GENETIC INDEPENDENCE BETWEEN TRAITS SEPARATED BY METAMORPHOSIS IS WIDESPREAD BUT VARIES WITH BIOLOGICAL FUNCTION. 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.

为何变态发育如此普遍？它是否能促进不同生命阶段的数量性状独立（微）进化，正如其对附肢、器官等离散性状所起到的作用一般？本研究通过对41个黑腹果蝇（Drosophila melanogaster）近交系在幼虫期与成虫期的6400个基因表达量进行检测，验证了上述假说。结果显示，仅32%的基因在幼虫与成虫的基因表达间存在显著遗传相关；与之相对，44%的性状在不同生命阶段间表现出一定程度的独立性。对各阶段受约束程度最高与最低的功能进行基因本体（Gene Ontology, GO）富集分析后发现，与免疫相关的性状在幼虫与成虫间整体呈现高度相关。与其他数据集的直接对比表明，阶段间的遗传约束水平低于性别间或跨环境的遗传约束。本研究结果证实，变态发育允许转录组的绝大部分在不同生命阶段独立进化，并明确了受高低遗传约束的生物学功能。

实验整体设计：DGRP品系与样本制备 本研究使用了果蝇遗传参考 panel（Drosophila Genetic Reference Panel, DGRP，文献18），该集合包含从一个野化黑腹果蝇种群中获得的近交系。我们筛选出50个无染色体倒位且覆盖既往研究[18]中测得的抗饥饿性范围的品系。在剔除无法成功培养或未达到质控标准的样本后，最终分析聚焦于41个DGRP品系。

所有品系均在25℃、12小时光暗循环的条件下，于玉米粉-糖蜜-琼脂培养基中饲养。祖父母代每组品系设置两个重复培养管，每管放入2只处女雄蝇与2只处女雌蝇，交配2天后将成虫转移至另一新培养管中再产卵2天。人工调控卵密度至每管约50枚。在子代世代中，将12只2~6日龄的处女雄蝇与12只处女雌蝇共同放置24小时以确保交配成功。随后从这12只成蝇中选取最多3只雄蝇与3只雌蝇（328个培养管中有36管因成虫数量不足而未达到该数量）置于同一培养管中48小时，再次将卵密度调控至每管约50枚。我们分别使用该培养管中孵化的幼虫与羽化的成虫进行RNA采样。并非所有品系的发育同步；为避免因发育时长差异导致品系间的方差，我们并未在同一天收集所有样本。

幼虫采集阶段：当培养管中首次观察到徘徊幼虫（即停止进食、开始寻找化蛹场所的成熟三龄幼虫）时，从培养基中收集每管最多10头非徘徊状态的幼虫，并用液氮快速冷冻。若条件允许，额外收集10头幼虫作为备份（该备份样本用于82份样本中的12份）。其余果蝇均被丢弃（即从未从幼虫培养管中收集成虫）。不同样本的冷冻时间随机分布于当日10:00至24:00之间。采集的幼虫未进行性别鉴定，因此我们认为样本为雌雄随机混合的群体。

成虫采集阶段：我们每日对培养管进行排查。当某一培养管中首次观察到成虫时，将其移除，并在随后48小时内收集该管中所有羽化的成虫。收集的成虫被转移至新鲜培养管中饲养4天以完成成熟，随后以与幼虫相同的方式进行液氮快速冷冻。