Previous cross-species comparisons between P. pacificus and C. elegans indicated strong differences in olfaction preferences that reflect their divergent evolutionary histories and ecology (Hong and Sommer, 2006). To identify the neurons that mediate gustation, we first compared the chemosensory profiles of these two species toward water-soluble ions. In this survey, we found ammonium salts to be the strongest attractants to wildtype P. pacificus J4 to adult hermaphrodites (NH₄Br, NH₄Cl, and NH₄I), with NH₄I significantly more attractive to P. pacificus compared to C. elegans (Figure 1). Notably, in contrast to C. elegans (Ward, 1973), we find that P. pacificus is less attracted to NaCl and LiCl compared to the ammonium salts (Figure 1). Also, P. pacificus is repulsed by acetate salts (NaAc and NH₄Ac), which induce attractive responses in C. elegans (Frøkjaer-Jensen et al., 2008). We conclude that P. pacificus and C. elegans display differences in their salt preferences.

The C. elegans che-1 mutant (chemotaxis-defective) was originally isolated for its inability to respond to a broad panel of salt tastants (Dusenbery et al., 1975; Ward, 1973), including those described above (Suzuki et al., 2008; Ortiz et al., 2009; Frøkjaer-Jensen et al., 2008). che-1 was found to encode for a Zn finger transcription factor that is exclusively expressed in the ASE neuron pair (Etchberger et al., 2007), which through laser ablations had been found to be the main salt receptor neurons (Bargmann et al., 1993). che-1 was found to control the entire differentiation of the ASE neurons, including the expression of putative receptors of the GCY receptor guanylyl cyclase family (Uchida et al., 2003; Chang et al., 2003; Etchberger et al., 2007; Etchberger et al., 2009).

To assess whether che-1 performs a similar function in salt perception for P. pacificus as for C. elegans, we analyzed the expression and function of the Ppa-che-1 ortholog. In a previous paper, we reported that the 5′ region of the sole Pristionchus che-1 ortholog directs reporter expression to the Ppa ASE and Ppa ASG neuron classes (Hong et al., 2019). Re-examination of our provisional cell identifications using a newly generated che-1p::GFP strain with stronger neurite expression revealed highly elaborated finger-like dendritic endings in the more anterior amphid neuron that could unambiguously be assigned to the AFD neurons (Figure 2A–C), prompting us to reassign expression of che-1 to ASE and AFD.

We confirmed that the che-1 reporter transgene indicates the full expression of the endogenous che-1 locus by tagging the endogenous che-1 locus with an ALFA-tag (Igreja et al., 2022). The che-1::ALFA animals showed staining in 2 pairs of head neurons whose position is consistent with being the ASE and AFD neurons (Figure 2D, E). By crossing the che-1 reporter transgene into a che-1 mutant background (see below), we also found that che-1 autoregulates its own expression especially in the ASE neurons (Figure 2F, GFP expression in 97% in WT (n = 36) vs 4% in che-1(−) (n = 48)), as it does in C. elegans (Etchberger et al., 2007). The reduction of che-1p::GFP in the AFDs was also observed (GFP expression in 100% in WT (n = 36) vs 33% in che-1(−) (n = 48)).

To provide further evidence that Ppa-CHE-1 is indeed expressed in the Ppa-AFD neurons, we analyzed the expression of the Ppa-ttx-1p::RFP reporter. In C. elegans, the OTX homeodomain transcription factor TTX-1 is a terminal selector expressed in the AFD neurons required for designating the AFD fate (Satterlee et al., 2001). We found ttx-1p::RFP to be strongly expressed in a pair of neurons with the hallmark finger-like dendritic ending of the AFD neurons (Figure 2G, H), as well as expression in other head and tail neurons (possibly RIP and RIB) and cells likely to be the pharyngeal marginal cells based on likely conservation with Cel-ttx-1 expression (Reilly et al., 2022). To confirm that endogenous TTX-1 protein expression is co-expressed with che-1p::GFP, we examined C-terminally tagged ttx-1:ALFA animals and found co-localization in the same anterior pair of amphid neurons, but no co-expression in the posterior pair of amphid neurons (Figure 2I, J, n = 15). Interestingly, the promoter expression in the posterior pair of amphid neurons in animals with both ttx-1p::RFP and gcy-22.3p::GFP reporters do co-localize in the ASER neurons (Figure 2—figure supplement 1), possibly due to differences between the expression patterns of the four possible ttx-1 splice forms. The transcriptional and protein co-expression of ttx-1 and che-1 in the AFD neurons unequivocally show the expression of che-1 in both AFD and ASE neurons in P. pacificus.

