DOI: 10.5061/dryad.2ngf1vhzp
title: Reciprocal costs of infection and reproduction in D. melanogaster

description: Trade-offs occur when an organism has to allocate limited resources into
 multiple biological processes. How organisms allocate their resources and
 whether one trait gets priority over another is poorly understood. Prior
 work has shown that reproductive investment reduces the capacity of D.
 melanogaster to mount an effective immune response against subsequent
 bacterial infection. However, it has not been tested whether the observed
 trade-off was unidirectional with reproductive fitness given primacy over
 immunity, or whether it might also occur in the reciprocal direction with
 an active prior immune response reducing reproductive output. In the
 present work, we delivered bacterial infection to female D. melanogaster
 prior to mating and tested whether reproductive capacity became reduced.
 We found that infected females produced the same number of eggs as
 uninfected females, but the eggs from infected females exhibited lower
 survivorship to adulthood. Additionally, we found that mating destabilizes
 chronic bacterial infections, stimulating additional host death and
 increasing variance in pathogen burden. Together, our results suggest the
 cost of reproduction and infection in Drosophila females is reciprocal,
 regardless of the order in which they occur.
description: Group of 10 male and 10 female <em>Drosophila
 melanogaster</em> flies from Canton S (CS) strain were reared at
 room temperature (22-23°C) with 12h:12h light:dark cycle, and allowed to
 lay eggs. Adult offspring were sorted by sex within 8 hours of emergence
 from the pupal case and were maintained separately before mating. The
 female offspring were divided into different treatment and control groups
 (listed below) and were then subjected to an infection with a
 Gram-negative bacterium <em>Providencia rettgeri</em> at 5 to
 7 days after eclosion: Tretament groups:
 i) M24I,  flies allowed to mate 24 hours prior to
 infection ii) I0M,  flies infected and immediately
 mated iii) I24M,  flies were infected and mated 24
 hours later iv) I120M,  flies infected then mated 5
 days later Control groups:  i) M24-
 , mated, held for 24 hours, then given sterile wound to act as controls
 for the M24I files  ii) -0M, given a sterile wound and
 immediately mated as a control for the I0M and I24M flies
  iii) M0, females mated on the day of infection but were not
 wounded as a control for the I0M  iv) -120M, given a
 sterile wound and then mated 5 days later as a control for the I120M
 flies  v) unmated virgin flies given a sterile
 wound. <strong>Survival
 assay</strong> Survival assay was carried out in
 three different experimental blocks with 5-7 vials per treatment per
 block. Each vial contained 5 females. All treatment and control groups
 listed above were used for survival assay. Survival of the female flies
 was observed over 10 days post infection. The flies that survived at the
 end of the experiment were censored. Survival analysis was carried out
 using the 'survival' package (v3.2-11) in R, for which a mixed
 effect model with treatment as fixed effect and experimental block as
 random effect. In order to compare survival between treatment groups, same
 Cox proportional hazard model within the emmeans() function from the
 package emmeans (v1.8.3) was used.  <strong>Egg
 and offspring count assay</strong> Egg and
 offspring count was carried out for all treatment and control conditions
 except foe unmated virgin flies across three experimental blocks. For
 which, 5 female flies were housed with 8 males in order to ensire mating
 and allowed to lay eggs. Females were transferred to new food vials every
 day and eggs were counted manually under a light microscope immediately.
 The vials were left at room temperature until adult offspring emerged.
 Adult offspring were transferred to empty vials 24 hours after the
 emergence of first adult offspring and frozen until they were counted. To
 estimate per-fly egg and offspring count, the total number of eggs and
 offsrping were divided by the total number of females in each treatment
 group within each experimental block. Egg-to-offspring ratio was
 calculated by dividing the total number of offspring by the total number
 of eggs in each experimental block. Two mixed effect models one with
 experimental block as a random effect and another one without experimental
 block were compared to test the effect of block using lme4 package in R.
 Multiple pairwise tests were then carried out with Bonferroni correction
 to evlauate paiwise differences between treatments.
