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Adult female mosquitoes rely on olfactory cues like carbon dioxide and other small molecules to find vertebrate hosts to acquire blood. The molecular physiology of the mosquito olfactory system is critical for their host preferences. Many laboratory strains of the yellow fever mosquito Aedes aegypti have been established since the late 19th century. These strains have been used for most molecular studies in this species. Some earlier comparative studies have identified significant physiological differences between different laboratory strains. In this study, we used a Y-tube olfactometer to determine the attraction of females of seven different strains of Ae. aegypti to a human host: UGAL, Rockefeller, Liverpool, Costa Rica, Puerto Rico, and two odorant receptor co-receptor (Orco) mutants Orco2 and Orco16. We performed RNA-seq using antennae of Rockefeller, Liverpool, Costa Rica, and Puerto Rico females. Our results showed that female Aedes aegypti from the Puerto Rico strain had significantly reduced attraction rates toward human hosts compared to all other strains. RNA-seq analyses of the antenna transcriptomes of Rockefeller, Liverpool, Costa Rica, and Puerto Rico strains revealed distinct differences in gene expression between the four strains, but conservation in gene expression patterns of known human-sensing genes. However, we identified several olfaction-related genes that significantly vary between strains, including receptors with significantly different expression in mosquitoes from the Puerto Rico strain and the other strains.

The fat body is considered the insect analog of vertebrate liver and fat tissue. In mosquitoes, a blood meal triggers a series of processes in the fat body that culminate in vitellogenesis, the process of yolk formation. Lipids are stored in the fat body in specialized organelles called lipid droplets that change in size depending on the nutritional and metabolic status of the insect. We surveyed lipid droplets in female Aedes aegypti fat body during a reproductive cycle using confocal microscopy and analyzed the dynamic changes in the fat body lipidome during this process using LC/MS. We found that lipid droplets underwent dynamic changes in volume after the mosquito took a blood meal. The lipid composition found in the fat body is quite complex with 117 distinct lipids that fall into 19 classes and sublcasses. Our results demonstrate that the lipid composition of the fat body is complex as most lipid classes underwent significant changes over the course of the vitellogenic cycle. This study lays the foundation for identifying unknown biochemical pathways active in the mosquito fat body, that are high-value targets for the development of novel mosquito control strategies.

Ticks are important vectors of human and veterinary diseases. A primary way ticks gain access to human hosts is by engaging to clothing. Repellents or acaricides sprayed onto fabric are used to deter ticks’ access to human hosts. However, there are a limited amount of standardized laboratory assays that can determine the potency and efficacy of repellents. We present a novel fabric-engagement assay referred to as the ‘Tick Carousel Assay’. This assay utilizes fabric brushing past ticks located on an artificial grass patch and measures tick engagements to fabric over time. After screening a variety of tick species, we used the lone star tick (Amblyomma americanum) to test the efficacy of four commonly used active ingredients in repellents: DEET, Picaridin, IR3535, and Oil of Lemon Eucalyptus. Repellency was tested immediately, after three hours, and six hours post application to fabric. Our data show that each repellent we tested significantly reduced the number of tick engagements to fabric for at least 6 hours. We did not find significant differences in repellent efficacy between the four active ingredients tested directly and three hours after application. After six hours, Oil of Lemon Eucalyptus repelled ticks more than the other active ingredients. We show that our Tick Carousel Assay provides an affordable, repeatable, and standardized way to compare and test repellent efficacy on treated fabrics. Our results confirm that commonly used repellents applied to fabric are an effective way to reduce tick engagement.

The use of insecticides has been a central approach to control disease-transmitting mosquitoes for the last century. The high prevalence of pyrethroid use as public health insecticides has resulted in the evolution of pyrethroid resistance in many populations of Aedes aegypti (Linnaeus) (Diptera: Culicidae), throughout its global distribution range. Insecticide resistance is often correlated with an associated fitness cost. In this project, we studied the phenotypes of hybrid mosquitoes derived from crossing a pyrethroid-resistant strain of Ae. aegypti (Puerto Rico [PR]) with a more susceptible one (Rockefeller [ROCK]). We first sequenced and compared the para gene of both original strains. We then crossed males from one strain with females of the other, creating two hybrids (Puertofeller, Rockorico). We used a Y-tube choice assay to measure the attraction of these strains towards a human host. We then compared the levels of pyrethroid resistance in the different strains. We found three known resistance mutations in the para gene sequence of the PR strain. In our attraction assays, PR females showed lower attraction to humans, than the ROCK females. Both hybrid strains showed strong attraction to a human host. In the insecticide resistance bottle assays, both hybrid strains showed marginal increases in resistance to permethrin compared to the more susceptible ROCK strain. These results suggest that hybrids of sensitive and permethrin-resistant mosquitoes have an incremental advantage compared to more susceptible mosquitoes when challenged with permethrin. This explains the rapid spread of permethrin resistance that was observed many times in the field.

