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The adult females of Aedes aegypti mosquitoes are facultative hematophagous insects but they are unable to feed on blood right after pupae emergence. The maturation process that takes place during the first post-emergence days, hereafter named hematophagic and gonotrophic capacitation, comprises a set of molecular and physiological changes that prepare the females for the first gonotrophic cycle. Notwithstanding, the molecular bases underlying mosquito hematophagic and gonotrophic capacitation remain obscure. Here, we investigated the molecular and biochemical changes in adult Ae. aegypti along the first four days post-emergence, prior to a blood meal. We performed a RNA-Seq analysis of the head and body, comparing male and female gene expression time courses. A total of 811 and 203 genes were differentially expressed, respectively in the body and head, and both body parts showed early, mid, and late female-specific expression profiles. Female-specific up-regulation of genes involved in muscle development and the oxidative phosphorylation pathway were remarkable features observed in the head. Functional assessment of mitochondrial oxygen consumption in heads showed a gradual increase in respiratory capacity and ATP-linked respiration as a consequence of induced mitochondrial biogenesis and content over time. This pattern strongly suggests that boosting oxidative phosphorylation in heads is a required step towards blood sucking habit. Several salivary gland genes, proteases, and genes involved in DNA replication and repair, ribosome biogenesis, and juvenile hormone signaling were up-regulated specifically in the female body, which may reflect the gonotrophic capacitation. This comprehensive description of molecular and biochemical mechanisms of the hematophagic and gonotrophic capacitation in mosquitoes unravels potentially new targets for vector control., Author summary Aedes aegypti is a vector of several human diseases such as Dengue, Zika, Chikungunya, and Yellow Fever. Only the adult females can feed on blood, important for the production of their offspring, and therefore transmitting these pathogens for the human host. Interestingly, right after their emergence as adults, females show a preference of feeding on nectar, and only after three days, they are fully capacitated to perform a blood meal. Understanding the molecular mechanisms that orchestrate this physiological and biochemical switch is the main topic of the present study. The transcriptome analysis of males and females during the first hours post-emergence reveals that the gene expression in females is tightly regulated, showing three time-dependent profiles of expression. There is an increase in genes related to the oxidative phosphorylation pathway in the female head. Assessment of mitochondrial function revealed a gradual increase in respiration linked to ATP synthesis and mitochondrial content. Also, there is an increase in genes related to the muscular function, suggesting a putative role in muscle development and contraction, required, for example, for pumping of blood. The female body showed an increase in salivary gland related genes and serine proteases, probably related to the ingestion and digestion of the blood. Also, the increase in DNA replication, ribosome biogenesis, and juvenile hormone signaling pathways together with changes in cell cycle control suggest the gonotrophic capacitation for the massive synthesis of yolk proteins precursors during vitellogenesis. All these genes were enhanced even before the first blood meal, which reveals insightful information about the biology of anautogenous mosquitoes. Our data showed the molecular machinery that stands the females hematophagy and gonotrophy, leading to potential new targets for vector control.

Aedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. Using CRISPR-Cas9 we generated loss-of-function mutations in several genes hypothesized to control flight in mosquitoes, including actin (AeAct-4) and myosin (myo-fem) genes expressed specifically in the female flight muscle. Genetic knockout of these genes resulted in 100% flightless females, with homozygous males able to fly, mate, and produce offspring, albeit at a reduced rate when compared to wild type males. Interestingly, we found that while AeAct-4 was haplosufficient, with most heterozygous individuals capable of flight, this was not the case for myo-fem, where about half of individuals carrying only one intact copy could not fly. These findings lay the groundwork for developing novel mechanisms of controlling Ae. aegypti populations, and our results suggest that this mechanism could be applicable to other vector species of mosquito., Author summary Ae. aegypti is the most important vector of arboviruses throughout the world, and new strategies are urgently needed to add to our existing arsenal of control methods to prevent or halt disease transmission. Importantly, only female Ae. aegypti mosquitoes transmit arboviruses due to their need for vertebrate blood to support egg production. Here, we identify several genes that are critical only for female mosquito flight; genetic ablation of each gene resulted in flightless females, but flying males. Importantly, the female flightless phenotype was completely penetrant (100%), and the use of sex-biased flight muscle proteins appeared to be conserved throughout mosquito evolution. These data could be used to support the development of novel genetic control approaches to reduce the number of biting females across a wide range of disease vector species.

