RESUMO
The mosquito, Aedes aegypti, is the principal vector for several arboviruses. The mosquito midgut is the initial tissue that gets infected with an arbovirus acquired along with a blood meal from a vertebrate host. Blood meal ingestion leads to midgut tissue distention thereby increasing the pore size of the surrounding basal lamina. This allows newly synthesized virions to exit the midgut by traversing the distended basal lamina to infect secondary tissues of the mosquito. We conducted a quantitative label-free proteomic time course analysis with saline meal-fed Ae. aegypti females to identify host factors involved in midgut tissue distention. Around 2000 proteins were detected during each of the seven sampling time points and 164 of those were uniquely expressed. Forty-five of 97 differentially expressed proteins were upregulated during the 96-h time course and most of those were involved in cytoskeleton modulation, metabolic activity, and vesicle/vacuole formation. The F-actin-modulating Ae. aegypti (Aa)-gelsolin was selected for further functional studies. Stable knockout of Aa-gelsolin resulted in a mosquito line, which showed distorted actin filaments in midgut-associated tissues likely due to diminished F-actin processing by gelsolin. Zika virus dissemination from the midgut of these mosquitoes was diminished and delayed. The loss of Aa-gelsolin function was associated with an increased induction of apoptosis in midgut tissue indicating an involvement of Aa-gelsolin in apoptotic signaling in mosquitoes. Here, we used proteomics to discover a novel host factor, Aa-gelsolin, which affects the midgut escape barrier for arboviruses in mosquitoes and apoptotic signaling in the midgut.
Assuntos
Aedes , Arbovírus , Gelsolina , Proteínas de Insetos , Animais , Aedes/virologia , Aedes/metabolismo , Gelsolina/metabolismo , Gelsolina/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Arbovírus/fisiologia , Citoesqueleto/metabolismo , Feminino , Mosquitos Vetores/virologia , Mosquitos Vetores/metabolismo , Proteômica/métodos , Zika virus/fisiologiaRESUMO
Aedes albopictus, also known as the Asian tiger mosquito, is indigenous to the tropical forests of Southeast Asia. Ae. albopictus is expanding across the globe at alarming rates, raising concern over the transmission of mosquito-borne diseases, such as dengue, West Nile fever, yellow fever, and chikungunya fever. Since Ae. albopictus was reported in Houston (Harris County, Texas) in 1985, this species has rapidly expanded to at least 32 states across the United States. Public health efforts aimed at controlling Ae. albopictus, including surveillance and adulticide spraying operations, occur regularly in Harris County. Despite rotation of insecticides to mitigate the development of resistance, multiple mosquito species including Culex quinquefasciatus and Aedes aegypti in Harris County show organophosphate and pyrethroid resistance. Aedes albopictus shows relatively low resistance levels as compared to Ae. aegypti, but kdr-mutation and the expression of detoxification genes have been reported in Ae. albopictus populations elsewhere. To identify potential candidate detoxification genes contributing to metabolic resistance, we used RNA sequencing of field-collected malathion-resistant and malathion-susceptible, and laboratory-maintained susceptible colonies of Ae. albopictus by comparing the relative expression of transcripts from three major detoxification superfamilies involved in malathion resistance due to metabolic detoxification. Between these groups, we identified 12 candidate malathion resistance genes and among these, most genes correlated with metabolic detoxification of malathion, including four P450 and one alpha esterase. Our results reveal the metabolic detoxification and potential cuticular-based resistance mechanisms associated with malathion resistance in Ae. albopictus in Harris County, Texas.
