登革病毒转基因载体pB［PUBnls_EGFP_prM］的构建及其在C6/36细胞中整合作用的检测

利用转基因技术来探索新的蚊媒疾病防治方法,将登革病毒前膜蛋白基因prM重组入以转座子piggyBac因子为基础的载体,构建了昆虫转基因载体pB［PUBnls-EGFP-prM］,在辅助质粒的作用下共同转染白纹伊蚊Aedes albopictus C6/36细胞。PCR和Southern blot证明构建的转基因载体可以将EGFP-prM基因整合入蚊虫基因组中。验证了转座子piggyBac因子、启动子polyubiquitin可以在白纹伊蚊中发挥功能,为进一步构建不传播登革病毒的转基因白纹伊蚊奠定了基础。     

Post-integration stability of piggyBac in Aedes aegypti

The post-integration activity of piggyBac transposable element gene vectors in Aedes aegypti mosquitoes was tested under a variety of conditions. The embryos from five independent transgenic lines of Ae. aegypti, each with a single integrated non-autonomous piggyBac transposable element gene vector, were injected with plasmids containing the piggyBac transposase open-reading frame under the regulatory control of the Drosophila melanogaster hsp70 promoter. No evidence for somatic remobilization was detected in the subsequent adults whereas somatic remobilization was readily detected when similar lines of transgenic D. melanogaster were injected with the same piggyBac transposase-expressing plasmid. Ae. aegypti heterozygotes of piggyBac reporter-containing transgenes and piggyBac transposase-expressing transgenes showed no evidence of somatic and germ-line remobilization based on phenotypic and molecular detection methods. The post-integration mobility properties of piggyBac in Ae. aegypti enhance the utility of this gene vector for certain applications, particularly those where any level of vector remobilization is unacceptable.     

piggyBac转座子及其在转基因昆虫中的应用

piggyBac是一种从粉纹夜蛾Trichoplusiani.中分离到的、具有TTAA插入位点特异性的DNA转座子。piggyBac可在昆虫基因组中准确切离,转化频率较高,并且不受宿主因子的限制,是目前转基因昆虫研究中应用最广的转座子载体。近年来的研究发现,piggyBac类转座子广泛分布于昆虫和其他生物基因组中。文章从piggyBac的结构、转座特性、在转基因昆虫中的应用以及piggyBac类转座子的分布等几个方面综述了piggyBac的研究进展。     

Excision and transposition of piggyBac transposable element in tobacco budworm embryos

The TTAA-specific lepidopteran transposon piggyBac has already proved useful as a gene-transfer vector for efficient transformation of a wide variety of insects. Transposable element excision and transposition assays are useful indicators of an element's ability to be mobilized in vivo and, thus, potentially serve as a transforming vector. Here, we report that this transposon is capable of excision and transposition in tobacco budworm embryos with relatively low frequency.     

Post-integration behavior of a Mos1 mariner gene vector in Aedes aegypti

The post-integration behavior of insect gene vectors will determine the types of applications for which they can be used. Transposon mutagenesis, enhancer trapping, and the use of transposable elements as genetic drive systems in insects requires transposable elements with high rates of remobilization in the presence of transposase. We investigated the post-integration behavior of the Mos1 mariner element in transgenic Aedes aegypti by examining both germ-line and somatic transpositions of a non-autonomous element in the presence of Mos1 transposase. Somatic transpositions were occasionally detected while germ-line transposition was only rarely observed. Only a single germ-line transposition event was recovered after screening 14,000 progeny. The observed patterns of transposition suggest that Mos1 movement takes place between the S phase and anaphase. The data reported here indicate that Mos1 will be a useful vector in Ae. aegypti for applications requiring a very high degree of vector stability but will have limited use in the construction of genetic drive, enhancer trap, or transposon tagging systems in this species.     

