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Gene-editing technologies have revolutionized the field of mosquito sensory biology. These technologies have been used to knock in reporter genes in-frame with neuronal genes and tag specific mosquito neurons to detect their activities using binary expression systems. Despite these advances, novel tools still need to be developed to elucidate the transmission of olfactory signals from the periphery to the brain. Here, we propose the development of a set of tools, including novel driver lines as well as sensors of neuromodulatory activities, which can advance our knowledge of how sensory input triggers behavioral outputs. This information can change our understanding of mosquito neurobiology and lead to the development of strategies for mosquito behavioral manipulation to reduce bites and disease transmission.

Pyrethroid insecticides frequently contaminate urban runoff and are transported to downstream waterways. Due to their high hydrophobicity, these insecticides have a propensity to partition from surface water into sediment, where they may be slow to degrade. Past studies have shown adverse impacts to aquatic ecosystems, where many fish and invertebrates are especially susceptible to pyrethroids. Apart from non-target toxicity, pyrethroid pollution risks exposure to larval mosquitoes, which are nuisance pests and vectors for disease across the world. Urban species of mosquitoes often lay their eggs in underground storm drain systems due to their tendency to accumulate standing water and detritus. In this exposure scenario, selection pressure may be exerted on populations of larvae, increasing their resistance to pyrethroids. Pyrethroids are one of the few types of insecticides used for adult mosquito control in the United States, and a reduction of their efficacy imposes costs on pest management operations. Though pyrethroid contamination of urban water bodies, like streams, is well-documented, specific information on their occurrence within storm drain systems along with their influence on current mosquito populations is lacking. In the first study of this dissertation, the prevalence of pyrethroid residues in water from urban catch basins, which are storm drain structures that accept runoff, was determined. Results showed that pyrethroids were present in nearly every sample, with the pyrethroid bifenthrin being the most frequently detected analyte. In the second study, pyrethroid distribution across catch basins, open channels, and stormwater outfalls in a storm drain system was assessed. Pyrethroids were detected in every sediment, algae, and biofilm sample, and bifenthrin concentrations in catch basin water were notably elevated compared to those in other structures. In the third study, larval mosquitoes of strains established from field-collected populations were

This research synthesizes and extends the large body of work on the distribution, ecology, and physiology of the three mosquito vectors of Ross River virus, Culex annulirostris Skuse, Aedes vigilax (Skuse) and Aedes camptorhynchus (Thomson), using mathematical modelling and simulation methods. Computational techniques developed for malaria vector development and physiological research of other mosquito-borne disease vectors have been adapted and applied to a temperate saltmarsh environment located in the Swan River catchment in Southwest Western Australia. The simulation has two compartments, a hydrological compartment which predicts water-body height and water temperature using a one-layer energy budget model developed for small scale shallow-water bodies and modifies it to minimise error attributable to parametrisation of latent heat flux. Real world performance was assessed against on-site field measurement for water depth and temperature, and against three other methods for estimation of water temperature; the original unmodified energy balance model, estimation of water temperature based on air temperature and waterbody volume, and by using air-temperature as a direct proxy for water temperature. The second compartment models mosquito development from egg through larval and adult stages, using a series of equations. Species-specific physiological parameter values were applied from the large body of laboratory and field studies for these three mosquito species. The main driver of mosquito development examined was thermal development. The simulation model accurately predicts hourly shallow-water body temperature, and the timing and relative magnitude of mosquito population peaks for the three species across seasons and years. The main limitations identified were due to insufficient studies on the thermal development and mortality limits, particularly for the two Aedes species. The model is sensitive to the minimum egg-hatching temperature thresholds and maximum t

Aedes albopictus is an invasive mosquito species that can maintain and transmit several arboviruses causing disease in humans. Understanding the determinants of its ecology and population dynamics to predict its abundance was the main objective of this study. Adult mosquitoes were captured weekly between 2009 and 2016 with BG sentinel traps baited with BG-Lure outdoors at a collection site within the urban area of Palermo (southern Italy). In parallel, between 2012 and 2016, we monitored the uninterrupted weekly abundance of Ae. albopictus at four additional sites nearby over an area of about two hectares. Catches were collected three times per week and mosquitoes were identified morphologically. To identify the determinants of mosquito abundance, seasonal autoregressive integrated moving-average and Poisson regression models were fitted to the weekly abundance of Ae. albopictus with a series of weather predictors that potentially modulate its activity and population dynamics. The time lag of the influence of predictors was analysed to identify the intergenerational environmental determinants of Ae. albopictus population dynamics. A cross-validation of the predictive accuracy of the different models was carried out to select the best predictive model. Over 7 years we captured 12,152 Ae. albopictus in the first trap and another 58,710 in four years of trapping in four additional traps. Aedes albopictus abundance was highly seasonal, with activity between mid-March and late December, highest abundances between July and September, and peak abundances in autumn. The predictive potential of the best model was further externally validated with four years of data from the other four traps, showing a high predictive capacity and a very good fit of seasonality and abundance peaks. Relative humidity, vapour saturation deficit and wind speed were identified as the main determinants of the weekly abundance of Ae. albopictus. The results obtained will allow accurate prediction o

Female mosquitoes require blood from their host for egg development. However, the relationship between the composition of host blood and mosquito reproduction, and whether and how this is linked to host selection, remain unclear. A better understanding of these issues is beneficial for mass‐rearing of mosquitoes for vector control. This review provides an overview of the currently known effects of blood constituents on mosquito reproduction. Furthermore, it highlights knowledge gaps and proposes new avenues for investigation. We recommend that research efforts be focused on physiological differences between generalist and specialist mosquito species as models to investigate if and how host preference correlates with reproductive output.

