DOI: 10.5061/dryad.05qfttf2z
title: Enemies make you stronger: Coevolution between fruit fly host and bacterial pathogen increases post-infection survivorship in the host

description: Multiple laboratory studies have evolved hosts against a non-evolving
 pathogen to address questions about evolution of immune responses.
 However, an ecologically more relevant scenario is one where hosts and
 pathogens can coevolve. Such coevolution between the antagonists,
 depending on the mutual selection pressure and additive variance in the
 respective populations, can potentially lead to a different pattern of
 evolution in the hosts compared to a situation where the host evolves
 against a non-evolving pathogen. In the present study, we used Drosophila
 melanogaster as the host and Pseudomonas entomophila as the pathogen. We
 let the host populations to either evolve against a non-evolving pathogen,
 or coevolve with the same pathogen. We found that the coevolving hosts on
 average evolved higher survivorship against the coevolving pathogen and
 ancestral (non-evolving) pathogen relative to the hosts evolving against a
 non-evolving pathogen. The coevolving pathogens evolved greater ability to
 induce host mortality even in non-local (novel hosts) hosts compared to
 infection by an ancestral (non-evolving) pathogen. Thus, our results
 clearly show that the evolved traits in the host and the pathogen under
 coevolution can be different from one-sided adaptation. In addition, our
 results also show that the coevolving host-pathogen interactions can
 involve certain general mechanisms in the pathogen, leading to increased
 mortality induction in non-local or novel hosts. 
description: This file ("Raw_data.xlsx") was generated in 2019 by
 Neetika Ahlawat. GENERAL INFORMATION
 1. Title of Dataset: "Enemies make you stronger: Coevolution
 between fruit fly host and bacterial pathogen increases post-infection
 survivorship in the host" 2. Author
 Information<br>     A. Principal Investigator Contact
 Information<br>         Name: Prof. N. G. Prasad<br>       
  Institution: Indian Institute of Science Education and Research,
 Mohali<br>         Address: IISER Mohali, Sector 81, Knowledge City,
 SAS Nagar, Punjab - 140306, India.<br>         Email:
 prasad@iisermohali.ac.in     B. Associate or
 Co-investigator Contact Information<br>         Name: Neetika
 Ahlawat<br>         Institution: Indian Institute of Science
 Education and Research, Mohali<br>         Address: IISER Mohali,
 Sector 81, Knowledge City, SAS Nagar, Punjab - 140306, India.<br>   
      Email: neetika.ahlawat@gmail.com 3. Duration of
 data collection: 2018-2019 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. #######SHARING/ACCESS
 INFORMATION<br>  <br> Links to publications that cite or use
 the data:  DATA & FILE OVERVIEW
 1. File List:"Raw_data.xlsx" Note:
 This file contains four separate data sheets (please see below for
 methodological details) in the following four tabs: Tab
 1. "Experiment1" <br> Tab 2.
 "Experiment2" <br> Tab 3. "Selection
 experiment"<br> Tab 4. "Notes"
 METHODOLOGICAL INFORMATION Maintenance of
 populations  Selection Regimes – Experimental evolution
 lines with four different selection regimes –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 selection regimes were derived
 from laboratory adapted baseline BRB populations. From four blocks
 (replicates) of BRB populations, four blocks of these selection lines were
 derived. Replicate populations bearing the same numerical subscript were
 derived from same ancestral populations. For example, Coev 1 and Adapt 1
 were derived from BRB 1 population, Coev 2 and Adapt 2 from BRB 2
 population and so on. Hence populations carrying the same numerical
 subscript (e.g., Coev 1 and Adapt 1) are more closely related to each
 other than to any other population with a different numerical subscript.
 Also, populations related by ancestry (e.g. Coev 1, Adapt 1, Co.S 1 and
 Co.U 1) are 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 from the four selection
 regimes (Coev, Adapt, Co.S and Co.U), each having 4 replicates, were used
 for this study. Populations belonging to both regimes are maintained in an
 identical manner except that they differ in the type of bacterial
 infection. All the bacterial infection were done using a fine minutium 1mm
 needle dipped in bacterial slurry, at the lateral side of the thorax. A
 discrete 16-day generation cycle is followed for population maintenance at
 25C and ~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). 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. Fly
 mortality in each cage was monitored till 96hours. 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 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 next
 generation.  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.<br> 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.  Experimental design
 Experiment (1) Host-pathogen coevolution experiment (Data in Tab
 1 of "Raw_data.xlsx")<br> Host survivorship assay - After
 20 generations of coevolution cycle, evolved changes in Coev and Adapt
 hosts were measured in terms of better survival against coevolving and
 ancestral pathogens.This experiment for each of the four blocks were
 conducted on seperate days. For each block, eggs were collected from the
 standardised flies of each selection regime On 12th day post egg
 collection, flies from each selection regime were divided into three
 treatments- (a) Ancestral Pe treatment: Flies from each selection regime
 were infected with ancestral pathogen, a non-evolving pathogen against
 which Adapt host had evolved; (b) Coevlving Pe treatment: Flies from each
 selection regime were infected with coevolving pathogen of their
 respective block; (c) Sham treatment: Flies from each selection regime
 were pricked with needle MgSO4 solution. For the experiment, 50 males and
 50 females were randomly chosen from each selection regime and for each
 infection treatment. Post infection, flies were transferred into cages and
 mortality was recoded for each cage. For first 48 hours, mortality was
 recorded after 3-4 hours after that, mortality was recorded after 6-8
 hours untill 96 hours.  Experiment (2) Mortality of
 non-local hosts (Data in Tab 2 of "Raw_data.xlsx")<br> To
 measure if the change in coevolving pathogen’s ability to induce host
 mortality, we used two laboratory adapted baseline populations (different
 hosts) that had never experienced bacterial infection-BRB-5 and Canton-S.
