Social feeding in C. elegans is induced by neurons that
detect aversive stimuli
Mario de Bono*†, David M. Tobin*, M. Wayne Davis‡§, Leon Avery‡ and Cornelia
I. Bargmann*
* HHMI, Programs in Developmental Biology, Neuroscience, and Genetics,
Departments of Anatomy and Biochemistry & Biophysics, UCSF CA 94143-0452,
USA;
†MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.;
‡
Department of Molecular Biology, University of Texas Southwestern Medical Center,
6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA;
§Present address: Department of Biology, University of Utah,257 South 1400 East, Salt Lake City, UT
84112-0840, USA
Supplementary information
Strains
Strains were obtained from the Caenorhabditis Genetics Center. Most mutations have
been described previously1. Strains used were: CB1141, cat-4(e1141) V; CB1124, che3(e1124) I; CB3323, che-13(e1805) I; CB1391, daf-2(e1391ts) III; CB1376, daf3(e1376) X; CB1386, daf-5(e1386) II; CB1372, daf-7(e1372ts) III; CB1393, daf8(e1393ts) I; DR26, daf-16(m26) I; DR476, daf-22(m130) II; CB1338, mec-3(e1338) IV;
CB3274, lon-2(e678) mec-4(e1497) X; CX4057, npr-1(g320) X; AX201, npr-1(ky13) X;
AX203, lon-2(e678) npr-1(ad609) X; AX204, npr-1(ad609) X; CX4546, ocr-2(ak47) IV;
CX2335, odr-1(n1936) odr-7(ky4) X; CX2304, odr-2(n2145) V; CX2205, odr-3(n2150)
V; CX2675, odr-4(n2144) III; CX31, odr-8(ky31) IV; CX3073, odr-8(ky173) IV; PR808,
1
osm-1(p808) X; PR802, osm-3(p802) IV; MT3665, osm-9(n1601) IV; CX10, osm9(ky10) IV; FK134, ttx-3(ks5) X; CB156, unc-25(e156) III; CB251, unc-36(e251) III.
Strains generated in this work include: AX1013, cat-4(e1141) V; npr-1(ad609) X;
AX986, che-3(e1124) I; npr-1(ad609) X; AX298, daf-2(e1391ts) III; lon-2(e678) npr1(ad609) X; AX1020, daf-3(e1376) lon-2(e678) npr-1(ad609) X; AX988 daf-5(e1386)
II; npr-1(ad609) X; AX990, daf-7(e1372ts) III; npr-1(g320) X; AX992 daf-7(e1372ts)
III; npr-1(ky13) X; AX208, dpy-5(e61) daf-8(e1393ts) I; npr-1(ad609) X; AX289, daf16(m26) I; lon-2(e678) npr-1(ad609) X; AX994, daf-22(m130) II; npr-1(ad609) X;
AX996, mec-3(e1338) III; npr-1(ad609) X; AX998 npr-1(ad609) mec-4(e1497) X;
CX4650, ocr-2(ak47) IV; npr-1(ad609) X; AX1000, lon-2(e678) npr-1(ad609) odr1(n1936) odr-7(ky4) X; AX1003, odr-2(n2145) V; npr-1(ad609) X; AX623, odr4(n2144) III; npr-1(ad609) X; AX215, odr-4(n2144) III; npr-1(ad609) lin-15(n765ts) X;
AX433, odr-8(ky31) IV; npr-1(ad609) X; AX434, odr-8(ky173) IV; npr-1(ad609) X;
AX256, npr-1(ad609) osm-1(p808) X; AX295, osm-3(p802) IV; npr-1(ad609) X;
AX218, osm-9(n1601) IV; npr-1(ad609) X; AX485, osm-9(ky10) IV; npr-1(ad609) X;
AX1005, lon-2(e678) npr-1(ad609) ttx-3(ks5) X; AX821, osm-3(p802) ocr-2(ak47) IV;
npr-1(ad609) X; AX636, odr-4(n2144) III; osm-3(p802) IV; npr-1(ad609) X; AX265,
dpy-20(e1282ts) IV; npr-1(ad609) X; CX4827 ocr-2(ak47) IV; npr-1(ad609) lin15(n765ts) X, AX1101, unc-25(e156) III; npr-1(ad609) X; AX1100, unc-36(e251) III;
npr-1(ad609) X.