West Nile virus (WNV) invaded the continental United States over 20 years ago and continues to cause yearly seasonal outbreaks of human and veterinary disease. In the suburbs of Chicago, Illinois, ultra-low volume (ULV) truck-mounted adulticide spraying frequently is performed to reduce populations of Culex restuans Theobald and Cx. pipiens L. mosquitoes (Diptera: Culicidae) in an effort to lower the risk of WNV transmission. The effectiveness of this control method has not been rigorously evaluated, and evidence for Culex population reduction after ULV adulticide spraying has been inconclusive. Therefore, we evaluated the results of 5 sequential weekly truck-mounted adulticide applications of Zenivex® E20 (etofenprox) in 2 paired sites located in Cook County, IL, during the summer of 2018. Mosquito population abundance, age structure, and WNV infection prevalence were monitored and compared between paired treatment and nearby control sites. Adulticide treatment did not result in consistent short-term or long-term reductions in target WNV vector Culex abundance. However, there was a significant increase in the proportion of nulliparous females in the treated sites compared to control sites and a decrease in Cx. pipiens WNV infection rates at one of the treated sites. This evidence that ULV adulticide spraying altered the age structure and WNV infection prevalence in a vector population has important implications for WNV transmission risk management. Our findings also underscore the importance of measuring these important indicators in addition to abundance metrics when evaluating the efficacy of control methods.

Developing effective tools to control mosquito populations is essential for reducing the incidence of diseases like malaria and dengue. Biopesticides of microbial origin are a rich, underexplored source of mosquitocidal compounds. We previously developed a biopesticide from the bacterium Chromobacterium sp. Panama that rapidly kills vector mosquito larvae, including Aedes aegypti and Anopheles gambiae. Here, we demonstrate that two independent Ae. aegypti colonies exposed to a sublethal dose of that biopesticide over consecutive generations persistently exhibited high mortality and developmental delays, indicating that resistance did not develop during the study period. Critically, the descendants of biopesticide-exposed mosquitoes experienced decreased longevity and did not display increased susceptibility to dengue virus or decreased susceptibility to common chemical insecticides. Through RNA sequencing, we observed no link between biopesticide exposure and the increased activity of xenobiotic metabolism and detoxification genes typically associated with insecticide resistance. These findings indicate that the Chromobacterium biopesticide is an exciting, emerging mosquito control tool. IMPORTANCE: Vector control is an essential part of mitigating diseases caused by pathogens that mosquitoes spread. Modern vector control is highly reliant on using synthetic insecticides to eliminate mosquito populations before they can cause disease. However, many of these populations have become resistant to commonly used insecticides. There is a strong need to explore alternative vector control strategies that aim to mitigate disease burden. Biopesticides, insecticides of biological origin, can have unique mosquitocidal activities, meaning they can effectively kill mosquitoes that are already resistant to other insecticides. We previously developed a highly effective mosquito biopesticide from the bacterium Chromobacterium sp. Csp_P. Here, we investigate whether exposure to a sub

BackgroundMosquitoes are vectors of many pathogens, such as malaria, dengue virus, yellow fever virus, filaria and Japanese encephalitis virus. Wolbachia are capable of inducing a wide range of reproductive abnormalities in their hosts, such as cytoplasmic incompatibility. Wolbachia has been proposed as a tool to modify mosquitoes that are resistant to pathogen infection as an alternative vector control strategy. This study aimed to determine natural Wolbachia infections in different mosquito species across Hainan Province, China.MethodsAdult mosquitoes were collected using light traps, human landing catches and aspirators in five areas in Hainan Province from May 2020 to November 2021. Species were identified based on morphological characteristics, species-specific PCR and DNA barcoding of cox1 assays. Molecular classification of species and phylogenetic analyses of Wolbachia infections were conducted based on the sequences from PCR products of cox1, wsp, 16S rRNA and FtsZ gene segments.ResultsA total of 413 female adult mosquitoes representing 15 species were identified molecularly and analyzed. Four mosquito species (Aedes albopictus, Culex quinquefasciatus, Armigeres subalbatus and Culex gelidus) were positive for Wolbachia infection. The overall Wolbachia infection rate for all mosquitoes tested in this study was 36.1% but varied among species. Wolbachia types A, B and mixed infections of A × B were detected in Ae. albopictus mosquitoes. A total of five wsp haplotypes, six FtsZ haplotypes and six 16S rRNA haplotypes were detected from Wolbachia infections. Phylogenetic tree analysis of wsp sequences classified them into three groups (type A, B and C) of Wolbachia strains compared to two groups each for FtsZ and 16S rRNA sequences. A novel type C Wolbachia strain was detected in Cx. gelidus by both single locus wsp gene and the combination of three genes.ConclusionOur study revealed the prevalence and distribution of Wolbachia in mosquitoes from Hainan Province

Knowledge of insect dispersal is relevant to the control of agricultural pests, vector-borne transmission of human and veterinary pathogens, and insect biodiversity. Previous studies in a malaria endemic area of the Sahel region in West Africa revealed high-altitude, long-distance migration of insects and various mosquito species. The objective of the current study was to assess whether similar behavior is exhibited by mosquitoes and other insects around the Lake Victoria basin region of Kenya in East Africa. Insects were sampled monthly from dusk to dawn over 1 year using sticky nets suspended on a tethered helium-filled balloon. A total of 17,883 insects were caught on nets tethered at 90, 120, and 160 m above ground level; 818 insects were caught in control nets. Small insects (<0.5 cm, n = 15,250) were predominant regardless of height compared with large insects (>0.5 cm, n = 2,334) and mosquitoes (n = 299). Seven orders were identified; dipteran was the most common. Barcoding molecular assays of 184 mosquitoes identified 7 genera, with Culex being the most common (65.8%) and Anopheles being the least common (5.4%). The survival rate of mosquitoes, experimentally exposed to high-altitude overnight, was significantly lower than controls maintained in the laboratory (19% vs. 85%). There were no significant differences in mosquito survival and oviposition rate according to capture height. These data suggest that windborne dispersal activity of mosquito vectors of malaria and other diseases occurs on a broad scale in sub-Saharan Africa.

Here, we report on a serendipitous finding of a chryso-like virus associated with Culex pipiens mosquitos in the course of study aimed to detect and characterize West Nile virus (WNV) circulating in mosquitos in Serbia, Southern Europe. Upon initial detection of unexpected product in a PCR protocol for partial WNV NS5 gene amplification, further confirmation and identification was obtained through additional PCR and Sanger sequencing experiments. Bioinformatic and phylogenetic analysis identified the obtained sequences as Xanthi chryso-like virus (XCLV). The finding is particular for the fact that it associates XCLV with a new potential vector species and documents a novel geographical area of its distribution.

Developing effective tools to control mosquito populations is essential for reducing the incidence of diseases like malaria and dengue. Biopesticides of microbial origin are a rich, underexplored source of mosquitocidal compounds. We previously developed a biopesticide from the bacterium Chromobacterium sp. Panama that rapidly kills vector mosquito larvae, including Aedes aegypti and Anopheles gambiae. Here, we demonstrate that two independent Ae. aegypti colonies exposed to a sublethal dose of that biopesticide over consecutive generations persistently exhibited high mortality and developmental delays, indicating that resistance did not develop during the study period. Critically, the descendants of biopesticide-exposed mosquitoes experienced decreased longevity and did not display increased susceptibility to dengue virus or decreased susceptibility to common chemical insecticides. Through RNA sequencing, we observed no link between biopesticide exposure and the increased activity of xenobiotic metabolism and detoxification genes typically associated with insecticide resistance. These findings indicate that the Chromobacterium biopesticide is an exciting, emerging mosquito control tool. IMPORTANCE: Vector control is an essential part of mitigating diseases caused by pathogens that mosquitoes spread. Modern vector control is highly reliant on using synthetic insecticides to eliminate mosquito populations before they can cause disease. However, many of these populations have become resistant to commonly used insecticides. There is a strong need to explore alternative vector control strategies that aim to mitigate disease burden. Biopesticides, insecticides of biological origin, can have unique mosquitocidal activities, meaning they can effectively kill mosquitoes that are already resistant to other insecticides. We previously developed a highly effective mosquito biopesticide from the bacterium Chromobacterium sp. Csp_P. Here, we investigate whether exposure to a sub

Developing effective tools to control mosquito populations is essential for reducing the incidence of diseases like malaria and dengue. Biopesticides of microbial origin are a rich, underexplored source of mosquitocidal compounds. We previously developed a biopesticide from the bacterium Chromobacterium sp. Panama that rapidly kills vector mosquito larvae, including Aedes aegypti and Anopheles gambiae. Here, we demonstrate that two independent Ae. aegypti colonies exposed to a sublethal dose of that biopesticide over consecutive generations persistently exhibited high mortality and developmental delays, indicating that resistance did not develop during the study period. Critically, the descendants of biopesticide-exposed mosquitoes experienced decreased longevity and did not display increased susceptibility to dengue virus or decreased susceptibility to common chemical insecticides. Through RNA sequencing, we observed no link between biopesticide exposure and the increased activity of xenobiotic metabolism and detoxification genes typically associated with insecticide resistance. These findings indicate that the Chromobacterium biopesticide is an exciting, emerging mosquito control tool. IMPORTANCE: Vector control is an essential part of mitigating diseases caused by pathogens that mosquitoes spread. Modern vector control is highly reliant on using synthetic insecticides to eliminate mosquito populations before they can cause disease. However, many of these populations have become resistant to commonly used insecticides. There is a strong need to explore alternative vector control strategies that aim to mitigate disease burden. Biopesticides, insecticides of biological origin, can have unique mosquitocidal activities, meaning they can effectively kill mosquitoes that are already resistant to other insecticides. We previously developed a highly effective mosquito biopesticide from the bacterium Chromobacterium sp. Csp_P. Here, we investigate whether exposure to a sub

Developing effective tools to control mosquito populations is essential for reducing the incidence of diseases like malaria and dengue. Biopesticides of microbial origin are a rich, underexplored source of mosquitocidal compounds. We previously developed a biopesticide from the bacterium Chromobacterium sp. Panama that rapidly kills vector mosquito larvae, including Aedes aegypti and Anopheles gambiae. Here, we demonstrate that two independent Ae. aegypti colonies exposed to a sublethal dose of that biopesticide over consecutive generations persistently exhibited high mortality and developmental delays, indicating that resistance did not develop during the study period. Critically, the descendants of biopesticide-exposed mosquitoes experienced decreased longevity and did not display increased susceptibility to dengue virus or decreased susceptibility to common chemical insecticides. Through RNA sequencing, we observed no link between biopesticide exposure and the increased activity of xenobiotic metabolism and detoxification genes typically associated with insecticide resistance. These findings indicate that the Chromobacterium biopesticide is an exciting, emerging mosquito control tool. IMPORTANCE: Vector control is an essential part of mitigating diseases caused by pathogens that mosquitoes spread. Modern vector control is highly reliant on using synthetic insecticides to eliminate mosquito populations before they can cause disease. However, many of these populations have become resistant to commonly used insecticides. There is a strong need to explore alternative vector control strategies that aim to mitigate disease burden. Biopesticides, insecticides of biological origin, can have unique mosquitocidal activities, meaning they can effectively kill mosquitoes that are already resistant to other insecticides. We previously developed a highly effective mosquito biopesticide from the bacterium Chromobacterium sp. Csp_P. Here, we investigate whether exposure to a sub

Mosquitoes are considered to be the deadliest animals in the world due to the diseases they spread. Naturally, most research centers around anthropophilic disease vector mosquitoes such as Aedes aegypti or Anopheles gambiae. However, mosquitoes can feed on, and pose a threat to, more than humans. In fact, mosquitoes can specialize in feeding on a wide variety of hosts, including ectotherms such as amphibians and reptiles. Culex territans is a mosquito that feeds primarily on amphibians and reptiles and is known to transmit parasites to its hosts. However, little else is known about this mosquito. This work dives into the biology, physiology, and vector potential of this mosquito species to provide critical insights into understanding the evolution of feeding on endotherms as well as the importance of Cx. territans to amphibian health. The first study investigates activity patterns and feeding behaviors as well as the potential cues that may attract this mosquito to its hosts. We also compare transcriptomics and anatomy of the heads of three Culex species. The second study compares three Culex species' thermopreference and host-seeking to understand the effects of the differences of geographical distribution and preferred hosts. The last three studies determine the presence of ranaviruses, giant anuran trypanosomes, and the fungus Batrachochytrium dendrobatidis at Mountain Lake Biological Station and explore the potential role of Cx. territans in transmitting these pathogens. Overall, this work provides insights into the biology of Cx. territans and the implications it may have for understanding endotherm-feeding mosquitoes and amphibian disease epidemiology.

Culex species mosquitoes are responsible for the transmission of West Nile virus (WNV) and other encephalitic viruses within the United States. In Arizona, Culex quinquefasciatus and Culex tarsalis are the principal vectors of WNV and St. Louis encephalitis (SLEV). This research examines the desert ecology of these mosquitoes in the context of vector control. Specifically, the study examines how human land-use and vector control activities influence both mosquito abundance and WNV and SLEV presence. Chapter 1 investigates the efficacy of larvicide applications to public drainage features. Chapter 2 analyzes the relationship between land-use and female mosquito density and virus presence. Chapter 3 assesses the susceptibility of Cx. quinquefasciatus adults to the insecticides commonly used for WNV prevention in Maricopa County, Arizona which is home to the Phoenix metropolitan area. We found that dry well drainage features are an important habitat for Cx. quinquefasciatus mosquitoes, but standard larvicide application techniques have limited efficacy due to the water depth. Suspending either of the larvicides Altosid or Bti/Ls at the same water level as the mosquito immatures caused significant reduction on viable larvae. There are many significant associations in adult Cx. quinquefasciatus and Cx. tarsalis density with designated land use throughout the greater Phoenix area. Notably, land areas designated as agriculture and active open space were positively associated with mosquito density, while single and multi-family residential areas, golf courses, and vacant space were negatively associated. The odds of finding virus-infected mosquito pools were higher residential areas, agriculture, and active open space land use categories and lower in industrial, commercial, and business areas, golf courses, inactive open space areas, and vacant areas. Pyrethroid class adulticides have been used exclusively in Maricopa County for mosquito control purposes. Mosquito immatures

Developing effective tools to control mosquito populations is essential for reducing the incidence of diseases like malaria and dengue. Biopesticides of microbial origin are a rich, underexplored source of mosquitocidal compounds. We previously developed a biopesticide from the bacterium Chromobacterium sp. Panama that rapidly kills vector mosquito larvae, including Aedes aegypti and Anopheles gambiae. Here, we demonstrate that two independent Ae. aegypti colonies exposed to a sublethal dose of that biopesticide over consecutive generations persistently exhibited high mortality and developmental delays, indicating that resistance did not develop during the study period. Critically, the descendants of biopesticide-exposed mosquitoes experienced decreased longevity and did not display increased susceptibility to dengue virus or decreased susceptibility to common chemical insecticides. Through RNA sequencing, we observed no link between biopesticide exposure and the increased activity of xenobiotic metabolism and detoxification genes typically associated with insecticide resistance. These findings indicate that the Chromobacterium biopesticide is an exciting, emerging mosquito control tool. IMPORTANCE: Vector control is an essential part of mitigating diseases caused by pathogens that mosquitoes spread. Modern vector control is highly reliant on using synthetic insecticides to eliminate mosquito populations before they can cause disease. However, many of these populations have become resistant to commonly used insecticides. There is a strong need to explore alternative vector control strategies that aim to mitigate disease burden. Biopesticides, insecticides of biological origin, can have unique mosquitocidal activities, meaning they can effectively kill mosquitoes that are already resistant to other insecticides. We previously developed a highly effective mosquito biopesticide from the bacterium Chromobacterium sp. Csp_P. Here, we investigate whether exposure to a sub

Mosquito disease vector control relies mostly on toxic insecticides. A more environmentally friendly alternative is to make use of light-based behavioral manipulation to attract pests to traps and repel pests away from human habitation. The present technology is based on the assumption that mosquito UV light detection occurs solely through opsin-based photoreception in the eyes. The Holmes Lab has recently found additional UV and short wavelength photoreceptive elements expressed in central brain neurons that strongly modulate complex insect behavioral responses to light. Therefore, there is a need to incorporate these additional elements in disease vector control designs for improved efficiency. Namely, CRYPTOCHROME (CRY), which is classically associated with its role in circadian clock resetting, activates with blue- and UV- light and increases the electrical excitability of circadian/arousal neurons. I hypothesized that CRY and other photoreceptor systems mediate photic input to the circadian/arousal circuit of flies in order to mediate specific light-based behavioral responses. To test this, I modified and adapted light-evoked electrophysiology protocols to test the photoexcitability of primary arousal neurons in response to different stimuli of light using several photoreceptor mutants that lack one of three photoreceptor systems, as well as transgenic flies expressing mosquito CRY1 from a diurnal and nocturnal species. I also modified and adapted several behavioral assays to determine light attraction/avoidance as well as light-pulse arousal behavioral response to these different mutants. These modifications involved developing a new light emitting apparatus that improved on intensity control and spatiotemporal light exposure with a finer tuned spectral emission for UV, violet, blue, and red light. In this dissertation, I found that CRY coordinates with other photoreceptors to mediate light-induced electrical excitability of neurons, which underlie complex sle

The deltamethrin impregnated mosquito nets not only prevent but also control malaria, dengue fever and other vector-bone diseases. However, the deltamethrin concentration and bio-efficacy of mosquito nets can reduce due to washing. The purpose of this study is to investigate the effect of washing on deltamethrin concentration and bio-efficacy of military insecticide-treated mosquito nets. High Performance Liquid Chromatography method was used for the determination of deltamethrin in mosquito-nets. Then, the WHO recommended cone and excito-chamber methods were used to determine the bio-efficacy of mosquito nets. The deltamethrin concentrations in the mosquito-net were after no wash (4590±58.54 ppm), 1× wash (4020±47.39 ppm), 2× wash (3225±45.22 ppm), 3× wash (2771±107.5 ppm), 4× wash (2657±58.59 ppm), 5× wash (1835±47.55 ppm), 10× wash (957±34.85 ppm), 15× wash (197±22.54 ppm) and 20× wash (87±23.75 ppm). The mosquito knockdown effects were reduced by washing of net (no wash to 20× wash) after 60 minutes and 6 hours exposure range from 90-40% and 100-70%. The mosquito mortality rate was reduced by washing of net (no wash to 20× wash) after 24 hours exposure were reduced range from 100-80%. The mosquito repellent action was reduced by washing of net (no wash to 20× wash) after 10 minutes and 24 hours exposure, range from 90-30% and 100-60%. The effective bio-efficacy level was found in 5×wash (1835±47.55 ppm of deltamethrin).Therefore, mosquito net should be impregnated again after the 5× wash to obtain the best protection on the public from mosquito bite.

type:Article, Species of the genus Spiroplasma are common within arthropods and plants worldwide. Mosquito-associated Spiroplasma spp. have been reported to show pathogenicity toward mosquitoes, which serve as vectors of several infectious diseases that have detrimental effects on public health. Although Spiroplasma spp. are expected to have potential use as biological vector-control tools, characteristics such as their distribution, host species, and cytopathogenic effects (CPEs) are not well understood. In this study, we isolated a Spiroplasma sp. from a female Aedes japonicus collected in Hokkaido, northern Japan. Phylogenetic analysis based on the 16S rRNA gene sequence indicated our isolate was closely related to S. cantharicola. We screened 103 mosquito pools consisting of 3 genera and 9 species, but only detected S. cantharicola in the first isolation. In an in vitro assay, our isolate grew well at 28 °C, but no propagation was observed at 37 °C. Furthermore, the isolate showed strong CPE on a mosquito-derived cultured cell line (C6/36), and its propagation slightly increased when co-cultured with C6/36 cells. To our knowledge, this is the third report of the isolation of S. cantharicola from mosquitoes and the first case in Asia. Our findings provide epidemiological data on S. cantharicola distribution in the region.

The deltamethrin impregnated mosquito nets not only prevent but also control malaria, dengue fever and other vector-bone diseases. However, the deltamethrin concentration and bio-efficacy of mosquito nets can reduce due to washing. The purpose of this study is to investigate the effect of washing on deltamethrin concentration and bio-efficacy of military insecticide-treated mosquito nets. High Performance Liquid Chromatography method was used for the determination of deltamethrin in mosquito-nets. Then, the WHO recommended cone and excito-chamber methods were used to determine the bio-efficacy of mosquito nets. The deltamethrin concentrations in the mosquito-net were after no wash (4590±58.54 ppm), 1× wash (4020±47.39 ppm), 2× wash (3225±45.22 ppm), 3× wash (2771±107.5 ppm), 4× wash (2657±58.59 ppm), 5× wash (1835±47.55 ppm), 10× wash (957±34.85 ppm), 15× wash (197±22.54 ppm) and 20× wash (87±23.75 ppm). The mosquito knockdown effects were reduced by washing of net (no wash to 20× wash) after 60 minutes and 6 hours exposure range from 90-40% and 100-70%. The mosquito mortality rate was reduced by washing of net (no wash to 20× wash) after 24 hours exposure were reduced range from 100-80%. The mosquito repellent action was reduced by washing of net (no wash to 20× wash) after 10 minutes and 24 hours exposure, range from 90-30% and 100-60%. The effective bio-efficacy level was found in 5×wash (1835±47.55 ppm of deltamethrin).Therefore, mosquito net should be impregnated again after the 5× wash to obtain the best protection on the public from mosquito bite.

Insect epithelial cells contain unique cellular extensions such as bristles, hairs, and scales. In contrast to bristle and hair, which are not divergent in their shape, scale morphology shows high diversity. In our attempt to characterize the role of the insect-specific gene, Spindle-F (spn-F), in mosquito development, we revealed a scale-type specific requirement for the mosquito Aedes aegypti spn-F homologue. Using CRISPR-Cas9, we generated Ae-spn-F mutants and found that Ae-spn-F is an essential gene, but we were able to recover a few adult escapers. These escapers could not fly nor move, and died after 3 to 4 days. We found that in Ae-spn-F mutants, only the tip part of the bristle was affected with bulbous with misoriented ribs. We also show that in Ae-spn-F mutants, only in falcate scales, which are curved with a sharp or narrowly rounded apex, and not in other scale types, the tip region is strongly affected. Our analysis also revealed that in contrast to Drosophila spn-F, which show strong defects in both the actin and microtubule (MT) network in the bristle, the Ae-spn-F gene is required only for MT organization in scales and bristles. In summary, our results reveal that Ae-spn-F is required for shaping tapered epithelial cellular extension structures, namely, the bristle and falcate scales by affecting MT organization.

eLife digest Mosquitoes are often referred to as the deadliest animals on earth because some species spread malaria, West Nile virus or other dangerous diseases when they bite humans and other animals. Adult mosquitoes fly to streams, ponds and other freshwater environments to lay their eggs. When the eggs hatch, the young mosquitoes live in the water until they are ready to grow wings and transform into adults. In the water, the young mosquitoes are particularly vulnerable to being eaten by dragonfly larvae, fish and other predators. When adult females are choosing where to lay their eggs, they can use their sense of smell to detect these predators and attempt to avoid them. Along with eating the mosquitoes, the predators may also reduce mosquito populations in other ways. For example, predators can disrupt feeding among young mosquitoes, which may affect the time that it takes for them to grow into adults or the size of their bodies once they reach the adult stage. Although the impacts of different predators have been tested separately in multiple settings, the overall effects of predators on the ability of mosquitoes to spread diseases to humans remain unclear. To address this question, Russell, Herzog et al. used an approach called meta-analysis on data from previous studies. The analysis found that along with increasing the death rates of mosquitoes, the presence of predators also leads to a reduction in the body size of those mosquitoes that survive, causing them to have shorter lifespans and fewer offspring. Russell, Herzog et al. found that one type of mosquito known as Culex - which carries West Nile virus - avoided laying its eggs near predators. During droughts, increased predation in streams, ponds and other aquatic environments may lead adult female Culex mosquitoes to lay their eggs closer to residential areas with fewer predators. Russell, Herzog et al. propose that this may be one reason why outbreaks of West Nile virus in humans are more likely to o

Copper is an essential metal that occurs chronically in the environment and affects the development and physiology of aquatic insects. In excess amounts, it can impair their nervous system and behaviour. We tested the anti-predatory behaviour of Cx. pipiens larvae after seven days exposure with several concentrations of copper up to 500 mg L-1. We measured responses to non- consumptive (predation cues) and consumptive predation (dragonfly larvae) across two generations. We also tested the accumulated effect of copper on AChE enzyme activity. We exposed half of treated and control larvae to predation cues (water with predator odour and crushed conspecifics) and the other half to water without predation cues. We evaluated total distance moved and velocity. Copper reduced the distance moved and velocity, with stronger effects in the second generation. Copper had no significant effect on larvae eaten by dragonflies. Copper inhibited the AChE enzyme across both generations at 500 µg L-1. Copper can affect the nervous system directly by inhibiting AChE activity, and possibly also by impairing the olfaction sensors of the larvae, resulting in larval inability to detect predation cues.

Alphaviruses are a group of medically relevant arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that are maintained by mosquito vectors. These zoonotic viruses are clustered into two groups: New World and Old World, depending on their geographical origin/distribution and clinical manifestations. Both of these groups cause disease symptoms of an acute febrile illness; however, each group has a distinct, hallmark disease symptom; New World alphaviruses, such as Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively), present with severe encephalitis while Old World alphaviruses, such as Sindbis, chikungunya, and Mayaro viruses (SINV, CHIKV, and MAYV, respectively) present with an incapacitating polyarthralgia that can persist for years following initial infection. To date, the most effective means of controlling these arboviral infections is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. Given the close phylogenetic and antigenic relationship between MAYV and CHIKV, we hypothesized that prior CHIKV immunity may affect the outcome of MAYV disease and/or limit its emergence in humans. Our work has shown that anti-CHIKV neutralizing antibodies can provide cross-protective immunity against MAYV disease. Alongside these studies, we have characterized the potency of a camelid-derived single-domain antibody (sdAb) that neutralizes a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express two anti-CHIKV sdAbs to target infection, dissemination, and transmission of MAYV and CHIKV within this deadly vector. These findings are particularly significant because they highlight the ability to co-target two emerging alphaviruses that are crippl

The risk of autochthonous transmission of Zika virus (ZIKV) in Europe greatly depends on the capacity of Aedes albopictus to transmit ZIKV. Experimental studies of the vector competence of European populations of this invasive mosquito species provided contrasting results, that may suggest differences between mosquito populations in ZIKV competence and/or differences in the methodological procedures used on the experimental studies. We systematically reviewed published studies of experimental mosquito infections and show that differences detected in the infection and transmission rates of Ae. albopictus are mostly due to differences between studies in the temperature, the viral load used to inoculate mosquitoes, and the number of days post-exposure (dpe) to the virus at which mosquitoes were analysed. In particular, the proportion of infected mosquitoes was affected by the viral load inoculated and the dpe, whereas the transmission rate was influenced by the viral load and temperature. Thus, the methodological differences used in these studies greatly affect the conclusions obtained regarding the role of Ae. albopictus in Europe as a competent vector for ZIKV and its epidemiological relevance.

Totivirus-like viruses are a group of non-segmented double-stranded (ds)RNA viruses with two open reading frames, which were recently discovered and provisionally assigned to the Totiviridae family. Unlike yeast and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first infectious full-length cDNA clone of a totivirus-like virus, the Omono River virus (OmRV), and produced infectious particles using an RNA -transcript-based method. Compared with the parent wild-type particles from nature, the infectious-cloning OmRV particles have presented strong cytopathic effects, infectivity and similar morphology. Thus far, the established system is one of the few reported systems for generating a non-segmented dsRNA virus cDNA clone.

Background: Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families Togaviridae, genus Alphavirus (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Peribunyaviridae, genus Orthobunyavirus, California serogroup (Inkoo, Batai, Tahyna). The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway, Sweden, Finland, and some parts of Russia. These viruses are associated with morbidity in humans. However, there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of INKV and SINV in blood sucking insects and seroprevalence for INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway. Materials and methods: In total, 213 pools containing about 25 blood sucking insects (BSI) each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013–2015. The pools were analysed by RT-PCR to detect INKV and by RT-real-time PCR for SINV. Reindeer sera were analysed for INKV-specific IgG by an Indirect Immunofluorescence Assay (n = 480, IIFA) and a Plaque Reduction Neutralization Test (n = 60, PRNT). Results: Aedes spp. were the most dominant species among the collected BSI. Two of the pools were positive for INKV-RNA by RT-PCR and were confirmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. None of the analysed pools were positive for SINV. Overall IgG seroprevalence in reindeer was 62% positive for INKV by IIFA. Of the 60 reindeer sera- analysed by PRNT for INKV, 80% were confirmed positive, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snowshoe hare virus (SSHV). Conclusion: The occurrence and prevalence of INKV in BSI and the high seroprevalence against the virus among semi-domesticated reindeer in Norw

The mosquito Aedes aegypti is the primary vector of many viruses that cause a major burden on human health worldwide, including dengue, Zika, yellow fever, and chikungunya viruses. Currently, a widely adopted vaccine is available only for yellow fever virus, and thus mitigating the burden of diseases caused by these viruses predominantly relies on avoiding mosquito bites and controlling mosquito populations. Mosquito-borne virus transmission risk models can help mosquito control decision-makers efficiently use limited resources and reduce the use of chemical insecticides that can lead to resistance and therefore less effective control. As the Zika virus (ZIKV) pandemic emerged in 2016, estimates for ZIKV transmission risk were based on proxy evidence from closely related dengue virus. To improve risk estimates, we first studied how temperature affects ZIKV extrinsic incubation period (EIP). We concluded that, in agreement with findings for other mosquito-borne viruses, ZIKV EIP decreased as temperature increased and ZIKV EIP was relatively shorter than for dengue virus across temperatures. We then sought to further improve ZIKV risk estimates by studying thermal preferences of Ae. aegypti mosquitoes in the laboratory and in the field. Current mosquito-borne pathogen risk models primarily use temperatures from weather stations or thermal imagery as a proxy for the temperatures mosquitoes experience; however, such approaches do not account for the range of local environments or microclimates available to adult mosquitoes nor the microhabitats mosquitoes select and therefore may lead to an inaccurate estimate of risk. In the lab, Ae. aegypti generally preferred the coldest temperatures available (<20°C) and avoided the hottest temperatures (>31°C) on a gradient ranging from 17.5°C to 36.5°C. However, mosquitoes reared at cooler temperatures (22°C) were larger and rested at warmer temperatures compared to mosquitoes reared at warm temperatures (26°C and 30°C). In

An experimental infection approach was used to estimate the competence of the common mosquito, Culex pipiens, for hepatitis E virus replication and transmission, using an isolate of hepatitis E virus genotype 3 of human origin in varying infectious doses. The experimental approach was carried out in biosafety level 2 conditions on three batches of 120 Cx. pipiens females, each using an artificial feeding system containing the virus in aliquots of fresh avian blood. Mosquitoes from each batch were collected 1, 7, 14, and 21 days post-infection (dpi) and dissected. The proboscis was subjected to forced excretion of saliva to estimate potential virus transmission. HEV RNA presence in abdomen, thorax, and saliva samples was analyzed by PCR at the selected post-infection times. HEV RNA was detected in the abdomens of Cx. pipiens females collected 1 dpi in the two experimentally-infected batches, but not in the saliva or thorax. None of the samples collected 7–21 dpi were positive. Our results show that Cx. pipiens is not a competent vector for HEV, at least for zoonotic genotype 3.

eLife digest Mosquitoes are often referred to as the deadliest animals on earth because some species spread malaria, West Nile virus or other dangerous diseases when they bite humans and other animals. Adult mosquitoes fly to streams, ponds and other freshwater environments to lay their eggs. When the eggs hatch, the young mosquitoes live in the water until they are ready to grow wings and transform into adults. In the water, the young mosquitoes are particularly vulnerable to being eaten by dragonfly larvae, fish and other predators. When adult females are choosing where to lay their eggs, they can use their sense of smell to detect these predators and attempt to avoid them. Along with eating the mosquitoes, the predators may also reduce mosquito populations in other ways. For example, predators can disrupt feeding among young mosquitoes, which may affect the time that it takes for them to grow into adults or the size of their bodies once they reach the adult stage. Although the impacts of different predators have been tested separately in multiple settings, the overall effects of predators on the ability of mosquitoes to spread diseases to humans remain unclear. To address this question, Russell, Herzog et al. used an approach called meta-analysis on data from previous studies. The analysis found that along with increasing the death rates of mosquitoes, the presence of predators also leads to a reduction in the body size of those mosquitoes that survive, causing them to have shorter lifespans and fewer offspring. Russell, Herzog et al. found that one type of mosquito known as Culex - which carries West Nile virus - avoided laying its eggs near predators. During droughts, increased predation in streams, ponds and other aquatic environments may lead adult female Culex mosquitoes to lay their eggs closer to residential areas with fewer predators. Russell, Herzog et al. propose that this may be one reason why outbreaks of West Nile virus in humans are more likely to o

Totivirus-like viruses are a group of non-segmented double-stranded (ds)RNA viruses with two open reading frames, which were recently discovered and provisionally assigned to the Totiviridae family. Unlike yeast and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first infectious full-length cDNA clone of a totivirus-like virus, the Omono River virus (OmRV), and produced infectious particles using an RNA -transcript-based method. Compared with the parent wild-type particles from nature, the infectious-cloning OmRV particles have presented strong cytopathic effects, infectivity and similar morphology. Thus far, the established system is one of the few reported systems for generating a non-segmented dsRNA virus cDNA clone.

Totivirus-like viruses are a group of non-segmented double-stranded (ds)RNA viruses with two open reading frames, which were recently discovered and provisionally assigned to the Totiviridae family. Unlike yeast and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first infectious full-length cDNA clone of a totivirus-like virus, the Omono River virus (OmRV), and produced infectious particles using an RNA -transcript-based method. Compared with the parent wild-type particles from nature, the infectious-cloning OmRV particles have presented strong cytopathic effects, infectivity and similar morphology. Thus far, the established system is one of the few reported systems for generating a non-segmented dsRNA virus cDNA clone.

Background: Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families Togaviridae, genus Alphavirus (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Peribunyaviridae, genus Orthobunyavirus, California serogroup (Inkoo, Batai, Tahyna). The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway, Sweden, Finland, and some parts of Russia. These viruses are associated with morbidity in humans. However, there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of INKV and SINV in blood sucking insects and seroprevalence for INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway. Materials and methods: In total, 213 pools containing about 25 blood sucking insects (BSI) each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013–2015. The pools were analysed by RT-PCR to detect INKV and by RT-real-time PCR for SINV. Reindeer sera were analysed for INKV-specific IgG by an Indirect Immunofluorescence Assay (n = 480, IIFA) and a Plaque Reduction Neutralization Test (n = 60, PRNT). Results: Aedes spp. were the most dominant species among the collected BSI. Two of the pools were positive for INKV-RNA by RT-PCR and were confirmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. None of the analysed pools were positive for SINV. Overall IgG seroprevalence in reindeer was 62% positive for INKV by IIFA. Of the 60 reindeer sera- analysed by PRNT for INKV, 80% were confirmed positive, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snowshoe hare virus (SSHV). Conclusion: The occurrence and prevalence of INKV in BSI and the high seroprevalence against the virus among semi-domesticated reindeer in Norw

Totivirus-like viruses are a group of non-segmented double-stranded (ds)RNA viruses with two open reading frames, which were recently discovered and provisionally assigned to the Totiviridae family. Unlike yeast and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first infectious full-length cDNA clone of a totivirus-like virus, the Omono River virus (OmRV), and produced infectious particles using an RNA -transcript-based method. Compared with the parent wild-type particles from nature, the infectious-cloning OmRV particles have presented strong cytopathic effects, infectivity and similar morphology. Thus far, the established system is one of the few reported systems for generating a non-segmented dsRNA virus cDNA clone.

Background: Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families Togaviridae, genus Alphavirus (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Peribunyaviridae, genus Orthobunyavirus, California serogroup (Inkoo, Batai, Tahyna). The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway, Sweden, Finland, and some parts of Russia. These viruses are associated with morbidity in humans. However, there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of INKV and SINV in blood sucking insects and seroprevalence for INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway. Materials and methods: In total, 213 pools containing about 25 blood sucking insects (BSI) each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013–2015. The pools were analysed by RT-PCR to detect INKV and by RT-real-time PCR for SINV. Reindeer sera were analysed for INKV-specific IgG by an Indirect Immunofluorescence Assay (n = 480, IIFA) and a Plaque Reduction Neutralization Test (n = 60, PRNT). Results: Aedes spp. were the most dominant species among the collected BSI. Two of the pools were positive for INKV-RNA by RT-PCR and were confirmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. None of the analysed pools were positive for SINV. Overall IgG seroprevalence in reindeer was 62% positive for INKV by IIFA. Of the 60 reindeer sera- analysed by PRNT for INKV, 80% were confirmed positive, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snowshoe hare virus (SSHV). Conclusion: The occurrence and prevalence of INKV in BSI and the high seroprevalence against the virus among semi-domesticated reindeer in Norw

Totivirus-like viruses are a group of non-segmented double-stranded (ds)RNA viruses with two open reading frames, which were recently discovered and provisionally assigned to the Totiviridae family. Unlike yeast and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first infectious full-length cDNA clone of a totivirus-like virus, the Omono River virus (OmRV), and produced infectious particles using an RNA -transcript-based method. Compared with the parent wild-type particles from nature, the infectious-cloning OmRV particles have presented strong cytopathic effects, infectivity and similar morphology. Thus far, the established system is one of the few reported systems for generating a non-segmented dsRNA virus cDNA clone.

Background: Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families Togaviridae, genus Alphavirus (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Peribunyaviridae, genus Orthobunyavirus, California serogroup (Inkoo, Batai, Tahyna). The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway, Sweden, Finland, and some parts of Russia. These viruses are associated with morbidity in humans. However, there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of INKV and SINV in blood sucking insects and seroprevalence for INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway. Materials and methods: In total, 213 pools containing about 25 blood sucking insects (BSI) each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013–2015. The pools were analysed by RT-PCR to detect INKV and by RT-real-time PCR for SINV. Reindeer sera were analysed for INKV-specific IgG by an Indirect Immunofluorescence Assay (n = 480, IIFA) and a Plaque Reduction Neutralization Test (n = 60, PRNT). Results: Aedes spp. were the most dominant species among the collected BSI. Two of the pools were positive for INKV-RNA by RT-PCR and were confirmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. None of the analysed pools were positive for SINV. Overall IgG seroprevalence in reindeer was 62% positive for INKV by IIFA. Of the 60 reindeer sera- analysed by PRNT for INKV, 80% were confirmed positive, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snowshoe hare virus (SSHV). Conclusion: The occurrence and prevalence of INKV in BSI and the high seroprevalence against the virus among semi-domesticated reindeer in Norw