Phototaxis was tested on day 1 adult worms unless otherwise indicated. Worms were transferred to NGM plates (one worm per plate) covered with a thin layer of freshly spread OP50 bacteria 2–5 min before the test. To quantify the percent responding, we tested each worm five times with an 8–10-min interval between each test and tabulated a percentage score for each worm. To quantify response delay, response amplitude and response duration, we tested each worm only once. The number of head swings was determined according to the definition created in a previous study37 (link). Light pulses from an Arc lamp (EXFO Xcite) were delivered to the worm head or tail via a 10× objective in combination with a 1–8× zoom lens on a Zeiss microscope (Zeiss Discovery) and the entire event was recorded with a digital camera (Cohu 7800) at 16 frames per s. To direct light to the worm head, we manually moved the stage (plate) such that only the head of the worm appeared in the field of view. A positive response was scored if the worm topped forward movement within 3 s after the cessation of light illumination and also initiated backward movement that lasted at least half of a head swing. In most cases, a 2-s light pulse was used to trigger responses unless otherwise indicated. When light was directed to the worm tail or body, it usually stimulated forward movement. Light intensity was determined with a radiometric sensor head (268S for UV-A light and 268LP for visible light) coupled to an optometer (S471, UDT Instruments). The intensities of UV-A, violet, blue, green-1, green-2 and yellow light were sampled at 340, 430, 470, 500, 550 and 580 nm, respectively. The background light used to visualize worms was filtered into red with a red filter. Io was set as 20 mW mm−2 for all wavelengths. A software package was developed in the laboratory by modifying one reported previously to control the shutter and the camera, as well as to process images and quantify behavioral parameters38 (link),39 (link). Laser ablation was performed on L2 worms using standard protocols40 (link) and phototaxis was analyzed at day 1 or 2 adulthood. A GFP transgene under the control of the tax-2Δ promoter was expressed in the worm to aid laser ablation17 (link).
Statistical analysis was carried out using the Statistica (StatSoft). P values were generated by ANOVA using the Bonferroni test. P < 0.05 was considered to be significant.