Figures and data
A comparison of chemotaxis responses to water-soluble ions between P. pacificus and C. elegans.
J4 to Adult hermaphrodites from the two species responded to NH4I, LiCl, and acetates significantly differently. Using two-way ANOVA, significant difference found between wildtype P. pacificus and C. elegans for the same salt is indicated above each pair (*p<0.05, ****p<0.0001), while the differences within P. pacificus is as follows: all salts showed difference when compared to NaAc and to NH4Ac (****p<0.0001), but not between NH4Ac and NaAc. Both LiCl and NaCl attraction are significantly lower than NH4Cl (*p<0.05) and NH4I (***p<0.001). Error bars denote standard error of the mean and the sample sizes are indicated on the bottom.
P. pacificus che-1 expression in ASE and AFD amphid neurons.
(A-B) The che-1::GFP marker in the che-1::RCaMP reporter strain is expressed in the ASE and AFD amphid neurons. (C) che-1::GFP expression is detectable in the morphologically distinct AFD neurons with ‘finger’-like dendritic endings. (D-E) Immunostaining of CHE-1::ALFA shows two pairs of amphid neuron cell bodies corresponding to the ASE and AFD neurons; dorsal-ventral view (n=114). (F) The loss of che-1 results in loss che-1::RFP expression in the ASE (circle) while retaining reduced AFD expression. (G) ttx-1p::RFP expression in the AFD neurons with ‘finger’-like dendritic endings. Inset shows expanded inverted black-white image of the AFD dendritic ending. (H) AFD expression of the same ttx-1::RFP animal shown in (G) in a different plane with cell body in focus. (I-J) Immunostaining of TTX-1::ALFA (red) shows one pair of amphid neuron cell bodies co-localizing with the anterior pair of che-1::GFP-expressing AFD neurons (yellow); dorsal-ventral view (n=13). Anterior is left and the scale bar in (C) represents 50 µm for all panels except for the G inset, which is 5 µm.
che-1 expressing amphid neurons are required for sensing water-soluble ions in P. pacificus.
(A) The che-1 locus with CRISPR/Cas9-induced mutations in Exon 1. (B) The che-1 mutants show defects in attraction towards NH4Br, NH4Cl, and LiCl. Sample sizes are indicated below for attractants and above for repellent. (C) The che-1p::HisCl1 animals lose attraction towards NH4Br, NH4Cl, and NH4I in a histamine dependent manner. Sample sizes are indicated at the base of each bar. (D) A schematic map of the P. pacificus AM7(ASE) and AM12(AFD) amphid neurons that express the che-1p::RCaMP used in calcium imaging. The ASE axons are the only amphid axons in P. pacificus to cross each other over the dorsal lateral midline contralaterally. **P<0.01, *P<0.05 Two-way ANOVA with Dunnett’s post-hoc comparison showing alleles with significant difference to wildtype P. pacificus (PS312). ****P<0.0001 Two-way ANOVA showing significant difference between water control and histamine treatment.
ASEL and ASER responses to different concentrations of NH4Cl, NaCl, and NH4I.
Average percent change in RCaMP fluorescence (dF/F) over time (seconds) of tracked ASE (left and right) sensory neurons in P. pacificus. Salts were presented at 10s (“ON”, left vertical line) for a duration of 20s, and then removed (“OFF”, right vertical line) for the remaining 30s; the total recording time was 60s. (A-B) ASEL and ASER neuron responses to high (250 mM, red) compared to low (25 mM, blue) concentrations of NH4Cl. (C-D) ASEL and ASER neuron responses to NaCl. (E-F) ASEL and ASER neuron responses to NH4I. Shaded ribbons represent 95% confidence intervals. Shaded ribbons in (E-F) have been cropped to maintain consistent y-axes across the plots, allowing for easier comparison. Sample sizes are indicated (n).
Combined AFD responses in comparison to ASE left or right neuron responses to NH4Cl, NaCl, and NH4I.
Average percent change in RCaMP fluorescence (dF/F) over time (seconds) as described in Fig. 4. Averaged combined AFD (left and right neurons, orange) compared to left or right ASE (green) responses to (A-B) 250mM NH4Cl (C-D) 25mM NH4Cl (E-F) 250mM NaCl (G-H) 25mM NaCl (I-J) 250mM NH4I (K-L) 25mM NH4I. Shaded ribbons represent 95% confidence intervals. Shaded ribbons in (J-K) have been cropped to maintain consistent y-axes across the plots, allowing for easier comparison. Sample sizes are indicated (n).
The laterally asymmetric expression of gcy-22.3 is dependent on the zinc finger transcription factor CHE-1.
(A) The gcy-22.3::GFP marker is expressed exclusively in the right ASE neuron (ASER)(n>200). (B) The gcy-22.3::GFP marker co-localizes with che-1::RFP expression in the ASER. (C) gcy-22.3::GFP expression is absent in the che-1(ot5012) mutant (n=55). Anterior is left and dorsal is top. Scale bar in (A) represents 50 µm for all panels.
ASE and AFD responses in wildtype compared to gcy-22.3 mutants.
