DOI: 10.5061/dryad.fttdz08vn
title: Data for: The effect of brief or prolonged bouts of winning or losing male-male contests on plasticity in sexually selected traits

description: Fight outcomes often affect male fitness by determining their access to
 mates. Thus ‘winner-loser’ effects, where winners often win their next
 contest, while losers tend to lose, can influence how males allocate
 resources towards pre- and post-copulatory traits. We experimentally
 manipulated the winning/losing experiences of pairs of size-matched male
 Gambusia holbrooki for either a day, a week or three weeks to test whether
 prior winning/losing experiences differentially affect the plasticity of
 male investment into either mating effort (pre-copulatory) or ejaculates
 (post-copulatory). When winner/loser pairs directly competed for a female,
 winners had better pre-copulatory outcomes than losers for three of the
 four traits we measured: mating attempts, successful attempts, and time
 spent with the female (but not aggression). However, winners and losers
 did not differ in either their total sperm counts nor sperm velocity.
 Interestingly, absolute male size, an important predictor of fighting
 success, mediated winner-loser effects on how long males then spent near a
 female. Compared to losers, smaller winners spent more time with the
 female than did larger winners, suggesting that how males respond to prior
 social experiences is size-dependent. We discuss the general importance of
 controlling for inherent male condition when comparing male investment
 into condition-dependent traits.
description: We experimentally manipulated the contest experiences (winning or
 losing) of males for either 1 day, 1 week, or 3 weeks. Winners were made
 to face smaller rivals while losers faced larger rivals continuously
 throughout their contest duration period. We then measured a set of key
 traits on focal males that are under pre- or post-copulatory sexual
 selection. We experimentally created winners and losers by randomly
 assigning size-matched focal male pairs to compete against either a
 smaller (winners) or larger (losers) competitor male. Winning/losing
 experiences were staggered such that each contest experience treatment
 ended on the same day for a given block of males. Contest experiences were
 broken up into 20 blocks to measure pre-copulatory investment and 21
 blocks to measure post-copulatory investment. In each winning/losing trial
 a focal and a competitor male interacted freely in a 6 L aquarium with a
 stimulus female (randomly chosen from the stock population) present behind
 a mesh barrier to encourage agonistic interactions but prevent mating.
 Competitor males were rotated every ~3 days to ensure that focal males
 were continually winning/losing contests, while stimulus females were
 rotated every seven days to keep males motivated to fight. At the end of
 their contest experience winners and losers from the same contest duration
 treatment were randomly assigned to either compete directly for a female
 to measure pre-copulatory investment or to have their sperm traits
 measured (post-copulatory investment).
 <em>Pre-copulatory investment</em>
 To compare male investment into pre-copulatory mating behaviours,
 we placed size-matched focal male pairs (winner and loser from the same
 contest duration treatment) together in a new, 6 L aquarium with a stock
 female. All females were only used once. Male interactions were observed
 for 20 mins where we recorded: a) time spent near the female, b) number of
 mating attempts, c) number of successful mating attempts, and d)
 aggression directed towards the rival. Mating attempts were recorded each
 time a male swung his gonopodium forwards towards the female’s gonopore.
 These mating attempts are unambiguous and easy to quantify. Successful
 mating attempts were recorded when the gonopodium touched the gonopore,
 potentially transferring spermatophores. Successful mating attempts
 involve the male twisting his body and the female attempting to roll away
 from him. We used stopwatches to record the time each male spent within ~5
 cm of the female (interacting with or guarding her from rival approaches).
 Finally, aggression was recorded as how often the male displayed
 aggressively, nipped, or chased his rival. 
 <em>Post-copulatory investment</em>
 To compare male investment into post-copulatory traits, focal
 males were isolated and stripped of their sperm to determine how their
 sperm reserves were affected by winning or losing. They were then stripped
 again seven days later to measure the effect of winning/losing on rates of
 sperm replenishment or sperm traits. Sperm collected immediately
 post-treatment provided baseline measures of the number and velocity of
 sperm produced by males prior to or during the contest treatment, while
 replenished sperm are presumably directly influenced by the male’s contest
 experience. As such, we expected a quantifiable difference between the two
 measures. We measured three key indicators of ejaculate quality: sperm
 count, sperm velocity (swimming speed) and sperm replenishment rates
 (comparing current and baseline counts). a) <em>
    Sperm collection</em> At the end of their
 contest experiences, focal males were anaesthetised briefly in ice slurry
 and sperm bundles were then stripped by gently massaging the ventral area
 directly above the base of the gonopodium. This process removes most
 sperm, while a seven-day period thereafter allows males enough time to
 replenish sperm reserves to measure sperm replenishment rates. Two samples
 of three sperm bundles each were collected and set aside for sperm
 velocity analysis. The remaining bundles were pipetted into an Eppendorf
 tube containing 100-1100 µL of extender medium (pH 7.5 with composition:
 207 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 0.49 mM MgCl2, 0.41 mM MgSO4, 10 mM
 Tris (Cl)) to count sperm. Sperm collection and subsequent trait
 measurements were performed blind to male contest treatment.
