Infection Incidence and Relative Density of the Bacteriophage WO-B in Aedes albopictus Mosquitoes from Fields in Thailand

We have used real-time quantitative PCR to measure, for the first time, the relative phage WO-B orf7 density and infection incidence in Aedes albopictus mosquitoes from fields in Thailand. Our results showed that the infection incidence of phage WO-B in this mosquito, sampled from geographically different places in Thailand, was 97.9%. Average relative densities of the offspring were different when collected from diverse parts and reared under the same conditions in the laboratory. Our results also revealed that geographical differences within Thailand did not influence the maternal transmission rate of bacteriophage WO-B. In addition, the orf7 loci might not be strictly associated with Wolbachia, because less than 100% of them were maternally inherited. This discovery does not support the hypothesis that bacteriophage WO-B is involved in Aedes albopictus' cytoplasmic incompatibility. Whether this bacteriophage actually is involved in Wolbachia-induced cytoplasmic incompatibility in this mosquito thus needs further investigation, and additional densities of phage WO-B loci should be integrated.     

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections.     

Bacteriophage WO-B and Wolbachia in natural mosquito hosts: infection incidence, transmission mode and relative density

Bacteriophages of Wolbachia bacteria have been proposed as a potential transformation tool for genetically modifying mosquito vectors. In this study, we report the presence of the WO-B class of Wolbachia-associated phages among natural populations of several mosquito hosts. Eighty-eight percent (22/25) of Wolbachia-infected mosquito species surveyed were found to contain WO-B phages. WO-B phage orf7 sequence analysis suggested that a single strain of WO-B phage was found in most singly (23/24) or doubly (1/1) Wolbachia-infected mosquitoes. However, the single Wolbachia strain infecting Aedes perplexus was found to harbour at least two different WO-B phages. Phylogenetic analysis suggested that horizontal transmission of WO-B phages has occurred on an evolutionary scale between the Wolbachia residing in mosquitoes. On an ecological scale, a low trend of co-transmission occurred among specific WO-B phages within Wolbachia of each mosquito species. Assessment of the density of WO-B phage by real-time quantitative polymerase chain reaction (RTQ-PCR) revealed an average relative density of 7.76 x 10(5)+/- 1.61 x 10(5) orf7 copies per individual mosquito for a single Wolbachia strain infecting mosquitoes, but a threefold higher density in the doubly Wolbachia-infected Aedes albopictus. However, the average combined density of WO-B phage(s) did not correlate with that of their Wolbachia hosts, which varied in different mosquito species. We also confirmed the presence of WO-B-like virus particles in the laboratory colony of Ae. albopictus (KLPP) morphologically, by transmission electron microscopy (TEM). The viral-like particles were detected after purification and filtration of Ae. albopictus ovary extract, suggesting that at least one WO-B-like phage is active (temperate) within the Wolbachia of this mosquito vector. Nevertheless, the idea of utilizing these bacteriophages as transformation vectors still needs more investigation and is likely to be unfeasible.     

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.     

Effects of temephos and temperature on Wolbachia load and life history traits of Aedes albopictus

Maternally inherited Wolbachia (gram-negative bacteria) often affect the reproductive fitness of their arthropod hosts and may cause cytoplasmic incompatibility (CI). Comparing Wolbachia-infected and uninfected strains of the mosquito Aedes albopictus (Skuse) (Diptera: Culicidae), we assessed the effects on fitness of two stressors: temperature elevation (25 degrees C vs. 37 degrees C) and exposure to temephos insecticide (concentration range 0.0017-0.0167 mg/L) during larval development. Fitness was measured in terms of life history traits: percentage survival, development time and wing size. Insecticide treatment was associated with reduction in survival rates and wing size in both sexes, but did not affect development time or Wolbachia load. Temperature elevation by 12 degrees C significantly reduced all four bionomic parameters observed in both sexes. Wolbachia density within individual adult mosquitoes was determined by using real-time quantitative polymerase chain reaction (PCR) based on the wsp gene. Both male and female adults had significantly lower densities of Wolbachia after larval rearing at the higher temperature.     

