ubr-1 exhibits IVM-resistant phenotypes.

(A) Left: Schematic representation of the IVM resistance test in C. elegans. Well-fed L4 stage animals were transferred to plates containing OP50 bacteria with or without macrocyclic lactones (MLs) for 20 hours. Right: Representative grid plots illustrating the viability of wild-type and ubr-1 animals in response to ivermectin (IVM, 5 ng/mL), avermectin (AVM, 5 ng/mL), and doramectin (DOM, 5 ng/mL). We used shades of red to represent worm viability on each experimental plate (n = 50 animals per plate), with darker shades indicating lower survival rates. The viability test was repeated at least 5 times (5 trials). (B) Quantification analysis of the viability of wild-type and ubr-1(hp684) mutants exposed to different MLs. ubr-1(hp684) mutants exhibit resistance to various MLs. (C) Dose-response curve depicting the viability of wild-type and ubr-1(hp684) mutants in the presence of varying concentrations of IVM. The IC50 was 5.7 ng/mL for the wild type and 11.6 ng/mL for the ubr-1 mutants. (D) Time-dependent effect of IVM exposure on animal viability. The IC50 values were 20.2 hours for the wild type and 42.1 hours for the ubr-1 mutants. (E) Representative images of worm size in the wild type and ubr-1(hp684) mutants with or without IVM treatment. Scale bar, 50 µm. (F) Quantitative analysis of body length in different genotypes with or without IVM. (G) Quantification of the average pharynx pump number in animals with or without IVM. (H) Raster plots illustrating the locomotion velocity of individual animals in the absence (0 ng/mL) and presence (5 ng/mL) of IVM. n = 10 animals in each group. (I) Quantification of the average velocity in different genotypes with or without IVM treatment. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. The error bars represent the SEM.

Glutamate mimics IVM resistance in wild-type N2 animals, and ceftriaxone fully reverses IVM resistance in ubr-1 mutants.

(A) Modified schematic representation of the pretreatment approach in the IVM resistance test in C. elegans. Synchronized L1 animals were cultured on plates containing OP50 bacteria and pretreated with glutamate (Glu, 5 mM), γ-aminobutyric acid (GABA, 5 mM), or aspartate (Asp, 5 mM) until they reached the L4 stage. Pretreated animals were then transferred to plates containing additional IVM (5 ng/mL) for 20 hours. (B) Representative grid plots illustrating the viability of wild-type and ubr-1 animals pretreated with different glutamate metabolites. (C-F) Quantitative analysis of viability, body length, pharyngeal pump rate, and locomotion velocity in wild-type and ubr-1 mutants following exposure to various glutamate metabolites. Wild-type worms treated with glutamate displayed resistance to IVM. However, glutamate did not affect the IVM resistance of the ubr-1 mutant. ns, not significant, ****p < 0.0001 by one-way ANOVA in C-F. (G) Diagram illustrating the ceftriaxone pretreatment procedure in the IVM resistance test. Synchronized L1 animals were cultured on OP50-fed plates with or without ceftriaxone (50 μg/mL) until the L4 stage. The animals were subsequently transferred to plates seeded with additional IVM (5 ng/mL) for 20 hours. (H) Upper: Representative grid plots depicting the viability of ubr-1 animals at different concentrations of ceftriaxone. Bottom: Dose-response curve illustrating the amount of ceftriaxone required for viability in ubr-1(hp684) mutants (IC50 = 5.1 μg/mL). (I-L) Quantification of viability, body length, pharyngeal pumping, and locomotion velocity in the wild type and ubr-1 mutants in the absence and presence of ceftriaxone. Ceftriaxone fully restored the sensitivity of ubr-1 mutants to IVM, comparable to the levels observed in wild-type N2 animals. ns, not significant, ***p < 0.001 by Student’s t test in I-L. Error bars represent SEM.

Deletion of got-1 and eat-4 suppresses ubr-1’s IVM resistance.

