Edited by: Martin Giurfa, UMR5169 Centre de Recherches sur la Cognition Animale (CRCA), France
Reviewed by: Dennis Pauls, University of Würzburg, Germany; E. Axel Gorostiza, University of Regensburg, Germany
*Correspondence: Simon G. Sprecher
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Animals use various environmental cues as key determinant for their behavioral decisions. Visual systems are hereby responsible to translate light-dependent stimuli into neuronal encoded information. Even though the larval eyes of the fruit fly
The ability of an animal to navigate in response to distinct environmental cues depends on proper perception and processing of sensory stimuli. Light is perceived by specialized photoreceptor neurons (PRs) in the eye, which transmit this information to defined neural circuits in the brain. In many animal species light is perceived as a highly attractive or aversive cue, depending on their life style. The larva of the fruit fly
In the current study, we investigate age- and wavelength-dependency of visual navigation and identify a role of Rh6-PRs for phototaxis in natural lighting conditions. First studies on larval phototaxis were performed over a century ago and several different hypotheses on age-dependency have been supported in the past years. More than 35 years ago it was reported that
While Rh5 is tuned towards blue and Rh6 towards green, the calculated absorption spectra of the two Rhodopsins are overlapping and also include absorption peaks at different wavelengths (Salcedo et al.,
Behavioral experiments were performed with larvae of the following lines: wild-type (WT)
All experiments were performed on 2% agarose plates. 2% agarose (Agarose Standard, Roth) was filled into 24.5 × 24.5 cm plates (BD Falcon BioDishXL, BD Biosciences). At the bottom of these plates we placed a black aluminum plate, in order to increase the contrast. The agarose had to cool down to room temperature before experiments were performed.
At least 20 min before the experiment started food vials (containing larvae) were stored in darkness. With a spoon larvae were transferred into a petri dish. With tap water the larvae were cleaned from the food. With a fine paintbrush 30 larvae were collected and kept in a water drop for up to 10 min. The 30 larvae were transferred into the middle of the agarose plate. All experiments were prepared under red illumination. We performed 10 trials per genotype and assay. The only exception were the outdoor experiments, in which we were comparing light avoidance in the morning (3 trials) and in the afternoon (4 trails).
We used a computer-based tracking-system comparable to one described earlier (Gershow et al.,
For image acquisition we used a customized LabView software (Gershow et al.,
In the tracking system, we used for visual stimulation of the larvae either a projector (EX7200 Multimedia Projector, EPSON) equipped with a bandpass filter (335–610 nm, BG40, Thorlabs) or different LEDs emitting nearly monochromatic light. The projector produced light with an intensity of 2687 μW/cm2. Maximum intensity peaks were at 438 nm with half-widths of 12 nm (blue) and at 549 nm with half-widths of 12 nm (green). The projector was 38 cm away from the middle of the testing plate in a height of 26 cm and orientated with an angle of 40° with respect to the plate.
The colors, intensity peaks and half-widths of the LEDs were: UV-A (368 nm, half-widths: 7 nm), blue (466 nm, half-widths: 11 nm), green (514 nm, half-widths: 17 nm), yellow (595 nm, half-widths: 7 nm) and white (441 nm, half-widths: 13 nm; 586 nm, half-widths: 62 nm). The LEDs were placed 14 cm away from the middle of the agarose plate in a height of 10 cm over its surface and were orientated with an angle of 40° with respect to the plate. The different types of LEDs emitted light with an intensity of 72 μW/cm2 (UV-A), 71 μW/cm2 (blue, yellow and white) and 69 μW/cm2 (green). The “no stimulus groups” are WT larvae (4 days AEL, if not indicated differently), which were not stimulated with a directional light source.
From the recordings generated in the tracking system, we extracted and determined larval position, bearing, body contour, center of mass, position of head and tail and midline of the larvae as previously described (Scantlebury et al.,
We used a compass for describing the heading direction of larvae. 0° indicates heading towards the light source and 180° indicates heading away from the light source. ±90° indicates heading perpendicular to the light source. All four directions were binned in 90°. Like this heading between −45° and +45° was defined as heading towards the light source (0°, +x direction), whereas heading between +135° and −135° was defined as heading away from the light source (180°, −x direction). Heading between +45° and +135° was defined as heading perpendicular to the light source (+90°, +y direction). And heading between −135° and −45° was defined as heading perpendicular to the light source (−90°, −y direction). We calculated a navigation index, in order to analyze the general phototaxis navigational performance. Therefore, the velocity of all larvae in x-direction was divided by the mean run speed in all directions. The navigation index would be −1, if all larvae would navigate uniformly away from the light source. If all larvae would run uniformly towards the light source, the navigation index would be +1. In case the run direction would be unbiased away and towards the light source the navigation index would by 0.
