Muscle calcium responses were measured by detecting fluorescence intensity changes of GCaMP3. C. elegans expressing GCaMP3 in the body-wall muscle (Pmyo-3::GCaMP3) and Chrimson in the DB and VB neurons(Pacr-5::Chrimson) were used for calcium imaging experiments. Young adult animals fed with 1.6 mM ATR (in 100 µl E. coli OP 50) were immobilized on 10% agarose pads by polystyrene microbeads. Fluorescence images were captured using a Nikon 60 × 1.4 NA objective on a Nikon spinning-disk confocal system (Yokogawa CSU-W1) at 10 ms per frame. We used wide-field illumination with a nominal wavelength at 640 nm for Chrimson activation. The GCaMP signals were captured using 488 nm laser excitation and were analyzed by ImageJ software.

Calcium responses in the soma of AWB sensory neurons were measured by detecting fluorescence intensity changes of GCaMP6f. A home-made microfluidic device was used for calcium imaging as previously described (Liu et al., 2019; Zou et al., 2018). Briefly, a young adult animal was rinsed by M9 buffer and loaded into a home-made microfluidic device with its nose exposed to buffer under laminar flow. Diluted metabolites of N2 hermaphrodite and him-5 mutants was delivered using a programmable automatic drug feeding equipment (MPS-2, InBio Life Science Instrument Co. Ltd, Wuhan, China). For Ca2+ fluorescence imaging in AWB, the neurons were exposed under fluorescent excitation light for 30 s before recording, to eliminate the light-evoked calcium transients. During the recording process, for the first 5 s, we gave the M9 solution and then switched hermaphrodite excretome or male excretome for 30 s, removing extract liquid from 35 s and washing for 25 s. The AWB neurons were imaged with a 60× objective (Olympus) and EMCCD camera (Andor iXonEM) at 100 ms/frame. The imaging sequences were subsequently analyzed using Image-Pro Plus6 (Media Cybernetics, Inc, Rockville, MD).

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