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.

A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations.

Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.

Characterization of essential eggshell proteins from Aedes aegypti mosquitoes.

BACKGROUND: Up to 40% of the world population live in areas where mosquitoes capable of transmitting the dengue virus, including Aedes aegypti, coexist with humans. Understanding how mosquito egg development and oviposition are regulated at the molecular level may provide new insights into novel mosquito control strategies. Previously, we identified a protein named eggshell organizing factor 1 (EOF1) that when knocked down with RNA interference (RNAi) resulted in non-melanized and fragile eggs that did not contain viable embryos. RESULTS: In this current study, we performed a comprehensive RNAi screen of putative A. aegypti eggshell proteins to identify additional proteins that interact with intracellular EOF1. We identified several proteins essential for eggshell formation in A. aegypti and characterized their phenotypes through a combination of molecular and biochemical approaches. We found that Nasrat, Closca, and Polehole structural proteins, together with the Nudel serine protease, are indispensable for eggshell melanization and egg viability. While all four proteins are predominantly expressed in ovaries of adult females, Nudel messenger RNA (mRNA) expression is highly upregulated in response to blood feeding. Furthermore, we identified four additional secreted eggshell enzymes that regulated mosquito eggshell formation and melanization. These enzymes included three dopachrome-converting enzymes (DCEs) and one cysteine protease. All eight of these eggshell proteins were essential for proper eggshell formation. Interestingly, their eggshell surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1, suggesting that additional mechanisms may influence how EOF1 regulates eggshell formation and melanization. CONCLUSIONS: While our studies did not identify a definitive regulator of EOF1, we did identify eight additional proteins involved in mosquito eggshell formation that may be leveraged for future control strategies.

Exposure to ultraviolet-B radiation increases the susceptibility of mosquitoes to infection with dengue virus.

By 2100, greenhouse gases are predicted to reduce ozone and cloud cover over the tropics causing increased exposure of organisms to harmful ultraviolet-B radiation (UVBR). UVBR damages DNA and is an important modulator of immune function and disease susceptibility in humans and other vertebrates. The effect of UVBR on invertebrate immune function is largely unknown, but UVBR together with ultraviolet-A radiation impairs an insect immune response that utilizes melanin, a pigment that also protects against UVBR-induced DNA damage. If UVBR weakens insect immunity, then it may make insect disease vectors more susceptible to infection with pathogens of socioeconomic and public health importance. In the tropics, where UVBR is predicted to increase, the mosquito-borne dengue virus (DENV), is prevalent and a growing threat to humans. We therefore examined the effect of UVBR on the mosquito Aedes aegypti, the primary vector for DENV, to better understand the potential implications of increased tropical UVBR for mosquito-borne disease risk. We found that exposure to a UVBR dose that caused significant larval mortality approximately doubled the probability that surviving females would become infected with DENV, despite this UVBR dose having no effect on the expression of an effector gene involved in antiviral immunity. We also found that females exposed to a lower UVBR dose were more likely to have low fecundity even though this UVBR dose had no effect on larval size or activity, pupal cuticular melanin content, or adult mass, metabolic rate, or flight capacity. We conclude that future increases in tropical UVBR associated with anthropogenic global change may have the benefit of reducing mosquito-borne disease risk for humans by reducing mosquito fitness, but this benefit may be eroded if it also makes mosquitoes more likely to be infected with deadly pathogens.

In Silico Characterisation of the Aedes aegypti Gustatory Receptors.