我国蚊虫体内感染的Wolbachia的wsp基因序列测定与分析

测定了我国尖音库蚊复合组和白纹伊蚊蚊虫体内感染的Wolbachia株的wsp基因序列。核苷酸和氨基酸的同源性及系统关系分析表明,我国尖音库蚊复合组和白纹伊蚊中Wolbachia株的wsp基因序列与Pip组其它株的核苷酸及氨基酸同源性分别为98%～100%和97%～100%, 属B大组Wolbachia中的Pip组。     

Interspecific transfer of Wolbachia into the mosquito disease vector Aedes albopictus

Intracellular Wolbachia bacteria are obligate, maternally inherited endosymbionts found frequently in insects and other invertebrates. The evolutionary success of Wolbachia is due in part to an ability to manipulate reproduction. In mosquitoes and many other insects, Wolbachia causes a form of sterility known as cytoplasmic incompatibility (CI). Wolbachia-induced CI has attracted interest as a potential agent for affecting medically important disease vectors. However, application of the approach has been restricted by an absence of appropriate, naturally occurring Wolbachia infections. Here, we report the interspecific transfer of Wolbachia infection into a medically important mosquito. Using embryonic microinjection, Wolbachia is transferred from Drosophila simulans into the invasive pest and disease vector: Aedes albopictus (Asian tiger mosquito). The resulting infection is stably maintained and displays a unique pattern of bidirectional CI in crosses with naturally infected mosquitoes. Laboratory population cage experiments examine a strategy in which releases of Wolbachia-infected males are used to suppress mosquito egg hatch. We discuss the results in relation to developing appropriate Wolbachia-infected mosquito strains for population replacement and population suppression strategies.     

Wolbachia strain wAlbB enhances infection by the rodent malaria parasite Plasmodium berghei in Anopheles gambiae mosquitoes

Wolbachia, a common bacterial endosymbiont of insects, has been shown to protect its hosts against a wide range of pathogens. However, not all strains exert a protective effect on their host. Here we assess the effects of two divergent Wolbachia strains, wAlbB from Aedes albopictus and wMelPop from Drosophila melanogaster, on the vector competence of Anopheles gambiae challenged with Plasmodium berghei. We show that the wAlbB strain significantly increases P. berghei oocyst levels in the mosquito midgut while wMelPop modestly suppresses oocyst levels. The wAlbB strain is avirulent to mosquitoes while wMelPop is moderately virulent to mosquitoes pre-blood meal and highly virulent after mosquitoes have fed on mice. These various effects on P. berghei levels suggest that Wolbachia strains differ in their interactions with the host and/or pathogen, and these differences could be used to dissect the molecular mechanisms that cause interference of pathogen development in mosquitoes.     

Bacterial diversity of field-caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar

Symbiotic bacteria are known to play important roles in the biology of insects, but the current knowledge of bacterial communities associated with mosquitoes is very limited and consequently their contribution to host behaviors is mostly unknown. In this study, we explored the composition and diversity of mosquito-associated bacteria in relation with mosquitoes' habitats. Wild Aedes albopictus and Aedes aegypti were collected in three different geographic regions of Madagascar. Culturing methods and denaturing gradient gel electrophoresis (DGGE) and sequencing of the rrs amplicons revealed that Proteobacteria and Firmicutes were the major phyla. Isolated bacterial genera were dominated by Bacillus, followed by Acinetobacter, Agrobacterium and Enterobacter. Common DGGE bands belonged to Acinetobacter, Asaia, Delftia, Pseudomonas, Enterobacteriaceae and an uncultured Gammaproteobacterium. Double infection by maternally inherited Wolbachia pipientis prevailed in 98% of males (n=272) and 99% of females (n=413); few individuals were found to be monoinfected with Wolbachia wAlbB strain. Bacterial diversity (Shannon-Weaver and Simpson indices) differed significantly per habitat whereas evenness (Pielou index) was similar. Overall, the bacterial composition and diversity were influenced both by the sex of individuals and by the environment inhabited by the mosquitoes; the latter might be related to both the vegetation and the animal host populations that Aedes used as food sources.     

