Behavior of Nymphs and Adults of the Black-Legged Tick Ixodes scapularis and the Lone Star Tick Ambylomma americanum in Response to Thermal Stimuli.

Ticks use chemical and thermal signals emitted by humans and other vertebrates to locate suitable hosts for a blood meal. Here, we study the behavior of black-legged Ixodes scapularis and the lone star ticks Amblyomma americanum exposed to heat sources held at temperatures near those of vertebrate hosts (32 °C). First, we used a locomotion compensator to test behavioral responses of ticks to an infrared light emitting diode (LED). The servosphere allowed us to measure parameters such as velocity, acceleration, linearity, and orientation. Then a heating element (Peltier) located in one of the extremes of a double-choice vertical rod (flying T), was employed to observe upward movement of the ticks toward such a heat source. While both species oriented toward the LED, only lone star ticks were attracted to the Peltier element while climbing upward. In conclusion, we showed that heat attracted ticks from short distances up to several centimeters on a the servosphere, and those responses differed between the two species of ticks on the flying T. We discuss our results in the context of the ecology of both tick species and their potential in tick survey and management.

Membrane Proteins Mediating Reception and Transduction in Chemosensory Neurons in Mosquitoes.

Mosquitoes use chemical cues to modulate important behaviors such as feeding, mating, and egg laying. The primary chemosensory organs comprising the paired antennae, maxillary palps and labial palps are adorned with porous sensilla that house primary sensory neurons. Dendrites of these neurons provide an interface between the chemical environment and higher order neuronal processing. Diverse proteins located on outer membranes interact with chemicals, ions, and soluble proteins outside the cell and within the lumen of sensilla. Here, we review the repertoire of chemosensory receptors and other membrane proteins involved in transduction and discuss the outlook for their functional characterization. We also provide a brief overview of select ion channels, their role in mammalian taste, and potential involvement in mosquito taste. These chemosensory proteins represent targets for the disruption of harmful biting behavior and disease transmission by mosquito vectors.

Mini review: Gustatory reception of chemicals affecting host feeding in aedine mosquitoes.

Mosquitoes vector dangerous human diseases during blood feeding. Gustatory (taste) receptor neurons in the mosquito provide important chemical information including the nature and suitability of a potential host. Here we discuss the behavior, neurophysiology and molecular mechanisms associated with feeding in aedine mosquitoes, important vectors of emerging diseases including Zika fever, chikungunya and dengue fever. We describe how interactions between feeding stimulation and deterrency at the peripheral neural receptor level provide input to higher order neural processing centers affecting decisions to feed. A better understanding of gustatory mechanisms involved in the female's decision to bite will provide the framework for novel strategies aimed at preventing the spread of vector-borne disease.

Chemosensory Responses to the Repellent Nepeta Essential Oil and Its Major Component Nepetalactone by Aedes aegypti (Diptera: Culicidae), a Vector of Zika Virus.

Nepeta essential oil (Neo; catnip) and its major component, nepetalactone, have long been known to repel insects including mosquitoes. However, the neural mechanisms through which these repellents are detected by mosquitoes, including the yellow fever mosquito Aedes aegypti (L.), an important vector of Zika virus, were poorly understood. Here we show that Neo volatiles activate olfactory receptor neurons within the basiconic sensilla on the maxillary palps of female Ae. aegypti. A gustatory receptor neuron sensitive to the feeding deterrent quinine and housed within sensilla on the labella of females was activated by both Neo and nepetalactone. Activity of a second gustatory receptor neuron sensitive to the feeding stimulant sucrose was suppressed by both repellents. Our results provide neural pathways for the reported spatial repellency and feeding deterrence of these repellents. A better understanding of the neural input through which female mosquitoes make decisions to feed will facilitate design of new repellents and management strategies involving their use.

Electrophysiological Responses of Gustatory Receptor Neurons on the Labella of the Common Malaria Mosquito, Anopheles quadrimaculatus (Diptera: Culicidae).