As a key vector for major arthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya, control of Aedes aegypti represents a major challenge in public health. Bloodmeal acquisition is necessary for the reproduction of vector mosquitoes and pathogen transmission. Blood contains potentially toxic amounts of iron while it provides nutrients for mosquito offspring; disruption of iron homeostasis in the mosquito may therefore lead to novel control strategies. We previously described a potential iron exporter in Ae. aegypti after a targeted functional screen of ZIP (zinc-regulated transporter/Iron-regulated transporter-like) and ZnT (zinc transporter) family genes. In this study, we performed an RNAseq-based screen in an Ae. aegypti cell line cultured under iron-deficient and iron-excess conditions. A subset of differentially expressed genes were analyzed via a cytosolic iron-sensitive dual-luciferase reporter assay with several gene candidates potentially involved in iron transport. In vivo gene silencing resulted in significant reduction of fecundity (egg number) and fertility (hatch rate) for one gene, termed dyspepsia. Silencing of dyspepsia reduced the induction of ferritin expression in the midgut and also resulted in delayed/impaired excretion and digestion. Further characterization of this gene, including a more direct confirmation of its substrate (iron or otherwise), could inform vector control strategies as well as to contribute to the field of metal biology., Author summary Aedes aegypti is the major vector of dengue, Zika, chikungunya and yellow fever viruses. One of the most effective ways to reduce transmission of these viruses is to control Ae. aegypti populations. However, these mosquitoes continue to develop resistance to conventional insecticides. Therefore, alternative methods to control mosquitoes are urgently needed. Most mosquitoes require a bloodmeal greater than their body weight in order to produce eggs. The nutrient-rich blood also contains a large amount of iron, which in such large quantities could be toxic. This implies that the regulation of iron homeostasis is critical to mosquito fitness, and suggests that the mechanisms for how mosquitoes achieve this balancing act with regards to iron may lead to novel mosquito control approaches. Here, we performed a screen to identify transporters potentially involved in mosquito iron homeostasis. After identifying differentially expressed genes via RNAseq and validating a subset in cell culture assays, gene silencing experiments in whole mosquitoes revealed a gene whose reduction resulted in a change in ferritin gene expression and a drastic reduction of egg numbers and hatch rate. Further investigation of this gene may lead to novel mosquito control approaches.

Aedes mosquitoes can transmit dengue and several other severe vector-borne viral diseases, thereby influencing millions of people worldwide. Insects primarily control and clear the viral infections via their innate immune systems. Mitogen-Activated Protein Kinases (MAPKs) and antimicrobial peptides (AMPs) are both evolutionarily conserved components of the innate immune systems. In this study, we investigated the role of MAPKs in Aedes mosquitoes following DENV infection by using genetic and pharmacological approaches. We demonstrated that knockdown of ERK, but not of JNK or p38, significantly enhances the viral replication in Aedes mosquito cells. The Ras/ERK signaling is activated in both the cells and midguts of Aedes mosquitoes following DENV infection, and thus plays a role in restricting the viral infection, as both genetic and pharmacological activation of the Ras/ERK pathway significantly decreases the viral titers. In contrast, inhibition of the Ras/ERK pathway enhances DENV infection. In addition, we identified a signaling crosstalk between the Ras/ERK pathway and DENV-induced AMPs in which defensin C participates in restricting DENV infection in Aedes mosquitoes. Our results reveal that the Ras/ERK signaling pathway couples AMPs to mediate the resistance of Aedes mosquitoes to DENV infection, which provides a new insight into understanding the crosstalk between MAPKs and AMPs in the innate immunity of mosquito vectors during the viral infection., Author summary Aedes mosquitoes are the vectors that can transmit dengue fever and several other severe vector-borne viral diseases in the human population. Insects control and clear viral infections via their innate immune systems. Studies of the genes and molecules in mosquitos that control pathogen infection may provide insights into finding novel targets for controlling the transmission of mosquito-borne diseases. In this study, we used genetic and pharmacological approaches to investigate the role of MAPKs in Aedes mosquitoes following DENV2 infection in vitro and in vivo. Activation of the Ras/ERK pathway via insulin treatment and overexpression of Ras significantly decreased the DENV titers, whereas inhibition of the Ras/ERK pathway by either U0126 treatment or by Ras knockdown enhanced DENV infection. We demonstrated that the Ras/ERK signaling couples AMPs to mediate the resistance of Aedes mosquitoes to DENV infection, which reveals a novel antiviral mechanism regulated by multiple innate immune components in mosquitoes.

