DOI: 10.5061/dryad.kkwh70sb5
title: A single amino acid polymorphism in natural Metchnikowin alleles of Drosophila results in systemic immunity and life history tradeoffs

description: Antimicrobial peptides (AMPs) are at the interface of interactions between
 hosts and microbes and are therefore expected to be fast evolving in a
 coevolutionary arms race with pathogens. In contrast, previous work
 demonstrated that one AMP, Metchikowin (Mtk), has a single residue that
 segregates as either proline (P) or arginine (R) in populations of four
 different Drosophila species, some of which diverged more than 10 million
 years ago. The recurrent finding of this polymorphism regardless of
 geography or host species, coupled with evidence of balancing selection in
 Drosophila AMPs, suggest there is a distinct functional importance to each
 allele. The most likely hypotheses involve alleles having specificity to
 different pathogens or the more potent allele conferring a cost on the
 host. To assess their functional differences, we created D. melanogaster
 lines with the P allele, R allele, or Mtk null mutation using CRISPR/Cas9
 genome editing. Here, we report results from experiments assessing the two
 hypotheses using these lines. In males, testing of systemic immune
 responses to a repertoire of bacteria and fungi demonstrated that the R
 allele performs as well or better than the P and null alleles with most
 infections. With some pathogens, however, females show results in contrast
 with males where Mtk alleles either do not contribute to survival or where
 the P allele outperforms the R allele. In addition, measurements of life
 history traits demonstrate that the R allele is more costly in the absence
 of infection for both sexes. These results provide strong in vivo evidence
 that differential fitness with or without infection and sex-based
 functional differences in alleles may be adaptive mechanisms of
 maintaining immune gene polymorphisms in contrast with expectations of
 rapid evolution. Therefore, a complex interplay of forces including
 pathogen species and host sex may lead to balancing selection for immune
 genotypes. Strikingly, this selection may act on even a single amino acid
 polymorphism in an AMP.
description: Datasets were collected from laboratory infection and life
 history experiments performed from 2020-2023 at the University of Kansas.
 Data was processed (graphics and statistics) using R/RStudio. Details are
 provided in the README file. Datasets
 Included Data from life history and
 longevity of <em>Drosophila melanogaster</em> with various
 <em>Metchnikowin </em>genotypes and R code used to analyze
 data Egg lay numbers from single females
 Egg hatch rates from single females
 Larval-to-pupal survival rates
 Pupal-to-adult survival rates Adult sex
 ratios Adult longevity 
 Infection data from <em>Drosophila
 melanogaster</em> with various <em>Metchnikowin</em>
 genotypes and R code used to analyze data Male and
 female survival rates from infections with various pathogens over
 time Pathogen load data
 from<em> Drosophila melanogaster</em> with various
 <em>Metchnikowin</em> genotypes and R code used to analyze
 data CFU/mL of various pathogens from male flies
 after 24 h infection 
 Infection data from <em>Drosophila melanogaster</em>
 with various <em>dnaK </em>genotypes and R code used to
 analyze data Survival rates of male flies with intact
 <em>dnaK</em> or a <em>dnaK</em> deletion with
 infection 
 <em>Metchnikowin</em> gene expression data from male and
 female flies with or without infection and R code used to analyze data
 qPCR 2^-(deltaCt) values for <em>mtk</em>
 in different fly genotypes and treatment conditions after 16 h
description: This version of the README file was generated on 07-22-24 by Jessamyn
 Perlmutter. ## General Information 1. Title of Dataset: A single amino
 acid polymorphism in natural Metchnikowin alleles of *Drosophila* results
 in systemic immunity and life history tradeoffs 2. Author Information: 1.
 Principal investigator contact information Name: Robert Unckless
 Institution: University of Kansas Address: Lawrence, KS, USA Email:
 [unckless@ku.edu](mailto:unckless@ku.edu) 2. First author contact
 information Name: Jessamyn Perlmutter Institution: University of Kansas
 Address: Lawrence, KS, USA Email:
 [jessie.perlmutter@gmail.com](mailto:jessie.perlmutter@gmail.com) 3. Dates
 of data collection: 2020-2023 4. Location of data collection: University
 of Kansas, Lawrence 5. Funding sources that supported the collection of
 the data: NIH R01 AI139154, P30 GM110761, K-INBRE P20 GM103418, NSF DBI
 2109772 6. General description of data from this data set
 ([https://doi.org/10.5061/dryad.kkwh70sb5).