Next, we examined whether P. pacificus salt responsiveness shows similar che-1 dependence as in C. elegans. We generated two putative null alleles in the Ppa-che-1 homolog using CRISPR/Cas9 genome engineering, through introduction of small deletions in the first exon of the gene, thereby resulting in frameshift and premature stops (Figure 3A; Figure 3—figure supplement 2). Both Ppa-che-1 alleles exhibited defects in attraction toward NH₄Cl and LiCl compared to wildtype (Figure 3B). However, unlike in C. elegans, Ppa-che-1 mutants showed no detectable difference in responses to NH₄I, NaCl, and NaAc.

We considered two different possibilities for the behavioral differences of Ppa-che-1 and Cel-che-1 mutants. P. pacificus may use sensory neurons other than ASE to sense these cues, or alternatively, Ppa-che-1 may not have the same fundamental impact on ASE function in P. pacificus as it does in C. elegans. To explore these different possibilities, we silenced che-1 expressing neurons by expressing codon-optimized HisCl1 channel under control of the che-1 promoter. Histamine-treated che-1p::HisCl1 animals showed complete loss of attraction to NH₄Br, NH₄Cl, and NH₄I but did not significantly alter their repulsive response to NaAc (Figure 3C). As a control, we show that the presence of the che-1p::HisCl1 transgene was necessary for the knockdown of NH₄Br attraction (Figure 3—figure supplement 1). These findings corroborate that NaAc is sensed by neurons other than ASE (or AFD, in which the che-1 promoter also drives HisCl1). Since NH₄I sensation is affected by silencing of che-1(+) neurons but is unaffected in che-1 mutants, ASE function may be more greatly impacted by the silencing of ASE than by the loss of che-1.

To assess whether Ppa ASE neurons show the same lateralized response to salt ions as Cel ASE neurons, we generated transgenic P. pacificus lines that express RCaMP in ASE neurons and assessed calcium responses to attractive salts (Figure 4, Figure 4—figure supplements 1 and 2). Specifically, we looked for changes in calcium levels immediately after the addition and removal of specific salts. When 250 mM NH₄Cl is administered, an ‘ON’ response is observed as calcium transiently increases in the left ASE neuron (ASEL). In contrast, an ‘OFF’ response was observed as calcium sharply dips in the right ASE neuron (ASER) before quickly returning to baseline when the salt was removed, which suggests hyperpolarization of this neuron (Figure 4A, B; Suzuki et al., 2008). However, when presented with a tenfold lower concentration of 25 mM NH₄Cl, the ‘OFF’ response completely disappeared in ASER while the ‘ON’ response became more pronounced in ASEL. The ASER responses to 250 and 25 mM NaCl (Figure 4D) were very similar to the ‘OFF’ response (including hyperpolarization) observed for NH₄Cl, but the ASEL responses differ between the two salts: instead of the ‘ON’ response expected of ASEL, we observed a relatively weak ‘OFF’ response without the characteristic hyperpolarization but accompanied by an attenuated ‘bump’, a profile that we classify as an ‘OFF-2’ response (Figure 4C). Finally, we examined the response to NH₄I and found that it also elicited laterally asymmetric responses, but yet again distinct from the responses to both NH₄Cl and NaCl, with ASEL showing a strong ‘ON’ response, and ASER showing an ‘ON–OFF’ biphasic response to 250 mM NH₄I (Figure 4E, F; Kato et al., 2014; Wang et al., 2015). Interestingly, the ASER exhibited an ‘OFF’-only response to 25 mM NH₄I, which was not observed for the same concentration of NH₄Cl and NaCl, and thus may reflect the higher response to NH₄I than to NH₄Cl and NaCl in the behavior assays. Altogether, P. pacificus ASE neurons clearly show left/right asymmetric responses to salt attractants and these asymmetric responses show similarities and differences to the C. elegans ASE taste neurons (see Discussion).

