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. 

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
    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

    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

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
 
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" 
Tab 2. "Experiment2" 
Tab 3. "Selection experiment"
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.
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")
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")
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- 
a) Ancestral Pe infection treatment, 
b) infection with coevolving Pe from block 1,
c) infection with coevolving Pe from block 2, 
d) infection with coevolving Pe from block 3, 
e) infection with coevolving Pe from block 4 and, 
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")
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)
Block (4 independent replicates of each selection regime; "1", "2", "3" or "4")
Selection (3 Selection Regime of the selection line; "Coev", "Adapt", "Co.S")
Treatment (2 different bacterial treatments used to infect flies from each selection regime; "Coev Pe", "Anc Pe")
Sex (Survivorship of "Males" and "Females" from each selection regime)
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: 
Time (Time in "Hours", upto where mortality readings were taken i.e. 96 hours)
Block (2 independent replicates conducted on different days; "1" or "2")
Population (2 different non-local host populations; "Canton S" and "BRB 5")
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")
Sex (Survivorship of "Males" and "Females" from each non-local host populations)
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")
Block (4 independent replicates of each selection regime; "1", "2", "3" or "4")
Sex ("Male" and "Female" from each selection regime)
Selection (4 Selection Regime of the selection line; "Coev", "Adapt", "Co.S" and "Co.U")
Dead (Total number of dead individuals till 96 hours)
Alive (Total number of individuals alive after 96 hours)
Proportion Survivorship (Proportion of individuals alive)

Tab 4. "Notes"

This contains some notes about the data in the other three tabs.