DOI: 10.5061/dryad.6q573n66r
title: Data from: Timing of starvation determines its effects on susceptibility to bacterial infection in female fruit flies independent of host evolutionary history

description: An organism's susceptibility to pathogens is contingent on various
 environmental factors, including the availability of nutrition. Starvation
 can alter host susceptibility to infections, either directly via depletion
 of resources essential for proper functioning of the immune system, or
 indirectly via the various physiological changes it induces within the
 host body. We tested if the susceptibility of Drosophila
 melanogaster populations to Enterococcus
 faecalis infection is affected by (a) whether the hosts are
 starved before or after the infection, and (b) the evolutionary history of
 the host. Hosts from laboratory fly populations that have been
 experimentally evolved to be more resistant to E. faecalis, and
 their corresponding control populations, were subjected to infection with
 or without being starved prior to and after being infected. We found that
 the effect of starvation on susceptibility to E. faecalis changed
 with the timing of starvation: starvation after infection improved
 survival of infected hosts, irrespective of how they were treated before
 infection, while starving only prior to infection (and not after)
 compromised post-infection survival. The changes in infection
 susceptibility were uniform in both the evolved and the control
 populations, suggesting that the effects of starvation are not dependent
 on pre-existing resistance to the infecting pathogen.
description: <strong>1. EPN experimental evolution
 set-up</strong> The experiments reported in this
 study were carried out using flies from the EPN experimental evolution
 set-up consisting of twelve populations distributed across three selection
 regimes (previously described in <strong>Singh et al.,
 2021</strong>). <strong>E regime:
 </strong>The E<sub>1-4</sub> populations are selected
 for better survival following infection with the Gram-positive bacterium
 <em>Enterococcus faecalis</em>. 2–3-days old adult flies (200
 females and 200 males) are subjected to infection with <em>E.
 faecalis</em> every generation, and 96-hours post-infection, the
 survivors are allowed to reproduce and contribute towards the next
 generation. At the end of 96 hours, on average 100 females and 100 males
 are left alive in each of the E<sub>1-4</sub>
 populations. <strong>P regime: </strong>The
 P<sub>1-4</sub> populations are procedural (sham-infected)
 control populations. 2–3-days old adult flies (100 females and 100 males)
 are subjected to sham-infections every generation, and 96-hours
 post-sham-infection, the survivors are allowed to reproduce and contribute
 towards the next generation. <strong>N regime:
 </strong>The N<sub>1-4</sub> populations are uninfected
 control populations. 2–3-day-old adult flies (100 females and 100 males)
 are subjected only to light CO<sub>2</sub> anesthesia every
 generation, and 96 hours post-handling, the survivors are allowed to
 reproduce and contribute towards the next generation. (Under usual
 circumstances, negligible mortality occurs in the
 P<sub>1-4</sub> (&lt; 2%) and N<sub>1-4</sub>
 (0%) populations during maintenance of the selection regimes.)
 These populations were derived from the ancestral Blue Ridge
 Baseline (BRB<sub>1-4</sub>) populations (<strong>Singh
 et al., 2015</strong>). The E<sub>1</sub>,
 P<sub>1</sub>, and N<sub>1</sub> populations were
 derived from the BRB<sub>1</sub> population and constitute
 ‘block 1’ of the experimental evolution regime. Similarly,
 E<sub>2</sub>, P<sub>2</sub>, and
 N<sub>2</sub> populations were derived from the
 BRB<sub>2</sub> population and constitute ‘block 2’, and so
 on. This block design implies that E<sub>1</sub>,
 P<sub>1</sub>, and N<sub>1</sub> have a more
 recent common ancestor, compared to E<sub>1</sub> and
 E<sub>2</sub>, or P<sub>1</sub> and
 P<sub>2</sub>, and so on. Populations belonging to each block
 were handled together, both during maintenance of the populations and
 during experiments. Please refer to <strong>supplementary figure
 1</strong> for a graphical depiction of the block design.