The piggyBac element is capable of precise excision and transposition in cells and embryos of the mosquito, Anopheles gambiae

The piggyBac transposable element was tested for transposition activity in plasmid-based excision and inter-plasmid transposition assays to determine if this element would function in Anopheles gambiae cells and embryos. In the Mos55 cell line, precise excision of the piggyBac element was observed only in the presence of a helper plasmid. Excision occurred at a rate of 1 event per 1000 donor plasmids screened. Precise excision of the piggyBac element was also observed in injected An. gambiae embryos, but at a lower rate of 1 excision per 5000 donor plasmids. Transposition of the marked piggyBac element into a target plasmid occurred in An. gambiae cells at a rate of 1 transposition event per 24,000 donor plasmids. The piggyBac element transposed in a precise manner, with the TTAA target site being duplicated upon insertion, in 56% of transpositions observed, and only in the presence of the piggyBac helper. The remaining transpositions resulted in a deletion of target sequence, a novel observation for the phenomenon of piggyBac element insertion. 'Hot spots' for insertion into the target plasmid were observed, with 25 of 34 events involving one particular site. These results are the first demonstration of the precise mobility of piggyBac in this malaria vector and suggest that the lepidopteran piggyBac transposon is a candidate element for germline transformation of anopheline mosquitoes.     

Use of the piggyBac transposon for germ-line transformation of insects

Germ-line transformation of insects is now possible with four independent transposable element vector systems. Among these, the TTAA-insertion site specific transposon, piggyBac, discovered in Trichoplusia ni, is one of the most widely used. Transformations have been achieved in a wide variety of dipterans, lepidopterans, and a coleopteran, and for many species, piggyBac transposition was first tested by plasmid-based mobility assays in cell lines and embryos. All plasmid and genomic insertions are consistent with the duplication of a TTAA insertion site, and most germ-line integrations appear to be stable, though this is largely based on stable marker phenotypes. Of the vector systems presently in use for non-drosophilids, piggyBac is the only one not currently associated with a superfamily of transposable elements, though other elements exist that share its TTAA insertion site specificity. While functional piggyBac elements have only been isolated from T. ni, nearly identical elements have been discovered in a dipteran species, Bactrocera dorsalis, and closely related elements exist in another moth species, Spodoptera frugiperda. It appears that piggyBac has recently traversed insect orders by horizontal transmission, possibly mediated by a baculovirus or other viral system. This interspecies movement has important implications for the practical use of piggyBac to create transgenic insect strains for field release.     

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.     

Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni

The Lepidopteran transposable element piggyBac is being recognized as a useful vector for genetic engineering in a variety of insect species. This transposon can mediate transformation in the Dipteran species Ceratitis capitata, and can potentially serve as a versatile vector for transformation of a wide variety of insect species. Using a plasmid-based interplasmid transposition assay, we have demonstrated that this transposon, of the short inverted terminal repeat type, is capable of transposition in embryos of three different insect species, Drosophila melanogaster, the yellow fever mosquito Aedes aegypti, and its host of origin, Trichoplusia ni. This assay can confirm the potential utility of piggyBac as a gene transfer tool in a given insect species, and provides an experimental model for assessing molecular mechanisms of transposon movement.     

Aedes albopictus, vector of chikungunya and dengue viruses in Reunion Island: biology and control

Chikungunya virus (CHIKV) and dengue virus (DENV) are mosquito-borne viruses transmitted by the Aedes genus. Dengue is considered as the most important arbovirus disease throughout the World. Chikungunya, known from epidemics in continental Africa and Asia, has up to now been poorly studied. It has been recently responsible for the severe 2004-2007 epidemic reported in the Indian Ocean (IO), which has caused several serious health and economic problems. This unprecedented epidemic of the IO has shown severe health troubles with morbidity and death associated, which had never been observed before. The two major vectors of those arboviruses in the IO area are Aedes aegypti and Aedes albopictus. The latest is considered as the main vector in most of the islands of the area, especially in Reunion Island. Ae. albopictus showed strong ecological plasticity. Small disposable containers were the principal urban breeding sites, and preferred natural developmental sites were bamboo stumps and rock holes in peri-urban and gully areas. The virus has been isolated from field collected Ae. albopictus females, and in two out of 500 pools of larvae, demonstrating vertical transmission. Experimental works showed that both Ae. albopictus and Ae. aegypti from west IO islands are efficient vectors of dengue and chikungunya viruses. Since 2006 and all along the epidemic of CHIKV, measures for the control of larvae (temephos then Bacillus thuringiensis) and adults (fenitrothion, then deltamethrine) of Ae. albopictus where applied along with individual and collective actions (by the use of repellents, and removal of breeding sites around houses) in Reunion Island. In order to prevent such epidemics, a preventive plan for arboviruses upsurge is ongoing processed. This plan would allow a quicker response to the threat and adapt it according to the virus and its specific vector.     

