The impact of yeast-encapsulated orange oil in Aedes aegypti oviposition.

The yeast-encapsulated orange oil (YEOO) is a novel larvicide under development against vector mosquitoes. Despite its efficiency against Aedes aegypti (L.) in small scale experiments, its applicability in vector control can be influenced by other effects on mosquito behaviour or physiology. For this reason, the impact of YEOO particles in mosquito oviposition was evaluated in laboratory and semi-field conditions. Oviposition assays with one gravid Aedes aegypti female were carried under laboratory and semi-field conditions with natural light and temperature fluctuation. For all ovitraps, the number of eggs was manually counted in the wooden paddle and in the solution of each ovitrap. The proportion of eggs between substrates (wooden paddle and solution) varied between conditions, with females in laboratory presenting a lower preference to lay eggs in paddles when compared with studies in semi-field. This behaviour shifts in laboratory can create challenges to extrapolate results from laboratory to the field. Here, studies in both conditions indicate a similar impact of YEOO particles in Aedes aegypti oviposition. The potential treatment concentration of YEOO particles presents a strong repellent/deterrent effect (-0.559 > OAI > -0.760) within the initial 72h of application when compared with water, and weak repellent/deterrent signal (OAI = -0.220) when compared against inactivated yeast. Control ovitraps with water were more positive for egg presence than treated ovitraps, while ovitraps with YEOO particles and inactivated yeast present similar number of positive ovitraps. It is possible that the repellent/deterrent action is partially driven by the delivery system, since most times Citrus sinensis EO oviposition repellent/deterrent signal is weak, and it seem influenced by solvent/delivery used. However, it is unclear how the yeast wall that protect/surrounds the orange oil will negatively affect oviposition since live yeast are normally consider an attractant for mosquito oviposition.

Evaluation of Insect Growth Regulators (IGRs) as biological pesticides for control of Aedes aegypti mosquitoes.

BACKGROUND OBJECTIVES: Insect growth regulators (IGRs) are biological hormone analogue or mimics used as pesticides to inhibit the growth of larva during their molting and skin shedding. This study aimed to test the effect of IGRs on the eggs hatching and post-hatching inhibition of Aedes mosquitoes and understanding its effect in the mosquito breeding habitats for reduction in adult emergence. METHODS: Experiments on the evaluation of three insect growth regulators (IGRs) for the control of different stages of Aedes aegypti was carried out during 2020-21. Each experiment consisted of four treatments viz., Pyriproxyfen, Novaluron, and Larvicol at 1.0 ppm and distilled water as a control. All experiments were carried out in completely randomized design (CRD) except eggs which were carried out in factorial design each with three replications. RESULTS: All tested IGRs performed better in affecting eggs, larval and pupal stages of Ae. aegypti. Highest eggs hatching inhibition (80%) of fresh eggs occurred in Pyriproxyfen followed by Novaluron (66%) and lowest in Larvicol (62%). Eggs hatch inhibition of embryonated eggs was lower than fresh eggs. Pyriproxyfen caused 69%, Novaluron 59% and Larvicol 39% eggs hatch inhibition of embryonated eggs. Both Pyriproxyfen and Novaluron performed better in causing 98-100% larval mortality followed by Larvicol (39%). Larval development to pupal stage was completely prevented by both Pyriproxyfen and Novaluron. Although Larvicol resulted in lowest eggs hatch and larval inhibition but prevented pupae to emerge as adults. Results further showed 70-89% mortality of 3rd instar larvae of Ae. aegypti when exposed to Pyriproxyfen and Novaluron solutions after 30 days storage at lab. temperature (27±2°C), RH 70±5. INTERPRETATION CONCLUSION: None of the IGRs was more effective at the pupal stage but showed carry-on activity of growth inhibition and mortality of the successive stages of development when used against eggs stages. Therefore, we recommend early application of IGRs at mosquito habitats during the beginning and onset of the season when very early stages of mosquitoes are available in the field.

Biological effects of Lippia alba essential oil against Anopheles gambiae and Aedes aegypti.

