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1.
J Vis Exp ; (174)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34515676

RESUMO

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.


Assuntos
Aedes , Neuropeptídeos , Aedes/metabolismo , Animais , Humanos , Transporte de Íons , Túbulos de Malpighi , Mosquitos Vetores , Neuropeptídeos/metabolismo
2.
PLoS Negl Trop Dis ; 15(7): e0009549, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34252110

RESUMO

Aedes aegypti is a primary vector of viral pathogens and is responsible for millions of human infections annually that represent critical public health and economic costs. Pyrethroids are one of the most commonly used classes of insecticides to control adult A. aegypti. The insecticidal activity of pyrethroids depends on their ability to bind and disrupt the voltage-sensitive sodium channel (VSSC). In mosquitoes, a common mechanism of resistance to pyrethroids is due to mutations in Vssc (hereafter referred as knockdown resistance, kdr). In this study, we found that a kdr (410L+V1016I+1534C) allele was the main mechanism of resistance in a pyrethroid-resistant strain of A. aegypti collected in Colombia. To characterize the level of resistance these mutations confer, we isolated a pyrethroid resistant strain (LMRKDR:RK, LKR) that was congenic to the susceptible Rockefeller (ROCK) strain. The full-length cDNA of Vssc was cloned from LKR and no additional resistance mutations were present. The levels of resistance to different pyrethroids varied from 3.9- to 56-fold. We compared the levels of resistance to pyrethroids, DCJW and DDT between LKR and what was previously reported in two other congenic strains that share the same pyrethroid-susceptible background (the ROCK strain), but carry different kdr alleles (F1534C or S989P + V1016G). The resistance conferred by kdr alleles can vary depending on the stereochemistry of the pyrethroid. The 410L+1016I+1534C kdr allele does not confer higher levels of resistance to six of ten pyrethroids, relative to the 1534C allele. The importance of these results to understand the evolution of insecticide resistance and mosquito control are discussed.


Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Proteínas de Insetos/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Piretrinas/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Aedes/metabolismo , Alelos , Animais , Colômbia , Técnicas de Silenciamento de Genes , Proteínas de Insetos/metabolismo , Mutação , Canais de Sódio Disparados por Voltagem/metabolismo
3.
RNA ; 27(10): 1155-1172, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34210890

RESUMO

PIWI-interacting (pi)RNAs are small silencing RNAs that are crucial for the defense against transposable elements in germline tissues of animals. In Aedes aegypti mosquitoes, the piRNA pathway also contributes to gene regulation in somatic tissues, illustrating additional roles for piRNAs and PIWI proteins besides transposon repression. Here, we identify a highly abundant endogenous piRNA (propiR1) that associates with both Piwi4 and Piwi5. PropiR1-mediated target silencing requires base-pairing in the seed region with supplemental base-pairing at the piRNA 3' end. Yet, propiR1 represses a limited set of targets, among which is the lncRNA AAEL027353 (lnc027353). Slicing of lnc027353 initiates production of responder and trailer piRNAs from the cleavage fragment. Expression of propiR1 commences early during embryonic development and mediates degradation of maternally provided lnc027353 Both propiR1 and its lncRNA target are conserved in the closely related Aedes albopictus mosquito, underscoring the importance of this regulatory network for mosquito development.


Assuntos
Aedes/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Aedes/embriologia , Aedes/metabolismo , Animais , Pareamento de Bases , Sequência de Bases , Sequência Conservada , Embrião não Mamífero , Redes Reguladoras de Genes , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , RNA Longo não Codificante/metabolismo
4.
PLoS Negl Trop Dis ; 15(7): e0009546, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34237076