 ](https://doi.org/10.5061/dryad.kkwh70sb5)This dataset contains the
 supplemental information associated with the manuscript publishes in PLOS
 Genetics
 ([10.1371/journal.pgen.1011155)](https://doi.org/10.1371%2Fjournal.pgen.1011155), with an earlier preprint on bioRxiv ([https://doi.org/10.1101/2023.01.16.524277)](https://doi.org/10.1101/2023.01.16.524277). The contents include raw data underlying graphs, R code used to analyze results, and tables with statistical outputs of all analyses. All R code is accompanied by corresponding .csv files with the data. ## Sharing/Access information 1. Licenses/restrictions placed on this data: CC0 1.0 Universal (CC0 1.0) Public Domain 2. Links to publications that cite or use the data: Perlmutter, J. I., Chapman, J. R., Wilkinson, M. C., Nevarez-Saenz, I., & Unckless, R. L. (2024). [A single amino acid polymorphism in natural Metchnikowin alleles of *Drosophila* results in systemic immunity and life history tradeoffs](https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011155). PLOS Genetics. ([https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011155](https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011155)) 3. Links to other publicly accessible locations of the data: None 4. Was data derived from another source? No A. If yes, list source(s): N/A 5. Recommended citation for this dataset: Perlmutter, J. I., Chapman, J. R., Wilkinson, M. C., Nevarez-Saenz, I., & Unckless, R. L. (2024). Data from: A single amino acid polymorphism in natural Metchnikowin alleles of *Drosophila* results in systemic immunity and life history tradeoffs. Dryad Digital Repository. [https://doi.org/10.5061/dryad.kkwh70sb5](https://doi.org/10.5061/dryad.kkwh70sb5) ## Data & File Overview 1\. File List: A) Figures1BS2A-D.LifeHistory.R B) Figures1CS3A_B.Longevity.R C) Figures2A-F3A-D.MaleInfections.R D) Figures4A_B.Male_FemaleInfections.R E) Figures5A-C.MicrobialLoad.R F) FiguresS4_S6_S10.SurvivalPlot.R G) FigureS5.AdditionalInfections.R H) FigureS7.FemaleInfections.R I) FigureS8.FemaleSurvivalPlots.R J) FigureS9.Male_FemaleSurvivalCurves.R K) FigureS11B.GeneExpression.R L) Figures1BS2A-D.LifeHistory.csv M) Figures1CS3A.LongevityBlockA.csv N) Figures1CS3B.LongevityBlockB.csv O) MtkLongevityDates.BlockA.csv P) MtkLongevityDates.BlockB.csv Q) Figures2A-F3A-D.MaleInfections.csv R) Figures4A_B.Male_FemaleInfections.csv S) Figures5A-C.MicrobialLoad.csv T) FigureS5.AdditionalInfections.csv U) FigureS7.FemaleInfections.csv V) FigureS11B.GeneExpression.csv 2\. Relationship between files, if important: A) Data underlying all figures is included in .csv files (Files L-V, above). B) R code used to analyze all of the data .csv files is included (Files A-K, above). C) List of corresponding data and analysis files: 1. Figures 1B and S2: files A and L 2. Figures 1C and S3: files B, M-P 3. Figures 2 & 3: files C & Q 4. Figure 4: files D & R 5. Figure 5: files E & S 6. Figures S4, S6, & S10: files F and C 7. Figure S5: files G & T 8. Figure S7: files H & U 9. Figure S8: files I & U 10. Figure S9: files J & R 11. Figure S11B: files K & V 3\. Additional related data collected that was not included in the current data package: None 4\. Are there multiple versions of the dataset? No ## Data-Specific Information Software and packages used: Data analysis and figure generation were performed in R v4.2.2, using several packages: ggplot2 (version 3.4.0), cowplot (version 1.1.1), car (version 3.1.1), SurvMiner (version 0.4.9), MultComp (version 1.4.20), ggExtra (version 0.10.0), ggtext (version 0.1.2), emmeans (version 1.8.3), stringr (version 1.5.0), and SurvMisc (version 0.5.6). Exact packages used for each figure may be found in the corresponding R file. Refer to [PLOS Genetics publication](https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011155) for citations of these packages. #### Information for Files A-K: Data analysis code in .R files 1. Description: All .R files used to analyze data from .csv files, including creation of figures and statistical analysis. 2. Variables, data codes, formats, & abbreviations: 1. For data corresponding to each .R file, see "Data and File Overview" section, part 2. 