Fig 1.
C. elegans performs multisensory integration to leave food paired with a repulsive odorant 2-nonanone.
(A) A schematic of 2-nonanone-dependent food leaving assay.(B) The time course for worms leaving an OP50 lawn that is paired with 2-nonanone of different concentrations over 60 minutes, n = 2 assays for 10% and n = 3 assays each for 30%, 50% and 100%. (C) More worms leave the OP50 food lawn paired with 100% 2-nonanone (n = 4 assays) than the OP50 lawn paired with 10% 2-nonanone (n = 5 assays). Bar graph represents the percentage of worms outside the lawn 15 minutes after the assay starts. (D) The time taken for worms to reach the edge of the OP50 food lawn when the lawn is paired with either 10% or 100% 2-nonanone, n = 2 assays each. (E) I–IV, Sample images of wild-type animals leaving an OP50 lawn that is paired with 100% 2-nonanone at different time points of the 60-minute assay. For B-D, Mean ± SEM, Student’s t test, ** p ≤ 0.01, n.s., not significant.
Fig 2.
Several sensory neurons modulate 2-nonanone-dependent food leaving.
(A-D) The transgenic animals that either lack the functional AWB sensory neurons by selectively expressing the gain-of-function isoform of an amiloride-sensitive sodium channel MEC-4 (A, pAWB::mec-4(d), n = 5 assays each) or lack the ASI sensory neurons by expressing a cell death promoting molecule caspase (B, pASI::caspase, n = 7 assays for wild type and 8 assays for the transgenic animals) or are defective in the synaptic transmission of the ADL sensory neurons by expressing the tetanus toxin (D, pADL::TeTx, n = 5 assays for wild type, 4 assays for the transgenic animals, and 3 assays for non-transgenic siblings) display a delayed decision to leave the OP50 lawn paired with 100% 2-nonanone; while the transgenic animals that express caspase in the ASK sensory neurons (C, pASK::caspase, n = 9 assays each) display a faster decision to leave. Each bar graph reports the average percentage of worms outside the lawn 15 minutes after the assay starts. Mean ± SEM, Student’s t-test, * p ≤ 0.05, ***p ≤ 0.001.
Table 1.
Wild type, mutants and transgenic animals are examined for avoiding 100% 2-nonanone as previously described (Troemel et al., 1997 and S1 Fig). Avoidance Index was calculated as described in S1 Fig. The avoidance in each genotype is represented by the average avoidance index of individual assays. n = 2–4 assays each genotype, 75–100 animals tested in each assay, mutants and transgenic animals are compared with wild-type animals or the non-transgenic siblings tested on the same days with student’s t test, Mean ± SEM.
Table 2.
Time to reach the edge of the food lawn during multisensory integration.
Wild type, mutants and transgenic animals are examined for the time taken to reach the edge of the food lawn away from the repellent. The average time taken for 90% of the worms in one assay to reach the edge of a E. coli OP50 food lawn during exposure to 100% 2-nonanone is presented for each genotype (Experimental Procedures and S1 Fig). n = 2–4 assays for each genotype, 20–25 animals tested in each assay, mutants or transgenic animals are compared with wild-type animals or non-transgenic siblings tested on the same days with student’s t test, Mean ± SEM.
Table 3.
Spontaneous food leaving from an OP50 lawn without pairing with 2-nonanone.
Wild type, mutant animals and transgenic animals are examined for food leaving for 1 hour. Young adult worms are placed on an OP50 food lawn for 10 minutes and the number of worms on food lawn is counted every 5 minutes for a total assay time of 60 minutes. The percentage of worms off the food lawn at 15 minutes is reported. n = 2–4 assays for each genotype and 20–25 worms in each assay, mutants or transgenic animals are compared with wild-type animals tested in parallel with student’s t test, Mean ± SEM.
Table 4.
Many signaling mutants show no phenotype in 2-nonanone-dependent food leaving.
Wild type, mutant and transgenic animals are examined for leaving an E. coli OP50 food lawn paired with 100% 2-nonanone. The average percentage of worms outside the food lawn at 15 minutes is reported. Mutants or transgenic animals are compared with the wild-type control tested on the same days with student’s t test, n = 2–4 assays for each genotype, 20–25 animals in each assay, Mean ± SEM.
Fig 3.
NLP-7 and TGF-β/DAF-7 modulate the decision to leave the OP50 food lawn paired with 2-nonanone.