The RCaMP line that we used to assess calcium responses in ASE is also expressed in AFD, allowing us to simultaneously examine the calcium responses in the AFD (AM12) neurons (Figure 5, Figure 2—figure supplement 1, Figure 5—figure supplements 1 and 2, Figure 7—figure supplement 1). Surprisingly, we detected a distinctly ‘ON–OFF’ biphasic response to all three salt types at both concentrations. Specifically, although we observed weaker or comparable responses in AFD neurons when compared to either ASE neuron’s response toward 250 mM NH₄Cl, NaCl, and NH₄I (Figure 5A, B, E, F, I, J), the AFD responses were more robust than ASER toward 25 mM NH₄Cl, NaCl, and NH₄I (Figure 5D, H, L). Specifically, AFD neurons responded strongly to 25 mM NaCl when neither one of the ASE neurons showed a positive response (Figure 5G, H). Averaging the calcium transients separately by AFD left versus right did not result in significant differences in the shape of the neuronal calcium responses, with the exception of the AFDR responses to higher versus lower concentrations of NH₄I (Figure 5—figure supplement 2, Figure 7—figure supplement 1). We have not further pursued whether these AFD responses are a reflection of a direct perception of salt or a secondary consequence of communication of salt-perceptive neurons (like ASE) to AFD.

We further explored the asymmetric salt perception by the ASE neurons, which in C. elegans is largely mediated through distinct receptor-type guanylyl cyclases (rGC proteins, encoded by gcy genes), which confer salt specificity via their extracellular domains (Ortiz et al., 2009). Our previous genome survey of Ppa homologs of gcy genes has revealed patterns that made us question whether Ppa gcy genes are convincing candidates for lateralized chemotactic responses. Specifically, we noted that ASER-expressed C. elegans gcy genes and ASEL-expressed C. elegans gcy genes have only expanded in the Caenorhabditis genus (Hong et al., 2019). One outlier to this pattern is the Cel-gcy-22 gene, which is expressed in ASER, but has not expanded in C. elegans (Hong et al., 2019; Ortiz et al., 2006). However, this gene has duplicated several times in P. pacificus, resulting in 5 putative Ppa-gcy-22 paralogs (Hong et al., 2019). We fused the promoter of one of these paralogs, Ppa-gcy-22.3, to gfp and found that transgenic animals express GFP exclusively in ASER (Figure 6A), identical to the C. elegans gcy-22 ortholog (Ortiz et al., 2006). We confirmed its expression in ASER by analyzing animals that carry both the Ppa-gcy-22p::gfp reporter and the Ppa-che-1p::rfp reporter, showing a unilateral overlap of these reporters in ASER (Figure 6B).

To assess whether Ppa-gcy-22.3 is a potential effector of Ppa-che-1 function, we crossed the gcy-22.3 reporter into che-1(ot5012) mutants. We found expression of gcy-22.3 was eliminated (Figure 6C), leading us to conclude that gcy-22.3 is a potential effector of che-1 function, identical to its homolog in C. elegans.

To determine whether and which of the observed salt responses is mediated by the ASER-expressing gcy-22.3, we generated a putative gcy-22.3 null mutant through CRISPR/Cas9 genome editing (2 bp complex deletion that introduces a frameshift) (Figure 7A) and examined its response to the higher salt concentration. The ‘OFF’ response to 250 mM NH₄Cl was notably abolished in the ASER neuron in the loss-of-function gcy-22.3 mutant, while the ‘ON’ response in the ASEL remained intact (Figure 7B, C). However, the responses to 250 mM NaCl were not significantly reduced in either the ASEL or ASER neuron in the gcy-22.3 mutant (Figure 7D, E). Furthermore, the gcy-22.3 mutation also reduced the ‘ON’ portion of the AFD biphasic response following presentation of 250 mM NH₄Cl and NaCl (Figure 7F, G). We also examined the behavioral responses toward individual salt ions in gcy-22.3 mutants, including a second loss-of-function allele csu182. Overall, we found no significant differences between wildtype and mutants in responses toward individual ions, and only the attraction to NH₄Cl was slightly enhanced in both alleles (Figure 8), which was unexpected given the lack of calcium response observed in gcy-22.3(csu181) toward NH₄Cl. Our findings show that while proper ASE function is critical for salt attraction, defects in individual gcy genes do not lead to a major impact on the worms’ ability to track toward attractive salts.

