. York (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2022-10-27

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Microbial taxa (amplicon sequence variant or ASV) statistical analyses for two seagrass genotypes from wasting disease mesocosm experiments at Bodega Marine Laboratory in July-Sept of 2015

Abstract:

This dataset includes outputs from statistical analyses of differences in microbial taxa (amplicon sequence variant or ASV) abundance among two groups of seagrass, Zostera marina, genotypes: those that showed reduced Labyrinthula zosterae parasites when warmed vs those that showed increased L. zosterae parasites when warmed; and two seawater temperature treatments: ambient or elevated +3.2oC. Data were collected as part of a mesocosm study at the Bodega Marine Laboratory examining the independent and interactive effects of warming, host genotypic identity, and host genotypic diversity on the prevalence and intensity of infections of seagrass by the wasting disease parasite L. zosterae. These data were published in Schenck et al (2022). Related sequence data from this experiment is accessible from the National Center for Biotechnology Information (NCBI) BioProject PRJNA716355.

Expand/Collapse All

Archival Copy

Version Date	Archive	Persistent Identifier	Date Assigned
2022-10-27	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.26008/1912/bco-dmo.883070.1	2022-11-22

Methods & Sampling

We used a substitutive design to test the effects of eelgrass (Zostera marina) genotypic identity (eight genotypes), diversity (monocultures of 1 genotype vs. polycultures of 4 genotypes), and temperature (ambient or + 3.2° C) on the prevalence and intensity of Labyrinthula over eight weeks in an array of flow-through 120-L mesocosms at the Bodega Marine Laboratory in Bodega Bay, CA. In July 2015, we created ten unique polyculture combinations of four genotypes (4 genotypes per experimental pot) randomly drawn from a pool of eight genotypes; all eight genotypes were also grown in monoculture (1 genotype per pot). We filled pots (8.9 x 8.9 cm) with coarsely sieved sediment collected from Bodega Harbor, and planted 4 shoots per pot, matching the lower range of average field densities reported for Bodega Harbor (Ha and Williams 2018) to allow for growth during the experiment. Plants were originally collected in Bodega Harbor, CA in 2012, confirmed to be unique genotypes using 11 DNA microsatellite loci developed specifically for Z. marina (Abbott et al. 2018), and propagated in separate flow through mesocosms at BML.

At the end of the experiment (10 weeks), we collected and preserved the top half of the focal leaf in individual plastic bags sealed with 30 ml of silica (Flower Drying Art Silica Gel; Activa) for subsequent DNA extraction and quantitative PCR to estimate Labyrinthula zosterae cells as a proxy for infection (Bergmann et al. 2011, Bockelmann et al. 2013, Groner et al. 2021).

We collected 2 cm of the focal leaf from directly below the midpoint and stored the tissue at -80˚C for later microbiome analyses to assess whether particular leaf microbial taxa changed in association with Labyrinthula presence on the focal leaf. From these samples, we selected a subset of 84 leaf microbial samples evenly distributed across temperature treatments and across genotypes in monoculture. We did not assess leaf microbiomes in genotypic polycultures. We extracted the surface community of these leaf segments with a modified protocol for the DNeasy Powersoil Kit (Qiagen) and sequenced the V4-V5 region of the 16S rRNA gene on an Illumina MiSeq to analyze differences in the leaf microbiome between treatments and among genotypes. Specifically, to extract the surface community of leaf segments we used a modified protocol for the DNeasy Powersoil Kit (Qiagen) where we first vortexed leaf samples in 500ul of MilliQ water, and then extracted the supernate of each sample. Extraction success was verified via Qubit dsDNA HS assay kit. We PCR-amplified the V4-V5 region of the 16S rRNA gene and then sequenced pooled barcoded fragments on an Illumina MiSeq through the Integrated Microbiome Resource at Dalhousie University (Halifax, NS; Comeau et al. 2017).

