Non-canonical odor coding in the mosquito.

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

Conserved and novel enhancers in the Aedes aegypti single-minded locus recapitulate embryonic ventral midline gene expression.

Transcriptional cis-regulatory modules, e.g., enhancers, control the time and location of metazoan gene expression. While changes in enhancers can provide a powerful force for evolution, there is also significant deep conservation of enhancers for developmentally important genes, with function and sequence characteristics maintained over hundreds of millions of years of divergence. Not well understood, however, is how the overall regulatory composition of a locus evolves, with important outstanding questions such as how many enhancers are conserved vs. novel, and to what extent are the locations of conserved enhancers within a locus maintained? We begin here to address these questions with a comparison of the respective single-minded (sim) loci in the two dipteran species Drosophila melanogaster (fruit fly) and Aedes aegypti (mosquito). sim encodes a highly conserved transcription factor that mediates development of the arthropod embryonic ventral midline. We identify two enhancers in the A. aegypti sim locus and demonstrate that they function equivalently in both transgenic flies and transgenic mosquitoes. One A. aegypti enhancer is highly similar to known Drosophila counterparts in its activity, location, and autoregulatory capability. The other differs from any known Drosophila sim enhancers with a novel location, failure to autoregulate, and regulation of expression in a unique subset of midline cells. Our results suggest that the conserved pattern of sim expression in the two species is the result of both conserved and novel regulatory sequences. Further examination of this locus will help to illuminate how the overall regulatory landscape of a conserved developmental gene evolves.

Ecdysone-controlled nuclear receptor ERR regulates metabolic homeostasis in the disease vector mosquito Aedes aegypti.

Hematophagous mosquitoes require vertebrate blood for their reproductive cycles, making them effective vectors for transmitting dangerous human diseases. Thus, high-intensity metabolism is needed to support reproductive events of female mosquitoes. However, the regulatory mechanism linking metabolism and reproduction in mosquitoes remains largely unclear. In this study, we found that the expression of estrogen-related receptor (ERR), a nuclear receptor, is activated by the direct binding of 20-hydroxyecdysone (20E) and ecdysone receptor (EcR) to the ecdysone response element (EcRE) in the ERR promoter region during the gonadotropic cycle of Aedes aegypti (named AaERR). RNA interference (RNAi) of AaERR in female mosquitoes led to delayed development of ovaries. mRNA abundance of genes encoding key enzymes involved in carbohydrate metabolism (CM)-glucose-6-phosphate isomerase (GPI) and pyruvate kinase (PYK)-was significantly decreased in AaERR knockdown mosquitoes, while the levels of metabolites, such as glycogen, glucose, and trehalose, were elevated. The expression of fatty acid synthase (FAS) was notably downregulated, and lipid accumulation was reduced in response to AaERR depletion. Dual luciferase reporter assays and electrophoretic mobility shift assays (EMSA) determined that AaERR directly activated the expression of metabolic genes, such as GPI, PYK, and FAS, by binding to the corresponding AaERR-responsive motif in the promoter region of these genes. Our results have revealed an important role of AaERR in the regulation of metabolism during mosquito reproduction and offer a novel target for mosquito control.

Precise coordination between nutrient transporters ensures fertility in the malaria mosquito Anopheles gambiae.

Females from many mosquito species feed on blood to acquire nutrients for egg development. The oogenetic cycle has been characterized in the arboviral vector Aedes aegypti, where after a bloodmeal, the lipid transporter lipophorin (Lp) shuttles lipids from the midgut and fat body to the ovaries, and a yolk precursor protein, vitellogenin (Vg), is deposited into the oocyte by receptor-mediated endocytosis. Our understanding of how the roles of these two nutrient transporters are mutually coordinated is however limited in this and other mosquito species. Here, we demonstrate that in the malaria mosquito Anopheles gambiae, Lp and Vg are reciprocally regulated in a timely manner to optimize egg development and ensure fertility. Defective lipid transport via Lp knockdown triggers abortive ovarian follicle development, leading to misregulation of Vg and aberrant yolk granules. Conversely, depletion of Vg causes an upregulation of Lp in the fat body in a manner that appears to be at least partially dependent on target of rapamycin (TOR) signaling, resulting in excess lipid accumulation in the developing follicles. Embryos deposited by Vg-depleted mothers are completely inviable, and are arrested early during development, likely due to severely reduced amino acid levels and protein synthesis. Our findings demonstrate that the mutual regulation of these two nutrient transporters is essential to safeguard fertility by ensuring correct nutrient balance in the developing oocyte, and validate Vg and Lp as two potential candidates for mosquito control.

An 11-point time course midgut transcriptome across 72 h after bloodfeeding provides detailed temporal resolution of transcript expression in the arbovirus vector, Aedes aegypti.

