DOI: 10.5061/dryad.jdfn2z394
title: Divergence of responses to variable socio-sexual environments in laboratory populations of Drosophila melanogaster evolving under altered operational sex-ratios

description: Post-copulatory sexual selection (PSS) is an important selective force
 that determines fitness in polyandrous species. PSS can be intense in some
 cases and can drive the evolution of remarkable ejaculate properties. In
 males, investment in ejaculate plays an important role in the outcome of
 PSS. Thus, males are expected to adaptively tailor their ejaculate
 according to the perceived competition in their vicinity. Plastic
 responses in ejaculate investment to variation in intra-sexual competition
 are disparate and widespread in males. We investigated the evolution of
 plasticity in reproductive traits using Drosophila melanogaster
 populations evolving for more than 150 generations under male- or
 female-biased sex ratios. When exposed to different numbers of competitors
 early in their life, males from these two regimes responded differently in
 terms of their copulation duration and sperm competitive ability. In
 addition, the effect of this early life experience wore off at different
 rates in males of male-biased and female-biased regimes with increasing
 time from the removal of competitive cues. Furthermore, our study finds
 that males change their reproductive strategies depending upon the
 identity of rival males. Together, our results provide evidence of the
 evolution of male reproductive investment that depends on socio-sexual
 cues experienced early in life.
description: METHODOLOGICAL INFORMATION Maintenance of
 populations  Baseline population – LHst is a large
 laboratory-adapted population of D. melanogaster that was derived from the
 LH population by introgressing a benign, autosomal recessive scarlet eye
 colour mutation (Nandy et al. 2013a). The maintenance protocol of both LH
 and LHst populations is described elsewhere (Nandy et al. 2012). Briefly,
 both LH and LHst are maintained on a 14-day discrete generation cycle, at
 25⁰C, on standard cornmeal-molasses-yeast food and on a 12 hours: 12hours
 light-dark cycle. Maintenance of LHst and LH population is similar to M,
 and F populations (described below) except that (1) Flies are not
 collected as virgins for LHst and LH populations, (2) Equal sex ratios are
 maintained in LHst and LH populations, i.e. 16 males: 16 females per vial,
 hence a total of 32 flies/vial. <br> Selection Regimes –
 Experimental evolution lines with two different selection regimes –M or
 male biased and F or female-biased were used for all the experiments.
 First, three replicate populations (C1-3) were derived from LHst and
 maintained under equal sex ratio (16 males:16 females) (Nandy et al.
 2013a). After 5 generations, M and F populations (M1-M3 and F1-F3) were
 derived from each of the C populations (C1-C3). Replicate populations
 bearing the same numerical subscript were derived from same ancestral
 populations. For example, M1 and F1 were derived from C1 population, M2
 and F2 from C2 population and similarly, M3 and F3 were derived from C3
 population. Hence populations carrying the same numerical subscript (e.g.,
 M1 and F1) are more closely related to each other than to any other
 population with a different numerical subscript. Also, populations related
 by ancestry (e.g. M1 and F1) are handled on the same day every generation
 during regular maintenance as well as during the assays performed in this
 study. Thus, M1 and F1 populations correspond to block-1, M2 and F2 to
 block-2 and M3 and F3 to block-3, making a total of 3 statistical “blocks”
 for our analyses. <br> Hence, a total of six populations from the
 two selection regimes (the male-biased or M regime and the female-biased
 or F regime), each having 3 replicates:   (M1, M2, M3 and F1, F2, F3) were
 used for this study. Populations belonging to both regimes are maintained
 in an identical manner except that they have different adult sex ratios -
 male: female ratio is 3:1 for the M populations and 1:3 for the F
 populations. A discrete 14- day generation cycle is followed for
 population maintenance at 250C and ~60% relative humidity (RH) and 12:12
 hours light/dark cycle. Flies are provided with standard
 cornmeal–molasses–yeast food in standard vials (90-mm length × 25-mm
 diameter). They are reared at a controlled larval density of 140– 160 per
 vial. Virgin flies are collected each generation and held in single-sex
 vials (eight individuals per vial) till the 12th day after egg collection.
 Therefore, during regular maintenance, the early-life adult densities are
 identical for both M and F selection regimes. On the 12th day, flies are
 then combined in fresh food vials following the respective sex ratio
 regimes i.e. 8 males: 24 females for F regime and 24 males: 8 females for
 M regime, making a total of 32 flies per vial. Every generation, a total
 of 20 such vials are maintained for each population from both male-biased
 and female-biased regimes (hence, making adult population size as
 20×32=640 flies for both M and F regimes). A fixed quantity of live yeast
 per female, i.e. 0.47 mg is provided to flies in food vials. After 2 days,
 flies are transferred to new vials for oviposition. The eggs laid during
 the next 18 hours are used to start the next generation.