Aedes aegypti, also known as the dengue mosquito or the yellow fewer mosquito, is the vector of dengue, chikungunya, Zika, Mayaro and yellow fever viruses. The A. aegypti genome contains an array of gustatory receptor (GR) proteins that are related to the recognition of taste. In this study, we performed in silico molecular characterization of all 72 A. aegypti GRs reported in the latest version of A. aegypti genome AaegL5. Phylogenetic analysis classified the receptors into three major clads. Multiple GRs were found to encode multiple transcripts. Physicochemical attributes such as the aliphatic index, hydropathicity index and isoelectric point indicated that A. aegypti gustatory receptors are highly stable and are tailored to perform under a variety of cellular environments. Analysis for subcellular localization indicated that all the GRs are located either in the extracellular matrix or the plasma membrane. Results also indicated that the GRs are distributed mainly on chromosomes 2 and 3, which house 22 and 49 GRs, respectively, whereas chromosome 1 houses only one GR. NCBI-CDD analysis showed the presence of a highly conserved 7tm_7 chemosensory receptor protein superfamily that includes gustatory and odorant receptors from insect species Anopheles gambiae and Drosophila melanogaster. Further, three significantly enriched ungapped motifs in the protein sequence of all 72 A. aegypti gustatory receptors were found. High-quality 3D models for the tertiary structures were predicted with significantly higher confidence, along with ligand-binding residues. Prediction of S-nitrosylation sites indicated the presence of target cysteines in all the GRs with close proximity to the ligand-bindings sites within the 3D structure of the receptors. In addition, two highly conserved motifs inside the GR proteins were discovered that house a tyrosine (Y) and a cysteine (C) residue which may serve as targets for NO-mediated tyrosine nitration and S-nitrosylation, respectively. This study will help devise strategies for functional genomic studies of these important receptor molecules in A. aegypti and other mosquito species through in vitro and in vivo studies.

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.

Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis.

Cry11 proteins are toxic to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of Bacillus thuringiensis and subjected to median-lethal concentration (LC50) tests on first-instar larvae of A. aegypti. LC50 analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL-1), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against A. aegypti. This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.

OTU7B Modulates the Mosquito Immune Response to Beauveria bassiana Infection via Deubiquitination of the Toll Adaptor TRAF4.

The Aedes aegypti mosquito transmits devastating flaviviruses, such as Zika, dengue, and yellow fever viruses. For more effective control of the vector, the pathogenicity of Beauveria bassiana, a fungus commonly used for biological control of pest insects, may be enhanced based on in-depth knowledge of molecular interactions between the pathogen and its host. Here, we identified a mechanism employed by B. bassiana, which efficiently blocks the Ae. aegypti antifungal immune response by a protease that contains an ovarian tumor (OTU) domain. RNA-sequencing analysis showed that the depletion of OTU7B significantly upregulates the mRNA level of immunity-related genes after a challenge of the fungus. CRISPR-Cas9 knockout of OTU7B conferred a higher resistance of mosquitoes to the fungus B. bassiana. OTU7B suppressed activation of the immune response by preventing nuclear translocation of the NF-κB transcription factor Rel1, a mosquito orthologue of Drosophila Dorsal. Further studies identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as an interacting protein of OTU7B. TRAF4-deficient mosquitoes were more sensitive to fungal infection, indicating TRAF4 to be the adaptor protein that activates the Toll pathway. TRAF4 is K63-link polyubiquitinated at K338 residue upon immune challenge. However, OTU7B inhibited the immune signaling by enzymatically removing the polyubiquitin chains of mosquito TRAF4. Thus, this study has uncovered a novel mechanism of fungal action against the host innate immunity, providing a platform for further improvement of fungal pathogen effectiveness. IMPORTANCE Insects use innate immunity to defend against microbial infection. The Toll pathway is a major immune signaling pathway that is associated with the antifungal immune response in mosquitoes. Our study identified a fungal-induced deubiquitinase, OTU7B, which, when knocked out, promotes the translocation of the NF-κB factor Rel1 into the nucleus and confers enhanced resistance to fungal infection. We further found the counterpart of OTU7B, TRAF4, which is a component of the Toll pathway and acts as an adaptor protein. OTU7B enzymatically removes K63-linked polyubiquitin chains from TRAF4. The immune response is suppressed, and mosquitoes become much more sensitive to the Beauveria bassiana infection. Our findings reveal a novel mechanism of fungal action against the host innate immunity.

Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.).

We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60-75kDa and ~80-95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.

Aedes aegypti CLIPB9 activates prophenoloxidase-3 in the presence of CLIPA14 after fungal infection.

