Intergenerational effects on offspring phenotypes occur in response to variation in both maternal and paternal nutrition. Because the combined maternal and paternal effects are rarely considered together however, their relative contributions, and the capacity for interactions between parental diets to shape offspring life history and physiology are not understood. To address this, we altered sucrose levels of adult fruit flies (Drosophila melanogaster) prior to mating, across two generations, producing parent-parent and parent-offspring combinations that were either matched or mismatched in dietary sucrose. We then measured lifespan, fecundity, body mass, and triglyceride levels in parents and offspring. We reveal complex non-additive interactions, that involve diets of each parent and offspring to shape offspring phenotypes, but the effects were generally not consistent with an adaptive response to parental diet. Notably, we find that interacting parental flies (sires and dams) lived longer when their sucrose treatments were matched, but they produced shorter-lived offspring.

Male and female virgin flies were assigned to one of the two dietary treatments prior to mating (we refer to this generation of flies as F0), and then the offspring produced (we refer to this as the F1 generation) were also assigned to one of the two treatments. All possible combinations of dam × sire × offspring diet treatment were represented (= 2 × 2 × 2 = 8 combinations). Specifically, we collected flies of the F0 generation as virgins and placed them in vials of 10 flies across 30 vial replicates per treatment (1300 flies, 650 of each sex), in their respective sexes, onto either the high sucrose (20%) or the low sucrose (2.5%) diets for the first 6 days of their adult life. We transferred flies to vials containing fresh food of the designated diet every 48 hours during this 6 day period.

At day 6, we randomly sampled six vials from each treatment, and snap froze (using liquid nitrogen) the flies of these vials, storing them at -80°C for later triglyceride and body-weight assays. Cohorts of flies in the remaining vials then entered a cohabitation phase to enable female and male flies to mate. Cohorts of males and female flies were combined, in vials of 10 pairs, in each of all four possible diet combinations: Lower sucrose females × lower sucrose males; higher sucrose females × higher sucrose males; lower sucrose females × higher sucrose males; higher sucrose females × lower sucrose males. During this phase, flies cohabited for 96 hours, allowing them to mate. They were transferred to a new vial with fresh food of standard 5% sucrose diet every 24 hours during this time.

Following the cohabitation phase of 96 h, the F0 flies were separated back into their respective sex-specific cohorts, and placed back onto the high or low sucrose diets that they were originally assigned prior to the cohabitation phase, in vials of 20 flies. Flies of these vials were then monitored for longevity (the longevity assay is described below). The vials from the 6 day old F0 flies (i.e., the vials from day 1 of the 96 h cohabitation phase) were retained, and the eggs that had been laid by females of the respective vials were trimmed to 80 per vial (by removing excess eggs with a spatula). The remaining eggs were left to develop into adult offspring over 10 days at 25°C (on a 12:12 light/dark cycle in a temperature-controlled cabinet; Panasonic MLR-352H-PE incubator). These adult flies constituted the F1 offspring in the experiment, all F1 were reared on standard media (5% sucrose). We collected virgin F1 adults from each of the four combinations of parental diet treatments, and placed them in their respective sexes in vials of 10 flies, across 30 vial replicates per treatment per sex (2400 flies). We then assigned these F1 flies, produced by each dietary treatment combination of F0 flies, to either the lower sucrose or higher sucrose diet. At day 6 of adulthood, we snap froze F1 flies of six randomly chosen vials per dam × sire × offspring diet combination. On the same day, 10 virgin focal F1 flies were placed together with 10 tester flies of the opposite sex (age standardised), collected from the Dahomey stock population, entering into a cohabitation phase of 96 h (during which time the number of eggs laid by females of each vial was assessed). After 96 hours flies were separated again into their respective sexes (in vials of 20 flies), and assigned back onto either the lower sucrose or higher sucrose diets that they had been on prior to cohabitation, and a longevity assay carried out.