RESUMO

BACKGROUND: Volatile pyrethroid insecticides, such as transfluthrin, have received increasing attention for their potent repellent activities in recent years for controlling human disease vectors. It has been long understood that pyrethroids kill insects by promoting activation and inhibiting inactivation of voltage-gated sodium channels. However, the mechanism of pyrethroid repellency remains poorly understood and controversial. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that transfluthrin repels Aedes aegypti in a hand-in-cage assay at nonlethal concentrations as low as 1 ppm. Contrary to a previous report, transfluthrin does not elicit any electroantennogram (EAG) responses, indicating that it does not activate olfactory receptor neurons (ORNs). The 1S-cis isomer of transfluthrin, which does not activate sodium channels, does not elicit repellency. Mutations in the sodium channel gene that reduce the potency of transfluthrin on sodium channels decrease transfluthrin repellency but do not affect repellency by DEET. Furthermore, transfluthrin enhances DEET repellency. CONCLUSIONS/SIGNIFICANCE: These results provide a surprising example that sodium channel activation alone is sufficient to potently repel mosquitoes. Our findings of sodium channel activation as the principal mechanism of transfluthrin repellency and potentiation of DEET repellency have broad implications in future development of a new generation of dual-target repellent formulations to more effectively repel a variety of human disease vectors.


Assuntos
Aedes/efeitos dos fármacos , Ciclopropanos/farmacologia , Fluorbenzenos/farmacologia , Proteínas de Insetos/metabolismo , Repelentes de Insetos/farmacologia , Canais de Sódio/metabolismo , Aedes/genética , Aedes/metabolismo , Animais , Ciclopropanos/química , Fluorbenzenos/química , Proteínas de Insetos/genética , Repelentes de Insetos/química , Isomerismo , Canais de Sódio/genética
5.
PLoS Negl Trop Dis ; 15(7): e0009587, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34270558

RESUMO

BACKGROUND: Aedes aegypti (L.) is an urban mosquito, vector of several arboviruses that cause severe diseases in hundreds of million people each year. The resistance to synthetic insecticides developed by Ae. aegypti populations worldwide has contributed to failures in vector control campaigns, increasing the impact of arbovirus diseases. In this context, plant-derived essential oils with larvicidal activity could be an attractive alternative for vector control. However, the mode of action and the detoxificant response of mosquitoes to plant derived compounds have not been established, impairing the optimization of their use. METHODS AND FINDINGS: Here we compare gene expression in Ae. aegypti larvae after 14 hrs of exposure to Eucalyptus camaldulensis essential oil with a control group exposed to vehicle (acetone) for the same lapse, by using RNA-Seq. We found differentially expressed genes encoding for cuticle proteins, fatty-acid synthesis, membrane transporters and detoxificant related gene families (i.e. heat shock proteins, cytochromes P450, glutathione transferases, UDP-glycosyltransferases and ABC transporters). Finally, our RNA-Seq and molecular docking results provide evidence pointing to a central involvement of chemosensory proteins in the detoxificant response in mosquitoes. CONCLUSIONS AND SIGNIFICANCE: Our work contributes to the understanding of the physiological response of Ae. aegypti larvae to an intoxication with a natural toxic distilled from Eucalyptus leafs. The results suggest an involvement of most of the gene families associated to detoxification of xenobiotics in insects. Noteworthy, this work provides important information regarding the implication of chemosensory proteins in the detoxification of a natural larvicide. Understanding the mode of detoxification of Eucalyptus distilled compounds could contribute to their implementation as a tool in mosquito control.


Assuntos
Aedes/efeitos dos fármacos , Eucalyptus/química , Simulação de Acoplamento Molecular , Óleos Voláteis/farmacologia , Óleos Vegetais/farmacologia , Transcriptoma , Aedes/metabolismo , Animais , Sequência de Bases , Biologia Computacional , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Modelos Moleculares , Óleos Voláteis/química , Óleos Vegetais/química , Conformação Proteica , RNA/genética
6.
Commun Biol ; 4(1): 856, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244602

RESUMO

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/genética
7.
Biochimie ; 187: 131-143, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34082040