Melanization is an integral part of the insect defense system and is often induced by pathogen invasion. Phenoloxidases (POs) are critical enzymes that catalyze melanin formation. PO3 is associated with the antifungal response of the mosquito, Aedes aegypti, but the molecular mechanism of the prophenoloxidase-3 (PPO3) activation is unclear. Here we report that PPO3 cleavage activation is mediated by a clip-domain serine protease, CLIPB9. We purified recombinant CLIPB9 and found that it cleaved PPO3 and increased PO activity in the hemolymph. We then identified CLIPA14 (a serine protease homolog) by co-immunoprecipitation using anti-CLIPB9 antibody. After being cleaved by CLIPB9, Ae. aegypti CLIPA14 acted as a cofactor for PPO3 activation. In addition, dsRNA co-silencing of CLIPB9 and CLIPA14 genes reduced melanization after infection with the entomopathogen, Beauveria bassiana, making the adult mosquitoes more sensitive to fungal infection. These results illustrate the roles of CLIPB9 and CLIPA14 in the PPO activation pathway and revealed the complexity of the upstream serine protease network controlling melanization.

N6-methyladenosine modification of the Aedes aegypti transcriptome and its alteration upon dengue virus infection in Aag2 cell line.

The N6-methyladenosine (m6A) modification of RNA has been reported to affect viral infections. Studies have confirmed the role of m6A in replication of several vector-borne flaviviruses, including dengue virus (DENV), in mammalian cells. Here, we explored the role of m6A in DENV replication in the mosquito Aedes aegypti Aag2 cell line. We first determined the presence of m6A on the RNAs from mosquito cells and using methylated RNA immunoprecipitation and sequencing (MeRIP-Seq) identified m6A modification of the mosquito transcriptome and those that changed upon DENV infection. Depletion of m6A methyltransferases and the m6A binding protein YTHDF3 RNAs decreased the replication of DENV. In particular, we found that the Ae. aegypti ubiquitin carrier protein 9 (Ubc9) is m6A modified and its expression increases after DENV infection. Silencing of the gene and ectopic expression of Ubc9 led to reduced and increased DENV replication, respectively. The abundance of Ubc9 mRNA and its stability were reduced with the inhibition of m6A modification, implying that m6A modification of Ubc9 might enhance expression of the gene. We also show that the genome of DENV is m6A modified at five sites in mosquito cells. Altogether, this work reveals the involvement of m6A modification in Ae. aegypti-DENV interaction.

High DDT resistance without apparent association to kdr and Glutathione-S-transferase (GST) gene mutations in Aedes aegypti population at hotel compounds in Zanzibar.

Global efforts to control Aedes mosquito-transmitted pathogens still rely heavily on insecticides. However, available information on vector resistance is mainly restricted to mosquito populations located in residential and public areas, whereas commercial settings, such as hotels are overlooked. This may obscure the real magnitude of the insecticide resistance problem and lead to ineffective vector control and resistance management. We investigated the profile of insecticide susceptibility of Aedes aegypti mosquitoes occurring at selected hotel compounds on Zanzibar Island. At least 100 adults Ae. aegypti females from larvae collected at four hotel compounds were exposed to papers impregnated with discriminant concentrations of DDT (4%), permethrin (0.75%), 0.05 deltamethrin (0.05%), propoxur (0.1%) and bendiocarb (0.1%) to determine their susceptibility profile. Allele-specific qPCR and sequencing analysis were applied to determine the possible association between observed resistance and presence of single nucleotide polymorphisms (SNPs) in the voltage-gated sodium channel gene (VGSC) linked to DDT/pyrethroid cross-resistance. Additionally, we explored the possible involvement of Glutathione-S-Transferase gene (GSTe2) mutations for the observed resistance profile. In vivo resistance bioassay indicated that Ae. aegypti at studied sites were highly resistant to DDT, mortality rate ranged from 26.3% to 55.3% and, moderately resistant to deltamethrin with a mortality rate between 79% to and 100%. However, genotyping of kdr mutations affecting the voltage-gated sodium channel only showed a low frequency of the V1016G mutation (n = 5; 0.97%). Moreover, for GSTe2, seven non-synonymous SNPs were detected (L111S, C115F, P117S, E132A, I150V, E178A and A198E) across two distinct haplotypes, but none of these were significantly associated with the observed resistance to DDT. Our findings suggest that cross-resistance to DDT/deltamethrin at hotel compounds in Zanzibar is not primarily mediated by mutations in VGSC. Moreover, the role of identified GSTe2 mutations in the resistance against DDT remains inconclusive. We encourage further studies to investigate the role of other potential insecticide resistance markers.

