Mosquito feeding-deterrent screening assay
This protocol is extracted from research article:
Bacteria: A novel source for potent mosquito feeding-deterrents
Sci Adv, Jan 16, 2019; DOI: 10.1126/sciadv.aau6141

Mosquitoes were exposed to Xbu feeding-deterrent compounds or test repellents for 30 min at room temperature (incandescent light, 25 to 26°C) between 10:00 a.m. and 4:00 p.m. (U.S. Central Time) using a modified membrane-feeding apparatus described in Fig. 1.

Briefly, the Hemotek temperature controller was set to a constant temperature of 37°C 5 to 10 min ahead of the assay. A 2.5 cm × 2.5 cm piece of a fresh, pre-cut, and thoroughly water-washed collagen casing membrane was secured to the metal feeder using an O-ring. Approximately 2.5 ml of the cocktail diet containing 2% (v/v) red food dye (McCormick & Company Inc., MD, USA) was introduced from the opening at the back of the metal feeder. A cotton cloth was placed on top of the collagen casing and secured by rubber bands. Test compound or water was then applied by immersing the feeder assembly in 1 ml of the respective test solution, and the metal feeder assembly was then secured to the feeder housing and exposed to mosquitoes housed in a screened container. Several types of cloth materials varying in thickness and texture/thread count were evaluated with an objective of finding the one that provided optimal mosquito feeding success. Eventually, a double-layer cotton cloth obtained from JOANN Fabrics (Madison, WI) was used with A. aegypti. The features and dimensions of the chosen cotton cloth were as follows: thread count = 14.9 × 17.0 threads in 1 cm2 (average of 10 measurements), diameter of the circular cloth applied to metal feeder = 3.67 cm (see Fig. 1A), and total area of the circular cloth area exposed to mosquitoes = ~10.57 cm2. However, for A. gambiae and C. pipiens, one layer of muslin cloth (thread count = 24.3 × 33.9 threads in 1 cm2; average of 10 measurements) was used, as this cloth worked well with these mosquitoes. Both types of cloth materials are shown in fig. S8.

Because the Xbu compounds were dissolved in water for testing, DEET and picaridin were also prepared in 0.2-μm filtered double-distilled water. DEET was diluted from SC Johnson’s OFF! Deep Woods containing 25% DEET and picaridin from Clean Insect Repellent (purchased from Walgreens, USA) containing 7% picaridin. One percent stock solutions of DEET and picaridin (equivalent to 10 mg/ml) were dissolved in water from which a range of dilutions was prepared for determination of feeding-deterrence dose. Both of these repellents were tested at a concentration range of 1 to 0.01% (v/v) corresponding to 0.95 to 0.0095 mg/cm2. Both compounds dissolved well in water at the tested concentrations. DEET and picaridin were tested on the same day with a 4-hour gap in between tests. The HPLC-separated, lyophilized bacterial sample was dissolved in water and filtered through a 0.2-μm filter. For an accurate assessment, protein content in this sample was determined via two methods: (i) bicinchoninic acid (BCA) assay according to the manufacturer’s instructions (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, USA) and (ii) total amino acid analysis (Molecular Structure Facility, University of California, Davis, CA). The difference in protein content determined by these two methods was negligible. Thus, for routine measurements, the BCA method was used. A dosage range from 0.73 to 0.048 mg/ml (v/v) corresponding to 69.5 to 4.57 μg/cm2 of the mosquito feeding-deterrent active compounds was tested with A. aegypti. Testing with A. gambiae and C. pipiens was done only with one concentration of Xbu compounds that yielded a feeding-deterrence dose of 90% with A. aegypti [i.e., 0.060% (v/v), corresponding to 0.057 mg/cm2; Table 1]. Three replicate feeding experiments were conducted for each compound with mosquitoes hatched from different egg batches (to account for cohort bias) over a period of 3 weeks. One replicate consisted of 20 mosquitoes tested on the same day with each concentration of the dilution range. For example, one replication of samples with a concentration range of 69.5 to 4.57 μg/cm2 was tested on the same day. Extensive washing of the metal feeders, replenishing of collagen membrane, cotton cloths, and feeding solution for each exposure test and time gaps between assays assured that there was no carryover of the compounds or contamination of the feeders in between assays. As an extra precaution, feeding-deterrence assays with bacterial compounds were purposely performed on a different day than assays with DEET and picaridin. After the completion of the screening assay (30 min), mosquitoes were killed by freezing at −20°C. Fed versus unfed mosquitoes were counted under a dissecting microscope and verified by at least two people. Fed mosquitoes could be easily distinguished as they were engorged and had red abdomens, which were clearly visible even to an unaided eye (fully fed or partially fed), while unfed mosquitos were lean and did not have red abdomens. Mosquitoes that were not engorged and had no red color were considered as unfed and hence deterred (Figs. 1C and 2B). This distinction provided a reliable, quantitative means of assessing the feeding success of mosquitoes with different compounds and across assays and allowed a robust comparison. Count data (proportion of unfed mosquitoes from a total) were used for statistical analysis. Several tests with Xbu Peak#3 from different batches exhibited consistent, concentration-dependent feeding-deterrent activity. Here, results of three replications are presented. Figures 1B and 2A and Tables 1 and 2 present results of all of the feeding-deterrence screening experiments.

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