Reduced expression of voltage-gated chloride channel genes in Caenorhabditis elegans: Implications for the mode of action of chloride channel-directed nematicides

Caenorhabditis elegans nematodes (fourth-stage larvae) were exposed for 24 h to long (4 μg/μl) or short (5 μg/μl) double-stranded RNA (dsRNA) of two voltage-gated chloride channel (VGCC) genes, ceclc-1 and ceclc-2, coding for two VGCC proteins, CeClC-1 and CeClC-2. The treatments reduced expression of these genes in F₁ progeny in a time- and concentration-dependent manner. Progeny with reduced expression of ceclc-2 (but not ceclc-1) showed significant phenotypic effects, such as increased pharyngeal contractions and decreased locomotion, which were maximal in 48 h old worms. Generally, there was little effect on the worms when expression of ceclc-1 alone was reduced. F₁ progeny with reduced expression of both ceclc-1 and ceclc-2 had a higher percentage showing affected locomotion than were present with reduced expression of ceclc-2 alone, whereas the increase in frequency of pharyngeal contractions was not different from that obtained with reduced expression of ceclc-2 alone. These phenotypic effects were consistent with the known expression patterns of ceclc-1 and ceclc-2 in C. elegans tissues. A strong negative correlation was observed between in vitro concentration of long dsRNA and ceclc-2 expression level, as well as between ceclc-2 expression level and corresponding phenotypic effects. The effects of dsRNA exposure are similar to previous studies in which 24-h incubation in 50 ppm of anion transporter (AT) blockers DIDS, 9-AC, IAA-94, or NPPB increased pharyngeal contractions and the proportion of nematodes exhibiting impaired locomotion. These findings confirm that the principal mechanism of nematicidal activity of AT blockers may arise from the inhibition of CeClC-2.