(A) Schematic of the pathway for glutamate synthesis through transaminase GOT-1 and loading via vesicular transport VGLUT/EAT-4. (B-E) Quantification of viability, body length, pharyngeal pump rate, and locomotion velocity in different genotypes. The removal of got-1 and eat-4 significantly restored sensitivity to IVM (5 ng/mL) in ubr-1 mutants, whereas the re-expression of GOT-1 (+GOT-1) and EAT-4 (+EAT-4) partially reinstated IVM resistance in the respective double mutants. (F-I) IVM resistance phenotypes were rescued by expression of UBR-1 (+UBR-1) driven by its own promoter (Pubr-1) or the pharynx muscle-specific promoter (Pmyo-2). For the viability test in F, n = 50 animals per plate and repeated at least 5 times (5 trials), n ≥ 10 animals in G-I, *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA. All statistical analyses were performed against ubr-1 mutant. Error bars, SEM.

Downregulation of AVR-15 in ubr-1 mutants.

(A) Representative fluorescence intensity of AVR-15 in different genotypes. (B) Quantitative analysis of normalized fluorescence intensity revealed a reduction in AVR-15::GFP expression in ubr-1 mutants. The diminished AVR-15 levels were rescued by overexpression of UBR-1 and genetically suppressed in got-1 and eat-4 mutants. Pharmacological intervention with ceftriaxone (50 μg/mL) successfully reinstated the pharyngeal expression of AVR-15::GFP. Conversely, glutamate administration reduced AVR-15 expression in the wild-type N2 strain. ROI, region of interest; n ≥ 10 animals in each group. **p < 0.01, ****p < 0.0001 by one-way ANOVA. Error bars, SEM.

Reduced inhibition of serotonin-stimulated pharyngeal activity by IVM in ubr-1 mutants.

(A) Left: Schematic diagram of the pharynx motor circuit composed of an excitatory cholinergic motor neuron MC, an inhibitory glutamatergic motor neuron M3 and a group of compact muscles. Both types of motor neurons could be activated by serotonin (5-HT). Right: Representative pharynx muscle Ca2+ response and kymograph evoked by 5-HT (20 mM). The dashed lines aligned with the arrows denote the initiation of perfusion. (B) Average Ca2+ traces evoked by different concentrations of 5-HT. (C) Dose-response curve of 5-HT for the pharynx muscle Ca2+ response in wild-type animals (EC50 = 20.1 mM). n ≥ 5 animals in each group. (D) Baseline-subtracted kymograph of the 5-HT-evoked pharynx muscle Ca2+ response in wild-type and ubr-1 mutants. IVM (3 μM) completely inhibited the wild-type pharynx Ca2+ response, which was significantly reduced in ubr-1 mutants. (E) Average Ca2+ traces evoked by 5-HT in different genotypes with or without IVM. (F) The dose-dependent inhibitory effect of IVM on pharynx Ca2+ was attenuated in ubr-1 mutants (blue line) compared with wild-type animals (black line) (IC50 = 0.5 μM in WT, while IC50 = 10.1 μM in ubr-1). (G) The decreased IVM inhibition of serotonin-evoked pharynx activity in ubr-1 was rescued by the restoration of UBR-1 expression (+ UBR-1), genetic removal of got-1 and eat-4, and pretreatment with ceftriaxone (+ Cef, 50 μg/mL). (H) IVM-mediated inhibition of serotonin-evoked pharynx activity in wild-type animals was blocked by glutamate feeding (WT + Glu, 20 mM). n = 10 animals in each group. (I) Quantification of the 5-HT-evoked peak amplitude of the pharynx muscle Ca2+ response in the different genotypes. Black stars: ****p < 0.0001 was used to compare intragroup differences or pharmacological groups via Student’s t test; apricot stars: ****p < 0.0001 was used to compare the differences with ubr-1 in the “+ 5-HT & IVM” group via one-way ANOVA. Error bars, SEM.

A working model.

In wild-type animals, functional UBR-1 helps maintain balanced glutamate levels by inhibiting transaminase GOT-1 activity. In contrast, in ubr-1 loss-of-function mutants, the absence of GOT-1 inhibition leads to excessive glutamate production. This glutamate excess, which can be reduced by ceftriaxone treatment, induces compensatory downregulation of IVM-targeted GluCls. This downregulation results in a diminished inhibitory response to IVM in the pharyngeal region, ultimately causing IVM resistance in ubr-1 mutants.