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 ferritin light-chain homologue promoter in Aedes aegypti

Promoters that direct the expression of antipathogenic molecules to primary sites of pathogenic invasions provide a means to interfere with these invasions. Thus, they have the potential to be used in mosquito control. However, exogenous elements are known to lower the fitness of most insects, and given the ability of insects to evolve rapidly, all currently known promoters could be rendered useless. As transgenic mosquitoes may be a major component in the fight against mosquito-borne diseases, the identification of new mosquito promoters is needed. The promoter of the Aedes aegypti ferritin light-chain homologue (LCH) gene, a gene whose expression is induced in gut tissues during blood feeding has been identified and mapped. Transfection data indicate that the ferritin LCH promoter is a strong promoter. DNase I footprinting data and Transfac analyses suggest that the ferritin LCH promoter contains putative GATA, E2F, NIT2, TATA and DPE sites. These data together provide the first detailed map of a known ferritin LCH gene.     

Using bacteria to treat diseases

INTRODUCTION: Mosquito-borne diseases such as malaria and dengue fever result in significant morbidity and mortality in developing countries. Vector control is often the most effective strategy to prevent disease transmission and novel methods are required to complement existing insecticide-based strategies. Biological control uses natural predators or pathogens to kill mosquitoes or reduce their capacity to transmit disease. Bacteria such as Wolbachia have been proposed to have the potential to provide effective biological control of mosquitoes. AREAS COVERED: A review of the potential role of bacteria in the control of mosquito-borne diseases highlighting the advantages and disadvantages of each strategy. In particular, a comprehensive summary of the progress made using the bacterial endosymbiont Wolbachia for dengue control. EXPERT OPINION: Pathogenic bacteria such as Bti can be used to kill mosquito larvae and several endosymbiotic bacteria such as Asaia could be genetically transformed to alter the mosquito's ability to transmit pathogens. The endosymbiotic bacterium Wolbachia has been successfully introduced into the principal vector of dengue, Aedes aegypti, and induces a variety of phenotypic effects that are predicted to reduce dengue transmission. The release of Wolbachia-infected mosquitoes has been undertaken as part of preliminary trials to determine the applied use of this bacterium for mosquito-borne disease control.     

Green fluorescent protein as a genetic marker in transgenic Aedes aegypti

We report here the use of the enhanced green fluorescent protein (EGFP) from the jellyfish, Aequorea victoria, as a genetic marker for the genetic transformation of mosquitoes. The EGFP gene, under the control of the actin5C promoter of Drosophila melanogaster was inserted into the Hermes transposable element. Preblastoderm embryos of a wild-type strain of the yellow fever mosquito, Aedes aegypti, were microinjected with this plasmid, together with a helper plasmid containing the Hermes transposase placed under the control of the D. melanogaster hsp70 promoter. Somatic EGFP expression was observed during early instars in approximately one-half of all G0 individuals. Two G1 individuals arising from a G0 female displayed high levels of EGFP gene expression during all stages of development. EGFP was transmitted in a Mendelian fashion to the G2 and G3 generations and molecular analysis confirmed the presence of the Hermes[actin5C:EGFP] gene in these insects. These results clearly demonstrate that EGFP can be used as an effective genetic marker in wild-type Ae. aegypti and most likely in other mosquito species as well.     

Modulation of Anopheles gambiae gene expression in response to o'nyong-nyong virus infection

To determine if gene expression of An. gambiae is modulated in response to o'nyong-nyong virus (ONNV) infection, we utilized cDNA microarrays including about 20 000 cDNAs. Gene expression levels of ONNV-infected female mosquitoes were compared to that of the uninfected control females harvested at 14 days postinfection. In response to ONNV infection, expression levels of 18 genes were significantly modulated, being at least two-fold up- or down-regulated. Quantitative real-time PCR analysis (qRT-PCR) further substantiated the differential expression of six of these genes in response to ONNV infection. These genes have similarity to a putative heat shock protein 70, DAN4, agglutinin attachment subunit, elongation factor 1 alpha and ribosomal protein L35. One gene, with sequence similarity to mitochondrial ribosomal protein L7, was down-regulated in infected mosquitoes. The expression levels and annotation of the differentially expressed genes are discussed in the context of host/virus interaction including host translation/replication factors, and intracellular transport pathways.     

Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vector

Mosquito-vectored diseases such as yellow fever and dengue fever continue to have a substantial impact on human populations world-wide. Novel strategies for control of these mosquito vectored diseases can arise through the development of reliable systems for genetic manipulation of the insect vector. A piggyBac vector marked with the Drosophila melanogaster cinnabar (cn) gene was used to transform the white-eyed khw strain of Aedes aegypti. Microinjection of preblastoderm embryos resulted in four families of cinnabar transformed insects. An overall transformation frequency of 4%, with a range of 0% to as high as 13% for individual experiments, was achieved when using a heat-shock induced transposase providing helper plasmid. Southern hybridizations indicated multiple insertion events in three of four transgenic lines, while the presence of duplicated target TTAA sites at either ends of individual insertions confirmed characteristic piggyBac transposition events in these three transgenic lines. The transgenic phenotype has remained stable for more than twenty generations. The transformations effected using the piggyBac element establish the potential of this element as a germ-line transformation vector for Aedine mosquitoes.     

A p38 MAP kinase regulates the expression of the Aedes aegypti defensin gene in mosquito cells

An Aedes aegypti p38 (Aap38) mitogen-activated protein kinase was isolated and characterized in this study. The 1761 bp long full-length Aap38 cDNA encodes an open reading frame of 358 amino acids, exhibiting characteristics of Thr/Tyr dual kinase specificities. We showed that bacteria activate both the kinase activity of Aap38 and the expression of the Aedes aegypti defensin A (AaDefA) gene, which is inhibited by a p38 kinase inhibitor SB203580 and dsRNA interference of Aap38. A similar result was obtained by a reporter construct containing the AaDefA regulatory region linked to Ds-Red. The lipopolysaccharide-activated reporter gene was inhibited by SB203580. In addition, Aap38 translocated to the nucleus after lipopolysaccharide induction. Our findings suggest that the p38 protein kinase pathway is involved in the antibacterial peptide synthesis in mosquitoes.     

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.     

2006年中国登革热疫情监测分析

目的　分析中国（未包括香港、澳门和台湾地区）登革热疫情监测资料,为加强登革热的预防控制提供科学依据。方法　对2006年全国网络直报登革热疫情及登革热监测点资料用描述流行病学方法进行统计分析。结果 2006年全国共14个省份报告登革热病例1044例,无死亡报告。除广东省有本地感染外,其他地区均为输入性病例;输入性病例全年均有分布,主要来自东南亚和美洲等地区;输入病例最多的前3位国家是柬埔寨、缅甸和新加坡。广东省登革热多点暴发,发病时间集中在8-11月。媒介伊蚊监测显示广西、海南、云南地区所有监测点和福建省（86.7%）、广东省（55.8%）布雷图指数（BI）均超过5的安全水平;尤其6-10月,BI普遍较高。福建、广东、云南和广西地区媒介蚊种均为白纹伊蚊,海南省媒介为白纹伊蚊和埃及伊蚊。监测点健康人群登革热抗体阳性率为0%～14.5%。开展病原学检测的监测点没有从媒介伊蚊中检测到登革病毒核酸或分离到病毒。 结论　中国南方登革热监测省份具有登革热流行的媒介伊蚊和易感人群,夏秋季节可由输入病例引发本地暴发流行。应普遍建立灵敏的早期登革热预警监测系统,早期发现病例,防蚊灭蚊,控制疫情的扩散。