Laboratory colonization of Aedes japonicus japonicus (Diptera: Culicidae) collected in Narita, Japan and the biological properties of the established colony

A laboratory colony of the mosquito Aedes japonicus japonicus, which has recently invaded the United States and is recognized as a highly competent vector of West Nile virus, was established from larvae collected in Narita, Japan. The mosquitoes were maintained with induced insemination, blood-feeding on humans, and oviposition in water provided from the original collection site during the first few generations, then the colony was transferred to a large cage (40×40×100 cm in height) and adapted to conditions in which specimens were allowed to mate freely. White mice were provided as the blood source, and deionized water was available for oviposition. Approximately 185 eggs, most of which were tolerant to desiccation for at least 1 month, with some surviving for up to 2.5 months, were obtained per female following a single blood-feeding. The rate of successful emergence was nearly 90%, although this rate decreased significantly at high larval densities. The colony has been maintained for 5 years, and developmental profiles of the species have been obtained during that time.     

Laboratory and semi-field evaluation of Mosquito Dunks against Aedes aegypti and Aedes albopictus larvae (Diptera: Culicidae)

Laboratory bioassays and semi-field studies were conducted on the efficacy and longevity of Mosquito Dunks (7,000 ITU/mg Bti) in order to determine the concentration-response relationship and the effectiveness on the potency of the Bti product against Aedes mosquito species based on the WHO protocol standard methods and to determine the longevity of release for this product against Ae. aegypti mosquito larvae in water storage containers. This bio-potency study with the late 3rd instar larvae of Ae. aegypti and Ae. albopictus was carried out according to WHO standard protocols. The six concentrations of the Bti product used in each test were replicated 4 times with 25 mosquito larvae. Probit analysis was then used to determine the LC50 and LC95 which was equated with dosages of 1.02 and 1.86 ppm for Ae. aegypti; and 0.39 and 0.84 ppm for Ae. albopictus, which reveals a potency of 382.95 and 303.74 ITU/mg, respectively. The semi-field evaluation of this product in 200-liter earthen jars against 3rd instar larvae of Ae. aegypti showed satisfactory control of greater than 80% at 11 weeks post-treatment.     

Wolbachia uses host microRNAs to manipulate host gene expression and facilitate colonization of the dengue vector Aedes aegypti

The obligate endosymbiont Wolbachia pipientis is found in a wide range of invertebrates where they are best known for manipulating host reproduction. Recent studies have shown that Wolbachia also can modulate the lifespan of host insects and interfere with the development of human pathogens in mosquito vectors. Despite considerable study, very little is known about the molecular interactions between Wolbachia and its hosts that might mediate these effects. Using microarrays, we show that the microRNA (miRNA) profile of the mosquito, Aedes aegypti, is significantly altered by the wMelPop-CLA strain of W. pipientis. We found that a host miRNA (aae-miR-2940) is induced after Wolbachia infection in both mosquitoes and cell lines. One target of aae-miR-2940 is the Ae. aegypti metalloprotease gene. Interestingly, expression of the target gene was induced after Wolbachia infection, ectopic expression of the miRNA independent of Wolbachia, or transfection of an artificial mimic of the miRNA into mosquito cells. We also confirmed the interaction of aae-miR-2940 with the target sequences using GFP as a reporter gene. Silencing of the metalloprotease gene in both Wolbachia-infected cells and adult mosquitoes led to a significant reduction in Wolbachia density, as did inhibition of the miRNA in cells. These results indicate that manipulation of the mosquito metalloprotease gene via aae-miR-2940 is crucial for efficient maintenance of the endosymbiont. This report shows how Wolbachia alters the host miRNA profile and provides insight into the mechanisms of host manipulation used by this widespread endosymbiont.     

Laboratory colonization of Mansonia mosquitoes with an emphasis on Ma. annulata and Ma. bonneae

The present study records the first successful colonization of Mansonia annulata and describes colony maintenance with modification of rearing medium and host plants. Three species of Mansonia mosquitoes (Ma. uniformis, Ma. indiana and Ma annulifera) were successfully reared in ambient environments with adult emergence rates > 50%, while Ma. bonneae and Ma. dives yielded emergence rates > 30%. Colonization of Ma. annulata was modified and improved so that they were successfully raised to adult with emergence rates of 23%. Tube sedge, Lepironia articulata, was utilized as a host plant and peat swamp water was used as a rearing medium. Yeast and small lizard droppings were added daily to the larval medium to maintain microorganisms and pH in the infusion. However, identifying suitable culture medium remains an obstacle to establishing colonies of Ma. annulata, as the culture medium is difficult to mimic in the laboratory. Further study, focusing particularly on larval attachment substrates and rearing medium, is needed to develop a standardized and practical rearing technique for Mansonia mosquitoes.     

Laboratory studies of transovarial transmission of La Crosse and other arboviruses by Aedes albopictus and Culex fatigans.

