DOI: 10.5061/dryad.pnvx0k6qt
title: Idiosyncratic effects of bacterial infection on female fecundity in Drosophila melanogaster

description: Existing theories make different predictions regarding the effect of a
 pathogenic infection on the host capacity to reproduce. Terminal
 investment theory suggests that due to the increased risk of mortality,
 and the associated risk of losing future opportunity to reproduce,
 infected individuals would increase their investment towards reproduction.
 Life-history theory posits that due to energetic and resource costs
 associated with mounting an immune defense, hosts would decrease their
 investment towards reproduction, and reallocate resources towards defense
 and survival. Additionally, Somatic damage incurred by the host due to the
 infection is also expected to compromise the host capacity to reproduce.
 We explored these possibilities in Drosophila melanogaster
 females experimentally infected with pathogenic bacteria. We tested if the
 effect of infection on female fecundity is pathogen specific, determined
 by infection outcome, and variable between individual infected females. We
 observed that the mean, population level change in post-infection female
 fecundity was pathogen specific, but not correlated with mortality risk.
 Furthermore, infection outcome, i.e., if the infected female died or
 survived the infection, had no effect on fecundity at this level. At
 individual resolution, females that died after infection exhibited greater
 variation in fecundity compared to ones that survived the infection. This
 increased variation was bidirectional, with some females reproducing in
 excess while others reproducing less compared to the controls. Altogether,
 our results suggest that post-infection female fecundity is unlikely to be
 driven by risk of mortality and is probably determined by the precise
 physiological changes that an infected female undergoes when infected by a
 specific pathogen.
description: <strong>1. Host population and general
 handling</strong> Flies from BRB2 population - a
 large, lab adapted, outbred <em>Drosophila melanogaster
 </em>population - were used for the experiments. The
 Blue Ridge
 Baseline (BRB) population was originally established by
 hybridizing 19 wild-caught iso-female lines (Singh et al., 2015), and has
 been maintained since then as an outbred population on a 14-day discrete
 generation cycle with census size of about 2800 adults in each generation.
 Every generation, eggs are collected from population cages (plexiglass
 cages: 25 cm length × 20 cm width × 15 cm height) and dispensed into vials
 (25 mm diameter × 90 mm height) with 8 ml banana-jaggery-yeast food
 medium, at a density of 70 eggs per vial. 40 such vials are set up; the
 day of egg collection is demarcated as day 1. The vials are incubated at
 25 °C, 50-60% RH, 12:12 hour LD cycle; under these conditions the
 egg-to-adult development time for these flies is about 9-10 days. On day
 12 post egg collection all adults are transferred to population cage and
 provided with fresh food plates (banana-jaggery-yeast food medium in a 90
 mm Petri plate) supplemented with <em>ad libitum</em> live
 yeast paste. On day 14, the cage is provided with fresh food plate, and 18
 hours later eggs are collected from this plate to begin the next
 generation. <strong>2. Pathogen handling and
 infection protocol</strong> Four bacterial
 pathogens were used in this study for infecting the flies:
 <em>Bacillus thuringiensis</em> (<em>Bt</em>;
 obtained from DSMZ, Germany, catalogue number: DSM2046),
 <em>Pseudomonas aeruginosa</em> (<em>Pa</em>;
 obtained from MTCC, India, catalogue number: 4999), <em>Serratia
 marcescens </em>(<em>Sm</em>; Martins et al., 2013), and
 <em>Enterococcus faecalis</em> (<em>Ef</em>;
 Lazzaro et al., 2006). All four pathogens used for infection are natural
 pathogens of insects and are known to cause lethal infection in
 <em>D. melanogaster</em> flies following systemic infection
 (Shirasu-Hiza and Schneider 2007, Dionne and Schneider 2008). Two of the
 pathogens, <em>Pa</em> and <em>Sm</em>, are
 Gram-negative bacteria, while the rest of the two pathogens,
 <em>Bt</em> and<em> Ef</em>, are Gram-positive
 bacteria. Bacteria representing both Gram-character were used in the
 experiments, since in <em>D. melanogaster</em>, Gram-negative
 and Gram-positive bacterial pathogens are detected by separate immune
 surveillance components and are defended against by separate immune
 mechanisms, with some overlap and crosstalk between the elicited defense
 mechanisms (Lemaitre and Hoffman 2007, Dionne and Schneider 2008,