RESUMO

SGTs (small glutamine-rich TPR-containing proteins) are dimeric proteins that belong to the class of co-chaperones characterized by the presence of TPR domains (containing tetratricopeptide repeats). Human (SGTA) and yeast (Sgt2) SGTs are characterized by three distinct domains: an N-terminal dimerization domain, a central TPR-domain important for binding to other proteins (chaperones included) and a C-terminal domain involved in hydrophobic interactions. Both these SGTs are involved in the cellular PQC (protein quality control) system, as they interact with chaperones and have functions that aid stress recovery. However, there are differences between them, such as structural features and binding specificities, that could be better understood if other orthologous proteins were studied. Therefore, we produced and characterized a putative SGT protein, designated AaSGT, from the mosquito Aedes aegypti, which is a vector of several diseases, such as dengue and Zika. The protein was produced as a folded dimer which was stable up to 40 °C and was capable of binding to AaHsp90 and fully protecting a model protein, α-synuclein, from aggregation. The conformation of AaSGT was investigated by biophysical tools and small angle X-ray scattering, which showed that the protein had an elongated conformation and that its C-terminal domain was mainly disordered. The results with a C-terminal deletion mutant supported these observations. Altogether, these results are consistent with those from other functional SGT proteins and add to the understanding of the PQC system in Aedes aegypti, an important aim that may help to develop inhibitory strategies against this vector of neglected diseases.


Assuntos
Aedes/química , Proteínas de Insetos/química , Chaperonas Moleculares/química , Multimerização Proteica , Aedes/genética , Aedes/metabolismo , Animais , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
PLoS Genet ; 17(6): e1009606, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138859

RESUMO

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site-the voltage-gated sodium channel (VGSC)-and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.


Assuntos
Aedes/efeitos dos fármacos , Inativação Metabólica/genética , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Permetrina/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Aedes/genética , Aedes/metabolismo , Substituição de Aminoácidos , Animais , Expressão Gênica , Perfilação da Expressão Gênica , Proteínas de Insetos/classificação , Proteínas de Insetos/metabolismo , Inseticidas/metabolismo , Anotação de Sequência Molecular , Mosquitos Vetores , Mutação , Permetrina/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Canais de Sódio Disparados por Voltagem/metabolismo
9.
J Insect Physiol ; 132: 104269, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34174320

RESUMO

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 Hidroeletrolítico
10.
Sci Rep ; 11(1): 12038, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103581

RESUMO

We investigated the larvicidal potential of 10 plant essential oils (EOs) against the Asian tiger mosquito Aedes albopictus. Among the EOs, larvicidal activity against Ae. albopictus was strongest in those derived from massoia (Massoia aromatica) and nutmeg (Myristica fragrans). Larvicidal activities of massoia and nutmeg EOs against Ae. albopictus were 95.0% and 85.0% at 50 µg/mL, respectively. A total of 4 and 14 compounds were identified from massoia and nutmeg, respectively, and two massoia lactones, C10 and C12, were isolated from massoia EO. Among the identified compounds, benzyl salicylate, terpinolene, C12 massoia lactone, sabinene, benzyl benzoate, methyl eugenol, and C10 massoia lactone exhibited the strong larvicidal activity. Cellulose nanocrystal (CNC)-stabilized Pickering emulsions of massoia and nutmeg EOs were developed to overcome the insolubility of EOs in water. CNC/massoia and CNC/nutmeg emulsions were stable for at least 10 days, and larvicidal activities of CNC/massoia PE and CNC/nutmeg were higher than those of crude massoia and nutmeg EOs. This study presents a CNC-stabilized PE, a suitable formulation for EOs, as a potential larvicide against Ae. albopictus.