Multiple Salivary Proteins from Aedes aegypti Mosquito Bind to the Zika Virus Envelope Protein.

Aedes aegypti mosquitoes are important vectors of several debilitating and deadly arthropod-borne (arbo) viruses, including Yellow Fever virus, Dengue virus, West Nile virus and Zika virus (ZIKV). Arbovirus transmission occurs when an infected mosquito probes the host's skin in search of a blood meal. Salivary proteins from mosquitoes help to acquire blood and have also been shown to enhance pathogen transmission in vivo and in vitro. Here, we evaluated the interaction of mosquito salivary proteins with ZIKV by surface plasmon resonance and enzyme-linked immunosorbent assay. We found that three salivary proteins AAEL000793, AAEL007420, and AAEL006347 bind to the envelope protein of ZIKV with nanomolar affinities. Similar results were obtained using virus-like particles in binding assays. These interactions have no effect on viral replication in cultured endothelial cells and keratinocytes. Additionally, we found detectable antibody levels in ZIKV and DENV serum samples against the recombinant proteins that interact with ZIKV. These results highlight complex interactions between viruses, salivary proteins and antibodies that could be present during viral transmissions.

Effects of gamma radiation on the reproductive viability of Aedes aegypti and its descendants (Diptera: Culicidae).

This work evaluated the genetic damage in descendants of male pupae of Aedes (Stegomyia) aegypti (Diptera: Culicidae) separately exposed to 20, 30, and 40 Gy of gamma radiation in the context of Sterile Insect Technique (SIT). Despite the transmission of the dominant lethal mutation, the employed dose levels did not promote a marked reduction in adult mosquito emergence and fertility. This study emphasized that semi-sterilizing doses < 50 Gy for SIT of Aedes aegypti are not recommended.

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti.

BACKGROUND: Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immune responses are the first line of defense in mosquitoes against the invasion of pathogenic microorganisms. The Toll pathway, immunodeficiency (IMD) pathway and melanization are the main processes of innate immunity in Aedes aegypti. CLIPS are classified into five subfamilies-CLIPA, CLIPB, CLIPC, CLIPD, and CLIPE-based on their sequence specificity and phylogenetic relationships. We report the functional characterization of the genes that code for two CLIPs in Ae. aegypti (Ae): Ae-CLIPB15 and Ae-CLIPB22. METHODS: Clustal Omega was used for multiple amino acid sequence alignment of Ae-CLIPB15 and Ae-CLIPB22 with different CLIP genes from other insect species. The spatiotemporal expression profiles of Ae-CLIPB15 and Ae-CLIPB22 were examined. We determined whether Ae-CLIPB15 and Ae-CLIPB22 respond to microbial challenge and tissue injury. RNA interference (RNAi) was used to explore the function of Ae-CLIPB15 and Ae-CLIPB22 in the defense of Ae. aegypti against bacterial and fungal infections. The expression levels of nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2 in the Toll pathway and IMD pathway after bacterial infection were investigated. Finally, the change in phenoloxidase (PO) activity in Ae-CLIPB15 and Ae-CLIPB22 knockdown adults was investigated. RESULTS: We performed spatiotemporal gene expression profiling of Ae-CLIPB15 and Ae-CLIPB22 genes in Ae. aegypti using quantitative real-time polymerase chain reaction. These genes were expressed in different stages and tissues. The messenger RNA (mRNA) levels for both genes were also up-regulated by Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus and fungal Beauveria bassiana infections, as well as in the tissue injury experiments. RNAi-mediated knockdown of Ae-CLIPB15 led to a significant decrease of PO activity in the hemolymph of Ae. aegypti, while other RNAi experiments revealed that both Ae-CLIPB15 and Ae-CLIPB22 were involved in immune defense against bacterial and fungal infections. The mRNA expression of NF-κB transcription factors REL1 and REL2 in the Toll pathway and IMD pathway differed between Ae-CLIPB15 and Ae-CLIPB22 knockdown mosquitoes infected with bacteria and wild type mosquitoes infected with bacteria. CONCLUSIONS: Our findings suggest that Ae-CLIPB15 and Ae-CLIPB22 play a critical role in mosquito innate immunity, and that they are involved in immune responses to injury and infection. Their regulation of transcription factors and PO activity indicates that they also play a specific role in the regulation of innate immunity.