2. Variables are either created and defined in the code or details can be read below in the Information sections below for corresponding .csv data files. #### Information for File L: Figures1BS2A-D.LifeHistory.csv 1. Description: Life history data underlying Figures 1B and S2, including sample information, life history data, and calculations of survival rates and adult sex ratios. 2. Variables, data codes, formats, & abbreviations: 1. Strain: Unique strain code, with 3 strain codes for each genotype grouping: Unedit (P allele), Edit (R allele), deletion (null alleles) 2. StrainType: genotype groupings of the individual strains: Unedit (P allele), Edit (R allele), deletion (null alleles) 3. Vial: Biological replicate number within each strain and block. Each replicate follows all of the offspring of a single female from egg to death. 4. Eggs: Number of eggs laid by a single female 5. Hatched: Number of eggs hatched into larvae 6. Block: Experimental replicates (A or B), with each replicate performed on different days 7. hatchrate: Percent of eggs hatched into larvae (Hatched/Eggs*100) 8. pupaeD8: Number of pupae formed in a vial 9. L-P_Rate: Percent survival from larval to pupal stages (pupaeD8/Hatched*100) 10. E-P_Rate: Percent survival from egg to pupal stages (pupae D8/Eggs*100) 11. Adults: Total number of offspring reaching adulthood. 12. M: Of the adults, the number that are male. 13. F: Of the adults, the number that are female. 14. P-A_Rate: Percent survival from pupal to adult stages (Adults/pupaeD8*100) 15. L-A_Rate: Percent survival from larval to adult stages (Adults/Hatched*100) 16. E-A_Rate: Percent survival from egg to adult stages (Adults/Eggs*100) 17. NotHatched: Percent of eggs not hatched (100-hatchrate) 18. NotPupated: Percent of hatched larvae that did not pupate ((Hatched-pupaeD8)/Eggs*100) 19. NotEclosed: Percent of pupae that did not emerge as adults ((pupaeD8-Adults)/pupaeD8*100) 20. PropAdult: Percent of eggs that reached adulthood (Adults/Eggs*100) 21. sum: Percent of offspring that either survived to adulthood or died earlier, to check prior numbers (NotHatched+NotPupated+NotEclosed+PropAdult) 22. SexRatio: Male to female sex ratio among adults (M/F) 23. PropFem: Proportion of adults that are female (F/Adults) #### Information for File M: Figures1CS3A.LongevityBlockA.csv 1. Description: Life history data underlying Block A of offspring adult longevity, from day of emergence from pupae to death. Corresponds to figures 1C and S3A. 2. Variables, data codes, formats, & abbreviations: 1. Strain: Unique strain code, with 3 strain codes for each genotype grouping: Unedit (P allele), Edit (R allele), deletion (null alleles) 2. StrainType: genotype groupings of the individual strains: Unedit (P allele), Edit (R allele), deletion (null alleles) 3. Vial: Biological replicate number within each strain and block. Each replicate follows all of the offspring of a single female. 4. Remaining Columns: The remaining columns are in pairs of matching numbers indicating number of days into adulthood. For example, "0" indicates the starting number of adults that had just emerged on day 0, and "2" indicates 2 days into adulthood. This goes until day 122, when all flies were finally confirmed as dead. Columns are paired, with two columns for each day. The left column of each pair is the number of living males. The right column of each pair is the number of living females. Numbers were recorded until the first day that all flies were dead from a given replicate. Remaining days are left blank for a given replicate once all flies are dead. 5. Empty Data Cells: The data for these cells is empty as all flies were already recorded as dead. Code in the .R files for analyzing the data accounts for these empty cells, but could still work if the cells were filled with "0". #### Information for File N: Figures1CS3B.LongevityBlockB.csv 1. Description: Life history data underlying Block B of offspring adult longevity, from day of emergence from pupae to death. Corresponds to figures 1C and S3B. 2. Variables, data codes, formats, & abbreviations: 1. Strain: Unique strain code, with 3 strain codes for each genotype grouping: Unedit (P allele), Edit (R allele), deletion (null alleles) 2. StrainType: genotype groupings of the individual strains: Unedit (P allele), Edit (R allele), deletion (null alleles) 3. Vial: Biological replicate number within each strain and block. Each replicate follows all of the offspring of a single female. 