DOI: 10.5061/dryad.zcrjdfnmw
title: Data from: Nutrition, pesticide exposure, and virus infection interactions in honey bees

description: Declines in pollinator health are frequently hypothesized to be the
 combined result of multiple interacting biotic and abiotic stressors;
 namely, nutritional limitations, pesticide exposure, and pathogen
 infection. Despite this, most studies examining stressor interactions have
 been constrained to two concurrent factors, limiting our understanding of
 multi-stressor dynamics. Using honey bees as a model, we addressed this
 gap by studying how variable quality diet, field-realistic levels of
 multiple pesticides, and virus infection interact to affect survival,
 infection intensity, and immune and detoxification gene expression.
 Although we found evidence that agrochemical exposure (a field-derived
 mixture of chlorpyrifos and two fungicides) can exacerbate infection and
 increase virus-induced mortality, this result was nutritionally-dependent,
 only occurring when bees were provided artificial pollen. Provisioning
 with naturally-collected polyfloral pollen inverted the effect, reducing
 virus-induced mortality and suggesting a hormetic response. To test if the
 response was pesticide specific, we repeated our experiment with a
 pyrethroid (lambda-cyhalothrin) and a neonicotinoid (thiamethoxam),
 finding variable results. Finally, to understand the underpinnings of
 these effects, we measured viral load and expression of important immune
 and detoxification genes. Together, our results show that multi-stressor
 interactions are complex and highly context-dependent, but have great
 potential to affect bee health and physiology.
description: The code is provided as supporting data for &gt; Hsieh, E.M., Dolezal,
 A.G., 2024. Nutrition, pesticide exposure, and virus infection interact to
 produce context-dependent effects in honey bees (*Apis mellifera*).
 Science of The Total Environment 949, 175125. ### Project description:
 This project was designed to investigate the tripartite effects of
 variable nutrition, pesticide exposure, and virus infection in pollinators
 using honey bees as a model. Honey bees were exposed to different
 combinations of the three concurrent stressors and their resulting
 survival, virus titers, and relative expression of key immune and
 detoxification genes were recorded and compared. The data and code files
 contained within this repository present the complete data set and should
 allow for recreation of the results. All analyses performed using R
 version 4.3.2 and RStudio 2023.06.01 Build 524. Package versions below:
 survival version 3.5-7 survminer version 0.4.9 dplyr version 1.1.4 lme4
 version 1.1-35.1 emmeans version 1.10.0 ggplot2 version 3.4.4 ###
 Contents: Subfolder name and contents: Data and code are grouped by
 analysis type (relative gene expression, survival, absolutely virus titer
 quantification) and then split into pesticide (chlorpyrifos,
 lambda-cyhalothrin, thiamethoxam). See variable heading below for details
 on processed data within .csv files. `gene_expression_analyses` -- split
 by diet treatment (pollen, supplement, combined). CSV files contain
 log-transformed relative expression data, R files contain code for
 analyses and figure generation, subsets folders contain data isolated by
 gene. * `chlorpyrifos` -- contains all data subsets of relative gene
 expression results and analysis .R files for bees exposed to the
 chlorpyrifos + F combination. * `comb_subsets` -- contains all .csv files
 for relative expression data and treatment characteristics (diet,
 pesticide, and virus inoculation type) within the Experiment 3 subset.  *
 `factor` -- contains all treatment characteristics and relative expression
 data within the Experiment 3 subset split by target genes. *
 `trt_combo` -- contains all relative expression data within the Experiment
 3 subset split by target genes. * `poll_subsets` -- contains all .csv
 files for relative expression data and treatment characteristics (diet,
 pesticide, and virus inoculation type) within the Experiment 2 subset.  *
 `factor` -- contains all treatment characteristics and relative expression
 data within the Experiment 2 subset split by target genes. *
 `trt_combo` -- contains all relative expression data within the Experiment
 2 subset split by target genes. * `supp_subsets` -- contains all .csv
 files for relative expression data and treatment characteristics (diet,
 pesticide, and virus inoculation type) within the Experiment 1 subset.  *
 `factor` -- contains all treatment characteristics and relative expression
 data within the Experiment 1 subset split by target genes. *
 `trt_combo` -- contains all relative expression data within the Experiment
 31subset split by target genes. * `chlor_comb_expr_analysis.R` --
 Describes the necessary libraries, functions, and justifications for the
 analysis of the combined diet chlorpyrifos+F-exposed bees experimental
 subset (Experiment 3). See "Usage" heading below for details.  *
 `chlor_poll_expr_analysis.R` -- Same as above for the pollen diet
 chlorpyrifos+F-exposed bees experimental subset (Experiment 2). *
 `chlor_supp_expr_analysis.R` -- Same as above for the supplement diet
 chlorpyrifos+F-exposed bees experimental subset (Experiment 1). *
 `comb_expr_data.csv` -- Contains the processed relative expression data
 for import in `chlor_comb_expr_analysis.R` sorted by gene and treatment. 