 Vallet-Gely et al., 2008, Buchon et al., 2014). Previous work has
 demonstrated that oral infection with <em>Pa</em> increases
 female fecundity in <em>D. melanogaster </em>(Hudson et al.,
 2020) while infection via septic injury to the thorax with
 <em>Pa</em> decreases female fecundity in a genotype specific
 manner (Linder and Promislow 2009). Infection via septic injury to the
 abdomen with <em>Sm</em> has also been shown to compromise
 female fecundity in <em>D. melanogaster</em> (Brandt and
 Schneider 2007). All pathogens are maintained in the
 lab as glycerol stocks, and are cultured in Luria Bertani broth (Himedia,
 M1245); cultures are incubated at 30 °C for <em>Bt</em>, and
 37 °C for <em>Pa,</em> <em>Sm </em>and<em>
 Ef</em>. Overnight culture of bacteria grown from glycerol stocks
 was diluted (1:100) in fresh LB medium and incubated till confluency
 (optical density OD<sub>600</sub> = 1.0-1.2). The bacterial
 cells were pelleted down by centrifugation and re-suspended in sterile 10
 mM MgSO<sub>4</sub> buffer at OD<sub>600 </sub>=
 1.0. OD600 = 1.0 for <em>Bt</em> corresponds to
 10<sup>4</sup> cells/ml, for <em>Pa</em>
 corresponds to 10<sup>8</sup> cells/ml, for
 <em>Sm</em> corresponds to 10<sup>6</sup> cells
 /ml, and for <em>Ef</em> corresponds to
 10<sup>7</sup> cells/ml. Flies were infected via septic
 injury, by pricking them at the dorsolateral side of the thorax with a
 fine needle (Minutien pin, 0.1 mm, Fine Science Tools, CA, item no.
 26001-10) dipped in bacterial suspension under light
 CO<sub>2</sub> anesthesia. Flies for sham-infections were
 similarly treated but pricked with needle dipped in sterile 10 mM
 MgSO<sub>4</sub> buffer. Uninfected control flies were only
 subjected to CO<sub>2</sub> anesthesia.
 <strong>3. Generation of experimental
 flies</strong> Eggs were collected from BRB2
 population cages and distributed into food vials with 8 ml of standard
 food medium at a density of 70 eggs per vial. These vials were incubated
 as per the general maintenance regime. Twelve days post egg-laying flies
 were flipped into fresh food vials and hosted for two more days before
 experimentation. This ensured all focal females were 4-5 day old, sexually
 mature and inseminated, at the time of infections. Flies were again
 flipped into fresh food vials 6 hours before being subjected to
 experimental treatments (as described below).
 <strong>4. Experimental design</strong>
 <strong>Experiment 1. </strong>Focal females were
 randomly distributed into five treatments: (a) infected with
 <em>Bacillus thuringiensis</em> (<em>Bt</em>), (b)
 infected with <em>Pseudomonas aeruginosa</em>
 (<em>Pa</em>), (c) infected with <em>Serratia
 marcescens</em> (<em>Sm</em>), (d) sham-infected
 controls, and (e) uninfected controls. The entire experiment was
 independently replicated three times. Flies were placed in fresh food
 vials after being subjected to respective treatments. For each treatment
 10 vials were set up, each with 8 females for oviposition; each vial was
 used as a unit of replication. The vials were monitored every 2 hours to
 record any mortality, for 24 hours post-infection, divided into two
 consecutive 12-hour windows. Flies alive at the end of first 12-hour
 window were flipped into fresh food vials (one-to-one mapping of vial
 identity), and flies alive at the end of 24 hours were discarded
 (censored). The number of eggs in each vial was counted at the end of
 respective 12-hour windows. The vials were then incubated under standard
 maintenance conditions for the eggs to develop into adults, and 12 days
 after the oviposition period, all adult progeny were counted under light
 CO<sub>2</sub> anesthesia and transferred to fresh food
 vials. <strong>Experiment 2.</strong> Focal
 females were randomly distributed into two treatments: (a) infected with
 bacteria, and (b) sham-infected controls. Four pathogens were used in this
 experiment: the three used for experiment 1 and <em>Enterococcus
 faecalis</em> (<em>Ef</em>). For infected treatment, 120
 females were individually hosted in vials for oviposition, while for
 sham-infected controls 40 females were hosted individually. The experiment
 was replicated thrice with each pathogen. (Due to a handling accident, one
 replicate with Sm had sample size of 60 and 30 females for infected and
 sham-infected treatments, respectively.) The vials were monitored every 2
 hours for any mortality, for 48 hours post-infection, after which the
 alive flies were discarded. The vials were then incubated under standard
 maintenance conditions for the eggs to develop into adults, and 12 days
 later the number of adult progeny was counted for each individual
 female.
