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Drosophila melanogaster hosts coevolving with Pseudomonas entomophila pathogen show sex-specific patterns of local adaptation


Prasad, N. G. et al. (2022), Drosophila melanogaster hosts coevolving with Pseudomonas entomophila pathogen show sex-specific patterns of local adaptation, Dryad, Dataset,



In spatially structured populations, local adaptation improves organisms’ fitness in their native environment. Host and pathogens can rapidly adapt to their local antagonist. Since males and females can differ in their immunocompetence, the patterns of local adaptation can be different between the sexes. However, there is little information about sex differences in local adaptation in host-pathogen systems.


 In the current study, we experimentally coevolved four different replicate populations of Drosophila melanogaster (host) and Pseudomonas entomophila (pathogen) along with appropriate controls. We used the four host-pathogen coevolution populations to investigate the occurrence of local adaptation separately in males and females of the coevolving hosts. We also assessed local adaptation in pathogens. We set up a reciprocal infection experiment where we infected each of the four coevolving hosts with their local pathogen or non-local pathogens from the other three replicate populations. We found that overall, male and female hosts had better survivorship when infected with local pathogens, indicating that they were locally adapted. Interestingly, males were more susceptible to non-local pathogens compared to females. In addition, we found no fecundity cost in females infected with either local or non-local pathogens. We found no evidence of local adaptation among the pathogens.


Our study showed sex-specific adaptation in the coevolving hosts where female hosts had a broader response against allopatric coevolving pathogens with no cost in fecundity. Thus, our results might suggest a novel mechanism that can maintain variation in susceptibility in spatially structured populations.


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 25C 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.

Within 24-48h post infection when flies from Coev populations were dying, 10-15 dead flies per sex were collected and stored. These dead flies were later used to extract the coevolving pathogen to infect the next generation coevolving host. A random sample of five dead flies were taken and were homogenised in sterile 10mM MgSO4 solution. This homogenate was plated on LB agar plates containing ampicillin and were incubated. Later, to infect the next generation host, we randomly picked 11-12 colonies from different regions and use an overnight culture to infect the flies. Please note that for each of the four coevolving hosts (Coev 1, Coev 2, Coev 3 and Coev 4) we had four matched coevolving Pseudomonas entomophila pathogens designated as ‘B1Pe or Pe1’, ‘B2Pe or Pe2’, ‘B3Pe or Pe3’ and ‘B4Pe or Pe4’. It is important to note that the pathogen isolated from dead flies of Coev 1 population was used to infect the next generation host of Coev 1 only (and not Coev 2, 3 or 4). Similarly, the pathogen isolated from dead flies of Coev 2 population was used to infect the next generation host of Coev 2 only (and not Coev 1, 3 or 4) and so on. Thus, the host and the pathogen formed a coevolving pair. Therefore, we had four such matched pairs of coevolving host and pathogen (Coev 1 with B1Pe; Coev 2 with B2Pe etc). These four matched pairs are independent replicate populations of the coevolutionary experiment. It is also important to note that each of the four matched pairs were isolated from all the other pairs and hence formed a Sympatric pair.

The current study was performed using 4 different Coev populations (Coev 1, Coev 2, Coev 3 and Coev 4) and populations from other three selection regimes were not used. These 4 coevolving host-pathogen populations were labelled as Coev 1, Coev 2, Coev 3 and Coev 4.

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 each of the four Coev populations (Coev 1, Coev 2, Coev 3 and Coev 4), all on the same day. 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

In this experiment we investigated if the coevolving host or pathogen were locally adapted. We assessed two traits in hosts by conducting two different assays- 

(1) survivorship post infection and (2) fecundity post infection

(1) Survivorship post infection: For a coevolving host to show patterns of local adaptation, the host survives better against their sympatric pathogens relative to allopatric or non-local pathogens. In other words, if Coev 1 has higher survivorship when infected with B1Pe relative to B2Pe, B3Pe and B4Pe, this indicate host local adaptation. Similarly, if the coevolving pathogen causes higher mortality in its local or sympatric host (compared to mortality induced in non-local hosts), it would indicate pathogen local adaptation. For example, if B1Pe causes higher mortality in Coev 1 host as compared to allopatric (or non-local) hosts (Coev 2, Coev 3 and Coev 4), it would indicate that B1Pe or Pe1 was locally adapted. Therefore, to measure local adaptation, each of the four Coev hosts were infected individually with their sympatric or allopatric coevolving pathogens (B1Pe, B2Pe, B3Pe and B4Pe). 

For this, we had a combination of 4 hosts X 4 coevolving pathogens with a total of 16 treatments along with four sham control treatments. Different infection treatments in this experiment represented either sympatric combinations or allopatric combinations. Thus, there were 4 sympatric treatments (Coev 1 flies infected with B1Pe, Coev 2 infected with B2Pe and so on) and 12 allopatric treatments (Coev 1 flies infected with B2Pe or B3Pe or B4Pe and so on). In sham control treatments, the experimental flies were injured with a needle dipped in sterile 10mM MgSO4 solution. This complete experiment was conducted three times i.e. in three experimental replicates, keeping all the experimental conditions same, on three separate days. 

The experimental flies were generated from the standardized flies (descrived above) from each of the Coev populations (Coev 1, Coev 2, Coev 3 and Coev 4). For the experiment, eggs were collected from the standardized flies at a density of 70 eggs per vial containing 6-7 ml banana-jaggery food. Forty such vials were collected for each of the Coev populations and these vials were incubated under standard laboratory conditions (mentioned above). 

75 males and 75 females were randomly chosen for each infection treatment (one sympatric and three allopatric infection treatments for each Coev population) and sham control treatment. Post-infection mortality was recorded in each of these experimental cages every 3-4 hours for the first 48 hours, and then every 6-8 hours till 120 hours. 

(2) Fecundity post infection: Fecundity in female hosts was estimated in infected Coev hosts (with sympatric or allopatric pathogens) and sham treated coev hosts, through the same experimental set-up from the host survivorship assay. Post-infection, a fresh food plate was provided to each cage for 6 hours. Fecundity plates in each cage were provided for fixed time points to account for the fecundity peak that was observe in these flies when switching to the dark part of the light cycle. After 6 hours, plates from each cage were replaced with new food plates. These fecundity plates were provided daily to each cage, up until the 120 hour time-point. For each cage, mortality was recorded before and after the start of egg laying window, in each cage.


Indian Institute of Science Education and Research Mohali