(A-D) The mutant animals that are defective in the biosynthesis of the neurotransmitter serotonin (A, tph-1(mg280), n = 2 assays each), or dopamine (B, cat-2(e1112), n = 4 assays each), or tyramine and octopamine (C, tdc-1(n3419), n = 2 assays each), or octopamine (D, tbh-1(n3247), n = 3 and 4 assays for wild type and tbh-1 mutants, respectively) display a normal decision to leave the OP50 food lawn that is paired with 2-nonanone. (E-J) Mutations in the genes encoding the neuropeptide processing enzymes, kpc-1 (E, n = 4 and 5 assays for wild type and kpc-1 mutant, respectively), or egl-3 (F, n = 4 and 6 assays for wild type and egl-3 mutant, respectively), or in a TGF-β-encoding gene daf-7 (G, n = 6 and 5 assays for wild type and daf-7 mutant, respectively), or in a neuropeptide-encoding gene nlp-7 (H, n = 7 and 8 assays for wild type and nlp-7 mutant, respectively) generate a delayed decision to leave the OP50 food lawn paired with 2-nonanone, and expressing the genomic DNA of nlp-7 (I, n = 6, 7 and 4 assays for wild type, transgenic animals and non-transgenic siblings, respectively) or the genomic DNA of daf-7 (J, n = 4 assays each for wild type, transgenic animals and non-transgenic siblings) rescues the delayed food leaving phenotype of the respective mutant animals. (K) Expressing the wild-type daf-7 cDNA in the sensory neurons ADE rescues the delayed decision in the daf-7(e1372) mutant animals, n = 4 assays each for wild type, transgenic animals and non-transgenic siblings, respectively. (L) Expressing the wild-type daf-7 cDNA in the sensory neurons OLQ also rescues the delayed decision in the daf-7(e1372) mutant animals, n = 3 assays for wild type, 3 assays for transgenic animals and 2 assays for non-transgenic siblings, respectively. (M) Expressing a wild-type daf-7 cDNA in the sensory neurons ASI does not rescue the delayed decision phenotype in the daf-7(ok3125) mutant animals (n = 4, 3 and 4 assays for wild type, transgenic animals and non-transgenic siblings, respectively). Each bar graph reports the average percentage of worms outside the lawn 15 minutes after the start of the assay, mutants are compared with wild-type animals and transgenic animals are compared with non-transgenic siblings using Student’s t-test, * p<=0.05, ** p<=0.01, *** p<=0.001, n.s., not significant.
Fig 4.
The TGF-β receptor DAF-1 acts in the RIM and RIC neurons to mediate 2-nonanone-dependent food leaving.
(A-D) Mutating daf-1 that encodes the type I TGF-β receptor delays the decision to leave the OP50 lawn paired with 100% 2-nonanone (A, n = 8 and 9 assays for wild type and daf-1 mutant, respectively), and expressing the genomic DNA of daf-1 (B, n = 6 assays each) or a wild-type daf-1 cDNA in the RIM and RIC neurons (C, n = 6, 6 and 5 assays for wild type, transgenic animals and non-transgenic siblings, respectively) in the daf-1(m40) mutant animals rescues the delayed decision, but expressing wild-type daf-1 in the sensory neurons (D, n = 3, 5 and 2 assays for wild type, transgenic animals and non-transgenic siblings, respectively) does not rescue. Mutants are compared with wild type and transgenic animals are compared with non-transgenic siblings with Student t-test. (E-F) Inhibiting the activity of the RIM and RIC neurons by selectively expressing a histamine-gated chloride channel (E, n = 2 and 4 assays for wild type and transgenic animals, respectively) or blocking the synaptic release from these neurons by selectively expressing the tetanus toxin (F, n = 2 assays each) does not alter the decision to leave the OP50 food lawn paired with 100% 2-nonanone. Transgenic animals are compared with wild type. (G-I) Removing octopamine signaling in the daf-7(e1372) or daf-1(m40) mutants with a mutation that disrupts biosynthesis of octopamine tbh-1(ok1196) does not suppress the delayed leaving from the OP50 lawn paired with 100% 2-nonanone (G, n = 6 assays for wild type; n = 4 assays for daf-7 mutants; n = 2 assays for daf-1 mutants; n = 3 assays for daf-7;tbh-1 double mutants; n = 2 assays for daf-1;tbh-1 double mutants), but removing the tyramine and the octopamine signals with the mutation in tdc-1(ok914) in either the daf-7(e1372) (H, n = 6, 5 and 4 assays for wild type, daf-7 mutants and daf-7;tdc-1 double mutants, respectively) or the daf-1(m40) (I, n = 5, 4 and 5 assays for wild type, daf-1 mutants and daf-1;tdc-1 double mutants, respectively) mutant animals suppresses the delayed-decision phenotype in either of the mutant animals. Double mutants were compared with the respective single mutants using student’s t test. Each bar graph reports the average percentage of worms outside the lawn 15 minutes after the start of the assay. Mean ± SEM, ** p ≤ 0.01, *** p ≤ 0.001, n.s.; not significant.
