Investigation of the nicotinic receptor EAT-2 as a novel target to mitigate plant parasitic nematode infections

33 Plant parasitic nematodes (PPNs) are microscopic soil dwelling pests that infect crops, using a 34 lance-like organ , the stylet, to hatch, invade plant roots, and establish feeding sites. Stylet 35 function is underpinned by pharyngeal muscle contraction and relaxation cycles, making it an 36 attractive route to disrupt the PPN lifecycle. However, knowledge of pharyngeal regulation in 37 PPNs is relatively limited. In the free-living nematode Caenorhabditis elegans, the nicotinic 38 receptor EAT-2 stimulates pharyngeal contraction to facilitate feeding. Here we hypothesize 39 that EAT -2 orthologues may regulate a similar function in PPNs. A p hylogenetic analysis 40 reveals that EAT-2 and its orthologues in other nematode species cluster as a distinct group 41 suggesting that EAT-2 is exclusive of other animal species. We identified eat-2 in the genome 42 of the potato cyst nematode Globodera rostochiensis and used in situ hybridization to establish 43 an anterior expression pattern consistent with a pharyngeal function. In vitro pharmacological 44 assays directly compared the response of C. elegans pharynx and G. rostochiensis stylet to 45 cholinergic compounds. Both pharyngeal and stylet activity were stimulated by acetylcholine 46 and nicotine , and th ese responses were blocked by the nicotinic receptor antagonists, 47 mecamylamine and tubocurarine. Th ese data are consistent with a conserved cholinergic 48 pathway mediated by EAT-2 regulating pharyngeal muscle function. It highlights EAT-2 as a 49 potential determinant of stylet thrusting and a promising pharmacological target to selectively 50 mitigate PPN infections. 51 Key words: Cholinergic signaling, EAT-2 receptor , Globodera rostochiensis, plant 52 parasitic nematodes, pharyngeal function, pharmacological target, stylet thrusting 53 54 55 56 57 58 59 60 61 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint Plant parasitic nematodes (PPNs) are global economic pests that account for significant 62 annual losses in agriculture (Lilley et al., 2024) . Current control strategies rely on farming 63 practices like crop rotation, fallowing, use of resistant crop varieties and chemical strategies 64 that disrupt the parasitic life cycle (Li et al., 2015; Mesa-Valle et al., 2020; Pires et al., 2022). 65 Unfortunately, several available nematicides are either restricted or banned because of their 66 broad actions and negative environmental impacts (Ngala et al., 2021) . Thus, finding novel 67 pesticide targets an d new products that meet required regulatory standards is of high 68 importance (EPA, 2023) . Generally, the nervous system of pests is an attractive target for 69 pesticides because they are comprised of several targets that are rapidly and readily druggable 70 (Hirata, 2016). Pest control has been well served by targeting ion channels whose disruption 71 impairs signal transduction resulting in muscle paralysis, spasms and death of invertebrate pests 72 (Crisford et al., 2015; Hirata, 2016; Raisch and Raunser, 2023) . Although not widely used, 73 integrating the biological understanding of mechanisms unique to behaviors that support 74 parasitism might refine the focus on pesticide selectivity and reduce the limiting negative 75 environmental impacts. 76 Investigating the molecular determinants of core behaviors in PPNs is challenging 77 given the lack of genetic tools. In this regard, studies in the model nematode Caenorhabditis 78 elegans can be informative (Holden-Dye and Walker, 2014; Coke et al., 2024) . The nervous 79 system receives several sensory modalities that co -ordinate and generate distinct phenotypic 80 and behavioral outputs which regulate essential processes. This means that behaviors like 81 feeding, locomotion and reproduction can be templated in C. elegans and translated to other 82 nematodes (Alcedo et al., 2013; Chew et al., 2013; Gjorgjieva et al., 2014; Thapliyal and Babu, 83 2018). The pharynx of C. elegans acts as a neuromuscular pump that regulates ingestion of 84 bacteria. In C. elegans 20 radial muscle cells organized into 8 pharyngeal muscles regulate 85 feeding i n a two -part process involving pharyngeal pumping and peristalsis . The rate and 86 pattern of pharyngeal pumping is controlled by the pharyngeal nervous system (McKay et al., 87 2004; Avery and You, 2012). The presence of food triggers serotonergic neurons to release 5-88 HT resulting in elevated pharyngeal pumping rates (Avery and Horvitz, 1990; Song et al., 89 2013). The biogenic amine 5-HT acts on cholinergic neurons MC and M4 to induce essential 90 muscle contractions that are necessary for feeding (Niacaris and Avery, 2003; Ishita et al., 91 2020). A core determinant for this response is EAT -2, the receptor that utilizes ACh released 92 from the MC neuron to initiate pharyngeal muscle contraction that opens the pharyngeal lumen 93 to ingest bacteria (McKay et al., 2004). 94 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint Interestingly, EAT-2 was initially classified as a non-alpha nicotinic receptor subunit (McKay 95 et al., 2004) because it is missing the signature vicinal cysteines amino acid residues known to 96 be a major molecular determinant of agonist acetylcholine binding (Kao and Karlin, 1986; 97 Blum et al., 2011). Surprisingly, despite lacking this motif, both the C. elegans EAT-2 and its 98 orthologue from the animal parasitic nematode Ascaris suum have been shown to function as 99 homo-oligomeric receptors made up of the co -assembly of five identical EAT -2 receptor 100 subunits (Choudhary et al., 2020) . Moreover, functional expression of EAT -2 requires co -101 expression with EAT-18, a single transmembrane domain protein that functions as an auxiliary 102 subunit. Intriguingly, and of relevance with respect to potential as a target for selective 103 nematicides, EAT-18 has no known homology to previously described proteins (McKay et al., 104 2004). EAT-18’s unique and essential contribution is proposed to be via an association with 105 the EAT-2 pentamer at the muscle plasma membrane (Choudhary et al., 2020). 