Lobeline

Poly-D-Lysine (P7886), Nicotine (N3876), Varenicline (PZ0004), Lobeline (141879), Epibatidine (E1145), Mecamylamine (M9020), Ammonium Chloride (A0171), Chloroquine (C6628) and Bafilomycin A1 (B1793) were purchased from Sigma, MO. Dihydro beta-erythroidine (DHβE) (2349) was purchased from Tocris, MN. Neurobasal medium, B27, HBSS and DMEM were purchased from Life technologies (Thermofisher scientific, MA).

Animals were anesthetized with sodium pentobarbital (60 mg/kg, IP). The levels of anesthesia were maintained with a 20% solution (v/v) of the same anesthetic after assessing the withdrawal response. Rectal temperature was maintained through a homoeothermic blanket (Harvard Apparatus, Cambourne, UK). The trachea was cannulated below the larynx to record tracheal pressure. The femoral artery and vein were cannulated for blood pressure (BP) monitoring and injection of saline and drugs, respectively. The electrocardiogram (ECG) was recorded from subcutaneous electrodes placed into three limbs, and the heart rate was derived from the ECG recording (Neurology, Digitimer, Welwyn Garden City, UK).
The right carotid artery was cannulated, and chemoreceptors were stimulated by lobeline injection (0.2 mL, 25 µg/mL, Sigma, St. Louis, MO, USA). Baroreflexes were stimulated by phenylephrine injection (0.2 mL, 25 µg/mL, Sigma, St. Louis, MO, USA) in the femoral vein [29 (link),30 (link),31 (link)]. At the end of the above-mentioned acute experience, the animal was sacrificed with an overdose of anesthetic. The heart was then removed and placed in 4% paraformaldehyde (Sigma-Aldrich) at 4 °C for further histological studies.

After behavioural and metabolic evaluation, animals were anaesthetized with sodium pentobarbital (60 mg/kg, IP). Anaesthesia levels were maintained using a 20% solution (v/v) of the same anaesthetic after assessing the withdrawal response. A homoeothermic blanket (Harvard Apparatus, Cambourne, UK) was used to keep the rectal temperature stable. To measure tracheal pressure, the trachea was cannulated below the larynx and the respiratory frequency (RF) was calculated from the measured pressure. The femoral artery and vein were cannulated to monitor blood pressure (BP) and inject saline and drugs, respectively. The electrocardiogram (ECG) was assessed using subcutaneous electrodes in three limbs, and the heart rate was calculated using the ECG data (Neurology, Digitimer, Welwyn Garden City, UK).
The right carotid artery was catheterized, and chemoreceptors were stimulated with lobeline (0.2 mL, 25 g/mL, Sigma, St. Louis, MO, USA) [51 (link)]. A phenylephrine injection (0.2 mL, 25 g/mL, Sigma, St. Louis, MO, USA) in the femoral vein was used to stimulate the baroreflexes [32 (link),48 (link),51 (link)].
Blood lead levels (BLL) were determined from the venous blood using an atomic absorption spectrophotometer (Shimadzu, Model no. AA 7000, Kyoto, Japan).

On the 60th day, the animals were re-anaesthetised (sodium pentobarbitone, 60 mg.kg⁻¹, IP), and the trachea, common carotid artery, femoral artery, and vein were cannulated. A heating blanket was used to monitor the rectal temperature (Harvard Apparatus). The ECG was recorded, and the heart rate (HR) was derived from this. The baroreceptor reflex was activated via phenylephrine administration (0.2 mL, 25 μg.mL⁻¹ iv; Sigma Aldrich, St. Louis, MO, USA). Retrogradely injected lobeline (0.2 mL, 25 μg.mL⁻¹ iv, Sigma Aldrich, St. Louis, MO, USA) through the external carotid artery triggered the peripheral chemoreceptor reflex. The baro- and peripheral chemoreceptor reflexes were activated twice, with a 5 min interval between the stimulations. The ECG, HR, and BP (systolic, diastolic, and mean) were continuously recorded throughout the experiment.

Stimulants were chosen from a list of known olfactory and/or gustatory stimuli of both Drosophila melanogaster and adult Ae. aegypti [26 (link), 27 (link), 36 (link)–41 (link)]. These included ethyl acetate (Sigma Cat# 319902), lactic acid (Sigma Cat# L1750), 1-octen-3-ol (Sigma Cat# O5284), butylamine (Sigma Cat# 471305), VUAA1 (Vitas-M Cat# STK047588), sucrose (Sigma Cat# S0389), lobeline (Sigma Cat# 141879), glutamate (Sigma Cat# 49621), and water (negative control). All stock solutions of odors were prepared as 5% solutions in water, with a final bioassayed concentration of 6 × 10⁻⁵ M. Food extract for larval experiments was prepared by mixing 0.5% fish food (Hikari Tropic First Bites: Petco, San Diego, CA, USA) in milliQ water. The solution was allowed to sit for 1 h, then filtered through a 0.2 μm sterile filter (#28145-477, VWR International, Radnor, PA, USA) to remove solid particulates. 1-octen-3-ol used in behavior experiments was prepared as a 10⁻⁴M solution in water, based on preliminary experiments testing several odor concentrations (data not shown).

Sucrose (CAS No. 57-50-1), tricholine citrate (TCC) (CAS No. 546-63-4), sulforhodamine B (CAS No. 3520-42-1), capsaicin (CAS No. 404-86-4), caffeine (CAS No. 58-08-2), CaCl₂ dihydrate (CAS No. 10035-04-8), KCl (CAS No. 7447-40-7), quinine (CAS No. 6119-47-7), strychnine (CAS No. 1421-86-9), lobeline (CAS No. 134-63-4), denatonium (CAS No. 6234-33-6), and coumarin (CAS No. 91-64-5) were purchased from Sigma-Aldrich (USA). Brilliant blue FCF (CAS No. 3844-45-9) was purchased from Wako Pure Chemical Industry (Japan). Paraformaldehyde (CAS No. 30525-89-4) was purchased from Electron Microscopy Sciences (USA). NaCl (CAS No. 7647-14-5) was purchased from LPS solution (Korea). NaBr (CAS No. 7647-15-6) was purchased from DUKSAN (Korea). Goat serum, New Zealand Origin was purchased from Gibco (USA).