Alternative splicing generates multiple transcripts of the inhibitor of apoptosis protein 1 in Aedes and Culex spp. mosquitoes

We determined the sequences of cDNA encoding Inhibitor of Apoptosis Protein 1 (IAP1) homologues from Aedes triseriatus, Aedes albopictus, Aedes aegypti, Culex pipiens and Culex tarsalis. The cDNAs encode translation products that share > or = 84% sequence similarity. The IAP1 mRNA of each mosquito species exists as 3-5 distinct variants due to the presence of heterogeneous sequences at the distal end of their 5'UTRs. Partial genomic sequencing upstream of the 5' end of the Ae. triseriatus IAP1 gene, and analysis of the Ae. aegypti genomic sequence, suggest that these mRNA variants are generated by alternative splicing. Each IAP1 mRNA variant from Ae. triseriatus and Cx. pipiens was detected by RT-PCR in all mosquito life-stages and adult tissues examined, and the relative concentration of each Ae. triseriatus IAP mRNA variant in various tissues was determined.     

Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni.

The Lepidopteran transposable element piggyBac is being recognized as a useful vector for genetic engineering in a variety of insect species. This transposon can mediate transformation in the Dipteran species Ceratitis capitata, and can potentially serve as a versatile vector for transformation of a wide variety of insect species. Using a plasmid-based interplasmid transposition assay, we have demonstrated that this transposon, of the short inverted terminal repeat type, is capable of transposition in embryos of three different insect species, Drosophila melanogaster, the yellow fever mosquito Aedes aegypti, and its host of origin, Trichoplusia ni. This assay can confirm the potential utility of piggyBac as a gene transfer tool in a given insect species, and provides an experimental model for assessing molecular mechanisms of transposon movement. Received: 19 November 1998 / Accepted: 1 March 1999     

云南省4种伊蚊的乙型脑炎病毒分离物的研究

在云南省西南边境９县市捕获伊蚊属雌性成蚊１６种１９３６７只,用细胞法和乳鼠法分离病毒。从１８５批６４９１只白纹伊蚊中分离到病毒２株,从５０批１６０５只剌扰伊蚊中分离到病毒２株,从２３批７７２只窄翅伊蚊中分离到病毒２株,从４批１０３只阿萨姆伊蚊中分离到病毒１株。其它１２种共１０３９６只伊蚊的病毒分离物为阴性。分离到的７株病毒经免疫荧光、酶免疫、血凝抑制和中和试验鉴定,均为乙型脑炎病毒（ＪＥｖｉｒｕｓ）。白纹伊蚊是野外竹林的优势蚊种。分析认为白纹伊蚊在当地乙型脑炎病毒保存和传播中起重要作用,刺扰伊蚊、窄翅伊蚊和阿萨姆伊蚊亦可参与该病毒的传播。     

Nature of Predation Risk Cues in Container Systems: Mosquito Responses to Solid Residues From Predation

In aquatic systems, prey animals associate predation risk with cues that originate either from the predator or from injured conspecifics. Sources and benefits of these cues have received considerable attention in river, lake, and pond ecosystems but are less well understood in small container ecosystems that can hold less than a liter of water. Mosquitoes Aedes triseriatus (Say) and Aedes albopictus (Skuse) encounter predatory Corethrella appendiculata (Grabham) and Toxorhynchites rutilus (Coquillett) in small containers and show antipredatory behavioral responses. We investigated the sources of the predation cues to which these prey larvae respond. We tested whether Ae. albopictus larvae show behavioral responses to cues emanating from the predator or from damage to prey caused by the act of predation. We also tested whether Ae. triseriatus respond to cues present in fluid or solid residues from predator activity. Ae. albopictus showed behavioral modifications only in response to waterborne cues from a feeding predator and not to cues from a starving predator, indicating that Ae. albopictus respond to cues created by the act of predation, which could include substances derived from damaged prey or substances in predator feces. Ae. triseriatus showed behavioral responses to solid residues from predation but not to fluid without those solids, indicating that the cues to which they respond originate in predator feces or uneaten prey body parts. Our results suggest that cues in this system may be primarily chemicals that are detected upon contact with solid residues that are products of the feeding processes of these predators.     

