Pheromones and other semiochemicals play a crucial role in today's integrated pest and vector management strategies. These semiochemicals are typically discovered by bioassay-guided approaches. Here, we applied a reverse chemical ecology approach; that is, we used olfactory proteins to lead us to putative semiochemicals. Specifically, we used 7 of the top 10 odorant receptors (ORs) most expressed in the antennae of the southern house mosquito, Culex quinquefasciatus, and which are yet to be deorphanized. We expressed these receptors in the Xenopus oocyte recording system and challenged them with a panel of 230 odorants, including physiologically and behaviorally active compounds. Six of the ORs were silent either because they are not functional or a key odorant was missing. CquiOR36, which showed the highest transcript levels of all OR genes in female antennae, was also silent to all odorants in the tested panel, but yielded robust responses when it was accidentally challenged with an old sample of nonanal in ethanol. After confirming that fresh samples were inactive and through a careful investigation of all possible "contaminants" in the old nonanal samples, we identified the active ligand as acetaldehyde. That acetaldehyde is activating CquiOR36 was further confirmed by electroantennogram recordings from antennae of fruit flies engineered to carry CquiOR36. Antennae of female mosquitoes also responded to acetaldehyde. Cage oviposition and dual-choice assays demonstrated that acetaldehyde is an oviposition attractant in a wide range of concentrations and thus of potential practical applications.southern house mosquito | CquiOR36 | acetaldehyde | odorant receptors | EAG

The World Health Organization (WHO) recommends that people travelling to or living in areas with Zika virus (ZIKV) outbreaks or epidemics adopt prophylactic measures to reduce or eliminate mosquito bites, including the use of insect repellents. It is, however, unknown whether repellents are effective against ZIKV-infected mosquitoes, in part because of the ethical concerns related to exposing a human subject’s arm to infected mosquitoes in the standard arm-in-cage assay. We used a previously developed, human subject-free behavioural assay, which mimics a human subject to evaluate the top two recommended insect repellents. Our measurements showed that DEET provided significantly higher protection than picaridin provided against noninfected, host-seeking females of the southern house mosquito, Culex quinquefasciatus, and the yellow fever mosquito, Aedes aegypti. When tested at lower doses, we observed a significant reduction in DEET-elicited protection against ZIKV-infected yellow fever mosquitoes from old and recent laboratory colonies. The reduction in protection is more likely associated with aging than the virus infection and could be compensated by applying a 5x higher dose of DEET. A substantial protection against ZIKV-infected and old noninfected mosquitoes was achieved with 5% DEET, which corresponds approximately to a 30% dose in the conventional arm-in-cage assays.

Reception of odorants by two main head appendages, antennae and maxillary palps, is essential for insects' survival and reproduction. There is growing evidence in the literature suggesting that the proboscis is also an olfactory appendage and its function as an additional “antenna” has been previously proposed. We surmised that movements of the labrum toward a blood vessel might be chemically oriented and, if so, there should be odorant receptors expressed in the labrum. To test this hypothesis, we first compared by quantitative PCR expression of odorant receptors (OR) from the Southern house mosquito, Culex quinquefasciatus in antennae and proboscis and, subsequently compared OR expression in various proboscis parts. Our data suggested that a receptor for the oviposition attractant, skatole, CquiOR21, was not expressed in proboscis, whereas a receptor for another oviposition attractant, 4EP (4-ethylphenol), CquiOR99, and a receptorf for the insect repellent DEET, CquiOR136, were expressed in the stylet of the proboscis, particularly in the tip of the labrum. In a dual-choice olfactometer, mosquitoes having the stylet coated with nail polish were attracted to 4EP in the same manner as the untreated mosquitoes. By contrast, in an oviposition assay, the stylet-treated mosquitoes did not discriminate 4EP from control oviposition cups, whereas the untreated mosquitoes (as well as mosquitoes having the labella coated) laid significantly more egg rafts in cups treated with 4EP. Ablation experiments confirmed that 4EP was sensed by the labrum where CquiOR99 is highly expressed. Stylet-coated, labella-coated, and untreated mosquitoes laid significantly more egg rafts in skatole-treated cups than in control cups. Likewise, coating of proboscis structures with nail polish had no effect on DEET-mediated oviposition deterrence. In a behavioral arena designed to mimic a human arm, mosquitoes showed significantly reduced probing time when blood was impregnated with 4EP, i.e., they engaged more rapidly in continuous blood feeding as compared to untreated blood. The time of engagement for feeding in skatole-containing blood vs. untreated blood did not differ significantly. Taken together, these data suggest that 4EP reception by the labrum is important not only for oviposition decisions, but also for reducing probing and initiation of blood feeding.

