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1.
PLoS Genet ; 16(5): e1008794, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463828

RESUMO

Although specific interactions between host and pathogen genotypes have been well documented in invertebrates, the identification of host genes involved in discriminating pathogen genotypes remains a challenge. In the mosquito Aedes aegypti, the main dengue virus (DENV) vector worldwide, statistical associations between host genetic markers and DENV types or strains were previously detected, but the host genes underlying this genetic specificity have not been identified. In particular, it is unknown whether DENV type- or strain-specific resistance relies on allelic variants of the same genes or on distinct gene sets. Here, we investigated the genetic architecture of DENV resistance in a population of Ae. aegypti from Bakoumba, Gabon, which displays a stronger resistance phenotype to DENV type 1 (DENV-1) than to DENV type 3 (DENV-3) infection. Following experimental exposure to either DENV-1 or DENV-3, we sequenced the exomes of large phenotypic pools of mosquitoes that are either resistant or susceptible to each DENV type. Using variation in single-nucleotide polymorphism (SNP) frequencies among the pools, we computed empirical p values based on average gene scores adjusted for the differences in SNP counts, to identify genes associated with infection in a DENV type-specific manner. Among the top 5% most significant genes, 263 genes were significantly associated with resistance to both DENV-1 and DENV-3, 287 genes were only associated with DENV-1 resistance and 290 were only associated with DENV-3 resistance. The shared significant genes were enriched in genes with ATP binding activity and sulfur compound transmembrane transporter activity, whereas the genes uniquely associated with DENV-3 resistance were enriched in genes with zinc ion binding activity. Together, these results indicate that specific resistance to different DENV types relies on largely non-overlapping sets of genes in this Ae. aegypti population and pave the way for further mechanistic studies.


Assuntos
Aedes/genética , Vírus da Dengue/classificação , Resistência à Doença , Proteínas de Insetos/genética , Sequenciamento Completo do Exoma/métodos , Aedes/virologia , Animais , Células Cultivadas , Vírus da Dengue/patogenicidade , Feminino , Gabão , Genótipo , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Especificidade da Espécie
2.
Parasitol Res ; 119(7): 2075-2083, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32458116

RESUMO

Aedes aegypti represents one of the main vectors of at least five relevant arthropod-borne viral infections in humans (i.e., Rift Valley fever, Dengue fever, Zika, chikungunya, and yellow fever) worldwide. Ae. aegypti control strategies are mostly based on using chemical insecticides (i.e., organophosphates, pyrethroids, carbamates, and organochlorines) and reducing larval sources. Furthermore, monitoring the growth activity and mapping the geographical distribution of insecticide resistance are mandatory, as recommended by the WHO. Accordingly, we conducted a study on the possible mechanism by which Ae. aegypti develops resistance to several frequently used chemical insecticides (i.e., λ-cyhalothrin, bendiocarb, cyfluthrin, deltamethrin, malathion, and permethrin) in the city of Makassar, Sulawesi, Indonesia. The results showed the progression of resistance toward the examined insecticides in Ae. aegypti populations in Makassar. The mortality rate of Ae. aegypti was less than 90%, with the highest resistance recorded against 0.75% permethrin. The molecular evaluation of the voltage-gated sodium channel gene (VGSC) showed a significant correlation of the V1016G gene mutation in the tested 0.75% permethrin-resistant Ae. aegypti phenotypes. Nevertheless, the F1534C point mutation in the VGSC gene of Ae. aegypti did not show a significant correlation with the phenotype exhibiting insecticide resistance to 0.75% permethrin. These results indicate that Ae. aegypti mosquitoes in Makassar City have developed resistance against the frequently used insecticide permethrin, which might spread to less-populated regions of Sulawesi. Therefore, we call for further entomological monitoring of insecticide resistance not only on Sulawesi but also on other closely located islands of the Indonesian archipelago to delay the spread of Ae. aegypti insecticide resistance.