106 In PPNs pharyngeal function controls critical behaviors using a specialized pharyngeal 107 structure called the stylet, a lance -like hollow structure important for their lifestyle. The 108 neurobiology underpinning this pharyngeal function is poorly understood (Grundler and 109 Böckenhoff, 1997) . In one modality pharyngeal muscles co -ordinate the protraction and 110 retraction of the stylet by J2s that it used to pierce the encasing eggshell thus driving hatching 111 (Perry and Clarke, 2000; Mkandawire et al., 2022) . This biology is later used by the hatched 112 J2s to pierce plant roots and facilitate their invasion of host plants (Bernard et al., 2017; 113 Pulavarty et al., 2021) . Additionally, a distinct pharyngeal function involving stylet thrusting 114 and median bulb pumping allows protruded stylets to ingest nutrient or release effectors and 115 cell-wall degrading enzymes into feeding sites of invaded host plants (Koga, 2014). 116 Despite the difference in the feeding style and habits of C. elegans and G. rostochiensis, 117 the organization of their pharyngeal muscles include s the corpus, isthmus and terminal bulb. 118 Furthermore, the exposure of the whole organism to 5 -HT drives pharyngeal pumping 119 responses from averages of about 60 pumps/minute to 250 pumps/minute in C. elegans and 120 stylet thrusting behaviors from about zero to 80 thrusts/minute in Globodera (Masler, 2007; 121 Song et al., 2013). A putative model based on the limited comparisons of pharyngeal structure 122 and pharmacological evidence suggests that serotonergic signaling regulates pharyngeal 123 function upstream of EAT-2 (Kearn et al., 2017; Ishita et al., 2020). Based on this information, 124 we have investigated the molecular details of EAT -2 in PPNs, its cellular expression and 125 developed pharmacological assays to resolve cholinergic function. The data supports the notion 126 that cholinergic modulation of pharyngeal function via EAT-2 underpins stylet function. This 127 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint provokes the idea that EAT -2’s function and its distinct molecular features make it a suitable 128 target for new nematicide development. 129

130 C. elegans maintenance: 131 C. elegans, N2 (Bristol strain) were obtained from the Caenorhabditis Genetics Centre (CGC), 132 grown and maintained under standard conditions (Brenner, 1974) and used as wild type (WT) 133 worms. 134 G. rostochiensis maintenance: 135 Infective juveniles (J2s) of G. rostochiensis were hatched by incubating nematode cysts in 136 potato root diffusate (PRD) over 7 days at room temperature (Gaihre et al., 2019) . Freshly 137 hatched J2s (1 day old) were washed in M9 buffer containing 0.01% w/v Bovine Serum 138 Albumin (BSA) prior to experimentation. Batch hatchings were performed for each experiment 139 to obtain freshly hatched juveniles. Unused worms were collected in batches and stored at -20 140 ºC before being used for RNA extraction. Nematode cysts used were from the James Hutton 141 Research Institute, Scotland and provided by Vivian C. Blok. The PRD used for hatching was 142 provided by Catherine Lilley, University of Leeds, UK. 143 Drugs and chemicals: 144 Serotonin creatinine sulphate monohydrate, Nicotine hydrogen tartrate salt, Mecamylamine 145 hydrochloride and Tubocurarine chloride were purchased from Sigma Aldrich, UK. To make 146 stock solutions that were used in experiments the drugs were dissolved in sterile ddH 2O and 147 stored at -20 ºC. These were used no longer than 1 month after preparation. 148 Characterizing C. elegans and G. rostochiensis response to 5-HT, ACh and nicotine: 149 C. elegans behavioral assay: 150 These assays were performed on solid NGM plates by supplementing molten NGM with 5-HT, 151 nicotine or ACh to achieve the indicated final concentration. A C. elegans pharyngeal pump 152 dose-response to 5-HT was established by testing concentrations up to 5mM. ACh and nicotine 153 were tested at 10 mM . 3 ml NGM containing vehicle or the indicated concentration of drug 154 was transferred into clear Corning Coster 6 -well plates. After setting the NGM plates were 155 stored at 4 ºC and used the following morning. Before experimenting, these plates were 156 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint equilibrated to room temperature for 30 minutes. Well -fed young adult C. elegans (L4+1s) 157 were washed in M9 buffer to remove bacteria and then picked onto test plates. Pharyngeal 158 pump rates were measured by making visual observations using a Nikon SMZ800 binocular 159 microscope. A pump was defined as a forward and backward movement of the grinder and was 160 recorded for a minute per worm (Raizen et al., 1995) . All C. elegans behavioral assays were 161 performed on NGM supplemented plate unless specified otherwise. 162 G. rostochiensis behavioral assay: 163 Stylet thrusting dose -response to 5 -HT was established by supplementing 20 mM HEPES 164 solution, pH 7.4 with indicated concentrations of 5-HT. Ten freshly hatched J2s washed 3 times 165 with ddH2O were pipetted in 2 µl and transferred into clear Corning Coster 96-well plates 166 containing 200 μl of 20 mM HEPES (control) or with an indicated 5-HT concentration. The 167 number of stylet thrusts were measured for 30 seconds per worm, 30 minutes after incubating 168 G. rostochiensis in 5 -HT and represented as number of thrusts per minute. A forward and 169 backward movement (extension and retraction) of the stylet was counted as one stylet thrust 170 (Kearn et al., 2017) . All G. rostochiensis behavioral assays were performed in liquid assays 171 unless specified otherwise. 172 Acute responses to selected drugs: 173 We investigated the acute effects of 10 mM nicotine, 200 μM mecamylamine and 300 μM 174 tubocurarine on 5-HT induced pharyngeal pumping and stylet thrusting. This was performed 175 by adding respective drug or requisite vehicle after establishing a steady -state stylet thrusting 176 response for 30 mins in 1mM 5 -HT. The acute response to these selected drugs on 5 -HT 177 induced pharyngeal pumping and stylet thrusting was measured for up to 2 hours 178 Chronic responses to selected drugs: 179 The chronic effect of the drugs was investigated by pre -incubating C. elegans or G. 180 rostochiensis for 24 hours in 10 mM nicotine, 200 μM mecamylamine and 300 μM 181 tubocurarine prior to transferring them onto assays plates. 