DOI: 10.5061/dryad.2280gb615
title: Pesticide exposure triggers sex-specific inter- and trans-generational effects conditioned by past sexual selection

description: Environmental variation often induces plastic responses in organisms that
 can trigger changes in subsequent generations through non-genetic
 inheritance mechanisms. Such transgenerational plasticity thus consists of
 environmentally-induced non-random phenotypic modifications that are
 transmitted through generations. Transgenerational effects may vary
 according to the sex of the organism experiencing the environmental
 perturbation, the sex of their descendants, or both, but whether they are
 affected by past sexual selection is unknown. Here we use experimental
 evolution on an insect model system to conduct a first test of the
 involvement of sexual selection history in shaping transgenerational
 plasticity in the face of rapid environmental change (exposure to
 pesticides). We manipulated evolutionary history in terms of the intensity
 of sexual selection for over 80 generations before exposing individuals to
 the toxicant. We found that sexual selection history constrained
 adaptation under rapid environmental change. We also detected
 intergenerational and transgenerational effects of pesticide exposure in
 the form of increased fitness and longevity. These cross-generational
 influences of toxicants were sex-dependent (they affected only male
 descendants), and intergenerational, but not transgenerational, plasticity
 was modulated by sexual selection history. Our results highlight the
 complexity of intragenerational, intergenerational, and transgenerational
 influences of past selection and environmental stress on phenotypic
 expression.
description: We tested, using the seed beetle <em>Callosobruchus
 maculatus</em>, if divergent regimes of sexual selection (after 84
 generations of continued selection) modulate transgenerational plasticity
 in response to environmental stress (pesticide exposure). The experimental
 design hence consisted of a 2 x 2 design in which we crossed the two
 selection regimes (monogamous, henceforth Mono, or polygamous, henceforth
 Poly) with exposure/lack thereof of non-lethal concentration of pesticide.
 Before beetles were subjected to environmental stress treatment, the
 different selection lines underwent two generations of common garden
 breeding. Common garden breeding was carried out, first by duplicating the
 number of inoculated beans collected from each line to give rise to the
 next generation, and then by establishing a spare set of eight populations
 that were all bred under polygamous conditions for two generations, while
 maintaining the selection experiment with the original populations. After
 those two common garden generations, and to obtain focal virgin
 individuals of known age, we isolated three times as many infested beans
 as beetles were needed in our pesticide experiment. The isolation
 procedure was identical to that employed by the selection experiment and
 consisted of extracting inoculated beans with only one egg on them from
 the containers 11 days after oviposition, and reallocating them
 individually in the perforated Eppendorf tubes to ensure the virginity of
 the adults. This isolation protocol also allowed us to know the exact age
 of the focal adult beetles since adult emergences from the bean were
 checked daily. Five virgin males and five virgin
 females were randomly selected within each of the eight selection lines
 that underwent common garden breeding as the focal individuals to be
 allocated to each of the pesticide exposure levels (exposed and control)
 in our experiment to non-lethal concentrations of the pesticide
 deltamethrin (COMBO Deltamethrin 2.5% w/v, Sarabia). Pesticide application
 was restricted to these beetles (the parental generation, F0), but
 subsequent effects were tracked over three generations (F0, F1, F2). Thus,
 160 virgin beetles of <em>C. maculatus </em>(80 females and 80
 males), with different evolutionary histories regarding sexual selection
 regimes (Mono, Poly), were exposed to different levels of previously
 tested non-lethal concentrations of the pesticide, inside a flow cabinet
 for 24 h, at 23 ± 1 °C, 12:12 L:D and 35% RH. Two different pesticide
 treatments were given: 1) control treatment (C), without pesticide,
 exposed to distilled water; 2) pesticide treatment, 2 g/L deltamethrin in
 aqueous solution (P). In short, the exposure was done randomly assigning 5
 individuals from each sex from each of the eight lines (4 Mono and 4 Poly)
 to each treatment, having a total sample size of 160 parental exposed
 beetles in our experiment. Individuals at the moment of pesticide/control
 exposure were between 1 and 4 days old. For the
 pesticide exposure, the ends of cotton swabs were impregnated with 30 uL
 of the pesticide or water and put individually into 2 mL opened Eppendorf
 tubes, which would be filled with the evaporation of the pesticide
 solution shortly after closing the tubes and throughout most of the 24 h
 of exposure. Next, beetles were introduced to a second perforated
 Eppendorf tube with the bottom removed and stacked onto the first tube,
 with a mesh barrier between them, keeping the cotton swab in the lower
 compartment and avoiding direct contact of the beetles with the pesticide.
