DOI: 10.5061/dryad.8kprr4xtg
title: Operational sex ratio bias due to sex-specific cohort splitting in response to predation

description: The operational sex ratio (OSR), i.e., the local ratio of fertilizable
 females to sexually active males at any given time, is of key importance
 for the strength of sexual selection and the reproduction of populations.
 We hypothesize that sex-specific cohort splitting, i.e., when one sex
 mostly metamorphoses while the other mostly enters diapause, may lead to
 OSR bias in nature. The OSR of an aquatic moth, Acentria ephemerella, has
 been shown to be strongly male-biased in situ. Here, we use a mesocosm
 experiment in which we determine the sexes of active, diapausing, and
 metamorphosing larvae to test whether the male bias in Acentria is due to
 sex-specific mortality or sex-specific cohort-splitting. Fish predation dd
 not result in a strong male bias of the whole population but increased
 male bias in pupae and female bias in diapausing larvae. The opposite
 effect of fish on pupal versus diapausing larval sex ratios suggests that
 fish-induced sex-specific cohort splitting, rather than sex-specific
 mortality, caused the OSR bias of Acentria observed in situ. Future
 research needs to study whether the OSR bias is an adaptive response to
 the presumably higher fish predation pressure on females or a maladaptive
 byproduct of sex-specific activity and growth responses to fish presence.
 Overall, shifts in OSR due to sex-specific cohort splitting could be a
 more common component of arthropod life-histories than previously thought.
description: The aquatic moth <em>Acentria ephemerella</em>
 (Crambidae) displays a pronounced sexual wing and size dimorphism (Berg,
 1942). Female pupae and adult life stages are larger than male pupae and
 adult life stages (Berg, 1942; Miler et al., 2014). The adult females are
 predominantly rudimentarily winged (brachyptery &gt; 99%), aquatic
 throughout their life and display morphological adaptations to the aquatic
 habitat such as swimming legs (Berg, 1942). Adult males in contrast are
 winged and emerge from the water column after metamorphosis (Berg, 1942).
 <em>Acentria</em> is a capital breeder with a short adult life
 span of 1–3 days and the number of eggs can be counted in advanced pupal
 stages (Berg, 1942; Miler et al., 2014). Mating occurs at the water
 surface, with the abdominal apex of the aquatic adult females reaching
 above the water surface (Berg, 1942). During mating, the terrestrial males
 transfer a spermatophore into the bursa copulatrix of the females which is
 located ventrally between the 7th and 8th abdominal segment (Berg, 1942).
 The larvae of <em>Acentria</em> feed from June to September on
 submerged aquatic macrophytes, mainly pondweed species
 <em>Potamogeton</em> spp. (Gross et al., 2002; Miler, 2009). A
 strong decrease in the biomass of its food plants occurs under high
 <em>Acentria</em> population densities during summer (Le
 Bagousse-Pinguet et al., 2012; Miler & Straile, 2010). The
 life-cycle of <em>Acentria</em> consists of one to three
 generations per year, of which one generation includes a diapause stage
 (Berg, 1942; Gross et al., 2002; Miler, 2009). Although information about
 the number of instars until metamorphosis of *Acentria *is scarce,
 previous research suggests five instars to be likely (Bänziger, 2000;
 Haenni, 1980), which fits to a typical Lepidoptera life cycle with five to
 seven instar stages (Williams & Feltmate, 1992). Second and third
 instar <em>Acentria</em> larvae of the last generation of the
 year enter a diapause stage in autumn, overwinter inside plant stems in a
 protective cocoon, the hibernaculum, and continue larval development until
 metamorphosis and emergence in the spring of the next year (Berg, 1942;
 Haenni, 1980; Gross et al., 2002; Miler et al., 2014).
 The experiment was conducted in a large outdoor tank
 (area = 52.5 m<sup>2</sup>, depth = 1.5 m) filled with fine
 sediment (~ 0.35 m) and water (~ 1 m) from Lake Constance (Fig. 1). 15
 mesocosms (experimental units) were placed in this tank and consisted of a
 transparent plastic tube (volume ~ 0.48 m<sup>3</sup>, surface
 ~ 0.48 m<sup>2</sup>, height ~ 1 m, length ~ 0.8 m, width
 ~ 0.6 m) attached to a metal frame in the sediment and to a floating frame
 used as a buoy to keep the plastic tube in a vertical position in the
 water column. Plastic tubes completely separated the experimental units
 from the surrounding water and there was no water exchange between the
 individual mesocosms and the tank. Frames were covered with metal gauze
 (mesh size ~ 1620 µm) approximately 0.1 m above the water surface, to
 allow for <em>Acentria</em> reproduction, but to prevent
 winged males from dispersing out of the replicates (for details see Miler
 et al., 2008). Both the plastic tubes and the gauze-covered frames (1)
 prevented any <em>Acentria</em> adults or larvae to move out
 of each of the mesocosms into the tank and vice versa and (2) prevented
 the exchange of any terrestrial organisms larger than 1620 µm in length or
 width and the exchange of any aquatic organisms between each of the
 mesocosms and the tank. Five mesocosms were sampled at
 the time of fish introductions (10 August, i.e. start conditions), and ten
 experimental units were assigned to the fish and control (no fish)
 treatment, respectively and sampled on 20 August, i.e., altogether 15
 mesocosms. The assignment of mesocosms to treatment and time of sampling
 was randomized using a random number generator.
