The molecular and cellular basis of olfactory-driven behavior in Anopheles gambiae larvae

Abstract

The mosquito Anopheles gambiae is the principal Afrotropical vector for human malaria. A central component of its vectorial capacity is the ability to maintain sufficient populations of adults. During both adult and preadult (larval) stages, the mosquitoes depend on the ability to recognize and respond to chemical cues that mediate feeding and survival. In this study, we used a behavioral assay to identify a range of odorant-specific responses of An. gambiae larvae that are dependent on the integrity of the larval antennae. Parallel molecular analyses have identified a subset of the An. gambiae odorant receptors (AgOrs) that are localized to discrete neurons within the larval antennae and facilitate odor-evoked responses in Xenopus oocytes that are consistent with the larval behavioral spectrum. These studies shed light on chemosensory-driven behaviors and represent molecular and cellular characterization of olfactory processes in mosquito larvae. These advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in larval-stage mosquitoes to reduce the catastrophic effects of malaria and other diseases.

Fig. 1.

Expression of AgOr genes in the larval antenna. (A) Whole-mount staining of An. gambiae larval antennae with AgOR7 antibody. The arrow indicates the dendrites projecting into the sensory cone. (B–J) AgOr FISH on 8-μm section results revealed that each individual conventional AgOr is solely coexpressed with AgOr7 in a single larva OSN. Arrows indicate the individual neuron (yellow) with AgOr7 (red) and one conventional AgOr (green) coexpressed. (Scale bar, 25 μm.)

Fig. 2.

An olfactory-based behavioral assay for mosquito larvae and odorant response profile for An. gambiae. (A) Schematic diagram of the experimental arena of the larval behavioral assay. (B–K) Response profiles of behaviorally active odorants: 2-methylphenol, 3-methylphenol, 4-methylphenol, acetophenone, indole, 1-octen-3-ol, 4-methylcyclohexanol, yeast, DEET, and isovaleric acid. Error bars indicate SEM for n ≥ 8 trials. For comparisons, two-tailed unpaired student's t tests were performed: **, P < 0.01; *, P < 0.05. (L) Ablation of the larval antenna reduces olfactory responses. Behavioral responses for unablated larvae (black bars, n = 8); sham/maxilla ablations (cross-hatched bars, n = 3); antennal ablations (gray bars, n = 3) and no odorant/unablated control larvae (open bars, n = 8). Both 2-methylphenol and DEET were used at 10⁻³ dilutions. Error bar indicates SEM. Two-tailed unpaired student's t tests were performed: **, P < 0.01; *, P < 0.05 relative to unablated larvae.

Fig. 3.

Odor-response spectra of larval AgORs. Response is measured as induced currents, expressed in nA. Error bars indicate the SEM (n = 5–8). The corresponding tuning curve for a given receptor is placed in the Insets. The 82 odorants are displayed along the x axis, with those eliciting the strongest responses being placed near the center, and those eliciting the weakest responses placed near the edges.

Fig. 4.

Combinatorial coding of odors in An. gambiae larvae. Filled circles represent the maximal response for each AgOR. Checkered circles represent 80–99% of the maximal response of given AgOR. Horizontally striped circles represent 60–79% of the maximal response of given AgOR. Vertically striped circles represent 40–59% of the maximal response of given AgOR. Cross-hatched circles represent 20–39% of the maximal response of given AgOR. Odorants are classified into different categories according to their functional groups (aromatics, heterocyclics, esters, ketones, alcohols, and acids). The odorants highlighted in bold were also evaluated in behavioral assays.