Generating and Validating Transgenic Mosquitoes with Transposon-Mediated Transgenesis.

Transposon-mediated transgenesis has revolutionized both basic and applied studies of mosquito vectors of disease. Currently, techniques such as enhancer traps and transposon tagging, which rely on remobilizable insertional mutagenesis, are only possible with transposon-based vector systems. Here, we provide general descriptions of methods and applications of transposon-based mosquito transgenesis. The exact procedures must be adapted to each mosquito species and comparisons of some differences among different mosquito species are outlined. A number of excellent publications showing detailed and specific protocols and methods are featured and referenced.

Mosquito Transposon-Mediated Transgenesis.

Transposon-mediated transgenesis of mosquito vectors of disease pathogens followed the early success of transgenesis in the vinegar fly, Drosophila melanogaster The P transposable element used in Drosophila does not function canonically in mosquitoes, and repeatable, routine transgenesis in mosquitoes was not accomplished until new transposable elements were discovered and validated. A number of distinct transposons were subsequently identified that mediate the introduction of exogenous DNA in a stable and heritable manner in mosquito species, including members of the genera Aedes, Anopheles, and Culex The most versatile element, piggyBac, is functional in all of these mosquito genera, as well as in many other insects in diverse orders, and has been used extensively outside the class. Transposon-mediated transgenesis of recessive and dominant marker genes and reporter systems has been used to define functional fragments of gene control sequences, introduce exogenous DNA encoding products beneficial to medical interests, and act as "enhancer traps" to identify endogenous genes with specific expression characteristics.

The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme-induced peroxidase.

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

CRISPR mediated transactivation in the human disease vector Aedes aegypti.

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme–induced peroxidase

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

Beyond the eye: Kynurenine pathway impairment causes midgut homeostasis dysfunction and survival and reproductive costs in blood-feeding mosquitoes.

Insect ommochrome biosynthesis pathways metabolize tryptophan to generate eye-color pigments and wild-type alleles of pathway genes are useful phenotypic markers in transgenesis studies. Pleiotropic effects of mutations in some genes exert a load on both survival and reproductive success in blood-feeding species. Here, we investigated the challenges imposed on mosquitoes by the increase of tryptophan metabolites resulting from blood meal digestion and the impact of disruptions of the ommochrome biosynthesis pathway. Female mosquitoes with spontaneous and induced mutations in the orthologs of the genes encoding kynurenine hydroxylase in Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus exhibited impaired survival and reproductive phenotypes that varied in type and severity among the species. A compromised midgut permeability barrier function was also observed in An. stephensi. Surprisingly, mutant mosquitoes displayed an increase in microbiota compared to controls that was not accompanied by a general induction of immune genes. Antibiotic treatment rescued some deleterious traits implicating a role for the kynurenine pathway (KP) in midgut homeostasis. Supplemental xanthurenic acid, a KP end-product, rescued lethality and limited microbiota proliferation in Ae. aegypti. These data implicate the KP in the regulation of the host/microbiota interface. These pleiotropic effects on mosquito physiology are important in the development of genetic strategies targeting vector mosquitoes.

Use of Insect Promoters in Genetic Engineering to Control Mosquito-Borne Diseases.

Mosquito transgenesis and gene-drive technologies provide the basis for developing promising new tools for vector-borne disease prevention by either suppressing wild mosquito populations or reducing their capacity from transmitting pathogens. Many studies of the regulatory DNA and promoters of genes with robust sex-, tissue- and stage-specific expression profiles have supported the development of new tools and strategies that could bring mosquito-borne diseases under control. Although the list of regulatory elements available is significant, only a limited set of those can reliably drive spatial-temporal expression. Here, we review the advances in our ability to express beneficial and other genes in mosquitoes, and highlight the information needed for the development of new mosquito-control and anti-disease strategies.

Aedes aegypti Infection With Trypanosomatid Strigomonas culicis Alters Midgut Redox Metabolism and Reduces Mosquito Reproductive Fitness.

Aedes aegypti mosquitoes transmit arboviruses of important global health impact, and their intestinal microbiota can influence vector competence by stimulating the innate immune system. Midgut epithelial cells also produce toxic reactive oxygen species (ROS) by dual oxidases (DUOXs) that are essential players in insect immunity. Strigomonas culicis is a monoxenous trypanosomatid that naturally inhabits mosquitoes; it hosts an endosymbiotic bacterium that completes essential biosynthetic pathways of the parasite and influences its oxidative metabolism. Our group previously showed that S. culicis hydrogen peroxide (H2O2)-resistant (WTR) strain is more infectious to A. aegypti mosquitoes than the wild-type (WT) strain. Here, we investigated the influence of both strains on the midgut oxidative environment and the effect of infection on mosquito fitness and immunity. WT stimulated the production of superoxide by mitochondrial metabolism of midgut epithelial cells after 4 days post-infection, while WTR exacerbated H2O2 production mediated by increased DUOX activity and impairment of antioxidant system. The infection with both strains also disrupted the fecundity and fertility of the females, with a greater impact on reproductive fitness of WTR-infected mosquitoes. The presence of these parasites induced specific transcriptional modulation of immune-related genes, such as attacin and defensin A during WTR infection (11.8- and 6.4-fold, respectively) and defensin C in WT infection (7.1-fold). Thus, we propose that A. aegypti oxidative response starts in early infection time and does not affect the survival of the H2O2-resistant strain, which has a more efficient antioxidant system. Our data provide new biological aspects of A. aegypti-S. culicis relationship that can be used later in alternative vector control strategies.