4. Remaining Columns: The remaining columns are in pairs of matching numbers indicating number of days into adulthood. For example, "0" indicates the starting number of adults that had just emerged on day 0, and "7" indicates 7 days into adulthood. This goes until day 82, when all flies were finally confirmed as dead. Columns are paired, with two columns for each day. The left column of each pair is the number of living males. The right column of each pair is the number of living females. Numbers were recorded until the first day that all flies were dead from a given replicate. Remaining days were filled in with "0". #### Information for File O: MtkLongevityDates.BlockA.csv 1. Description: File with "Remaining Columns" From File P copied over into additional file for ease of coding. Corresponds to Figures 1C and S3A, and Block A of the experiment. 2. Variables, data codes, formats, & abbreviations: 1. See "Remaining Columns" section for "Information for File M" for further information #### Information for File P: MtkLongevityDates.BlockB.csv 1. Description: File with "Remaining Columns" From File P copied over into additional file for ease of coding. Corresponds to Figures 1C and S3B, and Block B of the experiment. 2. Variables, data codes, formats, & abbreviations: 1. See "Remaining Columns" section for "Information for File Q" for further information #### Information for File Q: Figures2A-F3A-D.MaleInfections.csv 1. Description: Data for infections of male flies underlying Figures 2 & 3. Data indicates the number of surviving males in each replicate from a starting number of 10 over time. Leftmost columns indicate important data for each sample. 2. Variables, data codes, formats, & abbreviations: 1. Vial: Replicate number, out of total on spreadsheet, indicating a single unique biological replicate out of all data. 2. Gt: Genotype, grouped by general descriptor of Mtk or other relevant genotype 1. 1 bp del: Mtk null allele strain with single base pair deletion 2. 6 bp del: Mtk null allele strain with 6 base pair deletion 3. 17 bp del: Mtk null allele strain with 17 bp deletion 4. R1/R2/R3: Independently-derived CRISPR Mtk arginine (R, or edit) allele strains, 3 independent strains total (R1/R2/R3) 5. P1/P2/P3: Independently-derived CRISPR Mtk proline (P, or unedit) allele strains, 3 independent strains total (P1/P2/P3) 6. Spz: Strain with null allele for spatzle gene 7. MyD88: Strain with null allele for myd88 gene 3. Line: Unique number identifiers corresponding to each independent Mtk fly strain tested, or Spz or MyD88 strains. 4. Treatment: Infection or control treatment used for a given sample 1. JU2- not used in this study, indicates a likely *Penicillium* fungal strain isolated from a fly. 2. B. bassiana= Beauveria bassiana filamentous fungus 3. 20% Glycerol= 20% Glycerol negative infection control for filmentous fungi 4. F. oxysporum= Fusarium oxysporum filamentous fungus 5. A. fumigatus= Apergillus fumigatus filamentous fungus 6. A. flavus= Aspergillus flavus filamentous fungus 7. G. pseudocandidus= Galactomyces pseudocandidus yeast 8. C. glabrata= Candida glabrata yeast 9. PDB= Potato Dextrose Broth negative control for yeast 10. B. thuringiensis= Bacillus thuringiensis bacteria 11. P. rettgeri= Providencia rettgeri bacteria 12. LB= Luria Broth negative control for bacteria 13. E. faecalis= Enterococcus faecalis bacteria 14. S. marsescens= Serratia marcescens bacteria 15. E. faecalis dnaK= Enterococcus faecalis bacteria with null allele in dnak gene, k12 strain background 16. E. faecalis k12= Enterococcus faecalis bacteria of k12 WT strain 5. Replicate: Replicate number within each sample set of a given Gt x Treatment x Day combination 6. DayCollected: Date flies were collected and set aside for experiment 7. DayInfected: Date flies were initially infected (Day 0) 8. OD600: Optical density at 600 nm for a given culture (if bacteria or yeast) or conidia/mL for filamentous fungi. 