We recorded electrical responses from sensory cells associated with gustatory sensilla on the labella of female Anopheles quadrimaculatus Say to salt, sucrose, quinine (a feeding deterrent), and the insect repellent, N,N-diethyl-3-methylbenzamide (DEET). A salt-sensitive cell responded to increasing concentrations of sodium chloride. A second cell was activated by increasing sucrose concentrations, while quinine, DEET, or a mixture of quinine + DEET elicited spike activity from a third cell, an apparent bitter- or deterrent-sensitive cell. Both quinine and DEET suppressed activity of the sugar-sensitive cell; sucrose suppressed activity of the bitter- or deterrent-sensitive cell. These results demonstrate separate gustatory pathways for a feeding stimulant and aversive contact cues mediated through distinct sensory inputs on the labellum. This sensory appendage may serve as a useful target to disrupt feeding behavior in this and other anopheline species, which transmit diseases like malaria to human populations.

Bitter-sensitive gustatory receptor neuron responds to chemically diverse insect repellents in the common malaria mosquito Anopheles quadrimaculatus.

Female mosquitoes feed on blood from animal hosts to obtain nutritional resources used for egg production. These contacts facilitate the spread of harmful human diseases. Chemical repellents are used to disrupt mosquito host-seeking and blood-feeding behaviors; however, little is known about the gustatory sensitivity of mosquitoes to known repellents. Here, we recorded electrical responses from gustatory receptor neurons (GRNs) housed within the labellar sensilla of female Anopheles quadrimaculatus to N,N-diethyl-3-methylbenzamide (DEET), picaridin, IR3535, 2-undecanone, p-menthane-3,8-diol, geraniol, trans-2-hexen-1-ol, quinine, and quinidine. A bitter-sensitive GRN responded to all tested repellents and quinine, a known feeding deterrent. Responses of the bitter-sensitive neuron to quinine and an isomer, quinidine, did not differ. Delayed bursts of electrical activity were observed in response to continuous stimulation with synthetic repellents at high concentrations. Electrophysiological recordings from bitter-sensitive GRNs associated with mosquito gustatory sensilla represent a convenient model to evaluate candidate repellents.

Olfactory disruption: toward controlling important insect vectors of disease.

Chemical repellents are used to decrease contacts between insect disease vectors and their hosts, thus reducing the probability of disease transmission. The molecular mechanisms by which repellents have their effects are poorly understood and remain a controversial topic. Here, we present recent results of studies aimed at a more thorough understanding of the mode of action of repellents and discuss the implications of these findings for future research and development of novel or improved repellents.

Neurophysiological and behavioral responses of gypsy moth larvae to insect repellents: DEET, IR3535, and picaridin.

The interactions between insect repellents and the olfactory system have been widely studied, however relatively little is known about the effects of repellents on the gustatory system of insects. In this study, we show that the gustatory receptor neuron (GRN) located in the medial styloconic sensilla on the maxillary palps of gypsy moth larvae, and known to be sensitive to feeding deterrents, also responds to the insect repellents DEET, IR3535, and picaridin. These repellents did not elicit responses in the lateral styloconic sensilla. Moreover, behavioral studies demonstrated that each repellent deterred feeding. This is the first study to show perception of insect repellents by the gustatory system of a lepidopteran larva and suggests that detection of a range of bitter or aversive compounds may be a broadly conserved feature among insects.

The genetics of chemoreception in the labella and tarsi of Aedes aegypti.

The yellow-fever mosquito Aedes aegypti is a major vector of human diseases, such as dengue, yellow fever, chikungunya and West Nile viruses. Chemoreceptor organs on the labella and tarsi are involved in human host evaluation and thus serve as potential foci for the disruption of blood feeding behavior. In addition to host detection, these contact chemoreceptors mediate feeding, oviposition and conspecific recognition; however, the molecular landscape of chemoreception in these tissues remains mostly uncharacterized. Here we report the expression profile of all putative chemoreception genes in the labella and tarsi of both sexes of adult Ae. aegypti and discuss their possible roles in the physiology and behavior of this important disease vector.