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes.

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.

Non-immune Traits Triggered by Blood Intake Impact Vectorial Competence.

Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector-pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host-parasite relationships.

Non-canonical transcriptional regulation of heme oxygenase in Aedes aegypti.

Heme oxygenase (HO) is a ubiquitous enzyme responsible for heme breakdown, which yields carbon monoxide (CO), biliverdin (BV) and ferrous ion. Here we show that the Aedes aegypti heme oxygenase gene (AeHO - AAEL008136) is expressed in different developmental stages and tissues. AeHO expression increases after a blood meal in the midgut, and its maximal transcription levels overlaps with the maximal rate of the further modified A. aegypti biglutaminyl-biliverdin (AeBV) pigment production. HO is a classical component of stress response in eukaryotic cells, being activated under oxidative stress or increased heme levels. Indeed, the final product of HO activity in the mosquito midgut, AeBV, exerts a protective antioxidant activity. AeHO, however, does not seem to be under a classical redox-sensitive transcriptional regulation, being unresponsive to heme itself, and even down regulated when insects face a pro-oxidant insult. In contrast, AeHO gene expression responds to nutrient sensing mechanisms, through the target of rapamycin (TOR) pathway. This unusual transcriptional control of AeHO, together with the antioxidant properties of AeBV, suggests that heme degradation by HO, in addition to its important role in protection of Aedes aegypti against heme exposure, also acts as a digestive feature, being an essential adaptation to blood feeding.

Regulation of midgut cell proliferation impacts Aedes aegypti susceptibility to dengue virus.

Aedes aegypti is the vector of some of the most important vector-borne diseases like dengue, chikungunya, zika and yellow fever, affecting millions of people worldwide. The cellular processes that follow a blood meal in the mosquito midgut are directly associated with pathogen transmission. We studied the homeostatic response of the midgut against oxidative stress, as well as bacterial and dengue virus (DENV) infections, focusing on the proliferative ability of the intestinal stem cells (ISC). Inhibition of the peritrophic matrix (PM) formation led to an increase in reactive oxygen species (ROS) production by the epithelial cells in response to contact with the resident microbiota, suggesting that maintenance of low levels of ROS in the intestinal lumen is key to keep ISCs division in balance. We show that dengue virus infection induces midgut cell division in both DENV susceptible (Rockefeller) and refractory (Orlando) mosquito strains. However, the susceptible strain delays the activation of the regeneration process compared with the refractory strain. Impairment of the Delta/Notch signaling, by silencing the Notch ligand Delta using RNAi, significantly increased the susceptibility of the refractory strains to DENV infection of the midgut. We propose that this cell replenishment is essential to control viral infection in the mosquito. Our study demonstrates that the intestinal epithelium of the blood fed mosquito is able to respond and defend against different challenges, including virus infection. In addition, we provide unprecedented evidence that the activation of a cellular regenerative program in the midgut is important for the determination of the mosquito vectorial competence.

The redox-sensing gene Nrf2 affects intestinal homeostasis, insecticide resistance, and Zika virus susceptibility in the mosquito Aedes aegypti.

Production and degradation of reactive oxygen species (ROS) are extensively regulated to ensure proper cellular responses to various environmental stimuli and stresses. Moreover, physiologically generated ROS function as secondary messengers that can influence tissue homeostasis. The cap'n'collar transcription factor known as nuclear factor erythroid-derived factor 2 (Nrf2) coordinates an evolutionarily conserved transcriptional activation pathway that mediates antioxidant and detoxification responses in many animal species, including insects and mammals. Here, we show that Nrf2-mediated signaling affects embryo survival, midgut homeostasis, and redox biology in Aedes aegypti, a mosquito species vector of dengue, Zika, and other disease-causing viruses. We observed that AeNrf2 silencing increases ROS levels and stimulates intestinal stem cell proliferation. Because ROS production is a major aspect of innate immunity in mosquito gut, we found that a decrease in Nrf2 signaling results in reduced microbiota growth and Zika virus infection. Moreover, we provide evidence that AeNrf2 signaling also controls transcriptional adaptation of A. aegypti to insecticide challenge. Therefore, we conclude that Nrf2-mediated response regulates assorted gene clusters in A. aegypti that determine cellular and midgut redox balance, affecting overall xenobiotic resistance and vectorial adaptation of the mosquito.

