A. Jennifer Mordue

Identification of Human-Derived Volatile Chemicals that Interfere with Attraction of Aedes aegypti Mosquitoes

It is known that human individuals show different levels of attractiveness to mosquitoes. In this study, we investigated the chemical basis for low attractiveness. We recorded behaviors of Aedes aegypti toward the hands of human volunteers and toward the volatile chemicals produced by their bodies. Some individuals, and their corresponding volatiles, elicited low upwind flight, relative attraction, and probing activity. Analyzing the components by gas chromatography coupled to electrophysiological recordings from the antennae of Aedes aegypti, enabled the location of 33 physiologically relevant compounds. The results indicated that higher levels of specific compounds may be responsible for decreased “attractiveness.” In behavioral experiments, five of the compounds caused a significant reduction in upwind flight of Aedes aegypti to attractive human hands. Thus, unattractiveness of individuals may result from a repellent, or attractant “masking,” mechanism.

Actions of azadirachtin, a plant allelochemical, against insects

Investigations of the antifeedant mode of action of azadirachtin and four synthetic analogues, 22,23-dihydroazadirachtin, 3-tigloylazadirachtol, 11-methoxydihydroazadirachtin and 22,23-bromoethoxydihydroazadirachtin have revealed that both polyphagous and oligophagous insects are behaviourally responsive to azadirachtin, with the most responsive species being able to differentiate extremely small changes in the parent molecule. In Lepidoptera the antifeedant response is correlated also with increased neural activity of the chemoreceptors. When locusts are treated on crop plants, the antifeedant and physiological actions of azadirachtin and analogues work in concert and result in feeding deterrence, growth and moulting aberrations and mortality with the same order of potency as for antifeedancy. Specific binding studies using [3H]dihydroazadirachtin carried out on locust testes and Spodoptera Sf9 cells have shown that the competitive binding of the different analogues of azadirachtin to these binding sites occurs in a similar order of potency to that found with antifeedant and IGR bioassays. This suggests a causal link between specific binding to membrane proteins and the ability of the molecule to exert biological effects.

Repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale, against the maize weevil, Sitophilus zeamais.

The repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale (Zingiberaceae), against the maize weevil, Sitophilus zeamais (Coleoptera: Curculionidae), was investigated in four-way olfactometer bioassays. Results showed that vacuum distilled A. melegueta and Z. officinale extracts were repellent towards adult S. zeamais both in the absence and the presence of maize, Zea mays, grains. Bioassay-guided liquid chromatographic fractionation of the distillates showed that fractions containing oxygenated compounds accounted for the repellent activity. Coupled gas chromatography-mass spectrometry (GC-MS), followed by GC peak enhancement and enantioselective GC using authentic compounds, identified 3 major compounds in the behaviourally active fractions of A. melegueta and Z. officinale to be (S)-2-heptanol, (S)-2-heptyl acetate and (R)-linalool in a ratio of 1:6:3, and 1,8-cineole, neral and geranial in a ratio of 5.48:1:2.13, respectively. The identification of these behaviourally active compounds provides the scientific basis for the observed repellent properties of A. melegueta and Z. officinale, and demonstrates the potential for their use in stored-product protection at the small-scale farmer level in Africa.

Azadirachtin Disrupts Formation of Organised Microtubule Arrays during Microgametogenesis of Plasmodium berghei

Abstract Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of asexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.

Arm-in-cage testing of natural human-derived mosquito repellents

BackgroundIndividual human subjects are differentially attractive to mosquitoes and other biting insects. Previous investigations have demonstrated that this can be attributed partly to enhanced production of natural repellent chemicals by those individuals that attract few mosquitoes in the laboratory. The most important compounds in this respect include three aldehydes, octanal, nonanal and decanal, and two ketones, 6-methyl-5-hepten-2-one and geranylacetone [(E)-6,10-dimethylundeca-5,9-dien-2-one]. In olfactometer trials, these compounds interfered with attraction of mosquitoes to a host and consequently show promise as novel mosquito repellents.MethodsTo test whether these chemicals could provide protection against mosquitoes, laboratory repellency trials were carried out to test the chemicals individually at different concentrations and in different mixtures and ratios with three major disease vectors: Anopheles gambiae, Culex quinquefasciatus and Aedes aegypti.ResultsUp to 100% repellency was achieved depending on the type of repellent compound tested, the concentration and the relative composition of the mixture. The greatest effect was observed by mixing together two compounds, 6-methyl-5-hepten-2-one and geranylacetone in a 1:1 ratio. This mixture exceeded the repellency of DEET when presented at low concentrations. The repellent effect of this mixture was maintained over several hours. Altering the ratio of these compounds significantly affected the behavioural response of the mosquitoes, providing evidence for the ability of mosquitoes to detect and respond to specific mixtures and ratios of natural repellent compounds that are associated with host location.ConclusionThe optimum mixture of 6-methyl-5-hepten-2-one and geranylacetone was a 1:1 ratio and this provided the most effective protection against all species of mosquito tested. With further improvements in formulation, selected blends of these compounds have the potential to be exploited and developed as human-derived novel repellents for personal protection.