Assuntos
Aedes , Perfilação da Expressão Gênica , Resistência a Inseticidas , Inseticidas , Malation , Animais , Malation/farmacologia , Aedes/genética , Aedes/efeitos dos fármacos , Aedes/metabolismo , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mosquitos Vetores/genética , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/metabolismo , Análise de Sequência de RNA , Transcriptoma , Texas , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismoRESUMO
The steroid hormone ecdysone is essential for the reproduction and survival of insects. The hormone is synthesized from dietary sterols such as cholesterol, yielding ecdysone in a series of consecutive enzymatic reactions. In the insect orders Lepidoptera and Diptera a glutathione transferase called Noppera-bo (Nobo) plays an essential, but biochemically uncharacterized, role in ecdysteroid biosynthesis. The Nobo enzyme is consequently a possible target in harmful dipterans, such as disease-carrying mosquitoes. Flavonoid compounds inhibit Nobo and have larvicidal effects in the yellow-fever transmitting mosquito Aedes aegypti, but the enzyme is functionally incompletely characterized. We here report that within a set of glutathione transferase substrates the double-bond isomerase activity with 5-androsten-3,17-dione stands out with an extraordinary specific activity of 4000 µmol min-1 mg-1. We suggest that the authentic function of Nobo is catalysis of a chemically analogous ketosteroid isomerization in ecdysone biosynthesis.
Assuntos
Aedes , Aedes/enzimologia , Aedes/metabolismo , Animais , Glutationa Transferase/metabolismo , Glutationa/metabolismo , Ecdisona/metabolismo , Proteínas de Insetos/metabolismo , Especificidade por Substrato , Esteroide Isomerases/metabolismo , Esteroide Isomerases/genética , Mosquitos Vetores/metabolismo , Cetosteroides/metabolismo , Cetosteroides/químicaRESUMO
Since its origin, the emergence of vector-borne infections has taken a toll on incalculable human lives. The use of chemical insecticides is one of the early known methods of vector control and although their use is still a prevalent way to combat insect population sadly the perils of insects related transmission still persists. Most commonly, the existing insecticides face the wrath of getting resisted repeatedly, paying way to develop resilient, efficient, and cost-effective natural insecticides. In this study, computational screening was performed using homology modelling, E-pharmacophore feature mapping, molecular docking, Density Function Theory (DFT) assessment, Molecular mechanics generalized Born surface area (MM-GBSA) based binding free energy calculations and Molecular Dynamics (MD) simulation to identify a potential lead phytochemical out of a manually curated library from published literature. The protein target used under this study is insect Butyrylcholine esterase (BChE). Additionally, in vitro insect (Aedes aegypti) BChE inhibition assay was also performed with the top phytochemical identified from in silico assessments. Our research highlights that curcumin leads to inhibition of enzyme BChE of Ae. aegypti. The identified mode of action of curcumin as an insect BChE inhibitor indicates the possibility of its use as an environment friendly and natural futuristic insecticide.
Assuntos
Aedes , Curcumina , Inseticidas , Animais , Colina/análogos & derivados , Colinesterases/metabolismo , Curcumina/metabolismo , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Resistência a Inseticidas , Inseticidas/metabolismo , Inseticidas/farmacologia , Simulação de Acoplamento Molecular , Mosquitos Vetores/metabolismoRESUMO
SignificanceHematophagous Aedes aegypti mosquitoes spread devastating viral diseases. Upon blood feeding, a steroid hormone, 20-hydroxyecdysone (20E), initiates a reproductive program during which thousands of genes are differentially expressed. While 20E-mediated gene activation is well known, repressive action by this hormone remains poorly understood. Using bioinformatics and molecular biological approaches, we have identified the mechanisms of 20E-dependent direct and indirect transcriptional repression by the ecdysone receptor (EcR). While indirect repression involves E74, EcR binds to an ecdysone response element different from those utilized in 20E-mediated gene activation to exert direct repressive action. Moreover, liganded EcR recruits a corepressor Mi2, initiating chromatin compaction. This study advances our understanding of the 20E-EcR repression mechanism and could lead to improved vector control approaches.