Wolbachia and dengue virus infection in the mosquito Aedes fluviatilis (Diptera: Culicidae).

Dengue represents a serious threat to human health, with billions of people living at risk of the disease. Wolbachia pipientis is a bacterial endosymbiont common to many insect species. Wolbachia transinfections in mosquito disease vectors have great value for disease control given the bacterium's ability to spread into wild mosquito populations, and to interfere with infections of pathogens, such as dengue virus. Aedes fluviatilis is a mosquito with a widespread distribution in Latin America, but its status as a dengue vector has not been clarified. Ae. fluviatilis is also naturally infected by the wFlu Wolbachia strain, which has been demonstrated to enhance infection with the avian malarial parasite Plasmodium gallinaceum. We performed experimental infections of Ae. fluviatilis with DENV-2 and DENV-3 isolates from Brazil via injection or oral feeding to provide insight into its competence for the virus. We also examined the effect of the native Wolbachia infection on the virus using a mosquito line where the wFlu infection had been cleared by antibiotic treatment. Through RT-qPCR, we observed that Ae. fluviatilis could become infected with both viruses via either method of infection, although at a lower rate than Aedes aegypti, the primary dengue vector. We then detected DENV-2 and DENV-3 in the saliva of injected mosquitoes, and observed that injection of DENV-3-infected saliva produced subsequent infections in naïve Ae. aegypti. However, across our data we observed no difference in prevalence of infection and viral load between Wolbachia-infected and -uninfected mosquitoes, suggesting that there is no effect of wFlu on dengue virus. Our results highlight that Ae. fluviatilis could potentially serve as a dengue vector under the right circumstances, although further testing is required to determine if this occurs in the field.

The Dose Makes the Poison: Nutritional Overload Determines the Life Traits of Blood-Feeding Arthropods.

Vertebrate blood composition is heavily biased towards proteins, and hemoglobin, which is a hemeprotein, is by far the most abundant protein. Typically, hematophagous insects ingest blood volumes several times their weight before the blood meal. This barbarian feast offers an abundance of nutrients, but the degradation of blood proteins generates toxic concentrations of amino acids and heme, along with unparalleled microbiota growth. Despite this challenge, hematophagous arthropods have successfully developed mechanisms that bypass the toxicity of these molecules. While these adaptations allow hematophagous arthropods to tolerate their diet, they also constitute a unique mode of operation for cell signaling, immunity, and metabolism, the study of which may offer insights into the biology of disease vectors and may lead to novel vector-specific control methods.

The use of a chemically defined artificial diet as a tool to study Aedes aegypti physiology.

Aedes aegypti mosquitoes obtain from vertebrate blood nutrients that are essential to oogenesis, such as proteins and lipids. As with all insects, mosquitoes do not synthesize cholesterol but take it from the diet. Here, we used a chemically defined artificial diet, hereafter referred to as Substitute Blood Meal (SBM), that was supplemented with cholesterol to test the nutritional role of cholesterol. SBM-fed and blood-fed mosquitoes were compared regarding several aspects of the insect physiology that are influenced by a blood meal, including egg laying, peritrophic matrix formation, gut microbiota proliferation, generation of reactive oxygen species (ROS) and expression of antioxidant genes, such as catalase and ferritin. Our results show that SBM induced a physiological response that was very similar to a regular blood meal. Depending on the nutritional life history of the mosquito since the larval stage, the presence of cholesterol in the diet increased egg development, suggesting that the teneral reserves of cholesterol in the newly hatched female are determinant of reproductive performance. We propose here the use of SBM as a tool to study other aspects of the physiology of mosquitoes, including their interaction with microbiota and pathogens.

Heme Signaling Impacts Global Gene Expression, Immunity and Dengue Virus Infectivity in Aedes aegypti.

Blood-feeding mosquitoes are exposed to high levels of heme, the product of hemoglobin degradation. Heme is a pro-oxidant that influences a variety of cellular processes. We performed a global analysis of heme-regulated Aedes aegypti (yellow fever mosquito) transcriptional changes to better understand influence on mosquito physiology at the molecular level. We observed an iron- and reactive oxygen species (ROS)-independent signaling induced by heme that comprised genes related to redox metabolism. By modulating the abundance of these transcripts, heme possibly acts as a danger signaling molecule. Furthermore, heme triggered critical changes in the expression of energy metabolism and immune response genes, altering the susceptibility towards bacteria and dengue virus. These findings seem to have implications on the adaptation of mosquitoes to hematophagy and consequently on their ability to transmit diseases. Altogether, these results may also contribute to the understanding of heme cell biology in eukaryotic cells.