description: # Idiosyncratic effects of bacterial infection on female fecundity in
 Drosophila melanogaster
 [https://doi.org/10.5061/dryad.pnvx0k6qt](https://doi.org/10.5061/dryad.pnvx0k6qt) ## Description of the data and file structure The files ("infection_outcome_experiment.xlsx") and ("pathogen_identity_experiment.xlsx") were generated in 2019-2020 by Aabeer Basu and others at the Evolutionary Biology Lab, Department of Biological Sciences, IISER Mohali. **GENERAL INFORMATION** Title of Dataset: "Idiosyncratic effects of bacterial infection on female fecundity in *Drosophila melanogaster*" **Author Information** ``` A. Principal Investigator Contact Information Name: Prof. N. G. Prasad Institution: Indian Institute of Science Education and Research, Mohali Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India. Email: prasad@iisermohali.ac.in B. Associate or Co-investigator Contact Information Name: Aabeer Basu Institution: Indian Institute of Science Education and Research, Mohali Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India. Email: aabeerkbasu@gmail.com ``` Duration of data collection: November 2019 - February 2020; September 2020 - October 2020 Geographic location of data collection: Mohali, Punjab, India Information about funding sources that supported the collection of the data: IISER Mohali, Govt. of India. ### Files and variables #### File: pathogen\_identity\_experiment.xlsx ##### Variables Note: This file contains four separate tabs, each containing data from from different parts of Experiment 1: Tab 1. "survival_data" Tab 2. "reproductive_output_data" Tab 3. "pre-adult_viability_data" Tab 4. "offspring_fecundity_data" *Tab 1. "survival_data"* 1\. Number of variables: 4 2\. Number of cases/rows: 1203 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_treatment (Denotes whether the host was infected, and which pathogen was used for infection; "Bt" denotes Bacillus thuringiensis, "Pa" denotes Pseudomonas aeruginosa, "Sm" denotes Serratia marcescens, "Sham" denotes sham-infected, "Uninfected" denotes no infection) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) *Tab 2. "reproductive_output_data"* 1\. Number of variables: 7 2\. Number of cases/rows: 151 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_treatment (Denotes whether the host was infected, and which pathogen was used for infection; "Bt" denotes Bacillus thuringiensis, "Pa" denotes Pseudomonas aeruginosa, "Sm" denotes Serratia marcescens, "Sham" denotes sham-infected, "Uninfected" denotes no infection) TSH (Total surviving hours; summation of the hours survived (survival time right-censored at 24 hours post-infection for surviving females) by the females in each vial) NEgg (Number of eggs; total number of eggs in each vial) NProgeny (Number of progeny; total number of progeny in each vial) REprimary (Standardised reproductive output; total number of eggs in each vial divided with the summation of the hours survived (survival time right-censored at 24 hours post-infection for surviving females) by the females in that vial) REsecondary (Standardised reproductive output; total number of progeny in each vial divided with the summation of the hours survived (survival time right-censored at 24 hours post-infection for surviving females) by the females in that vial) *Tab 3. "pre-adult_viability_data"* 1\. Number of variables: 5 2\. Number of cases/rows: 151 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Maternal_treatment (Denotes whether the female parent of the was infected, and which pathogen was used for infection; "Bt" denotes Bacillus thuringiensis, "Pa" denotes Pseudomonas aeruginosa, "Sm" denotes Serratia marcescens, "Sham" denotes sham-infected, "Uninfected" denotes no infection) NEgg (Number of eggs; total number of eggs in each vial) NProgeny (Number of progeny; total number of progeny in each vial) Viability (Proportion of eggs that successfully developed into adult progeny; NProgeny divided by NEgg) *Tab 4. "offspring_fecundity_data"* 1\. Number of variables: 5 2\. Number of cases/rows: 901 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Maternal_treatment (Denotes whether the female parent of the was infected, and which pathogen was used for infection; "Bt" denotes Bacillus thuringiensis, "Pa" denotes Pseudomonas aeruginosa, "Sm" denotes Serratia marcescens, "Sham" denotes sham-infected, "Uninfected" denotes no infection) Day (Day of oviposition; ordinal data) VialID (Vial identity) Fecundity (Number of eggs laid) #### File: