Behavioural response of the malaria vector Anopheles gambiae to host plant volatiles and synthetic blends

Abstract

Background: Sugar feeding is critical for survival of malaria vectors and, although discriminative plant feeding previously has been shown to occur in Anopheles gambiae s.s., little is known about the cues mediating attraction to these plants. In this study, we investigated the role of olfaction in An. gambiae discriminative feeding behaviour.

Methods: Dual choice olfactometer assays were used to study odour discrimination by An. gambiae to three suspected host plants: Parthenium hysterophorus (Asteraceae), Bidens pilosa (Asteraceae) and Ricinus communis (Euphorbiaceae). Sugar content of the three plant species was determined by analysis of their trimethylsilyl derivatives by coupled gas chromatography-mass spectrometry (GC-MS) and confirmed with authentic standards. Volatiles from intact plants of the three species were collected on Super Q and analyzed by coupled GC-electroantennographic detection (GC-EAD) and GC-MS to identify electrophysiologically-active components whose identities were also confirmed with authentic standards. Active compounds and blends were formulated using dose-response olfactory bioassays. Responses of females were converted into preference indices and analyzed by chi-square tests. The amounts of common behaviourally-active components released by the three host plants were compared with one-way ANOVA.

Results: Overall, the sugar contents were similar in the two Asteraceae plants, P. hysterophorus and B. pilosa, but richer in R. communis. Odours released by P. hysterophorus were the most attractive, with those from B. pilosa being the least attractive to females in the olfactometer assays. Six EAD-active components identified were consistently detected by the antennae of adult females. The amounts of common antennally-active components released varied with the host plant, with the highest amounts released by P. hysterophorus. In dose-response assays, single compounds and blends of these components were attractive to females but to varying levels, with one of the blends recording a significantly attractive response from females when compared to volatiles released by either the most preferred plant, P. hysterophorus (χ2 = 5.23, df = 1, P < 0.05) or as a synthetic blend mimicking that released by P. hysterophorus.

Conclusions: Our results demonstrate that (a) a specific group of plant odours attract female An. gambiae (b) females use both qualitative and quantitative differences in volatile composition to associate and discriminate between different host plants, and (c) altering concentrations of individual EAD-active components in a blend provides a practical direction for developing effective plant-based lures for malaria vector management.

Figure 1

A schematic drawing of the dual choice olfactometer (not drawn to scale). X and Y are the glass chambers that hold intact plant while the broken arrows points to the direction of air flow. Air currents were drawn bidirectionally through the central chamber by applying a vacuum in the center of the chamber as shown in the figure. The tapering ends are made of aluminum sheet while the main olfactometer chamber is made of glass perspex.

Figure 2

Olfactometer responses of An. gambiae to odour of intact plants. A) intact plant odours versus blank control; B) plant odours from different species expressed as Preference Index (PI) ± SEM. Positive response indicates preference for the first odour source. The asterisks indicate the significance levels with * = significant at 0.05, and ** = significant at 0.01.

Figure 3

Coupled GC-electroantennographic responses of An. gambiae to volatiles of the three host plant species. A) P. hysterophorus; B) R. communis; and C) B. pilosa. The EAD-active compounds include hexanal (1), β-pinene (2), D-limonene (3), (Z)- β-ocimene (4), (E)- β-ocimene (5), (Z)-linalool oxide (6), (E)-linalool oxide (7) and (E)- β-farnesene (8) with their corresponding antennal response labelled as x.

Figure 4

Relative amounts of EAD-active components in volatiles of the three species. P. hysterophorus; R. communis and B. pilosa expressed as mean ± SEM. Bars capped with different letters are significantly different between the three plant species. The asterisks indicate the significance levels with * = significant at 0.05, and *** = significant at 0.001.

Figure 5

Olfactometric response of An. gambiae to synthetic compounds of EAD-active components. A) Individual EAD-active volatile components at different concentrations against solvent and B) intact P. hysterophorus volatiles against optimal attractive doses of EAD-active volatile components expressed as PI ± SEM. Positive response indicate preference for the first odour source. The asterisks indicate the significance levels with * = significant at 0.05, and ** = significant at 0.01.

Figure 6

Olfactometric responses of An. gambiae to synthetic blend of EAD-active volatile components against pentane and intact P. hysterophorus expressed as mean PI ± SEM. Positive PI indicates preference for the first odour source. The asterisks indicate the significance levels with * = significant at 0.05, and ** = significant at 0.01.