DOI: 10.5061/dryad.rjdfn2zp3
title: Data from: Winner-loser effects on life history traits

description: Ageing of adult males could be accelerated by both high
 mating/reproductive effort and fighting for mates. Testing the relative
 importance of these factors is challenging, however, because males that
 win fights also tend to have more mates. We used a 2 x 2 experimental
 design to test how a prolonged (9 week) period of either winning or losing
 fights, and either high or low reproductive effort (manipulating by
 varying access to females) interact to affect male ageing and future
 reproduction allocation in the mosquitofish, Gambusia holbrooki. We
 measured telomere length and several life-history traits, including mating
 effort and ejaculates (sperm count and velocity). After 9 weeks there were
 significant differences between winners and losers in their mating effort,
 but not in their ejaculates. Males with a higher past reproductive effort
 (i.e. access to females) had significantly lower current mating effort and
 grew more slowly. Males with a higher past reproductive effort also had
 slower swimming sperm, but only if they were smaller than average in body
 size. Surprisingly, neither males with a higher past reproductive effort
 nor males that repeatedly lost fights had shorter telomeres. Our findings
 show that past social dynamics affect how males allocate resources to
 reproduction and somatic maintenance.
description: <em>Creating winners and losers </em>
 We randomly selected focal males to assign to experimental
 treatments and followed them individually throughout the study
 (<em>n</em> = 176). Focal males were anaesthetized briefly to
 measure their standard body length (SL) and then marked with a
 subcutaneous elastomer tag (NorthWest Marine Technology, WA, USA). After
 one week of isolation, focal males were randomly assigned to be winners or
 losers by being paired with either a smaller or larger rival. This method
 controls for intrinsic differences in fighting ability between males that
 might otherwise determine contest outcomes or affect investment into
 reproduction and somatic maintenance. Contests took place in 6 L aquaria
 that contained gravel, plastic plants for refugia, and had black plastic
 on three sides to minimize disturbance. Focal and rival males freely
 interacted for one week, after which a female was introduced to the tank
 to apply the reproductive treatment for another 8 weeks. In half the
 tanks, the two males could freely interact with the female and therefore
 the focal male could fully invest in reproduction (i.e. chase the female
 and mate). In the other half of the tanks there was also a female present,
 but she was kept behind a mesh barrier to prevent the males from mating
 with her. Equal numbers of winner and loser focal males therefore either
 had full access to a female (‘contests and mating’) or only interacted
 with a female through the barrier (‘contest only’) (NB: ‘contest’ refers
 to the fact that all males competed with a rival for the full 9 weeks).
 Rival males and stimulus females were rotated every 3-5 days so that focal
 males continued to fight to establish dominance, and to approach
 females <em>Male mating
 behaviour</em> After 9 weeks, each focal male was
 placed in a new 6 L aquaria with a random stock female. Each female was
 used once. Male mating behaviour was observed for 20 mins. We recorded:
 (a) time spent near the female (&lt;5 cm and facing her); (b) the
 number of mating attempts; and (c) the number of successful attempts
 (i.e., those with the potential to transfer sperm).
 <em>Ejaculates</em> Immediately
 following the mating behaviour trials, focal males were anaesthetised in
 ice slurry to measure their body length (SL) and to strip their sperm.
 Males were then isolated for 5 days in 1 L aquaria to replenish their
 sperm reserves, after which we again stripped them. We had two ejaculate
 measures: total sperm count and sperm velocity. For
 sperm counts, we vortexed the sample to disperse sperm then pipetted 3 µL
 onto a 20 µm capillary slide (Leja), and used a CEROS Sperm Tracker
 (Hamilton Thorne Research, Beverly, MA, USA) to count sperm under x100
 magnification. We randomly counted five subsamples per sample. We
 estimated total sperm counts by adding the average sperm number per bundle
 to account for the six bundles removed for sperm velocity
 analyses. To measure sperm velocity, we took two
 samples of three sperm bundles from each male’s ejaculate and pipetted the
 bundles into two separate PCR tubes containing 2 µL extender medium. We
 then pipetted each sample onto a cell of a 12-cell multi-test slide (MP
 Biomedicals, Aurora, OH, USA) coated with 1% polyvinyl alcohol solution
 (PVA). Sperm was ‘activated’ with 3 µL of solution (125 mM KCL and 2 mg/mL
 bovine serum albumin). We used a CEROS Sperm Tracker to record two
 measures of sperm velocity: VAP (average path velocity) and VCL
 (curvilinear velocity). <em>Relative telomere
 length</em> Relative telomere length (rTL) was
 measured using real-time quantitative PCR, determined as the ratio (T/S)
 of telomere repeat length (T) to a single-copy reference gene length (S).