As the major vector for dengue, Zika, yellow fever, and chikungunya viruses, the mosquito Aedes aegypti is one of the most important insects in public health. These viruses are transmitted by bloodfeeding, which is also necessary for the reproduction of the mosquito. Thus, the midgut plays an essential role in mosquito physiology as the center for bloodmeal digestion and as an organ that serves as the first line of defense against viruses. Despite its importance, transcriptomic dynamics with fine temporal resolution across the entire digestion cycle have not yet been reported. To fill this gap, we conducted a transcriptomic analysis of A. aegypti female midguts across a 72-h bloodmeal digestion cycle for 11 time points, with a particular focus on the first 24 h. PCA analysis confirmed that 72 h is indeed a complete digestion cycle. Cluster and GO enrichment analysis showed the orchestrated modulation of thousands of genes to accomplish the midgut's role as the center for digestion, as well as nutrient transport with a clear progression with sequential emphasis on transcription, translation, energy production, nutrient metabolism, transport, and finally, autophagy by 24-36 h. We further determined that many serine proteases are robustly expressed as if to prepare for unexpected physiological challenges. This study provides a powerful resource for the analysis of genomic features that coordinate the rapid and complex transcriptional program induced by mosquito bloodfeeding.

A satellite repeat-derived piRNA controls embryonic development of Aedes.

Tandem repeat elements such as the diverse class of satellite repeats occupy large parts of eukaryotic chromosomes, mostly at centromeric, pericentromeric, telomeric and subtelomeric regions1. However, some elements are located in euchromatic regions throughout the genome and have been hypothesized to regulate gene expression in cis by modulating local chromatin structure, or in trans via transcripts derived from the repeats2-4. Here we show that a satellite repeat in the mosquito Aedes aegypti promotes sequence-specific gene silencing via the expression of two PIWI-interacting RNAs (piRNAs). Whereas satellite repeats and piRNA sequences generally evolve extremely quickly5-7, this locus was conserved for approximately 200 million years, suggesting that it has a central function in mosquito biology. piRNA production commenced shortly after egg laying, and inactivation of the more abundant piRNA resulted in failure to degrade maternally deposited transcripts in the zygote and developmental arrest. Our results reveal a mechanism by which satellite repeats regulate global gene expression in trans via piRNA-mediated gene silencing that is essential for embryonic development.

Efficient sex separation by exploiting differential alternative splicing of a dominant marker in Aedes aegypti.

Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.

On the Origin and Evolution of the Mosquito Male-determining Factor Nix.

The mosquito family Culicidae is divided into 2 subfamilies named the Culicinae and Anophelinae. Nix, the dominant male-determining factor, has only been found in the culicines Aedes aegypti and Aedes albopictus, 2 important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution of Nix. Except for the Culex genus, Nix homologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from 3 highly divergent tribes comprising 4 genera, suggesting Nix originated at least 133 to 165 million years ago (MYA). Heterologous expression of 1 of 3 divergent Nix open reading frames (ORFs) in Ae. aegypti resulted in partial masculinization of genetic females as evidenced by morphology and doublesex splicing. Phylogenetic analysis suggests Nix is related to femaleless (fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes. Nix from all species has a conserved structure, including 3 RNA-recognition motifs (RRMs), as does fle. However, Nix has evolved at a much faster rate than fle. The RRM3 of both Nix and fle are distantly related to the single RRM of a widely distributed and conserved splicing factor transformer-2 (tra2). The RRM3-based phylogenetic analysis suggests this domain in Nix and fle may have evolved from tra2 or a tra2-related gene in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex determination in mosquitoes and will inform broad applications of mosquito-control strategies based on manipulating sex ratios toward nonbiting males.

Long non-coding RNAs regulate Aedes aegypti vector competence for Zika virus and reproduction.

Long non-coding RNAs (lncRNAs) play critical regulatory roles in various cellular and metabolic processes in mosquitoes and all other organisms studied thus far. In particular, their involvement in essential processes such as reproduction makes them potential targets for the development of novel pest control approaches. However, their function in mosquito biology remains largely unexplored. To elucidate the role of lncRNAs in mosquitoes' reproduction and vector competence for arboviruses, we have implemented a computational and experimental pipeline to mine, screen, and characterize lncRNAs related to these two biological processes. Through analysis of publicly available Zika virus (ZIKV) infection-regulated Aedes aegypti transcriptomes, at least six lncRNAs were identified as being significantly upregulated in response to infection in various mosquito tissues. The roles of these ZIKV-regulated lncRNAs (designated Zinc1, Zinc2, Zinc3, Zinc9, Zinc10 and Zinc22), were further investigated by dsRNA-mediated silencing studies. Our results show that silencing of Zinc1, Zinc2, and Zinc22 renders mosquitoes significantly less permissive to ZIKV infection, while silencing of Zinc22 also reduces fecundity, indicating a potential role for Zinc22 in trade-offs between vector competence and reproduction. We also found that silencing of Zinc9 significantly increases fecundity but has no effect on ZIKV infection, suggesting that Zinc9 may be a negative regulator of oviposition. Our work demonstrates that some lncRNAs play host factor roles by facilitating viral infection in mosquitoes. We also show that lncRNAs can influence both mosquito reproduction and permissiveness to virus infection, two biological systems with important roles in mosquito vectorial capacity.