Genetics
Double mutants between npr-1 X and other loci were made by replacing marked balancer
chromosomes with chromosomes carrying the mutation of interest. The building of the
2
odr-4(n2144) III; npr-1(ad609) X double mutant strain is given as an example. odr-4 III
males were mated with unc-36 dpy-18 III; npr-1 X hermaphrodites and the F1 cross
progeny males of genotype + odr-4 +/unc-36 + dpy-18 III; npr-1/0 X backcrossed to unc36 dpy-18 III; npr-1 X hermaphrodites. Individual non-Unc non-Dpy hermaphrodite
cross-progeny of genotype unc-36 + dpy-18/+ odr-4 + III; npr-1 X were picked to a plate
and allowed to generate self-progeny. Nine non-Unc non-Dpy animals from among these
self-progeny were picked, of which on average three were of genotype odr-4 III; npr-1 X,
identified by failure to generate any Unc or Dpy progeny.
The genotype of constructed strains was confirmed either by phenotypic analysis
or by detection of the DNA lesions associated with the allele. When a mutation that
suppressed social feeding behavior was identified, a second allele of that gene was tested
to corroborate the suppression (ky10 and n1601 for osm-9; ky31 and ky173 for odr-8).
Only one allele of odr-4 is available.
Behavioral assays
In the bordering and aggregation time courses in Fig. 1a, the number of animals in the
assay was varied, but the size of the bacterial lawn was kept constant at 2cm diameter.
Aggregation and bordering were measured at the time intervals shown. To measure
aggregation and bordering in the absence of food (Fig. 1b) animals were trapped using
either a chemical barrier of 4M fructose, or a physical barrier in the form of a 1.7 cm
diameter copper washer (A. Davies and S. McIntire, personal communication). Assays in
the presence of food were run in parallel. The assays shown in Fig. 1e and 1f employed
the standard assay, except that 40 animals were put into the assay. The average group
3
size in Fig. 1d and 1f was calculated by dividing the total number of animals in groups by
the number of groups. In Fig. 2e and 2f, 2 test animals of the indicated genotypes were
assayed in the presence of 80 animals from strain AX265, dpy-20(e1282ts) IV; npr1(ad609) X that had been grown at 25 oC. Animals from strain AX265 grown at this
temperature are dumpy but aggregate to a similar degree as npr-1(ad609) animals. Test
animals can thus be distinguished by their wild type length from non-test dumpy animals,
and their ability to aggregate and border with npr-1 social animals determined.
Molecular Biology
ocr-2 fusion genes were built by constructing a promoterless ocr-2 cassette from a
rescuing ocr-2 genomic clone and then inserting the promoter sequences indicated in the
text. odr-4 fusion genes were built by replacing the odr-10 promoter in the p[odr10p::ODR-4-GFP] clone2, with the promoter sequences indicated in the text. Expression
of ODR-4-GFP in the appropriate predicted neuron was confirmed by fluorescence
microscopy for each transgene. The positive control for rescue was pODR-4-42.
References
1.
2.
Hodgkin, J. in C. elegans II (eds. Riddle, D. L., Blumenthal, T., Meyer, B. &
Priess, J. R.) 881-1047 (CSHL Press, Cold Spring Harbor, 1997).
Dwyer, N. D., Troemel, E. R., Sengupta, P. & Bargmann, C. I. Odorant receptor
localization to olfactory cilia is mediated by ODR-4, a novel membraneassociated protein. Cell 93, 455-66 (1998).
4