Aquaporins (AQP) are a family of plasma membrane proteins responsible for water transport through cell membranes. They are differentially expressed in different parts of the alimentary canal of insects where they regulate water transport. These proteins have been studied in detail in some insects, but few data are available for aquaporins of the honey bee, Apis mellifera. We used quantitative PCR to study the expression of six putative aquaporin genes in forager honey bees. We found differential expression of all putative AQP genes in crop, midgut, ileum, rectum and Malpighian tubules. We found the entomoglyceroporin Am_Eglp 1 expressed at extremely high levels in the midgut. We performed a functional characterization of Am_Eglp 1 using heterologous expression in Xenopus laevis oocyte followed by water uptake assays. Our results confirmed that the Am_Eglp 1 gene encodes a functional water transporter. This study shows that all putative honey bee aquaporin genes have complex expression patterns in the digestive and excretory organs of honey bee workers. Our results suggest that Am_Eglp 1 is the principal water transporter in the midgut of A. mellifera workers.

Mosquitoes of the Aedes genus are vectors for dengue, chikungunya, Zika, and yellow fever viruses. Mosquito repellents are an effective way to prevent mosquito bites and reduce the spread of mosquito-borne diseases. In the early 90s, the U.S. Environmental Protection Agency (EPA) published a list of active ingredients that pose minimum risk to human health that can be used as pesticides or repellents without passing the EPA registration process. The present study examined the efficacy of 21 of the active ingredients listed by the EPA 25 (B) exempt list and five commercially available sprays that only contained active ingredients from the EPA 25(B) list in repelling female Aedes aegypti (L.) females. We performed choice bioassays in a controlled laboratory environment, using a Y-tube olfactometer to determine attraction rates of humans to female Ae. aegypti in the presence of one of the 21 active ingredients and five commercially available repellent sprays. We found that cinnamon oil, peppermint oil, spearmint oil, lemongrass oil, and garlic oil reduced mosquito attraction to human odor. Of the five commercial repellent sprays, only one reduced mosquito attraction for up to 30 min in our assay. The EPA 25 (B) list contains active ingredients that under the conditions of our assay repel Ae. aegypti.

BackgroundAedes aegypti mosquitoes are vectors of a variety of emerging viral pathogens, including yellow fever, dengue, chikungunya, and Zika virus. This species has established endemic populations in all cities across southern New Mexico sampled to date. Presently, control of Aedes-borne viruses relies on deployment of insecticides to suppress mosquito populations, but the evolution of insecticide resistance threatens the success of vector control programs. While insecticide resistance is quite common in Ae. aegypti field populations across much of the U.S., the resistance status of this species in populations from New Mexico has not previously been assessed.ResultsFirst, we collected information on pesticide use in cities in southern New Mexico and found that the most commonly used active ingredients were pyrethroids. The use of insecticides with the same mode-of-action over multiple years is likely to promote the evolution of resistance. To determine if there was evidence of resistance in some cities in southern New Mexico, we collected Ae. aegypti from the same cities and established laboratory strains to assess resistance to pyrethroid insecticides and, for a subset of populations, to organophosphate insecticides. F2 or F4 generation mosquitoes were assessed for insecticide resistance using bottle test bioassays. The majority of the populations from New Mexico that we analyzed were resistant to the pyrethroids permethrin and deltamethrin. A notable exception to this trend were mosquitoes from Alamogordo, a city that did not report using pyrethroid insecticides for vector control. We screened individuals from each population for known knock down resistance (kdr) mutations via PCR and found a strong association between the presences of the F1534C kdr mutation in the para gene of Ae. aegypti (homologue to F1534C in Musca domestica L.) and pyrethroid resistance.ConclusionHigh-level pyrethroid resistance is common in Ae. aegypti from New Mexico and geographic variation in such resistance is likely associated with variation in usage of pyrethroids for vector control. Resistance monitoring and management is recommended in light of the potential for arbovirus outbreaks in this state. Also, alternative approaches to mosquito control that do not involve insecticides should be explored.

