Luciano Andrade Moreira

Wolbachia Blocks Currently Circulating Zika Virus Isolates in Brazilian Aedes aegypti Mosquitoes

Summary The recent association of Zika virus with cases of microcephaly has sparked a global health crisis and highlighted the need for mechanisms to combat the Zika vector, Aedes aegypti mosquitoes. Wolbachia pipientis, a bacterial endosymbiont of insect, has recently garnered attention as a mechanism for arbovirus control. Here we report that Aedes aegypti harboring Wolbachia are highly resistant to infection with two currently circulating Zika virus isolates from the recent Brazilian epidemic. Wolbachia-harboring mosquitoes displayed lower viral prevalence and intensity and decreased disseminated infection and, critically, did not carry infectious virus in the saliva, suggesting that viral transmission was blocked. Our data indicate that the use of Wolbachia-harboring mosquitoes could represent an effective mechanism to reduce Zika virus transmission and should be included as part of Zika control strategies.

Harnessing mosquito-Wolbachia symbiosis for vector and disease control

Mosquito species, members of the genera Aedes, Anopheles and Culex, are the major vectors of human pathogens including protozoa (Plasmodium sp.), filariae and of a variety of viruses (causing dengue, chikungunya, yellow fever, West Nile). There is lack of efficient methods and tools to treat many of the diseases caused by these major human pathogens, since no efficient vaccines or drugs are available; even in malaria where insecticide use and drug therapies have reduced incidence, 219 million cases still occurred in 2010. Therefore efforts are currently focused on the control of vector populations. Insecticides alone are insufficient to control mosquito populations since reduced susceptibility and even resistance is being observed more and more frequently. There is also increased concern about the toxic effects of insecticides on non-target (even beneficial) insect populations, on humans and the environment. During recent years, the role of symbionts in the biology, ecology and evolution of insect species has been well-documented and has led to suggestions that they could potentially be used as tools to control pests and therefore diseases. Wolbachia is perhaps the most renowned insect symbiont, mainly due to its ability to manipulate insect reproduction and to interfere with major human pathogens thus providing new avenues for pest control. We herein present recent achievements in the field of mosquito-Wolbachia symbiosis with an emphasis on Aedes albopictus. We also discuss how Wolbachia symbiosis can be harnessed for vector control as well as the potential to combine the sterile insect technique and Wolbachia-based approaches for the enhancement of population suppression programs.

wFlu: Characterization and Evaluation of a Native Wolbachia from the Mosquito Aedes fluviatilis as a Potential Vector Control Agent

There is currently considerable interest and practical progress in using the endosymbiotic bacteria Wolbachia as a vector control agent for human vector-borne diseases. Such vector control strategies may require the introduction of multiple, different Wolbachia strains into target vector populations, necessitating the identification and characterization of appropriate endosymbiont variants. Here, we report preliminary characterization of wFlu, a native Wolbachia from the neotropical mosquito Aedes fluviatilis, and evaluate its potential as a vector control agent by confirming its ability to cause cytoplasmic incompatibility, and measuring its effect on three parameters determining host fitness (survival, fecundity and fertility), as well as vector competence (susceptibility) for pathogen infection. Using an aposymbiotic strain of Ae. fluviatilis cured of its native Wolbachia by antibiotic treatment, we show that in its natural host wFlu causes incomplete, but high levels of, unidirectional cytoplasmic incompatibility, has high rates of maternal transmission, and no detectable fitness costs, indicating a high capacity to rapidly spread through host populations. However, wFlu does not inhibit, and even enhances, oocyst infection with the avian malaria parasite Plasmodium gallinaceum. The stage- and sex-specific density of wFlu was relatively low, and with limited tissue distribution, consistent with the lack of virulence and pathogen interference/symbiont-mediated protection observed. Unexpectedly, the density of wFlu was also shown to be specifically-reduced in the ovaries after bloodfeeding Ae. fluviatilis. Overall, our observations indicate that the Wolbachia strain wFlu has the potential to be used as a vector control agent, and suggests that appreciable mutualistic coevolution has occurred between this endosymbiont and its natural host. Future work will be needed to determine whether wFlu has virulent host effects and/or exhibits pathogen interference when artificially-transfected to the novel mosquito hosts that are the vectors of human pathogens.

