Larval environmental stress alters Aedes aegypti competence for Sindbis virus

Objective To evaluate how stress at the larval stage alters adult mosquito performance and susceptibility to viral infection. Methods We used a model system consisting of Sindbis virus (SINV) and the yellow fever mosquito Aedes aegypti. Larvae were either reared under optimal conditions (control) or exposed to one of four types of stressors; suboptimal nutrients, starvation, elevated temperature, and a low dose of the insecticide malathion and adult females were fed SINV infectious blood meal. Differential expressions of stress, immune‐specific and detoxification genes was measured in fourth instar larvae (HSP70, HSP83, cecropin, defensin, transferrin and CYP6Z6) and 3‐day‐old females (cecropin, defensin, transferrin) to identify plausible molecular mechanisms associated with mosquito response to stress. Results There were stress‐specific variations in mosquito performance (survival, development time, female size), but all stressors had a consistent effect of significantly increasing susceptibility to viral infection and dissemination relative to the controls. Three genes were up‐regulated in fourth instar larvae exposed to temperature stress (cecropin, defensin and CYP6Z6) compared to single genes in suboptimal nutrient (cecropin) and malathion (transferrin) stress treatments and down‐regulation of all the six genes in starvation treatments. In adult samples, transferrin was up‐regulated in all but starvation treatments while defensin was up‐regulated in starvation and temperature stress treatments. Conclusions Stress during larval development may cause alterations in adult mosquito phenotype and immunity that can increase their susceptibility to pathogens.     

Larval environmental temperature and insecticide exposure alter Aedes aegypti competence for arboviruses

Temperature is a key factor influencing mosquito growth and development and is also known to affect insecticide efficacy. We evaluated the effects of larval rearing temperature and exposure to insecticides on adult mosquito fitness and competence for arboviral infection using Sindbis virus (SINV). We exposed newly hatched larvae of Aedes aegypti to an environmentally realistic level of insecticide malathion at 20°C and 30°C and allowed the resulting adults to feed on SINV-infected blood meal. Exposure to malathion significantly reduced survival to adulthood. Statistically significant interactions between temperature and malathion were observed for body size, estimated population growth, and SINV infection and dissemination. Malathion-exposed Ae. aegypti cohorts had significantly higher population growth at 20°C than at 30°C. Body size decreased with higher temperature and malathion-exposed females were larger than unexposed females at 20°C but not at 30°C. Viral infection and dissemination increased with larval rearing temperature and were higher in malathion-exposed than unexposed females at 30°C but not at 20°C. These results show that environmental factors, including those factors used in controlling mosquitoes, experienced by immature stages have latent effects that continue to adulthood and alter vector competence to arboviruses.     

Larval habitats and distribution patterns of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse), in Thailand

This study was conducted to survey larval breeding habitats and to obtain larval abundance during the dry period covering all 5 geographical zones of Thailand. Our results indicated Aedes aegypri is prevalent all over the country, whereas Aedes albopictus is more restricted to the remote area of the south. Water storage containers, especially water jars, served as a main larval breeding habitats of Ae. aegypti, whereas broken cans and plastic containers are considered primary breeding sites for Ae. alpopictus during the dry period. In addition, Aedes larval indices, container index (CI), house index (HI), and Breteau index (BI) were measured. CI and HI values from the central part were significantly higher than those from other areas (p<0.01). BI values of all collection sites were greater than 50 (a maximum BI value accepted by the Ministry of Public Health, Thailand). In brief, Ae. aegypti and Ae. albopictus populations heavily infested many towns and residential areas of the country. Drought could not limit the density of Aedes mosquitos in Thailand. Systematic vector control and vector surveillance programs by public health organizations, if practical, should be continuously conducted to reduce or prevent dengue risk.     