Impacts of climate, land use, and biological invasion on the ecology of immature aedes mosquitoes: implications for la crosse emergence

Arthropod-borne viruses (arboviruses) cause many diseases worldwide and their transmission is likely to change with land use and climate changes. La Crosse virus (LACV) is historically transmitted by the native mosquito Aedes triseriatus (Say) in the upper Midwestern US, but the invasive congeners Aedes albopictus (Skuse) and A. japonicus (Theobald), which co-occur with A. triseriatus in water-holding containers, may be important accessory vectors in the Appalachian region where La Crosse encephalitis is an emerging disease. This review focuses on evidence for how climate, land use, and biological invasions may have direct abiotic and indirect community-level impacts on immature developmental stages (eggs and larvae) of Aedes mosquitoes. Because vector-borne diseases usually vary in space and time and are related to the ecology of the vector species, we propose that the ecology of its mosquito vectors, particularly at their immature stages, has played an important role in the emergence of La Crosse encephalitis in the Appalachian region and represents a model for investigating the effects of environmental changes on other vector-borne diseases. We summarize the health effects of LACV and associated socioeconomic costs that make it the most important native mosquito-borne disease in the US. We review of the transmission of LACV, and present evidence for the impacts of climate, land use, and biological invasions on Aedes mosquito communities. Finally, we discuss important questions about the ecology of LACV mosquito vectors that may improve our understanding of the impacts of environmental changes on LACV and other arboviruses.     

Germline transformation of Aedes fluviatilis (Diptera:Culicidae) with the piggyBac transposable element

The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.     

Effect of Novaluron (Rimon 10 EC) on the mosquitoes Anopheles albimanus, Anopheles pseudopunctipennis, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Chiapas, Mexico

Dengue fever is a serious problem in Mexico and vector control has not been effective enough at preventing outbreaks. Malaria is largely under control, but it is important that new control measures continue to be developed. Novaluron, a novel host-specific insect growth regulator and chitin synthesis inhibitor, has proved to be effective against agricultural pests, but its efficacy against larval mosquito vectors under field conditions remains unknown. In accordance with the World Health Organization Pesticide Evaluation Scheme, phase I, II and III studies were conducted to evaluate the efficacy and residual effect of Novaluron (Rimon 10 EC, Makhteshim, Beer-Sheva, Israel) on the malaria vectors Anopheles albimanus Wiedemann (Diptera: Culicidae) and Anopheles pseudopunctipennis Theobald, the dengue vectors Aedes aegypti (L) and Aedes albopictus Skuse and the nuisance mosquito Culex quinquefasciatus Say. Laboratory susceptibility tests yielded diagnostic concentrations for all five target species. Field trials to identify the optimum field dosage of Novaluron against Anopheles mosquitoes were carried out under semi-natural conditions in artificial plots and in vessels with wild mosquitoes. Efficacy was measured by monitoring mortality of larvae and pupae and the percentage of inhibition of emergence from floating cages. Dosages of Novaluron for field tests were based on pupal LC(99) (lethal concentration 99%) of An. pseudopunctipennis (0.166 mg/L) in plots and average pupal LC(99) of Ae. aegypti and Ae. albopictus (0.55 mg/L). At all dosages tested, Novaluron significantly reduced larval populations of An. albimanus, Culex coronator Dyar & Knab, Ae. albopictus and Cx. quinquefasciatus by approximately 90%, inhibited adult emergence of An. albimanus and An. pseudopunctipennis by approximately 97% for almost 4 months in experimental plots, and inhibited adult emergence of Ae. aegypti and Ae. albopictus by approximately 97% for up to 14 weeks. Recommended dosages of Novaluron for non-container breeding and container breeding mosquitoes are 0.166 mg/L and 0.55 mg/L, respectively. Overall, the residual effect was more sustained than that of temephos. The lowest dosage of Novaluron had less of an impact on non-target organisms than did temephos. Small-scale field trials in natural breeding sites treated with Novaluron at 0.6 L/ha eliminated adult emergence of An. albimanus and Cx. coronator for 8 weeks. For phase III studies, Novaluron was tested at the local and village levels, applying the optimum field rate to all natural breeding habitats within 1 km of a pair of neighbouring villages. Village-scale trials of Novaluron at 0.6 L/ha reduced An. albimanus larval populations for at least 8 weeks and, more importantly, sharply reduced the densities of adult host-seeking mosquitoes approaching houses. We conclude that Novaluron is effective and environmentally safer than temephos.     