 <br> 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 LHst population, i.e. they were not subjected to virgin
 collection and the sex ratio was not altered. Experimental flies were
 generated from these standardized populations.<br> For generating
 experimental flies, we collected eggs for our test males from M and F
 regimes, for females from LHst and for the second male for the sperm
 defense assay from LH, all on the same day. All the experimental flies
 were reared under controlled larval density and standard culture
 conditions (25⁰C, 60–80% RH, 12 hours–12 hours light / dark cycle). Eggs
 were cultured at a density of 150 eggs / vial in 8–10 mL of
 cornmeal-molasses-yeast food for each of the population. On the 9-10th day
 post egg collection, M and F males were collected as virgins by isolating
 them within 6 hours of eclosion, using light CO2 anaesthesia. Soon after
 eclosion, focal males from M and F regimes were assigned to experimental
 treatments. LHst females were collected as virgins and held in single-sex
 vials at a density of 8 females / vial. LH males were collected as adults
 from culture vials on 12th day using CO2 anaesthesia.
 <br> Experimental design Experiment (1)
 Effect of evolutionary history and number of competitors on reproductive
 traits (Data in Tab 1 "Response to coevolved rivals" of
 "Raw_data.xlsx")<br> Mating latency and Copulation
 duration assay - Virgin males were collected from the M and F populations
 and common virgin females were collected from the ancestral population
 LHst. Soon after virgin collection, both M and F males were assigned
 randomly to one of three experimental treatments- (a) 1 male/vial (b) 8
 males/vial (c) 32 males / vial. For each treatment, 30 replicates were
 sampled. Males were kept undisturbed in these treatments for 2 days. After
 2 days, one conditioned male from each treatment was pulled out at random,
 2 hours before the mating trials and kept singly in individual vials.
 Individual virgin LHst females were introduced into these vials containing
 single males. Each pair was observed individually for recording mating
 latency and copulation duration. In all the experiments, flies that did
 not mate within 2 hours were discarded.<br> Sperm defense ability
 assay (Proportion of progeny sired by the 1st male or P1) - After mating
 observations, males from the M and F selection regimes were discarded
 using light CO2 anaesthesia and females were retained. Following this,
 after about 1 hour, LHst females previously mated with M and F males were
 paired with control red-eyed LH males in new vials to remate. These vials
 were left undisturbed for the next 48 hours. Since the second mating of
 females with LH (red-eyed) males was not observed, our measure of P1
 incorporates a composite measure of actual P1 i.e. direct sperm
 competition between the males in the FRT as well as the first male’s
 ability to prevent the female from remating. After 48 hours, LH males were
 discarded and females were transferred individually to test tubes (12 mm ×
 75 mm) containing cornmeal-molasses-yeast food. Females were allowed to
 oviposit in these test tubes for 18 hours after which they were discarded.
 After 12 days, when all the flies had eclosed, progeny from each test tube
 were scored for eye colour to determine paternity.
 Experiment (2) Consequences of changing the identity of rival
 males (Data in Tab 2 "LH rivals" of
 "Raw_data.xlsx")<br> Virgin males were collected from M, F
 and LH populations, whereas common virgin females were collected from the
 ancestral population LHst. After virgin collection, single M/F males were
 assigned to one of three treatments: <br> (a) 1 M/F
 male/vial<br> (b) 1 M/F male + 7 LH males = 8 males/vial<br>
 (c) 1 M/F male + 31 LH males = 32 males/vial<br> Males were kept
 undisturbed in these treatments for 2 days. After 2 days, LH males were
 discarded and individual conditioned M/F males were pulled out using light
 CO2 anaesthesia before the mating assay. Then common virgin LHst females
 were presented to conditioned M/F males for recording mating latency and
 copulation duration. Flies that did not mate within 2 hours of observation
 were discarded. Sperm defense ability (P1) was measured in the same way as
 described previously in experiment (1). Experiment (3)
 Effect of increase in time lag between removal of competitive cues and
 assessment of reproductive traits (Data in Tab 3 "Assayed after 3
 days" and Tab 4 "Assayed after 5 days" of
 "Raw_data.xlsx")<br> <br> For this experiment, we
 collected 2 different sets of males to observe changes in the pattern of
 reproductive investment with an increase in the time between the removal
 of competitive cues and assessment of the reproductive traits. These 2
 sets of males were assayed at 2 different time periods after conditioning
 them with rival males for 2 days (same as experiment 1). Virgin males were
 collected from the M and F populations and common young virgin females
 were collected from the ancestral population LHst for each mating assay.