The management of mosquito resistance to chemical insecticides and the biting behaviour of some species are motivating the search for complementary and/or alternative control methods. The use of plants is increasingly considered as a sustainable biological solution for vector control. The aim of this study was to evaluate the biological effects of the essential oil (EO) of Lippia alba harvested in Abidjan (Côte d'Ivoire) against Anopheles gambiae and Aedes aegypti mosquitoes. Phytochemical compounds were identified by GC-MS. Knockdown and mortality were determined according to the WHO test tube protocol. Contact irritancy was assessed by observing the movement of mosquitoes from a treated WHO tube to a second untreated tube. Non-contact repellency was assessed using a standardised high-throughput screening system (HITSS). Blood meal inhibition was assessed using a membrane feeding assay treated with EO. The EO was identified as the citral chemotype. The EO gave 100% KD60 in both species at a concentration of 1%. Mortalities of 100% were recorded with An. gambiae and Ae. aegypti at concentrations of 1% and 5% respectively. The highest proportions of females escaping during the contact irritancy test were 100% for An. gambiae at 1% concentration and 94% for Ae. aegypti at 2.5% concentration. The 1% concentration produced the highest proportions of repelled mosquitoes in the non-contact repellency tests: 76.8% (An. gambiae) and 68.5% (Ae. aegypti). The blood meal inhibition rate at a dose of 10% was 98.4% in Ae. aegypti but only 15.5% in An. gambiae. The citral chemotype of L. alba EO has promising biological effects in both species that make it a potentially good candidate for its use in mosquito control. The results obtained in this study encourage the further evaluation of L. alba EOs from other localities and of different chemotypes, under laboratory and field conditions.

Adenosine triphosphate overrides the aversive effect of antifeedants and toxicants: a model alternative phagostimulant for sugar-based vector control tools.

BACKGROUND: Sugar, when used as the phagostimulant in attractive toxic bait control tools, limits the efficacy and selectivity of this technology. Thus, more potent and selective phagostimulants than sugar are required to improve this technology. The potency of adenosine triphosphate (ATP) as an alternative model phagostimulant was assessed to determine its capacity to override the aversive effects of select antifeedants and toxicants. How ATP and sucrose modulate the rate of toxicity in the yellow fever mosquito Aedes aegypti was also examined. METHODS: A no-choice feeding assay was used to investigate the phagostimulatory ability of ATP to override the aversive effects of structurally divergent antifeedant and toxicant compounds, and to modulate the rate of toxicity over 24 h. Binary combinations of antifeedant and toxicant compounds, at various concentrations, were similarly assessed for enhanced lethal potency. In comparison, no-choice open access and cotton wick feeding assays were used to determine the phagostimulatory role of sucrose in the ingestion of boric acid-laced diets. Dissections of the guts were performed to determine the diet destination as dependant on the phagostimulant. RESULTS: ATP is a potent phagostimulant that dose dependently overrides aversion to antifeedant and toxicant tastants. Feeding on antifeedant- or toxicant-laced diets that was induced by ATP selectively resulted in rapid knockdown (nicotine, lobeline and caffeine) or death (boric acid and propylene glycol), with a combination of the two lethal compounds inducing a synergistic effect at lower concentrations. ATP- and sucrose-induced feeding predominantly directed the antifeedant- or toxicant-laced meals to the midgut and the crop, respectively. CONCLUSIONS: ATP is an efficacious alternative model phagostimulant to sucrose that overrides the aversive effects of antifeedants and toxicants, resulting in rapid toxic effects. Furthermore, this study demonstrates that variation in the rate of toxicity between ATP- and sugar-induced feeding is at least partly regulated by the differential feeding response, volume imbibed and the destination of the meals. Additional research is needed to identify structurally related, stable analogues of ATP due to the ephemeral nature of this molecule. For future applications, the workflow presented in this study may be used to evaluate such analogues for their suitability for use in attractive bait stations designed to target a broad range of haematophagous arthropods and prevent off-target species' feeding.

Molecular and expression characterization of insulin-like signaling in development and metabolism of Aedes albopictus.

