Single sensillum electrophysiological recording

Electrophysiological recordings were conducted on single capitate peg (cp) sensillum along the maxillary palp of female mosquitoes that house three types of olfactory receptor neurons (ORNs) [35]. Here, gravid females of An. coluzzi (subsequently confirmed to contain mature Christopher stage IV or V embryos) were cold immobilized (~1 min at -20°C) and mounted on a double-sided tape on a microscope glass slide (25 × 75 × 1.0 mm). Two glass capillaries inserted with chloridized silver wire of appropriate size and filled with 0.1 M KCl saline were used as reference and recording electrode, respectively. The reference electrode was placed in the eye, and the recording electrode was connected to a preamplifier (10×, Syntech, Hilversum, The Netherlands) and inserted at the base of cp sensillum to record the extracellular activity of the ORNs. The signals were digitized by the IDAC4 interface box (Syntech, Hilversum, The Netherlands) and analyzed with Auto Spike v. 3.2 software (Syntech, Hilversum, The Netherlands).

Odor stimuli were diluted in DMSO in decadic steps, ranging from 10⁻⁵ M to 1 M and DMSO was used as controls. Odor cartridges were prepared by loading filter paper disk (ca. 12.7 mm ø) with 10 μl of test compounds and inserting them into glass Pasteur pipettes connected via silicone tubing to a stimulus controller (Syntech, Hilversum, The Netherlands). Odor stimulation (0.5 liter/min) was carried out for 500 ms by inserting the tip of the SSR odor cartridge into a glass tube with an a charcoal filtered, humidified air-flow (0.5 liter/min) directed towards the maxillary palp which was positioned 10 mm away from end of the glass tube.

The extracellular activity of an individual cp sensillum that houses three physiologically distinct ORNs, cpA, cpB, and cpC was characterized based on the spike amplitudes, spike frequency, and shape as described in S5B Fig and a previous study [35]. The response of an individual neuron to a stimulus was determined by manually counting number of spikes 1000 ms after the onset of neuronal response minus the number of spikes 1000 ms prior to stimulus onset. To rule out the solvent (DMSO) response, solvent spikes were subtracted from the odor induced spike counts. One sample t-test (p = 0.05, two-sided; JMP 8.01, SAS Institute, Cary, NC) was used to determine whether the normalized response value (i.e., solvent response subtracted) for each neuron was significantly different from zero for each concentration of odor stimuli. If the response value was significantly greater or smaller than zero, the neuronal response was considered as excitatory or inhibitory.