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.

Maintenance of populations 

Selection Regimes – An experimental set-up was derived from laboratory baseline BRB populations to investigate host-pathogen coevolution system using Drosophila melanogaster insect host and Pseudomonas entomophila 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 P. entomophila pathogen while the flies in Adapt regime experience infection by a non-evolving or ancestral p. entomophila. 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. 

Standardization and generation of experimental flies

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. 

Experimental design

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.

Longevity post-inf after 96h (Data in Tab 2 of "Raw_data.xlsx")

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. 

Prop surv post-inf (Data in Tab 3 of "Raw_data.xlsx")

Proportion of flies that were alive till 96 hours post-infection with ancestral Pe or coevoled Pe.

Longevity sham inf (Data in Tab 4 of "Raw_data.xlsx") 

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.

Basal longevity (Data in Tab 5 of "Raw_data.xlsx")

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.

Starvation resistance (Data in Tab 6 of "Raw_data.xlsx")

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.

Fecundity (Data in Tab 7 of "Raw_data.xlsx")

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. 

Development time (Data in Tab 8 of "Raw_data.xlsx")

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. 

Dry body weight (Data in Tab 9 of "Raw_data.xlsx")

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). 

Lipid content (Data in Tab 10 of "Raw_data.xlsx")

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. 

Coev Pe G15 Growth rate (Data in Tab 11 of "Raw_data.xlsx")

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. 

Coev Pe G20 Growth rate (Data in Tab 12 of "Raw_data.xlsx")

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.