Circadian Regulation of Light-Evoked Attraction and Avoidance Behaviors in Daytime- versus Nighttime-Biting Mosquitoes.

Mosquitoes pose widespread threats to humans and other animals as disease vectors [1]. Day- versus night-biting mosquitoes occupy distinct time-of-day niches [2, 3]. Here, we explore day- versus night-biting female and male mosquitoes' innate temporal attraction/avoidance behavioral responses to light and their regulation by circadian circuit and molecular mechanisms. Day-biting mosquitoes Aedes aegypti, particularly females, are attracted to light during the day regardless of spectra. In contrast, night-biting mosquitoes, Anopheles coluzzii, specifically avoid ultraviolet (UV) and blue light during the day. Behavioral attraction to/avoidance of light in both species change with time of day and show distinct sex and circadian neural circuit differences. Males of both diurnal and nocturnal mosquito species show reduced UV light avoidance in anticipation of evening onset relative to females. The circadian neural circuits of diurnal/day- and nocturnal/night-biting mosquitoes based on PERIOD (PER) and pigment-dispersing factor (PDF) expression show similar but distinct circuit organizations between species. The basis of diurnal versus nocturnal behaviors is driven by molecular clock timing, which cycles in anti-phase between day- versus night-biting mosquitoes. Observed differences at the neural circuit and protein levels provide insight into the fundamental basis underlying diurnality versus nocturnality. Molecular disruption of the circadian clock severely interferes with light-evoked attraction/avoidance behaviors in mosquitoes. In summary, attraction/avoidance behaviors show marked differences between day- versus night-biting mosquitoes, but both classes of mosquitoes are circadian and light regulated, which may be applied toward species-specific control of harmful mosquitoes.

Repellency Assessment of Nepeta cataria Essential Oils and Isolated Nepetalactones on Aedes aegypti.

There is an increased need for improved and affordable insect repellents to reduce transmission of rapidly spreading diseases with high mortality rates. Natural products are often used when DEET cannot be afforded or accessed and when consumers choose not to use a synthetic repellent. The essential oils from two newly bred Nepeta cataria (catnip) plants representing two different chemotypes and their respective isolated nepetalactone isomers were evaluated as mosquito repellents against Aedes aegypti mosquitoes that transmit the Zika and Dengue virus in a one choice landing rate inhibition assay. A dose response curve was generated for each treatment and a time course analysis of repellency was performed over 24 hours with a N. cataria essential oil sample. The results indicate that all essential oil samples and their respective purified nepetalactone isomers were able to achieve greater than 95% repellency. Between two and four hours, the ability to repel more than 95% of the mosquitoes diminished. At the lowest concentrations tested, the nepetalactones and crude essential oil samples were more effective than DEET at reducing the number of mosquito landings.

Odorants for surveillance and control of the Asian Citrus Psyllid (Diaphorina citri).

BACKGROUND: The Asian Citrus Psyllid (ACP), Diaphorina citri, can transmit the bacterium Candidatus Liberibacter while feeding on citrus flush shoots. This bacterium causes Huanglongbing (HLB), a major disease of citrus cultivation worldwide necessitating the development of new tools for ACP surveillance and control. The olfactory system of ACP is sensitive to variety of odorants released by citrus plants and offers an opportunity to develop new attractants and repellents. RESULTS: In this study, we performed single-unit electrophysiology to identify odorants that are strong activators, inhibitors, and prolonged activators of ACP odorant receptor neurons (ORNs). We identified a suite of odorants that activated the ORNs with high specificity and sensitivity, which may be useful in eliciting behavior such as attraction. In separate experiments, we also identified odorants that evoked prolonged ORN responses and antagonistic odorants able to suppress neuronal responses to activators, both of which can be useful in lowering attraction to hosts. In field trials, we tested the electrophysiologically identified activating odorants and identified a 3-odor blend that enhances trap catches by ∼230%. CONCLUSION: These findings provide a set of odorants that can be used to develop affordable and safe odor-based surveillance and masking strategies for this dangerous pest insect.

Targeting a dual detector of skin and CO2 to modify mosquito host seeking.