The mosquitoesAedes aegypti(L.) andAe. albopictusSkuse are the major vectors of dengue, Zika, yellow fever and chikungunya viruses worldwide.Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found thatWolbachiais commonly detected inAe.albopictusbut rarely detected inAe. aegypti. In this study, we used a two-step PCR assay to detectWolbachiain wild-collected samples ofAe. aegypti.The PCR products were sequenced to validate amplicons and identifyWolbachiastrains. A loop-mediated isothermal amplification (LAMP) assay was developed and used for detectingWolbachiain selected mosquito specimens as well. We foundWolbachiain 85/148 (57.4%) wildAe. aegyptispecimens from various cities in New Mexico and in 2/46 (4.3%) from St. Augustine, Florida. We did not detectWolbachiain 94 samples ofAe. aegyptifrom Deer Park, Harris County, Texas.Wolbachiadetected inAe. aegyptifrom both New Mexico and Florida was thewAlbB strain ofWolbachia pipientis.AWolbachiapositive colony ofAe. aegyptiwas established from pupae collected in Las Cruces, New Mexico in 2018. The infected females of this strain transmittedWolbachiato their progeny when crossed with males of Rockefeller strain ofAe. aegypti, which does not carryWolbachia.In contrast, none of the progeny of progeny of Las Cruces males mated to Rockefeller females were infected withWolbachia.

The introduction of Zika virus (ZIKV) to the Americas raised concern that the virus would spill back from human transmission, perpetuated by Aedes aegypti, into a sylvatic cycle maintained in wildlife and forest-living mosquitoes. In the Americas, Sabethes species are vectors of sylvatic yellow fever virus (YFV) and are therefore candidate vectors of a sylvatic ZIKV cycle. To test the potential of Sabethes cyaneus to transmit ZIKV, Sa. cyaneus and Ae. aegypti were fed on A129 mice one or two days post-infection (dpi) with a ZIKV isolate from Mexico. Sa. cyaneus were sampled at 3, 4, 5, 7, 14, and 21 days post-feeding (dpf) and Ae. aegypti were sampled at 14 and 21 dpf. ZIKV was quantified in mosquito bodies, legs, and saliva to measure infection, dissemination, and potential transmission, respectively. Of 69 Sa. cyaneus that fed, ZIKV was detected in only one, in all body compartments, at 21 dpf. In contrast, at 14 dpf 100% of 20 Ae. aegypti that fed on mice at 2 dpi were infected and 70% had virus in saliva. These data demonstrate that Sa. cyaneus is a competent vector for ZIKV, albeit much less competent than Ae. aegypti.

Sterile insect technique (SIT) is a promising, environmentally friendly alternative to the use of pesticides for insect pest control. However, implementing SIT with Aedes aegypti (Linnaeus) mosquitoes presents unique challenges. For example, during transport from the rearing facility to the release site and during the actual release in the field, damage to male mosquitoes should be minimized to preserve their reproductive competitiveness. The short flight range of male Ae. aegypti requires elaborate release strategies such as release via Unmanned Aircraft Systems, more commonly referred to as drones. Two key parameters during transport and release are storage temperature and compaction rate. We performed a set of laboratory experiments to identify the optimal temperatures and compaction rates for storage and transport of male Ae. aegypti. We then conducted shipping experiments to test our laboratory-derived results in a ‘real-life’ setting. The laboratory results indicate that male Ae. aegypti can survive at a broad range of storage temperatures ranging from 7 to 28°C, but storage time should not exceed 24 h. Male survival was high at all compaction rates we tested with a low at 40 males/cm3. Interestingly, results from our ‘real-life’ shipping experiment showed that high compaction rates were beneficial to survival. This study advances key understudied aspects of the practicalities of moving lab-reared insects into the field and lies the foundation for further studies on the effect of transport conditions on male reproductive fitness.

Public health research and vector control frequently require the rearing of large numbers of vector mosquitoes. All target vector mosquito species are anautogenous, meaning that females require vertebrate blood for egg production. Vertebrate blood, however, is costly, with a short shelf life. To overcome these constraints, we have developed SkitoSnack, an artificial blood meal replacement for the mosquito Aedes aegypti, the vector of dengue, Zika and chikungunya virus. SkitoSnack contains bovine serum albumin and hemoglobin as protein source as well as egg yolk and a bicarbonate buffer. SkitoSnack-raised females had comparable life history traits as blood-raised females. Mosquitoes reared from SkitoSnack-fed females had similar levels of infection and dissemination when orally challenged with dengue virus type 2 (DENV-2) and significantly lower infection with DENV-4. When SkitoSnack was used as a vehicle for DENV-2 delivery, blood-raised and SkitoSnack-raised females were equally susceptible. The midgut microbiota differed significantly between mosquitoes fed on SkitoSnack and mosquitoes fed on blood. By rearing 20 generations of Aedes exclusively on SkitoSnack, we have proven that this artificial diet can replace blood in mosquito mass rearing.

