Age- and Wavelength-Dependency of Drosophila Larval Phototaxis and Behavioral Responses to Natural Lighting Conditions

Abstract

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 Drosophila melanogaster are comparably simple, they comprise two types of photoreceptor neurons (PRs), defined by different Rhodopsin genes expressed. Recent findings support that for light avoidance Rhodopsin5 (Rh5) expressing photoreceptors are crucial, while Rhodopsin6 (Rh6) expressing photoreceptors are dispensable under laboratory conditions. However, it remains debated how animals change light preference during larval live. We show that larval negative phototaxis is age-independent as it persists in larvae from foraging to wandering developmental stages. Moreover, if spectrally different Rhodopsins are employed for the detection of different wavelength of light remains unexplored. We found that negative phototaxis can be elicit by light with wavelengths ranging from ultraviolet (UV) to green. This behavior is uniquely mediated by Rh5 expressing photoreceptors, and therefore suggest that this photoreceptor-type is able to perceive UV up to green light. In contrast to laboratory our field experiments revealed that Drosophila larvae uses both types of photoreceptors under natural lighting conditions. All our results, demonstrate that Drosophila larval eyes mediate avoidance of light stimuli with a wide, ecological relevant range of quantity (intensities) and quality (wavelengths). Thus, the two photoreceptor-types appear more likely to play a role in different aspects of phototaxis under natural lighting conditions, rather than color discrimination.

Keywords: Drosophila larva; navigation; photoreceptor; phototaxis; visual system.

Figure 1

Larval phototaxis is robust throughout all developmental stages. (A) Schematic illustration of the behavioral set-up and larval phototaxis strategies. 30 larvae were placed in the center of the testing plate. The light source (in this case a projector) was positioned on one side of the testing plate. We used a navigational compass. Larvae heading towards the light source would head towards 0°. Heading away from the light source would be heading to 180° and a perpendicular orientation towards the light source would be towards +90° or −90°. Red light illumination was needed for the camera, which recorded larval behavior from top. Orientated perpendicular towards the light source, larvae are able to bias their turn direction away from the light source. Larvae make greater turns, when previously heading towards the light, compared with turns, when previously heading away from the light. (B) Navigation indices of foraging larvae (2, 3, 4 or 5 days after egg laying (AEL)) and wandering larvae (6 days AEL). Rejection of the null hypothesis that the data set and the no stimulus data set have the same mean: ***p < 0.001, unpaired t test. (C) Probability of turn direction after runs heading perpendicular to the light source. The Fisher’s exact test was performed to test that the probabilitiy of turning away from the light source is significantly different from turn direction probability of the respective no stimulus group: ***p < 0.001. (D) The difference of turn size of reorientations where runs were heading previously towards and away from the light source (turn size previously heading towards the light source—turn size previously heading away from the light source). Rejection of the null hypothesis that the data set of the stimulated animals and of the no stimulus group have the same mean: **p < 0.01, ***p < 0.001, unpaired t test.

Figure 2

Larval locomotion is changing with larval body size and age. (A) Mean body size of different larval age groups. Mean run length (B), mean run speed (C) and the calculated ratio of mean run speed to body size (D) of different larval age groups. Pause rate (E) and turn rate (F) per min of the different aged larval groups. (A–F) Data show the mean and error bars indicate ± SEM. Rejection of the null hypothesis that the data set of two groups have the same mean: *p < 0.05, ^***p < 0.001, unpaired t test. (G) Two representative larval tracks of larvae corresponding to the age 6 days AEL. A larval track consists of all runs and turns the respective larvae is performing during the whole experiment. One track belongs to the category “normal locomotion” (G′), whereas the other track belongs to the category “decreased locomotion” (G″). “S” indicates the starting and “E” the end point of the respective larval track. Decreased locomotion was defined as loss of the ability to leave a virtual circle with 5 cm in diameter throughout the experiment. (H) Pie chart showing the percentage of larvae showing normal (green) and decreased (red) locomotion for larvae of age 5 and 6 days AEL.

Figure 3

Age-dependent decreased forward locomotion can impact on the outcome of light preference tests. (A) Schematic illustration of the tube-assays. Ten larvae were either placed in the middle of section “B” or close to the light-dark boundary. Larvae were stimulated with white light emitting diodes (LEDs) from top and were allowed to move freely in the tubes for 10 min. (B) Preference indices of wild-type (WT) 6 days AEL larvae after 5 and 10 min. (C) Preference indices of WT larvae for light section “D” against “B”. (D) Proportion of larvae forming a pupa within and after 6 h post experiment. (E) Proportion of larvae ended up in the different sections. Only larvae were taken into account, which were forming a pupa within 6 h post experiment. Data show the mean and error bars indicate ± SEM. Rejection of the null hypothesis that the mean of the data set is chance or that means of two groups are the same: *p < 0.05, **p < 0.01, ***p < 0.001, One sample t test for tests against chance, paired t test for testing the preference index after 5 and after 10 min against each other and unpaired t test for testing among different groups.

Figure 4

Light with wavelengths ranging from ultraviolet (UV) to green is robustly avoided by WT and Rhodopsin6 (Rh6) mutant larvae. (A) Spectra of UV (violet line), blue (blue line), green (green line) and yellow (yellow line) light emitted by LEDs, which were used for our behavioral experiments. The different sets of monochromatic and white LEDs emitted light with equal energy levels (69–72 μW/cm²). (B) Navigation indices of WT larvae stimulated with monochromatic and white light. (C) Probability of turn direction of WT larvae after runs heading perpendicular to the different LEDs. (D) The delta of reorientation magnitudes of turns where runs were heading previously towards and away from the LEDs. (E) Navigation indices of Rh6 and Rhodopsin5 (Rh5) single and double mutant larvae stimulated with monochromatic light. The mutant larvae turn direction probability (F) and delta of turn size (G). (B–G) Data show the mean and error bars indicate ± SEM. For UV, blue, green, yellow and white light stimulation the data sets are indicated by violet, blue, green, yellow background or black frame respectively. (B,D,E,G) Rejection of the null hypothesis that the data set and the no stimulus data set have the same mean: *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test. (C,F) Fisher’s exact test was performed to test that the probability of turning away from the light source is significantly different from the turn probability of the no stimulus control: *p < 0.05, **p < 0.01, ***p < 0.001. (B–G) The Benjamini Hochberg procedure was performed to adjuste p-values.

Figure 5

Drosophila larvae phototaxis under natural lighting conditions. (A) Schematic of the outdoor behavioral assay. Azimuth is the horizontal angle from north to the position of the sun. Azimuth defines in which direction the sun is. North has azimuth 0° and east has azimuth 90°. Altitude is the vertical angle from the horizon (0°) to center of the sun and defines the elevation of the sun. Zenith has altitude 90°. (B) Preference indices of WT larvae under cloudy conditions in the morning and evening respectivly. (C,D) Preference indices of WT, Rh6 single mutant larvae, Rh5 single mutant larvae, Rh5 and Rh6 double mutant larvae and Gr28b mutant larvae stimulated with either direct sun light (C) or with diffuse sun light under cloudy conditions (D). Data show the mean and error bars indicate ± SEM. Rejection of the null hypothesis that the mean of the data set is 0: *p < 0.05, **p < 0.01, ***p < 0.001, One sample t test. Or rejection of the null hypothesis that the mean of two data set is the same: *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.