 Virgin males from each of the 2 sets were assigned randomly to previously
 described 3 kinds of treatments (different number of competitors- 1, 8,
 32) in early life similar to experiment (1). These two sets of males from
 both M and F regimes were kept undisturbed for 2 days. After the
 conditioning period of 2 days, males from both the sets were removed from
 the treatments and were held singly in individual vials.<br>
 Following this, these 2 sets from both M and F regime were assayed at 2
 different time periods i.e; <br> (a) after 3 days of removal of
 cues <br> (b) after 5 days of the removal of cues. <br> Mating
 assays were executed in the same way as described above (experiment 1) for
 assaying mating latency, copulation duration and sperm defense ability
 (P1).
description: This file ("Raw_data.xlsx") was generated in 2016 and
 2017 by Komal Maggu. <br> GENERAL
 INFORMATION 1. Title of Dataset: "Divergence of
 responses to variable socio-sexual environments in laboratory populations
 of Drosophila melanogaster evolving under altered operational
 sex-ratios." 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: Komal Maggu<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: komal024maggu@gmail.com
 <br> 3. Duration of data collection: 2016-2017
 4. Geographic location of data collection: Mohali, Punjab,
 India  5. Information about funding sources that
 supported the collection of the data: IISER Mohali, University Grants
 Commission, Govt. of India. <br>
 #######SHARING/ACCESS INFORMATION<br>  <br> Links to
 publications that cite or use the data:  Manuscript id:
 20-0494.R1<br> Manuscript title: Divergence of responses to variable
 socio-sexual environments in laboratory populations of Drosophila
 melanogaster evolving under altered operational sex –ratios.
 <br> 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.
 "Response to coevolved rivals" (Data for Experiment 1)<br>
 Tab 2. "LH rivals" (Data for Experiment 2)<br> Tab 3.
 "Assayed after 3 days" (Data for Experiment 3)<br> Tab 4.
 "Assayed after 5 days" (Data for Experiment 3)
 <br> DATA-SPECIFIC INFORMATION FOR
 "Raw_data.xlsx" This file has four separate
 tabs. Below, we provide details separately for each tab.
 Tab 1. "Response to coevolved rivals" (Experiment
 1) 1. Number of variables: 6 2.
 Number of cases/rows: 360 3. Variable List: 
 Selection (Selection regime; male-biased "M" or
 female-biased "F")<br> Block (3 independent replicates of
 each selection regime; "1", "2" or
 "3")<br> Treatment (Number of early-life competitors;
 "1", "8" or "32")<br> Mating Latency
 (Time taken by focal males to begin copulating with a virgin female in
 minutes)<br> Copulation Duration (Time spent by focal males in
 copula with virgin females in minutes)<br> Sperm Defense Ability
 (P1) (Proportion of progeny sired by focal males) Tab
 2. "LH rivals" (Experiment 2) 1. Number of
 variables: 6 2. Number of cases/rows: 540
 3. Variable List:  Selection (Selection
 regime; male-biased "M" or female-biased
 "F")<br> Block (3 independent replicates of each selection
 regime; "1", "2" or "3")<br> Treatment
 (Number of early-life competitors; "1", "8" or
 "32")<br> Mating Latency (Time taken by focal males to
 begin copulating with a virgin female in minutes)<br> Copulation
 Duration (Time spent by focal males in copula with virgin females in
 minutes)<br> Sperm Defense Ability (P1) (Proportion of progeny sired
 by focal males) Tab 3. "Assayed after 3 days"
 (Experiment 3) 1. Number of variables: 6
 2. Number of cases/rows: 359 3. Variable
 List:  Selection (Selection regime; male-biased
 "M" or female-biased "F")<br> Block (3
 independent replicates of each selection regime; "1",
 "2" or "3")<br> Treatment (Number of early-life
 competitors; "1", "8" or "32")<br>
 Mating Latency (Time taken by focal males to begin copulating with a
 virgin female in minutes)<br> Copulation Duration (Time spent by
 focal males in copula with virgin females in minutes)<br> Sperm
 Defense Ability (P1) (Proportion of progeny sired by focal
 males) Tab 4. "Assayed after 5 days"
 (Experiment 4) 1. Number of variables: 6
 2. Number of cases/rows: 359 3. Variable
 List:  Selection (Selection regime; male-biased
 "M" or female-biased "F")<br> Block (3
 independent replicates of each selection regime; "1",
 "2" or "3")<br> Treatment (Number of early-life
 competitors; "1", "8" or "32")<br>
 Mating Latency (Time taken by focal males to begin copulating with a
 virgin female in minutes)<br> Copulation Duration (Time spent by
 focal males in copula with virgin females in minutes)<br> Sperm
 Defense Ability (P1) (Proportion of progeny sired by focal
 males) <br>