Life Sciences Identifiers (LSID) for taxonomic names:
Zostera marina (urn:lsid:marinespecies.org:taxname:145795)
Labyrinthula zosterae (urn:lsid:marinespecies.org:taxname:395093)
Labyrinthula (urn:lsid:marinespecies.org:taxname:119090)

Data Processing Description

To examine the potential contribution of the microbiome to host-parasite-warming interactions in this system, we ran 16S sequence data through a dada2 pipeline to de-noise sequence data, estimate error rates, identify amplicon sequence variants (ASVs) and remove chimeric sequences (Callahan et al. 2016). We assigned 16S rRNA gene taxonomy with the SILVA database (v. 128). After removing mitochondrial and chloroplast sequences we had 1,432,852 sequences in 3,195 ASVs with a median of 23,200 reads per sample and a minimum of 12,149 reads in a sample.

Analyses were done on unrarefied data. We compared the microbiome of two groups: genotypes that showed reduced parasites when warmed vs those that showed increased parasites when warmed. To identify differences among groups, we computed centered log ratio transformation on ASV counts and examined differences in Euclidian distance among treatments and genotype groups (i.e., the Aitchison distance; Aitchison et al. 2000, Gloor et al. 2017). We computed a permutational ANOVA on group-level differences with adonis2 in the R package ‘vegan 2.5’ (Oksanen et al. 2019). To identify which bacteria of the 2407 ASVs that we observed varied between genotypes with reduced vs. increased parasites when warmed, we built negative binomial models based on the geometric means of ASV counts in DESeq2 (Love et al. 2014). We separately assessed which ASVs varied (1) between treatments and (2) between genotype_group + temperature_treatment + genotype_group:treatment using a Wald test. We then applied a Benjamini-Hochberg correction to all reported p-values. All data analyses were performed in R (version 3.6.1, www.R-project.org).

Code for microbial analyses associated with the experiment: www.github.com/mkardish/Labyrinthula. See "Supplemental Files" section of this page for a zip package of commit 50d4b3f.

BCO-DMO Processing:

- Imported data from source file "Mesocosm_microbial_analyses_results.csv" into the BCO-DMO data system. Data file imported using missing data identifier "NA".
- Modified parameter (column) names to conform with BCO-DMO naming conventions.
- The repository https://github.com/mkardish/Labyrinthula (commit 50d4b3f) was forked to BCO-DMO Github Organization for preservation purposes. A release of the code was created, and the zip package was attached to this dataset as a supplemental file.

Data Files

File
mesocosm-analyses.csv (Comma Separated Values (.csv), 51.33 KB) MD5:9b3e60bb8a43bd916f37f33115722489 Primary data file for dataset ID 883070

Supplemental Files

File

Github respository: Labyrinthula
filename: Labyrinthula-883070_1.zip

(ZIP Archive (ZIP), 9.38 KB)
MD5:c74f12166ebe5baf84c0f1609185d7b5

R code for microbial community analysis accompanying Schenck, DuBois, Kardish, Stachowicz and Hughes 2021.

This zip file contains the contents of https://github.com/mkardish/Labyrinthula (commit 50d4b3f).

This repository https://github.com/mkardish/Labyrinthula (commit 50d4b3f) was forked to BCO-DMO for preservation purposes.

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1652320

Deployments

None available yet

Instruments

Aquarium

Supplied Name: flow through tanks

Supplied Description:

Instrument Type

Generic Name: Aquarium

Generic Description:

Aquarium - a vivarium consisting of at least one transparent side in which water-dwelling plants or animals are kept

Automated DNA Sequencer

Supplied Name: Illumina MiSeq

Supplied Description:

Instrument Type

Generic Name: Automated DNA Sequencer

Acronym: Automated Sequencer

Generic Description:

