Mosquitoes smell and avoid the insect repellent DEET

Abstract

The insect repellent DEET is effective against a variety of medically important pests, but its mode of action still draws considerable debate. The widely accepted hypothesis that DEET interferes with the detection of lactic acid has been challenged by demonstrated DEET-induced repellency in the absence of lactic acid. The most recent hypothesis suggests that DEET masks or jams the olfactory system by attenuating electrophysiological responses to 1-octen-3-ol. Our research shows that mosquitoes smell DEET directly and avoid it. We performed single-unit recordings from all functional ORNs on the antenna and maxillary palps of Culex quinquefasciatus and found an ORN in a short trichoid sensillum responding to DEET in a dose-dependent manner. The same ORN responded with higher sensitivity to terpenoid compounds. SPME and GC analysis showed that odorants were trapped in conventional stimulus cartridges upon addition of a DEET-impregnated filter paper strip thus leading to the observed reduced electrophysiological responses, as reported elsewhere. With a new stimulus delivery method releasing equal amounts of 1-octen-3-ol alone or in combination with DEET we found no difference in neuronal responses. When applied to human skin, DEET altered the chemical profile of emanations by a "fixative" effect that may also contribute to repellency. However, the main mode of action is the direct detection of DEET as indicated by the evidence that mosquitoes are endowed with DEET-detecting ORNs and corroborated by behavioral bioassays. In a sugar-feeding assay, both female and male mosquitoes avoided DEET. In addition, mosquitoes responding only to physical stimuli avoided DEET.

Fig. 1.

Excitatory responses from an ORN housed in a trichoid sensillum upon stimulation with increasing doses of DEET. (A) Single sensillum recordings with control, 1, 10, and 100 μg of DEET (top to bottom). (Scale bar: 500 ms.) (B) Dose-response curve (n = 5). (C) Scanning electronmicrograph of the second segment of the antenna displaying various types of olfactory sensilla. The white arrow in the micrograph and the black arrow in the Inset highlight the type of short trichoid sensillum housing ORN sensitive to DEET and other terpenoids.

Fig. 2.

Excitatory responses from DEET sensitive ORN to 10-μg dose of terpenoids (Upper traces) and 100 μg of DEET (Lower trace). Response of the second ORN, colocated in the DEET-detecting sensilla to 10 μg of 1-octen-3-ol. Note the larger spike amplitude of the DEET-sensitive ORN and the smaller spike amplitude of the neuron responding to 1-octen-3-ol. (Scale bar: 500 ms.) Dose-response curves indicating lower threshold and higher sensitivity of the DEET-detecting ORN to some terpenoid compounds compared with DEET (see Fig. 1B).

Fig. 3.

Responses to 1-octen-3-ol from ORNs in peg sensilla on the maxillary palps. The spike frequency decreased dramatically when 1-octen-3-ol was delivered from the same stimulus cartridge along with DEET. (Left) Significant decrease in spike frequency (P < 0.01, n = 5; Mean± SEM). (Right) Electrophysiological recordings depicting a typical response pattern. Upper and Lower traces in each recording are action potential and DC coupled sensillum potential, respectively. (Scale bars: spikes, 1 mV; DC, 4 mV; horizontal bar, 500 ms.)

Fig. 4.

Decrease in the amounts of 1-octen-3-ol collected by SPME at the tip of stimulus cartridges (Pasteur pipettes) and analyzed by GC. The pipettes containing equal amounts of 1-octen-3-ol (100 ng) loaded on filter paper strips. The DEET cartridge contained a second filter paper strip impregnated with 5 μl of pure DEET.

Fig. 5.

Neuronal responses recorded from an ORN in the maxillary palps of C. quinquefasciatus sensitive to 1-octen-3-ol. (A) Responses to stimulus (10 ng) in the presence and absence of DEET were indistinguishable. (B) Filter paper strips loaded with 1-octen-3-ol and DEET (or blank) were placed on separated syringes and puffed simultaneously. The compensatory flow cartridges are omitted from this diagram for clarity.

Fig. 6.

GC-MS traces of human-derived compounds extracted by SPME. Skin emanations were collected simultaneously from one arm treated with DEET (Lower, red trace) and an untreated arm (Upper, blue trace) of the same subject. Arrows indicate suppression in the amounts of five physiologically relevant compounds from the arm treated with DEET. The other peaks in the GC-MS trace from the treatment (Lower) are impurities from DEET, whereas an overshooting peak at 15.5 min for DEET was omitted.

Fig. 7.

DEET repellency in the absence of attractants. (A) Snapshots of the sugar-feeding bioassay. Although mosquitoes landed equally on solvent Petri dishes (Upper), they avoided landing on the side impregnated with DEET (Lower). (B) Mosquito landings were significantly higher in Petri dishes treated with solvent only compared with those treated with DEET, whereas there was no significant difference between the two sides of the arena.

Fig. 8.

DEET-induced repellency of mosquitoes responding to physical stimuli. Females landed preferentially on the solvent-treated side of this two-choice assay. (A) Total landings for 10 min. (B) Cumulative landing over time.