Assuntos
Aedes/genética , Resistência a Inseticidas/genética , Inseticidas , Mosquitos Vetores/genética , Mutação , Canais de Sódio Disparados por Voltagem/genética , Animais , Indonésia
3.
PLoS Negl Trop Dis ; 14(5): e0008279, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365059

RESUMO

Dengue is a highly endemic disease in Southeast Asia and is transmitted primarily by the mosquito, Aedes aegypti. The National Capital Region (NCR) of the Philippines, or Metropolitan Manila, is a highly urbanized area that is greatly affected by this arboviral disease. Urbanization has been shown to increase the dispersal of this mosquito vector. For this reason, we conducted a fine-scale population genetic study of Ae. aegypti in this region. We collected adult Ae. aegypti mosquitoes (n = 526 individuals) within the region (n = 21 study areas) and characterized the present population structure and the genetic relatedness among mosquito populations. We genotyped 11 microsatellite loci from all sampled mosquito individuals and analyzed their genetic diversity, differentiation and structure. The results revealed low genetic differentiation across mosquito populations which suggest high gene flow and/or weak genetic drift among mosquito populations. Bayesian analysis indicated multiple genetic structures (K = 3-6), with no clear genetically distinct population structures. This result implies the passive or long-distance dispersal capability nature Ae. aegypti possibly through human-mediated transportation. The constructed dendrogram in this study describes the potential passive dispersal patterns across Metropolitan Manila. Furthermore, spatial autocorrelation analysis showed the limited and active dispersal capability (<1km) of the mosquito vector. Our findings are consistent with previous studies that investigated the genetic structure and dual (active and passive) dispersal capability of Ae. aegypti in a fine-scale highly urbanized area.


Assuntos
Aedes/classificação , Aedes/genética , Genótipo , Mosquitos Vetores/classificação , Mosquitos Vetores/genética , Aedes/crescimento & desenvolvimento , Distribuição Animal , Animais , Cidades , Genética Populacional , Técnicas de Genotipagem , Repetições de Microssatélites , Mosquitos Vetores/crescimento & desenvolvimento , Filipinas , Análise Espacial
4.
PLoS Negl Trop Dis ; 14(5): e0008216, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32384079

RESUMO

The extensive use of insecticides for vector control has led to the development of insecticide resistance in Aedes aegypti populations on a global scale, which has significantly compromised control actions. Insecticide resistance, and its underlying mechanisms, has been investigated in several countries, mostly in South American and Asian countries. In Africa, however, studies reporting insecticide resistance are rare and data on resistance mechanisms, notably knockdown resistance (kdr) mutations, is scarce. In this study, the recently described V410L kdr mutation is reported for the first time in old world Ae. aegypti populations, namely from Angola and Madeira island. Two additional kdr mutations, V1016I and F1534C, are also reported for the first time in populations from Angola and Cape Verde. Significant associations with the resistance phenotype were found for both V410L and V1016I individually as well as for tri-locus genotypes in the Angolan population. However, no association was found in Madeira island, probably due to the presence of a complex pattern of multiple insecticide resistance mechanisms in the local Ae. aegypti population. These results suggest that populations carrying the same kdr mutations may respond differently to the same insecticide, stressing the need for complementary studies when assessing the impact of kdr resistance mechanisms in the outcome of insecticide-based control strategies.


Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Proteínas de Insetos/genética , Resistência a Inseticidas , Mosquitos Vetores/efeitos dos fármacos , Mutação de Sentido Incorreto , Aedes/metabolismo , Angola , Animais , Feminino , Genótipo , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Portugal
5.
PLoS Negl Trop Dis ; 14(5): e0007754, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32421713

RESUMO

Macroautophagy is an evolutionarily conserved cellular process critical for maintaining cellular homeostasis. It can additionally function as an innate immune response to viral infection as has been demonstrated for a number of arthropod-borne (arbo-) viruses. Arboviruses are maintained in a transmission cycle between vertebrate hosts and invertebrate vectors yet the majority of studies assessing autophagy-arbovirus interactions have been limited to the mammalian host. Therefore we evaluated the role of autophagy during arbovirus infection of the invertebrate vector using the tractable Aag2 Aedes aegypti mosquito cell culture system. Our data demonstrates that autophagy is significantly induced in mosquito cells upon infection with two divergent arboviruses: dengue virus-2 (DENV-2; Flaviviridae, Flavivirus) and chikungunya virus (CHIKV; Togaviridae, Alphavirus). While assessing the role of autophagy during arbovirus infection, we observed a somewhat paradoxical outcome. Both induction and suppression of autophagy via torin-1 and spautin-1, respectively, resulted in increased viral titers for both viruses, yet suppression of autophagy-related genes had no effect. Interestingly, chemical modulators of autophagy had either no effect or opposite effects in another widely used mosquito cell line, C6/36 Aedes albopictus cells. Together, our data reveals a limited role for autophagy during arbovirus infection of mosquito cells. Further, our findings suggest that commonly used chemical modulators of autophagy alter mosquito cells in such a way as to promote viral replication; however, it is unclear if this occurs directly through autophagic manipulation or other means.


