Wolbachia Infection Reduces Blood-Feeding Success in the Dengue Fever Mosquito, Aedes aegypti

Background

The mosquito Aedes aegypti was recently transinfected with a life-shortening strain of the endosymbiont Wolbachia pipientis (wMelPop) as the first step in developing a biocontrol strategy for dengue virus transmission. In addition to life-shortening, the wMelPop-infected mosquitoes also exhibit increased daytime activity and metabolic rates. Here we sought to quantify the blood-feeding behaviour of Wolbachia-infected females as an indicator of any virulence or energetic drain associated with Wolbachia infection.

Methodology/Principal Findings

In a series of blood-feeding trials in response to humans, we have shown that Wolbachia-infected mosquitoes do not differ in their response time to humans, but that as they age they obtain fewer and smaller blood meals than Wolbachia-uninfected controls. Lastly, we observed a behavioural characteristic in the Wolbachia infected mosquitoes best described as a “bendy” proboscis that may explain the decreased biting success.

Conclusions/Significance

Taken together the evidence suggests that wMelPop infection may be causing tissue damage in a manner that intensifies with mosquito age and that leads to reduced blood-feeding success. These behavioural changes require further investigation with respect to a possible physiological mechanism and their role in vectorial capacity of the insect. The selective decrease of feeding success in older mosquitoes may act synergistically with other Wolbachia-associated traits including life-shortening and viral protection in biocontrol strategies.     

Characterizing the Aedes aegypti Population in a Vietnamese Village in Preparation for a Wolbachia-Based Mosquito Control Strategy to Eliminate Dengue

Background

A life-shortening strain of the obligate intracellular bacteria Wolbachia, called wMelPop, is seen as a promising new tool for the control of Aedes aegypti. However, developing a vector control strategy based on the release of mosquitoes transinfected with wMelPop requires detailed knowledge of the demographics of the target population.

Methodology/Principal Findings

In Tri Nguyen village (611 households) on Hon Mieu Island in central Vietnam, we conducted nine quantitative entomologic surveys over 14 months to determine if Ae. aegypti populations were spatially and temporally homogenous, and to estimate population size. There was no obvious relationship between mosquito (larval, pupal or adult) abundance and temperature and rainfall, and no area of the village supported consistently high numbers of mosquitoes. In almost all surveys, key premises produced high numbers of Ae. aegypti. However, these premises were not consistent between surveys. For an intervention based on a single release of wMelPop-infected Ae. aegypti, release ratios of infected to uninfected adult mosquitoes of all age classes are estimated to be 1.8–6.7∶1 for gravid females (and similarly aged males) or teneral adults, respectively. We calculated that adult female mosquito abundance in Tri Nguyen village could range from 1.1 to 43.3 individuals of all age classes per house. Thus, an intervention could require the release of 2–78 wMelPop-infected gravid females and similarly aged males per house, or 7–290 infected teneral female and male mosquitoes per house.

Conclusions/Significance

Given the variability we encountered, this study highlights the importance of multiple entomologic surveys when evaluating the spatial structure of a vector population or estimating population size. If a single release of wMelPop-infected Ae. aegypti were to occur when wild Ae. aegypti abundance was at its maximum, a preintervention control program would be necessary to ensure that there was no net increase in mosquito numbers. However, because of the short-term temporal heterogeneity, the inconsistent spatial structure and the impact of transient key premises that we observed, the feasibility of multiple releases of smaller numbers of mosquitoes also needs to be considered. In either case, fewer wMelPop-infected mosquitoes would then need to be released, which will likely be more acceptable to householders.     

A Virulent Wolbachia Infection Decreases the Viability of the Dengue Vector Aedes aegypti during Periods of Embryonic Quiescence

A new approach for dengue control has been proposed that relies on life-shortening strains of the obligate intracellular bacterium Wolbachia pipientis to modify mosquito population age structure and reduce pathogen transmission. Previously we reported the stable transinfection of the major dengue vector Aedes aegypti with a life-shortening Wolbachia strain (wMelPop-CLA) from the vinegar fly Drosophila melanogaster. Here, we report a further characterization of the phenotypic effects of this virulent Wolbachia infection on several life-history traits of Ae. aegypti. Minor costs of wMelPop-CLA infection for pre-imaginal survivorship, development and adult size were found. However, we discovered that the wMelPop-CLA infection dramatically decreased the viability of desiccated Ae. aegypti eggs over time. Similarly, the reproductive fitness of wMelPop-CLA infected Ae. aegypti females declined with age. These results reveal a general pattern associated with wMelPop-CLA induced pathogenesis in this mosquito species, where host fitness costs increase during aging of both immature and adult life-history stages. In addition to influencing the invasion dynamics of this particular Wolbachia strain, we suggest that the negative impact of wMelPop-CLA on embryonic quiescence may have applied utility as a tool to reduce mosquito population size in regions with pronounced dry seasons or in regions that experience cool winters.     