Assuntos
Ecdisona/metabolismo , Regulação da Expressão Gênica , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Reprodução/genética , Sítios de Ligação , Ecdisterona/metabolismo , Genes Reporter , Especificidade de Órgãos , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Aedes/metabolismo , Proteínas de Insetos/metabolismo , Mosquitos Vetores/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Proteínas do Envelope Viral/metabolismo , Zika virus/metabolismo , Aedes/química , Aedes/genética , Aedes/virologia , Animais , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Proteínas de Insetos/química , Proteínas de Insetos/genética , Queratinócitos/metabolismo , Queratinócitos/virologia , Cinética , Mosquitos Vetores/química , Mosquitos Vetores/genética , Mosquitos Vetores/virologia , Ligação Proteica , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Replicação Viral , Zika virus/química , Zika virus/genéticaRESUMO
Identifying the species of the subfamily Anophelinae that are Plasmodium vectors is important to vector and malaria control. Despite the increase in cases, vector mosquitoes remain poorly known in Brazilian indigenous communities. This study explores Anophelinae mosquito diversity in the following areas: (1) a Yanomami reserve in the northwestern Amazon Brazil biome and (2) the Pantanal biome in southwestern Brazil. This is carried out by analyzing cytochrome c oxidase (COI) gene data using Refined Single Linkage (RESL), Assemble Species by Automatic Partitioning (ASAP), and tree-based multi-rate Poisson tree processes (mPTP) as species delimitation approaches. A total of 216 specimens collected from the Yanomami and Pantanal regions were sequenced and combined with 547 reference sequences for species delimitation analyses. The mPTP analysis for all sequences resulted in the delimitation of 45 species groups, while the ASAP analysis provided the partition of 48 groups. RESL analysis resulted in 63 operational taxonomic units (OTUs). This study expands our scant knowledge of anopheline species in the Yanomami and Pantanal regions. At least 18 species of Anophelinae mosquitoes were found in these study areas. Additional studies are now required to determine the species that transmit Plasmodium spp. in these regions.
Assuntos
Anopheles/genética , Mosquitos Vetores/genética , Plasmodium/parasitologia , Animais , Brasil/epidemiologia , Vetores de Doenças , Malária/transmissão , Mosquitos Vetores/metabolismo , Plasmodium/genética , Especificidade da EspécieRESUMO
Aedes aegypti is an important vector of human viral diseases. This mosquito is distributed globally and thrives in urban environments, making it a serious risk to human health. Pyrethroid insecticides have been the mainstay for control of adult A. aegypti for decades, but resistance has evolved, making control problematic in some areas. One major mechanism of pyrethroid resistance is detoxification by cytochrome P450 monooxygenases (CYPs), commonly associated with the overexpression of one or more CYPs. Unfortunately, the molecular basis underlying this mechanism remains unknown. We used a combination of RNA-seq and proteomic analysis to evaluate the molecular basis of pyrethroid resistance in the highly resistant CKR strain of A. aegypti. The CKR strain has the resistance mechanisms from the well-studied Singapore (SP) strain introgressed into the susceptible Rockefeller (ROCK) strain genome. The RNA-seq and proteomics data were complimentary; each offering insights that the other technique did not provide. However, transcriptomic results did not quantitatively mirror results of the proteomics. There were 10 CYPs which had increased expression of both transcripts and proteins. These CYPs appeared to be largely trans-regulated, except for some CYPs for which we could not rule out gene duplication. We identified 65 genes and lncRNAs as potentially being responsible for elevating the expression of CYPs in CKR. Resistance was associated with multiple loci on chromosome 1 and at least one locus on chromosome 3. We also identified five CYPs that were overexpressed only as proteins, suggesting that stabilization of CYP proteins could be a mechanism of resistance. Future studies to increase the resolution of the resistance loci, and to examine the candidate genes and lncRNAs identified here will greatly enhance our understanding of CYP-mediated resistance in A. aegypti.
Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Proteínas de Insetos/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Piretrinas/farmacologia , Aedes/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Insetos/metabolismo , Mosquitos Vetores/metabolismo , Proteômica , TranscriptomaRESUMO
Anopheline mosquitoes are the sole vectors for the Plasmodium pathogens responsible for malaria, which is among the oldest and most devastating of human diseases. The continuing global impact of malaria reflects the evolutionary success of a complex vector-pathogen relationship that accordingly has been the long-term focus of both debate and study. An open question in the biology of malaria transmission is the impact of naturally occurring low-level Plasmodium infections of the vector on the mosquito's health and longevity as well as critical behaviors such as host-preference/seeking. To begin to answer this, we have completed a comparative RNAseq-based transcriptome profile study examining the effect of biologically salient, salivary gland transmission-stage Plasmodium infection on the molecular physiology of Anopheles gambiae s.s. head, sensory appendages, and salivary glands. When compared with their uninfected counterparts, Plasmodium infected mosquitoes exhibit increased transcript abundance of genes associated with olfactory acuity as well as a range of synergistic processes that align with increased fitness based on both anti-aging and reproductive advantages. Taken together, these data argue against the long-held paradigm that malaria infection is pathogenic for anophelines and, instead suggests there are biological and evolutionary advantages for the mosquito that drive the preservation of its high vectorial capacity.
