DOI: 10.5061/dryad.8cz8w9gsx
title: Mating status affects female choice in Chloridea virescens

description: Sexual selection in animals has been mostly studied in species in which
 males are signallers and females are choosers. However, in many species
 females are (also) signallers. In species with non-signalling females,
 virgin females are hypothesized to be less choosy than mated females, as
 virgins must mate to realize fitness and the number of available males is
 generally limited. Yet, when females signal to attract males, mate
 limitation can be overcome. We tested how virgin and mated females differ
 in their calling behaviour, mating latency, and in mate choice, using the
 tobacco budworm Chloridea (Heliothis) virescens as an example for a
 species in which females are not only choosers but also signallers. We
 found that virgin females signalled longer than mated females, but virgin
 and mated signalling females were equally ready to mate, in contrast to
 non-signalling females. However, we found that virgin signalling females
 showed weaker mate preference than mated females, which can be explained
 by the fact that females increase their fitness with multiple matings.
 Mated females may thus further increase their fitness by more stringent
 mate selection. We conclude that signalling is a crucial aspect to
 consider when studying female mate choice, because signalling may affect
 the number of available mates to choose from.
description: excerpt from Zweerus, N. L., van Wijk, M., Smallegange, I. M.,
 & Groot, A. T. (2022). Mating status affects female choice when
 females are signalers. Ecology and Evolution, 12, e8864
 Study organism <em>Chloridea
 </em>(<em>Heliothis</em>) <em>virescens
 </em>populations<em> </em>originated from North Carolina
 State University (YDK strain) and the Max Planck Institute for Chemical
 Ecology, Jena, and have been reared at the Institute for Biodiversity and
 Ecosystem and Dynamics (IBED), University of Amsterdam since 2011. We
 conducted the experiment for this study between April 2018 and November
 2019. The moths were kept in a climate chamber at 60% relative humidity
 and 25 ± 1°C, with a 14 h light (photophase): 10 h dark (scotophase)
 photoperiod (lights off at 11 a.m. CET). Larvae were reared on artificial
 pinto bean diet (Burton, 1970) in individual plastic cups (37 ml, Solo,
 Lake Forest, Illinois). Pupae were checked daily for eclosion (i.e.,
 hatching of adult) and emerged adults were fed 10 % sucrose solution
 provided through 1 cm cotton dental wick. All experiments were conducted
 with 2–3-day old non-sibling individuals and were conducted under the same
 environmental conditions as those used for rearing.
 Procedure to obtain mated females and maintaining virgin
 females To obtain mated females, we placed an adult
 female (first day after eclosion) in a large clear plastic cup (473 ml,
 Solo) with an adult male that was randomly chosen from the adults of the
 standard rearing. At the same time, for the ‘virgin group’ females were
 also selected on the first day after eclosion and individually isolated.
 We next observed all pairs at 30 min intervals during the scotophase (dark
 period) until all pairs were mating, or until 9 hours of the scotophase
 had passed. Because <em>C. virescens</em> matings last for
 several hours, only females that mated for ≥ 60 min qualified for the
 ‘mated group’ of the experiment. In the photophase that followed the
 scotophase (i.e., light period on day 2), we individually isolated the
 mated females. We conducted the experiments on the third day after
 eclosion, under the same environmental conditions as the rearing and the
 preparatory matings. Procedure to obtain an extended
 range of male body sizes Previously we found that male
 body size affects female fitness: females mating with larger males had
 more offspring than when mating with smaller males (Zweerus et al., 2021).
 To assess female preference for males of different body sizes (a proxy for
 quality), we increased the range of male sizes by altering the larval
 diet, following Zweerus et al. (2021). Briefly, we obtained males of
 average to large size by rearing larvae on a standard pinto bean diet
 (Burton, 1970), and obtained males of smaller than average size by rearing
 larvae on diet which nutritional value was 25% of the standard diet. It is
 important to note that even on standard diet, male mass varies. The
 low-nutritional diet extended this ‘natural’ range of masses at the lower
 end (see Zweerus et al., 2021, Fig. S1). The diet treatment was thus a
 tool to increase the range of male masses without being a factor in the
 experimental design. For the experiments, we did not use males from one or
 the other diet, but rather males that differed in pupal mass.
 Mate-attraction effort experiment To compare
 mate attraction between virgin and mated females using their signalling
 behaviour as a proxy of mate attraction effort, we quantified the
 signalling activity of all females (virgin: n = 24 and mated: n = 21) in
 the third scotophase after eclosion as follows. In the hour prior to the
 scotophase, we placed single females into large clear plastic cups (473
 ml, Solo). From the onset of scotophase, we observed and scored the number
 of females signalling every 15 minutes. We stopped observing an individual
 if the female did not signal for at least 1 hour. To test if the
 proportion of signalling females differed between the virgin and mated
 group, we used a Chi-square test for independence. To assess if virgin
 females (n = 21) started signalling later than mated females (n = 17), we
 analyzed the normally-distributed data with equal variances for the onset
 time of signalling with a two-tailed t-test. To test if the duration of
 signalling differed between virgin and mated females, we used a
 Mann-Whitney-U-test, because the data were non-normally distributed
 (assessed by Shapiro-Wilk test and visual exploration of histograms). We
 visualized the data by fitting the proportion of signalling females per
 time point and mating status over the scotophase using the package ggplot2
 (Wickham, 2016) in the software R (version 4.0.5, R Core Team
 (2021)). No-choice experiment to assess readiness to
 mate in virgin and mated females To test the
 hypothesis that virgin females have a shorter mating latency (as a proxy
 of readiness to mate) than mated females, we measured their mating latency
 (the time from the pairing until copulation) in no-choice mating assays.
