Animal handling and maintenance
The strains used in this study are shown in Supplementary Table 1. C. elegans and P. pacificus were maintained at 20 °C on nematode growth medium (NGM) agar plates containing Escherichia coli OP50.
Transgenic animals
To generate the Ppa-myo-2p::RFP (JWL27) strain, we used the previously established protocol61. This construct was generated by PCR amplification of a 1,231-base pair (bp) upstream region in front of the first predicted ATG start codon of Ppa-myo-2 and subsequent cloning into the pZH009 containing the codon-optimized red fluorescent protein (TurboRFP) plasmid. NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) was employed to perform cloning. To generate the transcriptional reporters of Ppa-tdc-1, Ppa-ser-3, Ppa-ser-6 and Ppa-lgc-55, we cloned the upstream regions before their predicted start codon, including: Ppa-tdc-1: 1,585 bp, Ppa-ser-3: 1,996 bp, Ppa-ser-6: 1,996 bp and Ppa-lgc-55: 1,917 bp, to drive expression of the codon-optimized TurboRFP or GFP as required. Each injection mix contained 10 ng µl−1 of the PstI-HF-digested reporter plasmids, 10 ng µl−1 of the PstI-HF-digested Ppa-egl-20p::GFP plasmid as co-injection marker and 60 ng µl−1 of the PstI-HF-digested P. pacificus genomic carrier DNA. The mix was injected in the gonads of young adults. Between 50 and 3,000 animals were injected depending on the strain. The transgenic animals were screened using an epifluorescence microscope (Axio Zoom V16; Zeiss). The fluorescence images of the transgenic animals were obtained using a Leica SP8 confocal microscope.
Transgenic line integration
Ppa-myo-2p::RFP was integrated into the P. pacificus genome as previously described62. Briefly, ten NGM plates each containing 20 fluorescent Ppa-myo-2p::RFP animals were exposed to ultraviolet irradiation at 0.050 J cm−2 using a UVP Crosslinker (CL-3000 Analytik Jena). After 3–4 days F1 fluorescent animals were singled out onto 120 individual culture plates and after another 3–4 days the F2 progeny were screened for possible integration events. This was detected by observing an increase in the number of fluorescent animals to ≥75% of the population. Individual animals from these plates were isolated and screened for consistent 100% transmission. Integrated lines were subsequently outcrossed 4× to remove potential mutations caused by ultraviolet exposure.
Behavioural imaging
Ppa-myo-2p::RFP animals were recorded at ×1 effective magnification using an epifluorescence microscope (Axio Zoom V16; Zeiss) as previously described19. Recordings were made through a Basler camera (acA3088-57um; BASLER) with 15-ms exposure time. Animals were imaged at 30 frames per second for 10 min unless otherwise indicated. All animals that were in the field-of-view for at least 60 s were included in the analysis.
For tracking of animals on predatory assays, C. elegans prey were first maintained on OP50 bacteria until freshly starved, resulting in an abundance of young larvae. These plates were washed with M9, passed through two 20-µm filters, centrifuged and deposited onto the assay plate by pipetting 4 µl of worm pellet onto a 6-cm NGM unseeded plate. A copper arena (1.5 × 1.5 cm2) was placed in the middle of the assay plate to constrain predators in the recording field. Forty young adult P. pacificus predators (eurystomatous mouth form) were starved for 2 h and then added to assay plates inside the arena. After a recovery period of 15 min on the assay plate, their behaviours were recorded for 10 min.
For tracking of animals on bacterial assays, 300 µl of E. coli OP50 overnight culture was spotted onto an empty 6-cm NGM plate 24 h before the assay. A copper arena (1.5 × 1.5 cm2) was placed in the middle of the assay plate to contain the P. pacificus in the recording field. Forty young adult predators were starved for 2 h and then added to assay plates inside the arena. After a recovery period of 15 min on the assay plate their behaviours were recorded for 10 min.
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