182 Molecular biology: 183 Phylogenetic analysis: 184 We used the basic alignment search tool (BLAST) (Altschul et al., 1990) and the query 185 Ce.EAT-2 (Uniprot accession number: Q9U298) to search for EAT-2 homologues on Uniprot 186 and WormBase ParaSite. The resulting putative protein sequences were imported into the 187 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint MEGAx (Molecular Evolutionary Genetics Analysis) tool and aligned via MUSCLE alignment 188 (multiple sequence alignment by log -expectation) (Stecher et al., 2020) with default 189 parameters. The aligned sequences were used to generate a phylogenetic tree using the 190 Maximum Likelihood (ML) statistical method, a Jones -Taylor-Thornton (JTT) substitution 191 model and 500 bootstrap replications to assess node support. The tree interference options were 192 set to default parameters. The phylogenetic tree output was exported in a Newick format and 193 visualized using iTOL (interactive tree of life) (Letunic and Bork, 2024). The following protein 194 sequences were used in the analysis to build the phylogenetic tree: 195 Q9U298 (C. elegans); A0A261C1U6 (C. latens); A0A9P1MVH6 (C. angaria); A0A158PA63 196 (A. cantonensis ); A0A0N4XX74 ( N. brasiliensis ) A0AA36C8X6 ( M. spiculigera ); 197 A0AAF3EAP6 ( M. belari ); A0AA39GWV7 ( S. hermaphroditum ); A0A7E4V0M4 ( P. 198 redivivus); A0A2A6C485 ( P. pacificus ); A0A0K0DZ20 ( S. stercoralis ); A0A811LPF4 ( B. 199 okinawaensis); A0A1I7S0H9 (B. xylophilus); A0A914M6R1 (M. incognita); A0A6V7VQB4 200 (M. enterolobii ); A0A914I8V5 ( G. rostochiensis ); A0A8T0A120 ( M. graminicola ); 201 A0A914XFN7 ( P. sambesii ); A0A0N4UD55 ( D. medinensis ); A0A0B2W3Z2 ( T. canis ); 202 A0A4E9FW31 ( B. malayi ); W2TG30 ( N. americanus ); A0A4U8V1R9 ( S. carpocapsae ); 203 A0A2G9V6F1 ( T. circumcincta ); A0AA36MEL8 ( C. nassatus ); A0A0N4VD61 ( E. 204 vermicularis); A0A1I7V6L7 (L. loa); A0A0V0XKJ1 (T. pseudospinralis); A0A9C6WDJ3 (D. 205 albomicans); A0A8B8HQB2 ( V. tameamea ); M9PFD8 ( D. melanogaster ); A5H031 ( M. 206 domestica); A0A9P0BVQ3 ( C. includens ); A0A8R2AB14 ( A. pisum ); A0A8B7PB12 ( H. 207 azteca); A0A8I6SIA1 (C. lectularius); AgR003_g027 (A. suum); A0A915M3T9 (M. javanica); 208 tig00002135.g43658.t1 ( M. arenaria ); A0A1I8BSG2 ( M. hapla ); Hsc_gene_20958.t1 ( H. 209 schachtii), A0A183C532 ( G. pallida ); KAH7704458.1 ( A. avenae ); A0A158PA63 ( H. 210 sapiens). 211 RNA extraction: 212 Total RNA was extracted from one 1000 C. elegans young adults and 2000 infective G. 213 rostochiensis juveniles respectively. Nematodes were washed with diethyl pyrocarbonate 214 (DEPC) treated M9 buffer, centrifuged at 500 g and the supernatant discarded and leaving the 215 worms in minimal volumes of DEPC in the 2 ml eppendorf tubes. 1 ml of TRIzolTM was added 216 to each sample and homogenized for a minute using the hand -held VWR® VDI 12 217 homogenizer (VWR Collection). Homogenization was monitored via visual inspection and was 218 continued until all intact organisms were broken open . Total RNA was isolated using the 219 sequential TRIzolTM (Invitrogen) Qiagen RNA clean up protocol (Metzger, 2024). Total RNA 220 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint extracted was stored at -20 ºC or used to synthesize cDNA using the SuperScript® III First -221 Strand Synthesis System protocol (Invitrogen). 222 PCR amplification: 223 The sequences that encode the open reading frame (ORF) for Ce.EAT-2 (Uniprot accession 224 number: Q9U298 ) was used as input to perform a protein BLAST search on Uniprot and 225 WormBase ParaSite. We identified orthologues in sequenced parasitic nematodes and the 226 predicted ORF for Gr.EAT-2 (accession number: A0A914I8V5 ). Primers designed and 227 synthesized with Integrated DNA Technologies were used to amplify the predicted coding 228 sequence of both organisms. The following primers were used to amplify EAT -2 transcripts 229 for C. elegans and G. rostochiens; 230 FwCe.eat-2 5'- CGCACgaattcATGACCTTGAAAATCGCATT -3' 231 RvCe.eat-2 5’- CAGGCTAACAACTATAACTTATTcccgggCG - 3’ 232 FwGr.eat-2 5'- GCggtaccATGTTTTTGCGA -3' 233 Rv.Gr.eat-2 5'- GGTGGAGAGATTcccgggGA -3' 234 Restriction sites (underlined sequences) were added at primer ends to flank the amplified 235 sequence. The authenticity of the amplified sequence was confirmed by sequencing the forward 236 and reverse strands of the amplified gene (Eurofins Genomics) (see supplementary 1). 237 Hybridization Chain Reaction RNA-Fluorescence in-situ hybridization: 238 The expression patterns for the target mRNA Gr.EAT-2 (Uniprot accession number: 239 A0A914I8V5), Gr.MYO-3 (Uniprot accession number: A0A914HJT9) and Gr.UNC-17 240 (Uniprot accession number: A0A914GTZ9) were investigated using a modified HCRTM RNA-241 FISH (v3.0) protocol for whole-mount nematode larvae (Choi et al., 2016). 242 Probe sets, amplifiers and buffers: 243 Probes against target transcripts, probe hybridization buffers, probe wash and amplifier buffers 244 were purchased from Molecular Instruments. The number of probe binding sites for Gr.eat-2 245 was set to 40 to mitigate likely low and restricted expression levels based on exemplar data in 246 C. elegans (McKay et al., 2004). Probe binding sites for Gr.unc-17 and Gr.myo-3 were standard 247 20. Amplifiers and hairpins used were: B1H1, B1H2 for EAT -2 and B2H, B2H2 for UNC-17 248 and MYO-3. Other reagents used included: M9 buffer (22 mM KH 2PO4, 42 mM Na 2HPO4, 249 20.5 mM NaCl, 1 mM MgSO4), PBST (1X PBS, 0.1% Tween), 4% paraformaldehyde (PFA), 250 glycine solution (2 mg/mL glycine, PBST), proteinase K solution (100μg/mL). 251 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint Preparation of fixed whole-mount nematode larvae: 252 Approximately 1000 G. rostochiensis J2s were washed three times with 1 mL of M9. During 253 each wash the tube were centrifuged at 500 g for 2 min to bring larvae to the bottom and the 254 supernatant carefully removed. Washed J2s were aliquoted in minimal volumes of M9 buffer 255 and incubated in 1 mL of 4% paraformaldehyde (PFA) before immediately freezing at -80 ◦C 256 overnight. The following day the fixed J2s were thawed at room temperature for 45 minutes 257 washed twice in 1 mL of PBST and treated with 1 mL proteinase K (100 µg/mL) (Sigma 258 Merck) for 10 min at 37 ◦C. The proteinase K treated J2s were washed with PBST and incubated 259 in 1 mL of glycine solution (2 mg/mL) for 15 min on ice followed by 2 washes with PBST 260 (HCRTM RNA-FISH (v3.0) protocol). 