 We used standard telomere primers Tel1b
 (5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′) and Tel2b
 (5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′) and a
 <em>Gambusia</em>-specific region of the melanocortin 1
 receptor (MC1R) as our control single-copy reference gene with the primers
 MC1R.F (5’-CCTGTAGGCGTAGATGAGCG-3’) and MC1R.R
 (5’-CACCAGTCCCTTCTGCAACT-3’).  We ran qPCRs for each
 sample in triplicate on 96-well plates. Telomere and MC1R amplifications
 were run concurrently on separate plates using QuantStudio3 (Thermo Fisher
 Scientific, Waltham, USA). Each plate had three negative controls (9 µL
 reagent mix and 1 µL MilliQ purified water), two inter-plate control
 samples (run in triplicate, the same two individuals across all plates),
 and a golden sample at five DNA concentrations (0.05, 0.2, 1, 5 and 20
 ng/µL) to generate the standard curve and determine the amplification
 efficiency of each plate (telomere: 1.99-2.11; MC1R:
 1.93-2.01). 
description: # Winner-loser effects on life history traits
 [https://doi.org/10.5061/dryad.rjdfn2zp3](https://doi.org/10.5061/dryad.rjdfn2zp3) ## Description of the data and file structure There are 5 datasets (.csv files) used for analyses. The first dataset **mating_effort** is the main dataset used for analysis. Datasets 2-5 have been provided for transparency in our data collection. Dataset 2 **qpcr** is the raw output generated from qPCR used to generate data for telomere length determination. Dataset 3 **counts** is the dataset used to measure the repeatability of the 5 sperm count subsamples for each male. Dataset 4 **vcl_rep** is the data used to determine the repeatability of sperm velocity (vcl) for each male from two sperm samples. Dataset 5 **survival** is the dataset used to measure survival/mortality during the experimental period. All the missing data represented as NA. ### Files and variables #### File: data.xlsx **Description:** complete datasheet containing all the datasets (5 in total) used for analyses and retaining their names as used in the provided R code (submitted to Zenodo) ##### Variables Metadata (format - Column name: Description) **Dataset 1: mating_effort**          This is the main dataset used for analyses (see provided code file Rcode.r)         Row_ID: Unique row identification Male_ID: Unique male identification Block_ID: Block number used as random effect in models Date_Tagged: Date focal male was tagged with elastomer tag (see Methods) Tag_Colour: Colour of tag - recorded to check for potential behavioural biases Start_size_mm: Male standard length (SL) recorded when male was tagged using calipers Experience: Contest experience treatment (Winner or Loser) Treatment: Reproductive investment treatment (Contests Only or Contests + Reproduction) Start_date_social: Start date for initial contests - week before reproductive investment treatment began Start_date_treatment: Start date for reproductive investment treatment (week following contest treatment start - went for 8 weeks) End_date_treatment: End date for experimental treatments - date of behavioural data recording in mating assay End_size_mm : Male standard length recorded at the end of experimental period Male_growth: Life history trait 1 - Calculated as end size - start size Absolute_time_female_sec: Behavioural trait 1 - the total time male spent associating with female during mating assay Mating_attempts: Behavioural trait 2 - the total number of mating attempts (see Methods) made during mating assay Successful_attempts: Behavioural trait 3 - the total number of mating attempts that were successful (transfer spem) during mating assay Female_size_mm: Female standard length recorded at the end of mating assay Total_sperm: Ejaculate trait 1 - total number of sperm counted (average of 5 counts from 1 sample) from replenished sperm Weighted_avg_VAP: Ejaculate trait 2 - weighted average sperm velocity VAP (average path velocity) (see methods) from replenished sperm Weighted_avg_VCL: Ejaculate trait 3 - weighted average sperm velocity (curvilinear) VCL (see Methods) from replenished sperm Weighted_avg_VSL: Ejaculate trait 4 - weighted average sperm velocity (straight line) VSL (see Methods) from replenished sperm qPCR_Run_Number: Run number for qPCR qPCR_Date: Date qPCR for male sample was run Telomere_Cq1: Cq value from qPCR for telomeres - sample 1 MC1R_Cq1: Cq value from qPCR for MC1R reference gene - sample 1 Telomere_Cq2: Cq value from qPCR for telomeres - sample 2 MC1R_Cq2: Cq value from qPCR for MC1R reference gene - sample 2 Telomere_Cq3: Cq value from qPCR for telomeres - sample 2 MC1R_Cq3: Cq value from qPCR for MC1R reference gene - sample 2 rTL: Average relative telomere length averaged across the three samples