Sex-specific effects of antagonistic coevolution: Insights from an insect host and a bacterial pathogen coevolution system

Experimental host-pathogen coevolution provides an opportunity to understand the long-term consequences of adaptive interactions between hosts and pathogens. Studies using prokaryotic and eukaryotic hosts and their pathogens have explored the changing dynamics of antagonistic interactions with time, evolution of generalists or specialists, and potential costs associated with coevolution. However, the dependence of host-pathogen coevolutionary responses on sex of the host remains unexplored.

To address this, we chose a host species which allow us to compare coevolved traits between male and female hosts with their native pathogens. Towards this end, we conducted a laboratory experiment where we coevolved insect host Drosophila melanogaster with its bacterial pathogen Pseudomonas entomophila. Apart from the host-pathogen coevolution regimen, the experimental design included three other selection regimes - host adaptation to a non-evolving ancestral pathogen, and two control regimes. To study coevolved traits in hosts in pathogens across time and sex, we measured host survivorship post infection against pathogens from three evolutionary time points – ancestor, pathogen from the past, and present coevolution cycle. Our results showed that the coevolved hosts exhibited higher survivorship when exposed to pathogens, relative to the hosts adapted to non-evolved pathogen and control hosts. These results are true against pathogenic exposure from all three time points. Coevolved pathogens from the present time exhibited the highest virulence, which was variable across the replicate pathogen populations. We also observed that despite comparable mortality, the two sexes differ in the onset of mortality in the control regimes, a response not observed in the coevolved hosts.

Taken together, our results showed that pathogens and hosts had the greatest success against each other as they coevolved together, but the susceptibility of naïve hosts was sex-specific. These results provide important insights into the process of host-pathogen coevolution.

Methods
Maintenance of populations

Selection Regimes – There were four selection regimes in this experimental line- Coev (Coev 1-4) or Coevolution regime where host and pathogen coevolve with each other, Adapt (Adapt 1-4) or Adaptation where only the host evolved against a non-evolving pathogen, Co.S (Co.S 1-4) or Sham control regime where the host is pricked with needle but not with pathogen, and Co.U (Co.U 1-4) or unhandled control regime where the host is neither subjected to pathogen infection nor pricked with needle. These regimes were derived from a laboratory adapted populations called BRB (1-4) populations.These four regimes were derived from each of the four replicates of BRB(1-4) populations and populations in each replicate were kept isolated from one other. Thus, we had 16 different populations in this experimental lines. On day 12th post egg collection, when flies were roughly 2-3 days old as adults, 200 males and 200 females (20 males and 20 females from each of the 10 culture vials) from each population were infected with a needle dipped in a suspension (OD600 0.4) of coevolving P. entomophila pathogen. Flies uaually start to die after about 12 hours post infection and we observe a signifant host mortality by 24 hours. Fly mortality due to pathogen infection becomes constant by 96 hours. At this time, about 200 flies would survive the infection and these survived flies contributed to the next generation. A discrete 16-day generation cycle is followed for each population and at 25 degreeC and 50-60% relative humidity (RH) and 12:12 hours light/dark cycle. Flies are provided with standard banana–jaggery–yeast food in standard vials (90-mm length × 25-mm diameter). On the 12th day post egg collection, flies are provided bacterial infection, after which were transferred to cages and fly mortality was recorded as described above.

Coevlving pathogens were isolated from dead Coev flies that were collected within 24-48 hours post infection. Each generation, Adapt flies were evolved against a non-evolving P. entomophila pathogen that share its ancestory with the coevolved pathogens.

Standardization and generation of experimental flies

Before generating experimental flies, we followed one generation of standardization of populations (Rose 1984). This was done to eliminate any potential non-genetic parental effects between the two regimes. During standardization, populations were maintained just like the ancestral BRB populations, i.e. they were neither subjected to bacterial infections nor were they pricked with needle. Experimental flies were generated from these standardized populations.
For generating experimental flies, we collected eggs for populations from each of the four selection regimes. All the experimental flies were reared under controlled standard culture conditions (25⁰C, 50–60% RH, 12 hours–12 hours light / dark cycle). Eggs were cultured at a density of 70 eggs / vial in 6–7 mL of banana-jaggery-yeast food for each of the population.

Experimental design

The aim of this study was to assess host survivorship against pathogens from three evolutionary time points, and to understand how male and female hosts’ adaptation changes with time. Host survivorship was measured against ancestral pathogen or coevolved pathogens from the past i.e. 10th coevolution cycle (G10Pe), or present i.e. 20th coevolution cycle (G20Pe).
This experiment was conducted using flies from all 4 selection regimes and across all blocks. Coevolved pathogens were used to infect host populations from the same block i.e. coevolved pathogens (past and present time) from block 1 were used to infect flies from Coev 1, Adapt 1, Co.S 1 and Co.U 1; block 2 coevolved pathogens (past and present generation) were used to infect flies from Coev 2, Adapt 2, Co.S 2 and Co.U 2 and so on.
For the experimental set-up, eggs were collected from the standardized flies from each selection regime (Coev, Adapt, Co.S and Co.U) and for each block. On the 12th day post egg collection, when we had roughly 2-3 days old adult flies, 75 males and 75 females (15 males and 15 females from each vial) from each population were randomly picked and divided into three infection groups (and one control) as per the experimental treatments. Flies from each population were administered pathogen infections following systemic infection, with either (a) ancestral Pe, (b) coevolved pathogen from the past (G10Pe) or, (c) coevolved pathogen from the present (G20Pe), (d) sham treatment using sterile 10mM MgSO4 buffer solution. Immediately after infections, flies were transferred into cages, and each cage was provided with a food plate which was replaced every alternate day. Fly mortality was monitored separately for males and females till 96 hours post infection. Mortality readings were taken after every 4 hours till 48 hours, after which, readings were taken after every 6-7 hours till 96 hours. To account for the effect of environment, this experiment was conducted separately for each block, on different days.
The aim of this study is to assess host survivorship against pathogens from three evolutionary time points, and to understand how male and female hosts’ adaptation changes with time.

We used experimental evolution apporach to coevolve Drosophila melanogaster host with its native bacterial pathogen Pseudomonas entomophila, for ~ 20 coevolutionary cycles. There were three additional regimes, one-sided host adaptation against a non-evolving pathogen, an infection control regime and an unhandled control regime. Each four regimes were replicated as four independent blocks. 

To quantify host survivorship against ancestral pathogen or coevolved pathogens from the past i.e. 10th coevolution cycle (G10Pe), or present i.e. 20th coevolution cycle (G20Pe), we compared survivorship (post infection) of flies from all 4 selection regimes and across all blocks. 

For the experimental set-up, on the 12th day post egg collection, when we had roughly 2-3 days old adult flies, 75 males and 75 females (15 males and 15 females from each vial) from each population were randomly picked and divided into three infection groups (and one control) as per the experimental treatments. Flies from each population were administered pathogen infections following systemic infection, with either (a) ancestral Pe, (b) coevolved pathogen from the past (G10Pe) or, (c) coevolved pathogen from the present (G20Pe), (d) sham treatment using sterile 10mM MgSO4 buffer solution.  Fly mortality was monitored separately for males and females till 96 hours post infection. Mortality readings were taken after every 4 hours till 48 hours, after which, readings were taken after every 6-7 hours till 96 hours. 

The data was censored and was arranged accroding to mortality recorded at different time points. Dead individuals were marked as '0', and individuals that were alive till 96 hours post infection, were marked as '1'. This data was analysed using statistical tests such as cox proportional hazard test, binomial mixed effect model, Tukey's HSD non parameteric test.