Molecular studies with Aedes (Stegomyia) aegypti (Linnaeus, 1762), mosquito transmitting the dengue virus

Parasitology Research - Dengue is an infectious viral disease, which can present a wide clinical picture, ranging from oligo or asymptomatic forms, to bleeding and shock, and can progress to death....     

Comparative analysis of midgut bacterial communities of Aedes aegypti mosquito strains varying in vector competence to dengue virus

Differences in midgut bacterial communities of Aedes aegypti, the primary mosquito vector of dengue viruses (DENV), might influence the susceptibility of these mosquitoes to infection by DENV. As a first step toward addressing this hypothesis, comparative analysis of bacterial communities from midguts of mosquito strains with differential genetic susceptibility to DENV was performed. 16S rRNA gene libraries and real-time PCR approaches were used to characterize midgut bacterial community composition and abundance in three Aedes aegypti strains: MOYO, MOYO-R, and MOYO-S. Although Pseudomonas spp.-related clones were predominant across all libraries, some interesting and potentially significant differences were found in midgut bacterial communities among the three strains. Pedobacter sp.- and Janthinobacterium sp.-related phylotypes were identified only in the MOYO-R strain libraries, while Bacillus sp. was detected only in the MOYO-S strain. Rahnella sp. was found in MOYO-R and MOYO strains libraries but was absent in MOYO-S libraries. Both 16S rRNA gene library and real-time PCR approaches confirmed the presence of Pedobacter sp. only in the MOYO-R strain. Further, real-time PCR-based quantification of 16S rRNA gene copies showed bacterial abundance in midguts of the MOYO-R strain mosquitoes to be at least 10–100-folds higher than in the MOYO-S and MOYO strain mosquitoes. Our study identified some putative bacteria with characteristic physiological properties that could affect the infectivity of dengue virus. This analysis represents the first report of comparisons of midgut bacterial communities with respect to refractoriness and susceptibility of Aedes aegypti mosquitoes to DENV and will guide future efforts to address the potential interactive role of midgut bacteria of Aedes aegypti mosquitoes in determining vectorial capacity for DENV.     

Detection of insecticide resistance in Aedes aegypti (Diptera: Culicidae) from Cuba and Venezuela

Four strains of Aedes aegypti (L.), one from Cuba and three from Venezuela, were bioassayed for susceptibility to eight insecticides, including the organophosphates, temephos, malathion, fenthion, pirimiphos methyl, and chlorpyrifos, and the pyrethroids, deltamethrin, lambda cyhalothrin and cypermethrin, S, S, S,-tributyl phosphorotrithioate and piperonyl butoxide were used as synergists to assess the involvement of esterases and monooxygenases in organophosphate resistance. Venezuelan strains had low levels of resistance to fenthion and malathion, and moderate to high resistance to temephos, pyrimphos methyl, and chlorpiriphos. All strains were susceptible to the pyrethroids, except the Cuban strain, which had moderate levels of resistance to cypermethrin. Organophosphate resistance in Ae. aegypti is a serious threat to control operations. Integrated strategies for Ae. aegypti control to prevent or delay pyrethroid resistance in Venezuela and Cuba are discussed.     

Laboratory evaluation of the response of Aedes aegypti and Aedes albopictus uninfected and infected with dengue virus to deet

Laboratory studies were conducted to compare the response of Aedes aegypti (L.) and Aedes albopictus (Skuse) adults, uninfected and infected with four serotypes of dengue virus to a repellent containing 5% N, N-diethyl-3-methylbenzamide (deet). The results showed that mosquitoes infected with the four serotypes of dengue responded similarly to uninfected mosquitoes.     

Studies of the time of effectiveness and the characterization of compounds in insecticide tests with Aedes aegypti]

The contact-toxicity of 5 insecticides (2 chlorinated hydrocarbons, 2 organophosphate compounds 1 P-substance) are tested against Aedes aegypti L (adults). Because of the relationship between dosage (quantity of application) and time of effectiveness of a compound, we are able to design a specific curve of time-efficacy of each insecticide at graded intervals (= increasing dilutions). This curve may be characteristic of different pure compounds--possibly in connection with other features. The time measurement as a quantitative response was chosen according to the directions of Finney 1952 (mean time to response--dosage). The simple statistical analysis was possible after continuous observation until response-metameter as criterion for time of bioassay = time of exposure. Transformation into complete records (Finney) in feasible with a long period of observation, corresponding response-metameter, extreme values etc. This was found to be the only way to obtain a statistical analysis without complications, and only a few parameters in this model. For statistical evaluation of the differences of the mean reaction times for each dilution of DDT, gamma-BHC, Methylparathion and Trichlorofon the U-test according to Mann & Whitney has been applied. Percentages of 'inactivity' at each dilution are added (decreasing from D7). --The curve of time--efficacy is described, and the possibilities of characterization and indentification of the compounds discussed. General features of ET 50 values and mean reaction times are pointed out, and also the relation of time to response (= exposure time) and dosage, expressed by a simple hyperbolic equation (Haber's formula). The uptake of insecticides by "self-dosine" of insects (as practised in this bioassay) is the closest approximation to natural conditions, but the real dose is unknown [and will depend on the physiological condition of the insects]. Some values with Aedes aegypti (whole activity of the pure compound of each test object labelled with radioactive Trichlorfon) are added (exposure on 1 per cent deposite of insecticide).     

