Mir 154

Collections of Culicoides biting midges were made at farms in two areas in Switzerland, one in the Swiss Plateau (one site, 650 masl), the other in the pre-alpine region (two sites at around 1550 masl, referred to as pre-alpine I; one site at 2030 masl, referred to as pre-alpine II) (Table 1). Insects were trapped alive as described [38 (link)]. The light traps were operated from approximately 1–2 h before sunset to 1–2 h after sunrise, between June and October 2015-2017. Wet cotton pads were placed around the cages during transportation to the laboratory where the cages were transferred to incubators (Panasonic MIR-154, Gunma, Japan) with a fluctuating temperature (13–25 °C, mean 19 °C) and a relative humidity of 85–90% (Fig. 1). Cotton wool pads with 10% sucrose solution were supplemented.Table 1

Areas and features of the sites where Culicoides were collected

Area	Site (altitude, masl)	Coordinates	Predominant animal species at farm
Swiss Plateau	Adlisberg (650)	47.22298°N,008.34565°E	Horses
Pre-alpine I	Davos Wolfgang (1575)	46.82638°N,009.85732°E	Sheep and pigs
	Lenzerheide (1542)	46.73014°N,009.56091°E	Horses
Pre-alpine II	Davos, Clavadeler Alp (2030)	46.76650°N,009.81831°E	Cattle

Fig. 1

Fluctuating temperature regime during incubation of Culicoides

Gas sensing tests were carried out using an in-house gas characterisation system comprising an air-tight steel chamber housed in a Panasonic (MIR-154) cooled incubator with probes for connecting to the sensor pads, a Keithley 6487 picoammeter/voltage source for biasing and measuring the current through the sensor, a vacuum flow rates, and gas cylinders. The tests carried out spanned 9 h in each case for both dry air and humid air conditions. In every case, the RGO and RGO/MoS₂-coated fabric were placed on the sample holder. The probes were then brought into contact with it. The separation of the probes was kept constant at 2 cm all through the test cases to ensure uniformity of test situations. The voltage used was 3 V for biasing the sensor. For the dry air tests with increasing target gas concentration, the sensor was exposed to dry air until the resistance was constant. Then short time exposures of 10 min to NO₂, followed by dry air 30 min, and then to 20 ppb NO₂ with concentration increased by 20 ppb each time and the cycle repeated until 100 ppb. For humid air tests, the fabric was exposed to 65% RH air and allowed to reach stability before exposure to 100 ppb NO₂ for 10 min, and then exposed to humid air for 4 h to allow for recovery.

To record the sleep under short exposure to caffeine, 1-, 10-, 20-, and 30-day-old w¹¹¹⁸ male flies were transferred to locomotor activity glass tubes (65 mm length, 5 mm diameter) containing cornmeal dextrose medium for acclimatization 12 h prior to the start of the experiment. These flies were transferred into locomotor activity glass tubes containing cornmeal dextrose medium with different concentrations of caffeine (0.0, 0.5, 0.75, and 1 mg/mL) just before ZT 00 on the day of locomotor activity recording. Sleep was recorded by using the Drosophila Activity Monitors (Trikinetics, USA) for 24 h under LD in a cooled incubator (MIR-154, Panasonic, Japan). On the subsequent day, at ZT 00, these flies were transferred back to fresh cornmeal dextrose medium without caffeine to record their sleep rebound for the next 24 h. Activity counts were measured at every 1 min interval and sleep was defined as 5 min or more of continuous inactivity. Sleep parameters such as total sleep duration, sleep bout length, sleep bout number, sleep latency, and sleep in 1 h were analyzed using Sleep and Circadian Analysis MATLAB Program (SCAMP) [27 (link)]. To confirm the results, each experiment was replicated three times with a sample size of 25–32 flies in each replicate. The data obtained from one such biological replicate is used in the results and figures.

We used the reporter strains PE255 and MRS229 to measure larval developmental timing. In order to ensure that all animals started development at the same time, arrested L1s were first manually pipetted to a white 96-well plate, one worm per well, containing 100 μl of S-basal with 100 μM D-luciferin. Development was resumed by addition of 100 μl of S-basal with 20 g/L E. coli OP50 and 100 μM D-luciferin. Plates were sealed with a gas-permeable cover (Breathe Easier, Diversified Biotech, Dedham, MA, USA). We measured luminescence in a Berthold Centro LB960 XS3 (Berthold Technologies, Bad Wildbad, Germany) for 1 s, at 5 min intervals. Experiments were done inside temperature-controlled incubators (Panasonic MIR-154). We analysed the raw data from the luminometer as described in Olmedo et al. [48 (link)]. Briefly, the raw data was trend-corrected and thresholded using 75% of the moving average to produce a binarised output in order to determine onset and offset of the molts. The data was evaluated for onset and offset of molting by detecting the transitions in the binarised data. To assess statistics, we performed unpaired t tests using GraphPad Prism software.