Optimization of wrMTrck to monitor Drosophila larval locomotor activity

Abstract

An efficient and low-cost method of examining larval movement in Drosophila melanogaster is needed to study how mutations and/or alterations in the muscular, neural, and olfactory systems affect locomotor behavior. Here, we describe the implementation of wrMTrck, a freely available ImageJ plugin originally developed for examining multiple behavioral parameters in the nematode C. elegans. Our optimized method is rapid, reproducible and does not require automated microscope setups or the purchase of proprietary software. To demonstrate the utility of this method, we analyzed the velocity and crawling paths of two Drosophila mutants that affect muscle structure and/or function. Additionally, we show that this approach is useful for tracking the behavior of adult insects, including Tribolium castaneum and Drosophila melanogaster.

Keywords: Behavior assay; Drosophila; Larval locomotion; Muscle; Tribolium.

Fig. 1

Larval locomotion recording setup. (A, B) Wandering Drosophila L3 larvae (A) were selected and placed upon apple juice agar plates dyed with bromophenol blue (B, arrows indicate larvae). (C) The camera was mounted upside down in an enclosed container to eliminate stray light. Scale bar, 0.05 mm (A), 1.0 mm (B).

Fig. 2

ImageJ and WrMTrck analysis work flow. (A) After video collection, the resulting files were opened in ImageJ (panel 1), cropped to the area of interest (panel 2), and inverted so the larvae appear dark on a light background (panel 3). After applying the Kalman stack filter (panel 4), the image threshold was adjusted to increase the contrast of pixels corresponding to larvae (panel 5). Finally, application of the WrMTrck plugin assigned a number to each larva after data analysis.

Fig. 3

Locomotion is reduced in tn and parkin mutants. (A) Individuals homozygous for mutations in tn and parkin travel slower than their WT counterparts. (B) Diagram of path parameters, including the length (red) and distance (purple) from start to finish measured in WrMTrck. (C, D) Quantitation of the distance (C) and total length traveled (D). Both are reduced in tn, but not parkin mutants, compared to WT larvae. Data was analyzed using one-way ANOVA with Dunnett’s post hoc test (mean = +/− SEM) (* p < 0.05; **** p < 0.0001; ns = not significant).

Fig. 4

Muscle-specific RNAi knockdown of tn and parkin result in measurable locomotion defects. (A) qRT-PCR results demonstrate that tn and parkin transcripts are reduced upon ubiquitous induction of RNAi by the tubulin promoter (tub-Gal4). (B–D) A reduction in tn and parkin RNA levels by a muscle Gal4 driver (24B-Gal4) results in decreased larval locomotion speeds (B), a shorter distance traversed from start to end points (C), and a loss of total distance traveled (D) compared to 24B-Gal4/+ controls. Data was analyzed using one-way ANOVA with Kruskal-Wallis test (A) or Dunnett’s post hoc test (B–D) (mean = +/− SEM) (*** p < 0.0005; **** p < 0.0001; ns = not significant).

Fig. 5

Traces of larval path routes. (A–C) L3 larvae of the indicated genotypes were monitored for 120 sec and path lengths analyzed using ImageJ and WrMTrck. Compared to WT larvae which traverse relatively straight paths (A), both muscle-specific parkin RNAi (B) and tn RNAi (C) larvae make frequent turns and paths appear uncoordinated. (D) Quantitation of normal vs. abnormal paths traveled by the larvae of the indicated genotypes.

Fig. 6

Tracking and analysis of adult Drosophila and Tribolium. (A, C) wrMTrck can be used to monitor the velocity and paths traveled in Drosophila (A, B) and Tribolium (C, D) adults.