Assuntos
Anopheles/genética , Perfilação da Expressão Gênica , Malária Falciparum/genética , Mosquitos Vetores/genética , Plasmodium falciparum/patogenicidade , Transcriptoma , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Anopheles/metabolismo , Anopheles/parasitologia , Evolução Molecular , Aptidão Genética , Interações Hospedeiro-Parasita , Malária Falciparum/parasitologia , Mosquitos Vetores/metabolismo , Mosquitos Vetores/parasitologia , Odorantes , RNA-Seq , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Olfato/genéticaRESUMO
BACKGROUND: Aedes aegypti and Ae. albopictus are important vectors of human diseases such as dengue, chikungunya, and zika. In Sri Lanka, they have been responsible for transmitting dengue virus. One of the most important parameters influencing the likelihood of arbovirus transmission is the age structure of the mosquito population. However, mosquito age is difficult to measure with accuracy. This study aims to construct multivariate calibration models using the transcriptional abundance of three age-responsive genes: Ae15848 (calcium-binding protein), Ae8505 (structural component of cuticle), and Ae4274 (fizzy cell cycle/cell division cycle 20). METHODS: The transcriptional age-grading technique was applied to determine the chronological age of Ae. aegypti and Ae. albopictus female mosquito populations from Sri Lanka using the age-responsive genes Ae15848, Ae8505, and Ae4274. Furthermore, Ae. aegypti samples obtained from colonies reared at two temperatures (23 and 27 °C) were used to investigate the influence of temperature on this age-grading technique. Expression levels of these three genes were quantified using reverse transcription qualitative PCR (qRT-PCR), and results were normalized against the housekeeping gene ribosomal gene S17 (RpS17). RESULTS: The expression of Ae15848 and Ae8505 decreased with the age of mosquitoes and showed the most significant and consistent change while expression of Ae4274 increased with age. The multivariate calibration models showed > 80% correlation between expression of these age-responsive genes and the age of female mosquitoes at both temperatures. At 27 °C the accuracy of age predictions using the models was 2.19 (± 1.66) days and 2.58 (± 2.06) days for Ae. aegypti and Ae. albopictus females, respectively. The accuracy of the model for Ae. aegypti at 23 °C was 3.42 (± 2.74) days. CONCLUSIONS: An adult rearing temperature difference of 4 °C (23-27 °C) did not significantly affect the age predictions. The calibration models created during this study could be successfully used to estimate the age of wild Ae. aegypti and Ae. albopictus mosquitoes from Sri Lanka.
Assuntos
Aedes/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Mosquitos Vetores/crescimento & desenvolvimento , Aedes/genética , Aedes/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Sri Lanka , TemperaturaRESUMO
BACKGROUND: Anopheles sinensis is a dominant vector for malaria transmission in Asian countries. Voltage-gated sodium channel (VGSC) mutation-mediated knock-down resistance (kdr) has developed in many A. sinensis populations because of intensive and long-term use of pyrethroids. Our previous study showed that multiple mutations at position 1014 of the VGSC were heterogeneously distributed in A. sinensis populations across Sichuan, China. METHODS: To understand resistance genotypes at the haplotype level and reconstruct the phylogenetic relationship of VGSC haplotypes, a cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach was established to clarify haplotypes containing codon 1014 of the VGSC gene from a total of 446 adults collected in 12 locations of Sichuan, China. RESULTS: Nineteen (19) haplotypes were identified, including 11 wild 1014L, 6 resistance 1014F, and 2 resistance 1014C haplotypes. We found that resistance haplotypes of A. sinensis VGSC were widely distributed at frequencies ranging from 3.67 to 92.61%. The frequencies of the 1014C haplotype in the southeast of Sichuan (Luzhou, Guangan, and Suining) were relatively higher than those in other sampling locations. Phylogenetic analyses support that kdr-type mutation at position 1014 is not singly originated and resistance 1014C haplotypes evolve from TTT-encoding 1014F. CONCLUSIONS: A cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach has been established in this study. The data revealed the patchy distribution of VGSC resistance haplotypes with overall high frequencies in Sichuan, China. Phylogenetic analyses support multiple origins and sequential evolution (1014L â 1014F â 1014C) for kdr-type mutations in A. sinensis.