 <strong>Bacterial load assay</strong>
 Bacterial load assay was carried out to test whether mating has
 any impact on previously established pathogen burden. Individual females
 were anesthetized with CO<sub>2</sub> and homogenized in 500
 μl sterile 1X phosphate buffered saline (PBS). Fifty microliters of the
 homogenate were plated on an LB plate using a WASP2 spiral plater
 (Microbiology International). The plates were incubated overnight at 37°C
 and the bacterial colonies that developed were counted using the ProtoCOL
 plate counting system (Microbiology International). There were 13 plates
 that had colony densities too high to be resolved by the counting
 software. Those plates were assigned the highest measurable value (112500
 colony forming units (CFU) per fly) observed across all three experimental
 blocks. All of the analysis were carried out in R. CFU per fly was first
 calculated and then natural log transformed for subsequent analysis. Since
 some of the CFU per fly values were zero, 1 was added to all the values
 prior to the log transformation. Two mixed effect models with and without
 experimental block were compared to test the effect of experimental block
 using lme4 package in R. Both models had mating status as fixed effect
  Further, all three blocks were pooled together and Levene's test was
 used to compare the variance between treatments. Because treatments did
 not have equal variances, Welch’s t-test was performed to compare between
 means of mated and unmated treatments. Similarly, Fisher’s exact test was
 used to compare between the number of dead individuals between two
 treatment groups.
description: # Reciprocal costs of infection and reproduction in D.melanogaster
 [https://doi.org/10.5061/dryad.2ngf1vhzp](https://doi.org/10.5061/dryad.2ngf1vhzp) ## Description of the data and file structure There are five different datasets associated with this manuscripts. ### Files and variables #### File: Survival\_data.csv **Description:** This dataset contains the data generated from survival analysis. ##### Variables * Date: Date on which observations were carried out * Block: Experimental block * Treatment: Experimental/control treatment type * Hours: Observation time post infection * Flies: Number of flies dead or alive * Censor: Status of flies '1' represents dead individuals and '0' represents alive individuals. Dead individuals were counted each day and alive individuals were counted on last day of the observation #### File: Bacterial\_load.csv **Description:** This dataset contains the data generated for bacterial load assay. ##### Variables * Plate.ID: LB plate used * Time: Time post infection (hours) * Treatment: Experimental/control treatment type * Count.per.Frame: Data obtained from plate counting system * Count.per.ml: Data obtained from plate counting system * Spiral.Stop: Data obtained from plate counting system * Dilution.Factor: Dilution factor indicates in what ratio the ground infected fly was diluted or not * Flags: Flags obtained from plate counting system * Time.1: Time at which the plates were read by the plate counting system * Date: Date in which the plates were read * User: System User * Count_per_fly: Number of CFUs per fly * ln_count: Log transformed CFU * Block: Experimental block #### File: Egg\_death\_correlation\_data.csv **Description:** This dataset is used to generate supplemental figure S5 of manuscript ##### Variables * Tube: Experimental tube * Died.on: Day on which the flies died * Day_1: Number of eggs produced on Day 1 * Day_2: Number of eggs produced on Day 2 * Day_3: Number of eggs produced on Day 3 * Day_4: Number of eggs produced on Day 4 * NA: No egg counts possible as all the females in the vials were dead #### File: Egg\_to\_offspring\_data.csv **Description:** This dataset contains the data collected for egg to offspring analysis. ##### Variables * Batch: Experimental block * Treatment: Experimental group * Time: Observation time post infection (hours) * Tube: Experimental tube * Females: Number of females in the tube * Eggs: Number of eggs in tube * Offspring: Number of offspring enclosed in tube * Eggs_per_female: Number of eggs divided by number of females in tube * Offspring_per_female: Number of offspring divided by number of females in tube * Egg_to_offspring_ratio: Number of offspring divided by number of eggs in tube #### File: Correlation\_data.csv **Description:** This dataset is used to generate supplemental figures S3 and S4 in the manuscript ##### Variables * Conditions: Experimental condition * Tube: Experimental tube * Count_per_ml: bacterial load per ml * Count_per_fly: bacterial load per fly * Egg: Number of eggs * Offspring: Number of offspring * Egg_offspring.ratio: Number of offspring divided by number of eggs * ln: natural log of bacterial load per fly ## Code/software There are five different scripts written in R and were run with version 4.1.1. Survival.R : Script used to analyze survival_data.csv dataset Bacterial_load.R : Script used to analyze Bacterial_load.csv dataset Correlation.R : Script used to analyze Correlation_data.csv dataset Egg_offspring.R : Script used to analyze Egg_to_offspring_data.csv dataset Egg_death_correlation.R : Script used to analyze Egg_death_correlation_data.csv dataset