Background Mosquito-borne dengue virus (DENV), the agent of dengue hemorrhagic fever (DHF), is genetically diverse, and new strains regularly invade distant locations and displace existing strains. Invasive strains often cause higher rates of DHF than displaced strains, so it is critical to identify the mechanisms that enable invasion. We tested the hypothesis that invasive strains are less susceptible to RNA interference (RNAi), the major antiviral defense in mosquitoes, than displaced strains. Methods We knocked-down (KD) RNAi in vivo in Aedes aegypti, the DENV vector, by injecting mosquitoes with double-stranded RNA against Argonaute 2 (Ago2), a key enzyme in the RNAi pathway, or a control dsRNA. Ago2 KD and control mosquitoes were fed bloodmeals containing 1 of 3 isolates each of 3 different strains of DENV that had undergone sequential competitive displacement in Sri Lanka, termed, in order of displacement, Pre-DHF, Post-DHF and Ultra-DHF. We predicted that the Pre-DHF strain, which we have previously shown to be less infectious for mosquitoes than the other two strains, would show a greater increase in infectivity than those strains. Engorged mosquitoes were incubated for 10 days, homogenized, and assayed for virus. Results Ago2 KD efficiency ranged from 79% to 98%, as determined by semi-quantitative PCR and band densitometry. The percentage of mosquitoes infected following Ago2 vs. control KD was not significantly different (33% vs. 47%; paired t-test, DF = 8, P = 0.08). However, among infected mosquitoes, virus titer was significantly higher in Ago2 KD mosquitoes (3.98 vs. 3.38 log10 plaque forming units/body; t-test, DF = 14, P = 0.02). Contra our prediction, a two-factor ANOVA did not reveal a significant interaction between the effect of virus strain and treatment (DF = 5, P = 0.58), indicating that Pre-DHF viruses did not show a larger response to Ago2 KD than Post and Ultra-DHF viruses. Conclusion These data support the role of RNAi as a key mosquito defense against virus replication in mosquitoes but indicate that the differences in competitive success among the 3 DENV strains studied are not due to differences in interactions with Ago2 during initial stages of mosquito infection. Disclosures All authors: No reported disclosures.

Wolbachiais a genus of intracellular alpha-Proteobacteria that is maternally inherited and is capable of inducing a variety of reproductive alterations in host species. The host range ofWolbachiais not determined completely but is known to contain a number of arthropod taxa, including crustaceans.Wolbachiahas not been reported in crayfish, but sampling has been limited to date. We examine a species of crayfish,Procambarus clarkii, forWolbachiainfection using a suite ofWolbachia-specific primers in PCR assays. All specimens yielded negative results forWolbachiainfection and mathematical analysis of sample size shows a near 100% probability of detection for populations with greater than 0.1% infection rate.

The hematophagous bug Triatoma rubida is a species of kissing bug that has been marked as a potential vector for the transmission of Chagas disease in the Southern United States and Northern Mexico. However, information on the distribution of T. rubida in these areas is limited. Vector monitoring is crucial to assess disease risk, so effective trapping systems are required. Kissing bugs utilize extrinsic cues to guide host-seeking, aggregation, and dispersal behaviors. These cues have been recognized as high-value targets for exploitation by trapping systems. A modern video-tracking system was used with a four-port olfactometer system to quantitatively assess the behavioral response of T. rubida to cues of known significance. Also, response of T. rubida adults to seven wavelengths of light-emitting diodes (LED) in paired-choice pitfall was evaluated. Behavioral data gathered from these experiments indicate that T. rubida nymphs orient preferentially to airstreams at either 1600 or 3200 ppm carbon dioxide and prefer relative humidity levels of about 30%, while adults are most attracted to 470 nm light. These data may serve to help design an effective trapping system for T. rubida monitoring. Investigations described here also demonstrate the experimental power of combining an olfactometer with a video-tracking system for studying insect behavior.

