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The Aquaporin Gene Family of the Yellow Fever Mosquito, Aedes aegypti  [PDF]
Lisa L. Drake,Dmitri Y. Boudko,Osvaldo Marinotti,Victoria K. Carpenter,Angus L. Dawe,Immo A. Hansen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015578
Abstract: The mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti.
Analysis of cycle Gene Expression in Aedes aegypti Brains by In Situ Hybridization  [PDF]
Samira Chahad-Ehlers, Carla Gentile, José Bento Pereira Lima, Alexandre Afranio Peixoto, Rafaela Vieira Bruno
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0052559
Abstract: Even though the blood-sucking mosquito Aedes aegypti is one of the most important disease vectors, relatively little is known about the molecular mechanisms underlying processes involved in the temporal pattern of its activity and host seeking behavior. In this study, we analyzed the expression of the cycle (cyc) gene, one of the core components of the circadian clock, in Ae. aegypti brains by in situ hybridization at two different time points in light-dark conditions and compared the results with those obtained using a quantitative PCR assay (qPCR). Within the brain, differential labeling was detected according to distinct areas empirically pre-defined. Six out of seven of these areas showed significantly higher staining at ZT3 (three hours after light-on) compared to ZT11 (one before light-off), which is consistent with the qPCR data. Predominant staining was observed in three of those areas which correspond to positions of the optical and antennal lobes, as well as the region where the neurons controlling activity rhythms are presumably localized.
TALEN-Based Gene Disruption in the Dengue Vector Aedes aegypti  [PDF]
Azadeh Aryan, Michelle A. E. Anderson, Kevin M. Myles, Zach N. Adelman
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0060082
Abstract: In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20–40% of fertile survivors produced kmo alleles that failed to complement an existing khw mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1–7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector.
Gene Amplification, ABC Transporters and Cytochrome P450s: Unraveling the Molecular Basis of Pyrethroid Resistance in the Dengue Vector, Aedes aegypti  [PDF]
Vassiliki Bariami,Christopher M. Jones,Rodolphe Poupardin,John Vontas,Hilary Ranson
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001692
Abstract: Background Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance. Principal Findings Two previously described target site mutations, 1016I and 1534C were detected in pyrethroid resistant populations from Grand Cayman and Cuba. In addition between two and five per cent of the Ae. aegypti transcriptome was differentially expressed in the resistant populations compared to a laboratory susceptible population. Approximately 20 per cent of the genes over-expressed in resistant mosquitoes were up-regulated in both Caribbean populations (107 genes). Genes with putative monooxygenase activity were significantly over represented in the up-regulated subset, including five CYP9 P450 genes. Quantitative PCR was used to confirm the higher transcript levels of multiple cytochrome P450 genes from the CYP9J family and an ATP binding cassette transporter. Over expression of two genes, CYP9J26 and ABCB4, is due, at least in part, to gene amplification. Significance These results, and those from other studies, strongly suggest that increases in the amount of the CYP9J cytochrome P450s are an important mechanism of pyrethroid resistance in Ae. aegypti. The genetic redundancy resulting from the expansion of this gene family makes it unlikely that a single gene or mutation responsible for pyrethroid resistance will be identified in this mosquito species. However, the results from this study do pave the way for the development of new pyrethroid synergists and improved resistance diagnostics. The role of copy number polymorphisms in detoxification and transporter genes in providing protection against insecticide exposure requires further investigation.
Horizontal gene transfer between Wolbachia and the mosquito Aedes aegypti
Lisa Klasson, Zakaria Kambris, Peter E Cook, Thomas Walker, Steven P Sinkins
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-33
Abstract: We have discovered a case of HGT, involving two adjacent genes, between the genomes of Wolbachia and the currently Wolbachia-uninfected mosquito Aedes aegypti, an important human disease vector. The lower level of sequence identity between Wolbachia and insect, the transcription of all the genes involved, and the fact that we have identified homologs of the two genes in another Aedes species (Ae. mascarensis), suggest that these genes are being expressed after an extended evolutionary period since horizontal transfer, and therefore that the transfer has functional significance. The association of these genes with Wolbachia prophage regions also provides a mechanism for the transfer.The data support the argument that HGT between Wolbachia endosymbiotic bacteria and their hosts has produced evolutionary innovation.Wolbachia pipientis is an intracellular inherited bacterium found in arthropods, where it manipulates host reproduction using phenotypes such as cytoplasmic incompatibility (CI), male killing, parthenogenesis and feminization, and can spread rapidly through insect populations [1]. It is also an obligate mutualist of a number of filarial nematode species [2].Several cases where sections of the Wolbachia genome, sometimes large, have been transferred to the host chromosomes are now known in both insects and nematodes [3-5]. These are either recent events where Wolbachia and host sequences are highly similar or involve extensive pseudogenization [4]. Transcription was reported for 2% of the genes transferred to Drosophila ananassae but the levels were estimated to be 104 to 107 fold lower than for a control gene, act5C [5,6], and it has been argued that this could represent background transcriptional noise (as occurs for many pseudogenes) rather than functional expression [7,8] – translation has yet to be demonstrated. It has therefore been suggested that these fragments are on an evolutionary trajectory to degradation by neutral mutation and play no significan
Gene Flow, Subspecies Composition, and Dengue Virus-2 Susceptibility among Aedes aegypti Collections in Senegal  [PDF]
Massamba Sylla,Christopher Bosio,Ludmel Urdaneta-Marquez,Mady Ndiaye,William C. Black IV
PLOS Neglected Tropical Diseases , 2009, DOI: 10.1371/journal.pntd.0000408