BACKGROUND: Insulin-like signaling (IS) in insects is a conserved pathway that regulates development, reproduction and longevity. Insulin-like peptides (ILPs) activate the IS pathway by binding to the insulin receptor (InR) and trigger the ERK and AKT cascades. A varying number of ILPs were identified in Aedes aegypti mosquito and other insects. Aedes albopictus is an invasive mosquito which transmits dengue and Zika viruses worldwide. Until now, the molecular and expression characteristics of IS pathway in Ae. albopictus have not been investigated. METHODS: The orthologues of ILP in Ae. albopictus genome assembly was analyzed by using sequence blast. Phylogenetic analysis and molecular characterization were performed to identify the functional domains of ILPs. Quantitative analysis was performed to determine the expression characteristics of ILPs, InR as well as ERK and AKT in mosquito development and different tissues of female adults after blood-feeding. In addition, the knockdown of InR was achieved by feeding larvae with Escherichia coli-producing dsRNA to investigate the impact of IS pathway on mosquito development. RESULTS: We identified seven putative ILP genes in Ae. albopictus genome assembly, based on nucleotide similarity to the ILPs of Ae. aegypti and other insects. Bioinformatics and molecular analyses suggested that the ILPs contain the structural motif which is conserved in the insulin superfamily. Expression levels of ILPs, InR as well as ERK and AKT varied in Ae. albopictus development stages and between male and female adults. Quantitative analyses revealed that expression of ILP6, the putative orthologue of the insulin growth factor peptides, was highest in the midgut of female adults after blood-feeding. Knockdown of Ae. albopictus InR induces a significant decrease in the phosphorylation levels of ERK and AKT proteins and results in developmental delays and smaller body sizes. CONCLUSIONS: The IS pathway of Ae. albopictus mosquito contains ILP1-7, InR and ERK/AKT cascades, which exhibited different developmental and tissue expression characteristics. Feeding Ae. albopictus larvae with E. coli-producing InR dsRNA blocks the ERK and AKT cascades and interferes with the development of mosquito. Our data suggest that IS pathway plays an important role in the metabolism and developmental process and could represent a potential target for controlling mosquito-borne diseases.

Developing the radiation-based sterile insect technique (SIT) for controlling Aedes aegypti: identification of a sterilizing dose.

BACKGROUND: The sterile insect technique (SIT) is emerging as a tool to supplement traditional pesticide-based control of Aedes aegypti, a prominent mosquito vector of microbes that has increased the global burden of human morbidity and mortality over the past 50 years. SIT relies on rearing, sterilizing and releasing large numbers of male mosquitoes that will mate with fertile wild females, thus reducing production of offspring from the target population. In this study, we investigated the effects of ionizing radiation (gamma) on male and female survival, longevity, mating behavior, and sterility of Ae. aegypti in a dose-response design. This work is a first step towards developing an operational SIT field suppression program against Ae. aegypti in St. Augustine, Florida, USA. RESULTS: Exposing late-stage pupae to 50 Gy of radiation yielded 99% male sterility while maintaining similar survival of pupae to adult emergence, adult longevity and male mating competitiveness compared to unirradiated males. Females were completely sterilized at 30 Gy, and when females were dosed with 50 Gy, they had a lower incidence of blood-feeding than unirradiated females. CONCLUSION: Our work suggests that an ionizing radiation dose of 50 Gy should be used for future development of operational SIT in our program area because at this dose males are 99% sterile while maintaining mating competitiveness against unirradiated males. Furthermore, females that might be accidentally released with sterile males as a result of errors in sex sorting also are sterile and less likely to blood-feed than unirradiated females at our 50 Gy dose. © 2022 Society of Chemical Industry.

Aedes aegypti (L.) and Anopheles stephensi Liston (Diptera: Culicidae) Susceptibility and Response to Different Experimental Formulations of a Sodium Ascorbate Toxic Sugar Bait.

Attractive toxic sugar baits (ATSBs) require target insects to locate, orient toward, and feed on an insecticidal sugar solution to control populations. Formulating these baits with different attractants and phagostimulants can increase their efficacy by causing insects to choose the ATSB over competing natural sugar sources, and to ingest more of the bait solution. We tested formulations of a 20% sodium ascorbate (SA) ATSB solution using different sugars, adenosine triphosphate (ATP), gallic acid, and six plant volatile compounds to determine their effect on adult Aedes aegypti (L.) and Anopheles stephensi Liston mortality. Baits formulated with fructose or sucrose had no effect on either species, neither did the addition of ATP. Gallic acid increased the survival of Ae. aegypti. Four of the six volatile compounds increased mortality in at least one species. We also examined An. stephensi response to baits formulated with each of the six volatile compounds. Anisaldehyde significantly increased the number of mosquitoes responding toward the SA-ATSB, but increasing the amount had no effect. Addition of anisaldehyde also significantly increased An. stephensi feeding rates on the SA-ATSB, though mosquitoes will avoid the toxic bait if a nontoxic sugar source is available. Formulation of SA-ATSBs with synthetic blends of attractive compounds can increase bait efficacy and consistency, though further research is needed to assess their performance in the field in the presence of natural sugar sources.