登革病毒在白纹伊蚊细胞内免疫的研究

目的 研究白纹伊蚊对登革病毒产生细胞内免疫现象的分子机制。方法 扩增与克隆登革病毒NGC株的前膜蛋白基因prM，将prM基因以3种不同表达形式重组入昆虫表达载体pBh-spEGFP，以3种重组质粒分别转染白纹伊蚊C6／36细胞，测定转染后的细胞对登革病毒感染的免疫效果。结果 构建了包含prM基因的3种昆虫表达载体，Western blot和荧光显微镜观察证明在C6／36细胞中成功表达了prM-EGFP融合蛋白。MTT实验和空斑形成实验反映了C6／36细胞对登革病毒产生了细胞内免疫。结论 3种重组质粒均可诱导细胞内免疫现象，说明无论是否表达prM蛋白，只要有prM基因转录就可引起细胞内免疫现象；prM基因正义和反义转录形式均可诱导细胞内免疫；其形成机制可能是通过RNA干扰作用而产生。     

Vector competence of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) for dengue virus in the Florida Keys

In 2009-2011, Monroe County in southern Florida experienced locally acquired and traveler-imported focal dengue outbreaks. Aedes aegypti (L.) is the primary vector of dengue virus (DENV) worldwide, is prevalent in Monroe County, and is the suspected vector in Florida. Ae. albopictus (Skuse) is also known to be an important vector of DENV and this species is ubiquitous in Florida; however, it is not yet established in Monroe County. Florida Ae. aegypti (Key West and Stock Island geographic colonies) and Ae. albopictus (Vero Beach geographic colony) were fed blood containing 3.7 Log10 plaque-forming unit equivalents of DENV serotype 1 isolated from a patient involved in the Key West, FL, outbreak in 2010. Mosquitoes were maintained at extrinsic incubation temperatures of 28 or 30 degrees C for an incubation period of 14 d. Vector competence was assessed using rates of infection (percent with virus-positive bodies), dissemination (percent infected with virus-positive legs), and transmission (percent infected with virus-positive saliva). No significant differences were observed in rates of infection or dissemination between Ae. aegypti or Ae. albopictus at either extrinsic incubation temperature. Transmission was observed only at 28 degrees C in both Ae. aegypti (Key West) and Ae. albopictus. The assessment of local mosquito populations for their DENV vector competence is essential and will aid mosquito control operators interested in pinpointing specific vector populations for control. The extent to which vector competence is affected by seasonal changes in temperature is discussed and provides baseline risk assessment data to mosquito control agencies.     

Comparative laboratory study on the reaction of Aedes aegypti and Aedes albopictus to different attractive cues in a mosquito trap

The behavioral responses of Aedes aegypti (L.) and Aedes albopictus (Skuse) adults to several attractive cues, as reactions to mosquito traps, are compared in the laboratory, and differences in the primary attractive factors for both species are discussed. Target-attacking frequency of unfed Ae. aegypti females was >30 times that of unfed Ae. albopictus females under simulated conditions. Changes in the percentage of trapped mosquitoes under several attractive conditions using commercial mosquito traps showed that Ae. aegypti mosquitoes were trapped 2-3 times faster than Ae. albopictus. For Ae. aegypti, the combination of a visual cue + CO, alone enhanced attractiveness, whereas both a visual cue + CO, as well as a visual cue + octenol enhanced Ae. albopictus. The combination of at least three factors, such as a visual cue, CO2, and a chemical cue is thought to be valuable for trapping and estimating the relative adult population sizes of Ae. aegypti and Ae. albopictus     

A novel coding-region RNA element modulates infectious dengue virus particle production in both mammalian and mosquito cells and regulates viral replication in Aedes aegypti mosquitoes