Assuntos
Anopheles/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Insetos/genética , Mosquitos Vetores/genética , Filogenia , Canais de Sódio Disparados por Voltagem/genética , Animais , Anopheles/classificação , Anopheles/efeitos dos fármacos , Anopheles/metabolismo , China , Genótipo , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala/economia , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/farmacologia , Malária/transmissão , Mosquitos Vetores/classificação , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/metabolismo , Mutação , Piretrinas/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
The overexpression and overactivity of key cytochrome P450s (CYP450) genes are major drivers of metabolic resistance to insecticides in African malaria vectors such as Anopheles funestus s.s. Previous RNAseq-based transcription analyses revealed elevated expression of CYP325A specific to Central African populations but its role in conferring resistance has not previously been demonstrated. In this study, RT-qPCR consistently confirmed that CYP325A is highly over-expressed in pyrethroid-resistant An. funestus from Cameroon, compared with a control strain and insecticide-unexposed mosquitoes. A synergist bioassay with PBO significantly recovered susceptibility for permethrin and deltamethrin indicating P450-based metabolic resistance. Analyses of the coding sequence of CYP325A Africa-wide detected high-levels of polymorphism, but with no predominant alleles selected by pyrethroid resistance. Geographical amino acid changes were detected notably in Cameroon. In silico homology modelling and molecular docking simulations predicted that CYP325A binds and metabolises type I and type II pyrethroids. Heterologous expression of recombinant CYP325A and metabolic assays confirmed that the most-common Cameroonian haplotype metabolises both type I and type II pyrethroids with depletion rate twice that the of the DR Congo haplotype. Analysis of the 1 kb putative promoter of CYP325A revealed reduced diversity in resistant mosquitoes compared to susceptible ones, suggesting a potential selective sweep in this region. The establishment of CYP325A as a pyrethroid resistance metabolising gene further explains pyrethroid resistance in Central African populations of An. funestus. Our work will facilitate future efforts to detect the causative resistance markers in the promoter region of CYP325A to design field applicable DNA-based diagnostic tools.
Assuntos
Anopheles/genética , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mosquitos Vetores/genética , Piretrinas/farmacologia , África Central , Animais , Anopheles/metabolismo , Simulação por Computador , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Malária/transmissão , Simulação de Acoplamento Molecular , Mosquitos Vetores/metabolismoRESUMO
The mosquito continues to be the most lethal animal to humans due to the devastating diseases that it carries and transmits. Controlling mosquito-borne diseases relies heavily on vector management using neurotoxic insecticides with limited modes of action. This has led to the emergence of resistance to pyrethroids and other neurotoxic insecticides in mosquitoes, which has reduced the efficacy of chemical control agents. Moreover, many neurotoxic insecticides are not selective for mosquitoes and negatively impact beneficial insects such as honeybees. Developing new mosquitocides with novel mechanisms of action is a clear unmet medical need; this review covers the efforts made toward this end by targeting the renal inward rectifier potassium channel (Kir) of the mosquito.