We have shown that while C. elegans is attracted to acetate salts, P. pacificus avoids these acetates. Previous studies have shown that ammonium acetate (NH₄Ac) is sensed both as a water-soluble compound as well as a volatile odorant and is mediated by different signaling pathways (Frøkjaer-Jensen et al., 2008). Like C. elegans, it is therefore likely that ammonium and acetate ions involve a different set of neurons in P. pacificus (i.e. non-che-1 expressing neurons), based on the finding that histamine-treated che-1p::HisCl1 animals did not significantly attenuate their repulsive response to NaAc. We find that sodium chloride is a common attractant for both P. pacificus and C. elegans, although the magnitude of response is lower than previously published results (Suzuki et al., 2008). This result is likely due to differences in generating the salt gradients. Nevertheless, we find that P. pacificus neurons have a distinct response to this salt when compared to those observed in C. elegans neurons (Table 1). The P. pacificus ASEL neuron responds to a decrease in NaCl concentration (likely sodium), as evidenced by the ‘OFF-2’ response profile, whereas the C. elegans ASEL neuron responds to an increase in sodium concentration. However, in both nematodes, the ASER neuron responds to a decrease in chloride concentration. Additionally, the P. pacificus ASER neuron exhibits a unique ON–OFF response to ammonium iodide, whereas in C. elegans, no ON–OFF type of response is seen in the ASE neurons – the C. elegans ASER neuron responds only to a decrease in iodide concentration. Based on our calcium imaging and chemotaxis results on the gcy-22.3 mutants, attraction to ammonium ions (NH₄⁺) is likely mediated in part by the ASER neuron in P. pacificus. Pristionchus species are entomophilic and most frequently found to be associated with beetles in a necromenic manner and thus insect cadavers could be sources of ammonium in the soil (Koneru et al., 2016; Herrmann et al., 2007; Herrmann et al., 2006; Fielding et al., 2013). Additionally, ammonium salts could represent a biological signature of other nematodes that the predatory morph of P. pacificus could interpret as prey. In P. pacificus, nutritional state has a measurable role in the mouth-form polyphenism decision between predatory and non-predatory morphs (Piskobulu et al., 2025). Collectively, our findings highlight the divergence of the P. pacificus salt sensory neurons compared to those observed in C. elegans.

The sensitivity range of the P. pacificus ASER responses is significantly less compared to the ASEL responses, as well as to C. elegans ASER responses. Whereas the C. elegans ASER has a 40-fold sensitivity range in the ‘OFF’ response to the removal of various concentrations of NaCl (1–40 mM) (Suzuki et al., 2008; Rabinowitch et al., 2014; Shindou et al., 2019; Watteyne et al., 2020), the P. pacificus ASER showed the ‘OFF’ response only to 250 mM NH₄Cl but not to a tenfold reduction in concentration of NH₄Cl (25 mM). For the ASEL in contrast, the response to 25 mM was just as strong as to 250 mM NH₄Cl (tenfold) and comparable to the eightfold concentration range observed for C. elegans ASEL toward NaCl. Alternatively, the magnitude of these sensitivity differences may be also partially due to differences among calcium indicators (i.e. GCaMP and Cameleon), but multiple P. pacificus che-1p::GCaMP strains did not exhibit sufficient basal fluorescence to allow for image tracking and direct comparison. The narrower sensitivity range in P. pacificus chemosensation was also observed for attraction to volatile odors (Hong and Sommer, 2006), which span only 10-fold, versus up to 10,000-fold in attractive odors for C. elegans (Bargmann and Horvitz, 1991).

