The code is provided as supporting data for

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 inThiam_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 inThiam_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 parsing 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)