 * `poll_expr_data.csv` -- Contains the processed relative expression data
 for import in `chlor_poll_expr_analysis.R` sorted by gene and treatment. 
 * `supp_expr_data.csv` -- Contains the processed relative expression data
 for import in `chlor_supp_expr_analysis.R` sorted by gene and treatment. 
 `survival_analyses` -- split by diet treatment (pollen, supplement,
 combined). CSV files contains raw survival data, R files contains code for
 analyses and figure generation, parsing folders contains isolated
 treatment pairs from survival data for pairwise comparisons. *
 `chlorpyrifos` -- contains all data subsets of survival assay results and
 analysis .R files for bees exposed to the chlorpyrifos + F combination
 (Experiments 1-3) * `Chlor_comb_survival_parsing` -- contains all pairwise
 comparison .csv files of survival data and treatment characteristics
 within the Experiment 3 subset.  * `Chlor_poll_survival_parsing` --
 contains all pairwise comparison .csv files of survival data and treatment
 characteristics within the Experiment 2 subset.  *
 `Chlor_supp_survival_parsing` -- contains all pairwise comparison .csv
 files of survival data and treatment characteristics within the Experiment
 1 subset.  * `Chlor_comb_survival.csv` -- Contains the processed survival
 data for import in `Chlor_comb_survival_analysis.R` within the combined
 diet subset (Experiment 3) * `Chlor_comb_survival_analysis.R` -- Describes
 the necessary libraries, functions, and justifications for the survival
 analysis of the combined diet chlorpyrifos+F-exposed bees experimental
 subset (Experiment 3). See "Usage" heading below for details.  *
 `Chlor_poll_survival.csv` -- Contains the processed survival data for
 import in `Chlor_poll_survival_analysis.R` within the pollen diet subset
 (Experiment 2) * `Chlor_poll_survival_analysis.R` -- Describes the
 necessary libraries, functions, and justifications for the survival
 analysis of the pollen diet chlorpyrifos+F-exposed bees experimental
 subset (Experiment 2). See "Usage" heading below for details.  *
 `Chlor_supp_survival.csv` -- Contains the processed survival data for
 import in `Chlor_supp_survival_analysis.R` within the supplement diet
 subset (Experiment 1) * `Chlor_supp_survival_analysis.R` -- Describes the
 necessary libraries, functions, and justifications for the survival
 analysis of the supplement diet chlorpyrifos+F-exposed bees experimental
 subset (Experiment 1). See "Usage" heading below for details.  *
 `lambda-cyhalothrin`-- contains all data subsets of survival assay results
 and analysis .R files for bees exposed to lambda-cyhalothrin (Experiments
 4-5). * `Lambda_poll_survival_parsing` -- contains all pairwise comparison
 .csv files of survival data and treatment characteristics within the
 Experiment 5 subset.  * `Lambda_supp_survival_parsing` -- contains all
 pairwise comparison .csv files of survival data and treatment
 characteristics within the Experiment 4 subset.  *
 `Lambda_poll_survival.csv` -- Contains the processed survival data for
 import in `Lambda_poll_survival_analysis.R` within the supplement diet
 subset (Experiment 5) * `Lambda_poll_survival_analysis.R` -- Describes the
 necessary libraries, functions, and justifications for the survival
 analysis of the pollen diet lambda-cyhalothrin-exposed bees experimental
 subset (Experiment 5). See "Usage" heading below for details.  *
 `Lambda_supp_survival.csv` -- Contains the processed survival data for
 import in `Lambda_supp_survival_analysis.R` within the supplement diet
 subset (Experiment 4) * `Lambda_supp_survival_analysis.R` -- Describes the
 necessary libraries, functions, and justifications for the survival
 analysis of the supplement diet lambda-cyhalothrin-exposed bees
 experimental subset (Experiment 4). See "Usage" heading below
 for details.  * `thiamethoxam`-- contains all data subsets of survival
 assay results and analysis .R files for bees exposed to thiamethoxam
 (Experiments 6-7). * `Thiam_poll_survival_parsing` -- contains all
 pairwise comparison .csv files of survival data and treatment
 characteristics within the Experiment 7 subset.  *
 `Thiam_supp_survival_parsing` -- contains all pairwise comparison .csv
 files of survival data and treatment characteristics within the Experiment