Essential functions of mosquito ecdysone importers in development and reproduction.

The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. Here we report three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, -3, and -4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, our results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.

Ad libitum consumption of protein- or peptide-sucrose solutions stimulates egg formation by prolonging the vitellogenic phase of oogenesis in anautogenous mosquitoes.

BACKGROUND: Anautogenous mosquitoes commonly consume nectars and other solutions containing sugar but are thought to only produce eggs in discrete gonadotrophic cycles after blood-feeding on a vertebrate host. However, some anautogenous species are known to produce eggs if amino acids in the form of protein are added to a sugar solution. Unclear is how different sources of amino acids in sugar solutions affect the processes that regulate egg formation and whether responses vary among species. In this study, we addressed these questions by focusing on Aedes aegypti and conducting some comparative assays with Aedes albopictus, Anopheles gambiae, Anopheles stephensi and Culex quinquefasciatus. METHODS: Adult female mosquitoes were fed sugar solutions containing amino acids, peptides or protein. Markers for activation of a gonadotrophic cycle including yolk deposition into oocytes, oviposition, ovary ecdysteroidogenesis, expression of juvenile hormone and 20-hydroxyecdysone-responsive genes, and adult blood-feeding behavior were then measured. RESULTS: The five anautogenous species we studied produced eggs when fed two proteins (bovine serum albumin, hemoglobin) or a mixture of peptides (tryptone) in 10% sucrose but deposited only small amounts of yolk into oocytes when fed amino acids in 10% sucrose. Focusing on Ae. aegypti, cultures were maintained for multiple generations by feeding adult females protein- or tryptone-sugar meals. Ad libitum access to protein- or tryptone-sugar solutions protracted production of ecdysteroids by the ovaries, vitellogenin by the fat body and protease activity by the midgut albeit at levels that were lower than in blood-fed females. Females also exhibited semi-continual oogenesis and repressed host-seeking behavior. CONCLUSIONS: Several anautogenous mosquitoes produce eggs when provided ad libitum access to protein- or peptide-sugar meals, but several aspects of oogenesis also differ from females that blood-feed.

ATP-sensitive inward rectifier potassium channels reveal functional linkage between salivary gland function and blood feeding in the mosquito, Aedes aegypti.

Reducing saliva secretions into the vertebrate host reduces feeding efficacy by most hematophagous arthropods. However, seminal studies suggested saliva is not a prerequisite for blood feeding in Aedes aegypti. To test this paradigm, we manually transected the salivary duct of female A. aegypti and an inability to salivate was correlated to an inability to imbibe blood. These data justified testing the relevance of inwardly rectifying potassium (Kir) channels in the A. aegypti salivary gland as an antifeedant target site. Pharmacological activation of ATP-gated Kir (KATP) channels reduced the secretory activity of the salivary gland by 15-fold that led to near elimination of blood ingestion during feeding. The reduced salivation and feeding success nearly eliminated horizontal transmission and acquisition of Dengue virus-2 (DENV2). These data suggest mosquito salivation is a prerequisite for blood feeding and provide evidence that KATP channels are critical for salivation, feeding, and vector competency.

Insulin-like peptide 3 stimulates hemocytes to proliferate in anautogenous and facultatively autogenous mosquitoes.

Most mosquito species are anautogenous, which means they must blood feed on a vertebrate host to produce eggs, while a few are autogenous and can produce eggs without blood feeding. Egg formation is best understood in the anautogenous mosquito Aedes aegypti, where insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH) and 20-hydroxyecdysone (20E) interact to regulate gonadotrophic cycles. Circulating hemocytes also approximately double in abundance in conjunction with a gonadotrophic cycle, but the factors responsible for stimulating this increase remain unclear. Focusing on Ae. aegypti, we determined that hemocyte abundance similarly increased in intact blood-fed females and decapitated blood-fed females that were injected with ILP3, whereas OEH, 20E or heat-killed bacteria had no stimulatory activity. ILP3 upregulated insulin-insulin growth factor signaling in hemocytes, but few genes - including almost no transcripts for immune factors - were differentially expressed. ILP3 also stimulated circulating hemocytes to increase in two other anautogenous (Anopheles gambiae and Culex quinquefasciatus) and two facultatively autogenous mosquitoes (Aedes atropalpus and Culex pipiens molestus), but had no stimulatory activity in the obligately autogenous mosquito Toxorhynchites amboinensis. Altogether, our results identify ILPs as the primary regulators of hemocyte proliferation in association with egg formation, but also suggest this response has been lost in the evolution of obligate autogeny.