 After the exposure phase, beetles were individually allocated to 26 mL
 perforated plastic containers with <em>ad libitum</em> beans
 (around 60 beans per vial) to be used as an oviposition substrate. After
 the exposure phase, and until death, beetles were kept in walk-in climate
 chambers (Fitoclima 10000 EHF, Aralab) at a constant 29 °C temperature
 with 40% humidity and a 12 h/12 h light/dark cycle, which are the
 conditions at which all animals from the experimental evolution program
 had been cultured since the start of the selection experiment. Each
 individual shared the recipient with a non-exposed tester mate (male or
 female), all sourced from a standardized heterozygote tester line that was
 generated by crossing two near-isogenic lines that had been generated
 following 15 generations of full-sib mating (all individuals used in the
 generation of the near-isogenic lines were drawn from the stock
 population, i.e., they were individuals from outside the selection
 experiment). Mating and oviposition were allowed for 48 hours. Afterwards,
 focal males were individually reallocated to Eppendorf tubes to track
 their longevity, whereas mated females (focal females that were mated to
 tester males, or tester females who were mated to focal males) were kept
 in their container to determine longevity, fecundity, and lifetime
 reproductive success for each of them. We recorded
 life-history traits (longevity, fecundity, and lifetime reproductive
 success) on the generation exposed to pesticide treatment (F0), and on
 their offspring (F1) and grand offspring (F2). The F1 and F2 generations
 were not exposed to pesticides, but conditions for isolation, housing, and
 mating were similar to the parental generation. The sample size doubled in
 each generation because we selected both a son and a daughter from each
 cross in each generation (160 focal beetles in F0, 320 focal beetles in
 F1, and 640 focal beetles in F2), hence allowing us to test for
 differences between the sexes, in the F0, F1, and F2
 generations. We recorded the longevity of all beetles
 by individually monitoring survival on a daily basis from adult emergence
 until their death. We estimated total fecundity by carefully checking the
 presence of eggshells on each bean within every vial, on day
 11<sup>th</sup> after oviposition. Fecundity was measured for
 the F0 generation only as our focus was primarily on total number of adult
 offspring produced along life (lifetime reproductive success, LRS), and
 fecundity measures could not be attained for F1 and F2 generations due to
 the large sample sizes for individuals assessed in those generations. LRS
 could be measured as viable eggs hatched in a 28-day interval. As mating
 and oviposition times were controlled in our experiment, containers were
 frozen 28 days after the death of the female (the last possible day for
 oviposition). For those females who lived longer than 14 days, containers
 were frozen always before the predicted emergence date of the next
 generation (corresponding to day 42 from the first oviposition date). LRS
 was measured for all beetles in all generations by accounting for the
 lifetime number of adults produced by each female (either focal females or
 tester females mated to focal males). Dry body weight was measured after
 death for all individuals, focal or tester, and in all generations, with a
 Sartorius Cubis MSA 6.6S microbalance (accuracy 0.001mg, Sartorius,
 Goettingen, Germany). Beetles were frozen at -20 ºC when found dead, for a
 period of several weeks until the end of the experiments. The animals were
 then thawed and dried for 1 week at 40 ºC (so as to remove variation in
 weight due to time elapsed between death and freezing) before being
 weighed.
description: # Pesticide exposure triggers sex-specific inter- and trans-generational
 effects conditioned by past sexual selection
 [https://doi.org/10.5061/dryad.2280gb615](https://doi.org/10.5061/dryad.2280gb615) ## Description of the data and file structure Legend for the variables in the Excel file: * generation: Three generations, F0, F1 and F2. * id f0, id f1, id f2: These columns correspond to the identities of the individuals in generations F0, F1, and F2, respectively * Parent exposed: corresponds to the sex to the individual exposed to treatment in F0 generation. m=male and f = female * Sex focal: corresponds to the sex of the focal individual of that generation * Pesticide treatment corresponds to the levels of pesticide used in the experiment. c = control and p = 2g/L pesticide * Mating system corresponds to the evolutionary history of the beetles regarding sexual selection. poly = polygamous history and mono = monogamous history * line: selection line, 1-4 monogamous and 1-4 polygamous * Age at mating corresponds to the age (in days) at the time of mating, which was always established to happen one day after exposure to the pesticide or control. It applies to both focal and tester beetles. * Longevity (in days) corresponds to the difference between death and emergence day of the adult beetle. It applies to both focal and tester beetles. * Fecundity and LRS were the total count of eggs, and adult offspring across lifetime, respectively * Sex inbred: corresponds to the sex of the tester mate individual. * Age_mating_inbred: corresponds to the age (in days) at the time of mating of the tester individuals. * Longevity_inbred: corresponds to the difference between the death and emergence day of the adult tester beetle. * weight_fem and weight_male: correspond to the dry mass (in milligrams) of beetles after death. Empty cells correspond to no data (NA). Values of zero are specified as 0. Individuals for which information regarding response and predictor variables was not complete for each specific model did not enter the analysis