Protein restriction (PR) has established feasible trade-offs in Drosophila melanogaster to understand lifespan or aging in a nutritionally challenged environment. However, the phenotypes of body size, weight and wing length respond according to factors such as flies’ genotype, environmental exposure, and parental diet and hence their understanding is essential. Here, we demonstrate the effect of long-term PR diet on body size, weight, normal & dry wing length of flies subjected to PR50 and PR70 (50% and 70% protein content present in control food respectively) for 20 generations from pre-adult stage. We found that PR fed flies have lower body weight, relative water content (in males), unaltered (PR50%) and higher (PR70%) relative fat content in males, smaller normal and dry body size as compared to control and generations 1 and 2. Interestingly, wing size and pupal size of PR flies are smaller and showed significant effects of diet and generation. Thus, these traits are sex and generation dependent along with an interaction of diet, which is capable of modulating these results variably. Taken together, the trans-generational effect of PR on fitness and fitness-related traits might be helpful to understand the underpinning mechanisms of evolution and aging in fruit flies D. melanogaster.

We measured the normal and dry body weight of freshly eclosed flies collected in every 2 h intervals. The eggs were collected from the DR stocks over a 2 h window and kept under LD12:12 h. Post eclosion, the virgin male and female flies were separated by anesthetizing with CO₂. For weighing the normal body weight, flies were weighed post anesthetization using ether (to maintain the flies in the anesthetized state for longer duration), after which the flies were discarded. For the dry body weight assay, the virgin males and females were killed by freezing and were dried for 36 h at 70°C as per the protocol followed elsewhere. The normal and dry body weight assay was assessed by weighing a group of 10 males or 10 females per vial, and 5 such vials of randomly chosen flies from the control and the DR stocks were weighed. The body weight of flies was measured using a weighing balance from UniBloc (Shimadzu) AUX220. The relative water content of the flies was calculated by dividing the water content (normal body weight-dry weight) by the normal body weight of the flies as reported elsewhere. The relative fat content was assessed by dividing the fat content (dry weight-fat free dry weight) by the dry weight of the flies. The flies’ body size and wing length were measured under a microscope, wherein 30 virgin males and females from the control and DR stocks were assayed and averaged and the values were used to plot the graph. The body size and the wing length of the anesthetized males and females were measured using a microscope from Olympus with a normal ruler (least count 0.5 mm).