Cananga odorata (Magnoliales: Annonaceae) Essential Oil Produces Significant Avoidance Behavior in Mosquitoes.

Essential oil of Cananga odorata Hook. F. & Tomson is a source of insect repellent, but contact irritancy and noncontact repellency actions that stimulate insect's avoidance behavior (escape away from chemical source after direct physical contact or without making physical contact, respectively) have not been investigated. Therefore, an excito-repellency test chamber was used for measuring avoidance behavior of four insectary-reared mosquito species (Diptera: Culicidae) that escape from esposure to four concentrations (0.5, 1.0, 2.5, and 5.0% v/v) of C. odorata oil. The oil strongly repelled both Culex quinquefasciatus Say (85-97% escape) and Anopheles minimus Theobald (97-99%) at high concentrations (2.5-5.0%). For Anopheles dirus Peyton & Harrison and Aedes aegypti (L.), highest repellency (64 and 39% escape, respectively) was demonstrated at 2.5% concentration. For contact irritancy, the oil produced relatively high percent escape found in Cx. quinquefasciatus (90-100% escape) and An. minimus (83-100%). Whereas moderate contact irritancy was observed against An. dirus (40-50% escape) and Ae. aegypti (51-59%). The percent escape was then adjusted with repellency to estimate the effect of contact irritancy alone. We found that highest contact irritancy was presented at 0.5% concentration against An. minimus (67% escape). Knockdown and toxic actions were only found in Anopheles mosquitoes at 5.0% concentration. The results revealed that An. minimus and Cx. quinquefasciatus were more prone to be repelled by C. odorata oil. Detailed analysis of oil identified primary compounds as methyl benzoate (14.6%), α-gurjunene (12.8%), p-methyl-anisole (11.3%), and benzyl acetate (9.9%). Further investigations are needed to assess excito-repellency actions of these compounds alone or in combination.

Arboalvo: estratificação territorial para definição de áreas de pronta resposta para vigilância e controle de arboviroses urbanas em tempo oportuno / Arboalvo: territorial stratification for definition of areas for prompt response by surveillance and timely control of urban arbovirus infections / Arboalvo: estratificación territorial para la definición de áreas de respuesta rápida para la vigilancia y control de arbovirosis urbanas en el momento oportuno

O objetivo deste trabalho foi apresentar a proposta metodológica denominada de "Pronta Resposta" modelada nas cidades de Belo Horizonte (Minas Gerais) e Natal (Rio Grande do Norte), Brasil. A metodologia visa identificar e delimitar áreas prioritárias para o direcionamento das ações de vigilância em tempo oportuno, buscando a redução da intensidade e velocidade da dispersão de epidemias em áreas urbanas endêmicas. Para tanto, a metodologia utiliza três variáveis, que representam as causas necessárias para a produção e reprodução da dengue: casos notificados (vírus), ovos de Aedes (vetor) e população (hospedeiro). Trata-se de um estudo ecológico que utilizou os dados dos três planos de informações agregados em escalas temporais e espaciais mais finas, de três a quatro semanas e grades de 400 a 600 metros respectivamente. As áreas de pronta resposta foram definidas por meio de análise estatística de varredura Scan, com definição de clusters espaciais simultâneos para os três planos por meio do programa SaTScan. Os resultados observados foram: na cidade de Natal, as áreas definidas como pronta resposta ocuparam em média 15,2% do território do município e concentraram 67,77% dos casos de dengue do período posterior ao utilizado na delimitação das áreas de pronta resposta, e em Belo Horizonte, os números observados foram de 64,16% dos casos em 23,23% do território. Esses resultados foram obtidos em duas cidades com realidades socioambientais e geográficas diferentes e com perfis epidemiológicos também distintos, apontando que a metodologia pode ser aplicada em diferentes realidades urbanas, criando a possibilidade de os programas de controle atuarem em porções reduzidas do território e impactar num alto percentual de casos em tempo oportuno.

