DOI: 10.5061/dryad.9cnp5hqkx
title: No major cost of evolved survivorship in Drosophila melanogaster populations coevolving with Pseudomonas entomophila

description: Rapid exaggeration of host and pathogen traits via arms race dynamics is
 one possible outcome of host-pathogen coevolution. However, the
 exaggerated traits are expected to incur costs in terms of resource
 investment in other life-history traits. The current study investigated
 the costs associated with evolved traits in a host-pathogen coevolution
 system. We used the Drosophila melanogaster (host)-Pseudomonas entomophila
 (pathogen) system to experimentally derive two selection regimes, one
 where the host and pathogen both coevolved, and the other, where only the
 host evolved against a non-evolving pathogen. After 17 generations of
 selection, we found that hosts from both selected populations had better
 post-infection survivorship than controls. Even though the coevolving
 populations tended to have better survivorship post-infection, we found no
 clear evidence that the two selection regimes were significantly different
 from each other.. There was weak evidence for the coevolving pathogens
 being more virulent than the ancestral pathogen. We found no major cost of
 increased post-infection survivorship. The costs were not different
 between the coevolving hosts and the hosts evolving against a non-evolving
 pathogen. We found no evolved costs in the coevolving pathogens. Thus, our
 results suggest that increased host immunity and pathogen virulence may
 not be costly.
description: <strong>Maintenance of
 populations </strong> Selection Regimes – An
 experimental set-up was derived from laboratory baseline BRB populations
 to investigate host-pathogen coevolution system using <em>Drosophila
 melanogaster</em> insect host and <em>Pseudomonas entomophila
 </em>bacterial pathogen. Four different selection regimes were
 derived from BRB populations– 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 inoculated with
 10mM MgSO4 sterile solution 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 a needle. Four blocks (replicates) of BRB
 populations were used to derive four blocks of these experimental lines.
 Replicate populations with the same numeral were derived from same
 baseline populations. For example, Coev 1, Adapt 1, Co.S 1 and Co.U were
 derived from BRB 1 population, Coev 2, Adapt 2, Co.S 2 and Co.U 2 were
 derived from BRB 2 population and so on. Hence populations with the same
 numeral (i.e., Coev 1, Adapt 1, Co.S 1 and Co.U 1) are closely related to
 each other than to any other population with a different numeral value.
 Populations in each block ie. populations with same numeral were always
 handled on the same day every generation during regular maintenance as
 well as during the assays performed in this study. Thus, Coev 1, Adapt 1,
 Co.S 1 and Co.U 1 populations correspond to Block-1, Coev 2, Adapt 2, Co.S
 2 and Co.U 2 to Block-2, Coev 3, Adapt 3, Co.S 3 and Co.U 3 to Block-3 and
 Coev 4, Adapt 4, Co.S 4 and Co.U 4 to Block-4, making a total of 4
 statistical “blocks” for our analyses.  Hence, a total
 of sixteen populations i.e. four selection regimes (Coev, Adapt, Co.S and
 Co.U), each having 4 replicates, were used for this study. Coev and Adapt
 populations were maintained in an identical manner except that they differ
 in the type of bacterial infection. Flies in Coev regime are subjected to
 bacterial infection by coevolving <em>P. entomophila</em>
 pathogen while the flies in Adapt regime experience infection by a
 non-evolving or ancestral <em>p. entomophila</em>. Fly
 infections with bacteria were done using a fine minutium 1mm needle dipped
 in bacterial suspension, at the lateral side of the thorax. A discrete
 16-day generation cycle is followed for fly population under standard
 laboratory conditions of 25C temperature, ~60% relative humidity (RH) and
 12:12 hours light/dark cycle. Flies are cultured in standard
 banana–jaggery–yeast food in standard vials (90-mm length × 25-mm
 diameter). They are reared at a controlled larval density of 70 per vial.