 6 subset.  * `Thiam_poll_survival.csv` -- Contains the processed survival
 data for import in` Thiam_poll_survival_analysis.R` within the supplement
 diet subset (Experiment 7) * `Thiam_poll_survival_analysis.R` -- Describes
 the necessary libraries, functions, and justifications for the survival
 analysis of the pollen diet thiamethoxam-exposed bees experimental subset
 (Experiment 7). See "Usage" heading below for details.  *
 `Thiam_supp_survival.csv` -- Contains the processed survival data for
 import in` Thiam_poll_survival_analysis.R` within the supplement diet
 subset (Experiment 6) * `Thiam_supp_survival_analysis.R` -- Describes the
 necessary libraries, functions, and justifications for the survival
 analysis of the pollen diet thiamethoxam-exposed bees experimental subset
 (Experiment 6). See "Usage" heading below for details. 
 `virus_titer_analyses` -- chlorpyrifos data are split into three separate
 CSVs across diet types (pollen, supplement, combined); lambda-cy and
 thiamethoxam include two diet types (pollen, supplement) and are combined
 into the same CSV. * `Chlorpyrifos` -- contains all data subsets of viral
 titers and analysis .R files for bees exposed to the chlorpyrifos + F
 combination (Experiments 1-3). * `Chlor_titer_analysis.R` -- describes the
 necessary libraries, functions, and justifications for the viral titer
 analysis of each of the experiments involving bee exposed to chlorpyrifos
 + F (combined, pollen, and supplement). See "Usage" heading
 below for details.  * `comb_logquant.csv` -- Contains the processed viral
 titer data for import  in `Chlor_titer_analysis.R` of the combination diet
 trial (Experiment 3) * `poll_logquant.csv` -- Contains the processed viral
 titer data for import  in `Chlor_titer_analysis.R` of the pollen diet
 trial (Experiment 2) * `supp_logquant.csv` -- Contains the processed viral
 titer data for import  in `Chlor_titer_analysis.R` of the supplement diet
 trial (Experiment 1) * `Lambda-cyhalothrin` -- contains all data subsets
 of viral titers and analysis .R files for bees exposed to the
 lambda-cyhalothrin (Experiments 4-5). * `lambda_data.csv` -- Contains the
 processed viral titer data for import  in `Lambda_titer_analysis.R` of the
 supplement diet trial (Experiments 4 & 5) *
 `Lambda_titer_analysis.R` -- describes the necessary libraries, functions,
 and justifications for the viral titer analysis of each of the experiments
 within bees exposed to lambda-cyhalothrin (pollen, and supplement). See
 "Usage" heading below for details.  * `Thiamethoxam` -- contains
 all data subsets of viral titers and analysis .R files for bees exposed to
 the thiamethoxam (Experiments 6-7). * `thiam_data.csv` -- contains the
 processed viral titer data for import  in `Thiam_titer_analysis.R` of the
 supplement diet trial (Experiments 6 & 7) *
 `Thiam_titer_analysis.R` -- describes the necessary libraries, functions,
 and justifications for the viral titer analysis of each of the experiments
 within bees exposed to thiamethoxam (pollen, and supplement). See
 "Usage" heading below for details.  ### Usage: Analyses for each
 respective group can be run using the designated `_analysis.R` files in
 the RStudio environment to recreate the analyses performed in the project.
 Each .R file begins with a list of necessary libraries, which are also
 listed under the project description heading. Code annotations will
 describe every step and justification for specific commands (tests run,
 assumption checking, etc.), but the following should provide a general
 idea for the analysis flow. `gene_expression_analyses`:  1. Import
 expression values as log-transformed delta-delta CT values to reduce
 variance.  2. Fit models to linear mixed models, designating `plate_ID` as
 a random factor to account for plate-by-plate variability using the `lmer`
 and `lm` functions within the `lme4` package. 3. Compare the AIC scores of
 of linear models with and without random effects using the
 `compare_performance` function in the `performance` package. 4. Select
 preferred model, check assumptions using the `performance` package.  5. If
 differences are detected, use `emmeans` function within the `emmeans`
 package to determine pairwise differences between treatments and adjust
 for multiple comparisons using Tukey adjustments.  `survival_analyses`: 1.
