Department of Genetics and Biochemistry and Center for Human Genetics, Clemson University.
Department of Genetics and Biochemistry and Center for Human Genetics, Clemson University;
J Vis Exp. 2021 Jun 14(172). doi: 10.3791/62771.
Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.
在果蝇中定量食物摄入量可用于研究与消耗相关特征的遗传和生理基础、其环境因素以及许多物质的毒理学和药理学效应。目前实施的几种方法都不适用于高通量测量。微量板饲养测定法(MFA)是为了通过吸光度来定量测量单个蝇类消耗液体食物而开发的。在该测定法中,蝇类从 1536 孔微孔板的选定孔中消耗液体食物培养基。通过将稀释示踪染料掺入液体食物培养基中并将已知体积加载到每个孔中,消耗前后孔的吸光度测量反映了体积的变化(即消耗的体积)。为了使该方法能够进行高通量分析,设计了一种 3D 打印耦合器,可将蝇类单独分类到 96 孔微孔板中。该装置精确地将 96 孔和 1536 孔微孔板对准,使每只蝇类可以接触多达 4 个孔进行消耗,从而除了常规消耗外还可以定量食物偏好。此外,该装置具有阻隔条,可在打开和关闭位置之间切换,从而可以一次控制一组样品的容纳和释放。该方法可以同时对许多蝇类消耗水溶液进行高通量测量。它还有可能适用于其他昆虫,并用于筛选营养物质、毒素或药物的消耗。