From Lab to Field: The Influence of Urban Landscapes on the Invasive Potential of Wolbachia in Brazilian Aedes aegypti Mosquitoes

Background

The symbiotic bacterium Wolbachia is currently being trialled as a biocontrol agent in several countries to reduce dengue transmission. Wolbachia can invade and spread to infect all individuals within wild mosquito populations, but requires a high rate of maternal transmission, strong cytoplasmic incompatibility and low fitness costs in the host in order to do so. Additionally, extensive differences in climate, field-release protocols, urbanization level and human density amongst the sites where this bacterium has been deployed have limited comparison and analysis of Wolbachia’s invasive potential.

Methodology/Principal Findings

We examined key phenotypic effects of the wMel Wolbachia strain in laboratory Aedes aegypti mosquitoes with a Brazilian genetic background to characterize its invasive potential. We show that the wMel strain causes strong cytoplasmic incompatibility, a high rate of maternal transmission and has no evident detrimental effect on host fecundity or fertility. Next, to understand the effects of different urban landscapes on the likelihood of mosquito survival, we performed mark-release-recapture experiments using Wolbachia-uninfected Brazilian mosquitoes in two areas of Rio de Janeiro where Wolbachia will be deployed in the future. We characterized the mosquito populations in relation to the socio-demographic conditions at these sites, and at three other future release areas. We then constructed mathematical models using both the laboratory and field data, and used these to describe the influence of urban environmental conditions on the likelihood that the Wolbachia infection frequency could reach 100% following mosquito release. We predict successful invasion at all five field sites, however the conditions by which this occurs vary greatly between sites, and are strongly influenced by the size of the local mosquito population.

Conclusions/Significance

Through analysis of laboratory, field and mathematical data, we show that the wMel strain of Wolbachia possesses the characteristics required to spread effectively in different urban socio-demographic environments in Rio de Janeiro, including those where mosquito releases from the Eliminate Dengue Program will take place.     

Modulation of Host Learning in Aedes aegypti Mosquitoes

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Dispersal of Engineered Male Aedes aegypti Mosquitoes

Background

Aedes aegypti, the principal vector of dengue fever, have been genetically engineered for use in a sterile insect control programme. To improve our understanding of the dispersal ecology of mosquitoes and to inform appropriate release strategies of ‘genetically sterile’ male Aedes aegypti detailed knowledge of the dispersal ability of the released insects is needed.

Methodology/Principal Findings

The dispersal ability of released ‘genetically sterile’ male Aedes aegypti at a field site in Brazil has been estimated. Dispersal kernels embedded within a generalized linear model framework were used to analyse data collected from three large scale mark release recapture studies. The methodology has been applied to previously published dispersal data to compare the dispersal ability of ‘genetically sterile’ male Aedes aegypti in contrasting environments. We parameterised dispersal kernels and estimated the mean distance travelled for insects in Brazil: 52.8m (95% CI: 49.9m, 56.8m) and Malaysia: 58.0m (95% CI: 51.1m, 71.0m).

Conclusions/Significance

Our results provide specific, detailed estimates of the dispersal characteristics of released ‘genetically sterile’ male Aedes aegypti in the field. The comparative analysis indicates that despite differing environments and recapture rates, key features of the insects’ dispersal kernels are conserved across the two studies. The results can be used to inform both risk assessments and release programmes using ‘genetically sterile’ male Aedes aegypti.     

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     

A decade of stability for wMel Wolbachia in natural Aedes aegypti populations

Mosquitoes carrying Wolbachia endosymbionts are being released in many countries for arbovirus control. The wMel strain of Wolbachia blocks Aedes-borne virus transmission and can spread throughout mosquito populations by inducing cytoplasmic incompatibility. Aedes aegypti mosquitoes carrying wMel were first released into the field in Cairns, Australia, over a decade ago, and with wider releases have resulted in the near elimination of local dengue transmission. The long-term stability of Wolbachia effects is critical for ongoing disease suppression, requiring tracking of phenotypic and genomic changes in Wolbachia infections following releases. We used a combination of field surveys, phenotypic assessments, and Wolbachia genome sequencing to show that wMel has remained stable in its effects for up to a decade in Australian Ae. aegypti populations. Phenotypic comparisons of wMel-infected and uninfected mosquitoes from near-field and long-term laboratory populations suggest limited changes in the effects of wMel on mosquito fitness. Treating mosquitoes with antibiotics used to cure the wMel infection had limited effects on fitness in the next generation, supporting the use of tetracycline for generating uninfected mosquitoes without off-target effects. wMel has a temporally stable within-host density and continues to induce complete cytoplasmic incompatibility. A comparison of wMel genomes from pre-release (2010) and nine years post-release (2020) populations show few genomic differences and little divergence between release locations, consistent with the lack of phenotypic changes. These results indicate that releases of Wolbachia-infected mosquitoes for population replacement are likely to be effective for many years, but ongoing monitoring remains important to track potential evolutionary changes.     