1. Cells left blank for negative controls, which are at a baseline OD600 of 0 9. Day# Columns: Surviving flies on Day# post-infection. D0 indicates day 0, the day of infection. All vials start with 10 flies at D0. 1. Cells left blank indicate the end of a given experiment. Some experimental survival rates were recorded for longer than others, depending on the pathogen species and experimental replicate. Blank cells indicate days survival was no longer recorded. #### Information for File R: Figures4A\_B.Male\_FemaleInfections.csv 1. Description: Infection survival data corresponding to experiments done in Figures 4 and S9. 2. Variables, data codes, formats, & abbreviations: 1. Vial: Replicate number, out of total on spreadsheet, indicating a single unique biological replicate out of all data. 2. Gt: Genotype, grouped by general descriptor of Mtk or other relevant genotype 1. 1 bp del: Mtk null allele strain with single base pair deletion 2. 6 bp del: Mtk null allele strain with 6 base pair deletion 3. 17 bp del: Mtk null allele strain with 17 bp deletion 4. R1/R2/R3: Independently-derived CRISPR Mtk arginine (R, or edit) allele strains, 3 independent strains total (R1/R2/R3) 5. P1/P2/P3: Independently-derived CRISPR Mtk proline (P, or unedit) allele strains, 3 independent strains total (P1/P2/P3) 6. Spz: Strain with null allele for spatzle gene 7. MyD88: Strain with null allele for myd88 gene 3. MtkAllele: Broad grouping of similar allele types 1. Null= deletion or Mtk null strains 2. Edit= arginine (R) Mtk strains 3. Unedit= proline (P) Mtk strains 4. Spz= spatzle gene null strain 5. MyD88= myd88 gene null strain 4. Line: Unique number identifiers corresponding to each independent Mtk fly strain tested, or Spz or MyD88 strains. 5. Treatment: Infection or control treatment used for a given sample 1. B. bassiana= Beauveria bassiana filamentous fungus 2. A. fumigatus= Apergillus fumigatus filamentous fungus 3. 20% Glycerol= 20% Glycerol negative infection control for filmentous fungi 6. Replicate: Replicate number within each sample set of a given Gt x Treatment x Day x Sex combination 7. Sex: Sex of flies from the given sample (F for females, M for males) 8. DayCollected: Date flies were collected and set aside for experiment 9. DayInfected: Date flies were initially infected (Day 0) 10. Day# Columns: Surviving flies on Day# post-infection. D0 indicates day 0, the day of infection. All vials start with 10 flies at D0. #### Information for File S: Figures5A-C.MicrobialLoad.csv 1. Description: Data from microbial load experiment corresponding to Figure 5. 2. Variables, data codes, formats, & abbreviations: 1. Plate#: Indicates an individual sample across all replicate experiments. 2. Block: Indicates an experimental replicate, with the entire experiment replicated 3 times (Blocks A, B, C). 3. Replicate: Individual samples of a given FlyGenotype x Microbe x DateInfected combination (3 replicates for each grouping: a, b, or c). 4. FlyGenotype: 1. 1 bp del: Mtk null allele strain with single base pair deletion 2. 6 bp del: Mtk null allele strain with 6 base pair deletion 3. 17 bp del: Mtk null allele strain with 17 bp deletion 4. R1/R2/R3: Independently-derived CRISPR Mtk arginine (R, or edit) allele strains, 3 independent strains total (R1/R2/R3) 5. P1/P2/P3: Independently-derived CRISPR Mtk proline (P, or unedit) allele strains, 3 independent strains total (P1/P2/P3) 6. Spz: Strain with null allele for spatzle gene 7. MyD88: Strain with null allele for myd88 gene 5. MtkAllele: 1. Null= deletion or Mtk null strains 2. Edit= arginine (R) Mtk strains 3. Unedit= proline (P) Mtk strains 4. MyD88= myd88 gene null strain 6. FlyLine: Unique number identifiers corresponding to each independent Mtk fly strain tested or MyD88 strain. 