Body Size and Wing Shape Measurements as Quality Indicators of Aedes aegypti Mosquitoes Destined for Field Release

There is increasing interest in rearing modified mosquitoes for mass release to control vector-borne diseases, particularly Wolbachia-infected Aedes aegypti for suppression of dengue. Successful introductions require release of high quality mosquitoes into natural populations. Potential indicators of quality are body size and shape. We tested to determine if size, wing/thorax ratio, and wing shape are associated with field fitness of Wolbachia-infected Ae. aegypti. Compared with field-collected mosquitoes, released mosquitoes were larger in size, with lower size variance and different wing shape but similar in wing-thorax ratio and its associated variance. These differences were largely attributed to nutrition and to a minor extent to wMel Wolbachia infection. Survival potential of released female mosquitoes was similar to those from the field. Females at oviposition sites tended to be larger than those randomly collected from BG-Sentinel traps. Rearing conditions should thus aim for large size without affecting wing/thorax ratios.     

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.     

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.     

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.     

Phylogeography and population structure of Aedes aegypti in Arizona

Aedes aegypti, the mosquito responsible for transmitting dengue, has colonized many cities and towns throughout Arizona. Determining both the migration between, and the origin of, local Ae. aegypti populations is important for vector control and disease prevention purposes. Amplified fragment length polymorphism was used to infer geographic structure and local substructure, and effective migration rates (M, migrants per generation) between populations, and to determine genetic differentiation between populations (PhiPT). Three geographically and genetically differentiated groups of populations were identified. Population substructure was only detected in the border town of Nogales. Reliable estimates of M between regions ranged from 1.02 to 3.41 and between cities within regions from 1.66 to 4.44. In general, pairwise PhiPT were lowest between cities within regions. The observed patterns of genetic differentiation suggest infrequent migration between populations and are compatible with the idea of human transport facilitating dispersal between regions.     

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.     

Importance of ecology in Aedes aegypti control.

Aedes aegypti is one of the world's most widely distributed mosquitos and is of considerable medical importance as a vector of dengue and yellow fever. Not surprisingly therefore there has been more written on its biology than any other mosquito. The present paper summarizes ecological studies undertaken on this vector, including those on pre-adult mortalities, life-tables, adult dispersal and survival rates. In discussing surveillance techniques it is pointed out there are still no reliable methods for monitoring adult populations. The importance of the resting habits of adults and house construction in insecticidal control of Ae. aegypti is discussed. The question is, have ecological studies and population modeling resulted in any more understanding of the epidemiology of dengue, or helped formulate better control strategies? The answer seems to be not usually, possible because there have actually been relatively few good ecological studies on Ae. aegypti. Although mathematical modeling indicates that better control might be achieved if it were directed at the larvae, not adults, this has not usually been taken into consideration by those engaged in control. There is clearly need for greater collaboration between those practicing control, and ecologists and modelers.     

Limited potential for transmission of live dengue virus vaccine candidates by Aedes aegypti and Aedes albopictus

To evaluate the transmission risk of four live dengue (DEN) vaccine candidates developed by the U.S. Army (DEN-1, 45AZ5 PDK 20; DEN-2, S16803 PDK 50; DEN-3, CH53489 PDK 20; and DEN-4, 341750 PDK 20), we tested 3,010 Aedes aegypti and 1,576 Aedes albopictus mosquitoes blood-fed on 21 volunteers who had been administered one of the four vaccine candidates or the licensed yellow fever (YF) vaccine (17D). We used an indirect immunofluorescence assay (IFA) to detect DEN or YF viral antigen in the heads of mosquitoes. Corresponding to the lack of a detectable viremia among volunteers inoculated 8-13 days previously with live DEN-1 or DEN-2 vaccine candidates, only six mosquitoes developed disseminated infections after feeding on these volunteers. These six mosquitoes included 4 of 247 Ae. albopictus fed on volunteers inoculated with the DEN-1 vaccine candidate and 2 of 528 Ae. aegypti fed on volunteers inoculated with the DEN-2 vaccine candidate. Infection was confirmed in each of these IFA-positive mosquitoes by isolating infectious virus from the mosquito's body in Vero-cell culture. None of the 1,252 or the 969 mosquitoes fed on DEN-3 or DEN-4 recipients, respectively, were infected. Overall, dissemination rates in Ae. albopictus and Ae. aegypti were low. Dissemination rates were 0.5%, 0.3%, < 0.1%, and < 0.1% for the DEN-1 through DEN-4 vaccine candidates, respectively. Because of the observed low dissemination rates, it is unlikely that these vaccine viruses would be transmitted under natural conditions.