Post-Integration Silencing of piggyBac Transposable Elements in Aedes aegypti

The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites.     

Timing of resource input and larval competition between invasive and native container-inhabiting mosquitoes (Diptera: Culicidae).

Container-inhabiting mosquito species are subject to both intraspecific and interspecific competition during larval development in resource-limited habitats. The arrival of an invasive species, Aedes albopictus, in the U.S. has altered competitive interactions among container-inhabiting mosquito species and, in some cases, has led to displacement of these species. Resource enrichment of container habitats has been shown to alleviate competitive interactions and to promote species co-existence; however, the importance of the timing of enrichment has yet to be explored. Larval competition between Ae. albopictus and a native species, Ochlerotatus triseriatus, was explored when resources were added either gradually or in a single pulse. Replacement series experiments revealed that Ae. albopictus was able to outcompete and displace Oc. triseriatus via resource monopolization when all resources were made available simultaneously; however, when the same resource amount was added over time, survival was high for both species, leading to co-existence. Timing of resource input also had an effect in monospecific treatments, indicating that intraspecific competition impacts survival as well. Duration of larval development was influenced by both species presence and by timing of resource input for Oc. triseriatus. These results indicate competitive outcome is condition-specific and that timing of resource input can determine whether a dominant invasive competitor displaces a native species, or if the two species are able to co-exist. Both intraspecific and interspecific competition occur at different temporal scales due to species-specific differences in larval developmental time. Timing of resource availability in container habitats can impact mosquito survival via competitive interactions, which can ultimately influence vector population size and behavior, possibly influencing vector-borne disease transmission.     

Generation of a novel Wolbachia infection in Aedes albopictus (Asian tiger mosquito) via embryonic microinjection

Genetic strategies that reduce or block pathogen transmission by mosquitoes are being investigated as a means to augment current control measures. Strategies of vector suppression and replacement are based upon intracellular Wolbachia bacteria, which occur naturally in many insect populations. Maternally inherited Wolbachia have evolved diverse mechanisms to manipulate host insect reproduction and promote infection invasion. One mechanism is cytoplasmic incompatibility (CI) through which Wolbachia promotes infection spread by effectively sterilizing uninfected females. In a prior field test, releases of Wolbachia-infected males were used to suppress a field population of Culex pipiens. An additional strategy would employ Wolbachia as a vehicle to drive desired transgenes into vector populations (population replacement). Wolbachia-based population suppression and population replacement strategies require an ability to generate artificial Wolbachia associations in mosquitoes. Here, we demonstrate a technique for transferring Wolbachia (transfection) in a medically important mosquito species: Aedes albopictus (Asian tiger mosquito). Microinjection was used to transfer embryo cytoplasm from a double-infected Ae. albopictus line into an aposymbiotic line. The resulting mosquito line is single-infected with the wAlbB Wolbachia type. The artificially generated infection type is not known to occur naturally and displays a new CI crossing type and the first known example of bidirectional CI in Aedes mosquitoes. We discuss the results in relation to applied mosquito control strategies and the evolution of Wolbachia infections in Ae. albopictus.