白纹伊蚊和埃及伊蚊对基孔肯雅病毒的易感性和传播性的研究

用４株基孔肯雅病毒经口感染白纹伊蚊和埃及伊蚊,进行了易感性和传播性的研究。结果表明,这两种蚊虫对基孔肯雅病毒易感。无论白纹伊蚊或埃及伊蚊,感染后第５－６天即可通过吸血将病毒传播给乳鼠,至第８－１３天,传播率可高达５５．５５％－１００％。感染蚊亦可经叮咬将病毒传播给小鸡。埃及伊蚊的易感性和传播率高于白纹伊蚊。实验还发现,不同来源毒株之间存在一定差异,如分离自云南白纹伊蚊的Ｍ８１株的感染率和传播率均高于其它毒株。这些结果表明,白纹伊蚊和埃及伊蚊在基孔肯雅病毒的保存和传播中起重要作用。     

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.     

Winter Refuge for Aedes aegypti and Ae. albopictus Mosquitoes in Hanoi during Winter

Dengue occurs throughout the year in Hanoi, Vietnam, despite winter low temperatures <10°C. During July 2010 to March 2012, we surveyed monthly for Aedes larvae and pupae in 120 houses in 8 Hanoi districts. Aedes albopictus preferred discarded containers in summer and pupal density drastically decreased in winter. Aedes aegypti preferred concrete tanks and this preference increased in winter. Even in winter, the lowest water temperature found in concrete tanks was >14°C, exceeding the developmental zero point of Ae. aegypti. Although jars, drums and concrete tanks were the dominant containers previously (1994–97) in Hanoi, currently the percentage of residences with concrete tanks was still high while jars and drums were quite low. Our study showed that concrete tanks with broken lids allowing mosquitoes access were important winter refuge for Ae. aegypti. We also indicate a concern about concrete tanks serving as foci for Ae. aegypti to expand their distribution in cooler regions.     

Competition for Amino Acids Between Wolbachia and the Mosquito Host,Aedes aegypti

The endosymbiont Wolbachia represents a promising method of dengue control, as it reduces the ability of the primary vector, the mosquito Aedes aegypti, to transmit viruses. When mosquitoes infected with the virulent Wolbachia strain wMelPop are fed non-human blood, there is a drastic reduction in mosquito fecundity and egg viability. Wolbachia has a reduced genome and is clearly dependent on its host for a wide range of nutritional needs. The fitness defects seen in wMelPop-infected A. aegypti could be explained by competition between the mosquito and the symbiont for essential blood meal nutrients, the profiles of which are suboptimal in non-human blood. Here, we examine cholesterol and amino acids as candidate molecules for competition, as they have critical roles in egg structural development and are known to vary between blood sources. We found that Wolbachia infection reduces total cholesterol levels in mosquitoes by 15–25 %. We then showed that cholesterol supplementation of a rat blood meal did not improve fecundity or egg viability deficits. Conversely, amino acid supplementation of sucrose before and after a sheep blood meal led to statistically significant increases in fecundity of approximately 15–20 eggs per female and egg viability of 30–40 %. This mosquito system provides the first empirical evidence of competition between Wolbachia and a host over amino acids and may suggest a general feature of Wolbachia–insect associations. These competitive processes could affect many aspects of host physiology and potentially mosquito fitness, a key concern for Wolbachia-based mosquito biocontrol.     