 BRB-5 population, is genetically diverse and is related to BRB1-4, having
 been derived from the same ancestral population as BRB 1-4, and had
 remained an independent population for about 160 generations. Canton-S and
 BRB-5 eggs were collected at a density of 70 per vial containing 6-7 ml
 food. On the 12th day post egg collection, flies from each host population
 were divided into six infection treatments- <br> a) Ancestral Pe
 infection treatment, <br> b) infection with coevolving Pe from block
 1,<br> c) infection with coevolving Pe from block 2, <br> d)
 infection with coevolving Pe from block 3, <br> e) infection with
 coevolving Pe from block 4 and, <br> f) sham infection treatment
 using MgSO4. Mortality was recorded for 96 hours. For
 each infection treatment, 50 males and 50 females were randomly chosen
 from each non-local host populations. This experiment was replicated
 twice, on different days. Experiment (3) Selection
 Experiment (Data in Tab 3 of "Raw_data.xlsx")<br> To
 measure the response to selection in Coev hosts and Adapt hosts, selection
 experiments were conducted after coevolution generation 10, 15 and 20.
 Coev and Adapt hosts were infected with ancestral pathogen and their
 survivorship post infection was recorded for 96 hours. This experiment was
 conducted across all four blocks on separate days.
 DATA-SPECIFIC INFORMATION FOR "Raw_data.xlsx"
 This file has four separate tabs. Below, we provide details
 separately for each tab. Tab 1. "Experiment1"
 (Host-pathogen coevolution experiment) 1. Number of
 variables: 6 2. Number of cases/rows: 3200
 3. Variable List:  Time (Time in
 "Hours", upto where mortality readings were taken i.e. 96
 hours)<br> Block (4 independent replicates of each selection regime;
 "1", "2", "3" or "4")<br>
 Selection (3 Selection Regime of the selection line; "Coev",
 "Adapt", "Co.S")<br> Treatment (2 different
 bacterial treatments used to infect flies from each selection regime;
 "Coev Pe", "Anc Pe")<br> Sex (Survivorship of
 "Males" and "Females" from each selection
 regime)<br> Censor (status of the fly at the end of 96 hours
 observation window; "0" score represent mortality and
 "1" score represent number of alive individuals after 96
 hours) Tab 2. "Experiment2" (Mortality of
 non-local hosts) 1. Number of variables: 6
 2. Number of cases/rows: 2000 3. Variable
 List: <br> Time (Time in "Hours", upto where mortality
 readings were taken i.e. 96 hours)<br> Block (2 independent
 replicates conducted on different days; "1" or
 "2")<br> Population (2 different non-local host
 populations; "Canton S" and "BRB 5")<br>
 Bacteria (5 different bacterial treatments used to infect flies from each
 of the non-local host; "Anc Pe", "Block 1 coevolving
 Pe", "Block 2 coevolving Pe", "Block 3 coevolving
 Pe", "B4 coevolving Pe" and "B4 coevolving
 Pe")<br> Sex (Survivorship of "Males" and
 "Females" from each non-local host populations)<br> Censor
 (status of the fly at the end of 96 hours observation window;
 "0" score represent mortality and "1" score represent
 number of alive individuals after 96 hours) Tab 3.
 "Selection experiment" (Experiment 3) 1.
 Number of variables: 7 2. Number of cases/rows:
 97 3. Variable List:  Generation
 (host-pathogen coevolution generations after which selection experiments
 were conducted; "Gen 10", "Gen 15" or "Gen
 20")<br> Block (4 independent replicates of each selection
 regime; "1", "2", "3" or
 "4")<br> Sex ("Male" and "Female" from
 each selection regime)<br> Selection (4 Selection Regime of the
 selection line; "Coev", "Adapt", "Co.S" and
 "Co.U")<br> Dead (Total number of dead individuals till 96
 hours)<br> Alive (Total number of individuals alive after 96
 hours)<br> Proportion Survivorship (Proportion of individuals
 alive) Tab 4. "Notes" This
 contains some notes about the data in the other three tabs.