Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized <em>Drosophila</em>

Abstract

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO 2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO 2 in Drosophila melanogaster. The impact of CO 2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

The effect of gut microbiota elimination in Drosophila melanogaster: A how‐to guide for host–microbiota studies

“…In Drosophila , and other insect research, aspirators are commonly used to move individuals between treatments, as it allows for individuals to be manipulated without the use of carbon dioxide anesthesia, which has been shown to negatively impact on mating behavior in some species (e.g. Verspoor, Heys, & Price, ). Producing axenic or egg‐dechorionated individuals inhibits this ability to aspirate flies directly, in order to prevent external bacteria being transmitted onto the fly or their immediate environment, which could potentially confound experimental results.…”

“…In Drosophila, and other insect research, aspirators are commonly used to move individuals between treatments, as it allows for individuals to be manipulated without the use of carbon dioxide anesthesia, which has been shown to negatively impact on mating behavior in some species (e.g. Verspoor, Heys, & Price, 2015).…”

Ilex paraguariensis extracts extend the lifespan of Drosophila melanogaster fed a high-fat diet

“…The average body weight per fly in each group was recorded. To confirm food consumption, we used the dye method in accordance with the protocol described by Verspoor et al ( 15 ).…”

Preregistered Report: The Effects of Marking Methodology on Mate Choice in Drosophila melanogaster

“…Body paint involves the application of a small dot of acrylic paint on the thorax (e.g., Dukas, 2010;Tan et al, 2013), whereas wing marking uses a permanent marker to dot one or both wings (current authors, unpublished data). Food coloring can be dropped directly onto standard food media that is subsequently ingested by Drosophila (e.g., Avent et al, 2008;Verspoor, Heys et al, 2015). Flies can be placed on dyed food at eclosion or moved to this diet prior to mate choice trials, and color becomes visible in the abdomen within ~24 h of ingestion.…”

“…One of the biggest problems associated with marking methods such as wing clipping, permanent marker, and acrylic body paint is the requirement for anesthesia to allow application of the mark (Barron, 2000;Verspoor, Heys et al, 2015). In D. melanogaster, CO2 anesthesia has been shown to lead to a considerable increase in latency to mate, even following a 20-hour recovery period (Barron, 2000;Verspoor, Heys et al, 2015). This has led some researchers to suggest that CO2 anesthesia should be avoided when using Drosophila in behavioral studies (Barron, 2000).…”