Assuntos
Aedes/efeitos dos fármacos , Aedes/metabolismo , Celulose/química , Inseticidas/química , Nanopartículas/química , Óleos Voláteis/química , Animais , Benzoatos/química , Cromatografia Gasosa , Monoterpenos Cicloexânicos/química , Emulsões , Eugenol/análogos & derivados , Eugenol/química , Lactonas/química , Larva , Myristica , Óleos Vegetais/química , Salicilatos/química , Solubilidade
11.
Front Immunol ; 12: 670122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054842

RESUMO

Complement-like proteins in arthropods defend against invading pathogens in the early phases of infection. Thioester-containing proteins (TEPs), which exhibit high similarity to mammalian complement C3, are thought to play a key role in the innate immunity of arthropods. We identified and characterized anti-dengue virus (DENV) host factors, in particular complement-like proteins, in the mosquito Aedes aegypti. Our results indicate that TEP1 limits DENV infection in Ae. aegypti. We showed that TEP1 transcription is highly induced in mosquitoes following DENV infection. Silencing TEP1 resulted in the up-regulation of viral RNA and proteins. In addition, the production of infectious virus particles increased in the absence of TEP1. We generated a transgenic mosquito line with a TEP1 loss-of-function phenotype under a blood meal-inducible promoter. We showed that viral protein and titers increased in transgenic mosquitoes after an infectious blood meal. Interestingly, expression of transcription factor Rel2 and certain anti-microbial peptides (AMPs) were inhibited in transgenic mosquitoes. Overall, our results suggest that TEP1 regulates the immune response and consequently controls the replication of dengue virus in mosquitoes. This finding provides new insight into the molecular mechanisms of mosquito host factors in the regulation of DENV replication.


Assuntos
Aedes/virologia , Vírus da Dengue/patogenicidade , Dengue/prevenção & controle , Imunidade Inata , Proteínas de Insetos/metabolismo , Mosquitos Vetores , Aedes/genética , Aedes/imunologia , Aedes/metabolismo , Animais , Animais Geneticamente Modificados , Dengue/imunologia , Dengue/metabolismo , Dengue/virologia , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas de Insetos/genética , Replicação Viral
12.
Sci Rep ; 11(1): 9636, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953286

RESUMO

Understanding the molecular and biochemical basis of egg development is a central topic in mosquito reproductive biology. Lipids are a major source of energy and building blocks for the developing ovarian follicles. Ultra-High Resolution Mass Spectrometry (UHRMS) combined with in vivo metabolic labeling of follicle lipids with deuterated water (2H2O) can provide unequivocal identification of de novo lipid species during ovarian development. In the present study, we followed de novo triglyceride (TG) dynamics during the ovarian previtellogenic (PVG) stage (2-7 days post-eclosion) of female adult Aedes aegypti. The incorporation of stable isotopes from the diet was evaluated using liquid chromatography (LC) in tandem with the high accuracy (< 0.3 ppm) and high mass resolution (over 1 M) of a 14.5 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (14.5 T FT-ICR MS) equipped with hexapolar detection. LC-UHRMS provides effective lipid class separation and chemical formula identification based on the isotopic fine structure. The monitoring of stable isotope incorporation into de novo incorporated TGs suggests that ovarian lipids are consumed or recycled during the PVG stage, with variable time dynamics. These results provide further evidence of the complexity of the molecular mechanism of follicular lipid dynamics during oogenesis in mosquitoes.


Assuntos
Aedes/metabolismo , Ovário/metabolismo , Triglicerídeos/metabolismo , Animais , Cromatografia Líquida , Feminino , Espectrometria de Massas
13.
J Proteomics ; 242: 104257, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33957312

RESUMO

Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SIGNIFICANCE: Female Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.


Assuntos
Aedes , Febre Amarela , Infecção por Zika virus , Zika virus , Aedes/metabolismo , Animais , Cromatografia Líquida , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Hormônios Juvenis , Metoprene , Mosquitos Vetores , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem , Zika virus/metabolismo
14.
PLoS Negl Trop Dis ; 15(5): e0009393, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33970904

RESUMO

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.


