DOI: 10.5061/dryad.q573n5tm4
title: Paternal condition affects offspring reproduction and life history in a sex-specific manner in Drosophila melanogaster

description: Nongenetic parental effects can contribute to the adaptation of species to
 changing environments by circumventing some of the limitations of genetic
 inheritance. A clearer understanding of the influence of nongenetic
 inheritance and its potentially sex-specific responses in daughters and
 sons is needed to better predict the evolutionary trajectories of species.
 However, whereas nongenetic maternal effects have long been recognized and
 widely studied, comparatively little is known about corresponding paternal
 effects. Here, by following 30 isogenic lines of Drosophila melanogaster
 across two generations, each reared under two dietary regimes in each
 generation, we tested how protein restriction during larval development of
 the fathers affects the fitness and health of their daughters and sons. We
 then quantified genetic and non-genetic paternal, and direct
 environmental, effects across multiple axes of offspring fitness.
 Daughters and sons responded differently to their father’s developmental
 history. While isolines differed in mean trait values, their specific
 responses to protein restriction generally varied little. The sex- and
 trait-specific responses to paternal effects emphasize the complexity of
 inter-generational parental effects, which raise important questions about
 their mode of transmission and adaptive value, including the potential for
 conflict between the sexes.
description: <em>Study
 animals</em> Throughout this study, we followed 30 focal
 isolines (Parsons and
 Hosgood 1967; David et al. 2005) of <em>D</em>.
 <em>melanogaster</em> across two generations. Each isoline was
 derived from a single outbred population with 15 generations of
 full-sibling inbreeding (Lüpold et al. 2012; Lupold et al.
 2013), reaching a
 theoretical homozygosity of 96% (Falconer et al. 1996)⁠. Diet was manipulated during larval
 development. Since holometabolous insects grow only during their larval
 stages, adult size and relative investments in different tissues are
 determined before adult emergence, meaning that the developmental period
 can have dramatic long-term consequences for adult physiology and
 reproduction (e.g., McGraw
 et al. 2007; Andersen et al. 2010; De Nardo et al. 2021; Macartney et al.
 2021).
 Additionally, manipulating
 diet only during larval development appeared more ecologically relevant in
 that adult <em>Drosophila</em> are less likely to experience
 malnutrition than larvae in a natural setting due to their use of more
 diverse food sources (e.g., Shorrocks 1975). After adult emergence, offspring fitness was
 estimated across multiple axes to capture the complex and multifarious
 nature of fitness traits. Studies examining the effect of nutrition on
 different life-history traits often vary in their results depending on
 which specific nutrient is altered (Jensen et al. 2015). Here, we reduced the yeast content as the main
 protein source, keeping all other nutrients constant. This choice of diet
 manipulation was motivated by the fact that exposure to different
 protein:carbohydrate ratios has previously been shown to cause
 sex-specific variation in reproduction and lifespan (Jensen et al. 2015⁠; also see Lee et al. 2008) for a more generalizable context based on a
 geometric framework). In both the first and second generations
 (hereafter: F<sub>0</sub> and F<sub>1</sub>,
 respectively), we reared each genotype under two different dietary
 conditions, where larvae had <em>ad</em>
 <em>libitum</em> access to food (Fig. 1). Per liter of food
 medium, the normal larval diet consisted of 75 g glucose, 100 g fresh
 yeast, 55 g corn, 8 g agar, 10 g flour, and 15 ml Nipagin antimicrobial
 agent. The low-protein medium followed the same recipe, but only 25% of
 the yeast content. Replicated across 8 vials per isoline-diet combination,
 we transferred first-instar larvae to these diets at a density of 40
 larvae/vial in the F<sub>0</sub> and 30 larvae/vial in the F<sub>1</sub> generation. We transferred fewer larvae in the
 F1 generation to equalize larval densities across all vials instead of
 excluding entire isoline-diet combinations that consistently produced
 insufficient larvae in this generation for distribution across filial
 treatments (either few eggs or poor hatching rate). We note that increased
 larval densities could have increased larval competitive ability by
 increasing feeding rate, thereby reducing differences between the normal
 and restricted treatments (Joshi and Mueller 1996). At either density, larval competition should be
 relatively low, and so focal individuals are unlikely to have diverged in
 their internal states between treatments (Bentzur et al. 2021), given that all first-instar larvae were
 randomly assigned to one of the diet treatments at equal density within
 each generation. These low densities may be relatively conservative in
 terms of direct diet effects on larvae compared to crowded conditions on
 the same food. More crowded conditions, with their increased resource
 competition and accumulation of toxic waste (Henry et al. 2018; Klepsatel et al.
