A new chamber for studying the behavior of Drosophila

Abstract

Methods available for quickly and objectively quantifying the behavioral phenotypes of the fruit fly, Drosophila melanogaster, lag behind in sophistication the tools developed for manipulating their genotypes. We have developed a simple, easy-to-replicate, general-purpose experimental chamber for studying the ground-based behaviors of fruit flies. The major innovative feature of our design is that it restricts flies to a shallow volume of space, forcing all behavioral interactions to take place within a monolayer of individuals. The design lessens the frequency that flies occlude or obscure each other, limits the variability in their appearance, and promotes a greater number of flies to move throughout the center of the chamber, thereby increasing the frequency of their interactions. The new chamber design improves the quality of data collected by digital video and was conceived and designed to complement automated machine vision methodologies for studying behavior. Novel and improved methodologies for better quantifying the complex behavioral phenotypes of Drosophila will facilitate studies related to human disease and fundamental questions of behavioral neuroscience.

Figure 1. Side-view illustration of typical arrangements of flies in chambers with vertical walls.

Problematic conjunctions occur when a fly clings to the ceiling, partially occluding a fly standing on the floor, and when two flies stand one above the other on the wall.

Figure 2. Sloped walls lessened the probability of problematic conjunctions between flies.

(A) Photograph showing a typical distribution of 50 flies observed in chambers with sloped and vertical walls with associated drawings that depict the contour of the floors of the chambers along the cross section shown (arrow). (B) Comparison of the percent of problematic flies from groups of 50 individuals observed after 1 hour in chambers with sloped (red closed circles) and vertical walls (black open circles).

Figure 3. Trajectories of 50 flies moving for 10 minutes within a chamber designed with sloped walls.

(A) Superposition of the individual trajectories from the group of 50 flies. (B) Individual trajectories of the 50 flies making up the group shown in A with individual males (blue) and females (red) sorted along rows from the shortest to the longest distance traveled (Top left to bottom right).

Figure 4. Sloped walls reduced the variability in a fly's appearance.

(A) Cropped image taken from a movie that included only a small region surrounding the fly, a cropped background image from the same region of the chamber excluding the fly, a differenced image between the cropped image and the cropped background image, and a binerized representation of the difference between the images determined by a threshold. The total number of pixels from the binerized representation of the flies was calculated for each frame. (B, C) Examples using 100-second windows of movie illustrating the lower variability in the total number of pixels extracted from movies of flies recorded within the chambers with sloped walls, as compared to those with vertical walls. The median pixel area was calculated from the entire movie (gray line) and was approximately equal to when the fly was on the floor and stationary in the chambers with sloped walls. (B) Significant deviations from the median pixel area in the chamber with sloped walls corresponded to a hop (arrow) and a period when the fly was grooming (region between dashed lines). (C) Deviations in pixel area in the chamber with vertical walls were due to changes in the fly's appearance as it rotated on the wall between side and head on (astrisk) or moved from the wall onto the ceiling (region between dashed lines). (D) Normalized histogram of deviation in pixel area over the first hour of movie from flies observed in the chambers with sloped (red) and vertical walls (black). Numbers on the x-axis represent the deviation from the median pixel area, where 1 is no deviation and 0.5 and 1.5 are 50% deviation from the median pixel number.

Figure 5. Example of movie images of males courting females, including corresponding errors in automatic classifications by body orientation and identity.

(A) Examples of movie images with body orientations annotated (triangle apex denotes the position of the fly's head) that were extracted with the CADABRA software system. (B) Error rates of body orientations calculated for individual flies from movies recorded for males courting females within the chambers with sloped (red) and vertical (black) walls. Error rates from the chambers with vertical walls were decomposed into erroneous flips in orientation during periods when individual flies were either on the floor or on the wall. Medians (blue) and 25 and 75 percentiles are shown (black box). (C) Examples of movie images with identity annotated while males (blue) courted females (red). Trajectories represent the location of flies for the past 30 frames ( 1 s). Swaps in identity are denoted by the discontinuities in the trajectories and changes in color between triangles representing past locations of flies, and therefore can be compared to a movie image that has been corrected (left). (D) Error rates for the classification of identity between pairs of male and females from movies recorded within the chambers with sloped (red) and vertical (black) walls. Error rates from the chambers with vertical walls were decomposed into swaps in identity during periods when both flies were on the floor, both on the wall, and split with one fly on the floor and the second on the wall. Medians (blue) and 25 and 75 percentiles are shown (black open box). The rate of errors were similar between Wall vs. Split, Mann-Whitney U, p = 0.628; Wall vs. Vertical, Mann-Whitney U, p = 0.864; and Split vs. Vertical, Mann-Whitney U, p = 0.521.

Figure 6. Pairs of males and females in the chambers with sloped walls spend less time near the periphery of the chamber and spend more time near each other.

Normalized loitering probability prior to copulation and the locations when courting flies began copulating for (A) 18 pairs of flies in the chambers with sloped walls and (B) 18 pairs of flies in the chambers with vertical walls. Collective loitering probability normalized by area in 30 concentric regions for flies in the chambers with (C) sloped and (D) vertical walls. Concentric annuli making up the regions were 1 mm thick. (E) Collective mean percent total time (gray lines) and percent total time for individuals from pairs of flies spent near and on the wall in the chambers with sloped (red) and vertical (black) walls. Percentage of pairs of flies beginning to copulate on and near walls is also denoted (green arrowheads). (F) Collective medians (blue lines) of the average distance between pairs of flies prior to copulation in the chambers with sloped (red) and vertical (black) walls. The top and bottom of the boxes represent 25 and 75 percentiles (black open box). (G) Collective means (white lines) and average copulation latencies for pairs of flies in the chambers with sloped (red) and vertical (black) walls. The top and bottom of the boxes represent s.e.m. from collective means (gray filled box).

Figure 7. Drawings and photograph illustrating the new experimental chamber design and setup.

(A) Drawing from the side of the experimental chambers, highlighting the sloped wall of the new chamber design (red box) possessing a severely acute interior angle that prevents flies from moving up the wall and onto the ceiling. The ceiling of the chambers was made of glass coated with silicone paint to limit the frequency and duration flies could cling to its surface. (B) Photograph from a cross section wedge of the chamber showing that the height of the chambers provided sufficient room for flies to carry out their normal range of locomotor behaviors. (C) Technical drawing for the profile of the slope that is described within the text. The red line depicts the slope, the blue line represents the profile of the sigmoid curve near the ceiling that was not used in making the slope, and the dashed line denotes the line tangent to the sigmoid that was matched to linear segment of the slope. (D) Drawing from the side illustrating the experimental setup. Chambers were illuminated with standard fluorescent lights projecting through a screen and a cylinder with a lid made of translucent paper. The behavior of the flies was recorded with a camera mounted above the chambers. Chambers were mounted on an aluminum base to help prevent warping and to hold the chamber above lights used for backlighting.