Assuntos
Aedes/efeitos dos fármacos , Resistência a Inseticidas/genética , Mosquitos Vetores/efeitos dos fármacos , Aedes/genética , Aedes/metabolismo , Animais , Expressão Gênica , Inativação Metabólica/genética , Inseticidas , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Piretrinas , Senegal
15.
Sci Rep ; 11(1): 7301, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790374

RESUMO

Aedes aegypti is a major vector of Zika, dengue, and other arboviruses. Permethrin adulticidal spraying, which targets the voltage-gated sodium channel (VGSC), is commonly done to reduce local mosquito populations and protect humans from exposure to arbovirus pathogens transmitted by this dangerous pest. Permethrin resistance, however, is a growing problem and understanding its underlying molecular basis may identify avenues to combat it. We identified a single G:C polymorphism in pre-miR-33 that was genetically associated with permethrin resistance; resulting isoforms had structural differences that may affect DICER-1/pre-miRNA processing rates. We then assessed the effects of overexpression of pre-miR-33 isoforms on permethrin toxicological phenotypes, VGSC transcript abundance and protein levels for two genetically related mosquito strains. One strain had its naturally high permethrin resistance levels maintained by periodic treatment, and the other was released from selection. VGSC protein levels were lower in the permethrin resistant strain than in the related permethrin-susceptible strain. Overexpression of the G-pre-miR-33 isoform reduced VGSC expression levels in both strains. To further elucidate changes in gene expression associated with permethrin resistance, exome-capture gDNA deep sequencing, genetic association mapping and subsequent gene set enrichment analysis revealed that transport genes, in particular, were selected in resistant versus susceptible mosquitoes. Collectively, these data indicate that miR-33 regulates VGSC expression as part of a nuanced system of neuronal regulation that contributes to a network of heritable features determining permethrin resistance.


Assuntos
Aedes/genética , Proteínas de Insetos/genética , Resistência a Inseticidas , Inseticidas/toxicidade , MicroRNAs/metabolismo , Permetrina/toxicidade , Canais de Sódio/genética , Aedes/metabolismo , Animais , Proteínas de Insetos/metabolismo , MicroRNAs/genética , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais de Sódio/metabolismo
16.
PLoS Negl Trop Dis ; 15(4): e0009334, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33826624

RESUMO

As a key vector for major arthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya, control of Aedes aegypti represents a major challenge in public health. Bloodmeal acquisition is necessary for the reproduction of vector mosquitoes and pathogen transmission. Blood contains potentially toxic amounts of iron while it provides nutrients for mosquito offspring; disruption of iron homeostasis in the mosquito may therefore lead to novel control strategies. We previously described a potential iron exporter in Ae. aegypti after a targeted functional screen of ZIP (zinc-regulated transporter/Iron-regulated transporter-like) and ZnT (zinc transporter) family genes. In this study, we performed an RNAseq-based screen in an Ae. aegypti cell line cultured under iron-deficient and iron-excess conditions. A subset of differentially expressed genes were analyzed via a cytosolic iron-sensitive dual-luciferase reporter assay with several gene candidates potentially involved in iron transport. In vivo gene silencing resulted in significant reduction of fecundity (egg number) and fertility (hatch rate) for one gene, termed dyspepsia. Silencing of dyspepsia reduced the induction of ferritin expression in the midgut and also resulted in delayed/impaired excretion and digestion. Further characterization of this gene, including a more direct confirmation of its substrate (iron or otherwise), could inform vector control strategies as well as to contribute to the field of metal biology.


Assuntos
Aedes/genética , Dispepsia/genética , Proteínas de Insetos/genética , Proteínas de Membrana Transportadoras/genética , Aedes/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Dispepsia/metabolismo , Inativação Gênica , Aptidão Genética , Proteínas de Insetos/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Análise de Sequência de RNA , Zinco/metabolismo
17.
Insect Biochem Mol Biol ; 134: 103580, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33901693