Assuntos
Aedes/fisiologia , Aedes/virologia , Arbovirus/fisiologia , Autofagia , Aedes/genética , Animais , Linhagem Celular , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Replicação Viral
6.
Nature ; 580(7802): 274-277, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269344

RESUMO

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


Assuntos
Aedes/embriologia , Aedes/genética , DNA Satélite/genética , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Feminino , Inativação Gênica
7.
PLoS Negl Trop Dis ; 14(4): e0008219, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298261

RESUMO

Aedes aegypti is the primary vector of dengue, chikungunya, Zika, and urban yellow fever. Insecticides are often the most effective tools to rapidly decrease the density of vector populations, especially during arbovirus disease outbreaks. However, the intense use of insecticides, particularly pyrethroids, has selected for resistant mosquito populations worldwide. Mutations in the voltage gated sodium channel (NaV) are among the principal mechanisms of resistance to pyrethroids and DDT, also known as "knockdown resistance," kdr. Here we report studies on the origin and dispersion of kdr haplotypes in samples of Ae. aegypti from its worldwide distribution. We amplified the IIS6 and IIIS6 NaV segments from pools of Ae. aegypti populations from 15 countries, in South and North America, Africa, Asia, Pacific, and Australia. The amplicons were barcoded and sequenced using NGS Ion Torrent. Output data were filtered and analyzed using the bioinformatic pipeline Seekdeep to determine frequencies of the IIS6 and IIIS6 haplotypes per population. Phylogenetic relationships among the haplotypes were used to infer whether the kdr mutations have a single or multiple origin. We found 26 and 18 haplotypes, respectively for the IIS6 and IIIS6 segments, among which were the known kdr mutations 989P, 1011M, 1016I and 1016G (IIS6), 1520I, and 1534C (IIIS6). The highest diversity of haplotypes was found in African samples. Kdr mutations 1011M and 1016I were found only in American and African populations, 989P + 1016G and 1520I + 1534C in Asia, while 1534C was present in samples from all continents, except Australia. Based primarily on the intron sequence, IIS6 haplotypes were subdivided into two well-defined clades (A and B). Subsequent phasing of the IIS6 + IIIS6 haplotypes indicates two distinct origins for the 1534C kdr mutation. These results provide evidence of kdr mutations arising de novo at specific locations within the Ae. aegypti geographic distribution. In addition, our results suggest that the 1534C kdr mutation had at least two independent origins. We can thus conclude that insecticide selection pressure with DDT and more recently with pyrethroids is selecting for independent convergent mutations in NaV.


Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Genes de Insetos , Resistência a Inseticidas/genética , Mutação , Aedes/virologia , África , Alelos , Animais , Ásia , Austrália , Febre de Chikungunya/prevenção & controle , Febre de Chikungunya/transmissão , DNA/genética , Dengue/prevenção & controle , Dengue/transmissão , Frequência do Gene , Genótipo , Inseticidas , Mosquitos Vetores/genética , Mosquitos Vetores/virologia , América do Norte , Piretrinas , Febre Amarela/prevenção & controle , Febre Amarela/transmissão , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/transmissão
8.
PLoS Negl Trop Dis ; 14(4): e0008154, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32302303

RESUMO

BACKGROUND: Aedes aegypti is a globally distributed vector of human diseases including dengue, yellow fever, chikungunya, and Zika. Pyrethroid insecticides are the primary means of controlling adult A. aegypti populations to suppress arbovirus outbreaks, but resistance to pyrethroid insecticides has become a global problem. Mutations in the voltage-sensitive sodium channel (Vssc) gene are a major mechanism of pyrethroid resistance in A. aegypti. Vssc resistance alleles in A. aegypti commonly have more than one mutation. However, our understanding of the evolutionary dynamics of how alleles with multiple mutations arose is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We examined the geographic distribution and association between the common Vssc mutations (V410L, S989P, V1016G/I and F1534C) in A. aegypti by analyzing the relevant Vssc fragments in 25 collections, mainly from Asia and the Americas. Our results showed all 11 Asian populations had two types of resistance alleles: 1534C and 989P+1016G. The 1534C allele was more common with frequencies ranging from 0.31 to 0.88, while the 989P+1016G frequency ranged from 0.13 to 0.50. Four distinct alleles (410L, 1534C, 410L+1534C and 410L+1016I+1534C) were detected in populations from the Americas. The most common was 410L+1016I+1534C with frequencies ranging from 0.50 to 1.00, followed by 1534C with frequencies ranging from 0.13 to 0.50. Our phylogenetic analysis of Vssc supported multiple independent origins of the F1534C mutation. Our results indicated the 410L+1534C allele may have arisen by addition of the V410L mutation to the 1534C allele, or by a crossover event. The 410L+1016I+1534C allele was the result of one or two mutational steps from a 1534C background. CONCLUSIONS/SIGNIFICANCE: Our data corroborated previous geographic distributions of resistance mutations and provided evidence for both recombination and sequential accumulation of mutations contributing to the molecular evolution of resistance alleles in A. aegypti.


