Olfactory responses of the antennal trichoid sensilla to chemical repellents in the mosquito, Culex quinquefasciatus

Insect repellents are widely used to protect against insect bites and thus prevent allergic reaction and the spread of disease. To gain insight into the mosquito’s response to chemicals repellents, we investigated the interaction between the olfactory system of the mosquito Culex quinquefasciatus Say and chemical repellents using single sensillum recording. The interactions of 50 repellent chemicals with olfactory receptor neurons were measured in six different types of mosquito sensilla: long sharp trichoid (LST), short sharp trichoid (SST), short blunt trichoid I (SBT-I), short blunt trichoid II (SBT-II), short blunt trichoid-curved (SBT-C), and grooved peg (GP). A single olfactory neuron reacted to the chemical repellents in each of the sensilla except for SBT-I and SBT-II, where two neurons were involved. Other than LST and GP, which showed no or very weak responses to the repellents tested, all the sensilla showed significant excitatory responses to certain types of repellents. Terpene-derived chemicals such as eucalyptol, α-pinene, and camphor, stimulated olfactory receptor neurons in a dose-dependent manner and mosquitoes responded more strongly to terpene-derived chemical repellents than to non-terpene-derived chemicals such as dimethyl phthalate. Mosquitoes also exhibited a similar response to stereoisomers of chemicals such as (−)-β-pinene versus (+)-β-pinene, and (−)-menthone versus (+)-menthone. This study not only demonstrates the effects of chemical repellents on the mosquito olfactory system but also provides important information that will assist those screening new mosquito repellents and designing new mosquito control agents.     

Gustatory receptor expression in the labella and tarsi of Aedes aegypti

The yellow-fever mosquito, Aedes aegypti, infects a growing number of people every year with dengue, yellow fever and chikungunya viruses. Contact chemoreception in mosquitoes influences a number of behaviors including host-selection, oviposition and feeding. While these behaviors are in many instances well documented, the molecular mechanisms mediating them are not well understood. Here we report the results of sequencing total messenger RNA in the labella and tarsi of both male and female Ae. aegypti to reveal Gustatory Receptor (GR) gene expression profiles in these major gustatory appendages. Gene expression levels in each tissue were verified by RT-qPCR. We discuss potential functions for the GRs revealed here by considering homologous GRs in other insects. Specific GRs provide molecular targets for modification of gustatory-mediated behaviors in this important disease vector.     

Functional characterization of the octenol receptor neuron on the maxillary palps of the yellow fever mosquito, Aedes aegypti

Background

1-Octen-3-ol (octenol) is a common attractant released by vertebrates which in combination with carbon dioxide (CO₂) attracts hematophagous arthropods including mosquitoes. A receptor neuron contained within basiconic sensilla on the maxillary palps of adult mosquitoes responds selectively to 1-octen-3-ol. Recently, an odorant receptor (AaegOR8) known to occur on the maxillary palps was expressed in a heterologous system and demonstrated to be selectively sensitive to (R)-(−)-1-octen-3-ol, one of two enantiomeric forms. Lesser responses were elicited by stimulation with the (S)-enantiomer and various structural analogs.

Methodology/Principal Findings

Here we characterize the specificity of the octenol receptor neuron in the yellow fever mosquito, Aedes aegypti (L.), in vivo using single cell recordings. The octenol neuron is exquisitely sensitive to (R)-(−)-1-octen-3-ol; comparable responses to (S)-(+)-1-octen-3-ol were elicited only at stimulus doses over 100× that required for the (R)-enantiomer. An intermediate response closer to that elicited by the (R)-(−)-enantiomer was elicited by racemic 1-octen-3-ol. Small structural changes in (R)-(−)-1-octen-3-ol resulted in large decreases in responses. Increases in spike activity were also elicited in the octenol neuron by 2-undecanone, a known repellent; other repellents (DEET, IR3535 and picaridin) were inactive.

Conclusions/Significance

The results of our electrophysiological studies of the octenol receptor neuron in vivo approximates results of a previous study of the octenol receptor (AaegOR8 with its obligate partner Aaeg\ORco) expressed heterologously in Xenopus oocytes. By comparison of our current results with those of the heterologous expression study, we conclude that specificity of the octenol receptor neuron can be explained largely by characteristics of the OR alone without other associated proteins present in vivo. Our findings show that repellents may have specific stimulatory effects on receptor neurons and support the notion of repellents as modulators of mosquito odorant receptor activity.     