Aedes aegypti is the primary vector of dengue, chikungunya, Zika, and urban yellow fever. Insecticides are often the most effective tools to rapidly decrease the density of vector populations, especially during arbovirus disease outbreaks. However, the intense use of insecticides, particularly pyrethroids, has selected for resistant mosquito populations worldwide. Mutations in the voltage gated sodium channel (NaV) are among the principal mechanisms of resistance to pyrethroids and DDT, also known as “knockdown resistance,” kdr. Here we report studies on the origin and dispersion of kdr haplotypes in samples of Ae. aegypti from its worldwide distribution. We amplified the IIS6 and IIIS6 NaV segments from pools of Ae. aegypti populations from 15 countries, in South and North America, Africa, Asia, Pacific, and Australia. The amplicons were barcoded and sequenced using NGS Ion Torrent. Output data were filtered and analyzed using the bioinformatic pipeline Seekdeep to determine frequencies of the IIS6 and IIIS6 haplotypes per population. Phylogenetic relationships among the haplotypes were used to infer whether the kdr mutations have a single or multiple origin. We found 26 and 18 haplotypes, respectively for the IIS6 and IIIS6 segments, among which were the known kdr mutations 989P, 1011M, 1016I and 1016G (IIS6), 1520I, and 1534C (IIIS6). The highest diversity of haplotypes was found in African samples. Kdr mutations 1011M and 1016I were found only in American and African populations, 989P + 1016G and 1520I + 1534C in Asia, while 1534C was present in samples from all continents, except Australia. Based primarily on the intron sequence, IIS6 haplotypes were subdivided into two well-defined clades (A and B). Subsequent phasing of the IIS6 + IIIS6 haplotypes indicates two distinct origins for the 1534C kdr mutation. These results provide evidence of kdr mutations arising de novo at specific locations within the Ae. aegypti geographic distribution. In addition, our results suggest that the 1534C kdr mutation had at least two independent origins. We can thus conclude that insecticide selection pressure with DDT and more recently with pyrethroids is selecting for independent convergent mutations in NaV., Author summary Insecticide resistance is a global threat for the control of Aedes aegypti, the mosquito vector of aboviruses such as dengue, chikungunya and Zika. Mutations in the voltage gated sodium channel (NaV), known as kdr, are one of the principal mechanisms related to resistance to pyrethroids, the class of insecticide most employed worldwide inside and around residences. We investigate whether the same kdr mutations found in Ae. aegypti populations from distinct regions of the world have a common origin and subsequently dispersed or if they emerged in unrelated populations at distinct moments. By evaluating the sequences of two fragments of the NaV gene, obtained from DNA collections of Ae. aegypti from several countries, we found at least two independent origins for the F1534C kdr mutation in American, African and Asian populations. There was no evidence for multiple origins of the common kdr mutations V1016I and P989S + V1016G, which were exclusive to American and Asian populations. Our results increase our knowledge of insecticide resistance evolution in one of the main arboviral mosquito vectors of major global diseases.

Background Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown. Methodology/Principal findings V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNav1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNav1-1 sensitivity to PMT or DMT. However, the double mutant T1520I+F1534C was more resistant to PMT than F1534C, but remained sensitive to DMT. In contrast, the double mutant V1016I+F1534C was resistant to DMT and more resistant to PMT than F1534C. Furthermore, V1016I/G and F1534C channels, but not T1520I, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNav1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT. Conclusions/Significance Our data corroborated the previously proposed sequential selection of kdr mutations in Ae. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance., Author summary Intensive use of pyrethroids has led to the selection of resistance in mosquitoes, and knockdown resistance (kdr) is one of the major mechanisms of pyrethroid resistance. So far, eleven kdr mutations were identified to be associated with pyrethroid resistance in Aedes aegypti. Among the mutations, the V1016I and T1520I substitutions were found to be associated with F1534C but rarely found alone. F1534C confers sodium channel resistance to Type I pyrethroids including permethrin (PMT). However, whether V1016I or T1520I enhances the F1534C-mediated sodium channel resistance remain unknown. In this study, our electrophysiological results confirmed their involvement in kdr and corroborate the previously proposed sequential selection of kdr mutations in Ae. aegypti: F1534C likely emerged first in response to DDT and/or pyrethroids, whereas V1016I and T1520I appeared later under more intensive selection from pyrethroid use.