261 Probe hybridization, amplification and detection: 262 Fixed and permeabilized J2s were incubated in 1 mL of 50% PBST / 50% probe hybridization 263 buffer for 5 min at room temperature and centrifuged at 500 g for 2 min before removing the 264 supernatant. These processed J2s were then pre -hybridized in 300 µL of HCR ™ probe 265 hybridization buffer at 37 ◦C for 1 h. The indicated probe solutions were prepared just before 266 use by adding 2 µL of the 1 µM stock to 200 µL of probe hybridization buffer at 37 ◦C (See the 267 HCRTM RNA-FISH (v3.0) protocol for more detail) (Choi et al., 2016). The pre-hybridized J2s 268 were added to the freshly made probe solution and incubated overnight (>12 h) at 37 ◦C. After 269 the overnight incubation the probe solution was removed by washing the J2s 4 times in 1 mL 270 of probe wash buffer at 37 ◦C for 15 minutes. These washes were followed by two further 5 271 mins washes at room temperature in 1 mL of 5× SSCT. To facilitate pelleting of worms 272 between washes prepared worms were centrifuged at 800 g. Hybridized J2s were incubated in 273 300 µL of amplification buffer for 30 min before adding 200 µL of the hairpin solution and 274 incubating overnight (>12 h) in the dark , at room temperature. Following this incubation the 275 worms were washed 5 times with 1 mL 5× SSCT at room temperature. These samples were 276 treated with 0.01% DAPI (v/v) and stored at 4 ◦C protected from light before imaging. 277 Mounting and imaging: 278 The probe treated specimens were pipetted in 20 µL of SSCT (about 30-50 nematodes), placed 279 on a glass slide, secured with a cover slip and sealed with nail varnish. Slides were mounted 280 on the Olympus/Yokogawa Spinning Disk confocal microscope and imaged using the Alexa 281 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint fluor 488 for GFP and 594 for mcherry. Images were prepared using image J, with minor 282 adjustments on the brightness and contrast of the DIC images (Schindelin et al., 2012). 283 Statistical analysis: 284 Data analysis was performed with GraphPad Prism 10.4 and presented as the mean ± standard 285 error of the mean (SEM) for a number of observations (n). Statistical significance was tested 286 either by unpaired student’s t -test, one -way or two -way ANOVA followed by Bonferroni 287 multiple comparison where appropriate. Significance levels were set at p<0.05. Every 288 experiment was repeated in 3 or more independent occasions, unless stated otherwise. EC 50 289 values with 95% confidence intervals were determined by plotting log (agonist) vs. normalized 290 response-variable slope and fitted to the equation; Y=100/(1+10^((LogEC50-X)*HillSlope)) 291

292 Phylogenetic evidence reveals EAT-2 unique expression in the Nematoda: 293 Amino acid sequence for C. elegans EAT-2 was used as a query to run a protein blast on 294 Uniprot and WormBase ParaSite databases. The phylogenetic analysis revealed Ce.EAT-2 and 295 orthologues in other nematode species cluster as a distinct group suggesting that EAT-2 296 orthologues are exclusive of other animal species ( Fig. 1A). The EAT -2 orthologues predict 297 the classical nicotinic acetylcholine receptor transmembrane topology encompassing a large 298 extracellular N-terminal domain and four intervening transmembrane helices leading to a short 299 extracellular C-terminus. In the context of a nAChR the acetylcholine binding site lies at the 300 interface between the extended N -terminal extracellular domains of neighboring subunits. In 301 homomeric receptors like alpha-7 and EAT-2 this is at the principal (front) and complementary 302 (back) end of each subunit. The six-loop fold that comes together to contact the activating ACh 303 has a major and minor contact made up of discontinuous loops ABC and DEF respectively. 304 Comparing amino acid residues that make up the principal and complementary phases of the 305 ligand binding domain for Ce.EAT-2 and its closely related human orthologue the nicotinic 306 receptor alpha-7, revealed a 50% conservation of amino acid residues in these loops (Fig. 1B). 307 A multiple sequence alignment for Ce.EAT-2 and some selected nematode species with alpha-308 7 revealed the absence of vicinal cysteine residues in the loop -C region of these receptor 309 subunits (Fig. 1C). Interestingly these pivotal vicinal cysteines were originally identified as an 310 absolute requirement for nicotinic receptor alpha subunits acetylcholine binding, but emerging 311 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint data recognizes that activation can occur in the absence of this motif (Blount and Merlie, 1990; 312 Blum et al., 2011; Choudhary et al., 2020). 313 314 315 B .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint 316 Fig. 1 Maximum likelihood phylogeny of EAT-2. A. The tree was rooted with α-7, the closest 317 human orthologue of C. elegans EAT-2. Phylogenetic analysis groups EAT -2 of nematodes 318 (green) in a common branch separate from orthologues of arthropod and humans. B. A 319 schematic representation showing a 50% conservation of amino acid residues that make the 320 agonist binding site (ABS) of EAT-2 and α-7. Loops A, B and C make up the principal phase 321 of the binding domain and loops D, E and F, the complementary phase. C. A multiple sequence 322 alignment for C. elegans EAT-2 and orthologues in some economically important plant 323 parasitic nematodes shows the absence of vicinal cysteines in the loop-c when compared to the 324 human α-7. 325 EAT-2 expression pattern in G. rostochiensis: 326 Spatial expression of Gr.EAT-2 HCR RNA-FISH: 327 To resolve the organization of cholinergic transmission and the molecular features of Gr.eat-2 328 we first authenticated the predicted sequence. This involved extracting total RNA from G. 329 rostochiensis, reverse transcribing it to cDNA and then using specific primers to amplify the 330 predicted ORF for Gr.eat-2. The amplification product was authenticated through sequencing 331 and fully aligned with the predicted sequence . The amplified sequence was used to design 332 probes for HCR RNA-FISH, to investigate the spatial expression of Gr.eat-2 transcripts using 333 whole-mount in-situ hybridization. We extended this to the wider cholinergic system using the 334 predicted orthologues for Gr.unc-17 which encodes the vesicular ACh transporter in C. elegans 335 (Alfonso et al., 1993) and is found in all cholinergic releasing neurons and Gr.myo-3 which 336 encodes the myosin heavy chain protein highly expressed in longitudinal muscles of the body 337 wall muscle (Meissner et al., 2009). Previously developed methods for getting small molecules 338 like DAPI into worms suggested the need to physically break open the cuticle (Lilley et al., 339 2018). However, we used the HCRTM RNA-FISH (v3.0) protocol for whole-mount nematode 340 larvae (Choi et al., 2016) and achieved a 30% success rate (n=100). With probes for Gr.eat-2 341 and Gr.unc-17 in a co -localization hybridization reaction we identified a discrete expression 342 pattern for Gr.EAT-2 in pharyngeal muscles, specifically in the median bulb and further 343 C .