Transovarial transmission of La Crosse virus by experimentally infected Aedes albopictus females to 2.7% of their F1 generation offspring was demonstrated. Progeny of both sexes were infected. Mean virus titers in parent mosquitoes and infected F1 generation adults were 10(4.6) and 10(3.4) plaque forming units/insect, respectively. The La Crosse-infected offspring were randomly distributed among the female parents. After two serial passages in A. albopictus, a marked change occurred in the plaque morphology of the virus but this had no apparent effect on the subsequent vertical transmission rate. In contrast, transovarial transmission did not occur in La Crosse-infected Culex fatigans or in A. albopictus and C. fatigans infected with vesicular stomatitis-Indiana, Cache Valley, Batai, Arumowot, and Itaporanga viruses. Results of this experiment suggest that the La Crosse model might be useful in studying the mechanism of transovarial transmission in additional mosquito species.     

Laboratory evaluation of Bacillus thuringiensis H-14 against Aedes aegypti larvae in the northeast region of Thailand.

Laboratory bioassays using a preparation of Bacillus thuringiensis H-14 (Bt.H-14), namely Skeetal were conducted to determine their effectiveness against late 3rd/early 4th instar larvae of Aedes aegypti. The larvae were collected from municipal areas in 7 provinces, namely Burirum, Roi-Et, Khon Kaen, Ubol Ratchatani, Nakorn Phanom, Surin and Nakorn Ratchasima, in the Northeast of Thailand. It was found that for Skeetal, LC50 ranged from 128 to 151 nl/l (average 143) and LC90 ranged from 254 to 289 nl/l (average 275). The mortality rate of Ae. aegypti larvae in the 7 provinces did not differ significantly (p greater than 0.05) at a concentration of 300 nl/l. The result of the bioassays show that the preparation of Bt.H-14 is very effective against Ae. aegypti larvae in Northeast of Thailand and the mosquito larvae in the various areas were nearly equal in susceptibility to Bt.H-14.     

Transovarial transmission of dengue viruses by mosquitoes: Aedes albopictus and Aedes aegypti

Transovarial transmission of all four dengue serotypes was demonstrated in Aedes albopictus mosquitoes. The rates of such transmission varied with the serotype and strain of virus. In general, the highest rates were observed with strains of dengue type 1 and the lowest with dengue type 3. Surprisingly, despite the use of viral strains of the four dengue serotypes which gave the highest rates with Ae. albopictus, transovarial transmission was observed in Aedes aegypti only with dengue type 1, and then only at a relatively low rate. Five different strains of Ae. aegypti were employed, including one that was known to be relatively susceptible to oral infection with dengue viruses. The findings support the view that Ae. aegypti, while of major importance from the point of view of transmission of dengue to man, may be relatively unimportant in the overall natural history of dengue viruses.     

Laboratory evaluation of a juvenile hormone mimic, pyriproxyfen on Culex quinquefasciatus Say and Aedes aegypti Linn. at Mysore, India

Insect growth regulator (IGR), pyriproxyfen is a juvenile hormone analogue used in stored product insect pest and vector control programmes. It can be considered as an alternative source to conventional insecticides because of its specific activity against immature insects, low persistence in the environment and virtually nontoxic to mammals. So, in our laboratory the effect of Pyriproxyfen was evaluated against the late 3rd instar larvae of Culex quinquefasciatus and Aedes aegypti to study the Concentration-mortality response and inhibition of adult emergence. The results showed that LC50 was 0.00084 ppm and 0.00166 ppm for Cx. quinquefasciatus and A. aegypti respectively. Toxicity of this insect compound extended till the adult emergence by inhibiting pupae formation up to 61.0% and 95.6% of adult emergence against Cx. quinquefasciatus. Likewise it affected 65.0% of pupal formation and 98.9% of adult emergence against A. aegypti. These results represent a promising strategy to use insect growth regulator, Pyriproxyfen for the instant, safe and successful improvement in the integrated vector control programme.     

A mosquito densovirus infecting Aedes aegypti and Aedes albopictus from Thailand.

A previously undescribed mosquito densovirus was detected in colonies of Aedes aegypti and Ae. albopictus from Thailand, using a polymerase chain reaction (PCR)-based assay. Phylogenetic analysis of this virus showed it to be most closely related to ADNV isolated from Russian Ae. aegypti. Both Aedes species were susceptible to oral infection with the Thai-strain virus. Larval mortality for Ae. albopictus was higher (82%) than for Ae. aegypti (51%). Aedes aegypti were able to transmit the virus vertically to a high (58%) proportion of G1 progeny, and the virus was maintained persistently for up to six generations. A PCR survey of adult Ae. aegypti and Ae. albopictus in Thailand indicated that only Ae. aegypti are infected in the field, with an overall prevalence of 44%. Densovirus infection in adult Ae. aegypti showed distinct seasonal variation. The Thai strain densovirus may play a role in structuring Ae. albopictus and Ae. aegypti populations in nature.     