Although the left-ON and right-OFF responses are conserved in the ASE neurons in both species, the biphasic response of ASER to 250 mM NH₄I has not been observed in either of the ASE neurons toward salts. In C. elegans, hyperosmotic stimulus such as 1 M glycerol, or high concentrations or long duration of CuSO₄ exposure both result in biphasic responses by the ASH neurons that sense noxious chemicals (Wang et al., 2015; Chronis et al., 2007). Olfactory neurons that mediate avoidance behavior such as the AWB neurons can also exhibit a biphasic response to the presence and removal of a high concentration isoamyl alcohol that normally elicits avoidance behavior (Yoshida et al., 2012). The neuron-specific response can also be dependent on the concentration of the chemical compound, since the ASER to 25 mM NH₄I was a weak ‘OFF’ rather than a biphasic one. In contrast to the salt-dependent response types by P. pacificus ASE neurons, the P. pacificus AFD neurons also show exclusively biphasic responses with various amplitudes. Further characterization will help determine if biphasic responses can also be found in other sensory neuron types in P. pacificus, specifically those neurons mediating avoidance behavior.

Broadly speaking, C. elegans chemosensory neurons have been classically characterized as specialized neurons for dedicated modalities such as water-soluble chemicals (ASE), volatile odorants (AWA, AWB, and AWC), noxious chemicals (ASH), pheromones (ADL, ADF, and ASK), and light (ASJ) and temperature (AFD) (Bargmann, 2006; Bargmann et al., 1993; Mori and Ohshima, 1995; Sengupta et al., 1996; Macosko et al., 2009; Liu et al., 2010). Advances in multi-neuron calcium recordings have since shown that a given odor within a certain concentration range is detected by different ensembles of the 12 amphid neuron classes, including the AFD neurons (Leinwand and Chalasani, 2013; Lin et al., 2023; Yemini et al., 2021). Unexpectedly, we found that the P. pacificus AFD neurons exhibit a distinctive biphasic response to all three salts tested (NH₄Cl, NaCl, and NH₄I), which differ from the C. elegans AFD calcium responses (Yemini et al., 2021; Zaslaver et al., 2015). Moreover, the loss of the receptor gcy-22.3 reduced the AFD ‘ON’ response to NH₄Cl and NaCl, indicating ASER contributes to the AFD response. The strong positive AFD response to 25 mM NaCl that is absent in both ASE neurons further supports the likelihood that AFD receives inputs from other amphid neurons. The integration of thermosensation and chemosensation is important for memory-regulated behavior. In C. elegans, maximum chemotaxis indices toward NH₄Cl occurs when there is concordance between cultivation temperature and assay temperature (Kuhara et al., 2008). The C. elegans AFD neurons are also important for gustatory aversive learning in NaCl avoidance (Watteyne et al., 2020). Given the influence of environmental temperature on the P. pacificus mouth-form plasticity and the wide range of micro-climates that wild strains of P. pacificus have been isolated from Leaver et al., 2016; Sieriebriennikov et al., 2017; Leaver et al., 2022, temperature and taste preferences could be regulated at multiple genetic levels during crucial developmental decisions.

We found that the key regulator of Cel ASE identity, che-1, is also expressed in Ppa ASE and may play a similar role as a terminal selector in this neuron type, based on its effect on ASE-mediated behavior and regulation of the Ppa-gcy-22.3 gene. However, the stronger behavioral effect of silencing of che-1 expressing neurons compared to a che-1 mutant background could either indicate that che-1 does not have as broad a role in controlling ASE differentiation in P. pacificus versus C. elegans. It is possible that a developmental loss of ASE differentiation may result in compensatory changes in the chemosensory system during early development, as was observed in the C. elegans mating pheromone response by males (White et al., 2007). It is also worthwhile to note that Cel-gcy-22 stands out for being located on a separate chromosome (Chr. V) compared to the other ASER-type gcy genes (gcy-1, gcy-4, gcy-5 on Chr. II) as well as the Cel-gcy-22 mutant having defects in chemoattraction toward a wide range of salt ions (Ortiz et al., 2009; Ortiz et al., 2006). Given that the five P. pacificus gcy-22-like paralogs are located on three separate chromosomes (Chr. I, IV, and X), it is likely that they emerged from independent and repeated gene duplication events after the separation of Caenorhabditis and Pristionchus lineages. Although the promoter fusion reporters of other gcy genes have been uninformative due to lack of expression (Ppa-gcy-22.1, Ppa-gcy-7.1, Ppa-gcy-7.2, and Ppa-gcy-5), it is likely that ASEL-specific gcy genes as well as additional ASER-specific gcy-22-like genes exist. Finding other genes with left–right specific expression could help to identify genetic determinants affecting lateral asymmetry.