Aal-circRNA-407 regulates ovarian development of Aedes albopictus, a major arbovirus vector, via the miR-9a-5p/Foxl axis.

Aedes albopictus shows a rapid global expansion and dramatic vectorial capacity for various arboviruses, thus posing a severe threat to global health. Although many noncoding RNAs have been confirmed to play functional roles in various biological processes in Ae. albopictus, the roles of circRNA remain a mystery. In the present study, we first performed high-throughput circRNA sequencing in Ae. albopictus. Then, we identified a cysteine desulfurase (CsdA) superfamily gene-originated circRNA, named aal-circRNA-407, which was the third most abundant circRNA in adult females and displayed a fat body highly expressed manifestation and blood feeding-dependent onset. SiRNA-mediated knockdown of circRNA-407 resulted in a decrease in the number of developing follicles and a reduction in follicle size post blood meal. Furthermore, we demonstrated that circRNA-407 can act as a sponge of aal-miR-9a-5p to promote the expression of its target gene Foxl and eventually regulate ovarian development. Our study is the first to report a functional circRNA in mosquitoes, expanding our current understanding of important biological roles in mosquitoes and providing an alternative genetic strategy for mosquito control.

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.

MGSurvE: A framework to optimize trap placement for genetic surveillance of mosquito populations.

Genetic surveillance of mosquito populations is becoming increasingly relevant as genetics-based mosquito control strategies advance from laboratory to field testing. Especially applicable are mosquito gene drive projects, the potential scale of which leads monitoring to be a significant cost driver. For these projects, monitoring will be required to detect unintended spread of gene drive mosquitoes beyond field sites, and the emergence of alternative alleles, such as drive-resistant alleles or non-functional effector genes, within intervention sites. This entails the need to distribute mosquito traps efficiently such that an allele of interest is detected as quickly as possible-ideally when remediation is still viable. Additionally, insecticide-based tools such as bednets are compromised by insecticide-resistance alleles for which there is also a need to detect as quickly as possible. To this end, we present MGSurvE (Mosquito Gene SurveillancE): a computational framework that optimizes trap placement for genetic surveillance of mosquito populations such that the time to detection of an allele of interest is minimized. A key strength of MGSurvE is that it allows important biological features of mosquitoes and the landscapes they inhabit to be accounted for, namely: i) resources required by mosquitoes (e.g., food sources and aquatic breeding sites) can be explicitly distributed through a landscape, ii) movement of mosquitoes may depend on their sex, the current state of their gonotrophic cycle (if female) and resource attractiveness, and iii) traps may differ in their attractiveness profile. Example MGSurvE analyses are presented to demonstrate optimal trap placement for: i) an Aedes aegypti population in a suburban landscape in Queensland, Australia, and ii) an Anopheles gambiae population on the island of São Tomé, São Tomé and Príncipe. Further documentation and use examples are provided in project's documentation. MGSurvE is intended as a resource for both field and computational researchers interested in mosquito gene surveillance.

Genome sequencing and comparative analysis of Wolbachia strain wAlbA reveals Wolbachia-associated plasmids are common.

Wolbachia are widespread maternally-transmitted bacteria of arthropods that often spread by manipulating their host's reproduction through cytoplasmic incompatibility (CI). Their invasive potential is currently being harnessed in field trials aiming to control mosquito-borne diseases. Wolbachia genomes commonly harbour prophage regions encoding the cif genes which confer their ability to induce CI. Recently, a plasmid-like element was discovered in wPip, a Wolbachia strain infecting Culex mosquitoes; however, it is unclear how common such extra-chromosomal elements are in Wolbachia. Here we sequenced the complete genome of wAlbA, a strain of the symbiont found in Aedes albopictus, after eliminating the co-infecting and higher density wAlbB strain that previously made sequencing of wAlbA challenging. We show that wAlbA is associated with two new plasmids and identified additional Wolbachia plasmids and related chromosomal islands in over 20% of publicly available Wolbachia genome datasets. These plasmids encode a variety of accessory genes, including several phage-like DNA packaging genes as well as genes potentially contributing to host-symbiont interactions. In particular, we recovered divergent homologues of the cif genes in both Wolbachia- and Rickettsia-associated plasmids. Our results indicate that plasmids are common in Wolbachia and raise fundamental questions around their role in symbiosis. In addition, our comparative analysis provides useful information for the future development of genetic tools to manipulate and study Wolbachia symbionts.