Wolbachia enhances insect‐specific flavivirus infection in Aedes aegypti mosquitoes

Mosquitoes transmit a diverse group of human flaviviruses including West Nile, dengue, yellow fever, and Zika viruses. Mosquitoes are also naturally infected with insect‐specific flaviviruses (ISFs), a subgroup of the family not capable of infecting vertebrates. Although ISFs are not medically important, they are capable of altering the mosquito's susceptibility to flaviviruses and may alter host fitness. Wolbachia is an endosymbiotic bacterium of insects that when present in mosquitoes limits the replication of co‐infecting pathogens, including flaviviruses. Artificially created Wolbachia‐infected Aedes aegypti mosquitoes are being released into the wild in a series of trials around the globe with the hope of interrupting dengue and Zika virus transmission from mosquitoes to humans. Our work investigated the effect of Wolbachia on ISF infection in wild‐caught Ae. aegypti mosquitoes from field release zones. All field mosquitoes were screened for the presence of ISFs using general degenerate flavivirus primers and their PCR amplicons sequenced. ISFs were found to be common and widely distributed in Ae. aegypti populations. Field mosquitoes consistently had higher ISF infection rates and viral loads compared to laboratory colony material indicating that environmental conditions may modulate ISF infection in Ae. aegypti. Surprisingly, higher ISF infection rates and loads were found in Wolbachia‐infected mosquitoes compared to the Wolbachia‐free mosquitoes. Our findings demonstrate that the symbiont is capable of manipulating the mosquito virome and that Wolbachia‐mediated viral inhibition is not universal for flaviviruses. This may have implications for the Wolbachia‐based DENV control strategy if ISFs confer fitness effects or alter mosquito susceptibility to other flaviviruses.     

Urea Synthesis and Excretion in Aedes aegypti Mosquitoes Are Regulated by a Unique Cross-Talk Mechanism

Aedes aegypti mosquitoes do not have a typical functional urea cycle for ammonia disposal such as the one present in most terrestrial vertebrates. However, they can synthesize urea by two different pathways, argininolysis and uricolysis. We investigated how formation of urea by these two pathways is regulated in females of A. aegypti. The expression of arginase (AR) and urate oxidase (UO), either separately or simultaneously (ARUO) was silenced by RNAi. The amounts of several nitrogen compounds were quantified in excreta using mass spectrometry. Injection of mosquitoes with either dsRNA-AR or dsRNA-UO significantly decreased the expressions of AR or UO in the fat body (FB) and Malpighian tubules (MT). Surprisingly, the expression level of AR was increased when UO was silenced and vice versa, suggesting a cross-talk regulation between pathways. In agreement with these data, the amount of urea measured 48 h after blood feeding remained unchanged in those mosquitoes injected with dsRNA-AR or dsRNA-UO. However, allantoin significantly increased in the excreta of dsRNA-AR-injected females. The knockdown of ARUO mainly led to a decrease in urea and allantoin excretion, and an increase in arginine excretion. In addition, dsRNA-AR-injected mosquitoes treated with a specific nitric oxide synthase inhibitor showed an increase of UO expression in FB and MT and a significant increase in the excretion of nitrogen compounds. Interestingly, both a temporary delay in the digestion of a blood meal and a significant reduction in the expression of several genes involved in ammonia metabolism were observed in dsRNA-AR, UO or ARUO-injected females. These results reveal that urea synthesis and excretion in A. aegypti are tightly regulated by a unique cross-talk signaling mechanism. This process allows blood-fed mosquitoes to regulate the synthesis and/or excretion of nitrogen waste products, and avoid toxic effects that could result from a lethal concentration of ammonia in their tissues.     

Genetic Mapping of Specific Interactions between Aedes aegypti Mosquitoes and Dengue Viruses

Specific interactions between host genotypes and pathogen genotypes (G×G interactions) are commonly observed in invertebrate systems. Such specificity challenges our current understanding of invertebrate defenses against pathogens because it contrasts the limited discriminatory power of known invertebrate immune responses. Lack of a mechanistic explanation, however, has questioned the nature of host factors underlying G×G interactions. In this study, we aimed to determine whether G×G interactions observed between dengue viruses and their Aedes aegypti vectors in nature can be mapped to discrete loci in the mosquito genome and to document their genetic architecture. We developed an innovative genetic mapping strategy to survey G×G interactions using outbred mosquito families that were experimentally exposed to genetically distinct isolates of two dengue virus serotypes derived from human patients. Genetic loci associated with vector competence indices were detected in multiple regions of the mosquito genome. Importantly, correlation between genotype and phenotype was virus isolate-specific at several of these loci, indicating G×G interactions. The relatively high percentage of phenotypic variation explained by the markers associated with G×G interactions (ranging from 7.8% to 16.5%) is consistent with large-effect host genetic factors. Our data demonstrate that G×G interactions between dengue viruses and mosquito vectors can be assigned to physical regions of the mosquito genome, some of which have a large effect on the phenotype. This finding establishes the existence of tangible host genetic factors underlying specific interactions between invertebrates and their pathogens in a natural system. Fine mapping of the uncovered genetic loci will elucidate the molecular mechanisms of mosquito-virus specificity.     

