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.

Host populations

Replicate Drosophila melanogaster 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 Enterococcus faecalis. Every generation, 2–3-days old adult flies (200 females and 200 males) are subjected to infection with E. faecalis, 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 CO₂ 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 (<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. 

Standardization and derivation of experimental flies

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.

Pathogen handling and Infection protocol

Enterococcus faecalis (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 E. faecalis 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 CO₂ 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. 

Systemic pathogen load estimation

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 MgSO₄ (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 MgSO₄ (10 mM) buffer to begin with.

Post-infection survival assay

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.

Female fecundity assay (0–48 hours post-infection)

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.  

Female fecundity assay (96–120 hours post-infection)

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. 

Systemic bacterial load of dead flies

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.

Systemic bacterial load of alive files

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 E. faecalis, 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.