 Import survival data with date and mortality points recorded. 2. Fit cox
 proportional hazard model using the `coxph` function from the `survival`
 package.  3. Import the pairwise comparisons from each set from the
 relevant `parsin`g folder, detailed above. 4. Create the pairwise models
 using the same functions in Step 2. 5. Correct for multiple comparisons
 using the `p.adjust` function. `virus_titer_analyses`: 1. Import titer
 data with log-transformed viral quantities based on an absolute
 quantification standard curve.  2. Create linear model using the `lm`
 function.  3. Check assumptions using `shapiro.test`, qq plot
 visualizations, and `leveneTest` functions. 4. In the case of not meeting
 assumptions, analyze using `kruskal.test` and `pairwise.wilcox.test` to
 determine pairwise differences with Bejamini-Hochberg corrections for
 multiple comparisons.  ### Variables: All data files within
 the `gene_expression_analyses` subfolder: * `gene / target_name` -- the
 name of the target gene, apidacein, CYP6AS3, hopscotch, relish, or vg *
 `log_expr / logddct` -- the log-transformed value of the relative gene
 expression, based on the delta-delta CT method. Values are normalized
 against the largest absolute value of the delta CT within the control
 groups. * `mean` -- the mean log-transformed relative gene expression
 value for each treatment within each target gene. Used for figure
 generation. * `se` -- the standard error of the log-transformed value of
 relative gene expression for each treatment within each target gene. Used
 for figure generation. * `trt / trt_group` -- the treatment designation
 for each sample: MB / M = supplement, AZ / A = pollen, V- = sucrose, V+ =
 virus, Pe = chlorpyrifos, T = thiamethoxam, L = lambda-cyhalothrin, no Pe
 = control, C- = control-sucrose, C+ = control-virus, P- =
 pesticide-sucrose, P+ = pesticide-virus * `plate_ID` -- the 384 well plate
 identification number. Used as a random effect factor in the analysis. *
 `set_ID` -- which subset the data belong to, Comb = combined, MB =
 supplement, AZ = pollen * `diet` -- which diet type was consumed, AZ =
 pollen, MB = supplement * `pesticide` -- whether pesticide was present (Y)
 or absent (N) * `virus`-- whether virus was present (Y) or absent (N) All
 data files within the `survival_analyses` subfolder: * `treatment` -- the
 treatment designation, Con = no pesticide, pest = pesticide (chlorpyrifos,
 lambda-cyhalothrin, or thiamethoxam as designated by the destination
 folder), - = sucrose, + = virus. * `cage` -- cage number, treated as
 experimental unit * `date` -- timepoint of check, converted to `hpi` and
 `dpi` below for uniformity  * `status` -- mortality state, 1 = dead, 0 =
 alive * `trt` -- treatment designation code for R code analysis, 1 = Con-,
 2 = Con+, 3 = Pest-,  4 = Pest+ * `hpi` -- hours post infection, converted
 from date for uniformity and easier plotting - `dpi` -- days post
 infection, converted from date for uniformity and easier plotting All data
 files within the `virus_titer_analyses` subfolder: * `trt` -- the
 treatment designation, MB / M = supplement, AZ / A = pollen, V- = sucrose,
 V+ = virus, Pe = chlorpyrifos, T = thiamethoxam, L = lambda-cyhalothrin,
 no Pe = control, C- = control-sucrose, C+ = control-virus, P- =
 pesticide-sucrose, P+ = pesticide-virus * `log_quant` -- the base 10
 log-transformed viral titers, measured in viral genome equivalents using
 an absolute quantification standard curve method. * `pest` -- the
 pesticide type added to each diet mixture, control (acetone solvent) or
 pesticide (field-relevant combination of chlorpyrifos, azoxystrobin, and
 pyraclostrobin) * `virus` -- the inoculation type, virus (Israeli acute
 paralysis virus) or sucrose (30% sugar solution) * `diet` --  the diet
 type for each sample, fed either supplement (protein supplement) or pollen
 (naturally collected pollen)