DOI: 10.5061/dryad.rbnzs7hh8
title: Immune stimulation induced changes to the native gut microbiota of bumble bees

description: 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.
description: 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. 
description: 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.