 The regular maintenance of the EPN set-up has been previously
 described (<strong>Singh et al., 2021; Basu et al.,
 2024b</strong>). The populations are maintained on a diet of
 banana-jaggery-yeast food medium. Every generation, eggs are collected at
 a density of 60-80 eggs per vial, in 6-8 ml food medium. 10 such rearing
 vials (9 cm height × 2.5 cm diameter) are set up for each of the 12
 populations. These vials are incubated at 25 <sup>O</sup>C,
 60% RH, and a 12:12 LD cycle. Under these conditions, eggs develop into
 adults within 9-10 days of egg collection. The adults stay in the rearing
 vial till day 12 post-egg laying (PEL); by this point in time, the flies
 are sexually mature and have already mated. On day 12 PEL, flies from each
 population are handled according to their regime, as described above
 (<strong>see section 2 for details of the infection
 process</strong>). The flies are thereafter housed in plexiglass
 cages (14 cm × 16 cm × 13 cm); one cage for each population. The cages are
 provided with fresh food medium, on a 60 mm Petri plate on every alternate
 day. On day 16 PEL, the surviving flies are allowed to lay eggs for the
 next 18 hours on a fresh food plate in each cage. Eggs are collected from
 these oviposition plates to start the next generation.
 <strong>2. Pathogen handling and infection
 protocol</strong> <em>Enterococcus
 faecalis</em> (<strong>Lazzaro et al., 2006</strong>), a
 Gram-positive bacterium, was used in regular maintenance of the EPN
 populations and during the experiment. During regular maintenance of the
 EPN populations, the flies from the E<sub>1-4</sub>
 populations (<strong>see section 1</strong>) are infected with
 an <em>E. faecalis</em> suspension of
 OD<sub>600</sub> = 1.5. For all experimental infections
 (<strong>see section 4</strong>), flies were also infected
 with an <em>E. faecalis </em>suspension of
 OD<sub>600</sub> = 1.5 to keep experimental conditions
 consistent with that of regular maintenance. OD<sub>600</sub>
 = 1.0 corresponds to 10<sup>6</sup> cells/ml for <em>E.
 faecalis</em>. The bacteria are stored as
 glycerol stocks (17%) in -80 <sup>O</sup>C. To obtain live
 bacterial cells for infections, 10 ml lysogeny broth (Luria Bertani Broth,
 Miler, HiMedia) is inoculated with glycerol stocks and incubated overnight
 with aeration (150 rpm shaker incubator) at 37 <sup>O</sup>C.
 100 microliters from this primary culture is inoculated into 10 ml fresh
 lysogeny broth and incubated for the necessary amount of time to obtain
 confluent (OD<sub>600</sub> = 1.0-1.2) cultures. The bacterial
 cells are pelleted down using centrifugation and resuspended in sterile
 MgSO<sub>4</sub> (10 mM) buffer to obtain the required optical
 density (OD<sub>600</sub>) for infection. Flies are infected,
 under light CO<sub>2</sub> anaesthesia, by pricking them on
 the dorsolateral side of their thorax with a 0.1 mm Minutien pin (Fine
 Scientific Tools, USA) dipped in the bacterial suspension. Sham-infections
 (injury controls) are carried out in the same fashion, except by dipping
 the pins in sterile MgSO<sub>4</sub> (10 mM) buffer.
 <strong>3. Pre-experiment standardization and generation of
 experimental flies</strong> Prior to experiments,
 flies from the different selection regimes were reared for one generation
 under ancestral maintenance conditions to account for potential
 non-genetic parental effects (<strong>Rose 1984</strong>);
 flies thus generated are referred to as <em>standardized
 </em>flies. To generate standardized flies, eggs were collected from
 all populations at a density of 60-80 eggs per vial (with 6-8 ml food
 medium); 10 such vials were set up per population. The vials were
 incubated under standard maintenance conditions (<strong>section
 1</strong>). On day 12 PEL, the adults were transferred to
 plexiglass cages (14 cm × 16 cm × 13 cm) with food plates (Petri plates,
 60 mm diameter). Eggs for experimental flies were collected from these
 <em>standardized </em>population cages. To
 generate flies for the experiments, eggs were collected from
 <em>standardized</em> population cages at a density of 60-80
 eggs per vial (with 6-8 ml food medium); 60 vials were collected per
 regime per block. The vials were incubated under standard maintenance
 conditions. The eggs developed into adults by day 10 PEL. The adults
 continued to be housed in these vials till day 12 PEL, when they were
 distributed into respective experimental treatments (<strong>section
 4</strong>). The handling of the flies during the experiment was
 kept identical to how the populations are regularly maintained to keep
 their ecology consistent.  <strong>4. Experiment
 design</strong> Only flies from the N and E
 selection regimes were used in the experiment reported here. Previous
 works have reproducibly demonstrated that N (uninfected control regime)
 and P (procedural control regime) flies do not differ significantly in
 terms of post-infection survival (<strong>Singh et al., 2021; Singh
 et al., 2022a; Singh et al., 2022b;</strong> <strong>Basu et
 al., 2024b</strong>). This experiment was carried out after 80
 generations of forward selection. On day 12 PEL, 2-3
 day old flies from N and E regimes were randomly assigned to one of the
 four following treatments: a. <em>Fed/Fed
 </em>(FF): flies that were continuously fed their standard
 diet. b. <em>Fed/Starved </em>(FS): flies
 that were fed their standard diet before infection, and subjected to
 starvation post-infection. c. <em>Starved/Fed
 </em>(SF): flies that were subjected to starvation for 48 hours
 prior to infection, but fed their standard diet post-infection.
 d. <em>Starved/Starved</em> (SS): flies that were
 continuously starved starting from 48 hours before infection.
 15 vials worth of flies were allocated for each selection regime
 (N and E) × starvation treatment (FF, FS, SF, and SS) × block (1, 2, 3,
 and 4) combination. Flies in the FF and FS treatments were transferred
 into fresh food vials (with 2 ml standard food medium) from their rearing
 vials on day 12 PEL. Flies in the SF and SS treatments were transferred
 into <em>starvation </em>vials (with 2 ml non-nutritive 2%
 agar gel) from their rearing vials on day 12 PEL. Flies were held in these
 vials till the time of infection, which was 48-50 hours after this
 transfer. Flies were subjected to infection or sham-infection on day 14
 PEL when they were 4-5 days old as adults. Flies in each vial were
 anesthetized with CO<sub>2</sub>, and 8 females were infected
 (10 vials) or sham-infected (5 vials) from each vial (<strong>as
 described in section 2</strong>), and placed in a fresh vial after
 handling. Females in the FF and SF treatments were placed in fresh food
 vial,s while females in the FS and SS treatments were placed in
 fresh <em>starvation </em>vials. The females were housed in
 these vials for the next 48 hours while their mortality was recorded every
 4 to 6 hours. In total, 80 females (per selection regime per starvation
 treatment per block) were infected while 40 females (per selection regime
 per starvation treatment per block) were sham-infected. Experiments for
 each block were carried out on a separate day.
description: # Timing of starvation determines its effects on susceptibility to
 infection in Drosophila melanogaster females, independent of host
 evolutionary history
 [https://doi.org/10.5061/dryad.6q573n66r](https://doi.org/10.5061/dryad.6q573n66r) ## Description of the data and file structure 1\. Title of Dataset: "Timing of starvation determines its effects on susceptibility to infection in Drosophila melanogaster females independent of host evolutionary history" 2\. 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 ``` 3\. Duration of data collection: Aril, 2022 4\. Geographic location of data collection: Mohali, Punjab, India 5\. Information about funding sources that supported the collection of the data: IISER Mohali, Govt. of India. **DATA & FILE OVERVIEW** **File Name**: "survival_data.csv" Note: This file contains data for survival of females from the E and N populations when subjected to different starvation treatments and infected with Enterococcus faecalis (as described in the above cited bioRxiv pre-print). 1\. Number of variables: 6 2\. Number of cases/rows: 3836 3\. Variable List: * Block (Block identity of the host; also serves as an indicator of ancestry.) - Population (Population identity/selection history of the host. “E” denotes flies from the selected populations, “N” denotes flies from the un-infected control populations.) * Starvation_treatment (Denotes which starvation treatment the host was subjected to. "FF" denotes flies that were continuously fed their standard diet, "FS" denotes flies that were fed their standard diet prior to infection, and subjected to starvation post infection, "SF" denotes flies that were subjected to starvation for 48 hours prior to infection but fed their standard diet post infection, and "SS" denotes flies that were continuously starved starting from 48 hours prior to infection.) - Infection_treatment (Denotes whether the host was subjected to infection or was sham-infected. “Infected” denotes flies infected with Enterococcus faecalis, “Sham” denotes flies subjected to sham-infection.) * Time (Time in hours at which the event (death/censor) was recorded.) - Censor (Status of the fly at the time the event was recorded. "1" denotes mortality, "0" denotes right-censored data.) ## Code/software Not Applicable ## Access information Other publicly accessible locations of the data: * Not applicable Data was derived from the following sources: * Not Applicable