白纹伊蚊和埃及伊蚊经卵传递登革病毒的研究

白纹伊蚊和埃及伊蚊通过吸食病毒液或叮吸有病毒血症的小鸡血后,能感染登革１－４型病毒,并能在蚊体内增殖,对感染雌蚊了１和子２代幼虫,雌性或雄性成虫４５５９只,分１０１批进行了病毒检测,白纹伊蚊子１代的批阳性率;登革１型为１０％（１／１０）２型为２２．２２％（２／９）３型为３３．３３％（４／１２）,４型为２８．９５％（１１／３８）登革１～４型的最低子代感染率依次主国０．２０％,０．７１％,０．７０％和     

Evidence for genetic hitchhiking effect associated with insecticide resistance in Aedes aegypti.

Information on genetic variation within and between populations is critical for understanding the evolutionary history of mosquito populations and disease epidemiology. Previous studies with Drosophila suggest that genetic variation of selectively neutral loci in a large fraction of genome may be constrained by fixation of advantageous mutations associated with hitchhiking effect. This study examined restriction fragment length polymorphisms of four natural Aedes aegypti mosquito populations from Trinidad and Tobago, at 16 loci. These populations have been subjected to organophosphate (OP) insecticide treatments for more than two decades, while dichlor-diphenyltrichlor (DDT) was the insecticide of choice prior to this period. We predicted that genes closely linked to the OP target loci would exhibit reduced genetic variation as a result of the hitchhiking effect associated with intensive OP insecticide selection. We also predicted that genetic variability of the genes conferring resistance to DDT and loci near the target site would be similar to other unlinked loci. As predicted, reduced genetic variation was found for loci in the general chromosomal region of a putative OP target site, and these loci generally exhibited larger F(ST) values than other random loci. In contrast, the gene conferring resistance to DDT and its linked loci show polymorphisms and genetic differentiation similar to other random loci. The reduced genetic variability and apparent gene deletion in some regions of chromosome 1 likely reflect the hitchhiking effect associated with OP insecticide selection.     

Wolbachia infection alters the relative abundance of resident bacteria in adult Aedes aegypti mosquitoes,but not larvae

Insect–symbiont interactions are known to play key roles in host functions and fitness. The common insect endosymbiont Wolbachia can reduce the ability of several human pathogens, including arboviruses and the malaria parasite, to replicate in insect hosts. Wolbachia does not naturally infect Aedes aegypti, the primary vector of dengue virus, but transinfected Ae. aegypti have antidengue virus properties and are currently being trialled as a dengue biocontrol strategy. Here, we assess the impact of Wolbachia infection of Ae. aegypti on the microbiome of wild mosquito populations (adults and larvae) collected from release sites in Cairns, Australia, by profiling the 16S rRNA gene using next‐generation sequencing. Our data indicate that Wolbachia reduces the relative abundance of a large proportion of bacterial taxa in Ae. aegypti adults, that is in accordance with the known pathogen‐blocking effects of Wolbachia on a variety of bacteria and viruses. In adults, several of the most abundant bacterial genera were found to undergo significant shifts in relative abundance. However, the genera showing the greatest changes in relative abundance in Wolbachia‐infected adults represented a low proportion of the total microbiome. In addition, there was little effect of Wolbachia infection on the relative abundance of bacterial taxa in larvae, or on species diversity (accounting for species richness and evenness together) detected in adults or larvae. These results offer insight into the effects of Wolbachia on the Ae. aegypti microbiome in a native setting, an important consideration for field releases of Wolbachia into the population.     