The study aimed to present the methodological proposal entitled "Prompt Response", modelled in the cities of Belo Horizonte (Minas Gerais State) and Natal (Rio Grande do Norte State), Brazil. The proposal aims to identify and demarcate priority areas for timely targeting of surveillance activities, aiming to reduce the intensity and velocity in the spread of epidemics in endemic urban areas. The methodology uses three variables that represent the necessary causes for the production and reproduction of dengue: notified cases (virus), Aedes eggs (vector), and population (host). This was an ecological study that used data from three information planes aggregated in finer temporal and spatial scales of 3 to 4 weeks and 400 to 600-meter grids, respectively. The prompt response areas were defined by Scan statistical analysis with definition of simultaneous spatial clusters for the three planes via the SaTScan program. In Natal, the areas defined as prompt response occupied, on average, 15.2% of the city's territory and concentrated 67.77% of the dengue cases in the period following demarcation of the prompt response areas. In Belo Horizonte, the observed proportions were 64.16% of cases in 23.23% of the territory. These results were obtained in two cities with different socioenvironmental and geographic realities and distinct epidemiological profiles, indicating that the methodology can be applied to different urban realities, allowing control programs to concentrate on reduced portions of the territory and impacting a high percentage of cases in timely fashion.

El objetivo del trabajo fue presentar la propuesta metodológica, denominada de "Resposta Rápida", modelada en las ciudades de Belo Horizonte (Minas Gerais) y Natal (Rio Grande do Norte), Brasil. Esta última tiene como meta identificar y delimitar áreas prioritarias para la ejecución de acciones de vigilancia en el momento oportuno, buscando la reducción de la intensidad y velocidad de la dispersión de epidemias en áreas urbanas endémicas. Para tal fin, la metodología utiliza tres variables, que representan las causas necesarias para la producción y reproducción del dengue: casos notificados (virus), huevos de Aedes (vector) y población (huésped). Se trata de un estudio ecológico que utilizó los datos de los tres planos de información agregados en escalas temporales y espaciales más finas, de 3 a 4 semanas y tablas de 400 a 600 metros respectivamente. Las áreas de respuesta rápida se definieron a través del análisis estadístico de exploración Scan, con definición de clústeres espaciales simultáneos para los tres planos mediante el programa SaTScan. Los resultados observados fueron: en la ciudad de Natal, las áreas definidas como de respuesta rápida ocuparon de media un 15,2% del territorio del municipio y concentraron un 67,77% de los casos de dengue del período posterior al utilizado en la delimitación de las áreas de respuesta rápida y, en Belo Horizonte, los números observados fueron un 64,16% de los casos en un 23,23% del territorio. Estos resultados se obtuvieron en dos ciudades con realidades socioambientales y geográficas diferentes y con perfiles epidemiológicos también distintos, apuntando que la metodología se puede aplicar en diferentes realidades urbanas. Dando la posibilidad de que los programas de control actúen en secciones reducidas del territorio e impactar en un alto porcentaje de casos en el momento oportuno.

High resolution proteomics of Aedes aegypti salivary glands infected with either dengue, Zika or chikungunya viruses identify new virus specific and broad antiviral factors.

Arboviruses such as dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses infect close to half a billion people per year, and are primarily transmitted through Aedes aegypti bites. Infection-induced changes in mosquito salivary glands (SG) influence transmission by inducing antiviral immunity, which restricts virus replication in the vector, and by altering saliva composition, which influences skin infection. Here, we profiled SG proteome responses to DENV serotype 2 (DENV2), ZIKV and CHIKV infections by using high-resolution isobaric-tagged quantitative proteomics. We identified 218 proteins with putative functions in immunity, blood-feeding or related to the cellular machinery. We observed that 58, 27 and 29 proteins were regulated by DENV2, ZIKV and CHIKV infections, respectively. While the regulation patterns were mostly virus-specific, we separately depleted four uncharacterized proteins that were upregulated by all three viral infections to determine their effects on these viral infections. Our study suggests that gamma-interferon responsive lysosomal thiol-like (GILT-like) has an anti-ZIKV effect, adenosine deaminase (ADA) has an anti-CHIKV effect, salivary gland surface protein 1 (SGS1) has a pro-ZIKV effect and salivary gland broad-spectrum antiviral protein (SGBAP) has an antiviral effect against all three viruses. The comprehensive description of SG responses to three global pathogenic viruses and the identification of new restriction factors improves our understanding of the molecular mechanisms influencing transmission.

