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A high throughput method for egg size measurement in Drosophila.

ABSTRACT: Life-history traits are used as proxies of fitness in insects including Drosophila. Egg size is an adaptive and ecologically important trait potentially with genetic variation across different populations. However, the low throughput of manual measurement of egg size has hampered the widespread use of this trait in evolutionary biology and population genetics. We established a method for accurate and high throughput measurement of Drosophila egg size using large particle flow cytometry (LPFC). The size estimates using LPFC are accurate and highly correlated with the manual measurements. The measurement of egg size is high throughput (average of 214 eggs measured per minute) and viable eggs of a specific size can be sorted rapidly (average of 70 eggs per minute). Sorting by LPFC does not reduce the survival of eggs making it a suitable approach for sorting eggs for downstream analyses. This protocol can be applied to any organism within the detectable size range (10-1500 µm) of the large particle flow cytometers. We discuss the potential applications of this method and provide recommendations for optimizing the protocol for other organisms.

SUBMITTER: Barghi N

PROVIDER: S-EPMC9992389 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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A high throughput method for egg size measurement in Drosophila.

Barghi Neda N Ramirez-Lanzas Claudia C

Scientific reports 20230307 1

Life-history traits are used as proxies of fitness in insects including Drosophila. Egg size is an adaptive and ecologically important trait potentially with genetic variation across different populations. However, the low throughput of manual measurement of egg size has hampered the widespread use of this trait in evolutionary biology and population genetics. We established a method for accurate and high throughput measurement of Drosophila egg size using large particle flow cytometry (LPFC). T ...[more]

PMID: 36882448

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Project description:Drosophila melanogaster can be used to identify genes with novel functional roles in neuronal plasticity induced by repeated consumption of addictive drugs. Behavioral sensitization is a relatively simple behavioral output of plastic changes that occur in the brain after repeated exposures to drugs of abuse. The development of screening procedures for genes that control behavioral sensitization has stalled due to a lack of high-throughput behavioral tests that can be used in genetically tractable organism, such as Drosophila. We have developed a new behavioral test, FlyBong, which combines delivery of volatilized cocaine (vCOC) to individually housed flies with objective quantification of their locomotor activity. There are two main advantages of FlyBong: it is high-throughput and it allows for comparisons of locomotor activity of individual flies before and after single or multiple exposures. At the population level, exposure to vCOC leads to transient and concentration-dependent increase in locomotor activity, representing sensitivity to an acute dose. A second exposure leads to further increase in locomotion, representing locomotor sensitization. We validate FlyBong by showing that locomotor sensitization at either the population or individual level is absent in the mutants for circadian genes period (per), Clock (Clk), and cycle (cyc). The locomotor sensitization that is present in timeless (tim) and pigment dispersing factor (pdf) mutant flies is in large part not cocaine specific, but derived from increased sensitivity to warm air. Circadian genes are not only integral part of the neural mechanism that is required for development of locomotor sensitization, but in addition, they modulate the intensity of locomotor sensitization as a function of the time of day. Motor-activating effects of cocaine are sexually dimorphic and require a functional dopaminergic transporter. FlyBong is a new and improved method for inducing and measuring locomotor sensitization to cocaine in individual Drosophila. Because of its high-throughput nature, FlyBong can be used in genetic screens or in selection experiments aimed at the unbiased identification of functional genes involved in acute or chronic effects of volatilized psychoactive substances.

Project description:BackgroundLike most living organisms, the fruit fly Drosophila melanogaster exhibits strong and diverse behavioural reactions to light. Drosophila is a diurnal animal that displays both short- and long-term responses to light, important for, instance, in avoidance and light wavelength preference, regulation of eclosion, courtship, and activity, and provides an important model organism for understanding the regulation of circadian rhythms both at molecular and circuit levels. However, the assessment and comparison of light-based behaviours is still a challenge, mainly due to the lack of a standardised platform to measure behaviour and different protocols created across studies. Here, we describe the Drosophila Interactive System for Controlled Optical manipulations (DISCO), a low-cost, automated, high-throughput device that records the flies' activity using infrared beams while performing LED light manipulations.ResultsTo demonstrate the effectiveness of this tool and validate its potential as a standard platform, we developed a number of distinct assays, including measuring the locomotor response of flies exposed to sudden darkness (lights-off) stimuli. Both white-eyed and red-eyed wild-type flies exhibit increased activity after the application of stimuli, while no changes can be observed in Fmr1 null allele flies, a model of fragile X syndrome. Next, to demonstrate the use of DISCO in long-term protocols, we monitored the circadian rhythm of the flies for 48 h while performing an alcohol preference test. We show that increased alcohol consumption happens intermittently throughout the day, especially in the dark phases. Finally, we developed a feedback-loop algorithm to implement a place preference test based on the flies' innate aversion to blue light and preference for green light. We show that both white-eyed and red-eyed wild-type flies were able to learn to avoid the blue-illuminated zones.ConclusionsOur results demonstrate the versatility of DISCO for a range of protocols, indicating that this platform can be used in a variety of ways to study light-dependent behaviours in flies.

Dataset Information

A high throughput method for egg size measurement in Drosophila.

Publications

A high throughput method for egg size measurement in Drosophila.

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