 <em>Potamogeton perfoliatus</em> shoots were sampled
 on 14 June 2006 in an Upper Lake Constance macrophyte patch.
 Macrophyte-associated* Acentria* larvae and pupae were removed and
 subsequently eight macrophyte shoots were planted in each mesocosm, i.e.,
 a density of ~ 18 shoots m^-2^ typical for the early seasonal growing
 period (Wolfer & Straile, 2004). The shoots were allowed to root
 and establish new shoots for approximately five and a half weeks. From
 20 to 22 July, a total of 79 <em>Acentria</em> pupae from
 field samples in Lower Lake Constance were introduced into each mesocosm
 (i.e., 1580* Acentria* pupae in total for all 20 mesocosms taken
 together). Since <em>Acentria</em> larvae breathe through a
 thin membrane and removing this membrane would be necessary to identify
 the sex of the larvae (Berg, 1942), we did not know the sex of the pupae
 introduced into the mesocosms. Based on a field study from 2005 in Lower
 Lake Constance, we can assume the sex of the pupae introduced into the
 mesocosms was slightly male-biased (~ 54% males, Miler et al., 2014),
 whereas Upper Lake Constance sites showed a considerably higher proportion
 of male pupae (Miler et al., 2014). Starting on 20 July, from these pupae
 adult individuals hatched, mated and laid egg clutches and on 31 July,
 feeding damage and small larvae could be observed in all mesocosms. Twelve
 sticklebacks (<em>Gasterosteus aculeatus,</em> total length
 range: 4.3–6.2 cm) were introduced into each mesocosm of the fish
 treatment on 10 August, i.e., after allowing <em>Acentria</em>
 to develop for ten days. In a previous mesocosm experiment at Lake
 Constance, a strong and significant predation of sticklebacks on
 <em>Acentria</em> was observed, with
 <em>Acentria</em> specimens found in the guts of sticklebacks
 (Miler et al., 2008). Sticklebacks were caught in a small pond near the
 University of Konstanz (geographic coordinates in decimal degrees:
 latitude 47.687, longitude 9.190), where they occurred in high abundances
 and were easy to capture with minnow traps. To our knowledge, the density
 of sticklebacks in Lake Constance has not been studied, but the fish
 density of 24 individuals m^-2^ used in our experiment was comparable to
 stickleback densities observed in nature (Thiel et al., 1995; Ward
 & FitzGerald, 1983). Sampling occurred in 5
 mesocoms on 10 August to assess* Acentria* densities prior to the onset of
 fish predation as well as <em>Acentria</em> densities, sizes
 and life stage distributions on 20 August, after ten days of fish
 predation and when the first adult individuals were observed. We did not
 observe any direct predation of <em>Acentria</em> by
 sticklebacks, but any such events would have been unlikely to be observed,
 since the mesocosms were only occasionally and briefly checked between
 sampling events. It is likely that active <em>Acentria</em>
 larvae are more vulnerable to fish predation than diapausing larvae, since
 the latter are better protected inside a hibernaculum and the plant stem
 (Berg, 1942; Gross et al., 2002; Haenni, 1980), although active
 <em>Acentria</em> often construct simple protective covers
 from pieces of leaves (Berg, 1942). We distinguished three developmental
 life stages of <em>Acentria</em>: active larvae (larvae
 outside macrophyte stems), diapausing larvae (larvae inside macrophyte
 stems) and pupae. <em>Acentria</em> larvae were washed through
 a sieve (mesh size 200 µm) and fixed in Carnoy’s solution (ethanol,
 chloroform, acetic acid; 6:3:1) for subsequent sex determination. The
 remaining material washed from macrophytes, including
 <em>Acentria</em> larvae overlooked during the fixation in
 Carnoy’s solution, was preserved in 70% ethanol in 100 ml plastic bottles.
 Since <em>Acentria</em> pupae are closely attached to the
 stems of <em>P. perfoliatus</em> shoots and larvae overwinter
 inside the stems, the remaining plant material was stored at 5°C in
 plastic bags and searched through for pupae and diapausing larvae within
 one week after sampling. Pupae were fixed in 70% ethanol and diapausing
 larvae in Carnoy’s solution. Macrophytes were dried at 90°C for three days
 and densities/span&gt;of pupae and larvae were calculated as
 individuals per gram plant dry mass (Ind. g^-1^ dm).
