DOI: 10.5061/dryad.3n5tb2rvk
title: Sex-specific effects of antagonistic coevolution: Insights from an insect host and a bacterial pathogen coevolution system

description: 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.
description: <em>Maintenance of populations</em>
   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.  
 <em>Standardization and generation of experimental
 flies</em>   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.  
 <em>Experimental design</em>
   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 <em>Drosophila melanogaster</em> host
 with its native bacterial pathogen <em>Pseudomonas
 entomophila</em>, 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.
description: # Sex-specific effects of antagonistic coevolution: Insights from an
 insect host and a bacterial pathogen coevolution system This file
 ("data file_Evolutionary Ecology.xlsx") was generated in 2019 by
 Neetika Ahlawat. GENERAL INFORMATION 1. Title of Dataset:
 "Sex-specific effects of antagonistic coevolution: Insights from an
 insect host and a bacterial pathogen coevolution system" 2. Author
 Information A. Principal Investigator Contact Information Name: Prof. N.
 G. Prasad Institution: Indian Institute of Science Education and Research,
 Mohali Address: IISER Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab
 - 140306, India. Email:
 [prasad@iisermohali.ac.in](mailto:prasad@iisermohali.ac.in) B. Associate
 or Co-investigator Contact Information Name: Neetika Ahlawat Institution:
 Indian Institute of Science Education and Research, Mohali Address: IISER
 Mohali, Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India.
 Email: [neetika.ahlawat@gmail.com](mailto:neetika.ahlawat@gmail.com) 3.
 Duration of data collection: 2019 4. Geographic location of data
 collection: Mohali, Punjab, India 5. Information about funding sources
 that supported the collection of the data: IISER Mohali, Govt. of India.
 ### DATA & FILE OVERVIEW File List:"data file_Evolutionary
 Ecology.xlsx" Note: This file contains three separate data sheets
 created from the host survivorship assay conducted against pathogens from
 three different evolutionary time points. The file contains: Sheet 1.
 "survivorship across 96h" (Data for Assay 1: Host survivorship
 post infection against sympatric and allopatric pathogens) Sheet 2.
 "prop host mortality at 96h" (Data for Assay 1: Proportion
 survivorship of host post infection against symaptric and allopatric
 pathogens) Sheet 3. "prop mortality at 24h and 48h" (Data for
 Assay 2: Female fecundity post infection against sympatric and allopatric
 pathogens) ### DATA-SPECIFIC INFORMATION FOR "data file\_Evolutionary
 Ecology.xlsx" This file has three separate tabs. Below, we provide
 details separately for each tab. #### Sheet 1. survivorship across 96h 1.
 Number of variables: 6 2. Number of cases/rows: 9607 3. Variable List:
 Time (Time in "Hours"; time recorded after small interval (post
 infection) to observe death of individuals till the last observation, i.e.
 96 hours) Population (4 host populations; "Coev",
 "Adapt", "Co.S", "Co.U") Bacteria (3
 pathogens from three evolutionary time points: "Anc Pe",
 "G10Pe", "G20Pe" and an infection control treatment
 "Sham") Block (4 independent replicate populations of each
 selection regime. "1", "2", "3",
 "4" represent that populations with the same numeric value share
 ancestory) Sex (Survivorship of "M" (Males) and "F"
 (Females) from each of the four sets of hosts) Censor (status of the fly
 at the end of 96 hours observation window after infection; "0"
 represents dead and "1" represents that the individual was alive
 till 96 hours post infection) #### Sheet 2. prop host mortality at 96h 1.
 Number of variables: 7 2. Number of cases/rows: 97 3. Variable List:
 Population (4 host populations; "Coev", "Adapt",
 "Co.S", "Co.U") Bacteria (3 pathogens from three
 evolutionary time points: "Anc Pe", "G10Pe",
 "G20Pe") Block (4 independent replicate populations of each
 selection regime. "1", "2", "3",
 "4" represent that populations with the same numeric value share
 ancestory) Sex (Survivorship of "M" (Males) and "F"
 (Females) from each of the four Coev populations) No. of surviving
 individuals (Total number of males and females that were alive at 96th
 hour after infection) Total no. of individuals (Total number of male and
 female hosts randomly chosen from each population for three infection
 treatments) Prop Surv (Proportion of male and female hosts that were alive
 at 96th hour post infection out of the total number of flies at the start
 of the experiment) #### Sheet 3. prop mortality at 24h and 48h 1. Number
 of variables: 10 2. Number of cases/rows: 97 3. Variable List: Population
 (4 host populations; "Coev", "Adapt",
 "Co.S", "Co.U") Bacteria (3 pathogens from three
 evolutionary time points: "Anc Pe", "G10Pe",
 "G20Pe") Block (4 independent replicate populations of each
 selection regime. "1", "2", "3",
 "4" represent that populations with the same numeric value share
 ancestory) Sex (Survivorship of "M" (Males) and "F"
 (Females) from each of the four Coev populations) 0-48hrs (Total number of
 mortality in 48 hours post infection; number of individuals died in 48hrs
 post infection) 0-24hrs (Total number of mortality in 24 hours post
 infection; number of individuals died within 24hrs post infection)
 24-48hrs (Total number of mortality occurred between 24 to 48 hours post
 infection; total number of individuals died between 24 to 48hrs post
 infection) total individuals (Total number of male and female hosts
 randomly chosen from each population for three infection treatments) prop
 mort_24h (Proportion of male and female individuals died within 24 hours
 post infection, out of the total number of flies at the start of the
 experiment) prop mort_48h (Proportion of male and female individuals died
 between 24 to 48h post infection out of the total number of flies at the
 start of the experiment)