Predicting death by the loss of intestinal function

Populations Five large independent populations of Drosophila melanogaster were used in this experiment. Two of these populations, ACO and CO, are large, outbred populations that have been maintained on different age-at-reproduction schedules for hundreds of generations. The ACO population was maintained on 9 day discrete generation cycles. The CO population was kept on 28 day discrete generation cycles. The remaining populations, S93, A4 3852 and Canton S (CAS), were inbred lines raised on three week cycles in the Long lab at the University of California, Irvine. All populations were raised in identical conditions of temperature, food, cultures and density for three generations prior to these experiments . Mortality Assay Adult, 14 day old (from egg) flies were knocked out with CO2 gas and placed into individual plastic straws about 4 inches in length and capped with plastic pipette tips on both ends (Fig. 1). During anesthetization, a steady supply of CO2was flowing through a semi-porous plate. The flies were placed on the plate and separated by gender and each fly was gently swept into the plastic straw using a fine painters brush. An equal number of females and males were used per population. Food was provided to each fly at one end of the straw. Each fly was transferred to a new straw with new food and new pipette tips every 3 days to maintain a clean environment. The straw length and girth permitted individuals to fly from one end to the other. The process of transferring the flies, as well as daily checking of the flies, required a light tapping of the fly into the pipette tip. Cohorts of about 56 adult flies, equal numbers of males and females from each of the five populations were exposed to either control food or food with one of six dyes (table 1) added to their food. Substantial replication was used. Thus, the original dye, SPS Alfachem, was replicated in 5 different populations, and each population was replicated in 6 different dye environments. The use of different FDA FD&C Blue dye #1’s permitted us to determine if the development of the SMURF phenotype was sensitive to the particular dye used. By using a combination of different populations of D. melanogaster,which varied in levels of inbreeding, we could determine if the development of the SMURF phenotype was limited to inbred populations. The flies were exposed to the blue dyes from day 14 (from egg) continuously to their death. Each fly was individually checked underneath a microscope and light to see if it had become a ‘smurf’. Smurf status required that the entire body changed to any variation of a blue color. This was an important distinction as all the Drosophilaflies fed food with a blue dye would have visible blue coloring in only the gut portion when they weren’t a Smurf. Some of the dyes resulted in a slight variation in blue color in the Smurfs. Every day under a microscope with a light we looked for any change of color in the fly thorax, head and abdomen. If the fly was any shade of blue in all three sections, it was marked as a Smurf and was then checked daily to see when it died. We did not limit our observations to individual sections of the fly, such as only the thorax, for our evaluation of when a fly became a Smurf. Tapping We did the tapping experiment to see if the physical disruption, the process of tapping the fly into the pipette tip, affected the mean longevity and lifespan of the fly. A total of 164 ACO flies were chosen for this assay – 83 males and 81 females. The 164 flies were placed into regular food straws with no dye. A total of 84 flies (42 male and 42 female) were tapped 5 times daily, mimicking the checking that occurred in the original experiment, and the other 81 (41 males and 39 females) flies were not tapped. The flies were transferred to new straws, with fresh food and new pipette tips every 3 days. Each fly was checked daily for movement and if no movement was detected, the fly was classified as deceased on that day. Only ACO flies were used as the purpose of the Tapping experiment was to see if our methods for checking for Smurf flies would affect the mean longevity of the fly. Food & Dyes Flies were provided with banana-molasses food with one of the dyes added. The control flies received only banana molasses food in their respective straws. The recipe for the banana molasses food used in the lab, as well as the experiment, can be found in the Supplemental Portion. Food with dye was prepared by mixing 2.5 grams of each dye to create a 100 ml solution of the banana molasses food mixed with the dye (2.5% wt/vol). Food was always prepared the day before it was needed and stored in a refrigerator until it was used. The dyes were kept separate and carefully handled so no cross-contamination occurred during the preparation and food blending process. Statistical Analysis To analyze the effects of dye, sex and population on longevity we let yijklbe the age at death of the lth individual of sex-i(i=1 (female), 2 (male)), treatment-j (j=1,,..,7 (see table 1 of paper, 7=control)), and population-k(k=1,..,5 (see table 1 of paper)), whereds=0 if s=1, and 1 otherwise, eijklis an error term assumed to have normal distribution with mean 0 and variance s2. An initial test showed no significant differences between sexes so the final model tested did not include the bparameter. These tests were run with R (version 3.4.3, R Core team, 2017) and the lmfunction. Pairwise tests with Bonferroni corrections for simultaneous tests were conducted with the R emmeansfunction. At the time of death each fly was classified according to their sex, population, treatment, and Smurf status (blue: yes or no). Using hierarchical log-linear models (loglmfunction in the R MASS package) we tested in succession whether sex, treatment, and population would have an effect on Smurf status at the time of death. A t-test was performed on the Tapping Experiment results, comparing the mean longevity of the tapped flies versus the non-tapped flies to see if the mechanical disruption would affect their mean longevity.