Average percent change in RCaMP fluorescence (dF/F) over time (seconds) as described in Fig. 4. (A) www.pristionchus.org Genome Browser view of the gcy-22.3 locus with the CRISPR/Cas9-induced mutations indicated with a red bar (Exon 5 of PPA04454 or Exon 4 of Contig12-snapTAU.506). (B-C) ASEL and ASER neuron responses to 250 mM NH4Cl wildtype (gray) and gcy-22.3 mutants (magenta). (D-E) ASEL and ASER neuron responses to 250 mM NaCl in wildtype (gray) and gcy-22.3 mutants (magenta). AFD (combined left and right neurons) responses (F) to 250 mM NH4Cl and (G) to 250 mM NaCl in wildtype (orange) and gcy-22.3 mutants (magenta). Shaded ribbons represent 95% confidence intervals. Sample sizes are indicated (n). For ASEL/R comparisons, bar plots represent the difference between the minimum % dF/F value 10s pre-stimulus and maximum % dF/F value 10s post-stimulus for either the (B) ON or (C-E) OFF stimulus. For AFD comparisons, bar plots represent maximum % dF/F values 10s after the ON and OFF stimulus. *P<0.05, **P<0.01, ***P<0.001 indicate significant difference. “ns” indicate no significant difference. Comparisons between different genotypes were analyzed using unpaired t-test or Mann-Whitney test.
Chemotaxis responses to individual ions in gcy-22.3 mutants.
Young adult hermaphrodite responses to salt gradients with or without various salts in background. *P<0.05 Significant differences were found between wildtype PS312 and gcy-22.3 mutants to NH4Cl by two-way ANOVA and Dunnett’s test. Each assay involves a minimum of 10 animals. Sample sizes for each condition are indicated on the bottom. Error bars denote standard error of the mean.
Co-expression of gcy-22.3p::GFP and ttx-1p::RFP in ASER.
A representative F1 male animal from a cross between the two reporter strains (n=5). Scale bar in (C) represents 50 µm for all panels.
The che-1p::HisCl1 transgene is necessary for histamine-dependent knockdown of salt attraction.
Animals containing the che-1p::HisCl1 transgene and the co-injection marker egl-20p::RFP were scored separately on each assay plate. Unpaired t-test between histamine and water treatment prior to chemotaxis assays (****P<0.0001).
Negative controls of ASE and AFD neuron responses to green light.
Average percent change in fluorescence (dF/F) over time (seconds) of tracked sensory neurons in P. pacificus (black line). Individual animals are represented by colored lines. No salt was presented; vertical lines indicate time points where salt was presented and removed for experimental samples, shown for comparison. csuEx93[Ppa-che-1p::optRCaMP] worms experienced the control solution for the duration of the recording and were imaged under green light. (A) ASE (n=6). (B) AFD (n=5).
Individual traces and heatmaps of ASE responses to various salts.
Average percent change in fluorescence (dF/F) over time (seconds) of tracked ASEL and ASER neurons in P. pacificus (black line). Individual animals are represented by colored lines. For heatmaps, each row represents a single individual. Lighter colors (closer to 1) represent more positive dF/F values and darker colors (closer to 0) represent more negative dF/F values.
Individual traces and heatmaps of AFD responses to various salts.
Average percent change in fluorescence (dF/F) over time (seconds) of tracked left and right AFD neurons in P. pacificus (black line). Individual animals are represented by colored lines. For heatmaps, each row represents a single individual. Lighter colors (closer to 1) represent more positive dF/F values and darker colors (closer to 0) represent more negative dF/F values.
AFDL/R responses to different concentrations of NH4Cl, NaCl, and NH4I.
Average percent change in RCaMP fluorescence (dF/F) over time (seconds) as described in Fig. 4. Average AFDL and AFDR neuron responses to high (250mM, red) and low (25mM, blue) concentrations of salts: (A-B) NH4Cl, (C-D) NaCl, (E-F) NH4I. Shaded ribbons represent 95% confidence intervals. Shaded ribbons in (D) have been cropped to maintain consistent y-axes across the plots, allowing for easier comparison. Sample sizes are indicated (n).
AFDL and AFDR responses to NH4Cl and NaCl in wildtype and gcy-22.3 mutant.
The combined AFD responses shown in Figure 7F-7G have been separated into AFDL and AFDR. Average percent change in RCaMP fluorescence (dF/F) over time (seconds) as described in Fig. 4. AFDL and AFDR neuron responses to (A-B) 250mM NH4Cl and (C-D) 250mM NaCl in wildtype (orange) compared to gcy-22.3 mutants (magenta). Shaded ribbons represent 95% confidence intervals. Shaded ribbons in (D) have been cropped to maintain consistent y-axes across the plots. Sample sizes are indicated (n).
Molecular lesions for P. pacificus che-1 and gcy-22.3.
DNA alignment of the Ppa-che-1 and Ppa-gcy-22.3 alleles and their predicted amino acid changes.
(A) Schematic of the microfluidic apparatus to conduct calcium imaging while delivering stimuli directly to the nose of an immobilized worm. Syringes act as reservoirs for buffer solution (light blue), control solution (purple), and stimulant solution (red). Tubing inserts into the microfluidic PDMS chip. Panels (right) zoom in to depict a schematic of the microscope view of the PDMS chip: (top) fluid flow over the worm nose when stimulant is switched OFF (control solution flows over worm nose); (bottom) fluid flow over the worm nose when stimulant is switched ON (stimulant solution flows over worm nose). Created with BioRender.com. (B) Actual microscope view of PDMS chip design. The outer two channels hold buffer solution and can be switched open (ON) or closed (OFF) by the Valvebank. The inner two channels hold experimental solutions: the inner channel closer to the worm trap holds the control solution, and the inner channel farther from the worm trap holds the stimulant solution. (C) Actual microscope view of P. pacificus loaded into the PDMS chip while fluid is flowing. The PDMS chip features a U-shaped worm trap to facilitate loading the worm head-first, and a tapered opening to ensure the worm fits snugly and will not slide too far forward during recording.
Comparison of ASEL/R responses between P. pacificus and C. elegans.
Nematode strains.
Plasmids.