Generic insect repellent detector from the fruit fly Drosophila melanogaster

Background

Insect repellents are prophylactic tools against a number of vector-borne diseases. There is growing demand for repellents outperforming DEET in cost and safety, but with the current technologies R&D of a new product takes almost 10 years, with a prohibitive cost of $30 million dollar in part due to the demand for large-scale synthesis of thousands of test compounds of which only 1 may reach the market. R&D could be expedited and cost dramatically reduced with a molecular/physiological target to streamline putative repellents for final efficacy and toxicological tests.

Methodology

Using olfactory-based choice assay we show here that the fruit fly is repelled by not only DEET, but also IR3535 and picaridin thus suggesting they might have “generic repellent detector(s),” which may be of practical applications in new repellent screenings. We performed single unit recordings from all olfactory sensilla in the antennae and maxillary palps. Although the ab3A neuron in the wild type flies responded to picaridin, it was unresponsive to DEET and IR3535. By contrast, a neuron housed in the palp basiconic sensilla pb1 responded to DEET, IR3535, and picaridin, with apparent sensitivity higher than that of the DEET detectors in the mosquitoes Culex quinquefasciatus and Aedes aegypti. DmOr42a was transplanted from pb1 to the “empty neuron” and showed to be sensitive to the three insect repellents.

Conclusions

For the first time we have demonstrated that the fruit fly avoids not only DEET but also IR3535 and picaridin, and identified an olfactory receptor neuron (ORN), which is sensitive to these three major insect repellents. We have also identified the insect repellent-sensitive receptor, DmOr42a. This generic detector fulfils the requirements for a simplified bioassay for early screening of test insect repellents.     

Efficiency Evaluation of Nozawa-Style Black Light Trap for Control of Anopheline Mosquitoes

House-residual spraying and insecticide-treated bed nets have achieved some success in controlling anthropophilic and endophagic vectors. However, these methods have relatively low efficacy in Korea because Anopheles sinensis, the primary malaria vector, is highly zoophilic and exophilic. So, we focused our vector control efforts within livestock enclosures using ultraviolet black light traps as a mechanical control measure. We found that black light traps captured significantly more mosquitoes at 2 and 2.5 m above the ground (P < 0.05). We also evaluated the effectiveness of trap spacing within the livestock enclosure. In general, traps spaced between 4 and 7 m apart captured mosquitoes more efficiently than those spaced closer together (P > 0.05). Based on these findings, we concluded that each black light trap in the livestock enclosures killed 7,586 female mosquitoes per trap per night during the peak mosquito season (July-August). In May-August 2003, additional concurrent field trials were conducted in Ganghwa county. We got 74.9% reduction (P < 0.05) of An. sinensis in human dwellings and 61.5% reduction (P > 0.05) in the livestock enclosures. The black light trap operation in the livestock enclosures proved to be an effective control method and should be incorporated into existing control strategies in developed countries.     

Malaria Vector Surveillance in Ganghwa-do, a Malaria-Endemic Area in the Republic of Korea

We investigated the seasonality of Anopheles mosquitoes, including its species composition, density, parity, and population densities of mosquitoes infected with the parasite in Ganghwa-do (Island), a vivax malaria endemic area in the Republic of Korea. Mosquitoes were collected periodically with a dry-ice-tent trap and a blacklight trap during the mosquito season (April-October) in 2008. Anopheles sinensis (94.9%) was the most abundant species collected, followed by Anopheles belenrae (3.8%), Anopheles pullus (1.2%), and Anopheles lesteri (0.1%). Hibernating Anopheles mosquitoes were also collected from December 2007 to March 2008. An. pullus (72.1%) was the most frequently collected, followed by An. sinensis (18.4%) and An. belenrae (9.5%). The composition of Anopheles species differed between the mosquito season and hibernation seasons. The parous rate fluctuated from 0% to 92.9%, and the highest rate was recorded on 10 September 2008. Sporozoite infections were detected by PCR in the head and thorax of female Anopheles mosquitoes. The annual sporozoite rate of mosquitoes was 0.11% (2 of 1,845 mosquitoes). The 2 mosquitoes that tested positive for sporozoites were An. sinensis. Malarial infections in anopheline mosquitoes from a population pool were also tried irrespective of the mosquito species. Nine of 2,331 pools of Anopheles mosquitoes were positive. From our study, it can be concluded that An. sinensis, which was the predominant vector species and confirmed as sporozoite-infected, plays an important role in malaria transmission in Ganghwa-do.     

Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects’ genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.     

Genetic variation in the hTAS2R38 taste receptor and food consumption among Finnish adults

Genetic variation in bitter taste receptors, such as hTAS2R38, may affect food preferences and intake. The aim of the present study was to investigate the association between bitter taste receptor haplotypes and the consumption of vegetables, fruits, berries and sweet foods among an adult Finnish population. A cross-sectional design utilizing data from the Cardiovascular Risk in Young Finns cohort from 2007, which consisted of 1,903 men and women who were 30–45 years of age from five different regions in Finland, was employed. DNA was extracted from blood samples, and hTAS2R38 polymorphisms were determined based on three SNPs (rs713598, rs1726866 and rs10246939). Food consumption was assessed with a validated food frequency questionnaire. The prevalence of the bitter taste-sensitive (PAV/PAV) haplotype was 11.3 % and that of the insensitive (AVI/AVI) haplotype was 39.5 % among this Finnish population. PAV homozygotic women consumed fewer vegetables than did the AVI homozygotic women, 269 g/day (SD 131) versus 301 g/day (SD 187), respectively, p = 0.03 (multivariate ANOVA). Furthermore, the intake of sweet foods was higher among the PAV homozygotes of both genders. Fruit and berry consumption did not differ significantly between the haplotypes in either gender. Individuals perceive foods differently, and this may influence their patterns of food consumption. This study showed that the hTAS2R38 taste receptor gene variation was associated with vegetable and sweet food consumption among adults in a Finnish population.     

蚊虫嗅觉识别的神经机制

蚊虫主要依赖嗅觉系统与外界环境进行化学信息交流。蚊虫通过嗅觉感受系统寻找食物、 配偶和产卵场所, 进而做出相应的行为反应。本文综述了近年来蚊虫嗅觉系统对气味信号神经传导机制的研究进展。蚊虫的嗅觉感器主要位于触角和下颚须, 触角上的毛形感器和锥形感器感受氨水、 乳酸、 羧酸类化合物等人体和其他动物释放的微量气味物质, 下颚须上的锥形感器则感受呼出的二氧化碳以及一些其他的挥发性物质; 蚊虫嗅觉感器内部有受体神经细胞, 其上分布有嗅觉受体蛋白, 蚊虫对外界环境的化学感受就是通过气味物质与这些受体蛋白互作而得以实现; 根据对不同气味物质的反应谱差异, 嗅觉神经细胞被分为不同的功能类型; 来自嗅觉神经细胞的神经信号进一步从外周传导至中枢神经中脑触角叶内的神经小球, 在此对信息进行初步的处理, 通过评估嗅觉神经细胞的反应和触角叶内的神经小球相应被激活的区域, 不同小球被分别命名; 最后, 神经信号继续整合, 由投射神经传向前脑, 最终引发一系列昆虫行为反应。这些研究从理论上剖析了气味信号在蚊虫嗅觉系统中的神经转导通路, 对于我们深刻理解蚊虫的嗅觉系统具有重要意义, 同时也有助于进一步理解其他昆虫甚至人类的气味识别机制及进行更深层次神经科学的探索。     

Functional Analyses of Bitter Taste Receptors in Domestic Cats (Felis catus)