The maxillary palp of Aedes aegypti, a model of multisensory integration.

Female yellow-fever mosquitoes, Aedes aegypti, are obligate blood-feeders and vectors of the pathogens that cause dengue fever, yellow fever and Chikungunya. This feeding behavior concludes a series of multisensory events guiding the mosquito to its host from a distance. The antennae and maxillary palps play a major role in host detection and other sensory-mediated behaviors. Compared to the antennae, the maxillary palps are a relatively simple organ and thus an attractive model for exploration of the neuromolecular networks underlying chemo- and mechanosensation. In this study, we surveyed the expressed genetic components and examined their potential involvement with these sensory modalities. Using Illumina sequencing, we identified the transcriptome of the maxillary palps of physiologically mature female Ae. aegypti. Genes expressed in the maxillary palps included those involved in sensory reception, signal transduction and neuromodulation. In addition to previously reported chemosensory genes, we identified candidate transcripts potentially involved in mechanosensation and thermosensation. This survey lays the groundwork to explore sensory networks in an insect appendage. The identification of genes involved in thermosensation provides prospective molecular targets for the development of chemicals aimed at disrupting the behavior of this medically important insect.

Physiological recordings and RNA sequencing of the gustatory appendages of the yellow-fever mosquito Aedes aegypti.

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

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 Development of the Octenol Response in Aedes aegypti.

Attraction of female Aedes aegypti mosquitoes to 1-octen-3-ol (octenol), CO2, lactic acid, or ammonia emitted by vertebrate hosts is not only contingent on the presence of odorants in the environment, but is also influenced by the insect's physiological state. For anautogenous mosquito species, like A. aegypti, newly emerged adult females neither respond to host odors nor engage in blood-feeding; the bases for these behaviors are poorly understood. Here we investigated detection of two components of an attractant blend emitted by vertebrate hosts, octenol, and CO2, by female A. aegypti mosquitoes using electrophysiological, behavioral, and molecular approaches. An increase in sensitivity of octenol olfactory receptor neurons (ORNs) was correlated with an increase in odorant receptor gene (Or) expression and octenol-mediated attractive behavior from day 1 to day 6 post-emergence. While the sensitivity of octenol ORNs was maintained through day 10, behavioral responses to octenol decreased as did the ability of females to discriminate between octenol and octenol + CO2. Our results show differing age-related roles for the peripheral receptors for octenol and higher order neural processing in the behavior of female mosquitoes.

Gustatory receptor neuron responds to DEET and other insect repellents in the yellow-fever mosquito, Aedes aegypti.

Three gustatory receptor neurons were characterized for contact chemoreceptive sensilla on the labella of female yellow-fever mosquitoes, Aedes aegypti. The neuron with the smallest amplitude spike responded to the feeding deterrent, quinine, as well as N,N-diethyl-3-methylbenzamide and other insect repellents. Two other neurons with differing spikes responded to salt (NaCl) and sucrose. This is the first report of a gustatory receptor neuron specific for insect repellents in mosquitoes and may provide a tool for screening chemicals to discover novel or improved feeding deterrents and repellents for use in the management of arthropod disease vectors.

Olfactory cues are subordinate to visual stimuli in a neotropical generalist weevil.

The tropical root weevil Diaprepes abbreviatus is a major pest of multiple crops in the Caribbean Islands and has become a serious constraint to citrus production in the United States. Recent work has identified host and conspecific volatiles that mediate host- and mate-finding by D. abbreviatus. The interaction of light, color, and odors has not been studied in this species. The responses of male and female D. abbreviatus to narrow bandwidths of visible light emitted by LEDs offered alone and in combination with olfactory stimuli were studied in a specially-designed multiple choice arena combined with a locomotion compensator. Weevils were more attracted to wavelengths close to green and yellow compared with blue or ultraviolet, but preferred red and darkness over green. Additionally, dim green light was preferred over brighter green. Adult weevils were also attracted to the odor of its citrus host + conspecifics. However, the attractiveness of citrus + conspecific odors disappeared in the presence of a green light. Photic stimulation induced males but not females to increase their speed. In the presence of light emitted by LEDs, turning speed decreased and path straightness increased, indicating that weevils tended to walk less tortuously. Diaprepes abbreviatus showed a hierarchy between chemo- and photo-taxis in the series of experiments presented herein, where the presence of the green light abolished upwind anemotaxis elicited by the pheromone + host plant odor. Insight into the strong responses to visual stimuli of chemically stimulated insects may be provided when the amount of information supplied by vision and olfaction is compared, as the information transmission capacity of compound eyes is estimated to be several orders of magnitude higher compared with the olfactory system. Subordination of olfactory responses by photic stimuli should be considered in the design of strategies aimed at management of such insects.