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall. The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics. In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

A simple sensor for viral particles based on ionic conductivity through anodized alumina membranes was demonstrated using MS2 bacteriophage as an example. A facile two-point measuring scheme is geared toward realization using a computer’s sound card input/output capabilities suitable for a fast and inexpensive point of care testing. The lowest detection concentration down to ∼7 pfu/mL and a large dynamic range up to ∼2000 pfu/mL were obtained due to physical optimization that included proper length and diameter for the pores, removing the oxide layer at the electrode, as well as the chemical optimization of covalent binding of antibodies to the pore’s walls.

The current Zika health crisis in the Americas has created an intense interest in mosquito control methods and products. Mosquito vectors of Zika are of the genus Aedes, mainly the yellow fever mosquito, Aedes aegypti. L. The use of repellents to alter mosquito host seeking behavior is an effective method for the prevention of mosquito-borne diseases. A large number of different spray-on repellents and wearable repellent devices are commercially available. The efficacies of many repellents are unknown. This study focuses on the efficacy of eleven different repellents in reducing the number of Ae. aegypti female mosquitoes attracted to human bait. We performed attraction-inhibition assays using a taxis cage in a wind tunnel setting. One person was placed upwind of the taxis cage and the mosquito movement towards or away from the person was recorded. The person was treated with various spray-on repellents or equipped with different mosquito repellent devices. We found that the spray-on repellents containing N,N-Diethyl-meta-toluamide and p-menthane-3,8-diol had the highest efficacy in repelling mosquitoes compared to repellents with other ingredients. From the five wearable devices that we tested, only the one that releases Metofluthrin significantly reduced the numbers of attracted mosquitoes. The citronella candle had no effect. We conclude that many of the products that we tested that were marketed as repellents do not reduce mosquito attraction to humans.

In this paper, we report an experimental demonstration of malaria pathogen detection in a whole blood lysate using plasmonic nanostructures. The plasmon sensor utilizes extraordinary optical transmission through a nanostructure to directly probe antibody-antigen interactions. The measured refractive index sensitivity of the nanostructured sensor is 378 nm per refractive index unit in the visible range. The surface chemistry reported here provides highly site directed and stable antibody immobilization. To validate the observed response of the optical sensor, positive and negative control tests were performed. Results confirm that a refractive index change induced by the interaction between immobilized antibodies and malaria parasites is successfully detected by the fabricated sensor. The demonstrated plasmonic sensor is a compact, highly sensitive, cost effective, selective diagnostic tool for many portable biosensing applications, such as point-of-care diagnostics.

Mosquito-borne diseases are responsible for more than a million human deaths every year. Modern mosquito control strategies such as sterile insect technique (SIT), release of insects carrying a dominant lethal (RIDL), population replacement strategies (PR), and Wolbachia-based strategies require the rearing of large numbers of mosquitoes in culture for continuous release over an extended period of time. Anautogenous mosquitoes require essential nutrients for egg production, which they obtain through the acquisition and digestion of a protein-rich blood meal. Therefore, mosquito mass production in laboratories and other facilities relies on vertebrate blood from live animal hosts. However, vertebrate blood is expensive to acquire and hard to store for longer times especially under field conditions. This review discusses older and recent studies that were aimed at the development of artificial diets for mosquitoes in order to replace vertebrate blood.

An important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs.We identified 11 putative SLC7 transporters in the genome sequence of Aedes aegypti. Phylogenetic analysis puts five of these in the cationic AA transporter subfamily (CAT) and six in the heterodimeric AA transporter (HAT) subfamily. All 11 A. aegypti SLC7 genes are expressed in adult females. Expression profiles are dynamic after a blood meal. We knocked down six fat body-expressed SLC7 transporters using RNAi and found that these 'knockdowns' reduced AA-induced TOR signaling. We also determined the effect these knockdowns had on the number of eggs deposited following a blood meal.Our analysis stresses the importance of SLC7 transporters in TOR signaling pathway and mosquito reproduction.

Insect yolk protein precursor gene expression is regulated by nutritional and endocrine signals. A surge of amino acids in the hemolymph of blood-fed female mosquitoes activates a nutrient signaling system in the fat bodies, which subsequently derepresses yolk protein precursor genes and makes them responsive to activation by steroid hormones. Orphan transporters of the SLC7 family were identified as essential upstream components of the nutrient signaling system in the fat body of fruit flies and the yellow fever mosquito, Aedes aegypti. However, the transport function of these proteins was unknown. We report expression and functional characterization of AaCAT1, cloned from the fat body of A. aegypti. Expression of AaCAT1 transcript and protein undergoes dynamic changes during postembryonic development of the mosquito. Transcript expression was especially high in the third and fourth larval stages; however, the AaCAT1 protein was detected only in pupa and adult stages. Functional expression and analysis of AaCAT1 in Xenopus oocytes revealed that it acts as a sodium-independent cationic amino acid transporter, with unique selectivity to l-histidine at neutral pH (K0.5l-His = 0.34 ± 0.07 mm, pH 7.2). Acidification to pH 6.2 dramatically increases AaCAT1-specific His+-induced current. RNAi-mediated silencing of AaCAT1 reduces egg yield of subsequent ovipositions. Our data show that AaCAT1 has notable differences in its transport mechanism when compared with related mammalian cationic amino acid transporters. It may execute histidine-specific transport and signaling in mosquito tissues.