for each male rTL_1: relative telomere length calculated from sample 1 rTL_2: relative telomere length calculated from sample 2 rTL_3: relative telomere length calculated from sample 3                      **Dataset 2: qpcr**               This is the dataset generated from qPCR output used to generate data for telomere length determination    Row_ID: Unique row identification Male_ID: Unique male identification qPCR_Run_Number: Run number for qPCR qPCR_Date: Date qPCR for male sample was run Well_1: Well number for 1st sample Well_Position1: Plate position for 1st sample Telomere_Cq1: Cq value from qPCR for telomeres - sample 1 MC1R_Cq1: Cq value from qPCR for MC1R reference gene - sample 1 Well_2 : Well number for 2nd sample Well_Position2: Plate position for 2nd sample Telomere_Cq2: Cq value from qPCR for telomeres - sample 2 MC1R_Cq2: Cq value from qPCR for MC1R reference gene - sample 2 Well_3: Well number for 3rd sample Well_Position3: Plate position for 3rd sample Telomere_Cq3: Cq value from qPCR for telomeres - sample 2 MC1R_Cq3: Cq value from qPCR for MC1R reference gene - sample 2 Tel_Interplate_ControlS1_1: Telomere control cq - golden sample used across plates - sample 1 run 1 (run in triplicate) Tel_Interplate_ControlS1_2: Telomere control cq - golden sample used across plates - sample 1 run 2 (run in triplicate) Tel_Interplate_ControlS1_3: Telomere control cq - golden sample used across plates - sample 1 run 3 (run in triplicate) Tel_Interplate_ControlS2_1: Telomere control cq - golden sample used across plates - sample 2 run 1 (run in triplicate) Tel_Interplate_ControlS2_2: Telomere control cq - golden sample used across plates - sample 2 run 2 (run in triplicate) Tel_Interplate_ControlS2_3: Telomere control cq - golden sample used across plates - sample 2 run 3 (run in triplicate) MC1R_Interplate_ControlS1_1: MC1R control cq - golden sample used across plates - sample 1 run 1 (run in triplicate) MC1R_Interplate_ControlS1_2: MC1R control cq - golden sample used across plates - sample 1 run 2 (run in triplicate) MC1R_Interplate_ControlS1_3: MC1R control cq - golden sample used across plates - sample 1 run 3 (run in triplicate) MC1R_Interplate_ControlS2_1: MC1R control cq - golden sample used across plates - sample 2 run 1 (run in triplicate) MC1R_Interplate_ControlS2_2: MC1R control cq - golden sample used across plates - sample 2 run 2 (run in triplicate) MC1R_Interplate_ControlS2_3: MC1R control cq - golden sample used across plates - sample 2 run 3 (run in triplicate) Amplification_Efficiency_Telomere: Amplification efficiency for telomere Amplification_Efficiency_MC1R: Amplification efficiency for MC1R reference gene rTL: Average relative telomere length averaged across the three samples for each male AVG_TEL_CONTROL: Average Cq for the telomere controls (see Methods) AVG_MC1R_CONTROL: Average Cq for the MC1R controls (see Methods) rTL_1: relative telomere length calculated from sample 1 rTL_2: relative telomere length calculated from sample 2 rTL_3: relative telomere length calculated from sample 3 **Dataset 3: counts**         This is the dataset used to measure the repeatability of the 5 sperm count subsamples for each male            Male_ID: Unique male identification total_C1: sperm count subsample 1 total_C2: sperm count subsample 2 total_C3: sperm count subsample 3 total_C4: sperm count subsample 4 total_C5: sperm count subsample 5 **Dataset 4: vcl_rep**        *This is the data used to determine the repeatability of sperm velocity (vcl) for each male from two sperm samples. There are no raw videos associated with these data.*                 Male_ID: Unique male identification mean_vcl_1: mean sperm velocity (vcl) from sample 1 mean_vcl_2: mean sperm velocity (vcl) from sample 2 **Dataset 5: survival**       This is the dataset used to measure survival/mortality during the experimental period Male_ID: Unique male identification Block_ID: Block number used as random effect in models Start_date_social: Start date for initial contests - week before reproductive investment treatment began Tag_Colour: Colour of tag - recorded to check for potential behavioural biases Start_size_mm: Male standard length (SL) recorded when male was tagged using calipers Experience: Contest experience treatment (Winner or Loser) Treatment: Reproductive investment treatment (Contests Only or Contests + Reproduction) Time: Days alive Survival: Death = 1, Alive = 0 Data notes: Missing values recorded as NA ## Code/software *For data analysis:* R (version 4.0.2) ## Access information Other publicly accessible locations of the data: * Open Science Foundation study pre-registration ([https://osf.io/saj46/](https://osf.io/saj46/))  Data was derived from the following sources: * NA