RESUMO

Anautogenous female mosquitoes obtain the nutrients needed for egg development from vertebrate blood, and consequently they transmit numerous pathogens of devastating human diseases. Digestion of blood proteins into amino acids that are used for energy production, egg maturation and replenishment of maternal reserves is an essential part of the female mosquito reproductive cycle. However, the regulatory mechanisms underlying this process remain largely unknown. Here, we report that the transcription factor E93 is a critical factor promoting blood meal digestion in adult females of the major arboviral vector Aedes aegypti in response to the steroid hormone 20-hydroxyecdysone (20E). E93 was upregulated in the female mosquito midgut after a blood meal, and RNA interference (RNAi)-mediated knockdown of E93 inhibited midgut blood digestion. E93 RNAi depletion repressed late trypsin (LT), serine protease I (SPI), SPVI and SPVII, and activated early trypsin (ET) expression in the female mosquito midgut after a blood meal. Injection of 20E activated E93, LT, SPI, SPVI and SPVII, and repressed ET expression, whereas RNAi knockdown of the ecdysone receptor (EcR) repressed E93, LT, SPI, SPVI and SPVII, and activated ET expression in the midgut. Furthermore, E93 depletion resulted in a complete loss of 20E responsiveness of LT, SPVI and SPVII. Our findings reveal important mechanisms regulating blood meal digestion in disease-transmitting mosquitoes.


Assuntos
Aedes , Sangue/metabolismo , Sistema Digestório/metabolismo , Fatores de Transcrição/genética , Aedes/genética , Aedes/metabolismo , Aedes/fisiologia , Ração Animal , Animais , Proteínas Sanguíneas/metabolismo , Digestão , Proteínas de Drosophila/genética , Ecdisterona/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Insetos/metabolismo , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Mosquitos Vetores/fisiologia , Interferência de RNA , Serina Proteases/metabolismo
18.
Viruses ; 13(2)2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671824

RESUMO

Dengue virus (DENV), a member of the Flavivirus genus of the Flaviviridae family, can cause dengue fever (DF) and more serious diseases and thus imposes a heavy burden worldwide. As the main vector of DENV, mosquitoes are a serious hazard. After infection, they induce a complex host-pathogen interaction mechanism. Our goal is to further study the interaction mechanism of viruses in homologous, sensitive, and repeatable C6/36 cell vectors. Transcriptome sequencing (RNA-Seq) technology was applied to the host transcript profiles of C6/36 cells infected with DENV2. Then, bioinformatics analysis was used to identify significant differentially expressed genes and the associated biological processes. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to verify the sequencing data. A total of 1239 DEGs were found by transcriptional analysis of Aedes albopictus C6/36 cells that were infected and uninfected with dengue virus, among which 1133 were upregulated and 106 were downregulated. Further bioinformatics analysis showed that the upregulated DEGs were significantly enriched in signaling pathways such as the MAPK, Hippo, FoxO, Wnt, mTOR, and Notch; metabolic pathways and cellular physiological processes such as autophagy, endocytosis, and apoptosis. Downregulated DEGs were mainly enriched in DNA replication, pyrimidine metabolism, and repair pathways, including BER, NER, and MMR. The qRT-PCR results showed that the concordance between the RNA-Seq and RT-qPCR data was very high (92.3%). The results of this study provide more information about DENV2 infection of C6/36 cells at the transcriptome level, laying a foundation for further research on mosquito vector-virus interactions. These data provide candidate antiviral genes that can be used for further functional verification in the future.


Assuntos
Aedes/genética , Aedes/virologia , Vírus da Dengue/fisiologia , Proteínas de Insetos/genética , Mosquitos Vetores/genética , Mosquitos Vetores/virologia , Aedes/metabolismo , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas de Insetos/metabolismo , Mosquitos Vetores/metabolismo , Replicação Viral
19.
Parasit Vectors ; 14(1): 141, 2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33676552