Prostaglandins (PGs) are immuno-active lipids that mediate the immune response in invertebrates and vertebrates. In insects, PGs play a role on different physiological processes such as reproduction, ion transport and regulation of cellular immunity. However, it is unclear whether PGs play a role in invertebrate's humoral immunity, and, if so, which immune signaling pathways would be modulated by PGs. Here, we show that Aedes aegypti gut microbiota and Gram-negative bacteria challenge induces prostaglandin production sensitive to an irreversible inhibitor of the vertebrate cyclooxygenase, acetylsalicylic acid (ASA). ASA treatment reduced PG synthesis and is associated with decreased expression of components of the Toll and IMD immune pathways, thereby rendering mosquitoes more susceptible to both bacterial and viral infections. We also shown that a cytosolic phospholipase (PLAc), one of the upstream regulators of PG synthesis, is induced by the microbiota in the midgut after blood feeding. The knockdown of the PLAc decreased prostaglandin production and enhanced the replication of Dengue in the midgut. We conclude that in Ae. aegypti, PGs control the amplitude of the immune response to guarantee an efficient pathogen clearance., Author summary Mosquito immune responses work on an on/off switch model, where the recognition of microorganisms turns the switch on, and its clearance turns it off. Besides pathogen derived molecules, no other compounds are known to directly regulate the activation and the amplitude of immune responses. Here we described that prostaglandins, lipid mediators of the immunity in vertebrates, also modulate the amplitude of immune responses in mosquitoes as well. Prostaglandins regulate the production of antimicrobial peptides and other effector molecules and directly impact the susceptibility of mosquitoes to bacterial and viral infections. When prostaglandin production is impaired, immune activation is inefficient and renders the mosquito more susceptible to bacterial and Dengue infections.

Mosquitoes are incriminated as vectors for many crippling diseases, including malaria, West Nile fever, Dengue fever, and other neglected tropical diseases (NTDs). microRNAs (miRNAs) can interact with multiple target genes to elicit biological functions in the mosquitoes. However, characterization and function of individual miRNAs and their potential targets have not been fully determined to date. We conducted a systematic review of published literature following PRISMA guidelines. We summarize the information about miRNAs in mosquitoes to better understand their metabolism, development, and responses to microorganisms. Depending on the study, we found that miRNAs were dysregulated in a species-, sex-, stage-, and tissue/organ-specific manner. Aberrant miRNA expressions were observed in development, metabolism, host-pathogen interactions, and insecticide resistance. Of note, many miRNAs were down-regulated upon pathogen infection. The experimental studies have expanded the identification of miRNA target from the 3′ untranslated regions (UTRs) of mRNAs of mosquitoes to the 5′ UTRs of mRNAs of the virus. In addition, we discuss current trends in mosquito miRNA research and offer suggestions for future studies., Author summary Millions of human infections are caused by mosquito-borne diseases, so understanding the molecular and genetic mechanisms that determine variability in transmission efficiency and insect susceptibility may assist with novel vector control strategies. Recently, microRNAs (miRNAs) have been studied as post-transcriptional regulators of gene expression in vertebrates, invertebrates, and viruses. Because interactions between vectors and parasites are the keys to malarial transmission dynamics, identifying miRNAs involved in the developmental cycles of the mosquito and interactions between the mosquito and pathogens that allow the survival, proliferation, and disease transmission may help with disease control. In this review, we summarize the research on mosquito miRNAs regarding their expression and function in various physiological and pathological processes, and explore future research in this area.

The mosquito Aedes aegypti is the primary vector of human arboviral diseases caused by dengue, chikungunya and Zika viruses. Many studies have shown the potential roles of small RNA molecules such as microRNA, small interfering RNA and PIWI-interacting RNA in vector mosquitoes. The function of tRNA fragments (tRF), the newly discovered class of small RNAs, in mosquitoes is not known. In this study, we show that specific tRFs are expressed in significantly differential manner between males and females of Ae. aegypti strains. Specific tRFs also show differential response during developmental transition from larvae to adults, as well as after blood feeding of adult females. The expression pattern of tRFs upon blood feeding varied depending upon if the blood contained dengue virus, and also if the females were treated with antibiotic prior to feeding to cleanse of the gut bacteria. Our findings show that a single tRF derived from the precursor sequences of a tRNA-Gly was differentially expressed between males and females, developmental transitions and also upon blood feeding by females of two laboratory strains that vary in midgut susceptibility to dengue virus infection. The multifaceted functional implications of this specific tRF suggest that biogenesis of small regulatory molecules from a tRNA can have wide ranging effects on key aspects of Ae. aegypti vector biology., Author summary The mosquito Aedes aegypti is a major vector of arboviral diseases in subtropics and tropics. The confounding effects of immature development and adult microbiome on the ability of Ae. aegypti to transmit diseases (vector competence) have gained renewed attention in the recent years. However, the molecular nature of these links/ effects remains unknown. This is major gap in knowledge regarding how vector competence is regulated at molecular level, and how that regulation may be variable among different strains of this mosquito. In this study, we investigated expression of newly discovered class of small RNAs, called tRNA fragments (tRF) in Ae. aegypti strains. Based on small RNA sequencing and bioinformatics analyses, we show that tRFs are expressed in Ae. aegypti, and they are associated with significant changes in expression between males and females, during development stages, and post blood feeding responses. A single tRF showed association with sex-biased expression, developmental regulation and in response to blood meals between Moyo-S and Moyo-R strains that differ in midgut susceptibility to dengue virus. The findings of this study are expected to guide future research efforts directed toward examining detailed regulatory mechanisms of tRFs in vector competence of Ae. aegypti to disease transmission.