Assuntos
Aedes/genética , Alelos , Evolução Molecular , Resistência a Inseticidas/genética , Mutação , Recombinação Genética , Animais , Feminino , Haplótipos , Inseticidas/farmacologia , Masculino , Mosquitos Vetores/genética , Filogenia , Piretrinas/farmacologia
9.
PLoS Pathog ; 16(4): e1008433, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282862

RESUMO

The insect bacterium Wolbachia pipientis is being introgressed into Aedes aegypti populations as an intervention against the transmission of medically important arboviruses. Here we compare Ae. aegypti mosquitoes infected with wMelCS or wAlbB to the widely used wMel Wolbachia strain on an Australian nuclear genetic background for their susceptibility to infection by dengue virus (DENV) genotypes spanning all four serotypes. All Wolbachia-infected mosquitoes were more resistant to intrathoracic DENV challenge than their wildtype counterparts. Blocking of DENV replication was greatest by wMelCS. Conversely, wAlbB-infected mosquitoes were more susceptible to whole body infection than wMel and wMelCS. We extended these findings via mosquito oral feeding experiments, using viremic blood from 36 acute, hospitalised dengue cases in Vietnam, additionally including wMel and wildtype mosquitoes on a Vietnamese nuclear genetic background. As above, wAlbB was less effective at blocking DENV replication in the abdomen compared to wMel and wMelCS. The transmission potential of all Wolbachia-infected mosquito lines (measured by the presence/absence of infectious DENV in mosquito saliva) after 14 days, was significantly reduced compared to their wildtype counterparts, and lowest for wMelCS and wAlbB. These data support the use of wAlbB and wMelCS strains for introgression field trials and the biocontrol of DENV transmission. Furthermore, despite observing significant differences in transmission potential between wildtype mosquitoes from Australia and Vietnam, no difference was observed between wMel-infected mosquitoes from each background suggesting that Wolbachia may override any underlying variation in DENV transmission potential.


Assuntos
Aedes/microbiologia , Aedes/virologia , Vírus da Dengue/fisiologia , Mosquitos Vetores/microbiologia , Mosquitos Vetores/virologia , Wolbachia/fisiologia , Aedes/genética , Aedes/metabolismo , Animais , Feminino , Masculino , Mosquitos Vetores/genética , Mosquitos Vetores/metabolismo , Controle Biológico de Vetores , Replicação Viral
10.
PLoS One ; 15(4): e0231047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32282857

RESUMO

The mosquitoes Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) are two major vectors of arthropod-borne pathogens in Grenada, West Indies. As conventional vector control methods present many challenges, alternatives are urgently needed. Manipulation of mosquito microbiota is emerging as a field for the development of vector control strategies. Critical to this vector control approach is knowledge of the microbiota of these mosquitoes and finding candidate microorganisms that are common to the vectors with properties that could be used in microbiota modification studies. Results showed that bacteria genera including Asaia, Escherichia, Pantoea, Pseudomonas, and Serratia are common to both major arboviral vectors in Grenada and have previously been shown to be good candidates for transgenetic studies. Also, for the first time, the presence of Grenada mosquito rhabdovirus 1 is reported in C. quinquefasciatus.


