Paternal condition affects offspring reproduction and life history in a sex-specific manner in Drosophila melanogaster
Data files
Dec 05, 2022 version files 351.89 KB
Abstract
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.
Methods
Study animals
Throughout this study, we followed 30 focal isolines (Parsons and Hosgood 1967; David et al. 2005) of D. melanogaster 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 Drosophila 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: F0 and F1, respectively), we reared each genotype under two different dietary conditions, where larvae had ad libitum 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 F0 and 30 larvae/vial in the F1 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).
Filial fitness traits
To quantify the effect of the larval dietary conditions experienced by the sires and their F1 offspring, respectively, we determined the sex-specific F1 development time (in days) and the larval-to-adult survival rate (hereafter: viability) 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 F1 females and 8.0 ± 0.16 F1 males per isoline-diet combination (i.e., 30 isolines × 2 paternal × 2 filial conditions: Ntotal = 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 F1 males, quantified in sperm offence assays (hereafter P2) 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., Ntotal = 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.
Usage notes
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 females: "F0_f_single_progeny" or "F1_f_single_progeny"
2: the single assays with focal males: "F0_m_single_progeny" or "F1_m_single_progeny"
3: the competitive mating assays with focal males: "F0_comp" or "F1_comp"