Furthermore, we analyzed the turn direction of larvae which were previously running perpendicular to the light source. If the heading direction before the turn was +90° a turn towards the right was considered as turning towards the light source, whereas a turn to the left was contemplated as a turn away from the light source, and vice versa for previous heading direction to −90°. In these cases a turn to the right was considered as a turn away from the light source, whereas a turn to the left was contemplated as turn towards the light source.
To analyze the navigational parameter “turn magnitude”, we compared the turn size of larvae, which were running previously towards (0°) or away from the light source (180°). The difference between heading before and after the turn was defined as the turn size.
To reveal defects in forward locomotion, we draw manually a virtual circle of 5 cm in diameter around each larva of the 5 and 6 days AEL data sets. The center of the circle was positioned on the respective larva. Larvae which left the circle with the whole body at least once throughout the complete 11 min of the experiment were counted as larvae with normal locomotion whereas larvae which failed to leave the circle were counted as larvae with decreased locomotion.
All outdoor experiments were performed 636 m above the sea level at latitude N 46°47′34.64″ 46.79296° and longitude E 7°9′21.55″ 7.15599°. The experiments were performed between sun altitudes of 19° and 56°. For each experiment 30 larvae were prepared and placed on a 2% agarose plate like described above. The agarose plate was orientated with two borders perpendicular to the sun. Experiments were performed on either clear sunny days or when a layer of clouds were covering the sun. We divided the plate in a neutral zone and a solar and an antisolar zone. The neutral zone was the midline of the plate plus 1 cm in both directions and therefore also orientated perpendicular to the sun position. All larvae where placed at the beginning of the experiment in the neutral zone. Larvae which start in the neutral zone and were navigating away from the sun would end up in the antisolar zone. Larvae which were moving towards the sun would end up in the solar zone. At the end of the experiment we count how many larvae ended up in the different zones of the plate. During outdoor experiments larvae seemed to be less agile/slower than during indoor experiments. After 10 min larval light avoidance behavior was observable (for example for WT larvae), but larvae were still close to the starting region. To give to the larvae more time to navigate away from the starting point (in any direction), we increased experimental time to 20 min. For 20 min larvae could move freely on the plate. We did experiments during two distinct time windows in a day to minimize the effects of the environment. During our experiments the sun has an azimuth of 91°–140° in the morning and of 235°–261° in the afternoon. Between this two time windows the solar and antisolar side changed positions as the position of the sun changes during the day. For each experiment we calculated a preference index by subtracting the number of larvae in the antisolar side from the number of larvae in the solar side divided by the total number of larvae. The temperature of the testing plate was varying between the different experiments (cloudy conditions: 15–23°C and sunny conditions: 13–24°C) and sometimes also during single experiments by up to 4°C. However, the temperature was not varying at a given time point between different points of a testing plate. Experiments of WT and
We used glass tubes, which were 10 cm long and were 1.6 cm in diameter. Two glass tubes were fixed apposed together with transparent tape. We subdivided the two tubes in four sections (two dark areas and two enlighten areas) similar to the tube assay used by Sawin-McCormack et al. (
All data is presented as means and error bars indicate ± SEM. Statistical analyses were performed using standard statistic functions in MATLAB. An unpaired
When given a choice,
As a first assessment of phototaxis we calculated a navigation index. The navigation index is defined as the average velocity along the
During larval life, the animal drastically increases body length from 0.9 mm early to 3.2 mm at 4 days AEL (Figure
Further, we repeated the previously described tube assays with a few modifications (Sawin-McCormack et al.,
The larval eye is composed of two PR-subtypes expressing either Rh5 or Rh6, two Rhodopsins with distinct absorption spectra (Salcedo et al.,
Behavioral studies on phototaxis in
Adult flies progressively reduce their light preference as they age (Le Bourg and Badia,
Sawin-McCormack et al. (
Since
The complex organization of insect compound eye allows the animal to detect various types of visual cues including color vision, motion detection or polarized light vision (Borst,
Other authors suggested previously that larvae could use in dependence on lighting conditions klinotacticle or tropotacticle mechanisms (Hinnemann et al.,
T-HH and SGS designed the experiments; wrote the manuscript. T-HH performed the experiments and analyzed the data. SGS coordinated the study.
This work was funded by the Swiss National Science Foundation (31003A_149499 to SGS) and the European Research Council (ERC-2012-StG 309832-PhotoNaviNet to SGS).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank C. Desplan and the Bloomington