 On the 12th day post egg collection, flies are provided bacterial
 infection as per their respective selection, after which flies from each
 selection regime were transferred to cages containing banana-jaggery food
 plate. Fly mortality in each cage was monitored till 96 hours. Within
 24-48 hours post-infection, 10-15 dead flies of each sex were collected
 for Coev regime. Later, these flies were used to isolate the coevolving
 pathogen to infect next generation flies. The flies that survived
 bacterial infections after 96 hours in Coev and Adapt regimes, contributed
 their eggs to the next generation. 
 <strong>Standardization and generation of experimental
 flies</strong> Before generating experimental
 flies, one generation of standardization of populations (Rose 1984) was
 followed. This was followed in order to eliminate any potential
 non-genetic parental effects between the two selection regimes. During
 standardization, populations were neither subjected to bacterial
 infections nor were they pricked with needle. Flies from each regime were
 kept unhandled and were transferred into cages containing food plate
 smeared with yeast. 48 hours later, a fresh food plate was provided to
 each cage for collecting eggs for experiments. Experimental flies were
 generated from these standardized populations. For
 generating experimental flies, we collected eggs for each of the four
 selection regime (Coev, Adapt, Co.S and Co.U) in each block, all on the
 same day. All the experimental flies were reared under controlled standard
 culture conditions (25⁰C, 60–80% 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>
 Surv post-inf till 96h (Data in Tab 1 of
 "Raw_data.xlsx") Flies from each population
 were divided into three infection treatments- (1) infected with coevolved
 Pe, (2) infected with ancestral Pe, and (3) sham infection. For each
 infection/sham treatment, 75 males and 75 females were randomly chosen
 from each selection regime. In infection treatments (coevoled Pe and
 ancestral Pe), males and females were infected at an OD of 0.44.
 Similarly, in sham treatment, males and females from each selection regime
 were treated with 10mM MgSO4 solution. Hence, for each block there were
 twelve cages. In total, life-span of files from 48 cages were monitored (4
 selection regimes*3 treatments*4 blocks). For first 48h post-infection,
 mortality was monitored after every 4-5h; from 48h to 120h mortality was
 monitored after every 7-8h and later on mortality was monitored once in a
 day till the last fly died in each cage. Mortality over the first 96 hours
 post-infection was analysed separately to assess response to selection
 (since this period corresponds to the regular selection protocol). Tab 1
 of the data file contains the survivorship data of flies from each regime
 for 96 hours post-infection with coevolved and ancestral Pe.
 <strong>Longevity post-inf after 96h (Data in Tab 2 of
 "Raw_data.xlsx")</strong> Mortality
 data after 96 hours of infection till the death of the last fly was
 analysed separately to assess the long-term effects of infection on host
 longevity. A large number of Co.S and Co.U flies died within 96 hours
 post-infection with ancestral or coevolved Pe relative to flies in Coev
 and Adapt regimes. Therefore, longevity after 96 hours of infection was
 assessed between Coev and Adapt regimes. While analysing the results for
 longevity after 96 hours of infection, we did not include the results for
 block 2 as we lost one experimental cage (Adapt population infected with
 ancestral Pe) in an accident, one month later the set up of the
 experiment.  <strong>Prop surv post-inf (Data in
 Tab 3 of "Raw_data.xlsx")</strong>
 Proportion of flies that were alive till 96 hours post-infection
 with ancestral Pe or coevoled Pe.
 <strong>Longevity sham inf (Data in Tab 4 of
 "Raw_data.xlsx") </strong> Lifespan of
 flies from each selection regime was measured post sham treatment. Flies
 from each selection regime were treated with 10mM MgSO4 as described
 above. Each cage was tracked for fly mortality once a day, till the last
 fly died. <strong>Basal longevity (Data in Tab 5
 of "Raw_data.xlsx")</strong> Basal
 lifespan of the flies from each selection regime was measured in unhandled
 condition. 75 males and 75 females from each selection regime were
 randomly chosen and were transferred into cages. Fly mortality in each
 cage was recorded twice a day till the last fly died. This protocol was
 replicated across the populations in all four blocks.