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes.

In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3' end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3' ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3' ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3' end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3' end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.

Aedes aegypti SNAP and a calcium transporter ATPase influence dengue virus dissemination.

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world's population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.

Genotoxic and mutagenic effects of methyl ether dillapiole on the development of Aedes aegypti (Diptera: Culicidae).

Dillapiole, extracted from Piper aduncum essential oil and its derivatives, has been shown to be a potential alternative to the control of Aedes aegypti, which has become resistant to synthetic insecticides. Methyl ether dillapiole (MED) and temephos (TM) were compared to complement the data on the genotoxicity and developmental changes of Ae. aegypti. Over four generations (G1 -G4 ), third stage larvae were treated with MED at 60, 80 and 100 µg/mL and TM at 0.002, 0.005 and 0.007 µg/mL for 4 h. Adult females were separated to estimate oviposition and hatching rates, and total egg length. Over the four generations, a significant reduction was recorded in oviposition and hatching rates, and in mean egg length (Tukey, P < 0.05), compared with the negative control (NC). Cytological slide preparations were done from adult oocytes and larval neuroblasts. The cumulative effects of genotoxic (bridges, budding and nuclear fragmentation) and mutagenic (micronucleus and chromosomal breakage) damage was observed in the neuroblasts and oocytes of exposed mosquitoes. Developmental changes and damage to the genome of MED-treated Ae. aegypti were greater than those caused by TM. Further studies should focus on understanding the effects of the MED molecule on Ae. aegypti.

Population structure and ancestry prediction of Aedes aegypti (Diptera: Culicidae) supports a single African origin of Colombian populations

BACKGROUND A previous phylogeographic study revealed two Aedes aegypti African-related mitochondrial lineages distributed in Colombian's cities with different eco-epidemiologic characteristics with regard to dengue virus (DENV). It has been proposed these lineages might indicate independent invasion sources. OBJECTIVES Assessing to Colombian population structure and to support evidence of its probable source origin. METHODS We analysed a total of 267 individuals from cities of Bello, Riohacha and Villavicencio, which 241 were related to the West and East African mitochondrial lineages (termed here as WAL and EAL, respectively). Eight polymorphic microsatellite loci were analysed aiming population structure. FINDINGS Results indicate substantial gene flow among distant and low-connected cities composing a panmictic population with incipient local differentiation of Ae. aegypti is placed in Colombia. Likewise, genetic evidence indicates no significant differences among individuals related to WAL and EAL is placed. MAIN CONCLUSIONS Minimal genetic differentiation in low-connected Ae. aegypti populations of Colombia, and lack concordance between mitochondrial and nuclear genealogies suggest that Colombian Ae. aegypti shared a common demographic history. Under this scenario, we suggest current Ae. aegypti population structure reflects a single origin instead of contemporary migration, which founding populations have a single source from a mitochondrial polymorphic African ancient.

Mosquito cellular immunity at single-cell resolution.

Hemocytes limit the capacity of mosquitoes to transmit human pathogens. Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti mosquito vectors. Our data reveal the functional diversity of hemocytes, with different subtypes of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A previously unidentified cell type in An. gambiae, which we term "megacyte," is defined by a specific transmembrane protein marker (TM7318) and high expression of lipopolysaccharide-induced tumor necrosis factor-α transcription factor 3 (LL3). Knockdown experiments indicate that LL3 mediates hemocyte differentiation during immune priming. We identify and validate two main hemocyte lineages and find evidence of proliferating granulocyte populations. This atlas of medically relevant invertebrate immune cells at single-cell resolution identifies cellular events that underpin mosquito immunity to malaria infection.