DAF-25/Ankmy2 Functions in Ciliary cGMP Signaling Figure 1. daf-25 mutants have chemosensory defects. We assayed the ability of two daf-25 mutants to respond to four attractants. The daf-25 behavior was compared with N2 and with daf-11(m47), which is partially chemotaxis defective. Chemotaxis index scores were calculated as the number of adults at the attractant minus the number at the control, divided by the total number of adults [50]. Neither allele of daf-25 responded to any of the attractants, indicating an olfactory defect in daf-25 mutants that is more severe than that of the daf-11 guanylyl cyclase mutant. Benzaldehyde, trimethyl thiazole and isoamyl alcohol are detected by the AWC neurons, and pyrazine by the AWA neurons. Pyrazine: N2 n = 66, daf- 25(m98) n = 78, daf-25(m362) n = 123, daf-11(m47) n = 83. Benzaldehyde: N2 n = 91, daf-25(m98) n = 80, daf-25(m362) n = 115, daf-11(m47) n = 62. Isoamyl alchohol: N2 n = 66, daf-25(m98) n = 78, daf-25(m362) n = 78, daf-11(m47) n = 83. Trimethyl thiazole: N2 n = 91, daf-25(m98) n = 69, daf- 25(m362) n = 74, daf-11(m47) n = 93. doi:10.1371/journal.pgen.1001199.g001 odors isoamyl alcohol, trimethyl thiazole and benzaldehyde, and our results indicate that DAF-25 is also required in this pathway [28]. The AWA-detected scent, pyrazine, is not reported to be detected by the cGMP pathway, suggesting that DAF-25 participates in another signaling pathway in AWA neurons. Interestingly, alhough it has been shown that the cGMP pathway does not participate in AWA-mediated olfaction, the particular tested allele daf-11(m47) was previously shown to have reduced affinity for pyrazine [28], as we have seen here. To establish if the olfactory phenotypes are associated with ciliary defects, mixed-stage populations of daf-25 mutants and N2 were stained with the lipophillic dye, DiI. Mutants with cilia structure anomalies have abrogated dye filling of the olfactory neurons [29], whereas daf-25 mutants take up the dye normally at all ages, suggesting that they have structurally intact cilia (Figure S2). To confirm this possibility, we further examined the integrity of ciliary structures by transmission electron microscopy. Ciliary ultrastruc- tures in two daf-25(m362) L2 larvae—including transition zones, middle segments consisting of doublet microtubules, and distal segments composed of singlet microtubules—was indistinguishable from the two N2 controls (Figure S3). We conclude that daf-25 animals have no obvious defects in ciliogenesis or cilia ultrastructure. PLoS Genetics | www.plosgenetics.org 3 November 2010 | Volume 6 | Issue 11 | e1001199