Background Digestion of blood in the midgut of Aedes aegypti results in the release of pro-oxidant molecules that can be toxic to the mosquito. We hypothesized that after a blood meal, the antioxidant capacity of the midgut is increased to protect cells against oxidative stress. Concomitantly, pathogens present in the blood ingested by mosquitoes, such as the arboviruses Dengue and Zika, also have to overcome the same oxidative challenge, and the antioxidant program induced by the insect is likely to influence infection status of the mosquito and its vectorial competence. Methodology/Principal findings We found that blood-induced catalase mRNA and activity in the midgut peaked 24 h after feeding and returned to basal levels after the completion of digestion. RNAi-mediated silencing of catalase (AAEL013407-RB) reduced enzyme activity in the midgut epithelia, increased H2O2 leakage and decreased fecundity and lifespan when mosquitoes were fed H2O2. When infected with Dengue 4 and Zika virus, catalase-silenced mosquitoes showed no alteration in infection intensity (number of plaque forming units/midgut) 7 days after the infectious meal. However, catalase knockdown reduced Dengue 4, but not Zika, infection prevalence (percent of infected midguts). Conclusion/Significance Here, we showed that blood ingestion triggers an antioxidant response in the midgut through the induction of catalase. This protection facilitates the establishment of Dengue virus in the midgut. Importantly, this mechanism appears to be specific for Dengue because catalase silencing did not change Zika virus prevalence. In summary, our data suggest that redox balance in the midgut modulates mosquito vectorial competence to arboviral infections., Author summary Mosquitoes ingest large amounts of blood, a rich and abundant source of energy to sustain egg production. Blood digestion offers challenges to the insect, like managing high concentrations of heme and iron, pro-oxidant and potentially toxic molecules derived from hemoglobin. Mosquitoes and other blood-feeding arthropods have evolved adaptations to overcome this problem, such as the activation of catalase, an antioxidant enzyme that protect tissues against toxic free radicals. Mosquitoes act as important vectors of human diseases because during a blood meal they might also ingest microorganism circulating in our blood, such as dengue and zika virus. The adaptive antioxidant program that protects mosquito tissues against the oxidative challenge imposed by a blood meal might also influences the ability of virus to establish infection and disseminate from the midgut to the salivary glands. We show here that catalase differentially influences the number of infected midguts after mosquitoes were challenged with blood contaminated with virus, being beneficial to Dengue-4 but neutral do Zika, suggesting that redox metabolism may have distinct roles on mosquito vector competence towards different arbovirus.

We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role., Author Summary Dengue has represented a significant public health burden for a number of decades, and given the lack of dengue-specific drugs and limited availability of licensed vaccine, new methods for prevention and control are urgently needed. Here, we investigated whether genetic manipulation of the mosquitoes’ native JAK/STAT pathway-mediated anti-DENV defense system could be used to render mosquitoes more resistant to infection. We generated Ae. aegypti mosquitoes overexpressing the JAK/STAT pathway components Dome and Hop under the control of a bloodmeal-inducible, fat body-specific vitellogenin (Vg) promoter. These genetically modified mosquitoes showed an increased resistance to DENV infection, likely because of higher expression of DENV restriction factors and lower expression of DENV host factors, as indicated by transcriptome analyses. Expression of the transgenes had a minimal impact on mosquito longevity; however, it significantly impaired the mosquitoes’ fecundity. Interestingly, bloodmeal-inducible fat body-specific overexpression of either Hop or Dome did not affect mosquito permissiveness to either ZIKV or CHIKV infection, suggesting a possible specialization of JAK/STAT pathway antiviral defenses. Thus, our study is the first to provide a proof-of-concept that genetic engineering of the mosquitoes’ JAK/STAT immune pathway can be used to render this host more resistant to DENV infection.