Unexpectedly, we found that unlike in C. elegans, the Ppa-che-1 gene is also expressed in the AFD neurons. Since we cannot record neural activity in AFD in a che-1 mutant (the che-1p::RCaMP driver fails to be expressed sufficiently in che-1 mutants due to autoregulation), and do not yet have molecular markers for Ppa AFD neurons, we cannot assess whether che-1 affects AFD neuron differentiation.

Perhaps the most striking difference in the gene regulatory architecture of ASE neuron specification is the apparent lack of the key regulator of ASE asymmetry in P. pacificus, the miRNA lsy-6. In C. elegans, the expression of lsy-6 exclusively in ASEL is prepatterned via an early embryonic Notch signal (Cochella and Hobert, 2012) and serves to downregulate the homeodomain transcription factor cog-1 in the ASEL neuron (Johnston et al., 2005). Through a network of downstream regulatory events, asymmetry of rGCs eventually becomes established (Hobert, 2014). cog-1 and several asymmetrically expressed downstream effectors of cog-1, such as the die-1, lim-6, and fozi-1 transcription factors are conserved in P. pacificus, but whether the function of these factors in controlling P. pacificus ASE laterality is conserved remains to be determined. In this context it is intriguing to note that another prominent gene regulatory pathway that is controlled by miRNAs in C. elegans, the heterochronic pathway (let-7s and lin-4), appears to have diverged in P. pacificus as well, despite the conservation of the overall physiological readouts of this pathway (temporal patterning of cell lineage divisions) (Sharma et al., 2024). It is tempting to speculate that miRNA-meditated regulatory process is particularly labile.

In conclusion, our work illustrates how comparative behavioral and genetic analyses in nematodes is a powerful strategy to uncover substrates of evolutionary change in simple nervous systems.

P. pacificus and other nematode strains were maintained at ~20°C on NGM plates seeded with E. coli OP50 for food as described previously (Cinkornpumin et al., 2014); these are derived from standard C. elegans culture methods (Brenner, 1974). P. pacificus and other nematode strains used are listed in Supplementary file 1A.

The assay for assessing response to salt gradients was adapted from C. elegans and P. pacificus chemotaxis assays (Hong and Sommer, 2006; Bargmann and Horvitz, 1991; Ortiz et al., 2006). Overnight salt gradients were established on 10 cm chemotaxis plates containing 20 ml agar (5 mM KPO₄, 1 M CaCl₂, 3% Bacto-agar, and 1 mM MgSO₄) by adding 10 µl of 2.5 M salt solutions for 16 hr. Alternatively, agar containing 25 mM (NH₄Cl and NH₄I) or 50 mM (NaCl) were used to test for responses to individual salt ions. Following the establishment of the overnight point gradient, another 4 µl of the same salt solution or water control was added to reinforce the gradient 4 hr before the assay. Just prior to the assay, 1 µl of 1 M sodium azide was added to both the attractive salt (A) and the control (C) spots. P. pacificus J4 to adult hermaphrodites from near-saturated cultures were washed 3× with distilled water and collected by centrifuging at 2000 rpm for 2 min. Approximately 200 worms were loaded onto the edge of each assay plate between the gradient sources, and at least 10 combined worms have to reach the scoring arenas to be considered. At least 10 assays constituted each experimental trial, and multiple trials were conducted and averaged for each condition. The chemotaxis index for each end-point assay plate is defined as (A − C)/(A+C). To conduct conditional knockdowns of neurons, 5 M histamine dihydrochloride (Sigma-Aldrich H7250) stock solution in sterilized deionized water (Arrowhead CA) was filter-sterilized and top plated onto the agar plates to a final histamine concentration of 25 mM histamine approximately 10 min before loading the worms to commence the assay. Most assays lasted 3–4 hr at room temperature to allow P. pacificus sufficient time to reach the scoring arenas, with ~40% of the animals participating. We excluded an outlier due to likely scoring error (value = –0.64) in the chemotaxis assay for gcy-22.3(csu182) on NH₄Cl (Figure 8). When using the csuEx83[Ppa-che-1p::optHisCl1] strain, only animals expressing Ppa-egl-20p::RFP tail marker from the extrachromosomal array were scored. Because of P. pacificus’ strong aversion to acetate, we could not easily assess the individual contributions of salt ions in a saturated background of ammonium acetate as conventionally practiced in C. elegans studies (Ortiz et al., 2009).

