Identifying the genetic loci conferring resistance to anthelmintic compounds is of paramount importance in assessing and monitoring their effectiveness in the long term. Although Caenorhabditis elegans has been a useful reference point for research in parasitic nematodes, a comparative approach using additional genetically tractable nematodes could uncover other unknown factors that contribute to drug sensitivity. To better understand how diverse non-parasitic nematodes respond to known paralytic compounds with anthelmintic activity, we compared for the first time the response of an entomophilic nematode Pristionchus pacificus and C. elegans to betaine, levamisole, and ivermectin. In addition to paralytic compounds that require physical contact, we also examined the responses toward the volatile anesthetic halothane.
Betaine, a glycine derived amino acid, acts through the acetylcholine receptor ACR-23 expressed in the body muscles and neurons (Peden et al., 2013). The constant activation of the receptor in the presence of excessive exogenous betaine leads to hypercontraction in the muscles and the nervous system, which eventually results in death (Peden et al., 2013). We found that at 25 mM betaine delivered via the medium, almost every P. pacificus became paralyzed after four hours compared to ~30% of C. elegans.
Levamisole is an agonist for nicotinic acetylcholine receptors expressed in the neuromuscular junctions (nAChR): UNC-38, UNC-39, UNC-63, and LEV-1 (Lewis et al., 1980, Culetto et al., 2004). In C. elegans, loss-of-function mutations in several AChR subunits leads to reduced sensitivity to levamisole (Fleming et al., 1997). In vitro experiments have shown that Hco-ACR-8 is critical in levamisole sensitivity in Haemonchus contortus and could be a key component in other parasitic nematodes (Blanchard et al., 2018). We found that 0.50 mM levamisole caused ~90% of C. elegans and P. pacificus to become paralyzed after one hour. Interestingly, P. pacificus reached 90% paralysis within ten minutes of treatment, while C. elegans took between 10 minutes to an hour to reach the same percentage of paralysis. Although the initial paralysis of P. pacificus occurred faster than C. elegans, over the course of 6 hours P. pacificus was able to recover faster than C. elegans.
Ivermectin causes damage to the pharynx that lead to paralysis, starvation, and death by inhibiting pharyngeal pumping. Ivermectin targets
avr-14,
avr-15, and
glc-1 that encode glutamate chloride subunit channels (Dent et al., 1999). In C. elegans, the loss of function in glutamate-gated chloride channel subunits results in resistance to several avermectins (Ghosh et al., 2012). Rescuing the
avr-14 mutant with Hco-AVR-14B cDNA from Haemonchus contortus restored sensitivity towards ivermectin, suggesting that orthologous gene products from parasitic nematodes can function in C. elegans (Glendinning et al., 2011). We found that in liquid swim assays with 0.051 mg/ml ivermectin, C. elegans became paralyzed faster than P. pacificus.
Halothane, a volatile anesthetic, binds to the syntaxins and SNARE complex and acts on neurotransmitters (Nagele et al., 2005). We found that while most worms in both nematode species were paralyzed within the first hour, C. elegans recovered faster than P. pacificus.
Taken together, P. pacificus is more sensitive to betaine and halothane than C. elegans, while C. elegans is more sensitive to levamisole and ivermectin than P. pacificus. However, P. pacificus seems to recover faster than C. elegans after two hours in the presence of ivermectin, suggesting a possible divergence in nicotinic receptors. These results point to significant differences between the two species in drug uptake, receptor sensitivity, and metabolism.