Female mosquitoes that transmit deadly diseases locate human hosts by detecting exhaled CO2 and skin odor. The identities of olfactory neurons and receptors required for attraction to skin odor remain a mystery. Here, we show that the CO2-sensitive olfactory neuron is also a sensitive detector of human skin odorants in both Aedes aegypti and Anopheles gambiae. We demonstrate that activity of this neuron is important for attraction to skin odor, establishing it as a key target for intervention. We screen ~0.5 million compounds in silico and identify several CO2 receptor ligands, including an antagonist that reduces attraction to skin and an agonist that lures mosquitoes to traps as effectively as CO2. Analysis of the CO2 receptor ligand space provides a foundation for understanding mosquito host-seeking behavior and identifies odors that are potentially safe, pleasant, and affordable for use in a new generation of mosquito control strategies worldwide.

Odour receptors and neurons for DEET and new insect repellents.

There are major impediments to finding improved DEET alternatives because the receptors causing olfactory repellency are unknown, and new chemicals require exorbitant costs to determine safety for human use. Here we identify DEET-sensitive neurons in a pit-like structure in the Drosophila melanogaster antenna called the sacculus. They express a highly conserved receptor, Ir40a, and flies in which these neurons are silenced or Ir40a is knocked down lose avoidance to DEET. We used a computational structure-activity screen of >400,000 compounds that identified >100 natural compounds as candidate repellents. We tested several and found that most activate Ir40a(+) neurons and are repellents for Drosophila. These compounds are also strong repellents for mosquitoes. The candidates contain chemicals that do not dissolve plastic, are affordable and smell mildly like grapes, with three considered safe in human foods. Our findings pave the way to discover new generations of repellents that will help fight deadly insect-borne diseases worldwide.

Gene expression-based biomarkers for Anopheles gambiae age grading.

Information on population age structure of mosquitoes under natural conditions is fundamental to the understanding of vectorial capacity and crucial for assessing the impact of vector control measures on malaria transmission. Transcriptional profiling has been proposed as a method for predicting mosquito age for Aedes and Anopheles mosquitoes, however, whether this new method is adequate for natural conditions is unknown. This study tests the applicability of transcriptional profiling for age-grading of Anopheles gambiae, the most important malaria vector in Africa. The transcript abundance of two An. gambiae genes, AGAP009551 and AGAP011615, was measured during aging under laboratory and field conditions in three mosquito strains. Age-dependent monotonic changes in transcript levels were observed in all strains evaluated. These genes were validated as age-grading biomarkers using the mark, release and recapture (MRR) method. The MRR method determined a good correspondence between actual and predicted age, and thus demonstrated the value of age classifications derived from the transcriptional profiling of these two genes. The technique was used to establish the age structure of mosquito populations from two malaria-endemic areas in western Kenya. The population age structure determined by the transcriptional profiling method was consistent with that based on mosquito parity. This study demonstrates that the transcription profiling method based on two genes is valuable for age determination of natural mosquitoes, providing a new approach for determining a key life history trait of malaria vectors.

Ultra-prolonged activation of CO2-sensing neurons disorients mosquitoes.

Carbon dioxide (CO(2)) present in exhaled air is the most important sensory cue for female blood-feeding mosquitoes, causing activation of long-distance host-seeking flight, navigation towards the vertebrate host and, in the case of Aedes aegypti, increased sensitivity to skin odours. The CO(2) detection machinery is therefore an ideal target to disrupt host seeking. Here we use electrophysiological assays to identify a volatile odorant that causes an unusual, ultra-prolonged activation of CO(2)-detecting neurons in three major disease-transmitting mosquitoes: Anopheles gambiae, Culex quinquefasciatus and A. aegypti. Importantly, ultra-prolonged activation of these neurons severely compromises their ability subsequently to detect CO(2) for several minutes. We also identify odours that strongly inhibit CO(2)-sensitive neurons as candidates for use in disruption of host-seeking behaviour, as well as an odour that evokes CO(2)-like activity and thus has potential use as a lure in trapping devices. Analysis of responses to panels of structurally related odours across the three mosquitoes and Drosophila, which have related CO(2)-receptor proteins, reveals a pattern of inhibition that is often conserved. We use video tracking in wind-tunnel experiments to demonstrate that the novel ultra-prolonged activators can completely disrupt CO(2)-mediated activation as well as source-finding behaviour in Aedes mosquitoes, even after the odour is no longer present. Lastly, semi-field studies demonstrate that use of ultra-prolonged activators disrupts CO(2)-mediated hut entry behaviour of Culex mosquitoes. The three classes of CO(2)-response-modifying odours offer powerful instruments for developing new generations of insect repellents and lures, which even in small quantities can interfere with the ability of mosquitoes to seek humans.

