Reciprocal costs of infection and reproduction in D. melanogaster
Data files
Feb 05, 2025 version files 84.01 KB
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Bacterial_load.csv
41.45 KB
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Correlation_data.csv
4.69 KB
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Egg_death_correlation_data.csv
2.21 KB
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Egg_to_offspring_data.csv
28.14 KB
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README.md
3.67 KB
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Survival_data.csv
3.86 KB
Abstract
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.
README: Reciprocal costs of infection and reproduction in D.melanogaster
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
Methods
Group of 10 male and 10 female Drosophila melanogaster 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 Providencia rettgeri 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.
Survival assay
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.
Egg and offspring count assay
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.
Bacterial load assay
Bacterial load assay was carried out to test whether mating has any impact on previously established pathogen burden. Individual females were anesthetized with CO2 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.