DOI: 10.5061/dryad.xd2547dqr
title: Data from: Experimental evolution for improved post-infection survival selects for increased disease resistance in Drosophila melanogaster

description: Disease resistance (defined as the host capacity to limit systemic
 infection intensity) and disease tolerance (defined as the host capacity
 to limit infection-induced damage) are two complementary defense
 strategies that help the hosts maximize their survival and fitness when
 infected with pathogens and parasites. In addition to the underlying
 physiological mechanisms, existing theory postulates that these two
 strategies differ in terms of the conditions under which each strategy
 evolves in host populations, their evolutionary dynamics, and the
 ecological and epidemiological consequences of their evolution. Here we
 explored if one or both of these strategies evolve when host populations
 are subjected to selection for increased post-infection survival. We
 experimentally evolved Drosophila melanogaster populations, selecting for
 the flies that survived an infection with the entomopathogen Enterococcus
 faecalis, and found that the host populations evolved increased disease
 resistance in response. This was despite the physiological costs
 associated with increased resistance. We did not find evidence of any
 change in disease tolerance in the host populations. We have therefore
 demonstrated that in an experimental evolution set-up, where insect hosts
 must survive an infection with a pathogenic bacterium, the hosts evolve
 improved disease resistance but not disease tolerance.
description: <strong>Host populations</strong>
 Replicate <em>Drosophila melanogaster
 </em>populations were evolved parallelly, subjecting some of the
 populations to selection for increased post-infection survival, while
 maintaining the others as either procedural or uninfected controls. The
 experimental evolution set-up consisted of 12 populations in total,
 distributed into 3 selection regimes (figure 1, Singh et al.,
 2021):  1. E1-4: Populations selected for better
 survival following infection with the Gram-positive bacterium
 <em>Enterococcus faecalis</em>. Every generation, 2–3-days old
 adult flies (200 females and 200 males) are subjected to infection with
 <em>E. faecalis</em>, and 96-hours post-infection, the
 survivors are allowed to reproduce and contribute to the next generation.
 At the end of 96 hours, on average 100 females and 100 males are left
 alive in each of the E1-4 populations. 2. P1-4:
 Procedural (sham-infected) control populations. Every generation, 2–3-days
 old adult flies (100 females and 100 males) are subjected to
 sham-infection, and 96-hours post-sham-infection, the survivors are
 allowed to reproduce and contribute to the next generation.
 3. N1-4: Uninfected control populations. Every generation,
 2–3-days old adult flies (100 females and 100 males) are subjected to
 light CO<sub>2</sub> anesthesia only, and 96-hours
 post-procedure, the survivors are allowed to reproduce and contribute to
 the next generation. The EPN populations were derived
 from the ancestral Blue Ridge Baseline (BRB1-4) populations. The E1, P1,
 and N1 populations were derived from BRB1, the E2, P2, and N2 populations
 were derived from BRB2, and so on. Selected populations with the same
 numeric subscript therefore share a recent common ancestor and are part of
 the same ancestral 'block'. Blocks are handled on separate days
 for both regular maintenance and for experiments.  All
 the populations are maintained on a 16-day discrete generation cycle (day
 1 being the day of egg collection). Infections happen on day 12 of every
 generation. Flies are maintained on standard banana-jaggery-yeast medium,
 and are housed in an incubator at 25 °C with a 12:12 hours LD cycle and
 50% relative humidity. For experiments, flies were generated and handled
 in a fashion that closely resembles the regular maintenance regime for
 these populations. On day 1, eggs are collected from each of the 12
 populations, at a density of 60-80 eggs per vial (25 mm diameter × 90 mm
 height), in 6-8 ml of standard food medium; 10 vials are set-up per
 population. The vials are incubated under the constant environmental
 conditions detailed above, and the eggs develop into adults by day 10. The
 adults remain in these vials till day 12, when the adult flies are
 subjected to the corresponding selection regime according to their
 population identity. By this point in time, all adults are sexually mature
 and have mated at least once. After being subjected to selection
 (infected, sham-infected, or uninfected), adult flies are housed in
 plexiglass cages (14 cm length × 16 cm width × 13 cm height); each
 population is housed in a separate cage. Each cage is supplied with fresh
 food medium, poured into a 60 mm Petri plate. The flies remain in these
 cages for the next 96 hours, with fresh food medium provided to them every
 48 hours. On day 16 (96 hours after selection is imposed), each cage is
 provided with a fresh food plate for the surviving flies to lay eggs on.
