Zainulabeuddin Syed

Mosquitoes smell and avoid the insect repellent DEET

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.

Acute olfactory response of Culex mosquitoes to a human- and bird-derived attractant

West Nile virus, which is transmitted by Culex mosquitoes while feeding on birds and humans, has emerged as the dominant vector borne disease in North America. We have identified natural compounds from humans and birds, which are detected with extreme sensitivity by olfactory receptor neurons (ORNs) on the antennae of Culex pipiens quinquefasciatus (Cx. quinquefasciatus). One of these semiochemicals, nonanal, dominates the odorant spectrum of pigeons, chickens, and humans from various ethnic backgrounds. We determined the specificity and sensitivity of all ORN types housed in different sensilla types on Cx. quinquefasciatus antennae. Here, we present a comprehensive map of all antennal ORNs coding natural ligands and their dose-response functions. Nonanal is detected by a large array of sensilla and is by far the most potent stimulus; thus, supporting the assumption that Cx. quinquefasciatus can smell humans and birds. Nonanal and CO2 synergize, thus, leading to significantly higher catches of Culex mosquitoes in traps baited with binary than in those with individual lures.

Pheromone reception in fruit flies expressing a moth's odorant receptor

We have expressed a male-specific, pheromone-sensitive odorant receptor (OR), BmorOR1, from the silkworm moth Bombyx mori in an “empty neuron” housed in the ab3 sensilla of a Drosophila Δhalo mutant. Single-sensillum recordings showed that the BmorOR1-expressing neurons in the transgenic flies responded to the B. mori pheromone bombykol, albeit with low sensitivity. These transgenic flies responded to lower doses of bombykol in an altered stimulation method with direct delivery of pheromone into the sensillum milieu. We also expressed a B. mori pheromone-binding protein, BmorPBP, in the BmorOR1-expressing ab3 sensilla. Despite the low levels of BmorPBP expression, flies carrying both BmorOR1 and BmorPBP showed significantly higher electrophysiological responses than BmorOR1 flies. Both types of BmorOR1-expressing flies responded to bombykol, and to a lesser extent to a second compound, bombykal, even without the addition of organic solvents to the recording electrode buffer. When the semiochemicals were delivered by the conventional puffing of stimulus on the antennae, the receptor responded to bombykol but not to bombykal. The onset of response was remarkably slow, and neural activity extended for an unusually long time (>1 min) after the end of stimulus delivery. We hypothesize that BmorOR1-expressing ab3 sensilla lack a pheromone-degrading enzyme to rapidly inactivate bombykol and terminate the signal. We also found an endogenous receptor in one of the sensillum types on Drosophila antenna that responds to bombykol and bombykal with sensitivity comparable to the pheromone-detecting sensilla on B. mori male antennae.

Knockdown of a Mosquito Odorant-binding Protein Involved in the Sensitive Detection of Oviposition Attractants

Odorant-binding proteins (OBPs) were discovered almost three decades ago, but there is still considerable debate regarding their role(s) in insect olfaction, particularly due to our inability to knockdown OBPs and demonstrate their direct phenotypic effects. By using RNA interference (RNAi), we reduced transcription of a major OBP gene, CquiOBP1, in the antennae of the Southern house mosquito, Culex quinquefasciatus. Previously, we had demonstrated that the mosquito oviposition pheromone (MOP) binds to CquiOBP1, which is expressed in MOP-sensitive sensilla. Antennae of RNAi-treated mosquitoes showed significantly lower electrophysiological responses to known mosquito oviposition attractants than the antennae of water-injected, control mosquitoes. While electroantennogram (EAG) responses to MOP, skatole, and indole were reduced in the knockdowns, there was no significant difference in the EAG responses from RNAi-treated and water-injected mosquito antennae to nonanal at all doses tested. These data suggest that CquiOBP1 is involved in the reception of some oviposition attractants, and that high levels of OBPs expression are essential for the sensitivity of the insect’s olfactory system.

Reverse and Conventional Chemical Ecology Approaches for the Development of Oviposition Attractants for Culex Mosquitoes

Synthetic mosquito oviposition attractants are sorely needed for surveillance and control programs for Culex species, which are major vectors of pathogens causing various human diseases, including filariasis, encephalitis, and West Nile encephalomyelitis. We employed novel and conventional chemical ecology approaches to identify potential attractants, which were demonstrated in field tests to be effective for monitoring populations of Cx. p. quinquefasciatus in human dwellings. Immunohistochemistry studies showed that an odorant-binding protein from this species, CquiOBP1, is expressed in trichoid sensilla on the antennae, including short, sharp-tipped trichoid sensilla type, which house an olfactory receptor neuron sensitive to a previously identified mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide. CquiOBP1 exists in monomeric and dimeric forms. Monomeric CquiOBP1 bound MOP in a pH-dependent manner, with a change in secondary structure apparently related to the loss of binding at low pH. The pheromone antipode showed higher affinity than the natural stereoisomer. By using both CquiOBP1 as a molecular target in binding assays and gas chromatography-electroantennographic detection (GC-EAD), we identified nonanal, trimethylamine (TMA), and skatole as test compounds. Extensive field evaluations in Recife, Brazil, a region with high populations of Cx. p. quinquefasciatus, showed that a combination of TMA (0.9 µg/l) and nonanal (0.15 ng/µl) is equivalent in attraction to the currently used infusion-based lure, and superior in that the offensive smell of infusions was eliminated in the newly developed synthetic mixture.

Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae

We recently characterized 24-hr daily rhythmic patterns of gene expression in Anopheles gambiae mosquitoes. These include numerous odorant binding proteins (OBPs), soluble odorant carrying proteins enriched in olfactory organs. Here we demonstrate that multiple rhythmically expressed genes including OBPs and takeout proteins, involved in regulating blood feeding behavior, have corresponding rhythmic protein levels as measured by quantitative proteomics. This includes AgamOBP1, previously shown as important to An. gambiae odorant sensing. Further, electrophysiological investigations demonstrate time-of-day specific differences in olfactory sensitivity of antennae to major host-derived odorants. The pre-dusk/dusk peaks in OBPs and takeout gene expression correspond with peak protein abundance at night, and in turn coincide with the time of increased olfactory sensitivity to odorants requiring OBPs and times of increased blood-feeding behavior. This suggests an important role for OBPs in modulating temporal changes in odorant sensitivity, enabling the olfactory system to coordinate with the circadian niche of An. gambiae.

Chemical Ecology of Animal and Human Pathogen Vectors in a Changing Global Climate

Infectious diseases affecting livestock and human health that involve vector-borne pathogens are a global problem, unrestricted by borders or boundaries, which may be exacerbated by changing global climate. Thus, the availability of effective tools for control of pathogen vectors is of the utmost importance. The aim of this article is to review, selectively, current knowledge of the chemical ecology of pathogen vectors that affect livestock and human health in the developed and developing world, based on key note lectures presented in a symposium on “The Chemical Ecology of Disease Vectors” at the 25th Annual ISCE meeting in Neuchatel, Switzerland. The focus is on the deployment of semiochemicals for monitoring and control strategies, and discusses briefly future directions that such research should proceed along, bearing in mind the environmental challenges associated with climate change that we will face during the 21st century.

Bombykol receptors in the silkworm moth and the fruit fly

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the “empty neuron” system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor.

Volatile codes: Correlation of olfactory signals and reception in Drosophila-yeast chemical communication.

Drosophila have evolved strong mutualistic associations with yeast communities that best support their growth and survival, resulting in the development of novel niches. It has been suggested that flies recognize their cognate yeasts primarily based on the rich repertoire of volatile organic compounds (VOCs) derived from the yeasts. Thus, it remained an exciting avenue to study whether fly spp. detect and discriminate yeast strains based on odor alone, and if so, how such resolution is achieved by the olfactory system in flies. We used two fly species known to exploit different niches and harboring different yeasts, D. suzukii (a pest of fresh fruit) and D. melanogaster (a saprophytic fly and a neurogenetic model organism). We initially established the behavioral preference of both fly species to six Drosophila-associated yeasts; then chemically analyzed the VOC profile of each yeast which revealed quantitative and qualitative differences; and finally isolated and identified the physiologically active constituents from yeast VOCs for each drosophilid that potentially define attraction. By employing chemical, behavioral, and electrophysiological analyses, we provide a comprehensive portrait of the olfactory neuroethological correlates underlying fly-yeast coadaptation in two drosophilids with distinct habitats.

Pheromone Binding to General Odorant-binding Proteins from the Navel Orangeworm

General odorant-binding proteins (GOBPs) of moths are postulated to be involved in the reception of semiochemicals other than sex pheromones, the so-called “general odorants.” We have expressed two GOBPs, AtraGOBP1 and AtraGOBP2, which were previously isolated from the antennae of the navel orangeworm, Amyelois transitella. Surprisingly, these two proteins did not bind compounds that are known to attract adult moths, particularly females. The proper folding and functionality of the recombinant proteins was inferred from circular dichroism analysis and demonstration that both GOBPs bound nonanal in a pH-dependent manner. EAG experiments demonstrated that female attractants (1-phenylethanol, propionic acid phenyl ester, and isobutyric acid phenyl ester) are detected with high sensitivity by the antennae of day-0 to day-4 adult females, with response declining in older moths. The same age-dependence was shown for male antennae responding to constituents of the sex pheromone. Interestingly, AtraGOBP2 bound the major constituent of the sex pheromone, Z11Z13-16Ald, with affinity comparable to that shown by a pheromone-binding protein, AtraPBP1. The related alcohol bound to AtraPBP1 with higher affinity than to AtraGOBP2. AtraGOBP1 bound both ligands with low but nearly the same affinity.