Assuntos
Inseticidas/farmacologia , Mosquitos Vetores/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Animais , Humanos , Inseticidas/química , Estrutura Molecular , Mosquitos Vetores/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismoRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in controlling posttranscriptional gene regulation and have a profound effect on mosquito reproduction and metabolism. Juvenile hormone (JH) is critical for achieving reproductive competence in the main vector of human arboviral diseases, Aedes aegypti We report a JH-mediated mechanism governing miRNA expression. Using a transcription factor screen with multiple primary miRNA (pri-miRNA) promoters, we identified that the Ecdysone-induced protein E75 (E75) isoform (E75-RD) induced miRNA gene promoter activity. E75 binding sites were determined in miRNA promoters by means of cell transfection assay. E75-RD was found to be up-regulated by JH, as shown by the JH application and RNA interference (RNAi) of the JH receptor Methoprene-tolerant (Met). Small RNA sequencing from RNAi of Met and E75 displayed an overlapping miRNA cohort, suggesting E75 to be an intermediate component within the JH hierarchical network controlling miRNAs. Further experiments confirmed that E75-RD positively regulates several miRNAs including miR-2940. Reducing miR-2940 resulted in the arrest of follicle development and number of eggs laid. Performing miRNA target predictions and RT-qPCR from antagomir Ant-2940-3p-treated fat body tissues identified the mRNA target Clumsy (AAEL002518) The molecular interaction between this gene target and miR-2940 was confirmed using an in vitro dual luciferase assay in Drosophila S2 cells and in Ae. aegypti Aag2 cell lines. Finally, we performed a phenotypic rescue experiment to demonstrate that miR-2940/Clumsy is responsible for the disruption in egg development. Collectively, these results established the role of JH-mediated E75-RD in regulation of miRNA gene expression during the mosquito reproductive cycle.
Assuntos
Aedes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , MicroRNAs/genética , Mosquitos Vetores/metabolismo , Aedes/genética , Aedes/crescimento & desenvolvimento , Animais , Proteínas de Ligação a DNA/genética , Dengue/transmissão , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Insetos/genética , Masculino , MicroRNAs/metabolismo , Mosquitos Vetores/genética , Mosquitos Vetores/crescimento & desenvolvimento , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismoRESUMO
Mosquito vectors transmit various diseases through blood feeding, required for their egg development. Hence, blood feeding is a major physiological event in their life cycle, during which hundreds of genes are tightly regulated. Blood is a rich source of proteins for mosquitoes, but also contains many other molecules including microRNAs (miRNAs). Here, we found that human blood miRNAs are transported abundantly into the fat body tissue of Aedes aegypti, a key metabolic center in post-blood feeding reproductive events, where they target and regulate mosquito genes. Using an artificial diet spiked with the mimic of an abundant and stable human blood miRNA, hsa-miR-21-5p, and proteomics analysis, we found over 40 proteins showing differential expression in female Ae. aegypti mosquitoes after feeding. Of interest, we found that the miRNA positively regulates the vitellogenin gene, coding for a yolk protein produced in the mosquito fat body and then transported to the ovaries as a protein source for egg production. Inhibition of hsa-miR-21-5p followed by human blood feeding led to a statistically insignificant reduction in progeny production. The results provide another example of the involvement of small regulatory molecules in the interaction of taxonomically vastly different taxa.
Assuntos
Aedes/metabolismo , MicroRNAs/sangue , Mosquitos Vetores/metabolismo , Vitelogeninas/metabolismo , Aedes/citologia , Aedes/genética , Animais , Linhagem Celular , Cromatografia Líquida/métodos , Corpo Adiposo/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Proteínas de Insetos/metabolismo , MicroRNAs/genética , Mosquitos Vetores/genética , Proteômica/métodos , RNA-Seq/métodos , Espectrometria de Massas em Tandem/métodos , Vitelogeninas/genéticaRESUMO
In the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.