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint discrete but not always present staining in what looked to be the pharyngeal glands (Fig. 2A). 344 Gr.UNC-17 was identified as punctate staining in the anterior region and along the ventral side 345 of the worm, highlighting the possible locations of cholinergic motor neurons ( Fig. 2A). The 346 specific signals in the pharynx are suggested to highlight the neuronal cell bodies of the 347 cholinergic neurons that innervate pharyngeal muscles. The specificity of Gr.EAT-2’s 348 pharyngeal expression pattern was reinforced in a co-localization hybridization reaction with 349 Gr.MYO-3 (Fig. 2B). Worms fixed and prepared in the absence of target probes showed no 350 signal for our target genes. The sclerotized head and stylet were auto fluorescent and very 351 bright for both control and probe treated specimen (Fig. 2C). 352 353 Fig. 2 Spatial expression of EAT -2 in G. rostochiensis visualized with FISH. A. Whole-354 mount nematode visualizing the expression of Gr.EAT-2, seen as red puncta and Gr-UNC-17 355 seen as green puncta in the anterior region, consistent with pharyngeal expression. B. Gr.MYO-356 3 expressed in longitudinal muscles of the body wall muscle (green), C. Worms treated without 357 target probe show no cellular expression but a non-specific autofluorescence in the head region 358 and the stylet. Arrow heads point to the localization of indicated hybridization probe; the 359 asterisk represents an additional puncta pattern not always observed in worms that show the 360 arrow heads. The images are representative images from 2k prepared J2s in two independent 361 histology runs. Scale bar, 50 μm. 362 A ** .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint A comparative pharmacological analysis of C. elegans pharyngeal pumping and G. 363 rostochiensis stylet thrusting: 364 The functional control of the nematode pharynx is regulated by modulatory neurons that can 365 be probed by the external exposure to 5-HT (Hobson et al., 2006; Horvitz et al., 1982; Perry et 366 al., 2004). We compared the dose-dependence of 5-HT on C. elegans pharyngeal pumping and 367 G. rostochiensis stylet thrusting to benchmark pharmacological comparisons between these 368 nematode species. Increasing concentrations of 5-HT induced increased pharyngeal activity for 369 both nematode species. In C. elegans 5-HT had an EC 50 of 237 μM (95% confidence limit 370 168.5 - 315 μM) and 5 mM induced the highest pharyngeal pumping rate. Similarly, in G. 371 rostochiensis increasing 5 -HT concentrations induced increasing stylet thrusting responses 372 with an EC 50 of 409 μM (95% confidence limit 365.0 to 457 .5 μM) and a maximal stylet 373 thrusting rate at 1mM. 374 In solutions like M9 or 20 mM HEPES buffer, C. elegans moves by thrashing in a coordinated 375 manner (Buckingham and Sattelle, 2009) . In contrast, movement in G. rostochiensis is 376 relatively uncoordinated. Interestingly, when incubated in 1 mM 5-HT beyond 30 minutes they 377 became immobilized. This was similarly observed by incubating C. elegans in 30 mM 5 -HT 378 (Ranganathan et al., 2000) . C. elegans assumed a steady position with elevated pharyngeal 379 pump rates while G. rostochiensis adopted a kink position that was associated with the induced 380 stylet thrusting. 381 We also investigated known agonists of cholinergic systems, ACh and nicotine and observed 382 that these ligands activated pharyngeal pumping and stylet thrusting behaviors. The responses 383 were proportionately higher in C. elegans compared to G. rostochiensis (Fig. 3). The exposure 384 of C. elegans to 5-HT induced pharyngeal pumping in similar rates like ACh and nicotine (Fig. 385 3A). With G. rostochiensis , t he stylet thrusting response to ACh and nicotine was weak er 386 compared to 5 -HT induced responses ( Fig. 3B). In general, the modulation of pharyngeal 387 function by nicotinic receptor agonists supports an underlying contribution of cholinergic 388 transmission within the pharyngeal system of nematodes. 389 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint 390 Fig. 3 EAT-2 agonists induce pharyngeal behaviors in C. elegans and G. rostochiensis. A. 391 5-HT, ACh and nicotine activate pharyngeal pump responses in C. elegans (n>8) and B. stylet 392 thrusting in G. rostochiensis (n=10). Data represented as mean ± SEM 393 EAT-2 modulators inhibit 5-HT induced pharyngeal function in C. elegans and G. 394 rostochiensis: 395 There is a limited pharmacology of the EAT -2 receptor . However, micromolar 396 concentrations of the agonist nicotine and the antagonists mecamylamine and tubocurarine are 397 reported to be highly potent against the recombinantly expressed EAT-2 receptor (Choudhary 398 et al., 2020). Having established the 5 -HT dependence of stylet thrusting and pharyngeal 399 pumping, we performed comparative pharmacological assays to inform us on the organization 400 and role of EAT-2 in the pharyngeal system of PPNs. This involved comparing the acute (short-401 term) and chronic (long-term) exposure effects of these drugs on 5-HT stimulated pharyngeal 402 responses in both nematode species. 403 Acute exposure to cholinergic signaling modulators impact nematode pharyngeal 404 function: 405 The short-term exposure of C. elegans to nicotine rapid ly inhibited the pharyngeal 406 pump response induced by 5 -HT. This inhibitory effect was sustained over the 2 -hour 407 experimental period (Fig. 4A). This was an interesting effect because by itself, nicotine 408 activated pharyngeal pumping (Fig. 3A) but in combination with 5 -HT its effect was rather 409 inhibitory. The presence of antagonists, mecamylamine and tubocurarine induced varied 410 responses on 5-HT stimulated worms. Mecamylamine had a sustained inhibitory effect on 5-411 HT stimulated pharyngeal pumping in C. elegans. In contrast t ubocurarine had no effect on 412 this pharyngeal response (Fig. 4A). 413 0 60 120 0 100 200 300 Time [min] Pump rate[min-1] 10 mM ACh1 mM 5-HT 10 mM Nic A 0 60 120 0 30 60 90 Time [min] Stylet thrust [min-1] 1 mM 5-HT 10 mM ACh 10 mM Nic B .