General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
Sequence	Amplicon Sequence Variant (ASV) unique DNA base pair sequence of the V4-V5 region of the 16S rRNA gene; A = adenine; C = cytosine; G = guanine; T = thymine	unitless	sequence
Kingdom	ASV kingdom taxa assigned with the SILVA database (v. 128)	unitless	taxon
Phylum	ASV Phylum taxa assigned with the SILVA database (v. 128)	unitless	taxon
Class	ASV class taxa assigned with the SILVA database (v. 128)	unitless	taxon
Order	ASV order taxa assigned with the SILVA database (v. 128)	unitless	taxon
Family	ASV family taxa assigned with the SILVA database (v. 128)	unitless	taxon
Genus	ASV genus taxa assigned with the SILVA database (v. 128)	unitless	taxon
Species	ASV species taxa assigned with the SILVA database (v. 128)	unitless	taxon
baseMean	Average of the normalized ASV count values; dividing by size factors; taken over all samples	unitless	no_bcodmo_term
log2FoldChange_comp1	Differences in ASV abundance between genotypes that showed different responses of L. zosterae intensity to warming in elevated temperature treatment; positive change indicates greater ASV abundance in genotypes that showed increased L. zosterae intensity under warming; negative change indicates greater ASV abundance in genotypes that showed decreased L. zosterae intensity under warming; reported on a logarithmic scale to base 2	unitless	no_bcodmo_term
lfcSE_comp1	The standard error estimate for the log2 fold change estimate of ASV abundance between genotypes that showed increased L. zosterae intensity under warming and genotypes that showed decreased L. zosterae intensity under warming in elevated temperature treatment	unitless	no_bcodmo_term
padj_comp1	The P-value for difference in ASV abundance between genotypes that showed increased L. zosterae intensity under warming and genotypes that showed decreased L. zosterae intensity under warming in elevated temperature treatment adjusted for multiple testing using a Benjamini-Hochberg correction	unitless	no_bcodmo_term
log2FoldChange_comp2	Differences in ASV abundance between genotypes that showed different responses of L. zosterae intensity to warming in ambient temperature treatment; positive change indicates greater ASV abundance in genotypes that showed increased L. zosterae intensity under warming; negative change indicates greater ASV abundance in genotypes that showed decreased L. zosterae intensity under warming; reported on a logarithmic scale to base 2	unitless	no_bcodmo_term
lfcSE_comp2	The standard error estimate for the log2 fold change estimate of ASV abundance between genotypes that showed increased L. zosterae intensity under warming and genotypes that showed decreased L. zosterae intensity under warming in ambient temperature treatment	unitless	no_bcodmo_term
padj_comp2	The P-value for difference in ASV abundance between genotypes that showed increased L. zosterae intensity under warming and genotypes that showed decreased L. zosterae intensity under warming in ambient temperature treatment adjusted for multiple testing using a Benjamini-Hochberg correction	unitless	no_bcodmo_term
log2FoldChange_comp3	Differences in ASV abundance between temperature treatments in genotypes that showed increased L. zosterae intensity under warming; positive change indicates greater ASV abundance at elevated temperature; negative change indicates greater ASV abundance at ambient temperature; reported on a logarithmic scale to base 2	unitless	no_bcodmo_term
lfcSE_comp3	The standard error estimate for the log2 fold change estimate of ASV abundance between temperature treatments in genotypes that showed increased L. zosterae intensity under warming	unitless	no_bcodmo_term
padj_comp3	The P-value for difference in ASV abundance between temperature treatments in genotypes that showed increased L. zosterae intensity under warming adjusted for multiple testing using a Benjamini-Hochberg correction	unitless	no_bcodmo_term
log2FoldChange_comp4	Differences in ASV abundance between temperature treatments in genotypes that showed decreased L. zosterae intensity under warming; positive change indicates greater ASV abundance at elevated temperature; negative change indicates greater ASV abundance at ambient temperature; reported on a logarithmic scale to base 2	unitless	no_bcodmo_term
lfcSE_comp4	The standard error estimate for the log2 fold change estimate of ASV abundance between temperature treatments in genotypes that showed decreased L. zosterae intensity under warming	unitless	no_bcodmo_term
padj_comp4	The P-value for difference in ASV abundance between temperature treatments in genotypes that showed decreased L. zosterae intensity under warming adjusted for multiple testing using a Benjamini-Hochberg correction	unitless	no_bcodmo_term
log2FoldChange	Differences in ASV abundance between temperature treatments; positive change indicates greater ASV abundance at elevated temperature; negative change indicates greater ASV abundance at ambient temperature; reported on a logarithmic scale to base 2	unitless	no_bcodmo_term
lfcSE	The standard error estimate for the log2 fold change estimate of ASV abundance between temperature treatments	unitless	no_bcodmo_term
padj	The P-value for difference in ASV abundance between temperature adjusted for multiple testing using a Benjamini-Hochberg correction	unitless	no_bcodmo_term

Database

Contribute Data

Dataset: Mesocosm microbial analyses results