 Females of most lepidopteran species of the clades Ditrysia and
 Tischeriina (comprising together more than 98% of all species) contain a W
 sex chromosome forming a densely concentrated spot (the ‘W-body’) inside
 each somatic interphase nucleus, whereas males lack this feature (Fig. 2a,
 for reviews see Traut & Marec, 1996; Traut et al., 2007; Traut
 & Scholz, 1978). From each mesocosm up to 40 active and up to 40
 diapausing larvae (Appendix Table 1) were dissected and their tissue,
 preferentially Malpighian tubules and silk glands, was stained with 1.5%
 lactic acetic orcein (Traut et al., 1986). After staining, larvae were
 inspected for the presence of sex chromatin (W chromatin) in highly
 polyploid nuclei (Fig. 2a) under a light microscope (Zeiss Axioskop 40) at
 1000-fold magnification. In one of the five fish treatment mesocosms, the
 active larvae (n=8) could not be successfully sexed because these larvae
 were erroneously not preserved in Carnoy’s solution. In addition to active
 and diapausing larvae from the mesocosms, we analysed the primary sex
 ratio of 14 <em>Acentria</em> egg clutches that were sampled
 in Lake Constance and incubated at 20°C in the laboratory. From each
 clutch, the sex of 30 randomly selected freshly hatched larvae was
 determined within 24 h after hatching. <em>Acentria</em> pupae
 were sexed morphologically by measuring and comparing the length of wings
 and antennae as published in Berg (1942) (Fig. 2b). To convert length
 measurements into dry mass we established a head-capsule width – dry mass
 relationship for larvae (equation (1), n = 45,
 <em>R<sup>2</sup></em> = 0.93,
 <em>P</em> &lt; 0.0001) and a length – dry mass
 relationship for pupae (equation (2), n = 34,
 <em>R2</em> = 0.93,
 <em>P</em> &lt; 0.0001).
 log<sub>10</sub> (weight [mg]) =
 -12.17 + (4.17 * log<sub>10</sub>
 (head capsule width [µm]))   (1)
 log<sub>10</sub> (weight [mg]) =
 -1.96 + (2.93 * log<sub>10</sub> (body
 length [mm]))    (2) In the following analyses, we will
 abbreviate the weight of larvae and pupae in
 log<sub>10</sub> (weight [mg]) as log dm. The developmental
 stages of pupae were assessed based on their eye development and eggs from
 specimens in an advanced developmental stage were counted after
 dissection. All statistical analyses were performed
 with R 3.2.3 (R Development Core Team, 2015) and RStudio 0.99.489
 (RStudio Inc., 2015). We used Mixed model ANOVA to study a) the effect of
 fish presence and developmental stage on <em>Acentria</em>
 densities, and b) the effect of fish presence and sex on sizes of active
 larvae, diapausing larvae and pupae. The analysis of pupal sizes revealed
 a singularity problem for estimating the random effects. In order to
 confirm the results for the fixed effects, we additionally ran a simple
 linear model (lm() function) using mean sizes for each mesocosm). This
 only marginally altered the F and p-values. Hence, we report in the case
 of pupal sizes also the statistical results from the mixed model ANOVA.
 The effects of fish presence on the % densities of active larvae,
 diapausing larvae, and pupae was analysed using a multinominal generalized
 mixed model using a Bayesian approach. Finally, the effect of fish
 presence and developmental stage on the sex ratio was analysed with mixed
 logistic regression. Type II ANOVA (<em>F</em> or Wald
 Chi-square) tests were used to test for significance of model coefficients
 (function Anova() in R). In all mixed models, the mesocosm ID was included
 as a random factor to avoid pseudoreplication (R packages lme4, functions:
 lmer(), glmer(), Bates et al., 2015, nlme, function: lme(), Pinheiro et
 al., 2024, brms, function: brm(), Bürkner, 2017 and stats (base R),
 function: glm(), R Development Core Team, 2015). Diagnostic plots using
 the R package DHARMa (Hartig, 2022) suggest no violation of the
 assumptions for the statistical models used in this study (dispersion,
 homogeneity of variances). The sex ratio bias of freshly hatched larvae
 was analysed with a Χ<sup>22</sup> test (R package stats
 (base R), function: chisq.test(), R Development Core Team,
 2015).
