Regulation of the ferritin heavy-chain homologue gene in the yellow fever mosquito, Aedes aegypti

In the yellow fever mosquito Aedes aegypti, the ferritin heavy-chain homologue (HCH) gene is induced by blood feeding. This suggests that ferritin may serve as a cytotoxic protector against the oxidative challenge of the blood meal and may be essential for the survival of the insect. In this study, various cis-acting elements for the gene were identified and mapped. Transfection assays showed that the strength and activity of a subset of these elements are orientation-dependent. The shift observed for the ferritin HCH cis-acting elements is unique among known ferritin genes. DNase I footprinting data together with Transfac analyses identified a number of putative sites known for their involvement in developmental and cell proliferation processes.     

The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins

A cDNA cloned from Aedes aegypti (L.) (Aedae) female Malpighian tubule (AY596453) encodes a 584 amino acid residue protein (65.2 kDa) predicted as a G protein-coupled receptor and orthologue of the drosokinin receptor from Drosophila melanogaster and highly similar to the tick Boophilus microplus myokinin receptor (AF228521). Based on the similarity to this Aedes sequence, we also propose a correction for the Anopheles gambiae protein sequence EAA05450. When expressed in CHO-K1 cells, the Aedes receptor behaved as a multiligand receptor and functionally responded to concentrations > or = 1 nM of Aedae kinins 1-3, respectively, as determined by a calcium bioluminescence plate assay and single cell intracellular calcium measurements by confocal fluorescence cytometry. Estimates of EC50 values by the plate assay were 16.04 nM for Aedae-K-3, 26.6 nM for Aedae-K-2 and 48.8 nM for Aedae-K-1 and were statistically significantly different. These results suggest that the observed differences in physiological responses to the three Aedes kinins in the Aedes isolated Malpighian tubule reported elsewhere could now be explained by differences in intracellular signalling events triggered by the different peptides on the same receptor and not necessarily due to the existence of various receptors for the three Aedes kinins.     

Functional expression of insecticide-resistant GABA receptors from the mosquito Aedes aegypti

We are interested in cloning insecticide resistance genes from vector mosquitos for use as selectable markers in their genetic transformation. As a first step towards this goal, we here report the functional homo-muitimeric expression of a γ-aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin (Rdl) , from the yellow fever mosquito Aedes aegypti in baculovirus-infected insect cell lines. Replacement of alanine296 with a serine leads to approximately 100-fold insensitivity to picrotoxin as previously observed in Drosophila. This shows not only that the mosquito GABA receptor cDNA is functional but also that it can be simply mutated to resistance. Strategies for incorporation of this cDNA into a minigene for the genetic transformation of mosquitoes are discussed.     

The white gene from the yellow fever mosquito, Aedes aegypti

We report the cloning and primary characterization of both cDNA and genomic fragments from the white gene of the yellow fever mosquito, Aedes aegypti . Comparisons of the conceptual translation product with white genes from four other species within the order Diptera show that the Ae. aegypti gene is most similar to the white gene of the mosquito vector of human malaria, Anopheles gambiae (86% identity and 92% similarity). The analysis of the primary sequence of genomic DNA at the 5'-end of the coding region revealed the presence of an intron that is also present in An. gambiae , but not in the vinegar fly, Drosophila melanogaster . The isolated clones of the Ae. aegypti white gene will enable the construction of a marker gene for use in the development of a germline transformation system for this species.     

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.     

Intraspecific DNA variation in nuclear genes of the mosquito Aedes aegypti

Single nucleotide polymorphisms (SNPs) are an abundant source of genetic variation among individual organisms. To assess the usefulness of SNPs for genome analysis in the yellow fever mosquito, Aedes aegypti, we sequenced 25 nuclear genes in each of three strains and analysed nucleotide diversity. The average frequency of nucleotide variation was 12 SNPs per kilobase, indicating that nucleotide variation in Ae. aegypti is similar to that in other organisms, including Drosophila and the malaria vector Anopheles gambiae. Transition polymorphisms outnumbered transversion polymorphisms, at a ratio of about 2:1. We examined codon usage and confirmed that mutational bias favours G and C ending codons. Codon bias was most pronounced in highly expressed genes. Nucleotide diversity estimates indicated that substitution rates are positively correlated in coding and non-coding regions. Nucleotide diversity varied from one gene to another. The unequal distribution of SNPs among Ae. aegypti nuclear genes suggests that single base variations are non-neutral and are subject to selective constraints. Our analysis showed that ubiquitously expressed genes have lower polymorphism rates and are likely under strong purifying selection, whereas tissue specific genes and genes with a putative role in parasite defence exhibit higher levels of polymorphism that may be associated with diversifying selection.     

Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti

Pathogens that infect and/or are transmitted by mosquitoes typically are exposed to the body cavity, and to haemocytes circulating therein, during development or dissemination. Aedes aegypti haemocytes produce a range of immune response-related gene products, and an endpoint response of phagocytosis and/or melanization that is temporally and structurally distinct for the invading pathogen. Expressed sequence tags were generated from haemocyte libraries and then used to design oligonucleotide microarrays. Arrays were screened with haemocyte material collected 1-, 8- and 24-h post-inoculation with Escherichia coli or Micrococcus luteus bacteria. Data from these studies support the discovery of novel immune response-activated genes, provide an expanded understanding of antimicrobial peptide biology and highlight the coordination of immune factors that leads to an endpoint response.     

Improved transient silencing of gene expression in the mosquito female Aedes aegypti

Gene silencing using RNA interference (RNAi) has become a widely used genetic technique to study gene function in many organisms. In insects, this technique is often applied through the delivery of dsRNA. In the adult female Aedes aegypti, a main vector of human-infecting arboviruses, efficiency of gene silencing following dsRNA injection varies greatly according to targeted genes. Difficult knockdowns using dsRNA can thus hamper gene function analysis. Here, by analysing silencing of three different genes in female Ae. aegypti (p400, ago2 and E75), we show that gene silencing can indeed be dsRNA sequence dependent but different efficiencies do not correlate with dsRNA length. Our findings suggest that silencing is likely also gene dependent, probably due to gene-specific tissue expression and/or feedback mechanisms. We demonstrate that use of high doses of dsRNA can improve knockdown efficiency, and injection of a transfection reagent along with dsRNA reduces the variability in efficiency between replicates. Finally, we show that gene silencing cannot be achieved using siRNA injection in Ae. aegypti adult females. Overall, this work should help future gene function analyses using RNAi in adult females Ae. aegypti, leading toward a better understanding of physiological and infectious processes.     

Transcription of detoxification genes after permethrin selection in the mosquito Aedes aegypti

Changes in gene expression before, during and after five generations of permethrin laboratory selection were monitored in six strains of Aedes aegypti: five F(2)-F(3) collections from the Yucatán Peninsula of Mexico and one F(2) from Iquitos, Peru. Three biological replicate lines were generated for each strain. The response to selection was measured as changes in the lethal and knockdown permethrin concentrations (LC(50), KC(50)) and in the frequency of the Ile1,016 substitution in the voltage-gated sodium channel (para) gene. Changes in expression of 290 metabolic detoxification genes were measured using the 'Aedes Detox' microarray. Selection simultaneously increased the LC(50), KC(50) and Ile1,016 frequency. There was an inverse relationship between Ile1,016 frequency and the numbers of differentially transcribed genes. The Iquitos strain lacked the Ile1,016 allele and 51 genes were differentially transcribed after selection as compared with 10-18 genes in the Mexican strains. Very few of the same genes were differentially transcribed among field strains but 10 cytochrome P(450) genes were upregulated in more than one strain. Laboratory adaptation to permethrin in Ae. aegypti is genetically complex and largely conditioned by geographic origin and pre-existing target site insensitivity in the para gene. The lack of uniformity in the genes that responded to artificial selection as well as differences in the direction of their responses challenges the assumption that one or a few genes control permethrin metabolic resistance. Attempts to identify one or a few metabolic genes that are predictably associated with permethrin adaptation may be futile.     

Identification and initial characterization of matrix metalloproteinases in the yellow fever mosquito,Aedes aegypti

Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL probably requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1?9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut.