The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype-dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can have carry-over effects on adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we showed that the bacterial community structure differs between the two genotypes of Ae. aegypti when given identical microbiomes, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific larval microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti. We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Toward Implementation of Mosquito Sterile Insect Technique: The Effect of Storage Conditions on Survival of Male Aedes aegypti Mosquitoes (Diptera: Culicidae) During Transport.

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

Efficacy of Some Wearable Devices Compared with Spray-On Insect Repellents for the Yellow Fever Mosquito, Aedes aegypti (L.) (Diptera: Culicidae).

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

Dehydration induced AePer50 regulates midgut infection in Ae. aegypti.

In the face of climate change, mosquitoes will experience evolving climates including longer periods of drought. An important physiological response to dry environments is the protection against water loss or dehydration, here defined as desiccation tolerance. Various environmental factors including temperature are known to alter interactions between the mosquito, Aedes aegypti , and the arboviruses it transmits, but little is known about how low humidity impacts arboviral infection. Here, we report that a gene upregulated in response to desiccation is important for controlling midgut infection. We have identified two genetically diverse lines of Ae. aegypti with marked differences in desiccation tolerance. To understand if the genetic basis underlying desiccation tolerance is the same between the contrasting lines, we compared gene expression profiles between desiccant treated and non-desiccant treated individuals in both the desiccation tolerant and susceptible lines by RNAseq. Gene expression analysis demonstrates that different genes are differentially expressed in response to desiccation stress between desiccation tolerant and susceptible lines. The most highly expressed transcript under desiccation stress in the desiccation susceptible line encodes a peritrophin protein, Ae Per50. Peritrophins play a crucial role in peritrophic matrix formation after a bloodmeal. Gene silencing of Ae Per50 by RNAi demonstrates that expression of Ae Per50 is required for survival of the desiccation susceptible line under desiccation stress, but not for the desiccation tolerant line. Moreover, the knockdown of Ae Per50 results in higher infection rates and viral replication rates of ZIKV and higher infection rates of CHIKV. Finally, following a bloodmeal, the desiccation susceptible line develops a thicker peritrophic matrix than the desiccation tolerant line. Together these results provide a functional link between the protection against desiccation and midgut infection which has important implications in predicting how climate change will impact mosquito-borne viruses.

The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can influence adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we show that similarities in bacterial community structures when given identical microbiomes between different genetic backgrounds of Ae. aegypti was dependent on the source microbiome, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti . We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Variable microbiomes between mosquito lines are maintained across different environments.

The composition of the microbiome is shaped by both environment and host in most organisms, but in the mosquito Aedes aegypti the role of the host in shaping the microbiome is poorly understood. Previously, we had shown that four lines of Ae. aegypti harbored different microbiomes when reared in the same insectary under identical conditions. To determine whether these lines differed from each other across time and in different environments, we characterized the microbiome of the same four lines of Ae. aegypti reared in the original insectary and at another institution. While it was clear that the environment influenced the microbiomes of these lines, we did still observe distinct differences in the microbiome between lines within each insectary. Clear differences were observed in alpha diversity, beta diversity, and abundance of specific bacterial taxa. To determine if the line specific differences in the microbiome were maintained across environments, pair-wise differential abundances of taxa was compared between insectaries. Lines were most similar to other lines from the same insectary than to the same line reared in a different insectary. Additionally, relatively few differentially abundant taxa identified between pairs of lines were shared across insectaries, indicating that line specific properties of the microbiome are not conserved across environments, or that there were distinct microbiota within each insectary. Overall, these results demonstrate that mosquito lines under the same environmental conditions have different microbiomes across microbially- diverse environments and host by microbe interactions affecting microbiome composition and abundance is dependent on environmentally available bacteria.

A novel Tick Carousel Assay for testing efficacy of repellents on Amblyomma americanum L.

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

Efficacy of Active Ingredients From the EPA 25(B) List in Reducing Attraction of Aedes aegypti (Diptera: Culicidae) to Humans.

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

Widespread insecticide resistance in Aedes aegypti L. from New Mexico, U.S.A.

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

An online survey of personal mosquito-repellent strategies.

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

The Effect of SkitoSnack, an Artificial Blood Meal Replacement, on Aedes aegypti Life History Traits and Gut Microbiota.

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

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus.

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