Immune stimulation induced changes to the native gut microbiota of bumble bees
Data files
Jul 11, 2023 version files 754.31 KB
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AbsoluteCounts_06042023.csv
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Immune_perturbation_activation_06042023.csv
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Immune_perturbation_R_Code_06042023.R
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Immune_PerturbationData_06042023.csv
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PredictedTaxonomy_06042023.csv
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QIIME2_ImmunePerturbation_06042023
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README.md
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Sample_MetaData_06042023.csv
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Sauers_Gill_06042023.xls
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Sauers_Lacto_06042023.xls
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Sauers_Schmid_06042023.xls
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Sauers_Snod_06042023.xls
Jul 10, 2023 version files 754.22 KB
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AbsoluteCounts_06042023.csv
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Immune_perturbation_activation_06042023.csv
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Immune_perturbation_R_Code_06042023.R
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Immune_PerturbationData_06042023.csv
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PredictedTaxonomy_06042023.csv
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QIIME2_ImmunePerturbation_06042023
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README.md
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Sample_MetaData_06042023.csv
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Sauers_Gill_06042023.xls
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Sauers_Lacto_06042023.xls
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Sauers_Schmid_06042023.xls
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Sauers_Snod_06042023.xls
Abstract
Understanding factors influencing the maintenance and membership of beneficial host-associated microbial communities is central to understanding ecological, evolutionary, and health consequences of these communities for their hosts. Host immunity is often implicated as a potential regulator of these microbiota. However, conversely, immunity may play a disruptive role, with immune responses to infection causing collateral damage. Such effects may be more prominent from innate immune responses, with more rapid-acting and relatively non-specific components. We investigated how upregulation of antibacterial immunity in the bumble bee Bombus impatiens affects the core gut microbiota, testing the hypothesis of immunity-induced perturbation of the beneficial microbiota structure. Freshly emerged adult bees received a native microbiota inoculation before being subjected to non-pathogenic immune stimulation treatments. We quantified the microbial community using 16S rRNA amplicon sequencing and targeted qPCR. We find colonization of the core member Gilliamella is altered by immune stimulation treatment. Additionally, a positive association in communities between Gilliamella and another core bacteria, Snodgrassella alvi, is perturbed. These changes are indicative of immune-response-induced dysbiosis. As such, the potential for collateral perturbation of beneficial microbial communities upon a host's innate immune response may contribute to immune costs, shaping the evolutionary optimization of immune investment.
README
Explanation of files for Sauers et al. "Membership robustness but structural change of the native gut microbiota of bumble bees upon systemic immune induction"
File: Immune_PerturbationData_06042023.csv
Data sheet containing sample meta data, qPCR determined values for total bacterial 16S rRNA, downstream relative abundances calculated from 16S amplicon sequencing analysis with QIIME2 and phyloseq, the adjusted amplicon read values for core bacteria (Relative abundance * total bacteira 16S rRNA for the sample), and species-specific qPCR values.
Analytical script: Immune_perturbation_R_Code_06042023
sampleID: The unique sample id for each sample collected in the experiment.
Colony: The colony of origin for each sample
Bee_ID: The initial ID, nested within colony, before assignment of the unique ID
Block: Sampling block for each sample
ImmuneChallenge: The immune challenge treatment for each sample, N = naive, C = wounding, E = gram negative, and S = gram positive
TreatmentDay: The day the immune challenge treatment was given, 0 = day 0 after fecal inoculation, 4 = four days after bees received fecal inoculation
Date_emerged: The date (month/day/year) on which the sample was collected for fecal inoculation and assigned an immune challenge treatment
Total_qPCR_Mean: The mean of the qPCR determined total 16S rRNA for each sample from the duplicate 16S rRNA runs
Total_Adjusted: The total 16S rRNA from samples adjusted from the qPCR mean value to per bee values (Total_qPCR_Mean * 50ul)
SchmidAmplicon: The adjusted Schmidhempelia amplicon values (Total_Adjusted * Schmidhempelia relative abundance determined by QIIME2 and phyloseq)
Schmid_qPCR: The mean of the qPCR determined 16S rRNA for Schmidhempelia
LactoAmplicon: The adjusted Lactobacillus amplicon values (Total_Adjusted * Lactobacillus relative abundance determined from QIIME2 and phyloseq)
Lacto_qPCR: The mean of the qPCR determined 16S rRNA for Lactobacillus
SnodAmplicon: The adjusted Snodgrassella amplicon values (Total_Adjusted * Snodgrassella relative abundance)
Snod_qPCR: The mean of the qPCR determined 16S rRNA for Snodgrassella
GillAmplicon: The adjusted Gilliamella amplicon values (Total_Adjusted * Gilliamella relative abundance)
Gill_qPCR: The mean of the qPCR determined 16S rRNA for Gilliamella
PseudomonasAmplicon: The adjusted Pseudomonas amplicon values (Total_Adjusted * Pseudomonas relative abundance)
File: Immune_perturbation_activation_06042023.csv
Data from samples used to verify the activation of immune system following injection with the treatments used in this study.