High Wolbachia density correlates with cost of infection for insecticide resistant Culex pipiens mosquitoes

In the mosquito Culex pipiens, insecticide resistance genes alter many life-history traits and incur a fitness cost. Resistance to organophosphate insecticides involves two loci, with each locus coding for a different mechanism of resistance (degradation vs. insensitivity to insecticides). The density of intracellular Wolbachia bacteria has been found to be higher in resistant mosquitoes, regardless of the mechanism involved. To discriminate between costs of resistance due to resistance genes from those associated with elevated Wolbachia densities, we compared strains of mosquito sharing the same genetic background but differing in their resistance alleles and Wolbachia infection status. Life-history traits measured included strength of insecticide resistance, larval mortality, adult female size, fecundity, predation avoidance, mating competition, and strength of cytoplasmic incompatibility (CI). We found that: (1) when Wolbachia are removed, insecticide resistance genes still affect some life-history traits; (2) Wolbachia are capable of modifying the cost of resistance; (3) the cost of Wolbachia infections increases with their density; (4) different interactions occurred depending on the resistance alleles involved; and (5) high densities of Wolbachia do not increase the strength of CI or maternal transmission efficiency relative to low Wolbachia densities. Insecticide resistance genes generated variation in the costs of Wolbachia infections and provided an interesting opportunity to study how these costs evolve, a process generally operating when Wolbachia colonizes a new host.     

Phylogeography of Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) based on mitochondrial DNA variations

Aedes (Stegomyia) aegypti (l.) and Aedes (Stegomyia) albopictus (Skuse) are the most important vectors of the dengue and yellow-fever viruses. Both took advantage of trade developments to spread throughout the tropics from their native area: A. aegypti originated from Africa and a. albopictus from South-East Asia. We investigated the relationships between A. aegypti and A. albopictus mosquitoes based on three mitochondrial-DNA genes (cytochrome b, cytochrome oxidase I and NADH dehydrogenase subunit 5). Little genetic variation was observed for a. albopictus, probably owing to the recent spreading of the species via human activities. For A. aegypti, most populations from South America were found to be genetically similar to populations from South-East Asia (Thailand and Vietnam), except for one sample from Boa Vista (northern Amazonia), which was more closely related to samples from Africa (Guinea and Ivory Coast). This suggests that African populations of A. aegypti introduced during the slave trade have persisted in Boa Vista, resisting eradication campaigns.     

Wolbachia and cytoplasmic incompatibility in mosquitoes

Wolbachia are maternally inherited bacteria that induce cytoplasmic incompatibility in mosquitoes, and are able to use these patterns of sterility to spread themselves through populations. For this reason they have been proposed as a gene drive system for mosquito genetic replacement, as well as for the reduction of population size or for modulating population age structure in order to reduce disease transmission. Here, recent progress in the study of mosquito Wolbachia is reviewed. We now have much more comprehensive estimates of the parameters that can affect the spread of Wolbachia through natural populations from low starting frequencies, and for waves of spread to be maintained in the face of partial barriers to gene flow. In Aedes albopictus these dynamics are extremely favourable, with very high maternal transmission fidelity and levels of incompatibility recorded. Correspondence between measurements taken in the lab and field is much better than in the Drosophila simulans model system. Important research goals are also discussed, including Wolbachia transformation, interspecific transfer and the elucidation of the mechanisms of incompatibility and rescue; all will be aided by a wealth of new Wolbachia genome information.     

