C. elegans strains and ivermectin treatment

Strains were maintained on agar plates containing standard nematode growth media (NGM) seeded with E. coli OP50 at 20 °C. Small liquid cultures were used to arrest larval development (see below), and E. coli HB101 was used as food in these cultures. Standard genetic techniques were used to make combinations of alleles. The wild-type strain N2 (Bristol) and the following mutant and transgenic strains were used:

CB1370 daf-2(e1370)

CF1038 daf-16(mu86)

LRB268 avr-14(vu47); glc-3(ok321) avr-15(vu227) glc-1(pk54); dukIs9[Pmyo-2::avr-15, Pmyo-2::mCherry, Pajm-1::AJM-1::GFP]

LRB269 avr-14(vu47); glc-3(ok321) avr-15(vu227) glc-1(pk54); dukIs10[Pmyo-2::avr-15, Pmyo-2::mCherry, Phlh-8::GFP]

JD369 avr-14(vu47); glc-3(ok321) avr-15(vu227) glc-1(pk54)

NK1228 daf-16(mu86); unc-119(ed4); qyIs288 [Pdaf-16::GFP::DAF-16, unc-119(+)]

LRB338 daf-16(mu86); daf-28(tm2308); qyIs289[Pdaf-16::GFP::DAF-16, unc-119 (+)]

LRB339 daf-16(mu86); ins-4, ins-5, ins-6(hpDF761); daf-28(tm2308); qyIs289[Pdaf-16::GFP::DAF-16, unc-119(+)]

LRB340 daf-16(mu86); ins-4, ins-5, ins-6(hpDF761); qyIs289[Pdaf-16::GFP::DAF-16, unc-119(+)]

GR1455 mgls40[Pdaf-28::GFP]

dukIs9 injection mix contained the following: 1 ng/μL pCFJ90 (Pmyo-2::mCherry), 1 ng/μL pPD30_69_TK414_4A (Pmyo-2::avr-15), and 50 ng/μL pJS191 (Pajm-1::AJM-1::GFP). dukIs10 injection mix contained the following: 1 ng/μL pCFJ90 (Pmyo-2::mCherry), 1 ng/μL pPD30_69_TK414_4A (Pmyo-2::avr-15), and 50 ng/μL pJKL464 (Phlh-8::GFP).

We used LRB268 and LRB269 for our primary analyses. These strains are sensitive to ivermectin in the pharynx alone, providing a controlled way to manipulate pharyngeal pumping. However, these strains have complex genetics, with four chromosomal mutations and a transgenic extrachromosomal array. In addition, the array carries the pharynx-specific avr-15 transgene as well as a reporter gene. Consequently, these two strains were not amenable to analysis of other mutations and reporter genes. We determined that wild-type (N2) worms display similar responses to ivermectin and food (e.g., Fig. 3i), albeit at slightly different doses (see below). We proceeded to use the wild-type background for subsequent genetic analyses. The strain used is indicated for each figure or figure panel.

Ivermectin (Sigma) dissolved in DMSO was added to the appropriate cultures immediately following culture setup. Ivermectin dose was adjusted for different levels of resistance in different strains. We performed a dose response to ivermectin in each background and used the minimal effective dose that made each strain not eat, as assessed by growth (data not shown). LRB269 was treated with 20 ng/mL ivermectin, or 22.85 nM. LRB268 was treated with 50 ng/mL ivermectin. Strains in the N2 background, rather than the quadruple mutant background, were treated with 10 ng/mL. DMSO was added in equal amounts to control tubes. DMSO concentration ranged from 0.05 to 0.2%.