Unique_ID: The unique sample id for each sample collected in the experiment
Bee_ID: The sample id nested within colony before assignment of unique ID
Colony: The colony the sample was isolated from
Block: The injection block the sample was treated in, blocks correlated with date_emerged
Inject_trt: ImmuneChallenge: The immune challenge treatment for each sample, N = naive, C = wounding, E = gram negative, and S = gram positive
Inject_day: The days after sample collection that samples received the Inject_trt, either day 0 = 0 or day 4 = 4
Date_emerged: The date (month/day/year) the sample emerged as an adult
Date_injected: The date (month/day/year) the sample was injected
Date_bled: The date (month/day/year) hemolymph was collected from the sample
ZoneA_dia1_mm: The diameter (mm) from the first measure of the first replicate zone for the Arthrobacter zone of inhibition trial
ZoneA_dia2_mm: The diameter (mm) from the second measure of the first replicate zone for the Arthrobacter zone of inhibition trial
ZoneA_avg_dia: The average diameter (mm) of the first replicate zone determined from the duplicate measures
Tetracycline_A: The tetracycline equivalent (ug/mL) for the first replicate zone determined from standard curve
ZoneB_dia1_mm: The diameter (mm) from the first measure of the second replicate zone for the Arthrobacter zone of inhibition trial
ZoneB_dia2_mm: The diameter (mm) from the second measure of the second replicate zone for the Arthrobacter zone of inhibition trial
ZoneB_avg_dia: The average diameter (mm) of the second replicate zone determined from the duplicate measures
Tetracycline_B: The tetracycline equivalent (ug/mL) for the second replicate zone determined from standard curve
Average_tetra: The average tetracycline (ug/mL) equivalent determined from the values from the duplicate zones
Comments: Additional comments on the samples
Files: Sauers_Gill_06042023.xls, Sauers_Schmid_06042023.xls, Sauers_Snod_06042023.xls, Sauers_Lacto_06042023.xls
qPCR values and information from qPCR utilizing the species-specific primers. For Gilliamella (Sauers_Gill_06042023.xls), for Schmidhempelia (Sauers_Schmid_06042023.xls), Snodgrassella (Sauers_Snod_06042023.xls), and Lactobacillis (Sauers_Lacto_06042023.xls).
Well: The unique well ID
Well Position: The position the well is on the well plate
Sample Name: The unique ID of the sample
Task: The task of the sample in the well, either a dilution of the synthetic standard (STANDARD), a sample (UNKNOWN), or a negative control (NTC)
Reporter: The flourescent dye utilized in the qPCR chemistry
CT: The cycle where the flourescences can be detected from the background
Ct Mean: The mean ct determined from the duplicate reactions
Ct SD: The standard deviation of the duplicate reactions
Quantity: The quantity of the sample determined from a synthetic standard curve
Quantity Mean: The mean of the quantity determined from the duplicate reactions
Quantity SD: The standard deviation of the duplicates
Ct Threshold: The manually set threshold value
Y-intercept: The y-intercept of the fitted standard curve
R(superscript2): The R2 value for the fitted standard curve
Slope: The slope of the fitted standard curve
Efficiency: The efficiency of the fitted standard curve
Tm: The peak melting temperate (C) of the sample
File: AbsoluteCounts_06042023.csv
The absolute counts for each ASV across each sample determined by transforming the 16S amplicon determined relative abundance by the total 16S rRNA determined by qPCR. Bee samples are in the columns and ASVs are in the rows. Formmated for use in phyloseq with the files PredictedTaxonomy.csv and Sample_MetaData_06042023.csv
File: PredictedTaxonomy_06042023.csv
The predicted taxonomy for each ASV using the Silva database Formmated for use with phyloseq with the files AbsoluteCounts_06042023.csv and Sample_MetaData_06042023.csv
feature-id: The unique feature id of the ASV
Domain: Predicted domain
Phylum: Predicted phylum
Class: Predicted class
Order: Predicted order
Family: Predicted family
Genus: Predicted Genus
File: Sample_MetaData_06042023.csv
The metadata for the amplicon sequencing samples. Formatted for use with phyloseq with the files AbsoluteCounts_06042023.csv and PredictedTaxonomy_06042023.csv
sampleid: Unique sample id
Colony: Colony the sample was taken from
Bee_ID: The original sample ID nested within colony
Block: The block the sample was collected in
ImmuneChallenge: The immune challenge treatment for each sample, N = naive, C = wounding, E = gram negative, and S = gram positive
TreatmentDay: The day the immune challenge treatment was given, 0 = day 0 after fecal inoculation, 4 = four days after bees received fecal inoculation
Date_emerged: The day (month/day/year) on which the sample was collected for fecal inoculation and assigned an immune challenge treatment
File: Immune_Perturbation_R_Code_06042023
The R code used for downstream analysis of the sequencing data, analysis of species-specific bacterial qPCR values, and network analyses.
File: QIIME2_ImmunePerturbation_06042023
The code for QIIME2 used for quality control, trimming, demultiplexing, and analysis of the 16S amplicon sequences.
Usage notes
See the uploaded readme (ReadME.md). In addition to this, the latest version of R statistical software and the latest versions of the following packages: MASS, car, plyr, dplyr, lme4, msme, emmeans, phyloseq, ggplot2, cowplot, vegan, dplyr, metagMisc, glmmTMB, DHARMa, NetCoMi, AICcmodavg and a Linux system with the latest version of Ubuntu, Conda, and QIIME2 will be required to recreate the analysis.