Fig 5.
Downstream circuit that regulates 2-nonanone-dependent food leaving (A-C) Inhibiting the activity of the AIY interneurons by expressing the gain-of-function isoform of the potassium channel TWK-18 (A, Pttx-3::twk-18(gf), n = 3 assays each) or by blocking the synaptic outputs of AIY by expressing tetanus toxin (B, Pttx-3::TeTx, n = 4 assays each), or the mutation ttx-3(mg158) that generates development defects in AIY (C, n = 3 assays each) delays the decision to leave the OP50 lawn paired with 100% 2-nonanone.(D, E) Selectively expressing tetanus toxin (D, Pinx-1::TeTx, n = 2 assays each) or the inhibitory HisCl channel (E, Pinx-1::HisCl, n = 3 and 4 assays for wild type and transgenic animals, respectively) in the AIB interneurons does not significantly alter the lawn-leaving decision, when the OP50 lawn is paired with 100% 2-nonanone.(F, G) Blocking synaptic outputs from the nmr-1-expressing neurons (F, Pnmr-1::TeTx, n = 3 assays each) or the glr-1-expressing neurons (G, Pglr-1::TeTx, n = 4 and 3 assays for wild type and transgenic animals, respectively) enhances the 2-nonanone-dependent lawn leaving. Each bar graph reports the average percentage of worms outside the lawn 15 minutes after the start of the assay (A-F), unless otherwise specified (G). Mean ± SEM, mutants are compared with wild-type animals with student’s t test, transgenic animals are compared with non-transgenic siblings or wild type with student’s t-test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n.s., not significant.
Fig 6.
Integrated response to a repellent-paired food lawn is regulated by a common set of factors.
(A) Wild-type animals also leave the lawn of OP50 paired with 100% benzaldehyde; in contrast, paring an OP50 lawn with 100% 1-octanol does not repel worms (n = 2 assays for each condition).(B-E) Genetic ablation of the sensory neurons AWB (B, pAWB::mec-4(d), n = 3 assays each) or ASK (C, pASK::caspase, n = 3 assays each) or mutating the genetic components of the TGF-β/DAF-7 pathway (D, daf-7(e1372), n = 3 assays each; E, daf-1(m40), n = 4 assays each) alters the decision to leave the benzaldehyde-paired OP50 lawn. Each bar graph reports the average percentage of worms outside the lawn 25 minutes (B, D, E) or 5 minutes (C) after the start of the assay. Mean ± SEM, mutants or transgenic animals are compared with wild-type animals with Student’s t test, ** p ≤ 0.01, *** p ≤ 0.001, n.s., not significant.
Fig 7.
Integrated response to a repellent-paired food lawn requires a common set of factors.
(A-D) Genetic ablation of the sensory neurons AWB (A, pAWB::mec-4(d), n = 2 assays each) or ASK (B, pASK::caspase, n = 2 assays each), or mutating the genetic components of the TGF-β/DAF-7 pathway (C, daf-7(e1372), n = 4 assays each; D, daf-1(m40), n = 2 assays each) alters the decision to leave the 2-nonanone-paired Comamonas lawn. Each bar graph reports the average percentage of worms outside the lawn 25 minutes (A) or 20 minutes (B-D) after the start of the assay. Mean ± SEM, mutants or transgenic animals are compared with wild type with Student’s t test, * p ≤ 0.05, ** p ≤ 0.01.
Fig 8.
A summary of the identified regulators of a multisensory integration behavior.
Summary of the neuronal signaling molecules and neurons that regulate 2-nonanone-dependent food leaving. DAF-7, a TGF-β; NLP-7, a neuropeptide; RIM/RIC, AIY, interneurons; ASK, ASI, AWB, ADL, sensory neurons; ADE and OLQ, sensory neurons that express daf-7; command interneurons, AVA, AVD, AVB, PVC neurons. Green and red represent regulators that promote or suppress 2-nonanone-dependent food leaving, respectively.