种群
本实验使用了五个独立的大型黑腹果蝇（Drosophila melanogaster）种群。其中ACO与CO两个种群为大型远交种群，已在不同的繁殖年龄周期下维持数百代：ACO种群以9天离散世代周期饲养，CO种群则采用28天离散世代周期。剩余的S93、A4 3852及Canton S（CAS）为近交系，由加州大学欧文分校朗实验室（Long lab）以三周周期饲养。所有种群在实验前均在一致的温度、食物、培养条件及种群密度下饲养三代。

死亡率测定试验
将从卵孵化后14日龄的成年果蝇用二氧化碳气体麻醉，转移至长约4英寸的独立塑料吸管中，两端以塑料吸头密封（见图1）。麻醉过程中，持续向半多孔板通入稳定流量的二氧化碳，将果蝇置于板上并按性别分离，随后使用细画笔将每只果蝇轻轻扫入塑料吸管。每个种群使用等量的雌雄果蝇。在吸管一端为果蝇提供食物，每3天将果蝇转移至装有新鲜食物和新吸头的新吸管中，以维持清洁环境。吸管的长度与内径允许果蝇在两端间飞行。转移果蝇及每日检查果蝇时，需轻轻将果蝇弹入吸头。

从五个种群中各取等量雌雄果蝇组成约56只的队列，分别暴露于对照食物或添加了六种染料之一的食物（见表1）。实验设置了充分的重复：原始染料SPS Alfachem在5个不同种群中重复，每个种群在6种不同染料环境下重复。使用不同批次的FDA FD&C蓝染料#1，可探究SMURF表型的发生是否对所用染料存在敏感性。结合不同近交程度的黑腹果蝇种群，可判断SMURF表型的发生是否仅局限于近交种群。果蝇从卵孵化后第14天起持续暴露于蓝染料直至死亡。

在显微镜与光照下逐只检查果蝇是否变为“SMURF”。SMURF状态的判定标准为：果蝇全身变为任意深浅的蓝色。这一区分十分关键，因为未成为SMURF的喂食蓝色染料的果蝇，仅肠道部分可见蓝色着色。部分染料会使SMURF个体的蓝色色调产生细微差异。每日均在带光照的显微镜下观察果蝇胸部、头部和腹部的颜色变化，若果蝇三个部位均呈现任意深浅的蓝色，则标记为SMURF，随后每日检查直至其死亡。本次评估未局限于果蝇的单一部位（如仅胸部）来判定果蝇是否成为SMURF。

敲击试验
开展敲击试验旨在探究物理扰动——即“将果蝇弹入吸头”的操作——是否会影响果蝇的平均寿命与存活时长。本次试验共选取164只ACO种群果蝇，其中雄性83只、雌性81只。将164只果蝇置于无染料的常规食物吸管中：其中84只（雌雄各42只）每日被敲击5次，以模拟原试验中的检查操作；剩余81只（雄性41只、雌性39只）不进行敲击操作。每3天将果蝇转移至装有新鲜食物和新吸头的新吸管中。每日检查每只果蝇的活动情况，若未检测到活动，则判定该果蝇当日死亡。本次敲击试验仅使用ACO种群果蝇，目的是探究原试验中检查SMURF果蝇的操作是否会影响果蝇的平均寿命。

食物与染料
果蝇喂食添加了染料的香蕉-糖蜜食物，对照组果蝇仅喂食普通香蕉-糖蜜食物。本实验室及本试验所用的香蕉-糖蜜食物配方详见补充材料。添加染料的食物制备方式为：将2.5克每种染料与100毫升香蕉-糖蜜食物混合，配制成质量体积比为2.5%（wt/vol）的溶液。食物均在使用前一日制备并存放在冰箱中直至使用。染料单独存放并谨慎操作，以避免制备及混合食物过程中出现交叉污染。

统计分析
为分析染料、性别及种群对寿命的影响，设$y_{ijkl}$为第$i$个性别（$i=1$为雌性，$i=2$为雄性）、第$j$种处理（$j=1,…,7$，详见论文表1，其中$j=7$为对照组）、第$k$个种群（$k=1,…,5$，详见论文表1）的第$l$个个体的死亡年龄，其中当$s=1$时$d_s=0$，否则$d_s=1$，$e_{ijkl}$为服从均值为0、方差为$sigma^2$的正态分布的误差项。初步检验显示性别间无显著差异，因此最终模型未纳入性别参数。所有检验均使用R软件（版本3.4.3，R核心团队，2017）及`lm`函数完成。使用R的`emmeans`函数进行Bonferroni校正的两两比较检验。果蝇死亡时，需按其性别、种群、处理及SMURF状态（蓝色：是/否）进行分类。使用分层对数线性模型（R MASS包中的`loglm`函数），依次检验性别、处理及种群是否会对死亡时的SMURF状态产生影响。针对敲击试验的结果，采用t检验比较敲击组与非敲击组果蝇的平均寿命，以探究机械扰动是否会影响其平均寿命。