Lineage Divergence and Vector-Specific Adaptation Have Driven Chikungunya Virus onto Multiple Adaptive Landscapes.

Previous studies have shown that the adaptation of Indian Ocean lineage (IOL) chikungunya virus (CHIKV) strains for Aedes albopictus transmission was mediated by an E1-A226V substitution, followed by either a single substitution in E2 or synergistic substitutions in the E2 and E3 envelope glycoproteins. Here, we examined whether Asian lineage strains, including those that descended from the 2014 Caribbean introduction, are likely to acquire these A. albopictus-adaptive E2 substitutions. Because Asian lineage strains cannot adapt through the E1-A226V substitution due to an epistatic constraint, we first determined that the beneficial effect of these E2 mutations in IOL strains is independent of E1-A226V. We then introduced each of these E2 adaptive mutations into the Asian lineage backbone to determine if they improve infectivity for A. albopictus. Surprisingly, our results indicated that in the Asian lineage backbone, these E2 mutations significantly decreased CHIKV fitness in A. albopictus. Furthermore, we tested the effects of these mutations in Aedes aegypti and observed different results from those in A. albopictus, suggesting that mosquito species-specific factors that interact with the envelope proteins are involved in vector infection efficiency. Overall, our results indicate that the divergence between Asian lineage and IOL CHIKVs has led them onto different adaptive landscapes with differing potentials to expand their vector host range. IMPORTANCE Since its introduction into the Caribbean in October 2013, CHIKV has rapidly spread to almost the entire neotropical region. However, its potential to further spread globally, including into more temperate climates, depends in part on its ability to be transmitted efficiently by Aedes albopictus, which can survive colder winters than A. aegypti. We examined in an Asian lineage backbone A. albopictus-adaptive mutations that arose from 2005 to 2009 in Indian Ocean lineage (IOL) strains. Our results predict that the Asian CHIKV lineage now in the Americas will not readily adapt for enhanced A. albopictus transmission via the same mechanisms or adaptive mutations used previously by IOL strains. The vector species- and CHIKV lineage-specific effects caused by adaptive CHIKV envelope glycoprotein substitutions may elucidate our understanding of the mechanisms of mosquito infection and spread.

Recording central neurophysiological output from mosquito larvae for neuropharmacological and insecticide resistance studies.

Resistance to currently utilized chemical insecticidal agents represents a significant threat to public health and food security worldwide. Better understanding the neurophysiological effects of available and candidate insecticidal molecules is valuable for characterizing the mechanisms of insecticide resistance, as well as the design and study of novel control chemistries. In this paper, we describe a method of recording nerve firing from the central nervous system of Aedes aegypti fourth instar larvae. In short, mosquito larvae were immobilized by placing small pins through the head and siphon of the larvae in a wax dish, ventral side down. A single, longitudinal, dorsal incision from the distal abdomen to the pronotum of the larva was made, the alimentary canal removed, and the ventral nerve cord severed between the second and third abdominal ganglia. A recording suction electrode was connected directly to axons within the severed end of the connective in a novel way to record nerve firing in the ventral nerve cord at a high signal-to-noise ratio with conventional electrophysiological equipment. Using this novel method, we report the effects of four neuroactive compounds using this method: octopamine, pilocarpine, nicotine, and γ-aminobutyric acid (GABA). The utility of this recording technique for elucidating target site mechanisms involved in insecticide resistance is demonstrated with p,p'-dichlorodiphenyltrichlorethane (DDT) and its difluoro analog (DFDT).

Radio-frequency exposure of the yellow fever mosquito (A. aegypti) from 2 to 240 GHz.

Fifth generation networks (5G) will be associated with a partial shift to higher carrier frequencies, including wavelengths comparable in size to insects. This may lead to higher absorption of radio frequency (RF) electromagnetic fields (EMF) by insects and could cause dielectric heating. The yellow fever mosquito (Aedes aegypti), a vector for diseases such as yellow and dengue fever, favors warm climates. Being exposed to higher frequency RF EMFs causing possible dielectric heating, could have an influence on behavior, physiology and morphology, and could be a possible factor for introduction of the species in regions where the yellow fever mosquito normally does not appear. In this study, the influence of far field RF exposure on A. aegypti was examined between 2 and 240 GHz. Using Finite Difference Time Domain (FDTD) simulations, the distribution of the electric field in and around the insect and the absorbed RF power were found for six different mosquito models (three male, three female). The 3D models were created from micro-CT scans of real mosquitoes. The dielectric properties used in the simulation were measured from a mixture of homogenized A. aegypti. For a given incident RF power, the absorption increases with increasing frequency between 2 and 90 GHz with a maximum between 90 and 240 GHz. The absorption was maximal in the region where the wavelength matches the size of the mosquito. For a same incident field strength, the power absorption by the mosquito is 16 times higher at 60 GHz than at 6 GHz. The higher absorption of RF power by future technologies can result in dielectric heating and potentially influence the biology of this mosquito.

