ATG8 is conserved between Saccharomyces cerevisiae and psychrophilic, polar-collected fungi.

Autophagy is a conserved catabolic process by which eukaryotic cells respond to stress by targeting damaged or unneeded molecules or organelles for sequestration into specialized vesicles known as autophagosomes. Autophagosomes ultimately facilitate the digestion and recycling of their contents by fusing with the degradative organelle of the cell. Studies of the budding yeast Saccharomyces cerevisiae have revealed various types of stress that can regulate autophagy, including starvation and extreme temperatures. While autophagy has not yet been directly shown to confer the ability to survive extreme cold or freeze-thaw stress in yeast, upregulation of autophagy has been directly implicated in the ability of arctic insects to survive cold temperatures. We are interested in investigating the potential role of autophagy in polar habitat survival by cold-loving (psychrophilic) yeast like Mrakia blollopsis. To begin to examine the conservation of Atg machinery in polar-collected yeast, we focused on Atg8, a small, ubiquitin-like protein that plays an important role in autophagy. We report that Atg8 is conserved between S. cerevisiae and polar-collected yeast, using Atg8 from Mrakia blollopsis (strain TGK1-2) as an example. This study represents the first direct examination of autophagy machinery conservation across mesophilic and psychrophilic species of yeast.

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.

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.

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.

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.

The insect SNMP gene family.

SNMPs are membrane proteins observed to associate with chemosensory neurons in insects; in Drosophila melanogaster, SNMP1 has been shown to be essential for the detection of the pheromone cis-vaccenyl acetate (CVA). SNMPs are one of three insect gene clades related to the human fatty acid transporter CD36. We previously characterized the CD36 gene family in 4 insect Orders that effectively cover the Holometabola, or some 80% of known insect species and the 300 million years of evolution since this lineage emerged: Lepidoptera (e.g. Bombyx mori, Antheraea polyphemus, Manduca sexta, Heliothis virescens, Helicoverpa assulta, Helicoverpa armigera, Mamestra brassicae); Diptera (D. melanogaster, Drosophila pseudoobscura, Aedes aegypti, Anopheles gambiae, Culex pipiens quinquefasciatus); Hymenoptera (Apis mellifera); and Coleoptera (Tribolium castaneum). This previous study suggested a complex topography within the SNMP clade including a strongly supported SNMP1 sub-clade plus additional SNMP genes. To further resolve the SNMP clade here, we used cDNA sequences of SNMP1 and SNMP2 from various Lepidoptera species, D. melanogaster and Ae. aegypti, as well as BAC derived genomic sequences from Ae. aegypti as models for proposing corrected sequences of orthologues in the D. pseudoobscura and An. gambiae genomes, and for identifying orthologues in the B. mori and C. pipiens q. genomes. We then used these sequences to analyze the SNMP clade of the insect CD36 gene family, supporting the existence of two well supported sub-clades, SNMP1 and SNMP2, throughout the dipteran and lepidopteran lineages, and plausibly throughout the Holometabola and across a broad evolutionary time scale. We present indirect evidence based on evolutionary selection (dN/dS) that the dipteran SNMPs are expressed as functional proteins. We observed expansions of the SNMP1 sub-clade in C. pipiens q. and T. castaneum suggesting that the SNMP1s may have an expanded functional role in these species.