 Firstly, we placed one female with one male into a clear plastic cup (473
 ml, Solo) and covered the cup with a mesh. We then mounted each plastic
 cup in a hanging grid with a camera (GoPro Hero silver) underneath. The
 assay started at the beginning of the scotophase, after which we recorded
 a time-lapse series of 1pic / min. We collected the data on minimally 4
 and maximally 20 samples per time over 8 scotophases between
 11<sup>th</sup> and 20<sup>th</sup> of May 2018.
 To determine mating latency, we identified the timestamp of the picture
 showing a newly-formed mating pair in the time-lapse series. For
 individuals that did not mate, we censored their data by assigning a
 maximum time span of 600 min, which corresponds to an entire scotophase.
 To test if virgin females (n = 61) mated significantly earlier than mated
 females (n = 29), and whether male pupal mass or female pupal mass
 affected mating latency, we fitted a Cox proportional-hazards model with
 the explanatory variables <em>mating status</em>,
 <em>male pupal mass</em>, and <em>female pupal
 mass</em> as main effects and mating latency as the response
 variable in R using the packages survival (Therneau, 2021), and survminer
 (Kassambara et al., 2019). Two-choice experiment to
 compare virgin and mated female mate preferences To
 test the hypothesis that virgin females show a weaker preference for
 larger (i.e., higher-quality) males than mated females, we conducted
 two-choice tests, in which we placed one female (virgin: n =  61 or mated:
 n = 63) together with a larger and a smaller male into a BugDorm cage (H30
 cm x W30 cm x D30 cm), and scored which male a female mated. To
 distinguish between the two males, we marked each male by clipping the tip
 of one randomly chosen wing. The experiment started 10 min before the
 onset of the scotophase and we checked all cages at 30 min intervals. Once
 a mating pair had formed, we removed the unmated male from the cage. To
 check if the matings were successful, we isolated and froze all
 individuals in the next photophase, dissected the females, and quantified
 the number of spermatophores per individual. We collected the data over a
 total of 13 scotophases between 13<sup>th</sup> Augustus and
 8<sup>th</sup>November 2019. Since not all females mated in
 the two-choice assay (virgin: n = 56, mated: n = 45), we first tested for
 an association between mating status (virgin, mated) and mating occurrence
 (mating, no mating) with a Fisher’s exact test. Additionally, we confirmed
 that the mass range of larger and smaller males did not differ
 significantly between males offered to virgin females and to mated
 females, using a Welch’s two-sample t-test. Finally, we determined if the
 size range between virgin females and mated females differed using a
 Welch’s two-sample t-test. To assess whether the males
 that females chose to mate with were on average larger than the rejected
 males, we first tested if mean pupal mass differed significantly between
 the chosen and not chosen males by computing paired t-tests. To then test
 whether virgin and mated females differed in the strength of their mate
 preference, we first randomly selected the data of one male per cage
 (i.e., this male was either chosen or not chosen by the female). Since
 each female made one choice, this step ensured that the number of data
 points for the analysis corresponded to the actual number of choices made
 in the experiment. Subsequently, we modelled the response variable
 <em>female</em> <em>choice</em> as a function of
 the variable <em>larger/smaller </em>(male), indicating
 whether the male was larger or smaller compared to the other male in the
 same cage, the<em> difference in male pupal mass </em>between
 the two males per cag<em>e</em>, female<em> mating
 status</em>, and their three-way interaction. Including a three-way
 interaction in the model allowed us to let the slopes of the function vary
 independently, and thus enabled us to identify the differences in female
 mate choice with respect to female mating status. We fitted the model
 (glm) with a binomial error distribution and produced the ANOVA table
 using the package car (Fox & Weisberg, 2019) in R. The results
 were visualized using the package ggplot2 (Wickham, 2016).
description: EAD ME for data for manuscript ID: ECE-2021-12-02045_R1 Title: Mating
 status affects female choice when females are signallers Consists of three
 datasets and one README file. 1_The data set SIGNALLING contains a time
 series of signalling (calling) behaviour per individual in a binomial
 manner (0 = not calling, 1 = calling/signalling). The experiment started
 at 11am (time = 0). The time_min column thus states the number of minutes
 from the start of the observations. NA = not applicable / missing value
 Column names meaning: no = number for processing date_start = date of the
 start of the experiment sample = unique sample id female_status = female
 mating status time = actual time of day time_min = duration in minutes
 since start of the experiment calling = female behaviour as calling (yes =
 1, no =0) 2_The data set LATENCY reports the mating latencies of mated and
 virgin females. Male and female pupal mass were included in the analysis
 (see methods). The time from the start of the experiment to the moment of
 mating is reported as latency in minute (latency_min column). Column names
 meaning: Experiment_date = date of experiment Cup_id = unique identity of
 the testing cup female_status = female mating status feamle_mass_grams =
 mass of female measured in grams male_mass_grams = mass of male measured
 in grams Grid_Location = location of the cup in the experimental set up
 (i.e., grid) Mating latency_min = mating latency measured in minutes 3_The
 data set TWO-CHOICE reports the outcomes of the two-choice assay with
 virgin and mated female choosing from big or small males. Please note that
 the pupal masses are reported in 1/10.000 grams. Wing marks of males are
 reported as R = right and L = left. The variable delta states the
 difference in pupal mass between the two males per cage.