Cats are obligate carnivores and under most circumstances eat only animal products. Owing to the pseudogenization of one of two subunits of the sweet receptor gene, they are indifferent to sweeteners, presumably having no need to detect plant-based sugars in their diet. Following this reasoning and a recent report of a positive correlation between the proportion of dietary plants and the number of Tas2r (bitter receptor) genes in vertebrate species, we tested the hypothesis that if bitter perception exists primarily to protect animals from poisonous plant compounds, the genome of the domestic cat (Felis catus) should have lost functional bitter receptors and they should also have reduced bitter receptor function. To test functionality of cat bitter receptors, we expressed cat Tas2R receptors in cell-based assays. We found that they have at least 7 functional receptors with distinct receptive ranges, showing many similarities, along with some differences, with human bitter receptors. To provide a comparative perspective, we compared the cat repertoire of intact receptors with those of a restricted number of members of the order Carnivora, with a range of dietary habits as reported in the literature. The numbers of functional bitter receptors in the terrestrial Carnivora we examined, including omnivorous and herbivorous species, were roughly comparable to that of cats thereby providing no strong support for the hypothesis that a strict meat diet influences bitter receptor number or function. Maintenance of bitter receptor function in terrestrial obligate carnivores may be due to the presence of bitter compounds in vertebrate and invertebrate prey, to the necessary role these receptors play in non-oral perception, or to other unknown factors. We also found that the two aquatic Carnivora species examined had fewer intact bitter receptors. Further comparative studies of factors driving numbers and functions of bitter taste receptors will aid in understanding the forces shaping their repertoire.     

Bitter taste perception in Neanderthals through the analysis of the TAS2R38 gene

The bitter taste perception (associated with the ability or inability to taste phenylthiocarbamide) is mediated by the TAS2R38 gene. Most of the variation in this gene is explained by three common amino-acid polymorphisms at positions 49 (encoding proline or alanine), 262 (alanine or valine) and 296 (valine or isoleucine) that determine two common isoforms: proline–alanine–valine (PAV) and alanine–valine–isoleucine (AVI). PAV is the major taster haplotype (heterozygote and homozygote) and AVI is the major non-taster haplotype (homozygote). Amino acid 49 has the major effect on the distinction between tasters and non-tasters of all three variants. The sense of bitter taste protects us from ingesting toxic substances, present in some vegetables, that can affect the thyroid when ingested in large quantities. Balancing selection has been used to explain the current high non-taster frequency, by maintaining divergent TAS2R38 alleles in humans. We have amplified and sequenced the TAS2R38 amino acid 49 in the virtually uncontaminated Neanderthal sample of El Sidrón 1253 and have determined that it was heterozygous. Thus, this Neanderthal was a taster individual, although probably slightly less than a PAV homozygote. This indicates that variation in bitter taste perception pre-dates the divergence of the lineages leading to Neanderthals and modern humans.     

Silent,generic and plant kairomone sensitive odorant receptors from the Southern house mosquito

The Southern house mosquito Culex quinquefasciatus has the largest repertoire of odorant receptors (ORs) of all mosquitoes and dipteran species whose genomes have been sequenced to date. Previously, we have identified and de-orphanized two ORs expressed in female antennae, CquiOR2 and CquiOR10, which are sensitive to oviposition attractants. In view of a new nomenclature for the Culex genome (VectorBase) we renamed these ORs as CquiOR21 (formerly CquiOR10) and CquiOR121 (CquiOR2). In addition, we selected ORs from six different phylogenetic groups for deorphanization. We cloned four of them by using cDNA from female antennae as a template. Attempts to clone CquiOR87 and CquiOR110 were unsuccessful either because they are pseudogenes or are not expressed in adult female antennae, the main olfactory tissue. By contrast, CquiOR1, CquiOR44, CquiOR73, and CquiOR161 were highly expressed in female antennae. To de-orphanize these ORs, we employed the Xenopus oocyte recording system. CquiORx–CquiOrco-expressed oocytes were challenged with a panel of 90 compounds, including known oviposition attractants, human and vertebrate host odorants, plant kairomones, and naturally occurring repellents. While CquiOR161 did not respond to any test compound in two different laboratories, CquiOR1 showed the features of a generic OR, with strong responses to 1-octen-3-ol and other ligands. CquiOR44 and CquiOR73 showed preference to plant-derived terpenoids and phenolic compounds, respectively. While fenchone was the best ligand for the former, 3,5-dimethylphenol elicited the strongest responses in the latter. The newly de-orphanized ORs may be involved in reception of plant kairomones and/or natural repellents.