Co-occurrence Patterns of the Dengue Vector Aedes aegypti and Aedes mediovitattus, a Dengue Competent Mosquito in Puerto Rico

Aedes aegypti is implicated in dengue transmission in tropical and subtropical urban areas around the world. Ae. aegypti populations are controlled through integrative vector management. However, the efficacy of vector control may be undermined by the presence of alternative, competent species. In Puerto Rico, a native mosquito, Ae. mediovittatus, is a competent dengue vector in laboratory settings and spatially overlaps with Ae. aegypti. It has been proposed that Ae. mediovittatus may act as a dengue reservoir during inter-epidemic periods, perpetuating endemic dengue transmission in rural Puerto Rico. Dengue transmission dynamics may therefore be influenced by the spatial overlap of Ae. mediovittatus, Ae. aegypti, dengue viruses, and humans. We take a landscape epidemiology approach to examine the association between landscape composition and configuration and the distribution of each of these Aedes species and their co-occurrence. We used remotely sensed imagery from a newly launched satellite to map landscape features at very high spatial resolution. We found that the distribution of Ae. aegypti is positively predicted by urban density and by the number of tree patches, Ae. mediovittatus is positively predicted by the number of tree patches, but negatively predicted by large contiguous urban areas, and both species are predicted by urban density and the number of tree patches. This analysis provides evidence that landscape composition and configuration is a surrogate for mosquito community composition, and suggests that mapping landscape structure can be used to inform vector control efforts as well as to inform urban planning.     

Wolbachia strain wAlbA blocks Zika virus transmission in Aedes aegypti

Transinfections of the maternally transmitted endosymbiont Wolbachia pipientis can reduce RNA virus replication and prevent transmission by Aedes aegypti, and also have the capacity to invade wild-type populations, potentially reaching and maintaining high infection frequencies. Levels of virus transmission blocking are positively correlated with Wolbachia intracellular density. Despite reaching high densities in Ae. aegypti, transinfections of wAlbA, a strain native to Aedes albopictus, showed no blocking of Semliki Forest Virus in previous intrathoracic injection challenges. To further characterize wAlbA blocking in Ae. aegypti, adult females were intrathoracically challenged with Zika (ZIKV) and dengue viruses, and then fed a ZIKV-containing bloodmeal. No blocking was observed with either virus when challenged by intrathoracic injection. However, when ZIKV was delivered orally, wAlbA-infected females showed a significant reduction in viral replication and dissemination compared with uninfected controls, as well as a complete absence of virus in saliva. Although other Wolbachia strains have been shown to cause more robust viral blocking in Ae. aegypti, these findings demonstrate that, in principle, wAlbA could be used to reduce virus transmission in this species. Moreover, the results highlight the potential for underestimation of the strength of virus-blocking when based on intrathoracic injection compared with more natural oral challenges.     

Comparison of the insecticide susceptibilities of laboratory strains of Aedes aegypti and Aedes albopictus

A susceptible strain of Aedes albopictus derived from the Gainesville strain (Florida, USA) was established in our laboratory. The larvicidal efficacies of the neurotoxic insecticides temephos, permethrin and the pure cis and trans-permethrin isomers and the microbial insecticide Bacillus thuringiensis israelensis (Bti) against Ae. albopictus were estimated and compared to a susceptible strain of Aedes aegypti. The larvicidal effect of insect growth regulator pyriproxyfen was also evaluated in both mosquito strains. The median lethal concentration/median emergency inhibition values for Ae. aegypti and Ae. albopictus, respectively, were: temephos, 3.058 and 6.632 ppb, permethrin, 3.143 and 4.933 ppb, cis-permethrin, 4.457 and 10.068 ppb, trans-permethrin, 1.510 and 3.883 ppb, Bti, 0.655 and 0.880 ppb and pyriproxyfen, 0.00774 and 0.01642 ppb. Ae. albopictus was more tolerant than Ae. aegypti to all six larvicides evaluated. The order of susceptibility for Ae. aegypti was pyriproxyfen > Bti > trans-permethrin > temephos > permethrin > cis-permethrin and for Ae. albopictus was pyriproxyfen > Bti > trans-permethrin > permethrin > temephos > cis-permethrin. Because both species can be found together in common urban, suburban and rural breeding sites, the results of this work provide baseline data on the susceptibility of Ae. albopictus to insecticides commonly used for controlling Ae. aegypti in the field.     

Dengue infection increases the locomotor activity of Aedes aegypti females

Background

Aedes aegypti is the main vector of the virus causing Dengue fever, a disease that has increased dramatically in importance in recent decades, affecting many tropical and sub-tropical areas of the globe. It is known that viruses and other parasites can potentially alter vector behavior. We investigated whether infection with Dengue virus modifies the behavior of Aedes aegypti females with respect to their activity level.

Methods/Principal Findings

We carried out intrathoracic Dengue 2 virus (DENV-2) infections in Aedes aegypti females and recorded their locomotor activity behavior. We observed an increase of up to ∼50% in the activity of infected mosquitoes compared to the uninfected controls.