Anti-Anopheles darlingi saliva antibodies as marker of Plasmodium vivax infection and clinical immunity in the Brazilian Amazon

BackgroundDespite governmental and private efforts on providing malaria control, this disease continues to be a major health threat. Thus, innovative strategies are needed to reduce disease burden. The malaria vectors, through the injection of saliva into the host skin, play important role on disease transmission and may influence malaria morbidity. This study describes the humoral immune response against Anopheles (An.) darlingi saliva in volunteers from the Brazilian Amazon and addresses the association between levels of specific antibodies and clinical presentation of Plasmodium (P.) vivax infection.MethodsAdult volunteers from communities in the Rondônia State, Brazil, were screened in order to assess the presence of P. vivax infection by light microscopy and nested PCR. Non-infected volunteers and individuals with symptomatic or symptomless infection were randomly selected and plasma collected. An. darlingi salivary gland sonicates (SGS) were prepared and used to measure anti-saliva antibody levels. Plasma interleukin (IL)-10 and interferon (IFN)-γ levels were also estimated and correlated to anti-SGS levels.ResultsIndividuals infected with P. vivax presented higher levels of anti-SGS than non-infected individuals and antibody levels could discriminate infection. Furthermore, anti-saliva antibody measurement was also useful to distinguish asymptomatic infection from non-infection, with a high likelihood ratio. Interestingly, individuals with asymptomatic parasitaemia presented higher titers of anti-SGS and lower IFN-γ/IL-10 ratio than symptomatic ones. In P. vivax-infected asymptomatic individuals, the IFN-γ/IL-10 ratio was inversely correlated to anti-SGS titers, although not for while in symptomatic volunteers.ConclusionThe estimation of anti-An. darlingi antibody levels can indicate the probable P. vivax infection status and also could serve as a marker of disease severity in this region of Brazilian Amazon.

Driving midgut-specific expression and secretion of a foreign protein in transgenic mosquitoes with AgAper1 regulatory elements.

The Anopheles gambiae adult peritrophic matrix protein 1 (AgAper1) regulatory elements were used to drive the expression of phospholipase A2 (PLA2), a protein known to disrupt malaria parasite development in mosquitoes. These AgAper1 regulatory elements were sufficient to promote the accumulation of PLA2 in midgut epithelial cells before a blood meal and its release into the lumen upon blood ingestion. Plasmodium berghei oocyst formation was reduced by ∼80% (74–91% range) in transgenic mosquitoes. Blood‐seeking behaviour and survival of AgAper1‐PLA2 transgenic mosquitoes were comparable to sibling wild‐type mosquitoes, while fertility was substantially lower. Ultrastructural studies suggest that decreased fitness is a consequence of internal damage to midgut epithelial cells.

Exploiting Intimate Relationships: Controlling Mosquito-Transmitted Disease with Wolbachia

Mosquito-transmitted diseases impose a growing burden on human health, and current control strategies have proven insufficient to stem the tide. The bacterium Wolbachia is a novel and promising form of control for mosquito-transmitted disease. It manipulates host biology, restricts infection with dengue and other pathogens, and alters host reproduction to promote rapid spread in the field. In this review, we examine how the intimate and diverse relationships formed between Wolbachia and their mosquito hosts can be exploited for disease control purposes. We consider these relationships in the context of recent developments, including successful field trials with Wolbachia-infected mosquitoes to combat dengue, and new Wolbachia infections in key malaria vectors, which have enhanced the disease control prospects of this unique bacterium.

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.

Can Wolbachia be used to control malaria

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.

Expression of a mutated phospholipase A2 in transgenic Aedes fluviatilis mosquitoes impacts Plasmodium gallinaceum development

The genetic manipulation of mosquito vectors is an alternative strategy in the fight against malaria. It was previously shown that bee venom phospholipase A2 (PLA2) inhibits ookinete invasion of the mosquito midgut although mosquito fitness was reduced. To maintain the PLA2 blocking ability without compromising mosquito biology, we mutated the protein‐coding sequence to inactivate the enzyme while maintaining the proteins structure. DNA encoding the mutated PLA2 (mPLA2) was placed downstream of a mosquito midgut‐specific promoter (Anopheles gambiae peritrophin protein 1 promoter, AgPer1) and this construct used to transform Aedes fluviatilis mosquitoes. Four different transgenic lines were obtained and characterized and all lines significantly inhibited Plasmodium gallinaceum oocyst development (up to 68% fewer oocysts). No fitness cost was observed when this mosquito species expressed the mPLA2.

Why do we need alternative tools to control mosquito-borne diseases in Latin America?

In this opinion paper, we discuss the potential and challenges of using the symbiont Wolbachia to block mosquito transmitted diseases such as dengue, malaria and chikungunya in Latin America.