 <strong>Starvation resistance (Data in Tab 6 of
 "Raw_data.xlsx")</strong> This assay
 was conducted using two treatment- Mated and Virgin treatments. Virgin
 males and females from each selection regime were collected within 6 hours
 after emergence from pupae and were kept sepatate. Mated males and females
 were separated on the day of experiment. 75 males and 75 females were
 randomly chosen from each selection regime and for each treatment. These
 flies were transferred into cages with an access to water but no food.
 Mortality was recored every 7-8 hours till the last fly died. This
 protocol was followed across all four blocks.
 <strong>Fecundity (Data in Tab 7 of
 "Raw_data.xlsx")</strong> Fecundity
 data was obtained from the cages set-up for the longevity (post-infection
 and basal longevity) experiment. Fecundity was measured by providing a
 fresh food plate in each cage for 6 hours. Fecundity plates were provided
 every day for first five days. Later, fecundity was measured once every
 five days. These plates were stored at -20⁰C and were later thawed, and
 eggs were counted.  <strong>Development time
 (Data in Tab 8 of "Raw_data.xlsx")</strong>
 A fresh food plate was provided for 1h to the standardised flies
 so that females laid their stored eggs. After 1h, another fresh food plate
 was provided to each of these cages for 1.5h. Eggs that were laid during
 this period were collected at a density of seventy per vial. A total of
 ten vials were collected for each population and across all the four
 blocks. When flies started to emerge, they were collected every 4h till
 all the flies had eclosed. These flies were stored at -20°C. Later, flies
 in each vial were sexed and counted while maintaining their vial id and
 time of eclosion.  <strong>Dry body weight (Data
 in Tab 9 of "Raw_data.xlsx")</strong>
 The stored flies from the development time assay were used to
 measure dry body weight. 10 males and 10 females were randomly selected
 from the total number of flies from each vial and were put in 1.5ml
 micro-centrifuge tubes (MCTs). These flies were dried at 60°C in an
 incubator for 48h. Later, dry body weight of these flies were measured to
 nearest 0.01mg, using a sensitive microbalance (Sartorious, Germany,
 CPA225D).  <strong>Lipid content (Data in Tab 10
 of "Raw_data.xlsx")</strong> Subset of
 flies used to measure dry body weight were later used to measure lipid
 content. These flies in micro-centrifuge tube were provided with 1.5ml
 diethyl ether and were kept on a rotating shaker for 48h. Later, excess
 ether solution was drained and flies were dried at 60°C. After this, the
 body weight of these flies was measured. The difference between dry body
 weight and body weight after lipid extraction was taken as the amount of
 lipid content.  <strong>Coev Pe G15 Growth rate
 (Data in Tab 11 of "Raw_data.xlsx")</strong>
 Growth rate for ancestral Pe and coevolved Pe from the 15th
 generation was assessed across each of the four replicate populations of
 coevolving pathogen. LB broth was inoculated with ancestral Pe and
 coevolved Pe (from 15th genration of host-pathogen coevolution) and for
 all four blocks. Each of these flasks was incubated overnight at 27°C and
 at 150 rpm in order to have bacterial cells in an actively growing stage.
 On the following day, overnight grown culture was used to inoculate
 bacteria to fresh flasks containing LB media. The starting OD of this
 fresh culture was kept same for all flasks containing bacteria. These
 flasks were kept in the incubator shaker and the OD was recorded for each
 flask at 600nm, every hour until a stationary phase was attained. This
 assay was replicated thrice on different days with coevolved Pe of each of
 the 4 blocks and ancestral or non-evolving Pe. 
 <strong>Coev Pe G20 Growth rate (Data in Tab 12 of
 "Raw_data.xlsx")</strong> The
 experimental protocol described above (Coev Pe G15 Growth rate) was
 repeated using ancestral Pe and four different coevolving pathogens from
 20th generation of host-pathogen coevolution.