Assuntos
Anopheles/efeitos dos fármacos , Anopheles/genética , Inseticidas/farmacologia , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Acetilcolina/farmacologia , Acetilcolinesterase/genética , Animais , Anopheles/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Feminino , Genes de Insetos , Humanos , Técnicas In Vitro , Resistência a Inseticidas/genética , Malária/transmissão , Controle de Mosquitos/métodos , Mosquitos Vetores/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Mutação Puntual , Receptores Nicotínicos/metabolismo , Canais de Sódio/genéticaRESUMO
The anal papillae of mosquito larvae are osmoregulatory organs in direct contact with the external aquatic environment that actively sequester ions and take up water in dilute freshwater. In the disease vector Aedes aegypti mechanisms of ion, water and ammonia transport have only been partially resolved. Furthermore, A. aegypti larvae are known to reside in high ammonia sewage and high salt brackish waters, and understanding of anal papillae function in these conditions is in its infancy. The objective of this study was to identify the complement of ion and water transport genes expressed by the anal papillae of freshwater larvae by sequencing their transcriptome, and comparing their expression in anal papillae of larvae abruptly transferred to brackish water for 24 h. Results identified a number of ion and water transport proteins, ammonia detoxifying enzymes, a full suite of xenobiotic detoxifying enzymes and transporters, and G-protein coupled receptors of specific hormones. We identified a marked increase in transcript and protein abundance of aquaporin AaAQP2 in the anal papillae with abrupt transfer to brackish water. We present an updated and more comprehensive model for ion and water transport with additional putative transporters for Na+ and Cl- uptake in the anal papillae. These are organs which are actively engaged in Na+, Cl- and water uptake and regulation when the aquatic larvae encounter fluctuating salinities over the course of their development. Furthermore the transcriptome of the anal papillae includes a full set of xenobiotic detoxification genes suggesting that these are important detoxification organs which is particularly important when larvae reside in polluted water.
Assuntos
Aedes , Aquaporinas , Osmorregulação/genética , Receptores Acoplados a Proteínas G , Xenobióticos/metabolismo , Aedes/genética , Aedes/metabolismo , Aedes/fisiologia , Amônia/metabolismo , Canal Anal/metabolismo , Animais , Aquaporinas/genética , Aquaporinas/metabolismo , Transporte Biológico/genética , Genoma de Inseto , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Íons/metabolismo , Larva/genética , Larva/metabolismo , Larva/fisiologia , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Mosquitos Vetores/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Águas Salinas , Salinidade , Sódio/metabolismo , Transcriptoma , Água/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
BACKGROUND: Malaria control in Kenya is based on case management and vector control using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). However, the development of insecticide resistance compromises the effectiveness of insecticide-based vector control programs. The use of pesticides for agricultural purposes has been implicated as one of the sources driving the selection of resistance. The current study was undertaken to assess the status and mechanism of insecticide resistance in malaria vectors in irrigated and non-irrigated areas with varying agrochemical use in western Kenya. METHODS: The study was carried out in 2018-2019 in Homa Bay County, western Kenya. The bioassay was performed on adults reared from larvae collected from irrigated and non-irrigated fields in order to assess the susceptibility of malaria vectors to different classes of insecticides following the standard WHO guidelines. Characterization of knockdown resistance (kdr) and acetylcholinesterase-inhibiting enzyme/angiotensin-converting enzyme (Ace-1) mutations within Anopheles gambiae s.l. species was performed using the polymerase chain reaction (PCR) method. To determine the agricultural and public health insecticide usage pattern, a questionnaire was administered to farmers, households, and veterinary officers in the study area. RESULTS: Anopheles arabiensis was the predominant species in the irrigated (100%, n = 154) area and the dominant species in the non-irrigated areas (97.5%, n = 162), the rest being An. gambiae sensu stricto. In 2018, Anopheles arabiensis in the irrigated region were susceptible to all insecticides tested, while in the non-irrigated region reduced mortality was observed (84%) against deltamethrin. In 2019, phenotypic mortality was decreased (97.8-84% to 83.3-78.2%). In contrast, high mortality from malathion (100%), DDT (98.98%), and piperonyl butoxide (PBO)-deltamethrin (100%) was observed. Molecular analysis of the vectors from the irrigated and non-irrigated areas revealed low levels of leucine-serine/phenylalanine substitution at position 1014 (L1014S/L1014F), with mutation frequencies of 1-16%, and low-frequency mutation in the Ace-1R gene (0.7%). In addition to very high coverage of LLINs impregnated with pyrethroids and IRS with organophosphate insecticides, pyrethroids were the predominant chemical class of pesticides used for crop and animal protection. CONCLUSION: Anopheles arabiensis from irrigated areas showed increased phenotypic resistance, and the intensive use of pesticides for crop protection in this region may have contributed to the selection of resistance genes observed. The susceptibility of these malaria vectors to organophosphates and PBO synergists in pyrethroids offers a promising future for IRS and insecticide-treated net-based vector control interventions. These findings emphasize the need for integrated vector control strategies, with particular attention to agricultural practices to mitigate mosquito resistance to insecticides.