To make the Ppa-che-1p::optHisCl1, the P. pacificus codon-optimized histamine-gated chloride channel sequence used in C. elegans (HisCl1) (Pokala et al., 2014; Pokala and Flavell, 2022) was designed using (https://hallemlab.shinyapps.io/Wild_Worm_Codon_Adapter/; Bryant and Hallem, 2021), was custom synthesized (Twist Bioscience), and subsequently inserted behind the che-1 promoter (3.1 kb containing the first exon and intron) (Hong et al., 2019) to create the pHC30 plasmid construct. This Ppa-che-1p::optHisCl1 plasmid (2 ng/µl) along with PS312 genomic DNA (80 ng/µl) and Ppa-egl-20p::RFP co-injection marker (2 ng/µl) were individually digested with HindIII and assembled as the injection mix to create csuEx83.

To make the Ppa-che-1pei::optRCaMP, we generated a transgenic worm strain expressing the codon-optimized genetically encoded calcium indicator (GECI), jRCaMP1a, in the neurons of interest (Kerr et al., 2000; Nakai et al., 2001). jRCaMP1a is an improved red GECI based on mRuby with comparable sensitivity to GCaMP6 (Dana et al., 2016). The codon-optimized RCaMP sequence was custom synthesized (Twist Bioscience), and then subcloned using the plasmid pJET1.2/blunt (Thermo Fisher Scientific) to create the pMM2 plasmid construct. Using Gibson Assembly (E2611, New England Biolab), the codon-optimized RCaMP sequence was introduced downstream of the Ppa-che-1 promoter sequence with the first exon and intron sequences to create the pMM5 plasmid construct. Separately, we also generated a Ppa-che-1pei::optGFP transcriptional reporter with codon-optimized GFP (pMM3) to enable the localization of the che-1-expressing neurons during video acquisition (Han et al., 2020). The Ppa-che-1pei::optRCaMP (2 ng/µl) and Ppa-che-1pei::optGFP constructs (1 ng/µl), along with PS312 genomic DNA (80 ng/µl) and Ppa-egl-20p::RFP (1.5 ng/µl) were individually digested with HindIII and assembled as the injection mix to create csuEx93. Despite multiple attempts, we were unable to generate an equivalent che-1pei::GCaMP transgenic line with sufficient basal level of GCaMP expression for a comparison to the RCaMP calcium dynamics.

The Ppa-gcy-22.3p::GFP construct contains 899 bp of the upstream promoter of the PPA04554 transcript is fused to the codon-optimized GFP (pVL2). A shorter gcy-22.3 transcript with different first two exons is also predicted, which is more similar in length to other rGC genes in P. pacificus (Contig12-snapTAU.506). The Ppa-ttx-1pei::RFP construct contains 1935 bp of the upstream promoter along with the first exon and intron sequences of Ppa-ttx-1 (PPA26714) fused to the codon-optimized RFP, excluding the first two codons (pDC14). pVL2 (2 ng/µl) or pDC14 (2 ng/µl), along with PS312 genomic DNA (80 ng/µl) were individually digested with HindIII to create the injection mixes to generate the independent reporter strains, csuEx90[Ppa-gcy-22.3p::GFP], as well as Ppa-ttx-1pei::RFP(csuEx94) and Ppa-ttx-1pei::RFP(csuEx96). csuEx96 showed less gland cell expression (which can occlude neuronal expression) and stronger AFD expression than csuEx94.