Actions of Camptothecin Derivatives on Larvae and Adults of the Arboviral Vector Aedes aegypti.

Mosquito-borne viruses including dengue, Zika, and Chikungunya viruses, and parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe, and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility. We identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that reduced A. aegypti larval motility. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC50 of 25.5 ± 5.0 µM and 22.3 ± 5.4 µM, respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to A. aegypti adults at 100 µM and 10 µM, and completely blocked egg laying when fed at 100 µM. Camptothecin and its derivatives are inhibitors of topoisomerase I, have known activity against several agricultural pests, and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries.

Microencapsulation Preservation of the Stability and Efficacy of Citrus Grandis Oil-Based Repellent Formulation against Aedes aegypti during Storage.

Essential oils have been widely used as an active ingredient in mosquito repellent products. However, essential oils are highly unstable and prone to degradation when exposed to the environment during storage. Microencapsulation techniques help to maintain the stability of molecules in essential oils that are sensitive to environmental stress, and therefore improve shelf life. In this study, the physical stability and efficacy of a repellent formulation consisting of encapsulated Citrus grandis essential oil (CGEO) were evaluated under different storage conditions over a 12-month period by comparing the formulation with a non-encapsulated formulation. The formulations were both stored under two different storage conditions, i.e., 25 ± 2 °C/60% ± 5% relative humidity (RH) and 40 ± 2 °C/75% RH ± 5%, for 12 months. Droplet size, zeta potential, and pH value were measured after 1, 6, and 12 months of storage to determine their stability. For the study of efficacy, each formulation was tested against Aedes aegypti under laboratory conditions. We found that the microencapsulated formulation's physical characteristics showed insignificant changes as compared with the non-encapsulated formulation during storage. The microencapsulated formulation demonstrated better repellent effects, sustaining high protection (>80%) for 4 more hours of exposure after 12 months of storage as compared with the non-encapsulated formulation that demonstrated high protection for only an hour post application. Microencapsulation helped to preserve the stability of the formulation, which resulted in high protection being maintained for over 12 months of storage.

Neurochemical regulation of Aedes aegypti salivary gland function.

The salivary gland of hematophagous arthropods is critical for blood meal acquisition, blood vessel localization, and secretion of digestive enzymes. Thus, there is significant interest in the regulation of salivary gland function and mechanisms driving the secretion of saliva and digestive proteins. We aimed to gain a broader understanding of the regulatory role of aminergic, cholinergic, and octopaminergic neuromodulators to saliva and protein secretion from the female A. aegypti salivary gland. Quantification of saliva after injection with neuromodulators showed that dopamine, serotonin, and pilocarpine increased the secretory activity of the salivary gland with potency rankings dopamine = serotonin > pilocarpine. No change in saliva secretion was observed with octopamine or ergonovine, which indicates the A. aegypti salivary gland may be regulated by dopaminergic, serotonergic, and cholinergic systems, but are not likely regulated by octopaminergic or tryptaminergic systems. Next, we studied the regulatory control of dopamine-mediated salivation. Data indicate extracellular calcium flux, but not neural function, is critical for dopamine-mediated salivation, which suggests epithelial transport of ions and not neuronal control is responsible for dopamine-mediated salivation. For regulation of protein secretion, data indicate dopamine or serotonin exposure facilitates amylase secretion, whereas serotonin but not dopamine exposure increased apyrase concentrations in the secreted saliva. General immunoreactivity to anti-rat D1-dopamine receptor antibody was observed, yet immunoreactivity to the anti-rat D2-receptor antibody was identified in the proximal regions of the lateral lobes and slight immunoreactivity in the distal portion of the lateral lobe, with no expression in the medial lobe.