Stability of the wMel Wolbachia Infection following Invasion into Aedes aegypti Populations

The wMel infection of Drosophila melanogaster was successfully transferred into Aedes aegypti mosquitoes where it has the potential to suppress dengue and other arboviruses. The infection was subsequently spread into two natural populations at Yorkeys Knob and Gordonvale near Cairns, Queensland in 2011. Here we report on the stability of the infection following introduction and we characterize factors influencing the ongoing dynamics of the infection in these two populations. While the Wolbachia infection always remained high and near fixation in both locations, there was a persistent low frequency of uninfected mosquitoes. These uninfected mosquitoes showed weak spatial structure at both release sites although there was some clustering around two areas in Gordonvale. Infected females from both locations showed perfect maternal transmission consistent with patterns previously established pre-release in laboratory tests. After >2 years under field conditions, the infection continued to show complete cytoplasmic incompatibility across multiple gonotrophic cycles but persistent deleterious fitness effects, suggesting that host effects were stable over time. These results point to the stability of Wolbachia infections and their impact on hosts following local invasion, and also highlight the continued persistence of uninfected individuals at a low frequency most likely due to immigration.     

Suppression of a Field Population of Aedes aegypti in Brazil by Sustained Release of Transgenic Male Mosquitoes

The increasing burden of dengue, and the relative failure of traditional vector control programs highlight the need to develop new control methods. SIT using self-limiting genetic technology is one such promising method. A self-limiting strain of Aedes aegypti, OX513A, has already reached the stage of field evaluation. Sustained releases of OX513A Ae. aegypti males led to 80% suppression of a target wild Ae. aegypti population in the Cayman Islands in 2010. Here we describe sustained series of field releases of OX513A Ae. aegypti males in a suburb of Juazeiro, Bahia, Brazil. This study spanned over a year and reduced the local Ae. aegypti population by 95% (95% CI: 92.2%-97.5%) based on adult trap data and 81% (95% CI: 74.9-85.2%) based on ovitrap indices compared to the adjacent no-release control area. The mating competitiveness of the released males (0.031; 95% CI: 0.025-0.036) was similar to that estimated in the Cayman trials (0.059; 95% CI: 0.011 – 0.210), indicating that environmental and target-strain differences had little impact on the mating success of the OX513A males. We conclude that sustained release of OX513A males may be an effective and widely useful method for suppression of the key dengue vector Ae. aegypti. The observed level of suppression would likely be sufficient to prevent dengue epidemics in the locality tested and other areas with similar or lower transmission.     

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.     

Monoclonal antibodies as probes for Aedes aegypti trypsin

Two murine monoclonal cell lines secreting antibodies against mosquito trypsin were produced by the hybridoma technique. Selected clones of each line were expanded, injected into mice or rats and the immunoglobulins were purified from ascites fluid. Clones of one cell-line (Mab 1–7) recognized only the major 30 kD trypsin from imaginal midguts on SDS-western blots, while trypsin from larval midguts. bovine trypsin and trypsin from other mosquito species were not recognized. This antibody did not distinguish trypsin from 19 strains within the same species, collected around the world. The second line (Mab 11–12) exhibited crossreactivities with other species when applied to a native blot. Analysis of the digestive cycle with these antibodies revealed an increase in immunoreactivity after blood-feeding with both immunoblots and radioimmunoassays, confirming earlier reports based on enzymatic assays.     

Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida,USA

The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two‐step PCR assay to detect Wolbachia in wild‐collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop‐mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia‐positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.     

Limited Dengue Virus Replication in Field-Collected Aedes aegypti Mosquitoes Infected with Wolbachia

Introduction

Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus.

Methodology/Principal Findings

Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes.

Conclusions/Significance

Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.