Our study confirms and expands the evidence that odour cues released from graminoid plants play a role in the orientation of gravid An. gambiae females. Volatiles released from these plants add significant attraction to water vapour alone. Generally, all graminoid plant species tested, including the dry-land ornamental grass, Cenchrus setaceum, usually not associated with mosquito breeding sites, significantly attracted gravid females and behavioural differences in response to different test plants were not very pronounced especially under the more natural, longer-range trapping conditions.
Whilst the behavioural response of gravid An. gambiae mosquitoes appeared to be slightly stronger in reaction to the sedge, Cyperus rotundus, than to most other test plants, we were not able to exactly establish any unique differences in the chemical profiles that might explain this. This is likely, in part, due to the chemical sampling method. To the best of our knowledge, our bioassays are the first to use live plants rather than eluted headspace extracts for testing for attractiveness to gravid malaria vectors. Our aim was to test the behavioural response of gravid females to plant volatiles under as natural conditions as possible. Plant volatiles react differentially with atmospheric oxidants, such as ozone, resulting in odour plumes that do not only include the plant-emitted volatile chemicals but also gradually include a blend of degradation products , which might not be picked up during dynamic headspace sampling with filtered air. We had opted for headspace sampling since it is a non-destructive method for sampling the volatile profile emitted by plants which might consequently be detected by insects . The pooled analyses of our headspace samples suggest that there are variations between the chemical profiles of the different plant species. It is however unclear if these differences would be consistent over time and under different environmental conditions, and if they are responsible for the variations observed in attracting gravid females in the bioassays. Our GC results have been highly variable between replicate plant samples of the same species (supplementary data) with some samples not resulting in any detectable compounds. This is not unexpected, given that we have taken only a ‘snap-shot’ of volatiles released at a particular time point and without carefully standardizing plant age and development. Some volatiles may be emitted in quantities below technical detectability, yet these might be functionally relevant for insect attraction . Volatile organic chemicals emissions and concentrations are also affected by light, temperature, nutritional and soil-moisture conditions, and even by species composition of the neighbouring plant community [63–69]. Abiotic stresses, including stress induced by the air sampling itself when plant material is enclosed in plastic bags will also affect the volatile profile. Going forward, it will be desirable to sample under natural, yet varying environmental conditions and to compare results across different sampling strategies  for a better understanding of the composition and concentration of compounds in the headspace of plants that might affect natural mosquito behaviour.
In our study, and across published work, we see very little variation in the strengths of the behavioural response of gravid mosquitoes to varied graminoid plant species, despite the fact that volatile profiles appear variable. The behavioural response of gravid An. gambiae induced by the wild graminoid plants in our bioassays was in the same ranges as those reported previously for An. arabiensis and An. coluzzi in response to low release rates of headspace extracts from rice plants  and from the tropical African wetland grasses (Poaceae) Echinocloa. pyramidalis, Echinocloa stagnina and Typha latifolia . It was also in a similar range as observed for the attraction of unfed females to plant-based volatiles [58, 60, 70]. A limitation of our study was our inability to access equipment for electro-antennography to determine exactly which volatile chemicals released from the test plants were detected by the gravid female’s antenna. However, when comparing the volatile chemicals identified in our study with those published for rice plants and pollen from sugar cane and maize in the context of oviposition [22, 43, 44], as well as with those published for a range of plants preferentially visited by malaria vectors for sugar feeding [32, 58–60], it becomes apparent that there is significant overlap in the chemical compositions. Compounds reported here, such as limonene, α- and β-pinene, p-cymene, sulcatone, humulene, cedrene, β-myrcene, and β-caryophyllene, have previously been reported to elicit electrophysiological responses in gravid and unfed female Anopheles [22, 43, 44, 57–60] and many of them have been formulated into synthetic blends and shown to be attractive to unfed and gravid Anopheles under highly standardised experimental conditions [22, 43, 58, 71]. These compounds are among the most common VOCs emitted from plants  since they are synthesized through biosynthetic pathways common in most plants [39, 73, 74].
In our study, three volatile chemicals, namely 1,1-dimethyl-3-methylene-2-vinylcyclohexane, α-guaiene and β-elemene, have not been tested previously, yet were detected frequently in four out of the five test plants. It might be useful to explore their potential to manipulate odour-orientation of Anopheles mosquitoes in follow-up studies, since they have been implicated as semiochemicals for other insect species [75–79]. For example, 1,1-dimethyl-3-methylene-2-vinylcyclohexane was attractive to the beech leaf-mining weevil , guaiene has been suggested to play a role in the attraction of the litchi stem-end borer  and β-elemene has been implied to contribute to attraction of the gravid tobacco moths  and the white-spotted longhorn beetle .
Myrcene, γ-elemene, humulene epoxide II and hexahydrofarnesyl acetone were specific to headspace samples of Cyperus rotundus in our analysis. This does, however, not necessarily imply that these compounds contributed to the attractiveness in our bioassays. Information on these compounds as info-chemicals for insects and specifically mosquitoes is scant and none of them have been tested with gravid malaria vectors. Both, unfed Anopheles and unfed Aedes mosquitoes showed electrophysiological activity to β-myrcene in previous studies [60, 70]. It was observed that myrcene elicits an avoidance behaviour in unfed Anopheles gambiae when searching for sugar  or blood meals . γ-Elemene was identified from plant headspace and found to be electrophysiologically active for unfed An. gambiae but behavioural implications were not studied .
Gravid malaria vectors navigate a complex chemical environment in search for oviposition sites. It is plausible to assume that volatile chemical cues emanating from aquatic habitats and their surroundings are only used at relatively short-range, with visual cues and air movements guiding the gravid females’ flight towards a water body [1, 82]. Visual cues will include near-infrared radiation from slowly released heat from water bodies in the evening , polarized light from water surfaces  as well as ultraviolet light , all of which present strong long-range cues likely used by gravid mosquitoes to evaluate the location and quality of potential oviposition sites . In this context, it remains therefore unclear, if attractive, yet common, plant-based semiochemicals in odour-baited traps will be able to compete in an attract-and kill approach, with the complex interaction of cues provided by natural aquatic habitats. To date over 100 semiochemicals have been identified for mosquitoes of all physiological stages, yet synthetic odour-baited traps hardly play any role in contemporary surveillance and control of malaria vector mosquitoes . Synthetic odour-baits mimicking human body odour have shown to perform poorly in attracting host-seeking Anopheles mosquitoes when presented in close vicinity to natural human blood hosts  and field evaluations of the oviposition attractant cedrol, showed that visual cues provided by an open water surface were essential in combination with the chemical cue to attract wild oviposition-site searching females . In order to develop vector control interventions that manipulate the odour-orientation of malaria vectors in their natural environment, less emphasis might be placed in future on detecting more semiochemicals but more emphasis on how to formulate and present these chemicals in combination with other essential cues used by mosquitoes, to improve the efficacy of such interventions .