cGMP Signalling Mediates Water Sensation (Hydrosensation) and Hydrotaxis in Caenorhabditis elegans

Animals have developed the ability to sense the water content in their habitats, including hygrosensation (sensing humidity in the air) and hydrosensation (sensing the water content in other microenvironments), and they display preferences for specific water contents that influence their mating, reproduction and geographic distribution. We developed and employed four quantitative behavioural test paradigms to investigate the molecular and cellular mechanisms underlying sensing the water content in an agar substrate (hydrosensation) and hydrotaxis in Caenorhabditis elegans. By combining a reverse genetic screen with genetic manipulation, optogenetic neuronal manipulation and in vivo Ca2+ imaging, we demonstrate that adult worms avoid the wetter areas of agar plates and hypo-osmotic water droplets. We found that the cGMP signalling pathway in ciliated sensory neurons is involved in hydrosensation and hydrotaxis in Caenorhabditis elegans.

(wet weight -dry weight) / wet weight. A small slice of agar was taken from the behavioural plate and weighed as wet weight. The same slice of agar was weighed for dry weight after full drying by baking at 55 ℃ for 24h. For wedge-shaped agar, the agar slices were taken every 0.5 cm from area A (Fig. 1a).
Worm tracking and locomotion analysis. Briefly, 40 -50 washed worms were dripped on a piece of uniform or wedge-like agar plate 2.0 cm × 2.5 cm in size, then were imaged under a Zeiss Discovery V8 stereomicroscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Images were recorded at 1 frame per sec for 100 minutes with an Andor iXonEM+ DV-885K-CS0-#VP-500 EMCCD camera (Andor Technology plc., Springvale Business Park, Belfast, United Kingdom), which was controlled by Andor iQ2.2 software, and analysed with the Multi-Worm Tracker (MWT) 63 and custom-written scripts for MATLAB (The MathWorks, Natick, MA, USA).
The assay on 6 % -2 % agarose plate with long-term diffusion. The agarose plate was made of two layers of agarose. Each layer consisted of a semicircle of 6 % agarose and a semicircle of 2 % agarose. In the vertical direction, the two layers were made of 2 % (top) -6 % (bottom) agarose and 6 % (top) -2 % (bottom) agarose. Briefly, eight millilitres of hot 6 % agarose sol (W/V, in ultra-pure water) was poured into a 6 cm (in diameter) petri dish. When the 6 % agarose solidified, a semicircle was removed and refilled with 4 ml 2 % agarose sol. After the 2 % agarose cooled, another 8 ml of 6 % agarose sol was poured on the first layer. After cooling, the semicircle of the upper layer of 6 % agarose plate on top of 6 % agarose was carefully removed and refilled with 4 ml 2 % agarose sol. After the agarose solidified, the plates were sealed with parafilm and stored at 20 ℃ over 12h for diffusion. Then, the top layer of agarose was removed, and they were baked 20 min at 37 ℃. For the plates with food, 3 × OP50 was smeared on the surface of the agarose before baking. Washed animals were placed in the centre of a 6 % -2 % agarose plate and scored under a stereomicroscope at 40 min. The hydroaversive index (H.A. Index = (N 6 % -N 2 %) / (N 6 % + N 2 %)) was calculated. The assay plates were not sealed during the whole procedure.

Optogenetic Manipulation of Intracellular cGMP by Photoactivation of BlgC and
Behaviour Tests. To optogenetically manipulate intracellular cGMP levels in neurons, transgenes expressing the blue light activated guanylyl cyclase BlgC 27 , driven under promoters of daf-11, srh-11 (ASJ specific), sra-9 (ASK specific) and gpa-4 (ASI specific), were used and illuminated by blue light. Behaviour was assayed by the drop test and the wedge-shaped agar (WSA) tests. For the drop test, 40 -50 washed transgenics were dripped onto a 2 % pure agar plate, and tested for their hydroavoidance under a Zeiss fluorescent stereomicroscope after acclimation for 10 minutes on the test plate. The worms were illuminated by continuous blue laser light (450 nm, ~ 10 mW / cm 2 ) during the whole procedure. Identically treated daf-11(m47) or tax-2(p671) mutants were used as controls. For the WSA testing, the transgenics were illuminated with a round blue light with diameter of 9.5 cm sourced from a 100 W LED array were used, in which R-GECO 1.0 expression was driven by the promoters of srd-1, srh-11, sra-9 and gcy-8, respectively. For ASI, ASJ, and ASK Ca 2+ imaging in daf-11 mutants, R-GECO 1.0 was expressed under direction of the gpa-4 promoter (transgene ZXW961), srh-11 promoter (transgene ZXW974) and sra-9 promoter (transgene ZXW977). For the ASI and ASJ Ca 2+ imaging in daf-11 rescued worms, G-GECO 1.1 was expressed under the gpa-4 promoter (transgene ZXW962) and srh-11 promoter (transgene ZXW975). For the ASK Ca 2+ imaging in daf-11 rescued worms, G-CaMP 2.0 was expressed under direction of the sra-9 promoter (transgene ZXW978). A homemade microfluidic device was used for calcium imaging as previously described 34,64,65 .
Briefly, a worm was immobilized by trapping in a micro-channel of the microfluidic chip, and the head of the worm was exposed to water or air flow. Laminar flow controlled through two alternatively on-off laminar streams was used to control the delivery of stimuli. The flows of air, ultra-pure water and buffer were delivered using a diffusion. a and c, Schema of the assays on the 6% -2% agarose plates with long-term (> 12 hours) diffusion, in presence of (a) or in absence of (c) food. Two layers of 6% and 2% agarose was poured and diffused for over 12h. b, Hydroaversive indexes of daf-11(m47), tax-2(p671), tax-4(ks28), osm-6(p811) and the genetically rescued worms in assay model a (with food). d, Hydroaversive indexes of daf-11(m47), tax-2(p671), tax-4(ks28), osm-6(p811) and the genetically rescued worms in assay model c (without food).    Table   Supplymentary Table 1│Mutant and transgenic worms used in the study.

Strain
Genotype