Structure of hermes integrations in the germline of the yellow fever mosquito, Aedes aegypti

The Hermes transposable element is derived from the house fly, Musca domestica, and can incorporate into the germline of the yellow fever mosquito, Aedes aegypti. Preliminary Southern analyses indicated that Hermes integrated along with the marker gene into the mosquito genomic DNA. Here we show that Hermes integrations are accompanied by the integration of the donor plasmid as well. In addition, breaks in the donor plasmid DNAs do not occur precisely, or at the end of the terminal inverted repeats, and are accompanied by small deletions in the plasmids. Furthermore, integrations do not cause the typical 8-bp duplications of the target site DNA. No integrations are observed in the absence of a source of Hermes transposase. The Hermes transposase clearly did not catalyse precise cut-and-paste transposition in these transformed lines. It may have integrated the transposon through general recombination or through a partial replicative transposition mechanism. The imprecision of Hermes integration may result from interactions of the transposase with an endogenous hAT-like element in the mosquito genome.     

Transgene‐mediated suppression of dengue viruses in the salivary glands of the yellow fever mosquito,Aedes aegypti

Controlled sex‐, stage‐ and tissue‐specific expression of antipathogen effector molecules is important for genetic engineering strategies to control mosquito‐borne diseases. Adult female salivary glands are involved in pathogen transmission to human hosts and are target sites for expression of antipathogen effector molecules. The Aedes aegypti 30K a and 30K b genes are expressed exclusively in adult female salivary glands and are transcribed divergently from start sites separated by 263 nucleotides. The intergenic, 5′‐ and 3′‐end untranslated regions of both genes are sufficient to express simultaneously two different transgene products in the distal‐lateral lobes of the female salivary glands. An antidengue effector gene, membranes no protein (Mnp), driven by the 30K b promoter, expresses an inverted‐repeat RNA with sequences derived from the premembrane protein‐encoding region of the dengue virus serotype 2 genome and reduces significantly the prevalence and mean intensities of viral infection in mosquito salivary glands and saliva.     

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.     

Evidence of multiple chromosomal inversions in Aedes aegypti formosus from Senegal

Chromosomal inversions are prevalent in mosquito species but polytene chromosomes are difficult to prepare and visualize in members of the tribe Aedinii and thus there exists only indirect evidence of inversions. We constructed an F₁ intercross family using a P₁ female from a laboratory strain of Aedes aegypti aegypti ( Aaa ) and a P₁ male Aedes aegypti formosus ( Aaf ) from a strain collected from south-eastern Senegal. Recombination rates in the F₂ offspring were severely reduced and genotype ratios suggested a deleterious recessive allele on chromosome 3. The F₂ linkage map was incongruent in most respects with the established map for Aaa . Furthermore, no increased recombination was detected in F₅ offspring. Recombination rates and gene order were consistent with the presence in Aaf of at least four large inversions on chromosome 1, a single small inversion on chromosome 2 and three inversions on chromosome 3.     

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.     

Codon preference of Aedes aegypti and Aedes albopictus

The codon bias of two Aedes mosquito species was examined using a sign test. In general, there appeared to be some preference for C + G at the third base position, although this was not statistically significant. While amino acids such as phenylalanine and tyrosine clearly displayed biases, others such as valine and serine appeared to have little or no bias for any particular codon. Three homologous genes of Aedes aegypti and Drosophila melanogaster were compared using the chi-square test and the codon bias of the two species compared. Drosophila melanogaster was found to have a much stronger bias for C + G at the third base position compared to Aedes. The implications and usefulness of the codon bias are discussed.     

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.     

Immunity proteins from mosquito cell lines include three defensin A isoforms from Aedesaegypti and a defensin D from Aedesalbopictus

An Aedes aegypti mosquito cell line, Aag-2, exhibits a response to immune stimulation that is qualitatively similar to that of C7–10 cultured cells from the related mosquito, Aedes albopictus. Using SDS polyacrylamide gels, we found that a small peptide was preferentially induced by the treatment of growing cells with heat-killed, Gram-positive bacteria. By an analogy with other studies, this small peptide was postulated to be a member of the defensin family of insect immunity peptides. A differential display was used to obtain partial polymerase chain reaction products corresponding to mRNAs that were preferentially expressed in induced cells. One of these products, which contained the partial sequence of a defensin gene, was used to screen cDNA libraries from Ae. aegypti and Ae. albopictus cells. From Ae. aegypti cells, we found two previously described isoforms (A1 and A4) of mosquito defensin A, as well as a new isoform which we defined as A5. From Ae. albopictus cells, we found a new mature mosquito defensin, named D, which contains proline and isoleucine as the final amino acids. In both Ae. aegypti and Ae. albopictus cell lines, the expression of defensin mRNA was visible on Northern blots as early as 3 h after exposure to heat-killed bacteria, and defensin mRNA abundance was maximal at 12–36 h after induction.     

Toward a physical map of Aedes aegypti

Labelled recombinant cosmids were used as in situ hybridization probes to Aedes aegypti metaphase chromosomes. The cosmid probes yielded paired signals, one on each arm of sister chromatids, and they were ordered along the three chromosomes. In total, thirty-seven different probes were mapped to the three chromosomes of Ae. aegypti (2n = 6): twenty-eight to chromosome 1, six to chromosome 2, and six to chromosome 3. These results represent an initial stage in the generation of a physical map of the Ae. aegypti genome.     

Molecular characterization of the Aedes aegypti odorant receptor gene family

The olfactory-driven blood-feeding behaviour of female Aedes aegypti mosquitoes is the primary transmission mechanism by which the arboviruses causing dengue and yellow fevers affect over 40 million individuals worldwide. Bioinformatics analysis has been used to identify 131 putative odourant receptors from the A. aegypti genome that are likely to function in chemosensory perception in this mosquito. Comparison with the Anopheles gambiae olfactory subgenome demonstrates significant divergence of the odourant receptors that reflects a high degree of evolutionary activity potentially resulting from their critical roles during the mosquito life cycle. Expression analyses in the larval and adult olfactory chemosensory organs reveal that the ratio of odourant receptors to antennal glomeruli is not necessarily one to one in mosquitoes.     

Chemical and gamma-ray mutagenesis of the white gene in Aedes aegypti

A molecular understanding of an insect gene can be facilitated by analysing the phenotypes of mutants for that gene. Protocols were developed for both chemical and gamma-ray mutagenesis in Aedes aegypti using the white (w) gene as an assay. Wild-type adult males were subjected to varying doses of either ethyl methanesulphonate (0. 1%, 0.5% or 1%) or gamma rays (1500 R or 3000 R), mated to females homozygous for the recessive w mutation, and progeny screened for the w phenotype, indicating non-complementation. The expression of newly induced w alleles was either complete or mosaic. Gamma-ray mutagenesis resulted in high (1.65 or 6.39%, depending on dose) induction of mutant alleles for the w gene, but not for a different gene, red-eye (0.15%). Gamma-ray-induced w alleles did not revert at a reasonable frequency following additional irradiation, suggesting that the high rate of gamma-ray-induced w mutagenesis is not due to a transposon insertion event.     

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.