Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.
Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.
Here we describe a behavioral assay based on the larval preference for dark (or light). Larvae react with a strong and stereotypic photonegative response during foraging stages (L1 to early L3)1. The assay is aimed to assess the photophobic behavior of the larva and compares the light or dark preference of a group of larvae moving freely in a Petri dish coated with agar. This behavioral assay not only provides information about the sensitivity, integration and temporal plasticity of the visual system, it further provides hints on how light sensitivity and its process is controlled by the circadian system.
The Drosophila larval eye (also termed Bowlig Organ; BO), is the main organ for light perception. Each eye is composed of 12 photoreceptors (PR), eight PRs express the green-sensitive rhodopsin6 (rh6) and four PRs express the blue-sensitive rhodopsin5 (rh5)2,3. In addition to PRs, also class IV multidendritic neurons, which cover the larval body wall, have been identified to respond to noxious light intensities4,5. It is also known that pacemaker neurons situated in the central larval brain express the light sensitive protein Cryptochrome (Cry) that acts as clock intrinsic blue light sensor within the brain6,7. Intriguingly photophobicity of wild type animals shows a circadian component at different time points during the course of day and night when testing with this assay. Responses to light of foraging L3 larva showed stronger photophobicity at dawn and lower photophobicity at dusk when tested for light-dark preference7. Interestingly only Rh5-PRs are required for light avoidance, while Rh6-PRs are dispensable. Both, Rh5-PRs and Rh6-PRs are involved in resetting the molecular clock by light8. The Cry pathway must be coordinated with the other light-sensing pathways to orchestrate an appropriate behavioral output in the course of the day. Acetylcholine in PRs plays an essential role in light avoidance behavior as well as entrainment of the molecular clock. Blocking acetylcholine neurotransmission from PRs to circadian pacemaker neurons reduces the photophobic response in the light-darkness preference assay8. Employing the same assay, two symmetrical pairs of neurons have been recently identified to switch the light preference of the third larval instar of Drosophila9. These two pairs of neurons may be functioning during late larval stages, when animals leave the food to presumably find an appropriate pupariation site. However, the question of how the visual pathways interact and control larval visual behavior in a circadian manner remains largely unanswered. The light preference assay allows comparisons among circadian time points, fly lines and circadian state under different light qualities. The assay is easily prepared and inexpensive and has been useful previously in several labs to describe and study light derived behavior in the larva.
1. Larval Rearing
2. Test Setup
3. Plates Preparation
4. Light Preference Test
5. Data Analysis
Following the protocol described above, we tested light-dark preference in early third larval stadium of wild type Canton-S flies at two different circadian times CT0 and CT12. Adults were reared 12-hr light-12-hr dark and left to lay eggs for 12 hr. Larvae grow the first two days under the same light-dark regime. Since we wanted to test circadian modulation under constant conditions (free running of the circadian clock), larvae were then transferred to constant darkness for the next three days until test was performed (Figure 3A).
Here, we used 350 lux since it has been shown that this is the optimal light intensity to detect differences of light response of Drosophila larva along the circadian cycle in comparison to other intensities (70 and 600 lux)7. Differences in light sensitivity, and therefore in light response is observed when comparing CT0 with CT12. In Drosophila, CT0 coincides with dawn, the half cycle between CT0-CT12 is considered the subjective day and from CT12 to CT24 the subjective night12. Light photosensitivity is higher briefly after the transition from darkness to light, when almost 77% of the larvae prefer the darkness compared with almost 69% of dark preference at the early subjective night (Figure 3B). This is also reflected by the dark preference index (PREF(darkness)) calculated with the formula presented above for each experiment. Averaging all repetitions we obtained a preference index of 0.52 for CT0 and of 0.36 for CT12. Using the Wilcoxon test (Origin) a statistical significant difference between two time points is shown (p=0.0229).
Figure 1. (A) Marking quadrants in Petri dish lids. Quadrants can be marked in the Petri dish lid with help of a printed quadrant used as canvas. Posteriorly the first layer of aluminum foil can be glued on the external surface of the plate and be covered with black tape in the two opposite quadrants for darkness. (B) Schematic set up for the light-dark preference test. (C) A Petri dish covered with dark quadrants and filled with 2.5% agar, ready to be used for experiments. Click here to view larger figure.
Figure 2. Light-dark preference test, example of a dish right after setting larva on the test plate (Start) and after 5 min (End). Typically, we use a group of 30 animals for each experiment.
Figure 3. (A) Light regime followed to test light preference in feeding L3 larva. (B) Percentage of dark preference for both time points tested (CT0 and CT12). Percentage of larva that preferred darkness and light are counted for each repetition and all repetitions averaged. Larvae in the borders of the plate or digging on the agar are considered as neutral. (C) Dark preference index calculated for the same time points. Significant statistical difference between groups is shown by the Wilcoxon sum-rank test (p<0.05). (N=18 (540 larvae) per time point). Click here to view larger figure.
The light preference test described takes advantage of the larval innate photobehavior. The assay is easy to establish, allows many repetitions at low cost and delivers valuable information about light sensing and processing. The experimental paradigm allows relatively quick quantification of how many individuals prefer light or dark. Such preference can be displayed as crude percentages or alternatively as Preference Index (PREF). The PREF is expressed as the difference of animals that preferred light and animals that preferred darkness divided by the total of animals.
A crucial point for the light preference test is the duration of the experiments. Here, we tested five minutes, however it is possible that other genotypes or under other light qualities differences are not so clear using this experiment duration. For certain stimuli (gustatory) we have realized that longer experiments, with 20 min of duration, deliver cleaner preferences with smaller variability. Testing different experimental durations could be fair especially when using genotypes where locomotion or sensitivity is affected and larvae require longer time to explore the testing plate. In that case, the time should be equally adjusted for controls and experimental genotypes.
The assay also allows detecting differences among time points of the day through the circadian cycle as shown here and in previous reports7,13. Light responses to photic stimuli are strongly regulated by the circadian clock, presumably by modulating the sensitivity of photoreceptors. Larval responses to light decrease during the first two hours of the day and become stable during the remaining ten hours of the light phase. To compare different groups in the course of the day, it is crucial to make experiments at equivalent or identical time points of the circadian cycle for each group. In addition, it is also important to avoid changes that can affect larval behavior, as thermal fluctuations or light inputs previous to experiments if working in darkness. For this, it is important to rear the larvae at the same temperature and humidity than the ones used for experimentation and be careful by keeping animals in darkness when needed.
At least three recently described pathways contribute to light sensing in Drosophila larva5,14: 1) the rhodopsin dependent system mediated by the larval eye; 2) the multidendritic neurons class IV, spread along the larval body and 3) clock intrinsic blue light sensors expressing Cry. Contribution of distinct light-sensing pathways for visual behavior can be addressed by the preference test described here. Different sources of illumination with specific intensities and wavelengths can be used to test different light qualities. This possibility can depend on details about the sensitivity of individual elements of the light sensing system and how such light inputs are processed. For a more detailed experimentation regarding light stimulation, the assay can be easily modified to test different light qualities. Increasing and decreasing light intensities, as well as testing different light wavelengths is possible by using self-made LED lamps with specific wavelengths (many possibilities are available in the market). Control of such lamps with power supplies offers a good range of intensities. These possibilities for manipulating light qualities provide a wide range of variables to be tested with the light preference test.
Moreover, Drosophila larvae are able to associate punishment or rewards with either light or darkness, modifying their native photobehavior15. For this, an adaptation of the protocol described was employed showing the versatility of the assay. In summary, the light-dark preference test is a valuable instrument contributing with a better understanding of rapid and circadian behaviors derived from light stimuli and which elements of the light sensitive system and the circadian clock of the Drosophila larva are involved in this process.
The authors have nothing to disclose.
We thank our colleagues at the Department of Biology, University of Fribourg for fruitful discussions. We thank the Bloomington Stock Center for providing fly stocks. This work was financially supported by the Swiss National Science Foundation (PP00P3_123339) and the Velux Foundation to S.G.S.
Name of Reagent/Material | Company | Catalog Number | Comments |
Agar | Sigma-Aldrich | A5093-500G | 2.5%; Sigma-Aldrich, 9471 Buchs, Switzerland |
Petri dishes | Greiner Bio-One GmbH | 633180 | 90-mm diameter; Greiner Bio-One GmbH, 4550 Kremsmeinster, Austria |
LEDs Lamp | OSARAM | 80012 White | LED lamp, 80012 White |
Environment Meter | PCE | PCE EM882 | Lux, Temp, RH% |
Thermostatic cabinet | Aqua Lytic (Liebherr) | ET636-6 | |
Light timer | Timer T | 6185.104 | 230V/50HZ (check specifications for your country) |
Universal thermostat | Conrad | UT200 | |
Humidifier | Boneco | ||
Balck tape | Tesa | 5 cm | |
Glue | Uhu | ||
lncubator lamp | Phillips | Softtone | 5W |
Timer clock | Ziliss | Ziliss, Switzerland | |
Excel Software | Microsoft | Excel | |
Origin Software 8.5 | OriginLab | ||
Backer Yeast | Migros Switzerland | ||
Iron support stand 17X28CM | Fisher Scientific | S47808 | |
Acetic acid | Sigma Aldrich | A6283-100ML | 20% acetic acid dilluted in H2O |
Red light lamp | Phillips | PFE712E*8C | |
Spatula | Fisher Scientific | 14-373-25A | |
Power supply | EA | EA PS 2042-06B | Optional |
Aluminium foil | Prix Coop | ||
Heater | GOON | NSB200C | |
Microwave Oven | Intertronic | ||
Standard corn meal fly food | |||
Destilled water |