DOI: 10.5061/dryad.qz612jmt1
title: A suite of selective pressures supports the maintenance of alleles of a Drosophila immune peptide

description: The innate immune system provides hosts with a crucial first line of
 defense against pathogens. While immune genes are often among the fastest
 evolving genes in the genome, in Drosophila, antimicrobial peptides (AMPs)
 are notable exceptions. Instead, AMPs may be under balancing selection,
 such that over evolutionary timescales multiple alleles are maintained in
 populations. In this study, we focus on the Drosophila antimicrobial
 peptide Diptericin A, which has a segregating amino acid polymorphism
 associated with differential survival after infection with the
 Gram-negative bacteria Providencia rettgeri. Diptericin A also helps
 control opportunistic gut infections by common Drosophila gut microbes,
 especially those of Lactobacillus plantarum. In addition to genotypic
 effects on gut immunity, we also see strong sex-specific effects that are
 most prominent in flies without functional diptericin A. To further
 characterize differences in microbiomes between different diptericin
 genotypes, we used 16S metagenomics to look at the microbiome composition.
 We used both lab reared and wild caught flies for our sequencing and
 looked at overall composition as well as the differential abundance of
 individual bacterial families. Overall, we find flies that are homozygous
 for one allele of diptericin A are better equipped to survive a systemic
 infection from P. rettgeri, but in general have shorter lifespans
 after being fed common gut commensals. Our results suggest a possible
 mechanism for the maintenance of genetic variation of diptericin A through
 the complex interactions of sex, systemic immunity, and the maintenance of
 the gut microbiome.
description: # README for Mullinax *et al.* 2025 "A suite of selective pressures
 supports the maintenance of alleles of a Drosophila immune peptide"
 ## Figure 1 (Fig1\_data.csv) This is survival data for several genotypes
 after infection with several different pathogens. The columns are: | Title
 | Explanation | | ------------ |
 ------------------------------------------------------------------- | |
 Vial\_number | bookkeeping purposes and these values repeat so are not
 useful | | line | The Diptericin CRISPR/Cas9 edited stock (or other
 genotype) | | genotype | Specific edits to the Diptericin (or Imd) gene |
 | treatment | Microbes used for infection (see manuscript text for
 abbreviations or media below) | | start\_date | Date infection started
 (YYYYMMDD) | | day0-day5 | number of flies alive on each day | | OD600 |
 The optical density (600nm) of bacterial culture used for infection | *MRS
 = Man, Rogosa and Sharpe media; LB = Luria-Bertani broth media; ## Figure
 2 and Figure S3 (Fig2\_S3\_data.xlsx) Note that each tab is a different
 treatment. Data represents survival after association with different
 microbes in the food (not systemic infection). The columns are: | Title |
 Explanation | | ------------- |
 --------------------------------------------------------------------------------------- | | Vial\_number | bookkeeping purposes and these values repeat so are not useful | | line | The Diptericin CRISPR/Cas9 edited stock (or other genotype) | | genotype | Specific edits to the Diptericin (or Imd) gene | | gen\_genotype | Collapsed genotype | | sex | male of female flies | | treatment | Microbes used for infection (see manuscript text for abbreviations) | | start\_date | Date infection started (YYYYMMDD) | | day0-day... | number of flies alive on each day - note that the days counted may differ by experiment; NA indicates that flies were not counted on that day | ## Figure 3 and Figure S5 and S6 (Fig3\_S5\_data.xlsx) Note that each tab is a different treatment. Data represents bacterial load (CFU) after association with different microbes in the food (not systemic infection) after 2, 10 or 20 days. The columns are: | Title | Explanation | | ---------------- | --------------------------------------------------------------------------------------- | | SampleId | coded for line, sex and replicate | | line | The diptericin CRISPR/Cas9 edited stock (or other genotype) | | gen\_genotype | collapsed genotype | | sex | male or female flies | | replicate | replicate count | | counted.colonies | number of colonies counted on plater | | volume counted | the spiral plater counts a fraction of the plate; this corresponds to a specific volume | | dilution factor | dilution of the initial homogenate before plating | | Result.col.ml | colonies per ml | | exp | date experiment started (YYYYMMDD) | | treatment | microbe associated (see tab title for full name) | | Day | day homogenized (2, 10 or 20 days after association) | ## Figure 4 - Desiccation (Fig4\_data\_Dessication.xlsx) Each tab is a different date of the experiment, but because different hours were counted, the data is kept on separate tabs. | Title | Explanation | | ------------- | -------------------------------------------------------------------- | | Vial | bookkeeping purposes and these values repeat so are not useful | | line | The diptericin CRISPR/Cas9 edited stock (or other genotype) | | genotype | diptericin genotype associated with that line | | sex | male or female | | treatment | conventional (reared in standard vials) or axenic (reared germ-free) | | start_date | date experiment started (YYYYMMDD) | | hour0-hour... | the hour that surival was counted (number of flies alive at that time) | ## Figure 4 - Starvation (Fig4\_data\_Starvation.xlsx) Each tab is a different date of the experiment, but because different hours were counted, the data is kept on separate tabs. | Title | Explanation | | --------- | -------------------------------------------------------------------- | | Vial | bookkeeping purposes and these values repeat so are not useful | | line | The diptericin CRISPR/Cas9 edited stock (or other genotype) | | genotype | diptericin genotype associated with that line | | sex | male or female | | treatment | conventional (reared in standard vials) or axenic (reared germ-free) | | start_date | date experiment started (YYYYMMDD) | | 0-... | the hour that surival was counted (number of flies alive at that time) | ## Figure S2 (FigS2\_data\_male\_v\_female.csv) This is survival data for several genotypes and both sexes after infection with several different pathogens. The columns are: | Title | Explanation | | ------------ | -------------------------------------------------------------- | | Vial\_number | bookkeeping purposes and these values repeat so are not useful | | line | The Diptericin CRISPR/Cas9 edited stock (or other genotype) | | genotype | Specific edits to the Diptericin (or Imd) gene | | treatment | Microbes used for infection (see manuscript text for abbreviations) | | sex | male or female | | start\_date | Date infection started | | day0-day5 | number of flies alive on each day | ## Figure S3 (FigS3\_data.csv) This is absorbance data for measuring feeding. The columns are: | Title | Explanation | | ----------------- | ----------------------------------------------------------- | | Individual ID | countable index | | Genotype | The diptericin CRISPR/Cas9 edited stock (or other genotype) | | Sex | male or female | | Treatment | microbe/control/LB | | OD600 | concetration of bacteria used | | Date | date of experiment (MM/DD/YY) | | Absorbance\_630nm | absorbance at 630nm |