The mosquito midgut microbiota has been shown to influence vector competence for multiple human pathogens. The microbiota is highly variable in the field, and the sources of this variability are not well understood, which limits our ability to understand or predict its effects on pathogen transmission. In this work, we report significant variation in female adult midgut bacterial load between strains of A. aegypti which vary in their susceptibility to dengue virus. Composition of the midgut microbiome was similar overall between the strains, with 81–92% of reads coming from the same five bacterial families, though we did detect differences in the presence of some bacterial families including Flavobacteriaceae and Entobacteriaceae. We conducted transcriptomic analysis on the two mosquito strains that showed the greatest difference in bacterial load, and found that they differ in transcript abundance of many genes implicated in amino acid metabolism, in particular the branched chain amino acid degradation pathway. We then silenced this pathway by targeting multiple genes using RNA interference, which resulted in strain-specific bacterial proliferation, thereby eliminating the difference in midgut bacterial load between the strains. This suggests that the branched chain amino acid (BCAA) degradation pathway controls midgut bacterial load, though the mechanism underlying this remains unclear. Overall, our results indicate that amino acid metabolism can act to influence the midgut microbiota. Moreover, they suggest that genetic or physiological variation in BCAA degradation pathway activity may in part explain midgut microbiota variation in the field., Author summary The mosquito midgut microbiota plays an important role in mosquito susceptibility to human pathogens and therefore is an important component of mosquito disease transmission. The microbiota can be highly variable, however, and the sources of this variation are not well understood. In this work, we aimed to improve our understanding of the ways in which the mosquito can influence its microbiota. To do this, we utilized strains of Aedes aegypti that vary in their midgut microbial load. We compared the transcriptomes of two strains with highly disparate bacterial loads, and found that genes involved in amino acid metabolism (specifically branched chain amino acid degradation) were differentially regulated between the strains. We found that when we silenced these genes using an RNA interference approach, bacterial loads increased in a strain-specific manner, confirming a role for these genes in controlling proliferation of the midgut microbiota. Overall, these results suggest that differences in amino acid metabolism or catabolism in the mosquito could have important implications for the mosquito microbiota.

Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that Aedes aegypti larvae colonized by a native community of bacteria and gnotobiotic larvae colonized by only Escherichia coli develop very similarly into adults, whereas axenic larvae never molt and die as first instars. In this study, we extended these findings by first comparing the growth and abundance of bacteria in conventional, gnotobiotic, and axenic larvae during the first instar. Results showed that conventional and gnotobiotic larvae exhibited no differences in growth, timing of molting, or number of bacteria in their digestive tract. Axenic larvae in contrast grew minimally and never achieved the critical size associated with molting by conventional and gnotobiotic larvae. In the second part of the study we compared patterns of gene expression in conventional, gnotobiotic and axenic larvae by conducting an RNAseq analysis of gut and nongut tissues (carcass) at 22 h post-hatching. Approximately 12% of Ae. aegypti transcripts were differentially expressed in axenic versus conventional or gnotobiotic larvae. However, this profile consisted primarily of transcripts in seven categories that included the down-regulation of select peptidases in the gut and up-regulation of several genes in the gut and carcass with roles in amino acid transport, hormonal signaling, and metabolism. Overall, our results indicate that axenic larvae exhibit alterations in gene expression consistent with defects in acquisition and assimilation of nutrients required for growth., Author Summary Several mosquito species including Aedes aegypti transmit pathogens as adults that cause disease in humans and other vertebrates. It has also long been known that mosquitoes host bacteria in their digestive tract, which are primarily acquired during the larval stage and transstadially transmitted to adults. Our recent results indicate that axenic larvae, which lack bacteria, do not develop beyond the first instar, whereas larvae with living bacteria develop into adults. To better understand the effects of bacteria on mosquito development, we compared growth, molting and gene expression in larval Ae. aegypti that contained several species of bacteria, only one species of bacterium (Escherichia coli), or no bacteria. Results showed that larvae containing several species or only E. coli grew and molted very similarly while larvae with no bacteria grew minimally and never molted. A number of Ae. aegypti genes with roles in regulating growth were differentially expressed in larvae without bacteria when compared to larvae with bacteria. Overall, our results indicate that mosquito larvae without bacteria do not grow or molt because of defects in assimilating nutrients.