GENETIC INDEPENDENCE BETWEEN TRAITS SEPARATED BY METAMORPHOSIS IS WIDESPREAD BUT VARIES WITH BIOLOGICAL FUNCTION
Why is metamorphosis so pervasive? Does it facilitate the independent (micro)evolution of quantitative traits in distinct life-stages, like it does for discrete characters such as limbs and organs? We tested this hypothesis by measuring the expression of 6400 genes in 41 Drosophila melanogaster inbred lines at larval and adult stages. Only 32% of the genes showed significant genetic correlations between larval and adult expression. By contrast, 44% of the traits showed some level of independence between stages. Gene ontology terms enrichment of the functions most and least constrained among stages revealed that traits related to immunity emerged as largely correlated between larvae and adults. Direct comparisons with other datasets showed that inter-stage constraints were lower than inter-sexual or cross-environment genetic constraints. These results show that metamorphosis enables a large part of the transcriptome to evolve independently at different life-stages and identify biological functions under high and low genetic constraints. Overall design: DGRP lines and sample production We used the Drosophila Genetic Reference Panel [DGRP, 18], a set of inbred lines derived from an outbred population of Drosophila melanogaster. We chose 50 lines that showed no inversion and scored across the range of starvation resistance measured in a previous study [18]. Our final analyses focussed on 41 DGRP lines after removing samples that could not be produced or failed to meet the quality control analyses criteria. All lines were reared on cornmeal-molasses-agar medium at 25°C and 12-hour light and dark cycles. Grand-parental generation consisted of two replicate vials per line of two virgin males and two virgin females placed together for 2 days and further tipped in in another vial to lay for another two days. Egg density was manually regulated to approximate 50 eggs per vial. In the following generation, 12 virgin males and 12 virgin females aged two to six days were placed together for 24 hours to ensure mating occurred. Up to three males and three females (36 out of the 328 vials contained fewer adults as there were not enough flies available) of these 12 were subsequently placed together in a vial for 48 hours. Egg density was approximately kept to 50 per vial. We independently used the larvae and adults that emerged from those vials for RNA sampling. Not all of lines were synchronous; in order to avoid creating among-line variance resulting from difference in development times, we did not collect all flies on the same day. During larva collections, once the first 'wanderer' (fully grown 3rd instar larva that stop eating and start searching for a pupation site) was observed in the vial, up to 10 larvae per vial were collected from the medium (i.e. not wanderers) and flash frozen with liquid nitrogen. When possible, a further 10 larvae were collected as a backup supply (and used in 12 of the 82 samples). The remainder of the flies were discarded (i.e. adults were never collected from a larva vial). Freezing occurred randomly during the day for the different samples, between 10am and 12am. The collected larvae were not sexed and we expect our samples to be a random mix of males and females. During the collection of adult flies, we scanned vials every day. When the first adults were observed in a vial, we removed them and collected all emerged adults within the following 48 hours. The collected adult flies were placed in fresh vials for 4 days to finish maturing and flash frozen in the same manner as were the larvae.