Assuntos
Aedes/genética , Culex/genética , Genoma de Inseto/genética , Metagenômica , Aedes/microbiologia , Aedes/virologia , Animais , Culex/microbiologia , Culex/virologia , Feminino , Granada , Masculino , Reação em Cadeia da Polimerase
11.
PLoS One ; 15(4): e0232192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343725

RESUMO

The introduction of exotic disease vectors into a new habitat can drastically change the local epidemiological situation. During 2012-2015, larvae and an adult of the yellow-fever mosquito, Aedes aegypti, were captured alive at two international airports serving the Greater Tokyo Area, Japan. Because this species does not naturally distribute in this country, those mosquitoes were considered to be introduced from overseas via air-transportation. To infer the places of origin of those mosquitoes, we genotyped the 12 microsatellite loci for which the most comprehensive population genetic reference is currently available. Although clustering by Bayesian and multivariate methods both suggested that all those mosquitoes captured at the airports in Japan belonged to the Asia/Pacific populations, they were not clustered into a single cluster. Moreover, there was variation in mitochondrial cytochrome oxidase I gene (CoxI) haplotypes among mosquitoes collected in different incidents of discovery which indicated the existence of multiple maternal origins. We conclude there is little evidence to support the overwintering of Ae. aegypti at the airports; nevertheless, special attention is still needed to prevent the invasion of this prominent arbovirus vector.


Assuntos
Aedes/genética , Aeroportos , Mosquitos Vetores/genética , Aedes/classificação , Aedes/virologia , Animais , Infecções por Arbovirus/transmissão , Infecções por Arbovirus/virologia , Arbovirus/isolamento & purificação , Arbovirus/patogenicidade , Teorema de Bayes , Ecossistema , Complexo IV da Cadeia de Transporte de Elétrons/genética , Genes de Insetos , Genes Mitocondriais , Variação Genética , Genética Populacional , Genótipo , Haplótipos , Humanos , Proteínas de Insetos/genética , Repetições de Microssatélites , Mosquitos Vetores/classificação , Mosquitos Vetores/virologia , Tóquio
12.
Infect Dis Poverty ; 9(1): 30, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32183909

RESUMO

BACKGROUND: As of 2015 thousands of refugees are being hosted in temporary refugee camps in Greece. Displaced populations, travelling and living under poor conditions with limited access to healthcare are at a high risk of exposure to vector borne disease (VBD). This study sought to evaluate the risk for VBD transmission within refugee camps in Greece by analyzing the mosquito and sand fly populations present, in light of designing effective and efficient context specific vector and disease control programs. METHODS: A vector/pathogen surveillance network targeting mosquitoes and sand flies was deployed in four temporary refugee camps in Greece. Sample collections were conducted bi-weekly during June-September 2017 with the use of Centers for Disease Control (CDC) light traps and oviposition traps. Using conventional and molecular diagnostic tools we investigated the mosquito/sand fly species composition, population dynamics, pathogen infection rates, and insecticide resistance status in the major vector species. RESULTS: Important disease vectors including Anopheles sacharovi, Culex pipiens, Aedes albopictus and the Leishmania vectors Phlebotomus neglectus, P. perfiliewi and P. tobbi were recorded in the study refugee camps. No mosquito pathogens (Plasmodium parasites, flaviviruses) were detected in the analysed samples yet high sand fly Leishmania infection rates are reported. Culex pipiens mosquitoes displayed relatively high knock down resistance (kdr) mutation allelic frequencies (ranging from 41.0 to 63.3%) while kdr mutations were also detected in Ae. albopictus populations, but not in Anopheles and sand fly specimens. No diflubenzuron (DFB) mutations were detected in any of the mosquito species analysed. CONCLUSIONS: Important disease vectors and pathogens in vectors (Leishmania spp.) were recorded in the refugee camps indicating a situational risk factor for disease transmission. The Cx. pipiens and Ae. albopictus kdr mutation frequencies recorded pose a potential threat against the effectiveness of pyrethroid insecticides in these settings. In contrast, pyrethroids appear suitable for the control of Anopheles mosquitoes and sand flies and DFB for Cx. pipiens and Ae. albopictus larvicide applications. Targeted actions ensuring adequate living conditions and the establishment of integrated vector-borne disease surveillance programs in refugee settlements are essential for protecting refugee populations against VBDs.