Abstract: Background Aedes aegypti, the “yellow fever mosquito”, is the primary vector to humans of the four serotypes of dengue viruses (DENV1-4) and yellow fever virus (YFV) and is a known vector of Chikungunya virus. There are two recognized subspecies of Ae. aegypti sensu latu (s.l.): the presumed ancestral form, Ae. aegypti formosus (Aaf), a primarily sylvan mosquito in sub-Saharan Africa, and Ae. aegypti aegypti (Aaa), found globally in tropical and subtropical regions typically in association with humans. The designation of Ae. aegypti s.l. subspecies arose from observations made in East Africa in the late 1950s that the frequency of pale “forms” of Ae. aegypti was higher in populations in and around human dwellings than in those of the nearby bush. But few studies have been made of Ae. aegypti s.l. in West Africa. To address this deficiency we have been studying the population genetics, subspecies composition and vector competence for DENV-2 of Ae. aegypti s.l. in Senegal. Methods and Findings A population genetic analysis of gene flow was conducted among 1,040 Aedes aegypti s.l. from 19 collections distributed across the five phytogeographic regions of Senegal. Adults lacking pale scales on their first abdominal tergite were classified as Aedes aegypti formosus (Aaf) following the original description of the subspecies and the remainder were classified as Aedes aegypti aegypti (Aaa). There was a clear northwest–southeast cline in the abundance of Aaa and Aaf. Collections from the northern Sahelian region contained only Aaa while southern Forest gallery collections contained only Aaf. The two subspecies occurred in sympatry in four collections north of the Gambia in the central Savannah region and Aaa was a minor component of two collections from the Forest gallery area. Mosquitoes from 11 collections were orally challenged with DENV-2 virus. In agreement with the early literature, Aaf had significantly lower vector competence than Aaa. Among pure Aaa collections, the disseminated infection rate (DIR) was 73.9% with a midgut infection barrier (MIB) rate of 6.8%, and a midgut escape barrier (MEB) rate of 19.3%, while among pure Aaf collections, DIR = 34.2%, MIB rate = 7.4%, and MEB rate = 58.4%. Allele and genotype frequencies were analyzed at 11 nuclear single nucleotide polymorphism (SNP) loci using allele specific PCR and melting curve analysis. In agreement with a published isozyme gene flow study in Senegal, only a small and statistically insignificant percentage of the variance in allele frequencies was associated with subspecies. Conclusions These
Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes
James K Biedler, Zhijian Tu
BMC Evolutionary Biology , 2010, DOI: 10.1186/1471-2148-10-206
Abstract: Two novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1) in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the AaKLC2.1 start codon shows promoter activity at least as early as 3 hours in the developing Ae. aegypti embryo. The AaKLC2.1 promoter activity reached ~1600 fold over the negative control at 5 hr after egg deposition.Transcriptome profiling by use of high throughput sequencing technologies has proven to be a valuable method for the identification and discovery of early and transient zygotic genes. The evolutionary investigation of the KLC gene family reveals that duplication is a source for the evolution of new genes that play a role in the dynamic process of early embryonic development. AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.The early embryo is
siRNA-Mediated Gene Targeting in Aedes aegypti Embryos Reveals That Frazzled Regulates Vector Mosquito CNS Development  [PDF]
Anthony Clemons,Morgan Haugen,Christy Le,Akio Mori,Michael Tomchaney,David W. Severson,Molly Duman-Scheel
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0016730
Abstract: Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.
Gender-related family head schooling and Aedes aegypti larval breeding risk in Southern Mexico
Danis-Lozano,Rogelio; Rodríguez,Mario H; Hernández-Avila,Mauricio;
Salud Pública de México , 2002, DOI: 10.1590/S0036-36342002000300007
Abstract: objective. to investigate if family head genre-associated education is related to the risk of domiciliary aedes aegypti larval breeding in a dengue-endemic village of southern mexico. material and methods. a family head was considered to have a low education level if he/she had not completed elementary school. to estimate larval breeding risk within each household, a three-category maya index was constructed using a weighted estimation of controllable and disposable domestic water containers. a socio-economic index was constructed based on household construction characteristics. results. low-level education of either family head was associated to higher larval breeding risk. households with low-educated mothers had more larval breeding containers. these associations persisted after adjusting for household socio-economic level. conclusions. these results indicate that households with female family heads with low education levels accumulate more containers that favor ae. aegypti breeding, and that education campaigns for dengue control should be addressed to this part of the population.
Gender-related family head schooling and Aedes aegypti larval breeding risk in Southern Mexico  [cached]
Danis-Lozano Rogelio,Rodríguez Mario H,Hernández-Avila Mauricio
Salud Pública de México , 2002,
Abstract: Objective. To investigate if family head genre-associated education is related to the risk of domiciliary Aedes aegypti larval breeding in a dengue-endemic village of Southern Mexico. Material and Methods. A family head was considered to have a low education level if he/she had not completed elementary school. To estimate larval breeding risk within each household, a three-category Maya index was constructed using a weighted estimation of controllable and disposable domestic water containers. A socio-economic index was constructed based on household construction characteristics. Results. Low-level education of either family head was associated to higher larval breeding risk. Households with low-educated mothers had more larval breeding containers. These associations persisted after adjusting for household socio-economic level. Conclusions. These results indicate that households with female family heads with low education levels accumulate more containers that favor Ae. aegypti breeding, and that education campaigns for dengue control should be addressed to this part of the population.
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