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint With G. rostochiensis , short-term exposures to nicotine, mecamylamine and tubocurarine 414 failed to significantly inhibit ongoing 5-HT stimulated stylet thrusting. However, although not 415 significant, in the presence of nicotine there was an observable reduction in stylet thrusting 416 (Fig. 4B) 417 418 Fig. 4 Modulation of pharyngeal function in 5 -HT stimulated C. elegans and G. 419 rostochiensis. A. 5-HT induced pharyngeal pump rates in C. elegans were inhibited by nicotine 420 and mecamylamine. Tubocurarine had no inhibitory effect, n=10 B. In G. rostochiensis, these 421 modulators had no significant inhibitory effects on 5-HT induced stylet thrusting, n=13. Data 422 represented as mean ± SEM. The analysis uses the steady state rate of 5-HT as control or 5-HT 423 + ligand and significance tested by two-way ANOVA with Bonferroni’s multiple comparison 424 (nsp>0.05, ****p<0.0001). 425 Chronic exposure to cholinergic signaling modulators impact nematode pharyngeal 426 function: 427 A challenge with nematode drug exposure experiments is the cuticle that restricts the 428 entry and rate of accumulation of drug inside the worm . To overcome this limitation, we 429 prolonged the incubation time of C. elegans and G. rostochiensis in nicotine, mecamylamine 430 or tubocurarine prior to 5-HT exposure and saw an increase in drug effect. 431 Pre-incubating C. elegans for 24 h in nicotine markedly improved the drugs inhibitory 432 effect on 5 -HT stimulated pharyngeal pumping. Similarly, a prolonged incubation in 433 mecamylamine also reduced 5 -HT induced pump rates. In contrast prolonged incubations of 434 C. elegans in tubocurarine had no effect on the subsequent ability of 5-HT to induce pharyngeal 435 pumping (Fig. 5A). 436 0 100 200 300 2 hours post incubation Pump rate [min-1] 1 mM 5-HT 5-HT + 10 mM Nic 5-HT + 200 µM Mec 5-HT + 300 µM Tubo **** **** nsA Antagonists Agonist 0 20 40 60 80 100 2 hours post incubation Stylet thrust [min-1] 1 mM 5-HT 5-HT + 10 mM Nic 5-HT + 200 µM Mec 5-HT + 300 µM Tubo nsB Antagonists Agonist .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint Chronic exposures of G. rostochiensis to nicotine, mecamylamine or tubocurarine , 437 significantly improved their inhibitory effects on 5 -HT stimulated stylet thrusting. A 24-hour 438 incubation in nicotine prior to 5-HT exposure completely abolished stylet thrusting. Prolonged 439 incubations in mecamylamine also inhibited the 5-HT induced stylet thrusting response. 440 Interestingly, a prolonged incubation of G. rostochiensis in tubocurarine, which had no effect 441 on 5 -HT induced pharyngeal responses in C. elegans was quite potent. This significantly 442 inhibited 5-HT induced stylet thrusting (Fig. 5B). 443 444 Fig. 5 Chronic exposures to cholinergic modulators improves their inhibitory effects on 445 5-HT induced pharyngeal behaviors in C. elegans and G. rostochiensis . A. Prolonged 446 incubations of C. elegans in 10 mM nicotine and 200 μM mecamylamine prior to 5-HT inhibits 447 pharyngeal pumping. Incubations in 300 μM tubocurarine had no inhibitory effect (n=8). B. 448 Prolonged incubations of G. rostochiensis in 10 mM nicotine completely abolished 5-HT 449 induced stylet thrusting. Effects of mecamylamine and tubocurarine on 5 -HT induced stylet 450 thrusting responses in significantly reduced (n=30). The data are shown as mean ± SEM and 451 the analysis correspond to 5 -HT as control or 5 -HT + ligand and significance tested by one 452 way ANOVA with Bonferroni’s multiple comparison (nsp>0.05, ****p<0.0001). 453 Tubocurarine has been reported to completely block ectopically expressed Ce.EAT-2 454 receptors at a concentration 10-fold lower than what was used in our whole animal assay . By 455 breaking the cuticle of C. elegans, dissecting and exposing the pharyngeal muscles directly to 456 tubocurarine, we significantly improved the drug potency (Fig. 6A). Mutants of EAT-2 which 457 are physiologically defective in pharyngeal pumping were irresponsive to 5-HT stimulation 458 and tubocurarine inhibition (Fig. 6B). 459 0 100 200 300 24 hour post incubation Pump rate [min-1] 1 mM 5-HT 5-HT + 10 mM Nic 5-HT + 200 µM Mec 5-HT +300 µM Tubo Antagonists Agonist **** **** ns A 0 20 40 60 80 24 hours post incubation Stylet thrust [min-1] 5-HT + 10 mM Nic 5-HT + 200 µM Mec 5-HT + 300 µM Tubo 1 mM 5-HT Agonist Antagonists **** **** **** B .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint 460 Fig. 6 Inhibitory effect of tubocurarine on exposed C. elegans pharyngeal muscles. A. 461 Tubocurarine inhibits 5-HT induced pharyngeal pumping in cut -head wildtype worms (N2). 462 Pharyngeal pumping recovered after a 10 -minute wash and re -exposure to 5 -HT, n=10. B. 463 Mutants of eat-2(ad465) show no response to 5-HT stimulation, n=3. 464 Stylet thrusting and body posture in G. rostochiensis: 465 As indicated above, in addition to inducing pharyngeal function the exogenous application of 466 5-HT induced changes in G. rostochiensis motility and body posture. When incubated for 467 longer than 30 minutes in 1 mM 5-HT, G. rostochiensis became immobile and assumed a kink 468 posture in the head region , with elevated stylet thrusting activity ( Fig. 7A). However, G. 469 rostochiensis incubated for 24 h in nicotine prior to 5-HT exposure failed to adopt this kinked 470 posture and this coincided with the complete block of 5 -HT induced thrusting. Interestingly, 471 prolonged pre-incubations in mecamylamine and tubocurarine did not disrupt kinking but had 472 a selective inhibitory effect on stylet thrusting ( Fig. 7B). In summary, the comparison of the 473 consequence of 5-HT on posture and stylet function in face of distinct nicotinic acetylcholine 474 receptor agents suggests a potential uncoupling of the stylet thrusting and associated body 475 postures. 476 0 20 40 60 80 0 50 100 150 200 250 Time [min] Pump rate [min-1] 1 µM 5-HT 5-HT + 10 µM Tubo 5-HT + 20 µM Tubo Wash A 0 20 40 60 0 20 40 60 80 300 Time [min] Pump rate [min-1] 1 um 5-HT 5-HT + 10 µM Tubo 5-HT + 20 µM Tubo B .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint 477 Fig. 7 Comparing cholinergic drug effects on 5-HT induced stylet and posture states. A. 478 The effects of 10 mM nicotine, 200 μM mecamylamine and 300 μM tubocurarine on 5 -HT 479 induced stylet thrusting and associated kinked posture on G. rostochiensis (5-HT n= 13, 480 nicotine n=12, mecamylamine n=12 and tubocurarine n=30). J2s subjected to the indicated 481 treatments were scored for their kinked body posture and stylet thrusting activity and 482 represented as a percentage of kinked J2s and average (Av) stylet thrusts. B. Grayscale image 483 of G. rostochiensis in a kinked posture when incubated in 1mM 5-HT. 484