Assuntos
Insetos Vetores/efeitos dos fármacos , Insetos Vetores/genética , Resistência a Inseticidas/genética , Leishmania , Controle de Mosquitos , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Dinâmica Populacional , Campos de Refugiados , Aedes/efeitos dos fármacos , Aedes/genética , Animais , Anopheles/efeitos dos fármacos , Anopheles/genética , Culex/efeitos dos fármacos , Culex/genética , Feminino , Grécia , Leishmania/genética , Leishmania/patogenicidade , Leishmaniose/epidemiologia , Phlebotomus/efeitos dos fármacos , Phlebotomus/genética , Psychodidae
13.
PLoS Negl Trop Dis ; 14(3): e0008130, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130209

RESUMO

BACKGROUND: Yellow fever, dengue, chikungunya and Zika viruses are responsible for considerable morbidity and mortality in humans. Aedes aegypti and Aedes albopictus are the most important mosquito vectors involved in their transmission. Accurate identification of these species is essential for the implementation of control programs to limit arbovirus transmission, during suspected detections at ports of first entry, to delimit incursions or during presence/absence surveillance programs in regions vulnerable to invasion. We developed and evaluated simple and rapid colorimetric isothermal tests to detect these two mosquito species based on loop-mediated isothermal amplification (LAMP) targeting the ribosomal RNA internal transcribed spacer 1 (ITS1). METHODOLOGY/PRINCIPAL FINDINGS: Samples were prepared by homogenizing and heating at 99 oC for 10 min before an aliquot was added to the LAMP reaction. After 40 min incubation at 65 oC, a colour change indicated a positive result. The tests were 100% sensitive and species-specific, and demonstrated a limit of detection comparable with PCR-based detection (TaqMan chemistry). The LAMP assays were able to detect target species for various life stages tested (adult, 1st instar larva, 4th instar larva and pupa), and body components, such as legs, wings and pupal exuviae. Importantly, the LAMP assays could detect Ae. aegypti DNA in mosquitoes stored in Biogents Sentinel traps deployed in the field for 14 d. A single 1st instar Ae. aegypti larva could also be detected in a pool of 1,000 non-target 1st instar Aedes notoscriptus, thus expediting processing of ovitrap collections obtained during presence/absence surveys. A simple syringe-sponge protocol facilitated the concentration and collection of larvae from the ovitrap water post-hatch. CONCLUSIONS/SIGNIFICANCE: We describe the development of LAMP assays for species identification and demonstrate their direct application for surveillance in different field contexts. The LAMP assays described herein are useful adjuncts to laboratory diagnostic testing or could be employed as standalone tests. Their speed, ease-of-use, low cost and need for minimal equipment and training make the LAMP assays ideal for adoption in low-resource settings without the need to access diagnostic laboratory services.


Assuntos
Aedes/classificação , Aedes/crescimento & desenvolvimento , Colorimetria/métodos , Entomologia/métodos , Técnicas de Diagnóstico Molecular/métodos , Mosquitos Vetores/classificação , Mosquitos Vetores/crescimento & desenvolvimento , Técnicas de Amplificação de Ácido Nucleico/métodos , Aedes/genética , Animais , DNA Espaçador Ribossômico/genética , Feminino , Mosquitos Vetores/genética , Sensibilidade e Especificidade
14.
Nat Commun ; 11(1): 1425, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188851

RESUMO

A number of recent papers report that standing genetic variation in natural populations includes ubiquitous polymorphisms within target sites for Cas9-based gene drive (CGD) and that these "drive resistant alleles" (DRA) preclude the successful application of CGD for managing these populations. Here we report the results of a survey of 1280 genomes of the mosquitoes Anopheles gambiae, An. coluzzii, and Aedes aegypti in which we determine that ~90% of all protein-encoding CGD target genes in natural populations include at least one target site with no DRAs at a frequency of ≥1.0%. We conclude that the abundance of conserved target sites in mosquito genomes and the inherent flexibility in CGD design obviates the concern that DRAs present in the standing genetic variation of mosquito populations will be detrimental to the deployment of this technology for population modification strategies.


Assuntos
Aedes/genética , Anopheles/genética , Genoma de Inseto , Alelos , Animais , Sistemas CRISPR-Cas , Feminino , Frequência do Gene , Proteínas de Insetos/genética , Mosquitos Vetores/genética
15.
J Agric Food Chem ; 68(10): 3061-3070, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32059103