Identification of Human-Derived Volatile Chemicals That Interfere With Attraction of the Scottish Biting Midge and Their Potential Use as Repellents

ABSTRACT The Scottish biting midge, Culicoides impunctatus (Diptera: Ceratopogonidae), is a major pest in Scotland, causing a significant impact to the Scottish tourist and forestry industries. C. impunctatus is a generalist feeder, preferring to feed on large mammals, and is notorious for its attacks on humans. Until now, there was anecdotal evidence for differential attraction of female host-seeking C. impunctatus to individual human hosts, and the mechanism for this phenomenon was unknown. Using extracts of human odor collected by air entrainment, electroantennogram recordings to identify the physiologically active components, followed by behavioral assays, we show, for the first time, the differential attraction of female C. impunctatus to human odors and the chemical basis for this phenomenon. Certain chemicals, found in greater amounts in extracts that cause low attractiveness to midges, elicit a repellent effect in laboratory assays and repellency trials in the field. Differences in the production of these natural human-derived compounds could help to explain differential “attractiveness” between different human hosts. A mixture of two compounds in particular, 6-methyl-5-hepten-2-one and geranylacetone [(E)-6,10-dimethylundeca-5,9-dien-2-one], showed significant repellency (87, 77.4, 74.2, and 31.6% at hours 0, 1, 2, and 3, respectively) in the field and have the potential to be developed as novel repellents.

Laboratory and field responses of the mosquito, Culex quinquefasciatus, to plant-derived Culex spp. oviposition pheromone and the oviposition cue skatole.

Laboratory and field studies were conducted on the oviposition behavior of the pathogen-vectoring mosquito, Culex quinquefasciatus, in response to the oviposition pheromone 6-acetoxy-5-hexadecanolide, produced from a renewable plant resource, Kochia scoparia (Chenopodiaceae) (plant-derived pheromone, PDP), and via an established synthetic route (synthetic oviposition pheromone, SOP). Responses to the oviposition cue skatole (3-methylindole), presented individually and in combination with the plant-derived and synthetic oviposition pheromone, were also studied. Both laboratory and field assays showed that PDP and SOP were equally attractive. Synergistic effects were observed with one combination of PDP and skatole combinations in laboratory assays. Synergy was also observed under field conditions. SOP and skatole combinations showed additive effects in laboratory assays, but were not tested in field bioassays. Although synergism has been previously demonstrated with combinations of SOP and polluted waters, the work presented here is the first example of synergy between a specific oviposition attractant and the oviposition pheromone. Furthermore, the efficacy of mosquito pheromone produced from a cheap, renewable botanical source has been demonstrated.

Behavioural responses of the maize weevil, Sitophilus zeamais, to host (stored-grain) and non-host plant volatiles

BACKGROUND Four-arm olfactometer bioassays were conducted to assess the behavioural responses of the adult maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae), to harvested seeds of host plants, i.e. white maize, yellow maize (Zea mays L.) and winter wheat (Triticum aestivum L.) (Poaceae), and non-host plant materials, i.e. alligator pepper, Aframomum melegueta (Rosk) K. Schum (Zingiberaceae), rhizomes of ginger, Zingiber officinale (Roscoe) (Zingiberaceae), and West African black pepper, Piper guineense Thonn and Schum (Piperaceae). Additional bioassays with host plant volatiles were conducted in the presence of three doses of non-host plant materials. RESULTS Both sexes of the weevil showed strong attraction to maize and wheat seed volatiles, but were significantly repelled (P < 0.001) by odours from A. melegueta, Z. officinale and P. guineense. Furthermore, S. zeamais avoided maize and wheat seeds presented in combination with the non-host plant material at 10% (w/w) and 33% (w/w) levels. CONCLUSIONS A. melegueta, Z. officinale and P. guineense have the potential for use in the protection of stored grains by resource-poor farmers with local access to these plants.

Role of Semiochemicals in Mate Location by Parasitic Sea Louse, Lepeophtheirus salmonis

The role of olfaction and diffusible pheromones in mate location behavior of sea lice, Lepeophtheirus salmonis, was assessed with Y-tube behavioral bioassays. The pheromone “emitting” animals were located in a chamber in one arm of a Y-tube arena, with artificial seawater flowing through both arms. Adult male sea lice displayed both activation and directional responses to seawater conditioned with preadult II virgin females, but were only activated by mated adult female conditioned water. Further, when males were given the choice of preadult II virgin females or mated adult females, a significant number of males chose the arm with the preadult II virgin females. Adult males showed activation responses when presented with water conditioned with adult males but were not attracted to them. When presented with adult males, preadult II virgin females showed only directional responses, but not activation responses. Preadult II virgin female conditioned water was extracted using solid-phase extraction (SPE) protocols pioneered for semiochemical isolation. Adult male sea lice showed significant directional responses to the preadult II virgin female SPE extract. Distillation under vacuum was performed on the extract to give a distillate comprising components with a molecular weight range and physical properties comparable to those of compounds utilized as volatile semiochemicals by terrestrial organisms and a residue comprising components with higher molecular weight range comparable to those utilized as involatile semiochemicals. Adult males were found to be both significantly activated and attracted to the distillate, but not to the residue. This research provides evidence that small, lipophilic organic molecules are used by sea lice as sex pheromone signals to locate a member of the opposite sex.

Antifeedant effects of in vitro culture extracts of the neem tree, Azadirachta indica against the desert locust (Schistocerca gregaria (Forskål))

Callus and micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. A variety of whole plant and in vitro cell cultures from neem seedlings of Ghanian origin were tested for insect antifeedant compounds using the desert locust (Schistocerca gregaria (Forskål)). Feeding suppression occurred when whole extracts of seed, leaf, callus, suspension and shoot cultures were tested in no-choice feeding bioassays. Controls of sucrose, carrot callus and the plant growth medium showed no feeding deterrence. Azadirachtin, the main known antifeedant in neem seed kernels, was quantified from a seed extract by HPLC but was not detected in any of the other extracts. Antifeedancy was determined during batch growth of a suspension culture which had been in culture for 5 months; results indicated that antifeedants were still being formed and that levels increased after maximum biomass was attained.