description: # Operational sex ratio bias due to sex-specific cohort splitting in
 response to predation
 [https://doi.org/10.5061/dryad.8kprr4xtg](https://doi.org/10.5061/dryad.8kprr4xtg) ## Summary of dataset contents, contextualized in experimental procedures and results Operational sex ratio (OSR) is of key importance for the strength of sexual selection and the reproduction of populations. We suggest that n important mechanism causing OSR bias is sex-specific cohort splitting, i.e., when one sex mostly metamorphoses, whereas the other one mostly enters diapause. The OSR of an aquatic moth Acentria ephemerella, has been shown to be strongly male biased in situ. Here, we use a mesocosm experiment in which we determine the sexes of active, diapausing, and metamorphosing moths to test whether male bias in this key herbivore is due to sex-specific mortality or due to sex-specific cohort-splitting. Fish predation did not result in male bias of the whole population, but strongly enhanced male bias for pupae, and female bias for diapausing larvae. This suggests that fish-induced sex-specific cohort splitting caused the observed OSR bias of Acentria observed in situ. Future research needs to study whether the OSR bias is an adaptive response, or a maladaptive side-product of sex-specific activity and growth responses to fish presence. As Acentria shares key life history characteristics (size dimorphism, larval diapause) with other arthropods, shifts in OSR due to sex-specific cohort splitting might be more common in nature. ## Description of the data and file structure 4 R scripts are uploaded on Dryad: Fig6_clutch_sizes.r: Produces Figure 6, which shows the correlation between pupal dry mass and number of eggs in the fish treatment and control. `table1_and_2_Fig3_densities.r`: Produces Figure 3, which shows the percentage contribution of the density of active larvae, diapausing larvae and pupae on the overall density in the fish and control treatment at the end of the experiment. The R code calculates an ANOVA comparison of absolute Acentria densities in the fish and control treatments at the end compared to the start of the experiment and a linear mixed model comparing relative Acentria densities at the end of the experiment between treatment (fish, control) and life stage (active larvae, diapausing larvae, pupae) with a three-way interaction. `table3_Fig4_sex_ratio_analyses.r`: Produces Figure 4, which shows the percentage of males in the fish and control treatment of all life stages combined and separately for active larvae, diapausing larvae and pupae. The R code calculates a t-test comparison of sex-ratio (percent males) with all life stages taken together between fish and control treatments at the end of the experiment and a t-test comparison of sex-ratio (percent males) of diapausing larvae between fish and control treatments at the end of the experiment. The R code also calculates, if 40% of all active larvae would pupate, how much this would modify the % males of pupae in the fish and control treatments at the end of the experiment. The R code also calculates a linear mixed model comparing the % males at the end of the experiment between treatment (fish, control) and life stage (active larvae, diapausing larvae, pupae) with a three-way interaction. `table4_Fig5_size_analyses.r`: Produces Figure 5, which shows the mean male and female dry masses for active larvae, diapausing larvae, and pupae in control and fish treatments. The R code calculates how the reduction of mean female pupal size as observed in the fish treatment equals to a reduction of the clutch size. The R code also calculates a linear mixed model comparing the body sizes of active larvae, diapausing larvae and pupae at the end of the experiment between treatment (fish, control) and sex (males, females) with a three-way interaction. These contain the R code to produce the figures, tables and in-text statistics for the manuscript. Furthermore, the following input files are used in these R scripts: `Acentria_clutch_sizes.csv`: includes the variables meso (mesocosm number), fish (treatment; C = control = mesocoms without fish at the end of the experiment, F = fish = mesocoms with fish at the end of the experiment), length_mm (length in mm), weight_mg (weight in mg), nr_eggs (number of eggs), female_type (wf = winged, f = wingless) and date `Acentria_densities.csv`: includes the variables meso (mesocosm number), date, fish (treatment; start = mesocoms without fish at the start of the experiment, C = control = mesocoms without fish at the end of the experiment, F = fish = mesocoms with fish at the end of the experiment), stage (active larvae, diapausing larvae ("hib"), pupae), n_g_dm (density: number Acentria individuals per gram macrophyte dry mass), no_ind (absolute number of Acentria individuals in a sample) and dw (gram macrophyte dry mass in a sample) `Acentria_sizes.csv`: includes the variables: stage (active larvae, diapausing larvae ("hib"), pupae), meso (mesocosm number), date, sex (of Acentria, male, female), size (in micrometers head capsule width for larvae and mm body length for pupae), fish (treatment; C = control = mesocoms without fish at the end of the experiment, F = fish = mesocoms with fish at the end of the experiment), size_type (head capsule width, body length) and wings (y = winged, n = wingless, NA = not applicable (for larvae)) ## Links to other publicly accessible locations of the data: There are no other publicly accessible locations of the data. ## Data was derived from the following sources: We did not use any data other than the ones uploaded here that collected during the mesocosm experiment. ## Code/Software R and R Studio were used to run the code. All necessary functions and packages are included in the R scripts. Microsoft Excel and Notepad ++ were used to view the data files.