Assuntos
Anopheles/efeitos dos fármacos , Resistência a Inseticidas , Inseticidas/farmacologia , Irrigação Agrícola , Animais , Anopheles/genética , Anopheles/metabolismo , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Quênia , Masculino , Controle de Mosquitos , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Permetrina/farmacologia , Piretrinas/farmacologiaRESUMO
BACKGROUND: The pathogens transmitted by mosquitoes to humans and animals cause several emerging and resurgent infectious diseases. Increasing insecticide resistance requires rational action to control the target vector population. Chitin is indispensable for insect growth and development and absent from vertebrates and higher plants. Chitin synthase A (CHSA) is a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines chitin content may contribute to the development of novel vector control strategies. RESULTS: The injection of small interfering RNA targeting CHSA (siCHSA) to knockdown CHSA transcripts in larval, pupal and adult stages of Culex pipiens pallens resulted in the appearance of different lethal phenotypes. When larval and pupal stages were injected with siCHSA, CHSA knockdown prevented larval molting, pupation and adult eclosion, and affected the production of chitin and chitin degradation, which resulted in an ecdysis defect phenotype of mosquitoes. When siCHSA was injected into mosquitoes in the adult stage, CHSA knockdown also affected the laminar organization of the mesoderm and the formation of pseudo-orthogonal patterns of the large fibers of the endoderm. CONCLUSION: We provide a systematic and comprehensive description of the effects of CHSA on morphogenesis and metamorphosis. The results show that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our results further show that CHSA is important for the structural integrity of the adult mosquito cuticle.
Assuntos
Quitina Sintase , Culex , Animais , Quitina/biossíntese , Quitina Sintase/genética , Quitina Sintase/metabolismo , Culex/genética , Culex/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Metamorfose Biológica , Muda , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Interferência de RNARESUMO
Aedes aegypti is the main epidemic vector of arboviruses in Africa. In Senegal, control activities are mainly limited to mitigation of epidemics, with limited information available for Ae. aegypti populations. A better understanding of the current Ae. aegypti susceptibility status to various insecticides and relevant resistance mechanisms involved is needed for the implementation of effective vector control strategies. The present study focuses on the detection of insecticide resistance and reveals the related mechanisms in Ae. aegypti populations from Senegal. Bioassays were performed on Ae. aegypti adults from nine Senegalese localities (Matam, Louga, Barkedji, Ziguinchor, Mbour, Fatick, Dakar, Kédougou and Touba). Mosquitoes were exposed to four classes of insecticides using the standard WHO protocols. Resistance mechanisms were investigated by genotyping for pyrethroid target site resistance mutations (V1016G, V1016I, F1534C and S989P) and measuring gene expression levels of key detoxification genes (CYP6BB2, CYP9J26, CYP9J28, CYP9J32, CYP9M6, CCEae3a and GSTD4). All collected populations were resistant to DDT and carbamates except for the ones in Matam (Northern region). Resistance to permethrin was uniformly detected in mosquitoes from all areas. Except for Barkédji and Touba, all populations were characterized by a susceptibility to 0.75% Permethrin. Susceptibility to type II pyrethroids was detected only in the Southern regions (Kédougou and Ziguinchor). All mosquito populations were susceptible to 5% Malathion, but only Kédougou and Matam mosquitoes were susceptible to 0.8% Malathion. All populations were resistant to 0.05% Pirimiphos-methyl, whereas those from Louga, Mbour and Barkédji, also exhibited resistance to 1% Fenitrothion. None of the known target site pyrethroid resistance mutations was present in the mosquito samples included in the genotyping analysis (performed in > 1500 samples). In contrast, a remarkably high (20-70-fold) overexpression of major detoxification genes was observed, suggesting that insecticide resistance is mostly mediated through metabolic mechanisms. These data provide important evidence to support dengue vector control in Senegal.