Idiosyncratic effects of bacterial infection on female fecundity in Drosophila melanogaster
Data files
Oct 07, 2024 version files 150.77 KB
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infection_outcome_experiment.xlsx
77.29 KB
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pathogen_identity_experiment.xlsx
63.64 KB
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README.md
9.84 KB
Abstract
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.
README: Idiosyncratic effects of bacterial infection on female fecundity in Drosophila melanogaster
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)
Methods
1. Host population and general handling
Flies from BRB2 population - a large, lab adapted, outbred Drosophila melanogaster 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 ad libitum 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.
2. Pathogen handling and infection protocol
Four bacterial pathogens were used in this study for infecting the flies: Bacillus thuringiensis (Bt; obtained from DSMZ, Germany, catalogue number: DSM2046), Pseudomonas aeruginosa (Pa; obtained from MTCC, India, catalogue number: 4999), Serratia marcescens (Sm; Martins et al., 2013), and Enterococcus faecalis (Ef; Lazzaro et al., 2006). All four pathogens used for infection are natural pathogens of insects and are known to cause lethal infection in D. melanogaster flies following systemic infection (Shirasu-Hiza and Schneider 2007, Dionne and Schneider 2008). Two of the pathogens, Pa and Sm, are Gram-negative bacteria, while the rest of the two pathogens, Bt and Ef, are Gram-positive bacteria. Bacteria representing both Gram-character were used in the experiments, since in D. melanogaster, 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 Pa increases female fecundity in D. melanogaster (Hudson et al., 2020) while infection via septic injury to the thorax with Pa decreases female fecundity in a genotype specific manner (Linder and Promislow 2009). Infection via septic injury to the abdomen with Sm has also been shown to compromise female fecundity in D. melanogaster (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 Bt, and 37 °C for Pa, Sm and Ef. Overnight culture of bacteria grown from glycerol stocks was diluted (1:100) in fresh LB medium and incubated till confluency (optical density OD600 = 1.0-1.2). The bacterial cells were pelleted down by centrifugation and re-suspended in sterile 10 mM MgSO4 buffer at OD600 = 1.0. OD600 = 1.0 for Bt corresponds to 104 cells/ml, for Pa corresponds to 108 cells/ml, for Sm corresponds to 106 cells /ml, and for Ef corresponds to 107 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 CO2 anesthesia. Flies for sham-infections were similarly treated but pricked with needle dipped in sterile 10 mM MgSO4 buffer. Uninfected control flies were only subjected to CO2 anesthesia.
3. Generation of experimental flies
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).
4. Experimental design
Experiment 1. Focal females were randomly distributed into five treatments: (a) infected with Bacillus thuringiensis (Bt), (b) infected with Pseudomonas aeruginosa (Pa), (c) infected with Serratia marcescens (Sm), (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 CO2 anesthesia and transferred to fresh food vials.
Experiment 2. 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 Enterococcus faecalis (Ef). 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.