Stemi 508 stereo microscope

Adult males and females from all the treatments were stored at 4°C, and the size of the adults was estimated by measuring their right wing from the distal edge of the alula to the end of the radius vein excluding fringe scales, which is a reliable indicator of body size [20 (link)]. The measurement of the wings was carried out to a precision of 0.01 mm using ZEN 2.3 (blue edition) software for the Stemi 508 Stereomicroscope (Carl Zeiss), fitted with a digital camera.

The sample preparation following waterborne drug exposure is described elsewhere [26 (link),27 (link),28 (link),29 (link),55 (link)]. A nontoxic exposure concentration was chosen based on the survival rate as determined by in vivo maximum-tolerated concentration (MTC) experiments with 4 dpf (days post-fertilization) ZF larvae. To measure the heartbeat of the ZF larvae, the ZF larvae were anesthetized on ice and arranged. The heartbeat rates were determined using DanioScope software version 1.2.206 based on imaging using a stereo microscope (Zeiss Stemi 508 stereo microscope). For metabolite studies, 15 ZF larvae at 4 dpf were transferred to one well of a 6-well plate containing 3 mL of 0.3× Danieau’s medium with 300 µM naloxone. All exposure media contained a final concentration of 1% (v/v) DMSO, and the ZF larvae were treated for 24 h in an incubator at 28 °C. An additional 15 larvae were incubated in a compound-free medium containing only 1% (v/v) DMSO as a negative control (background masses). All treated larvae were rinsed twice with 1 mL of 0.3× Danieau’s solution prior to subsequent analyses.

The laboratory colonies of Ae. aegypti and Ae. albopictus used for all experiments originated from eggs collected in 2016 from twelve localities along the Pacific coast of Chiapas, Mexico (S1 File, sheet 1). The twelve populations were subjected to a process of introgression through backcrosses to obtain a genetically diverse strain for each species [25 (link)]. Colonies were maintained under controlled conditions at 28 ± 2°C, 80 ± 5% relative humidity (RH), and photoperiod of 14:10 h (light: dark). Larvae were reared at a density of 1.5 larvae/ml in 61x41x7.5 cm plastic trays containing 2000 ml dechlorinated water and were fed with powdered Laboratory Rodent Diet (LabDiet, Fort Worth, Texas, USA), as described previously [25 (link)]. Pupae were sexed as a function of body size using a plate separator (John W. Hock, Model 5412, Gainesville, Florida, USA) and the genital lobe was visually checked using a Stemi 508 Stereomicroscope (Carl Zeiss). Adults were placed in 30x30x30 cm acrylic cages with nylon mesh walls (BugDorm 1; Taichung, Taiwan) maintained at 26 ± 2°C, 80 ± 5% relative humidity (RH), and 14 h:10 h (light: dark), photoperiod and supplied ad libitum with 10% sucrose solution on a cotton pad. From 4 days post-emergence, bovine blood was provided for three consecutive days using a Hemotek membrane feeding system (PS6B, Hemotek Ltd., Great Harwood, UK).