Summary How chemical signals are integrated at the peripheral sensory system of insects is still an enigma. Here we show that when coexpressed with Orco in Xenopus oocytes, an odorant receptor from the southern house mosquito, CquiOR32, generated inward (regular) currents when challenged with cyclohexanone and methyl salicylate, whereas eucalyptol and fenchone elicited inhibitory (upward) currents. Responses of CquiOR32-CquiOrco-expressing oocytes to odorants were reduced in a dose-dependent fashion by coapplication of inhibitors. This intrareceptor inhibition was also manifested in vivo in fruit flies expressing the mosquito receptor CquiOR32, as well in neurons on the antennae of the southern house mosquito. Likewise, an orthologue from the yellow fever mosquito, AaegOR71, showed intrareceptor inhibition in the Xenopus oocyte recording system and corresponding inhibition in antennal neurons. Inhibition was also manifested in mosquito behavior. Blood-seeking females were repelled by methyl salicylate, but repellence was significantly reduced when methyl salicylate was coapplied with eucalyptol.

Anecdotes related to preferential mosquito bites are very common, but to date there is no complete explanation as to why one out of two people systematically receives more mosquito bites than the other when both are equally accessible. Here we tested the hypothesis that two constituents of skin emanations, 6-methyl-5-heptan-2-one (6-MHO) and geranylacetone (GA), are natural repellents and may account for differential attraction in different ratios. We studied skin emanations from two human subjects, confirmed in behavioral assays that female southern house mosquitoes are significantly more attracted to subject A (attractant) than to subject N (non-attractant), and tested their 6-MHO/GA ratios in a dual-choice olfactometer. Although repelling at high doses, 6-MHO/GA mixtures were not active at the levels emitted by human skin. We found, however, differential attraction elicited by the aldehydes in the ratios produced by subjects A and N. When tested in a dose commensurate with the level released from human skin and in the ratio produced by subject A, the aldehyde mixture significantly attracted mosquitoes. By contrast, an aldehyde mixture at the same ratio released by subject N did not attract mosquitoes. We, therefore, hypothesized that aldehydes may play a role in the commonly observed differential attraction.

Insect repellents are widely used to fend off nuisance mosquitoes and, more importantly, to reduce or eliminate mosquito bites in areas where viruses and other vector-borne diseases are circulating. Synthesized more than six decades ago, DEET is the most widely used insect repellent. Plant-derived compounds are used in a plethora of commercial formulations and natural recipes to repel mosquitoes. They are also used as fragrances. We analysed Bombshell® to identify the constituent(s) eliciting a previously reported “off- label” repellence activity. The two major fragrance ingredients in Bombshell, i.e., methyl dihydrojasmonate and lilial, demonstrated strong repellence against the southern house mosquito, Culex quinquefasciatus, in laboratory assays. Both compounds activated a previously identified DEET-sensitive odorant receptor, CquiOR136. These compounds were also major constituents of Ivanka Trump eau de parfum. The methyl dihydrojasmonate content was higher in the Ivanka Trump perfume than in Bombshell, the reverse being true for lilial. Both Bombshell and Ivanka Trump eaux de parfums retained activity for as long as 6 hours in laboratory assays. Although wearing these perfumes may repel nuisance mosquitoes, their use as “off-label” repellents against infected mosquitoes is not recommended.

Insect repellents are widely used as the first line of defense against mosquito bites and transmission of disease-causing agents. However, the cost of daily applications of even the most affordable and the gold standard of insect repellents, DEET, is still high for low-income populations where repellents are needed the most. An Indian clove-based homemade recipe has been presented as a panacea. We analyzed this homemade repellent and confirmed by behavioral measurements and odorant receptor responses that eugenol is the active ingredient in this formulation. Prepared as advertised, this homemade repellent is ineffective, whereas 5x more concentrated extracts from the brand most enriched in eugenol showed moderate repellency activity against Culex quinquefasciatus and Aedes aegypti. DEET showed higher performance when compared to the 5x concentrated formulation and is available in the same market at a lower price than the cost of the ingredients to prepare the homemade formulation.

THE HISTORY AND BIOLOGICAL MECHANISM OF AFLATOXIN Aflatoxin was first discovered in the United Kingdom in 1960, when many turkeys suddenly died as a result of an unknown "Turkey X Disease", which was later attributed to the consumption of moldy peanuts. Investigation showed that the mold on the peanuts was the toxic A. flavus. There are three classes of aflatoxins. Aflatoxins B and G are produced by A. flavus, while aflatoxin M is a metabolite of aflatoxin B. Aflatoxin M is produced by the liver and stored in the milk of humans and animals. The most prominent type of aflatoxin is aflatoxin B1. After ingestion of aflatoxin-contaminated maize, aflatoxin B1 is converted by hepatic CYP450 enzymes into aflatoxin B1 exo-8,9-epoxide, a more toxic molecule (Schmidt, 2013). Even though CYP450 enzymes are often associated with detoxification, they activate aflatoxin to become an electrophile to generate DNA adducts. Aflatoxin forms DNA adducts by interacting its exo-epoxide with DNA and therefore induces genetic mutation of the tumor suppressor gene p53, thus increasing risk of cancer (Leslie et al., 2008). This is especially true in individuals who are infected by Hepatitis B Virus (HBV) and are chronically exposed to aflatoxin. These individuals are 30-times more likely to contract liver cancer when compared to individuals not infected with HBV (Magnussen and Parsi, 2013).