S6d microscope

Automated worm tracking was performed essentially as previously described [30 (link)]. Briefly, 5–10 IJs from three replicate batches were placed on a chemotaxis plate and allowed to acclimate to room temperature for 1 hour. To measure average speed, IJ movement was recorded using an Olympus E-PM1 digital camera attached to a Leica S6 D microscope for 60 s. Recordings were analyzed using WormTracker and WormAnalyzer software (Miriam Goodman lab, Stanford University) [108 (link)]. Analysis was conducted as previously described [30 (link)], except that the following WormTracker settings were adjusted: sample rate = 15 frames/s; auto-thresholding correction factor = 0.20. The average speed of IJs that did not move during the assay period but were confirmed to be alive at the end of the assay was manually zeroed. We note that one Ste. carpocapsae IJ in the 25 °C to 15 °C temperature-swapped population crawled at a speed that was approximately 10 times faster than that of the other IJs tested, suggesting it was a “sprinter” [109 (link)]. Sprinters, which comprise less than 5 % of the IJ population, disperse farther than other IJs and are thought to facilitate long-range population dispersal in the absence of a host [109 (link)]. This IJ was not included in our population analysis.

Automated tracking was performed as described [22 (link)]. Briefly, recordings of iL3 movement were obtained with an Olympus E-PM1 digital camera attached to a Leica S6 D microscope. To quantify movement, 3–5 iL3s were placed in the center of a chemotaxis plate in a 5 μL drop of dH₂O. Once the drop dried, the iL3s were allowed to acclimate to the plate for 10 min. 20-s recordings were then obtained from each iL3, ensuring that each iL3 was only recorded once. Worm movement was quantified using WormTracker and WormAnalyzer software (Miriam Goodman lab, Stanford University) [21 (link)]. The following WormTracker settings were used: minimum single worm area = 20 pixels; maximum size change by worm between successive frames = 250 pixels; shortest valid track = 30 frames; auto-thresholding correction factor = 0.001. F₂ or F₃ wild-type (paralyzed) or unc (twitching) iL3s were recovered from 1% nicotine treatment overnight on chemotaxis plates and tested for crawling behavior the next day.

Automated tracking was performed as described [18 (link)]. For each recording session, 10–15 iL3s were placed on a chemotaxis plate and allowed to acclimate for 10 minutes. iL3 movement was then captured for 20 seconds using an Olympus E-PM1 digital camera attached to a Leica S6 D microscope. WormTracker and WormAnalyzer [35 (link)] were used to quantify crawling speed. WormTracker and WormAnalyzer settings were previously described [18 (link)].

Dynamic light-scattering (DLS) measurements were performed using a NaBiTec GmbH setup comprising a SpectroSize 302 (Molecular Dimensions) in combination with an S6D microscope (Leica). The purified protein sample was concentrated to 10 mg/mL as described earlier was illuminated in a 2 μl hanging drop using a 24-well crystallization plate (VDX Greased Plate, Hampton Research) covered with siliconized-glass circular cover slides (22 mm; Hampton Research). The well itself was filled with 400 μl SEC running buffer. Prior to the measurement, the protein solution was centrifuged at 18000 ×g, 30 min, 4°C to remove possible dust and other suspended particles. All measurements were done at 20°C. Ten consecutive measurements, each with an integration time of 20 s, were averaged. Hydrodynamic size of the particles was estimated with the instrument software using the following parameters: refractive index 1.33, viscosity 1.006, shape factor 1.0 and hydrated shell 0.2 nm.

Dynamic light-scattering (DLS) measurements were performed using a NaBiTec GmbH setup comprising a SpectroSize 302 (Molecular Dimensions) in combination with an S6D microscope (Leica). The purified protein sample (concentrated to 8 mg ml⁻¹ as described above) was illuminated in a 3 µl hanging drop using a 24-well crystallization plate (VDX Greased Plate, Hampton Research) covered with siliconized-glass circular cover slides (22 mm; Hampton Research). The well itself was filled with 600 µl SEC running buffer. Prior to the measurement, the protein solution was centrifuged (1000g, 30 min, 4°C) to remove possible dust particles. During the measurement, the temperature was set to 20°C. Ten consecutive measurements, each with an integration time of 20 s, were averaged. An estimate of the hydrodynamic size was obtained with the instrument software using the following parameters: refractive index 1.33, viscosity 1.006, shape factor 1.0, hydrated shell 0.2 nm.