7. Sex: M for males (all male in this experiment) 8. NumberFlies: Number of individual flies pooled for each sample (3 for all samples) 9. Microbe: Pathogen species used to infect the flies 1. C. glabrata= Candida glabrata yeast 2. P. rettgeri= Providencia rettgeri bacteria 3. E. faecalis= Enterococcus faecalis bacteria 10. MicrobeStrain: Strain number of each microbe, uniquely used within the Unckless lab. 11. Media: Media used to grow the indicated pathogen and the vehicle used for infection 1. PDB= Potato Dextrose Broth 2. LB= Luria Broth 12. Temp: Temperature used to grow pathogens 1. 30: 30 degrees Celsius 2. 37: 37 degrees Celsius 13. DateInfected: Date the flies were infected. 14. HomogenateVol: Volume of liquid PBS used to homogenize the pooled flies in each sample, in microliters 15. DilFactor: Dilution factor of a homogenized sample, if the sample was diluted in PBS before plating 16. Colonies: Total number of microbial colonies or CFU's from each sample. 1. Only filled if counted by hand. Blank cells indicate a colony counter was used (done for later replicates) 17. CFU.fly: Total number of microbial colonies or CFU's, calculated per fly 1. "-" symbol if no colonies grew 2. Blank cells for later replicates, as this calculation was no longer used. 18. CFU.mL: Total number of microbial colonies or CFU's, calculated per mL of homogenate 1. All samples from all replicates have this calculation, if microbes grew. This was the calculation used for the figure. Blank cells indicate no microbes grew from the sample. 19. CFU.mL (direct readout): Total number of microbial colonies or CFU's, calculated per mL of homogenate, as estimated by a robotic colony counter 1. Blank cells in the first replicate before the counter was used, and blank in later replicates if no microbes grew in the sample. 20. Final column: Replicate number for each given Microbe x DateInfected combination #### Information for File T: FigureS5.AdditionalInfections.csv 1. Description: Infection survival data for male flies infected with additional microbial pathogens, corresponding to Figure S5. 2. Variables, data codes, formats, & abbreviations: 1. Vial: Replicate number, out of total on spreadsheet, indicating a single unique biological replicate out of all data. 2. Gt: Genotype, grouped by general descriptor of Mtk or other relevant genotype 1. 1 bp del: Mtk null allele strain with single base pair deletion 2. 6 bp del: Mtk null allele strain with 6 base pair deletion 3. 17 bp del: Mtk null allele strain with 17 bp deletion 4. R1/R2/R3: Independently-derived CRISPR Mtk arginine (R, or edit) allele strains, 3 independent strains total (R1/R2/R3) 5. P1/P2/P3: Independently-derived CRISPR Mtk proline (P, or unedit) allele strains, 3 independent strains total (P1/P2/P3) 6. Spz: Strain with null allele for spatzle gene 7. MyD88: Strain with null allele for myd88 gene 3. MtkAllele: Broad grouping of similar allele types 1. Null= deletion or Mtk null strains 2. Edit= arginine (R) Mtk strains 3. Unedit= proline (P) Mtk strains 4. Spz= spatzle gene null strain 5. MyD88= myd88 gene null strain 4. Line: Unique number identifiers corresponding to each independent Mtk fly strain tested, or Spz or MyD88 strains. 5. Treatment: Infection or control treatment used for a given sample 1. Note: The spreadsheet contains data from infections that were not ultimately included in the publication. Abbreviations explained here are those that pertain to the published data. 2. Negative= Negative control 1. LB (Luria Broth) for bacteria corresponding to the "Negative control" (performed on same day) 2. YPD (Yeast extract, peptone, dextrose media) for yeast corresponding to the "Negative control" (performed on same day) 3. Ly. fusiformis= Lysinibacillus fusiformis bacteria 4. S. succinus= Staphylococcus succinus bacteria 5. S. sciuri= Staphylococcus sciuri bacteria 6. C. albicans= Candida albicans yeast 7. L. brevis= Lactococcus brevis bacteria 8. L. plantarum= Lactococcus plantarum bacteria 9. YPD= Yeast extract, peptone, dextrose media, negative control for C. albicans 10. LB= Luria broth media, negative control for most bacteria 11. MRS= deMan-Rogosa-Sharpe media, negative control for Lactococcus bacteria 6. Replicate: Replicate number within each sample set of a given Gt x Treatment x Day combination 7. DayCollected: Date flies were collected and set aside for experiment 8. DayInfected: Date flies were initially infected (Day 0) 9. OD600: Optical density at 600 nm for a given culture (if bacteria or yeast) or conidia/mL for filamentous fungi. 