Extended Data Fig. 6: Generation and validation of loss-of-function alleles of D. sechellia Ir genes.
From: Olfactory receptor and circuit evolution promote host specialization

a, Schematics depicting organization of Ir genes, the structure of mutant alleles and the sequences that encode antibody epitopes. For DsecIr8aRFP or DsecIr8aGFP, the fluorescent marker was integrated into the first coding exon. For DsecIr64aRFP, the marker replaces parts of exon 2. DsecIr75bRFP lacks parts of exons 3 and 4, and for DsecIr75bGFP the marker was integrated into exon 3. For both alleles of Ir75b, the fluorophore was removed via Cre-mediated recombination to produce Ir75b1 and Ir75b2. b, Immunofluorescence for Ir64a and Ir8a (as an internal staining control) on whole-mount antennae from wild-type and DsecIr64aRFP mutant flies. Arrowheads indicate the Ir64a-neuron dendrites that innervate sensilla in the sacculus (sac). Scale bars, 25 μm (main panels), 5 μm (insets). c, Left, immunofluorescence for Ir8a and Ir25a (as an internal staining control) on whole-mount antennae from wild-type and DsecIr8aGFP flies (top and middle). Left, immunofluorescence for Ir75b and RNA FISH for Or35a on whole-mount antennae from DsecIr75b2 mutant flies (bottom). Scale bars, 25 μm (main panels), 5 μm (insets). Right, electrophysiological responses in the ac3I sensillum (neurons housed are indicated in the cartoon) to noni juice, grape juice and odours present in noni (n = 4–11, female flies) in wild-type D. sechellia and olfactory-receptor mutants with the Ir75b neuron affected (DsecIr8aGFP and DsecIr75b2). Data points represent the summed solvent-corrected activities of the sensillum. Responses of wild-type D. sechellia are replotted from Fig. 2b. The Or35a neuron exhibits residual responses to hexanoic acid in the DsecIr8a and DsecIr75b olfactory-receptor mutants (Fig. 2b). d, Left, immunofluorescence for Ir8a and Ir25a (as an internal staining control) on whole-mount antennae from wild-type (same image as shown in c) and DsecIr8aRFP and DsecIr8aGFP (same image as shown in c) flies. Scale bars, 25 μm (main panels), 5 μm (insets). Right, electrophysiological responses in the ac2 sensillum (neurons housed are indicated in the cartoon) to noni juice, grape juice and odours present in noni (n = 3–11, female flies) in wild-type D. sechellia and olfactory-receptor mutants in which the Ir75a neuron is affected (DsecIr8aRFP and DsecIr8aGFP). Data points represent the summed solvent-corrected neuronal activities of the sensillum. Responses of wild-type D. sechellia to noni and grape juice are as shown in Extended Data Fig. 4e. e, Immunofluorescence for Ir75b and RNA FISH for Or35a on whole-mount antennae from wild-type and DsecIr75b1 and DsecIr75b2 (same image as shown in c) flies. Scale bars, 25 μm (main panels), 5 μm (insets).