Growth characteristics of ChimeriVax-Den vaccine viruses in Aedes aegypti and Aedes albopictus from Thailand

Four chimeric yellow fever (YF) 17D-dengue (DEN) candidate vaccine viruses (ChimeriVax-DEN; Acambis, Cambridge, MA) were characterized in Aedes aegypti and Ae. albopictus mosquitoes collected from Thailand. The four vaccine viruses contained the relevant prM and E genes of wild-type dengue viruses (DENV; serotypes 1-4) substituted for the equivalent genes in the YF vaccine virus (17D) backbone. Each chimera conferred protection against the homologous DENV serotype; a tetravalent mix of all four chimeras stimulates an immune response against all serotypes. Field-collected mosquitoes from Thailand were fed on blood containing each of the viruses under study and held 21 days after infection. Infection and dissemination rates were based on antigen detection in the body or head tissues, respectively. All four wild-type DENV serotypes infected and disseminated, but the candidate vaccine viruses were highly attenuated in mosquitoes with respect to infection and especially with respect to dissemination. Considering the low level viremias anticipated in humans vaccinated with these viruses, it is predicted that the risks of infection and transmission by mosquitoes in nature is minimal.     

Growth characteristics of ChimeriVax-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes

The chimeric yellow fever (YF) 17D-dengue type 2 (ChimeriVax-DEN2) vaccine virus developed by Acambis, Inc. (Cambridge, MA) contains the prM and E genes of wild-type (wt) dengue 2 (DEN-2) (strain PUO-218) virus in the YF vaccine virus (strain 17D) backbone. The potential of ChimeriVax-DEN2 virus to infect and be transmitted by Aedes aegypti, the principal DEN and YF virus mosquito vector, and Aedes albopictus, a species that occurs in areas of active transmission of YF and DEN viruses, was evaluated. Mosquitoes were intrathoracically (IT) inoculated with virus or were fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN2 were compared with the wt DEN-2 and YF 17D vaccine viruses. Replication of YF 17D virus is attenuated in cultured Ae. albopictus C6/36 mosquito cells and in Ae. aegypti and Ae. albopictus mosquitoes. Growth of ChimeriVax-DEN2 virus similarly was restricted in C6/36 cells and in mosquitoes. ChimeriVax-DEN2 replicated in 56% of IT inoculated Ae. aegypti, and virus disseminated to head tissue in 36%, with a mean viral titer of 1.8 log10 PFU/mosquito. Of mosquitoes, 16% of Ae. aegypti and 24% of Ae. albopictus were infected 14 days after a blood meal containing ChimeriVax-DEN2, but virus did not disseminate to head tissue. In contrast, DEN-2 replicated in all IT inoculated and orally infected Ae. aegypti (mean titer 5.5 log10 PFU/mosquito), and virus disseminated to head tissue in 95%. Of Ae. albopictus, 84% were infected after a blood meal containing DEN-2 virus; dissemination occurred in 36%. Replication of ChimeriVax-DEN2 virus in mosquitoes corresponded to that of YF 17D vaccine virus, which is restricted in its ability to infect and replicate in mosquitoes. Therefore, transmission of ChimeriVax-DEN2 virus by vector mosquitoes is unlikely.     

Laboratory and scaled up evaluation of cis-permethrin applied as a new ultra low volume formulation against Aedes aegypti (Diptera: Culicidae)

Ultra low volume (ULV) aerial spraying is a common methodology for spatial treatments for Aedes aegypti (L.) control. Previous studies from our laboratory indicated that the cis isomer of permethrin has an excellent efficacy for vector control, in particular for Triatoma infestans. It was of interest to determine the efficacy of this pure isomer in a ULV water-based formulation for adult A. aegypti control, since both vectors usually are found together in South America. A method is presented to make a quick measure at laboratory level of the knock down effect (KT50) of a new EC water-based formulation of cis-permethrin, in a small Peet Grady chamber of 0.34 m3, with a specially designed glass sprayer. A deltamethrin standard ULV formulation "CISLIN" was used for comparison. cis-Permethrin showed a significantly lower KT50 value than deltamethrin (7.50 and 9.65 min, respectively). When the mosquitoes were introduced into the chamber 10 min after spraying KT50 values were almost equal (15.59 and 15.88 min, respectively). In scaled up bioassays the ULV formulation of cis-permethrin showed 100% mortality of A. aegypti adult mosquitoes at the beginning of the treatment (t=0) and some mortality at 1h post treatment of freshly introduced mosquitoes. It was concluded that cis-permethrin could be an excellent tool for control of A. aegypti and other vectors of medical importance, with fewer side effects and better cost-effectiveness than with the cyanopyrethroids.     

Fungal colonization of the esophagus

A prospective study was conducted in 224 patients to determine the clinical significance of esophageal colonization with yeasts under different conditions. In accordance with the results of direct smear microscopic examination and culture of esophageal brushings, patients were divided into three groups: positive, negative, and the patients, in whom saprophytic forms were detected. A higher prevalence of positive findings was noted in patients with predisposing factors for yeast invasion than in patients free of underlying disease. Eleven percent of patients with an endoscopically normal appearing esophagus were positive. We have the impression that this situation may represent a preclinical condition of fungal esophagitis. Patients treated with H2-blocking agents showed a significantly higher incidence of positive findings, than did those without such treatment. Whether patients suffering from a refluxesophagitis resistant to long-term treatment with H2 blockers, but with a significant colonization by yeasts, could benefit by an additional treatment with antimycotics remains a controversial issue and should be studied in a controlled way.