 On day 17 (18 hours after the start of egg laying), eggs are collected
 from these plates to start the next generation. Day 17 of the previous
 generation becomes the day 1 of the following generation.
 During regular maintenance, there is negligible mortality in the
 P1-4 populations (&lt;1%) and no mortality in the N1-4 populations,
 from the point of handling of adults on day 12 till the start of
 oviposition window on day 16. During the selection process, the mortality
 of flies from E1-4 populations are maintained at about fifty percent. To
 this effect, the flies were infected at an infection dose of OD600 = 0.8
 (see below for more details) between generations 1 and 20 of forward
 selection. Thereafter, the flies were infected with OD600 = 1.0 from
 generation 21 to 40, with OD600 = 1.2 from generation 41 to 60, and with
 OD600 = 1.5 from generation 61 onwards. 
 <strong>Standardization and derivation of experimental
 flies</strong> Prior to experiments, flies from
 the three selection regimes were reared for a generation under ancestral
 maintenance conditions. This is done to account for any non-genetic
 parental effects (Rose 1984), and flies thus generated are referred to as
 standardized flies. To generate standardized flies, eggs were collected
 from all the 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 detailed above. On day 12
 after egg collection, 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 standardized population
 cages. <strong>Pathogen handling and Infection
 protocol</strong> <em>Enterococcus
 faecalis</em> (Lazzaro et al., 2006), a Gram-positive bacterium, and
 a known entomopathogen (Troha and Buchon 2019) was used for infecting the
 host individuals, both during regular population maintenance and for
 experiments. All experimental infections were done at an infection dose of
 OD600 = 1.5. A OD600 = 1.0 suspension of <em>E. faecalis</em>
 amounts to 106 CFUs per milliliter.  The bacteria are
 preserved as glycerol stocks (17%) in -80 °C. To obtain live bacterial
 cells for infections, 10 ml lysogeny broth (Luria Bertani Broth, Miler,
 HiMedia) is inoculated with glycerol stocks of the bacterium and incubated
 overnight with aeration (150 rpm in a shaker incubator) at suitable
 temperature (37 °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 (OD600 = 1.0-1.2) cultures. The
 bacterial cells are then pelleted down using centrifugation and
 resuspended in sterile MgSO4 (10 mM) buffer to obtain the required optical
 density (OD600) for infection. Flies are infected, under light
 CO<sub>2</sub> anesthesia, 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 MgSO4 (10 mM) buffer. 
 <strong>Systemic pathogen load
 estimation</strong> In this study, systemic
 pathogen load was estimated for both dead and alive flies. For estimation
 of Bacterial Load Upon Death (BLUD), the dead flies were surface
 sterilized twice with 70% ethanol (twice for 1 minute) and individually
 homogenized in 200 microliters of sterile MgSO<sub>4</sub> (10
 mM) buffer within an hour of their death. This homogenate was then
 serially 1:10 diluted eight times; 10 microliters of each dilution, along
 with 10 microliters of the original homogenate, was spotted onto Luria
 Bertani (Miller) agar (2%) plates. The plates were incubated for 8 hours
 at 37 °C. Colony forming units (CFUs) were counted for the dilution where
 CFUs ranged between 25 to 250, and the count was multiplied by the
 appropriate dilution factor to calculate the systemic pathogen load. For
 estimation of bacterial load of alive flies, a protocol similar to BLUD
 estimation was followed, except that the living flies were individually
 homogenized in 50 microliters sterile MgSO<sub>4</sub> (10 mM)
 buffer to begin with. <strong>Post-infection
 survival assay</strong> For assaying
 post-infection survival, 2–3-day old adult flies from N1-4, P1-4, and E1-4
 populations were either infected with Enterococcus faecalis (n = 100 males
 and 100 females per population per block) or sham-infected (n = 50 males
 and 50 females per population per block), and housed in individual cages
 (14 cm length × 16 cm width × 13 cm height) with food plates (Petri
 plates, 60 mm diameter). Fresh food was provided every 48 hours. The
 survival of these flies were monitored every hour for the first 48 hours
 and thereafter every 4-6 hours till 96 hours post-infection (HPI). Each
 block was handled separately on separate days. Survival assay was
 conducted twice, once after 65 generations of forward selection and again
 after 75 generations of forward selection.