BackgroundMosquito repellents can be an effective method for personal protection against mosquito bites that are a nuisance and carry the risk of transmission of mosquito-borne pathogens likeplasmodia, dengue virus, chikungunya virus, and Zika virus. A multitude of commercially available products are currently on the market, some of them highly effective while others have low or no efficacy. Many home remedies of unknown efficacy are also widely used.MethodsWe conducted a survey study to determine what kind of mosquito repellents and other mosquito control strategies people use. Our online survey was focused on unconventional methods and was answered by 5,209 participants.ResultsThe majority of participants resided in the United States, were female (67%), had higher education (81% had a university degree), and were 18 to 37 years old (50%). The most commonly used repellent was DEET spray (48%), followed closely by citronella candles (43%) and ‘natural’ repellent sprays (36%). We collected a plethora of home remedies and other strategies people use that warrant further research into their effectiveness.DiscussionOur study lays the foundation for future research in alternative, unconventional methods to repel mosquitoes that may be culturally acceptable and accessible for people.

Background The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses) may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal. Methodology/Principal findings We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses. Conclusion/Significance Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of Ae. albopictus.

In mosquitoes, yolk protein precursor (YPP) gene expression is activated after a blood meal through the synergistic action of a steroid hormone and the amino acid/Target of Rapamycin (TOR) signaling pathway in the fat body. We investigated the role of insulin signaling in the regulation of YPP gene expression. The presence of mosquito insulin receptor (InR) and the Protein kinase B (PKB/Akt) in the adult fat body of female mosquitoes was confirmed by means of the RNA interference (RNAi). Fat bodies stimulated with insulin were able to promote the phosphorylation of ribosomal S6 Kinase, a key protein of the TOR signaling pathway. Importantly, insulin in combination with 20-hydroxyecdysone activated transcription of the YPP gene vitellogenin (Vg), and this process was sensitive to the Phosphoinositide-3 kinase (PI-3k) inhibitor LY294002 as well as the TOR inhibitor rapamycin. RNAi-mediated knockdown of the mosquito InR, Akt, and TOR inhibited insulin-induced Vg gene expression as well as S6 Kinase phosphorylation in in vitro fat body culture assays.

Forkhead box (Fox) genes encode a family of transcription factors defined by a ‘winged helix’ DNA-binding domain. In this study we aimed to identify Fox factors that are expressed within the fat body of the yellow fever mosquito Aedes aegypti, and determine whether any of these are involved in the regulation of mosquito yolk protein gene expression. The Ae. aegypti genome contains eighteen loci that encode putative Fox factors. Our stringent cladistic analysis has profound implications for the use of Fox genes as phylogenetic markers. Twelve Ae. aegypti Fox genes are expressed within various tissues of adult females, six of which are expressed within the fat body. All six Fox genes expressed in the fat body displayed dynamic expression profiles following a blood meal. We knocked down the ’fat body Foxes’ through RNAi to determine whether these “knockdowns” hindered amino acid-induced vitellogenin gene expression. We also determined the effect of these knockdowns on the number of eggs deposited following a blood meal. Knockdown of FoxN1, FoxN2, FoxL, and FoxO, had a negative effect on amino acid- induced vitellogenin gene expression and resulted in significantly fewer eggs laid. Our analysis stresses the importance of Fox transcription factors in regulating mosquito reproduction.

Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino-acid transporter Slimfast (Slif) from the yellow-fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signalling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na+ dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction., Anautogenous mosquitoes need to obtain essential amino acids from a blood meal for reproduction. Here, the authors examine the amino acid transporter Slimfast from the yellow-fever mosquito and describe both its specificity and mechanism of action.

