Methylparaben

The study was carried out in the insectaries of the Department of Experimental Medicine, Functional Genomics Center of the University of Perugia. A total of six experimental cages were built in two large chambers, i.e. Insectary Field 1 measuring 6.68 × 3.80 × 3.00 m (all dimensions are L × W × H), and Insectary Field 2 measuring 8.45 × 3.80 × 3.00 m (Figure 1). These studies were carried out in three types of cages: (1) 30 cm on each side white plastic Bugdorms (BioQuip Products, Inc., Rancho Dominguez, CA, US); (2) custom-made large cages without swarming stimuli; (3) custom-made large cages with swarming stimuli. Large cages (5.00 × 1.22 × 2.60 m each, Figure 1) consisted of white-painted wooden frames with walls and ceiling made of polyester mesh (1,290 µm openings, US size 15). Part of the front panel of each cage was made of a 50 × 125 cm Plexiglas sheet provided with two 20 cm square entrances that allowed access to cages through affixed sleeves opening to the chamber. Each large cage was provided with a terracotta resting shelter which was kept humid and two 500 ml cups containing a 10% sucrose solution, 0.1% methylparaben as preservative [22 (link)] and approximately 4 ml acacia honey as an attractant. White absorbent paper placed in the cup allowed mosquitoes to land and get the sugar meal without drowning. In each large cage, swarming stimuli (Figure 1), consisted of (1) square arena made of four black plastic sheets (Correx equivalent, 122 cm long, 50 cm high and 0.5 cm thick), located at the back of the cages; (ii) a contrasting ground mark consisting of a black plastic square (40 × 40 cm) and white plastic square (20 × 20 cm) centred on top of it in the centre of the arena; (3) a 2.700 K 8 W compact fluorescent light located on the floor at the back of the cage hidden from the mosquitoes by a black plastic Correx equivalent sheet (122 cm long, 50 cm high and 0.5 cm thick), diffusing light onto the back wall of the chamber simulating twilight (horizon lights). Temperature and relative humidity were kept stable during the studies i.e. 28.01°C SD ± 0.57°C and 72.1% SD ± 2.0%.Figure 1

Illustration of the large cages. The typical arrangement of the swarming stimuli (left) is represented by the black-bordered swarm marker (M) surrounded by the black artificial horizon lining the interior of each cage. Above these and outside of the cage, the black baffle (B) is represented, preventing the three lights from shining directly into the cages but upward onto the wall (W). Also shown are the locations of sugar sources (S) and resting shelters (R) flat against the floor. The swarming stimuli and controls were switched between rooms to determine whether there was any effect of insectary.

Light in the chambers was provided by four or five ceiling light fixtures in Insectary Field 1 and Insectary Field 2, respectively, producing a diffused and evenly distributed light. Each light fixture was equipped with three T5 80 W linear fluorescent bulbs with different emissions: 6.400, 4.000 and 2.700 K. Emitting temperature, day duration, dusk and dawn fading of ceiling lights, were controlled by Easy Color Control software (OSRAM SpA, Società Riunite OSRAM Edison Clerici, Milan, Italy).
Length of daytime was the same for the two large cage treatments but light locations and durations differed. In both treatments, dawn lasted for 30 m from dark to full light, and full light lasted for 11:30 h. In the chamber where cages lacked the swarming stimuli, sunset lasted for 1 h: 30 m of fading ceiling from full light to minimum emission and 30 m of 2.700 K tubes only at minimum emission. In the chamber where cages where provided with swarming stimuli, sunset lasted for 1 h: 30 m of fading ceiling light from full light to minimum power, overlapping with 60 m of twilight provided by the horizon light. Different combinations of visual stimuli described above were tested, and the presence/absence of swarming males was detected using binoculars (7x35 mm roof prisms Foton, CCCP).

Drosophila were cultured at 25°C on a standard yeast/cornmeal medium containing 10 g.L⁻¹ agar (VWR, ref. #20768.361), 80 g.L⁻¹ cornmeal flour (Westhove, Farigel maize H1), 50 g.L⁻¹ inactivated dry yeast (Bio Springer, Springaline BA95/0-PW), 5.2 g.L⁻¹ Methylparaben sodium salt (MERCK, ref. #106756) and 4 ml.L⁻¹ of 99% propionic acid (CARLO ERBA, cref. #409553). Germ-free animals were obtained from bleached embryos cultured on autoclaved conventional medium. When needed GF stocks were maintained during few generations on antibiotic supplemented food (final concentration: 50 µg.L⁻¹ ampicilline, 50 µg.L⁻¹ kanamicine, 15 µg.L⁻¹ erythromycin, 50 µg.L⁻¹ tetracycline) to avoid bacterial contamination. Drosophila y,w flies were used as the reference strain in this work. The following mutant lines were used: y,w,Dredd^F64 and y,w;;Relish^E20[34] (link).

Carbopol 934, methylparaben, and propylparaben of analytical grade and sodium hydroxide pellets were purchased from Adwic Co. (Cairo, Egypt). Propylene glycol, Jasmine oil, and absolute ethanol were purchased from Agitech Co. (Cairo, Egypt).