Selectivity of odorant receptors in insects.

Insect olfactory receptors (ORs) detect chemicals, shape neuronal physiology, and regulate behavior. Although ORs have been categorized as "generalists" and "specialists" based on their ligand spectrum, both electrophysiological studies and recent pharmacological investigations show that ORs specifically recognize non-pheromonal compounds, and that our understanding of odorant-selectivity mirrors our knowledge of insect chemical ecology. As we are progressively becoming aware that ORs are activated through a variety of mechanisms, the molecular basis of odorant-selectivity and the corollary notion of broad-tuning need to be re-examined from a pharmacological and evolutionary perspective.

Identification and synthesis of a male-produced pheromone for the neotropical root weevil Diaprepes abbreviatus.

An unsaturated hydroxy-ester pheromone was isolated from the headspace and feces of male Diaprepes abbreviatus, identified, and synthesized. The pheromone, methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate, was discovered by gas chromatography-coupled electroantennogram detection (GC-EAD), and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). The synthesis yielded an 86:14 mixture of methyl (E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate (active) and methyl (Z)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate (inactive), along with a lactone breakdown product. The activity of the synthetic E-isomer was confirmed by GC-EAD, GC-MS, NMR, and bioassays. No antennal response was observed to the Z-isomer or the lactone. In a two-choice olfactometer bioassay, female D. abbreviatus moved upwind towards the synthetic pheromone or natural pheromone more often compared with clean air. Males showed no clear preference for the synthetic pheromone. This pheromone, alone or in combination with plant volatiles, may play a role in the location of males by female D. abbreviatus.

Odorant receptor modulation: ternary paradigm for mode of action of insect repellents.

The modulation of insect behavior for the purpose of controlling the spread of infectious diseases has been the task of a few insect repellents for which the mechanistic modes of action on odorant receptors (ORs) are unclear. Here, we study the effects of the repellents DEET and IR3535, and a novel OR co-receptor (Orco) agonist on odorant-evoked currents in Xenopus oocytes expressing two subtypes of Aedes aegypti ORs (AaORs). We show that DEET and IR3535 behave as insurmountable antagonists of ORs, and that modulation of OR activity is not restricted to antagonism and agonism, but also includes synergism. This knowledge of the molecular mechanisms underlying OR blockade, activation and hyperactivation will be fundamental to the development of novel strategies for the control of mosquito behavior.

Chemical Ecology of the Colorado Potato Beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), and Potential for Alternative Control Methods.

The Colorado potato beetle (CPB) has been a major insect pest to potato farming for over 150 years and various control methods have been established to reduce its impact on potato fields. Crop rotation and pesticide use are currently the most widely used approaches, although alternative methods are being developed. Here we review the role of various volatile and nonvolatile chemicals involved in behavior changes of CPB that may have potential for their control. First, we describe all volatile and nonvolatile chemicals involved in host plant localization and acceptance by CPB beetles, including glycoalcaloids and host plant volatiles used as kairomones. In the second section, we present the chemical signals used by CPB in intraspecific communication, including sex and aggregation pheromones. Some of these chemicals are used by natural enemies of CPBs to locate their prey and are presented in the third section. The last section of this review is devoted a discussion of the potential of some natural chemicals in biological control of CPB and to approaches that already reached efficient field applications.

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 (CO(2)) 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.