 2018), might enhance
 developmental differences between treatments, thus potentially shifting
 the relative importance of nongenetic paternal and direct larval effects
 on adult fitness traits. However, high densities might also introduce more
 noise by changes in the effective densities due to differential mortality
 (e.g., Moya and Botella
 1985; Henry et al. 2018).
 <em>Filial fitness
 traits</em> To quantify the effect of the larval dietary
 conditions experienced by the sires and their F<sub>1</sub> offspring, respectively, we determined the
 sex-specific F1 development time (in days) and the larval-to-adult
 survival rate (hereafter: <em>viability</em>) across the 8
 replicate-rearing vials per isoline-diet combination. For each of the 10
 females and males per genotype and treatment, we measured the thorax
 length as a proxy of body size, using a reticular eyepiece under a Leica
 MS5 stereomicroscope (Leica Microsystems, Heerbrugg, Switzerland) to the
 nearest 0.025 mm. Further, for a mean (± sd) of 7.5 ± 0.82
 F<sub>1</sub> females and 8.0 ± 0.16 F<sub>1</sub> males per isoline-diet combination (i.e., 30
 isolines × 2 paternal × 2 filial conditions:
 <em>N<sub>total</sub></em><sub>
 </sub>= 900 females and 963 males, respectively), we quantified
 female and male-induced fecundity as the number of progeny produced after
 a single mating with a standardized mate. To this end, we paired in
 individual food vials a 3- to 4-day-old focal fly with an individual of
 the opposite sex standardized for rearing condition (normal diet, approx.
 400 flies/bottle) and genotype (obtained by crossing virgin females of one
 isoline with males of another, creating heterozygous, quasi-clonal flies).
 For each pair, we recorded the latency to mating (from the introduction to
 the vial), which has previously been suggested to indicate female
 preference for a given male (Spieth 1974; Grillet et al. 2006; Hosken et al.
 2008; Sharma et al. 2010),
 and mating duration. After mating, we removed the male and allowed the
 female to oviposit in a fresh individual vial for three days for counts of
 offspring after their eclosion. Finally, we investigated the effect of paternal
 × filial conditions on the sperm competitiveness of F<sub>1</sub> males, quantified in sperm offence assays
 (hereafter P<sub>2</sub>) using standardized females and
 competitor males (also heterozygous, quasi-clonal). Using a separate
 subset of males (5.2 ± 1.00 focal males per isoline-diet combination,
 i.e.,
 <em>N<sub>total</sub></em> = 625), we repeated the mating trials as
 described above for the fecundity assay, but this time using 4- to
 6-day-old standard females that each had mated with a standard competitor
 male approximately 3 days before the focal mating. These standard males
 expressed green fluorescent protein (GFP) as well as ubiquitously
 (Manier et al. 2010;
 Lüpold et al. 2012), thus
 allowing easy paternity assignment under an Olympus SX12 fluorescent
 stereoscope (Olympus America, Melville, USA) equipped with a GFP filter.
 After the focal matings, we allowed females to oviposit for three days and
 then counted the focal and non-focal (GFP) offspring, respectively, after
 their eclosion.
description: There is one Excel file for each of the generations. Each of the
 two Excel files contains 3 sheets: 1: the single assays
 with focal <strong>females</strong>:
 "F0_f_single_progeny" or
 "F1_f_single_progeny" 2: the single assays
 with focal <strong>males:
 </strong>"F0_m_single_progeny" or
 "F1_m_single_progeny"<strong><br></strong> 3: the competitive mating assays with focal males: "F0_comp" or "F1_comp"