1. Cells filled with "0" for negative controls, which are at a baseline OD600 of 0. 10. Day# Columns: Surviving flies on Day# post-infection. D0 indicates day 0, the day of infection. All vials start with 10 flies at D0. 1. Cells left blank indicate the end of a given experiment. Some experimental survival rates were recorded for longer than others, depending on the pathogen species and experimental replicate. Blank cells indicate days survival was no longer recorded. #### Information for File U: FigureS7.FemaleInfections.csv 1. Description: Data for female infection survival, corresponding to Figures S7 and S8. 2. Variables, data codes, formats, & abbreviations: 1. Vial: Replicate number, out of total on spreadsheet, indicating a single unique biological replicate out of all data. 2. Gt: Genotype, grouped by general descriptor of Mtk or other relevant genotype 1. 1 bp del: Mtk null allele strain with single base pair deletion 2. 6 bp del: Mtk null allele strain with 6 base pair deletion 3. 17 bp del: Mtk null allele strain with 17 bp deletion 4. R1/R2/R3: Independently-derived CRISPR Mtk arginine (R, or edit) allele strains, 3 independent strains total (R1/R2/R3) 5. P1/P2/P3: Independently-derived CRISPR Mtk proline (P, or unedit) allele strains, 3 independent strains total (P1/P2/P3) 6. Spz: Strain with null allele for spatzle gene 7. MyD88: Strain with null allele for myd88 gene 3. MtkAllele: Broad grouping of similar allele types 1. Null= deletion or Mtk null strains 2. Edit= arginine (R) Mtk strains 3. Unedit= proline (P) Mtk strains 4. Spz= spatzle gene null strain 5. MyD88= myd88 gene null strain 4. Line: Unique number identifiers corresponding to each independent Mtk fly strain tested, or Spz or MyD88 strains. 5. Treatment: Infection or control treatment used for a given sample 1. P. rettgeri= Providencia rettgeri bacteria 2. E. faecalis= Enterococcus faecalis bacteria 3. LB= Luria Broth, negative control for bacteria 4. C. glabrata= Candida glabrata yeast 5. PDB= Potato dextrose broth, negative control for yeast 6. B. bassiana= Beauveria bassiana filamentous fungus 7. A. fumigatus= Aspergillus fumigatus filamentous fungus 8. 20% Glycerol= 20% glycerol negative control for filamentous fungi 6. Replicate: Replicate number within each sample set of a given Gt x Treatment x Day combination 7. DayCollected: Date flies were collected and set aside for experiment 8. DayInfected: Date flies were initially infected (Day 0) 9. OD600: Optical density at 600 nm for a given culture (if bacteria or yeast) or conidia/mL for filamentous fungi. 1. Cells left blank for negative controls, which are at a baseline OD600 of 0 10. Day# Columns: Surviving flies on Day# post-infection. D0 indicates day 0, the day of infection. All vials start with 10 flies at D0. 1. Cells left blank indicate the end of a given experiment. Some experimental survival rates were recorded for longer than others, depending on the pathogen species and experimental replicate. Blank cells indicate days survival was no longer recorded. #### Information for File V: FigureS11B.GeneExpression.csv 1. Description: Mtk gene expression data for experiment underlying Figure S11B. 2. Variables, data codes, formats, & abbreviations: 1. Sample#: Number for each unique sample measured 2. Allele: Mtk allele measured for all samples 3. Strain: Unique number identifiers corresponding to each independent Mtk fly strain tested 4. StrainType: Broad grouping of similar allele types 1. Deletion= Mtk null strains 2. Edit= arginine (R) Mtk strains 3. Unedit= proline (P) Mtk strains 5. Sex: Sex of flies from a given sample (F for female, M for male) 6. Treatment: Infection or control treatment used for a given sample 1. B. bassiana= Beauveria bassiana filamentous fungus 2. Control= negative control (not infected) 3. Deltact= 2^-(deltaCt) calculation for each of the samples from qPCR