infection\_outcome\_experiment.xlsx ##### Variables Note: This file contains three separate tabs, each containing data for a different pathogen from Experiment 2 (as described in the above cited bioRxiv pre-print): Tab 1. "b_thuringiensis" Tab 2. "p_aeruginosa" Tab 3. "s_marcescens" Tab 4. "e_faecalis" *Tab 1. "b_thuringiensis"* 1\. Number of variables: 8 2\. Number of cases/rows: 479 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_treatment (Denotes whether the host was infected, and which pathogen was used for infection; "Infected" denotes that a fly was infected with the corresponding pathogen, "Sham" denotes sham-infected) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) Progeny_count (Number of progeny produced by individual female in the vial) Status (Denotes whether the individual female died during the observation window; "Alive" denotes that the female was alive, "Dead" denotes that the female died) RE (Standardised reproductive output; number of progeny in each vial divided with the hours survived (survival time right-censored at 48 hours post-infection for surviving females) by the individual female in that vial) Category (A combined variable of Infection_treatment and Status; "InfA" indicates infected and alive, "InfD" indicates infected and dead, "Shm" indicates sham-infected and alive) *Tab 2. "p_aeruginosa"* 1\. Number of variables: 8 2\. Number of cases/rows: 478 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_treatment (Denotes whether the host was infected, and which pathogen was used for infection; "Infected" denotes that a fly was infected with the corresponding pathogen, "Sham" denotes sham-infected) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) Progeny_count (Number of progeny produced by individual female in the vial) Status (Denotes whether the individual female died during the observation window; "Alive" denotes that the female was alive, "Dead" denotes that the female died) RE (Standardised reproductive output; number of progeny in each vial divided with the hours survived (survival time right-censored at 48 hours post-infection for surviving females) by the individual female in that vial) Category (A combined variable of Infection_treatment and Status; "InfA" indicates infected and alive, "InfD" indicates infected and dead, "Shm" indicates sham-infected and alive) *Tab 3. "s_marcescens"* 1\. Number of variables: 8 2\. Number of cases/rows: 411 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_treatment (Denotes whether the host was infected, and which pathogen was used for infection; "Infected" denotes that a fly was infected with the corresponding pathogen, "Sham" denotes sham-infected) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) Progeny_count (Number of progeny produced by individual female in the vial) Status (Denotes whether the individual female died during the observation window; "Alive" denotes that the female was alive, "Dead" denotes that the female died) RE (Standardised reproductive output; number of progeny in each vial divided with the hours survived (survival time right-censored at 48 hours post-infection for surviving females) by the individual female in that vial) Category (A combined variable of Infection_treatment and Status; "InfA" indicates infected and alive, "InfD" indicates infected and dead, "Shm" indicates sham-infected and alive) *Tab 4. "e_faecalis"* 1\. Number of variables: 8 2\. Number of cases/rows: 359 3\. Variable List: Replicate (Replicate identity; "A", "B", "C") Infection_treatment (Denotes whether the host was infected, and which pathogen was used for infection; "Infected" denotes that a fly was infected with the corresponding pathogen, "Sham" denotes sham-infected) Time (Time in hours at which event (death/censor) was recorded) Censor (Status of the fly at the time event was recorded; "1" denotes mortality, "0" denotes right-censored data) Progeny_count (Number of progeny produced by individual female in the vial) Status (Denotes whether the individual female died during the observation window; "Alive" denotes that the female was alive, "Dead" denotes that the female died) RE (Standardised reproductive output; number of progeny in each vial divided with the hours survived (survival time right-censored at 48 hours post-infection for surviving females) by the individual female in that vial) Category (A combined variable of Infection_treatment and Status; "InfA" indicates infected and alive, "InfD" indicates infected and dead, "Shm" indicates sham-infected and alive)