RESUMO

Pyrethroids are one of the most commonly used classes of insecticides, and their acid and alcohol components are esterase degradation products, usually considered to be biologically inactive. In this study, it was found that several pyrethroid acids had a spatial repellent activity that was greater than DEET, often more active than the parent pyrethroids, and showed little cross resistance in a pyrethroid-resistant Puerto Rico strain of Aedes aegypti mosquitoes. Further investigation revealed that the acids can synergize not only contact repellent standards but also other pyrethroid components as well as the parent pyrethroids themselves. Synergism by the pyrethroid acids is expressed as both increased spatial repellency and vapor toxicity as well as human bite protection. Electrophysiological studies confirmed that pyrethroid acids (100 µM) had no effect on neuronal discharge in larval Drosophila melanogaster CNS and were detected by electroantennography, and there was little resistance to olfactory sensing of these acids in antennae from Puerto Rico strain mosquitoes carrying kdr mutations. Thus, the data suggest that the pyrethroid acids have a different mode of action than the parent pyrethroids, unrelated to the voltage-sensitive sodium channel. The results highlight the potential of pyrethroid acids to be useful in future repellent formulations.


Assuntos
Aedes/efeitos dos fármacos , Repelentes de Insetos/toxicidade , Piretrinas/química , Piretrinas/toxicidade , Ácidos/química , Ácidos/toxicidade , Aedes/genética , Álcoois/química , Álcoois/toxicidade , Animais , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/crescimento & desenvolvimento , Sinergismo Farmacológico , Repelentes de Insetos/química , Resistência a Inseticidas , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Estrutura Molecular , Controle de Mosquitos , Porto Rico
16.
PLoS Negl Trop Dis ; 14(2): e0007948, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32012156

RESUMO

Aedes cadherin (AaeCad, AAEL024535) has been characterized as a receptor for Bacillus thuringiensis subsp. israelensis (Bti) Cry11A toxins. However, its role in development is still unknown. In this study, we modified the cadherin gene using ZFN and TALEN. Even though we obtained heterozygous deletions, no homozygous mutants were viable. Because ZFN and TALEN have lower off-targets than CRISPR/Cas9, we conclude the cadherin gene is essential for Aedes development. In contrast, in lepidopteran insects loss of a homologous cadherin does not appear to be lethal, since homozygous mutants are viable. To analyze the role of AaeCad in vivo, we tagged this protein with EGFP using CRISPR-Cas9-mediated homologous recombination and obtained a homozygous AaeCad-EGFP line. Addition of Aedes Rad51 mRNA enhanced the rate of recombination. We then examined AaeCad protein expression in most tissues and protein dynamics during mosquito development. We observe that AaeCad is expressed in larval and adult midgut-specific manner and its expression pattern changed during the mosquito development. Confocal images showed AaeCad has high expression in larval caecae and posterior midgut, and also in adult midgut. Expression of AaeCad is observed primarily in the apical membranes of epithelial cells, and not in cell-cell junctions. The expression pattern observed suggests AaeCad does not appear to play a role in these junctions. However, we cannot exclude its role beyond cell-cell adhesion in the midgut. We also observed that Cry11A bound to the apical side of larval gastric caecae and posterior midgut cells exactly where AaeCad-EGFP was expressed. Their co-localization suggests that AaeCad is indeed a receptor for the Cry11A toxin. Using this mosquito line we also observed that low doses of Cry11A toxin caused the cells to slough off membranes, which likely represents a defense mechanism, to limit cell damage from Cry11A toxin pores formed in the cell membrane.


Assuntos
Aedes/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/metabolismo , Larva/crescimento & desenvolvimento , Aedes/genética , Aedes/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/genética , Caderinas/metabolismo , Sistema Digestório/crescimento & desenvolvimento , Sistema Digestório/metabolismo , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas de Insetos/genética , Larva/genética , Larva/metabolismo , Ligação Proteica
18.
Proc Natl Acad Sci U S A ; 117(7): 3711-3717, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015105

RESUMO

Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.


Assuntos
Aedes/imunologia , Aedes/parasitologia , Dirofilaria immitis/crescimento & desenvolvimento , Mosquitos Vetores/imunologia , Mosquitos Vetores/parasitologia , Aedes/genética , Animais , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Larva/crescimento & desenvolvimento , Mosquitos Vetores/genética
20.
PLoS Pathog ; 16(1): e1008103, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31945137

RESUMO

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.


Assuntos
Aedes/genética , Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Vírus da Dengue/imunologia , Aedes/virologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/genética , Anticorpos Amplamente Neutralizantes/biossíntese , Anticorpos Amplamente Neutralizantes/genética , Feminino , Humanos , Masculino , Engenharia de Proteínas , Anticorpos de Cadeia Única/genética
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