Fig. 2
Live calcium imaging of stimulus-evoked responses in GCaMP6s/+/+, GCaMP6s/orco5−/−, and GCaMP6s/Gr3−/−. Mosquito larval calcium responses to various stimulants were recorded using a Leica SP5 Confocal microscope. To secure the larval head for imaging while allowing free movement of the larval tail, the dorsal side of the larva’s head was adhered to a chambered cover glass using quick setting adhesive. The chamber was then filled with water and the larvae was allowed to rest before being introduced to stimulants (a). To compare temporal difference between the deuterocerebrum (DE, purple) and muscles (blue), the difference between time points at 50% of the maximum ∆F/F of the first response peak following the addition of stimuli (b, c). Stimuli, including 1-octen-3-ol, butylamine, and water were introduced to GCaMP6s/+/+, GCaMP6s/orco5−/−, and GCaMP6s/Gr3−/− larvae 15 s after the start of recording. Calcium responses within the DE and muscles were recorded at 0.645 frames/sec (d) and maximum fluorescence values were plotted (e). The temporal difference in seconds between the DE and muscle responses were calculated and plotted by comparing DE and muscle timepoints at 50% of maximum ∆F/F (f). The number of biological replicates used for each experiment were 3 or greater. Differences in ∆F/F and Latency were analyzed using a Welch’s T-test and a Mann–Whitney U test respectively. *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001. (GCaMP6s/+/+ DE: 1-octen-3-ol n = 6; butylamine n = 11; water n = 12. GCaMP6s/+/+ Muscle: 1-octen-3-ol n = 7; butylamine n = 15; water n = 12. GCaMP6s/orco5−/− DE: 1-octen-3-ol n = 4; butylamine n = 8; water n = 7. GCaMP6s/orco5−/− Muscle: 1-octen-3-ol n = 6; butylamine n = 10; water n = 9. GCaMP6s/Gr3−/−: 1-octen-3-ol n = 6; butylamine n = 11; water n = 12. GCaMP6s/Gr3−/− DE: 1-octen-3-ol n = 6; butylamine n = 5; water n = 4, GCaMP6s/Gr3−/− Muscle: 1-octen-3-ol n = 7; butylamine n = 11; water n = 10. Latency of GCaMP6s +/+ response: 1-octen-3-ol n = 6; butylamine n = 5. Latency of GCaMP6s/orco5−/− response: 1-octen-3-ol n = 4; butylamine n = 4. Latency of GCaMP6s/Gr3−/− response: 1-octen-3-ol n = 4; butylamine n = 6.)