485 The targeting of several distinct molecules and functional loci of the cholinergic 486 transmitter system has been a successful strategy for invertebrate pest control (Raisch and 487 Raunser, 2023) . These pesticides impact acetylcholinesterase, the vesicular acetylcholine 488 transporter or distinct classes of acetylcholine receptors (Costa et al., 2008; Čadež et al., 2021; 489 Goodchild et al., 2024) but their use is confounded by the off-target effects on other organisms 490 (Wan et al., 2025) . As a mitigation to this, nAChRs offered a more selective approach to 491 inhibiting the nervous system of invertebrate pests (Bradford et al., 2020) . However, despite 492 phyla selective potency for this receptor class it is still challenging to overcome the issue of 493 non-selective targeting of pests relative to non pest organisms (Li et al., 2025). 494 In this study we looked at specialized pharyngeal functions in two nematodes: stylet 495 thrusting in the plant parasitic nematode G. rostochiensis and pharyngeal pumping in the free-496 living model nematode C. elegans . This could offer a tissue -selective target underpinning 497 specialized life cycle behaviors as an approach to improve pesticide selectivity. We showed 498 1 mM 5-HT 5-HT + Nic 5-HT+ Mec 5-HT+ Tubo 0 50 100 % kinked Av stylet thrusts A B .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint that these pharyngeal functions in both nematode species were controlled by cholinergic and 499 serotonergic signaling. This is thought to be driven by the rather distinct, nematode -specific 500 nAChR EAT-2 which has previously been shown to be required for normal pumping in C. 501 elegans (McKay et al., 2004). It should be noted that whilst this role is important to C. elegans 502 it is non-essential as null mutants are merely retarded in development (Avery, 1993; McKay et 503 al., 2004). However, considering the multiplicity of life cycle selective functions like hatching, 504 host root invasion and feeding behaviors that the pharyngeal muscles play in the PPNs it can 505 be envisaged that targeting this receptor will be particularly pernicious for this nematode class. 506 Our study probed the molecular and structural determinants of pharyngeal muscles that regulate 507 pharyngeal function in G. rostochiensis. We investigated the possibility of translating existing 508 understanding of EAT-2 function in C. elegans to PPNs. 509 Initially, we established EAT -2 presence in other nematode species through a protein 510 blast and a phylogenetic compilation that revealed its conservation within Nematoda. This was 511 confirmed in the PPN G. rostochiensis by successfully amplifying the cDNA for the predicted 512 Gr.EAT-2 ORF. In C. elegans, EAT-2’s functional expression requires EAT -18, an auxiliary 513 protein that is under investigated. EAT-18 is a short single transmembrane domain protein with 514 an intracellular N -terminus and an extracellular C -terminus. Evidence suggests that in its 515 absence, EAT -2 is made, trafficked and localized in the plasma membrane but is a non -516 functional receptor (Choudhary et al., 2020). This makes EAT-18 a potential target to indirectly 517 disrupt EAT-2 function. We identified orthologues of Ce.EAT-18 in other nematode species, 518 and a multiple sequence alignment of these orthologues revealed a high degree of conservation 519 with a 59.09 % identity to the putative Gr.EAT-18 (see supplementary 2). 520 Fluorescence in-situ hybridization reveals Gr.EAT-2 expression in the pharynx: 521 Given the molecular conservation of EAT-2 across Nematoda, we probed its expression pattern 522 and function. Such gene expression histology has been challenging in nematode species even 523 with the increased availability of molecular information (Sperling and Eves -van den Akker, 524 2023). Our study took advantage of multiplex probes that facilitate the detection of gene 525 expression in whole mount nematodes to provide primary evidence on the expression pattern 526 of EAT-2 to underpin key signaling in stylet thrusting and possibly the median bulb pulsation. 527 Most FISH studies on PPNs have focused on effector genes (De Boer et al., 1998, 1999; Lilley 528 et al., 2018; Sperling and Eves -van den Akker, 2023) . Here, we show visual evidence of the 529 expression pattern for cholinergic neurons using probes for Gr.unc-17, the transporter gene 530 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint required for loading synthesized ACh into vesicles (Alfonso et al., 1993) , Gr.eat-2, a 531 cholinergic receptor and putative regulator for pharyngeal function in PPNs and Gr.myo-3 a 532 highly expressed gene in longitudinal muscles of the body -wall. With probes for Gr.eat-2 we 533 identified discrete and reproducible expression in pharyngeal structures. The tissue in which 534 we consistently found a hybridization pattern across several stained specimen was the median 535 bulb (metacorpus). In addition, there was a robust but less frequent staining in discrete 536 structures located in a position associated with the esophageal glands ( Fig. 2). These 537 observations resonate with expression patterns observed by McKay et al. (2004) and Cao et 538 al. (2023). Sense probes for Gr.unc-17 identified neurons in the pharynx and around the ventral 539 side of the worm. The expression pattern for Gr.UNC-17 was similar to transgenic expression 540 studies in C. elegans which showed Ce.UNC-17 expression in cholinergic neurons of the head 541 region and the ventral nerve cord motor neuron (Mathews et al., 2012; Haque and Nazir, 2016). 542 The selective distribution of cholinergic determinants is consistent with its key role in body 543 wall muscle transmission, pharyngeal transmissions and supports a discrete role for EAT -2 in 544 PPN pharyngeal function. Probes for Gr.myo-3 localized along longitudinal muscles of the 545 body wall muscle. 