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.

1. EPN experimental evolution set-up

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 Singh et al., 2021).

E regime: The E_1-4 populations are selected for better survival following infection with the Gram-positive bacterium Enterococcus faecalis. 2–3-days old adult flies (200 females and 200 males) are subjected to infection with E. faecalis 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_1-4 populations.

P regime: The P_1-4 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.

N regime: The N_1-4 populations are uninfected control populations. 2–3-day-old adult flies (100 females and 100 males) are subjected only to light CO₂ 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_1-4 (< 2%) and N_1-4 (0%) populations during maintenance of the selection regimes.)

These populations were derived from the ancestral Blue Ridge Baseline (BRB_1-4) populations (Singh et al., 2015). The E₁, P₁, and N₁ populations were derived from the BRB₁ population and constitute ‘block 1’ of the experimental evolution regime. Similarly, E₂, P₂, and N₂ populations were derived from the BRB₂ population and constitute ‘block 2’, and so on. This block design implies that E₁, P₁, and N₁ have a more recent common ancestor, compared to E₁ and E₂, or P₁ and P₂, and so on. Populations belonging to each block were handled together, both during maintenance of the populations and during experiments. Please refer to supplementary figure 1 for a graphical depiction of the block design.

The regular maintenance of the EPN set-up has been previously described (Singh et al., 2021; Basu et al., 2024b). 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 ^OC, 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 (see section 2 for details of the infection process). 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.

2. Pathogen handling and infection protocol

Enterococcus faecalis (Lazzaro et al., 2006), 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_1-4 populations (see section 1) are infected with an E. faecalis suspension of OD₆₀₀ = 1.5. For all experimental infections (see section 4), flies were also infected with an E. faecalis suspension of OD₆₀₀ = 1.5 to keep experimental conditions consistent with that of regular maintenance. OD₆₀₀ = 1.0 corresponds to 10⁶ cells/ml for E. faecalis.

The bacteria are stored as glycerol stocks (17%) in -80 ^OC. 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 ^OC. 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 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 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 MgSO₄ (10 mM) buffer.

3. Pre-experiment standardization and generation of experimental flies

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 (Rose 1984); flies thus generated are referred to as standardized 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 (section 1). 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 standardized population cages.

To generate flies for the experiments, eggs were collected from standardized 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 (section 4). The handling of the flies during the experiment was kept identical to how the populations are regularly maintained to keep their ecology consistent. 

4. Experiment design

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 (Singh et al., 2021; Singh et al., 2022a; Singh et al., 2022b; Basu et al., 2024b). 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. Fed/Fed (FF): flies that were continuously fed their standard diet.

b. Fed/Starved (FS): flies that were fed their standard diet before infection, and subjected to starvation post-infection.

c. Starved/Fed (SF): flies that were subjected to starvation for 48 hours prior to infection, but fed their standard diet post-infection.

d. Starved/Starved (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 starvation 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 CO₂, and 8 females were infected (10 vials) or sham-infected (5 vials) from each vial (as described in section 2), 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 starvation 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.