Fructose detection

Fructose concentration was measured using the cold anthrone colorimetric assay [45]. At room temperature, anthrone solution reacts with fructose, but not other sugars. The assay is indicative of plant feeding and does not measure blood sugars (primarily glucose) or stored sugars (trehalose), although non-sugar fed teneral mosquitoes contain small amounts of fructose.

Mosquitoes were homogenized in 1.7 ml microcentrifuge tubes using a lyser (FastPrep-24 Classic Instrument, MP Biomedicals, USA) at 4 m/s for 30 s with 50 μl of 2% sodium sulfate solution and glass beads (3 mm, Thermofisher). Chloroform methanol (1:2) solution (375 μl) was added to each tube and vortexed for 8 s and centrifuged for 15 min at 200 x g, extracting fructose into supernatant. Tubes were stored at -20°C until fructose quantification, at which time 10 μl of supernatant was transferred to two wells of a 96-well microplate.

To ensure consistency, standards were produced once via serial dilution and stored at 4°C for the duration of analysis. Two replicates of standards (10 μl of 0, 0.05, 0.1 and 0.2 μg /μl D-Fructose (Fisher Chemical, USA) in 25% ethanol) and samples (10 μl) were pipetted individually into wells on each plate. Thereafter, 240 μl anthrone solution (freshly prepared each day, 67.9 μl distilled water, 172.1 μl sulfuric acid, and 0.339 mg anthrone per sample) was added with a multichannel pipette. Samples were incubated at room temperature for 90 min in a chemical hood. The absorbance of light (630 nm) by the reaction of each sample was measured by the microplate reader (800 TS Absorbance Reader, BioTek, VT, USA) and compared against the standard curve to determine fructose concentration. The mean of the two experimental replicates of each sample was used in analyses, except when experimental replicates were dissimilar, the data were discarded or the sample was reanalyzed.

During the period of adult collection, pupae were collected from containers on a subset of properties and held in the laboratory until eclosion. Post-eclosion, adult Ae. albopictus (n = 78 male, 53 female) were held without sugar and frozen within 12 hrs of emergence followed by sugar analysis with the cold anthrone assay. One male and one female outlier were removed using the Median Absolute Deviation. The remaining mosquito data were used to establish a field baseline level of fructose in teneral Ae. albopictus [22]. Field-collected adults with fructose concentrations greater than one standard deviation above the sex-specific mean baseline concentration were considered to be fructose-positive.

To determine the time window of fructose detection in Ae. albopictus post-sugar meal consumption, we conducted an assay of fructose concentration in sugar fed females over digestion time [46]. Aedes albopictus (F6 from NY at 23.5°C and F8 from FL at 28°C) were vacuum hatched and provided with a pinch of pulverized fish food (crushed Cichlid Gold fish food pellets; Hikari, Himeji, Japan). One day later, they were separated into trays of 200 larvae with 1L of distilled water and 4 Cichlid Gold fish food pellets. Pupae were transferred to cages and fed 10% sucrose solution between 1–3 d post-eclosion. Males and females were removed before, immediately after, and at 24 hr intervals post sugar feeding. Between nine and twenty mosquitoes were removed per day. Fructose concentration was measured as described above. The assumption of constant variance was not met, so mean fructose concentrations were compared to concentration before feeding using the non-parametric Kruskal-Wallis test followed by a Dunn’s multiple comparisons test with Benjamini-Hochberg correction (reported as P_adj).