546 A common pathway may regulate pharyngeal pumping and stylet thrusting: 547 5-HT indirectly regulates pharyngeal pumping and stylet thrusting: 548 We proposed this molecular organization to investigate transmitter signaling in nematode 549 pharyngeal function. Consistent with other findings, we showed that exogenous exposure to 5-550 HT induced the pharyngeal behaviors, pharyngeal pumping and stylet thrusting in C. elegans 551 and G. rostochiensis respectively (Hobson et al., 2006; Horvitz et al., 1982; Perry et al., 2004). 552 Although this happens, it is worth mentioning that 5 -HT has no direct effect on EAT -2, the 553 cholinergic regulator of pharyngeal pumping in C. elegans. The biogenic amine activates a 554 cholinergic pathway through the MC neuron to initiate pharyngeal pumping via EAT-2 555 (McKay et al., 2004; Song and Avery, 2012) . Beyond pharyngeal function, 5 -HT 556 concentrations that induce pharyngeal behaviors in G. rostochiensis rendered them immotile 557 with a characteristic kinked -shape posture around the midbody or neck region. A supporting 558 interpretation for this kink is that worms generate enough hydrostatic pressure and tension to 559 act like a flex point for protractor muscles to drive stylet movements (Doncaster, 1966). 560 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint ACh and nicotine directly induce feeding behaviors: 561 The neuromuscular system within Nematoda shows a high degree of conservation, which 562 allows for generally valid hypotheses and conclusions to be made on physiological behaviors 563 like motility, egg laying, and feeding behaviors across different nematode species (Hahnel et 564 al., 2020). C. elegans requires cholinergic signaling to achieve muscle contractions that drive 565 pharyngeal functions like pharyngeal pumping and peristalsis. By utilizing the cholinergic 566 compounds ACh and nicotine, we induced stimulatory effects on pharyngeal function with C. 567 elegans and G. rostochiensis. Our findings complement the findings of Kozlova et al. (2019) 568 who observed that WT C. elegans and cha-1 mutants deficient in choline transferase activity 569 (Rand and Russell, 1984) exposed to nicotine showed induced pharyngeal pumps whilst eat-2 570 mutants were not significantly affected, suggesting that nicotine’s stimulatory effect on 571 pharyngeal pumping may be EAT -2 dependent. ACh and nicotine have also been reported as 572 agonist of recombinantly expressed EAT -2 receptors (Choudhary et al., 2020) . Per se, 573 comparing the pharmacologically induced pharyngeal behavior in G. rostochiens with the well-574 researched pharyngeal pump pathway in C. elegans, we propose that a cholinergic involvement 575 via EAT-2 drives pharyngeal function that can be manifested as stylet thrusting (Fig. 8). 576 577 Fig. 8 A putative signaling pathway for pharyngeal function in G. rostochiesis . 5-HT 578 modulates pharyngeal function through the MC neuron, activating a cholinergic release that 579 acts directly on EAT-2 and triggering pharyngeal function. Nicotine and ACh are agonists of 580 EAT-2 that induce stylet thrusting. Mecamylamine and tubocurarine block EAT -2 and inhibit 581 stylet thrusting functions. 582 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint The role of sensory inputs that innervate pharyngeal muscles and possibly controlling stylet 583 protractor muscles have long been recognized (Doncaster, 1966). Here we provide insights into 584 the downstream pharmacological and molecular determinants of this action. 585 Nicotine inhibits 5-HT stimulated pharynx: 586 Nicotine’s inhibitory effect on 5-HT stimulated pharyngeal responses was interesting because 587 nicotine in solution induced pharyngeal responses but in combination with 5 -HT was 588 inhibitory, suggesting that the overstimulation of pharyngeal muscles resulted in an activation 589 block and a subsequent inhibition in pharyngeal responses. Acute and chronic pre -exposures 590 of C. elegans and G. rostochiensis to nicotine inhibit 5-HT induced pharyngeal effects and this 591 may occur because the extended incubation leads to desensitization and an inhibitory block 592 (Liu et al., 2025) . This interpretation is in support of the notion that nicotinic responses act 593 down stream of 5HT (Kudelska, 2019). 594 Mecamylamine and tubocurarine directly pharyngeal function: 595 We designed experiments to help decipher the intersection between pharyngeal pumping and 596 stylet thrusting. The stimulatory effects of ACh and nicotine support the role of EAT-2 in these 597 responses. Probing this further by investigating the effect of EAT-2 antagonists mecamylamine 598 and tubocurarine on pharyngeal response s revealed their inhibitory significance on 5-HT 599 stimulated pharyngeal responses. Thus, based on two chemically distinct inhibitors we can 600 suggest that EAT-2 is an important mediator of stylet thrusting. The variation in drug potency 601 from acute to chronic exposures and between intact worms and those whose cuticle had been 602 broken open, reinforces the importance of the cuticle as a protective barrier (Johnstone, 1994). 603 The differential drug effects observed from our assays between C. elegans and G. rostochiensis 604 reflects on the differences in cuticle structure among nematode species (Decraemer and Hunt, 605 2013) 606 Our findings suggest that Gr.EAT-2 plays an important role in the signaling pathway 607 that drives pharyngeal function and stylet thrusting. It will be interesting to understand how the 608 discrete contexts that trigger stylet functions during the PPN lifestyle are integrated. Moreover, 609 considering the biological significance of stylet thrusting for PPNs, we propose Gr.EAT-2 to 610 be a valuable target to disrupt the lifecycle of this global economic agricultural pest. As such 611 the EAT-2 pharmacophore merits further investigation to resolve potential selective channel 612 modulators. 613 614 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 23, 2026. ; https://doi.org/10.64898/2026.03.20.711559doi: bioRxiv preprint LITERATURE CITED 615 Alcedo, J., Flatt, T., and Pasyukova, E. G. (2013). The role of the nervous system in aging 616 and longevity. Front Genet 4. doi: 10.3389/FGENE.2013.00124 617 Alfonso, A., Grundahl, K., Duerr, J. S., Han, H. P., and Rand, J. B. (1993). The 618 Caenorhabditis elegans unc-17 gene: A putative vesicular acetylcholine transporter. 619 Science (1979) 261, 617–619. doi: 10.1126/SCIENCE.8342028, 620 Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local 621 alignment search tool. 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