Conclusions

Dengue infection alters mosquito locomotor activity behavior. We speculate that the higher levels of activity observed in infected Aedes aegypti females might involve the circadian clock. Further studies are needed to assess whether this behavioral change could have implications for the dynamics of Dengue virus transmission.     

Dengue virus detection in Aedes aegypti larvae from southeastern Brazil

The transmission of dengue, the most important arthropod‐borne viral disease in Brazil, has been intensified over the past decades, along with the accompanying expansion and adaptation of its Aedes vectors. In the present study, we mapped dengue vectors in Ouro Preto and Ouro Branco, Minas Gerais, by installing ovitraps in 32 public schools. The traps were examined monthly between September, 2011 through July, 2012 and November, 2012 to April, 2013. The larvae were reared until the fourth stadium and identified according to species. The presence of dengue virus was detected by real time PCR and agarose gel electrophoresis. A total of 1,945 eggs was collected during the 17 months of the study. The Ovitrap Positivity Index (OPI) ranged from 0 to 28.13% and the Eggs Density Index (EDI) ranged from 0 to 59.9. The predominant species was Aedes aegypti, with 84.9% of the hatched larvae. Although the collection was low when compared to other ovitraps studies, vertical transmission could be detected. Of the 54 pools, dengue virus was detected in four Ae. aegypti pools.     

Comparison of Vector Competence of Aedes mediovittatus and Aedes aegypti for Dengue Virus: Implications for Dengue Control in the Caribbean

Background

Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes.

Methods

To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1–4 per os at viral titers of 5–6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution.

Results

Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus.

Conclusions

This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs.     

Temephos Resistance in Aedes aegypti in Colombia Compromises Dengue Vector Control

Background

Control and prevention of dengue relies heavily on the application of insecticides to control dengue vector mosquitoes. In Colombia, application of the larvicide temephos to the aquatic breeding sites of Aedes aegypti is a key part of the dengue control strategy. Resistance to temephos was recently detected in the dengue-endemic city of Cucuta, leading to questions about its efficacy as a control tool. Here, we characterize the underlying mechanisms and estimate the operational impact of this resistance.

Methodology/Principal Findings

Larval bioassays of Ae. aegypti larvae from Cucuta determined the temephos LC₅₀ to be 0.066 ppm (95% CI 0.06–0.074), approximately 15× higher than the value obtained from a susceptible laboratory colony. The efficacy of the field dose of temephos at killing this resistant Cucuta population was greatly reduced, with mortality rates <80% two weeks after application and <50% after 4 weeks. Neither biochemical assays nor partial sequencing of the ace-1 gene implicated target site resistance as the primary resistance mechanism. Synergism assays and microarray analysis suggested that metabolic mechanisms were most likely responsible for the temephos resistance. Interestingly, although the greatest synergism was observed with the carboxylesterase inhibitor, DEF, the primary candidate genes from the microarray analysis, and confirmed by quantitative PCR, were cytochrome P450 oxidases, notably CYP6N12, CYP6F3 and CYP6M11.

Conclusions/Significance

In Colombia, resistance to temephos in Ae. aegypti compromises the duration of its effect as a vector control tool. Several candidate genes potentially responsible for metabolic resistance to temephos were identified. Given the limited number of insecticides that are approved for vector control, future chemical-based control strategies should take into account the mechanisms underlying the resistance to discern which insecticides would likely lead to the greatest control efficacy while minimizing further selection of resistant phenotypes.     

Origin of the Dengue Fever Mosquito,Aedes aegypti,in California

Dengue fever is among the most widespread vector-borne infectious diseases. The primary vector of dengue is the Aedes aegypti mosquito. Ae. aegypti is prevalent in the tropics and sub-tropics and is closely associated with human habitats outside its native range of Africa. While long established in the southeastern United States of America where dengue is re-emerging, breeding populations have never been reported from California until the summer of 2013. Using 12 highly variable microsatellite loci and a database of reference populations, we have determined that the likely source of the California introduction is the southeastern United States, ruling out introductions from abroad, from the geographically closer Arizona or northern Mexico populations, or an accidental release from a research laboratory. The power to identify the origin of new introductions of invasive vectors of human disease relies heavily on the availability of a panel of reference populations. Our work demonstrates the importance of generating extensive reference databases of genetically fingerprinted human-disease vector populations to aid public health efforts to prevent the introduction and spread of vector-borne diseases.