 b)    <em> Sperm count</em>      
       To estimate total sperm count we vortexed the sperm sample for ~1
 min and then repeatedly pipetted the solution (10-20 times) to break up
 sperm bundles and disperse sperm throughout the sample. We pipetted 3 µL
 of the mixed sperm solution onto a 20-micron capillary slide (Leja) and
 counted sperm using a CEROS Sperm Tracker (Hamilton Thorne Research,
 Beverly, MA, USA) under x100 magnification. Threshold values defining cell
 detection were predetermined as elongation percentage 15-65 and head size
 5-15 µm (static tail filter set off). For sperm counts, we randomly
 counted five subsamples per sample and used the average. The repeatability
 of our count subsamples for each male was obtained using the R package
 <em>rpt</em>R. We then obtained the total sperm counts by
 adding the average sperm number per bundle for the six bundles removed for
 sperm velocity analyses. We measured the total sperm count of 205 males on
 Day 0 (baseline) and 220 males on Day 7 post-treatment (replenished);
 hereafter referred to as baseline and replenished sperm,
 respectively. c)      <em>Sperm
 velocity</em>             To measure sperm
 velocity, we used two samples from each male’s ejaculate (3 sperm bundles
 each in 3 µL of extender medium). We then pipetted each sample onto the
 centre of a cell of a 12-cell multi-test slide (MP Biomedicals, Aurora,
 OH, USA) previously coated with 1% polyvinyl alcohol solution (PVA) to
 prevent sperm from sticking to the slide. Each sample was then ‘activated’
 with 3 µL of activator solution (125 mM KCL and 2 mg/mL bovine serum
 albumin) to mimic the chemical environment of the reproductive tract of
 female <em>G. holbrooki</em> and covered with a coverslip. We
 recorded two standard measures of sperm velocity – VAP (average path
 velocity) and VCL (curvilinear velocity) using a CEROS Sperm Tracker.
 Threshold values for defining static cells was predetermined at 20 µm/s
 for VAP and 15 µm/s for VCL. We used VCL for our analysis because it is a
 more biologically relevant measure. Sperm velocity measures were obtained
 from 182 males for baseline sperm and 190 males for replenished
 sperm. Please see the Methods section of the associated
 manuscript for more details on experimental design and data
 collection. 
description: <strong>Metadata:</strong>
 <em>Sheet 1 - mating_behav</em>
 Male_ID: unique male identification  Tag_ID:
 tag colour and location Male_size_pre: male body length
 (standard length; mm) prior to starting experimental treatment
 Block_ID: block number for a given group of males and used as a
 random effect in all models Contest_treatment: Winner
 or Loser Treatment_length: 1 day (D1), 1 week (WK1) and
 3 weeks (WK3) Trial_date: Date males had their mating
 effort measured Pair_ID: identifier for a pair of
 competing males (size-matched winner and loser from same treatment length
 treatment) Female_size: Female SL (mm)
 Male_size_post: Male SL at the end of the treatment; mm (measured
 after mating trials) Total_attempts: Total number of
 mating attempts (see methods of paper for quantification)
 Total_successes: total number of successful mating attempts
 (attempts where sperm could be transferred)
 Absolute_aggression_rival: number of times male was aggressive to
 rival male (see methods of paper for quantification)
 Absolute_time_female_total: seconds; time male spent associating
 with the female during mating trials **NOTE: NA for a
 pair of males for absolute_time_female_total due to equipment recording
 failure. Issue affects no other trait or male**
   <em>Sheet 2 -
 sperm</em> Male_ID: unique male
 identification  Tag_ID: tag colour and
 location Block_ID: block number for a given group of
 males and used as a random effect in all models
 Male_size_pre: male body length (standard length; mm) prior to
 starting experimental treatment Contest_treatment:
 Winner or Loser Treatment_length: 1 day (D1), 1 week
 (WK1) and 3 weeks (WK3) Date_measured: date males had
 their sperm stripped and measured Day_post_treatment:
 Day 0 and Day 7; refers to baseline (Day 0) or replenished (Day 7) sperm
 measures Male_size_post: male body length (standard
 length; mm) at the end of experiments (taken on Day 0 and again on Day
 7) Total_sperm_count: total number of sperm. Average of
 5 subsamples from each male multiplied by the dilution of the sperm sample
 and the number of sperm lost to velocity measures (average number of sperm
 within 6 sperm bundles) Weighted_avg_VAP: sperm
 velocity VAP. The weighted average swimming performance of 2 sperm samples
 (3 bundles each; 6 total) from each male (mean of each sample x number of
 tracks)/ total tracks Weighted_avg_VCL: sperm velocity
 VCL. The weighted average swimming performance of 2 sperm samples (3
 bundles each; 6 total) from each male (mean of each sample x number of
 tracks)/ total tracks **NOTE: Some cells contain
 "NA" where we were unable to obtain any sperm (NA for all sperm
 measures), or there was not enough sperm to obtain a measure of velocity
 (NA for velocity but not for count), or sperm were not moving (NA for
 velocity). These cells containing "NA" were not used for further
 analysis.**    <em>Sheet 3 -
 counts</em> male_ID: unique male
 identification measurement_day: Day 0 or Day 7
 (baseline or replenished sperm measurements) total_c1:
 sperm counts subsample 1 total_c2: sperm counts
 subsample 2 total_c3: sperm counts subsample
 3 total_c4: sperm counts subsample 4
 total_c5: sperm counts subsample 5