Dengue virus (DENV) is an enveloped flavivirus with a positive-sense RNA genome transmitted by Aedes mosquitoes, causing the most important arthropod-borne viral disease affecting humans. Relatively few cis-acting RNA regulatory elements have been described in the DENV coding-region. Here, by introducing silent mutations into a DENV-2 infectious clone, we identify the conserved capsid-coding region 1 (CCR1), an RNA sequence element that regulates viral replication in mammalian cells and to a greater extent in Ae. albopictus mosquito cells. These defects were confirmed in vivo, resulting in decreased replication in Ae. aegypti mosquito bodies and dissemination to the salivary glands. Furthermore, CCR1 does not regulate translation, RNA synthesis or virion retention but likely modulates assembly, as mutations resulted in the release of non-infectious viral particles from both cell types. Understanding the role of CCR1 could help characterize the poorly-defined stage of assembly in the DENV life cycle and uncover novel anti-viral targets.     

Studies on the behavior of arboviruses in an Aedes aegypti mosquito cell line (Peleg)

Summary Aedes aegypti mosquito cell cultures inoculated with eastern equine encephalitis (EEE) virus, yielded at the height of infection about 50% virus-producing cells, and a calculated amount of 600 PFU per infected cell, yet CPE was not observed. Cultures co-infected with EEE and Semliki Forest (SF) viruses supported the proliferation of both viruses, but no CPE or other evidence for the enhancement of infection was manifested. The apparent absence of doubly infected cells in cultures exposed simultaneously to EEE and SF viruses suggested that the two viruses were proliferating in different target cells.This investigation was supported by Research Grant 06-325-01 from the US Public Health Service, Bureau of State Services.     

Genetic differentiation of Aedes aegypti (Diptera: Culicidae), the major dengue vector in Brazil

In 2000, Brazil reported 180,137 cases of dengue, approximately 80% of the total in the Americas. However, little is known about gene flow among the vector populations in Brazil. Random amplified polymorphic DNA (RAPD) was used to study the genetic structure of Aedes aegypti in 15 populations from five states, with a range extending 2,800 km. An analysis of 47 polymorphic RAPD loci estimated gene flow at the macro- (different states) and micro- (different cities) geographical levels. Genetic polymorphism was high (H(S) = 0.274), and high levels of genetic differentiation existed both between different states (G(ST) = 0.317) and between cities or neighborhoods in each state (G(ST) = 0.085-0.265). These values are higher than those described for any other populations of A. aegypti.     

Reinvestigation of an endogenous meiotic drive system in the mosquito, Aedes aegypti (Diptera: Culicidae)

We have initiated efforts to determine the molecular basis for the M(D) meiotic drive system in the mosquito, Aedes aegypti. The effect of the M(D) gene is a highly male-biased sex ratio, but varies depending on the frequency and sensitivity of a susceptible responder m(s) allele. The M(D) system has potential as a mechanism for driving trangenes for pathogen resistance into natural Ae. aegypti populations. Because all previously existing laboratory strains carrying the M(D) gene have been lost, we have selected for a new strain, T37, that carries a strong driver. Matings between T37 males and drive-susceptible m(s) females result in progeny with highly biased sex ratios, wherein only approximately 14.7% females are produced. We discuss the potential for identifying M(D) candidate genes based on comparisons with the well-described Drosophila melanogaster segregation distorter (SD) meiotic drive system and considerations for release of transgenic Ae. aegypti into natural populations where M(D) and insensitive m3 alleles are likely segregating.     

Identification of genomic changes associated with cisplatin resistance in testicular germ cell tumor cell lines

Since the introduction of cisplatin into the clinic, the treatment of patients with a variety of solid tumors including testicular germ cell tumors, ovarian and lung cancers, has dramatically improved. One of the main causes for therapeutic failure in these malignancies is the development of drug resistance. Testicular germ cell tumors (TGCTs), the most common malignancy in young men, exhibit extreme sensitivity to cisplatin-based chemotherapy, making them an ideal model for investigating the mechanisms of cisplatin chemo-sensitivity and resistance. TGCT development and pathogenesis have been well studied but little is known about the genetic background in chemo-resistant cases. We investigated genomic differences between three TGCT parental cell lines and their cisplatin resistant derivatives. Using 10K single nucleotide polymorphism (SNP) microarray analysis, we identified two small chromosomal regions with consistent copy number changes across all three pairs of resistant cell lines. These were an 8.7 Mb region at 6q26-27, which displayed consistent copy number gain and a 0.3 Mb deletion involving 4 SNPs at 10p14. Both the chromosomal gain and loss were confirmed by fluorescence in situ hybridization. The significance of these regions should be further investigated as they may contain key genes involved in the development of chemo- resistance to cisplatin-based treatment in TGCTs and other cancers.