Quantitative trait loci controlling refractoriness to Plasmodium falciparum in natural Anopheles gambiae mosquitoes from a malaria-endemic region in western Kenya.

Natural anopheline populations exhibit much variation in ability to support malaria parasite development, but the genetic mechanisms underlying this variation are not clear. Previous studies in Mali, West Africa, identified two quantitative trait loci (QTL) in Anopheles gambiae mosquitoes that confer refractoriness (failure of oocyst development in mosquito midguts) to natural Plasmodium falciparum parasites. We hypothesize that new QTL may be involved in mosquito refractoriness to malaria parasites and that the frequency of natural refractoriness genotypes may be higher in the basin region of Lake Victoria, East Africa, where malaria transmission intensity and parasite genetic diversity are among the highest in the world. Using field-derived F2 isofemale families and microsatellite marker genotyping, two loci significantly affecting oocyst density were identified: one on chromosome 2 between markers AG2H135 and AG2H603 and the second on chromosome 3 near marker AG3H93. The first locus was detected in three of the five isofemale families studied and colocalized to the same region as Pen3 and pfin1 described in other studies. The second locus was detected in two of the five isofemale families, and it appears to be a new QTL. QTL on chromosome 2 showed significant additive effects while those on chromosome 3 exhibited significant dominant effects. Identification of P. falciparum-refractoriness QTL in natural An. gambiae mosquitoes is critical to the identification of the genes involved in malaria parasite transmission in nature and for understanding the coevolution between malaria parasites and mosquito vectors.

Dynamics of gene introgression in the African malaria vector Anopheles gambiae.

Anopheles gambiae is a major malaria vector in Africa and a popular model species for a variety of ecological, evolutionary, and genetic studies on vector control. Genetic manipulation of mosquito vectorial capacity is a promising new weapon for the control of malaria. However, the release of exotic transgenic mosquitoes will bring in novel alleles in addition to the parasite-inhibiting genes, which may have unknown effects on the local population. Therefore, it is necessary to develop methodologies that can be used to evaluate the spread rate of introduced genes in A. gambiae. In this study, the effects and dynamics of genetic introgression between two geographically distinct A. gambiae populations from western Kenya (Mbita) and eastern Tanzania (Ifakara) were investigated with amplified fragment length polymorphisms (AFLPs) and microsatellite markers. Microsatellites and polymorphic cDNA markers revealed a large genetic differentiation between the two populations (average F(ST) = 0.093, P < 0.001). When the two strains were crossed in random mating between the two populations, significant differences in the rate of genetic introgression were found in the mixed populations. Allele frequencies of 18 AFLP markers (64.3%) for Mbita and of 26 markers (92.9%) for Ifakara varied significantly from F5 to F20. This study provides basic information on how a mosquito release program would alter the genetic makeup of natural populations, which is critical for pilot field testing and ecological risk evaluation of transgenic mosquitoes.

Fitness consequences of Anopheles gambiae population hybridization.

BACKGROUND: The use of transgenic mosquitoes with parasite inhibiting genes has been proposed as an integral strategy to control malaria transmission. However, release of exotic transgenic mosquitoes will bring in novel alleles along with parasite-inhibiting genes that may have unknown effects on native populations. Thus it is necessary to study the effects and dynamics of fitness traits in native mosquito populations in response to the introduction of novel genes. This study was designed to evaluate the dynamics of fitness traits in a simulation of introduction of novel alleles under laboratory conditions using two strains of Anopheles gambiae: Mbita strain from western Kenya and Ifakara strain from Tanzania. METHODS: The dynamics of fitness traits were evaluated under laboratory conditions using the two An. gambiae strains. These two geographically different strains were cross-bred and monitored for 20 generations to score fecundity, body size, blood-meal size, larval survival, and adult longevity, all of which are important determinants of the vector's potential in malaria transmission. Traits were analysed using pair-wise analysis of variance (ANOVA) for fecundity, body size, and blood-meal size while survival analysis was performed for larval survival and adult longevity. RESULTS: Fecundity and body size were significantly higher in the progeny up to the 20th generation compared to founder strains. Adult longevity had a significantly higher mean up to the 10th generation and average blood-meal size was significantly larger up to the 5th generation, indicating that hybrids fitness is enhanced over that of the founder strains. CONCLUSION: Hybridization of the two mosquito populations used in this study led to increased performance in the fitness traits studied. Given that the studied traits are important determinants of the vector's potential to transmit malaria, these results suggest the need to release genetically modified mosquitoes that have the same or very similar backgrounds to the native populations.