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。     

中华按蚊和白纹伊蚊接种马来丝虫微丝蚴后酚氧化酶活性的变化(英文)

酚氧化酶在昆虫抵抗外来异物黑化和成囊的免疫反应中起重要作用。中华按蚊和白纹伊蚊分别经胸内接种马来丝虫微丝蚴和生理盐水后,测定蚊血淋巴中酚氧化酶活性。结果表明:未接种的两种蚊,其1日龄蚊的酚氧化酶活性显著高于3、12和16日龄蚊。1日龄中华按蚊接种微丝蚴24h后,其酚氧化酶活性比未接种蚊和接种生理盐水蚊高2—3倍。1日龄白纹伊蚊接种微丝蚴24h后,其酚氧化酶活性是未接种蚊的7倍。白纹伊蚊酚氧化酶活性的免疫激活水平显著高于中华按蚊。讨论了中华按蚊和白纹伊蚊酚氧化酶活性的差异与其免疫力差异的关系。     

Development of a novel sticky trap for container-breeding mosquitoes and evaluation of its sampling properties to monitor urban populations of Aedes albopictus

Collection methods currently used for large-scale sampling of adult Stegomyia mosquitoes (Diptera: Culicidae) present several operational limitations, which constitute major drawbacks to the epidemiological surveillance of arboviruses, the evaluation of the impact of control strategies, and the surveillance of the spreading of allochthonous species into non-endemic regions. Here, we describe a new sticky trap designed to capture adult container-breeding mosquitoes and to monitor their population dynamics. We tested the sampling properties of the sticky trap in Rome, Italy, where Aedes (Stegomyia) albopictus is common. The results of our observations, and the comparison between sticky trap catches and catches made with the standard oviposition trap, are presented. The sticky trap collected significantly larger numbers of Ae. albopictus females than any other Culicidae species representing >90% of the total catches. A maximum of 83 An. albopictus females was collected in a single week. A high correlation (Pearson correlation coefficient r= 0.96) was found between the number of females and the number of eggs collected by the traps. The functional relationship between the number of eggs and the number of adult females was assessed by major axis regression fitted to log(1 +x)-transformed trap counts as y= 0.065 + 1.695x. Trap samples significantly departed from a random distribution; Taylor's power law was fitted to the trap samples to quantify the degree of aggregation in the catches, returning the equations s(2)= 2.401 m(1.325) for the sticky trap and s(2)= 13.068 m(1.441) for the ovitrap, with s(2) and m denoting the weekly catch variance and mean, respectively, indicating that eggs were significantly more aggregated than mosquitoes (P < 0.0001). Taylor's power law parameters were used to estimate the minimum number of sample units necessary to obtain sample estimates with a fixed degree of precision and sensitivity. For the range of densities encountered in our study area during the Ae. albopictus breeding season, the sticky trap was more precise and sensitive than the ovitrap. At low population densities (c. < 0.1 mosquito/trap), however, the ovitrap was more sensitive at detecting the presence of this species. Overall, our results indicate that our new model of sticky trap can be used to sample Ae. albopictus females in urban environments, and, possibly, other container-breeding Stegomyia mosquitoes (e.g. Aedes aegypti). The technical properties of the new trap are discussed with respect to its possible application in monitoring the population dynamics of container-breeding mosquitoes, in studying their bionomics, and in vector surveillance and, possibly, control.     

Trend of temephos resistance in Aedes (Stegomyia) mosquitoes in Thailand during 2003-2005

The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae. aegypti mosquitoes of one collection site each in Bangkok, Nakhon Sawan (northcentral), and Nakhon Ratchasrima (northeast) were resistant to temephos, with mortality ranging from 50.5 to 71.4%. Moreover, there was a trend of resistance to temephos among Ae. aegypti populations of all studied districts of Nakorn Ratchasima and most areas of Nakhon Sawan, of which those in one area were susceptible. However, various levels of temephos susceptibility were found in Bangkok populations, including resistance and incipient resistance. In Chonburi Province (eastern), all mosquitoes were susceptible to temephos with an indication of tolerance in one sample. Additionally, mosquitoes from Songkhla (south), Chiang Rai (north), Kanchanaburi (west), and Chanthaburi (east) remained susceptible to temephos during the sample collecting period. Bioassay tests on Aedes albopictus populations collected in this study from Nakhon Sawan, Nakorn Ratchasima, Songkhla, and Kanchanaburi revealed high susceptibility to temephos. Although the use of temephos seems to be potentially effective in many areas of the country, a noticeable trend of resistance indicated that alternative vector control methods should be periodically applied.