 <strong>Female fecundity assay (0–48 hours
 post-infection)</strong> Fecundity of females
 during 0–48 HPI was assayed after 70 generations of forward selection. For
 this assay, 4–5-day old, inseminated females  from N1-4 and E1-4
 populations were either infected with E. faecalis (n = 80 females per
 population per block) or sham-infected (n = 40 females per population per
 block), and thereafter housed individually in food vials where they could
 oviposit. The survival of these females was monitored every 2 hours till
 48 HPI. At the end of this window, all surviving females were discarded,
 and the vials were incubated under standard maintenance conditions, and
 the number of progenies eclosing out of these vials were counted 12 days
 after the end of oviposition period.  
 <strong>Female fecundity assay (96–120 hours
 post-infection)</strong> Fecundity of females
 during 96–120 HPI was assayed along with the post-infection survival assay
 conducted after 75 generations of forward selection. At 96 HPI, alive
 females from N1-4 and E1-4 populations, of both infected and sham-infected
 treatments, were aspirated out of their respective cages and housed
 individually in food vials (n = 30 females per population per treatment
 per block). These females were allowed to oviposit for 24 hours, after
 which they were discarded. The vials with laid eggs were incubated under
 standard maintenance conditions, and the number of progenies eclosing out
 of these vials were counted 12 days after the oviposition period. The
 assay design closely matches the regular maintenance regime, where 96
 hours after infection the flies are allowed to oviposit on fresh food
 medium, and these eggs are used to start the next generation. 
 <strong>Systemic bacterial load of dead
 flies</strong> Systemic bacterial load of dead
 flies (BLUD) was assayed along with the post-infection survival assay
 conducted after 75 generations of forward selection. Dead females and
 males of N1-4 and E1-4 populations were aspirated out of the cages within
 an hour of their death, and their systemic bacterial load was estimated
 using the above-described protocol. The sample size varied between 15-30
 flies per sex per population per block, depending upon how many flies
 perished due to infection. Since no death was recorded in sham-infected
 flies, systemic load of sham-infected flies was not monitored during this
 assay. <strong>Systemic bacterial load of alive
 files</strong> To assay for the time dependent
 changes in systemic bacterial load in living, infected females, 700 N1-4
 females and 350 E1-4 females (per block) were infected with <em>E.
 faecalis</em>, and housed in separate cages. Starting sample size of
 N females was double of that of the E females to ensure enough surviving
 flies were available for bacterial load measurements at later time points,
 given that N females are expected to have much greater mortality compared
 to E females. Starting from 3 hours post-infection (HPI), every 3 hours,
 the systemic bacterial load of the females were assayed for the first 48
 hours, after which the sampling frequency was reduced to 6-12 hours. The
 assay continued till 96 HPI. At every sampling time point, 10 alive
 females of each population were aspirated out of their respective cages,
 and their systemic bacterial load was estimated using the above-described
 protocol. Individual blocks were assayed on separate days. To assay for
 the time dependent changes in systemic bacterial load in living, infected
 males, we followed an identical experimental design, except that the
 starting sample sizes for N1-4 and E1-4 males were 240 and 120 (per
 block), respectively, and measurement of systemic bacterial load was
 carried out only at 4-, 10-, 48-, and 96-HPI. This assay was done for
 females after 78 generations of forward selection and for males after 80
 generations of forward selection. Systemic bacterial load for
 sham-infected flies were measured only at one time point: 3 HPI for
 females and 4 HPI for males. No sham-infected fly yielded any CFU during
 any of the experiments.
description: # Experimental evolution for improved post-infection survival selects for
 increased disease resistance in Drosophila melanogaster.
 [https://doi.org/10.5061/dryad.xd2547dqr](https://doi.org/10.5061/dryad.xd2547dqr) This consists of seven separate files of data from a set of experiments, addressing the evolutionary responses in disease resistance and disease tolerance. There are seven README files describing the seven data files. ## Description of the data and file structure There are seven data files in this set. Each file corresponds to a particular experiment. There are seven README files to help understand the data. ## Sharing/Access information These data come from a set of experiments described in the following pre-print: Links to publications/pre-prints that cite or use the data: (bioRxiv) [https://www.biorxiv.org/content/10.1101/2024.02.14.580293v1.full](https://www.biorxiv.org/content/10.1101/2024.02.14.580293v1.full) [https://doi.org/10.1101/2024.02.14.580293](https://doi.org/10.1101/2024.02.14.580293) Links to other publicly accessible locations of the data: * Not Applicable Data was derived from the following sources: * Not Applicable ## Code/Software Not Applicable