Background Environmental factors such as temperature, nutrient availability, and larval density determine the outcome of postembryonic development in mosquitoes. Suboptimal temperatures, crowding, and starvation during the larval phase reduce adult mosquito size, nutrient stores and affect vectorial capacity. Methods In this study we compared adult female Aedes aegypti, Rockefeller strain, raised under standard laboratory conditions (Large) with those raised under crowded and nutritionally deprived conditions (Small). To compare the gene expression and nutritional state of the major energy storage and metabolic organ, the fat body, we performed transcriptomics using Illumina based RNA-seq and metabolomics using GC/MS on females before and 24 hours following blood feeding. Results Analysis of fat body gene expression between the experimental groups revealed a large number of significantly differentially expressed genes. Transcripts related to immunity, reproduction, autophagy, several metabolic pathways; including amino acid degradation and metabolism; and membrane transport were differentially expressed. Metabolite profiling identified 60 metabolites within the fat body to be significantly affected between small and large mosquitoes, with the majority of detected free amino acids at a higher level in small mosquitoes compared to large. Conclusions Gene expression and metabolites in the adult fat body reflect the individual post-embryonic developmental history of a mosquito larva. These changes affect nutritional storage and utilization, immunity, and reproduction. Therefore, it is apparent that changes in larval environment due to weather conditions, nutrition availability, vector control efforts, and other factors can affect adult vectorial capacity in the field. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0863-9) contains supplementary material, which is available to authorized users.

After taking vertebrate blood, female mosquitoes quickly shed excess water and ions while retaining and concentrating the mostly proteinaceous nutrients. Aquaporins (AQPs) are an evolutionary conserved family of membrane transporter proteins that regulate the flow of water and in some cases glycerol and other small molecules across cellular membranes. In a previous study, we found six putative AQP genes in the genome of the yellow fever mosquito, Ae. aegypti, and demonstrated the involvement of three of them in the blood meal-induced diuresis. Here we characterized AQP expression in different tissues before and after a blood meal, explored the substrate specificity of AQPs expressed in the Malpighian tubules and performed RNAi-mediated knockdown and tested for changes in mosquito desiccation resistance. We found that AQPs are generally down-regulated 24 hrs after a blood meal. Ae. aegypti AQP 1 strictly transports water, AQP 2 and 5 demonstrate limited solute transport, but primarily function as water transporters. AQP 4 is an aquaglyceroporin with multiple substrates. Knockdown of AQPs expressed in the MTs increased survival of Ae. aegypti under dry conditions. We conclude that Malpighian tubules of adult female yellow fever mosquitoes utilize three distinct AQPs and one aquaglyceroporin in their osmoregulatory functions.

Control of insect species for the protection of crops, livestock, and prevention of disease such as dengue fever and malaria is a high priority in today’s global economy. Traditional methods such as pesticides have fallen out of favor because its effects are indiscriminate as well as adverse and unpredictable impacts on the environment. Modern novel techniques such as genetic modification have had trouble gaining traction due to ethics concerns and the potential for unforeseen side effects. One approach that has gained traction and has proven its efficacy is the use of ionizing radiation to affect sterility in insect species in order to scale back their population. Known as Sterile Insect Technique (SIT), it has proven very effective in eradicating certain dipteran insect populations. However, when standard sterilization methods developed for dipertans are applied to mosquito populations significant complications arise, such as an inability to compete with non-irradiated males and high mortality rates. We have investigated the effect of treatment with x-rays of different wavelengths on x-ray sterilized mosquito males. Our results have demonstrated that longer wavelength x-rays have a significant effect on the outcome of the sterile males’ longevity as well as an increase on the efficacy of sterilization while employing a substantially lower dose.

Recently, the bed bug, Cimex lectularius L. has re-emerged as a serious and growing problem in many parts of the world. Presence of resistant bed bugs and the difficulty to eliminate them has renewed interest in alternative control tactics. Similar to other haematophagous arthropods, bed bugs rely on their olfactory system to detect semiochemicals in the environment. Previous studies have morphologically characterized olfactory organs of bed bugs' antenna and have physiologically evaluated the responses of olfactory receptor neurons (ORNs) to host-derived chemicals. To date, odorant binding proteins (OBPs) and odorant receptors (ORs) associated with these olfaction processes have not been studied in bed bugs. Chemoreception in insects requires formation of heteromeric complexes of ORs and a universal OR coreceptor (Orco). Orco is the constant chain of every odorant receptor in insects and is critical for insect olfaction but does not directly bind to odorants. Orco agonists and antagonists have been suggested as high-value targets for the development of novel insect repellents. In this study, we have performed RNAseq of bed bug sensory organs and identified several odorant receptors as well as Orco. We characterized Orco expression and investigated the effect of chemicals targeting Orco on bed bug behavior and reproduction. We have identified partial cDNAs of six C. lectularius OBPs and 16 ORs. Full length bed bug Orco was cloned and sequenced. Orco is widely expressed in different parts of the bed bug including OR neurons and spermatozoa. Treatment of bed bugs with the agonist VUAA1 changed bed bug pheromone-induced aggregation behavior and inactivated spermatozoa. We have described and characterized for the first time OBPs, ORs and Orco in bed bugs. Given the importance of these molecules in chemoreception of this insect they are interesting targets for the development of novel insect behavior modifiers.