The AalNix3&4 isoform is required and sufficient to convert Aedes albopictus females into males.

Aedes albopictus is one of the most invasive insect species in the world and an effective vector for many important arboviruses. We reported previously that Ae. albopictus Nix (AalNix) is the male-determining factor of this species. However, whether AalNix alone is sufficient to initiate male development is unknown. Transgenic lines that express each of the three AalNix isoforms from the native promoter were obtained using piggyBac transformation. We verified the stable expression of AalNix isoforms in the transgenic lines and confirm that one isoform, AalNix3&4, is sufficient to convert females into fertile males (pseudo-males) that are indistinguishable from wild-type males. We also established a stable sex-converted female mosquito strain, AalNix3&4-♂4-pseudo-male. The pseudo-male mosquitoes can fly and mate normally with wild-type female, although their mating competitiveness is lower than wild-type. This work further clarifies the role of AalNix in the sex determination pathway and will facilitate the development of Ae. albopictus control strategies that rely on male-only releases such as SIT and sex-ratio distortion.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes.

BACKGROUND: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. METHODS: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus. RESULTS: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. CONCLUSION: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.

The accumulation of modular serine protease mediated by a novel circRNA sponging miRNA increases Aedes aegypti immunity to fungus.

BACKGROUND: Mosquitoes transmit many infectious diseases that affect human health. The fungus Beauveria bassiana is a biological pesticide that is pathogenic to mosquitoes but harmless to the environment. RESULTS: We found a microRNA (miRNA) that can modulate the antifungal immunity of Aedes aegypti by inhibiting its cognate serine protease. Fungal infection can induce the expression of modular serine protease (ModSP), and ModSP knockdown mosquitoes were more sensitive to B. bassiana infection. The novel miRNA-novel-53 is linked to antifungal immune response and was greatly diminished in infected mosquitoes. The miRNA-novel-53 could bind to the coding sequences of ModSP and impede its expression. Double fluorescence in situ hybridization (FISH) showed that this inhibition occurred in the cytoplasm. The amount of miRNA-novel-53 increased after miRNA agomir injection. This resulted in a significant decrease in ModSP transcript and a significant increase in mortality after fungal infection. An opposite effect was produced after antagomir injection. The miRNA-novel-53 was also knocked out using CRISPR-Cas9, which increased mosquito resistance to the fungus B. bassiana. Moreover, mosquito novel-circ-930 can affect ModSP mRNA by interacting with miRNA-novel-53 during transfection with siRNA or overexpression plasmid. CONCLUSIONS: Novel-circ-930 affects the expression level of ModSP by a novel-circ-930/miRNA-novel-53/ModSP mechanism to modulate antifungal immunity, revealing new information on innate immunity in insects.

Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein.

BACKGROUND: Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects. RESULTS: Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference. CONCLUSIONS: Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.

The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types.

Mosquitoes are prolific vectors of human pathogens, therefore a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.

Differential gene expression and microRNA profile in corpora allata-corpora cardiaca of Aedes aegypti mosquitoes with weak juvenile hormone signalling.

The corpora allata-corpora cardiaca (CA-CC) is an endocrine gland complex that regulates mosquito development and reproduction through the synthesis of juvenile hormone (JH). Epoxidase (Epox) is a key enzyme in the production of JH. We recently utilized CRISPR/Cas9 to establish an epoxidase-deficient (epox-/-) Aedes aegypti line. The CA from epox-/- mutants do not synthesize epoxidated JH III but methyl farneosate (MF), a weak agonist of the JH receptor, and therefore have reduced JH signalling. Illumina sequencing was used to examine the differences in gene expression between the CA-CC from wild type (WT) and epox-/- adult female mosquitoes. From 18,034 identified genes, 317 were significantly differentially expressed. These genes are involved in many biological processes, including the regulation of cell proliferation and apoptosis, energy metabolism, and nutritional uptake. In addition, the same CA-CC samples were also used to examine the microRNA (miRNA) profiles of epox-/- and WT mosquitoes. A total of 197 miRNAs were detected, 24 of which were differentially regulated in epox-/- mutants. miRNA binding sites for these particular miRNAs were identified using an in silico approach; they target a total of 101 differentially expressed genes. Our results suggest that a lack of epoxidase, besides affecting JH synthesis, results in the diminishing of JH signalling that have significant effects on Ae. aegypti CA-CC transcriptome profiles, as well as its miRNA repertoire.