RESUMO

BACKGROUND: Since their detection in 2013, Aedes aegypti has become a widespread urban pest in California. The availability of cryptic larval breeding sites in residential areas and resistance to insecticides pose significant challenges to control efforts. Resistance to pyrethroids is largely attributed to mutations in the voltage gated sodium channels (VGSC), the pyrethroid site of action. However, past studies have indicated that VGSC mutations may not be entirely predictive of the observed resistance phenotype. METHODS: To investigate the frequencies of VGSC mutations and the relationship with pyrethroid insecticide resistance in California, we sampled Ae. aegypti from four locations in the Central Valley, and the Greater Los Angeles area. Mosquitoes from each location were subjected to an individual pyrethrum bottle bioassay to determine knockdown times. A subset of assayed mosquitoes from each location was then analyzed to determine the composition of 5 single nucleotide polymorphism (SNP) loci within the VGSC gene. RESULTS: The distribution of knockdown times for each of the five Californian populations sampled was non-parametric with potentially bimodal distributions. One group succumbs to insecticidal effects around 35-45 min and the second group lasts up to and beyond the termination of the assay (120+ min). We detected 5 polymorphic VGSC SNPs within the sampled California populations. One is potentially new and alternatively spliced (I915K), and four are documented and associated with resistance: F1534C, V1016I, V410L and S723T. The Central Valley populations (Clovis, Dinuba, Sanger and Kingsburg) are fairly homogenous with only 5% of the mosquitoes showing heterozygosity at any given position. In the Greater LA mosquitoes, 55% had at least one susceptible allele at any of the five SNP loci. The known resistance allele F1534C was detected in almost all sampled mosquitoes (99.4%). We also observe significant heterogeneity in the knockdown phenotypes of individuals with the identical VGSC haplotypes suggesting the presence of additional undefined resistance mechanisms. CONCLUSIONS: Resistance associated VGSC SNPs are prevalent, particularly in the Central Valley. Interestingly, among mosquitoes carrying all 4 resistance associated SNPs, we observe significant heterogeneity in bottle bioassay profiles suggesting that other mechanisms are important to the individual resistance of Ae. aegypti in California.


Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Genótipo , Inseticidas/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Piretrinas/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Aedes/metabolismo , Animais , Bioensaio , Feminino , Resistência a Inseticidas/genética , Inseticidas/metabolismo , Mosquitos Vetores/genética , Fenótipo , Piretrinas/metabolismo , Canais de Sódio Disparados por Voltagem/classificação
20.
Acta Trop ; 218: 105895, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33753028

RESUMO

Most artificial dietary systems for feeding mosquitoes require a membrane feeder, host cues, phosphate saline buffers and a phagostimulant. These multicomponent feeders are complex, expansive and cumbersome that requires fully trained personnel. The objective of the present is to develop a simple sugar assisted protein (SAP) diet for the egg production of Aedes albopictus. The present study assessed the potential use of SAP dietary system on the engorgement, fecundity, preference of diet components and production of multiple generations of Ae. albopictus. Our data show that the female mosquitoes have strongly preferred a diet with: (i) a combination of sugar and protein over the individual component, and (ii) water over PBS (phosphate buffered saline) buffer as a carrier, whereas adenosine triphosphate (ATP) was not required as a phagostimulant. Based on our optimization data, the SAP diets (10-20% bovine serum albumin in 5% sucrose aqueous solution) do not require chemo-attractive lure, phagostimulant ATP, temperature and membrane feeding components. Female mosquitoes readily engorge on SAP diets and show similar rates of survival and fecundity compared to those when blood-fed on live animals. In addition, the number of eggs produced by female mosquitoes fed on SAP diets kept consistent for 10 consecutive generations. Our results indicate that SAP diet is a potential alternative against blood feeding that is simple and cost-effective diets for Ae. albopictus colony maintenance and to support large scale mass- production for experimental and other purposes.


Assuntos
Aedes/crescimento & desenvolvimento , Aedes/metabolismo , Ração Animal , Métodos de Alimentação , Proteínas/administração & dosagem , Açúcares/administração & dosagem , Animais , Tampões (Química) , Dieta , Comportamento Alimentar , Feminino , Fertilidade , Masculino
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