CRISPR/Cas9 mutagenesis was used to generate mutations (Han et al., 2020; Nakayama et al., 2020). crRNA and primer sequences, and induced mutations, are included in Supplementary file 1C and .

To conduct calcium imaging, worms were trapped and imaged within a microfluidic PDMS chip while delivering stimuli directly to the nose of an immobilized worm, as previously described (Chronis et al., 2007; Chalasani et al., 2007). The microfluidic chip was connected to a programmable valve controller (ValveBank) that enables the user to toggle between ‘stimulant ON’ and ‘stimulant OFF’ states. Specifically, the ValveBank allows controlled switching of flow from the two outer buffer channels in the chip such that either the control solution (stimulant OFF) or the stimulant solution (stimulant ON) flows over the worm nose (Figure 4—figure supplement 3). Buffer solution (for the outer two channels) consisted of M9 buffer with 0.1% Tween-20 and 1 µg/ml fluorescein (Stiernagle, 2006; Liu et al., 2018). The worm loading solution consisted of M9 buffer with 0.1% Tween-20 and 1.5 mM tetramisole hydrochloride to immobilize the animal. Water-soluble stimulant solutions consisted of the following water-soluble compounds dissolved in nanopure/milli-Q water: ammonium chloride (NH₄Cl), ammonium iodide (NH₄I), and sodium chloride (NaCl) at concentrations of 250 and 25 mM. 750 mM NaCl elicited very inconsistent responses. The water-soluble stimulant solution was made by diluting the water-soluble compound in nanopure/milli-Q water with 0.1% Tween-20. The control solution consisted of 0.1% Tween-20 nanopure/milli-Q water. Worms were exposed to the control solution, stimulant solution, and then control solution using a 60-s program: 10 s stimulant OFF, 20 s stimulant ON, and 30 s stimulant OFF. As a negative control, animals were exposed only to the control solution, without switching channels, for the duration of the recording. For each animal, the orientation of the nose and vulva were recorded and used as a guide to determine the ventral and dorsal sides of the worm, and subsequently, the left and right sides of the worm. Accounting for the plane of focus of the neuron pairs as viewed through the microscope, it was then determined whether the imaged neuron was the worm’s left or right neuron of each pair. Images were captured using a Zeiss Axio Observer Z1 inverted fluorescence microscope and a pco.panda 4.2 sCMOS camera. Changes in fluorescence intensity were measured in the neurons of interest while the worm was exposed to green light. Images were processed using MetaMorph software version 7.10.5.476. Images were captured at 500 ms exposure time at 2 fps because the baseline fluorescence in Ppa-che-1p::optRCaMP worms was too dim to capture viable data using the standard 100 ms exposure time. Baseline F₀ was measured as the average background-subtracted fluorescence from the first 9 s of each recording and change in fluorescence intensity was calculated as dF/F = (F − F₀)/F₀, as described (Liu et al., 2018). Data were analyzed and plotted using custom scripts generated in MATLAB versions R2021a–R2024a. This code is available at https://github.com/honglabcsun/Calcium-Imaging, copy archived at Mackie and Hong Lab CSUN, 2025.

For bar plot comparisons between wildtype and gcy-22.3 mutants, we calculated minimum pre-stimulus and maximum post-stimulus % dF/F values using custom scripts generated in MATLAB. Min–Max data were exported as text files, manually converted and organized into an XLSX file (Microsoft Excel Office16) and imported into Prism GraphPad software (version 10) for data visualization and statistical analysis.

Throughout the results section, P. pacificus genes will be referred to without the Ppa- prefix; if necessary for comparison to another species such as C. elegans (Cel-) the Ppa- prefix will then be used.

The plasmids and viable nematode strains will be made available upon request.