Anautogeny is a successful reproductive strategy utilized by many mosquito species and other disease-transmitting arthropod vectors. Developing an understanding of the mechanisms underlying anautogeny in mosquitoes is very important because this reproductive strategy is the driving force behind the transmission of disease to millions of people. Information gained from mosquito studies may also be applicable to other blood feeding insect vectors. The conversion of protein from blood into yolk protein precursors for the developing oocytes is an essential part of the reproductive cycle, and understanding how this process is regulated could lead to safe, specific, and effective ways to block reproduction in blood feeding insects. Great gains have been made in elucidating the mechanisms that regulate vitellogenesis in mosquitoes, especially Ae. aegypti. However, a number of questions remain to be answered to make the picture more complete. In this review, we summarize what is currently known about the nutritional regulation of vitellogenesis in mosquitoes and the questions that remain to be answered about this important biological phenomenon.

Female mosquitoes are effective disease vectors, because they take blood from vertebrate hosts to obtain nutrients for egg development. Amino acid signaling via the target of rapamycin (TOR) pathway has been identified as a key requirement for the activation of egg development after a blood meal. We report the characterization of the TOR kinase and one of its major downstream targets, S6 kinase, of the yellow fever mosquito Aedes aegypti during egg development in adult females. Both TOR and S6K mRNA are expressed at high levels in the ovaries and in lower levels in fat body and other tissues. After a blood meal, the subcellular localization of TOR shifts from the cytoplasm to the plasma membrane of fat body cells. By detecting phosphothreonine 388 of mosquito S6 kinase, we show that TOR activity strongly increases in fat body and ovaries after a blood meal in vivo. Furthermore, phosphorylation of S6 kinase increases in in vitro cultured fat bodies after stimulation with amino acids. This increase is sensitive to the TOR inhibitor rapamycin in a concentration-dependent manner but not to the phosphatidylinositol 3-kinase/phosphatidylinositol 3-kinase-related kinase inhibitor LY294002, the MAPK inhibitor PD98059, or the translational inhibitor cycloheximide. RNA interference-mediated reduction of S6 kinase strongly inhibits the amino acid-induced up-regulation of the major yolk protein vitellogenin in vitro and effectively disrupts egg development after a blood meal in vivo. Our data show that TOR-dependent activation of S6 kinase is a central step in the transduction of nutritional information during egg development in mosquitoes.

The activity of acid phosphatase (ACP) in insect fat bodies is stimulated by the steroid hormone 20-hydoxyecdysone (20E) in vivo. However, in fat bodies kept in culture, a factor from the hemolymph is required to enhance the ACP activity. We identified the factor as a protein with a molecular mass of 19 kDa (HP19) from the hemolymph of a lepidopteran insect, the rice moth, Corcyra cephalonica. Western analysis of hemolymph proteins with denaturing and non-denaturing PAGE using antibodies raised against HP19 suggest that this protein exists as a monomer. It is synthesized by the hind gut-associated lobular fat body of the larvae and is released into the hemolymph. The stimulatory effect of HP19 on the ACP activity is developmentally regulated and exhibits its maximal effect shortly before the onset of metamorphosis. We cloned the HP19 cDNA by immunoscreening a hind gut-associated lobular fat body cDNA expression library. Analysis of the amino acid sequence shows that HP19 belongs to the family of glutathione S-transferase (GST) like proteins. However, affinity-purified GST from Corcyra failed to show any mediation effect on 20E-stimulated ACP activity, and HP19 lacks GST enzymatic activity. Notably, HP19 mediates the hormone-stimulated ACP activity in intact fat body tissue and homogenates even in the presence of inhibitors of transcription and translation, suggesting a nongenomic mode of action. In addition, we show that HP19 inhibits the 20E-induced phosphorylation of the hexamerin receptor protein.