DOI: 10.5061/dryad.mw6m905zz
title: Drosophila melanogaster hosts coevolving with Pseudomonas entomophila pathogen show sex-specific patterns of local adaptation

description: Background: 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. Results:
  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. Conclusion: 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.
description: <strong>Maintenance of
 populations </strong> 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. <strong>Standardization and
 generation of experimental flies</strong> 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. 
 <strong>Experimental design</strong>
 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.