Bacteria isolated from Aedes aegypti with potential vector control applications.

Highly anthropophilic and adapted to urban environments, Aedes aegypti mosquitoes are the main vectors of arboviruses that cause human diseases such as dengue, zika, and chikungunya fever, especially in countries with tropical and subtropical climates. Microorganisms with mosquitocidal and larvicidal activities have been suggested as environmentally safe alternatives to chemical or mechanical mosquito control methods. Here, we analyzed cultivable bacteria isolated from all stages of the mosquito life cycle for their larvicidal activity against Ae. aegypti. A total of 424 bacterial strains isolated from eggs, larvae, pupae, or adult Ae. aegypti were analyzed for the pathogenic potential of their crude cultures against larvae of this same mosquito species. Nine strains displayed larvicidal activity comparable to the strain AM65-52, reisolated from commercial BTi-based product VectoBac® WG. 16S rRNA gene sequencing revealed that the set of larvicidal strains contains two representatives of the genus Bacillus, five Enterobacter, and two Stenotrophomonas. This study demonstrates that some bacteria isolated from Ae. aegypti are pathogenic for the mosquito from which they were isolated. The data are promising for developing novel bioinsecticides for the control of these medically important mosquitoes.

Biotechnological Potential of Microorganisms for Mosquito Population Control and Reduction in Vector Competence.

Mosquitoes transmit pathogens that cause human diseases such as malaria, dengue fever, chikungunya, yellow fever, Zika fever, and filariasis. Biotechnological approaches using microorganisms have a significant potential to control mosquito populations and reduce their vector competence, making them alternatives to synthetic insecticides. Ongoing research has identified many microorganisms that can be used effectively to control mosquito populations and disease transmission. However, the successful implementation of these newly proposed approaches requires a thorough understanding of the multipronged microorganism-mosquito-pathogen-environment interactions. Although much has been achieved in discovering new entomopathogenic microorganisms, antipathogen compounds, and their mechanisms of action, only a few have been turned into viable products for mosquito control. There is a discrepancy between the number of microorganisms with the potential for the development of new insecticides and/or antipathogen products and the actual available products, highlighting the need for investments in the intersection of basic research and biotechnology.

Comparative assessment of the bacterial communities associated with Anopheles darlingi immature stages and their breeding sites in the Brazilian Amazon.

BACKGROUND: The neotropical anopheline mosquito Anopheles darlingi is a major malaria vector in the Americas. Studies on mosquito-associated microbiota have shown that symbiotic bacteria play a major role in host biology. Mosquitoes acquire and transmit microorganisms over their life cycle. Specifically, the microbiota of immature forms is largely acquired from their aquatic environment. Therefore, our study aimed to describe the microbial communities associated with An. darlingi immature forms and their breeding sites in the Coari municipality, Brazilian Amazon. METHODS: Larvae, pupae, and breeding water were collected in two different geographical locations. Samples were submitted for DNA extraction and high-throughput 16S rRNA gene sequencing was conducted. Microbial ecology analyses were performed to explore and compare the bacterial profiles of An. darlingi and their aquatic habitats. RESULTS: We found lower richness and diversity in An. darlingi microbiota than in water samples, which suggests that larvae are colonized by a subset of the bacterial community present in their breeding sites. Moreover, the bacterial community composition of the immature mosquitoes and their breeding water differed according to their collection sites, i.e., the microbiota associated with An. darlingi reflected that in the aquatic habitats where they developed. The three most abundant bacterial classes across the An. darlingi samples were Betaproteobacteria, Clostridia, and Gammaproteobacteria, while across the water samples they were Gammaproteobacteria, Bacilli, and Alphaproteobacteria. CONCLUSIONS: Our findings reinforce the current evidence that the environment strongly shapes the composition and diversity of mosquito microbiota. A better understanding of mosquito-microbe interactions will contribute to identifying microbial candidates impacting host fitness and disease transmission.

Culturable bacteria associated with Anopheles darlingi and their paratransgenesis potential.

BACKGROUND: Malaria remains a major public health problem in South America, mostly in the Amazon region. Among newly proposed ways of controlling malaria transmission to humans, paratransgenesis is a promising alternative. Paratransgenesis aims to inhibit the development of parasites within the vector through the action of genetically modified bacteria. The first step towards successful paratransgenesis in the Amazon is the identification of Anopheles darlingi symbiotic bacteria, which are transmitted vertically among mosquitoes, and are not pathogenic to humans. METHODS: Culturable bacteria associated with An. darlingi and their breeding sites were isolated by conventional microbiological techniques. Isolated strains were transformed with a GFP expressing plasmid, pSPT-1-GFP, and reintroduced in mosquitoes by feeding. Their survival and persistence in the next generation was assessed by the isolation of fluorescent bacteria from eggs, larvae, pupae and adult homogenates. RESULTS: A total of 179 bacterial strains were isolated from samples from two locations, Coari and Manaus. The predominant genera identified in this study were Acinetobacter, Enterobacter, Klebsiella, Serratia, Bacillus, Elizabethkingia, Stenotrophomonas and Pantoea. Two isolated strains, Serratia-Adu40 and Pantoea-Ovo3, were successfully transformed with the pSPT-1-GFP plasmid and expressed GFP. The fluorescent bacteria fed to adult females were transferred to their eggs, which persisted in larvae and throughout metamorphosis, and were detected in adult mosquitoes of the next generation. CONCLUSION: Serratia-Adu40 and Pantoea-Ovo3 are promising candidates for paratransgenesis in An. darlingi. Further research is needed to determine if these bacteria are vertically transferred in nature.

Vector-Focused Approaches to Curb Malaria Transmission in the Brazilian Amazon: An Overview of Current and Future Challenges and Strategies.

In Brazil, malaria transmission is mostly confined to the Amazon, where substantial progress has been made towards disease control in the past decade. Vector control has been historically considered a fundamental part of the main malaria control programs implemented in Brazil. However, the conventional vector-control tools have been insufficient to control or eliminate local vector populations due to the complexity of the Amazonian rainforest environment and ecological features of malaria vector species in the Amazon, especially Anopheles darlingi. Malaria elimination in Brazil and worldwide eradication will require a combination of conventional and new approaches that takes into account the regional specificities of vector populations and malaria transmission dynamics. Here we present an overview on both conventional and novel promising vector-focused tools to curb malaria transmission in the Brazilian Amazon. If well designed and employed, vector-based approaches may improve the implementation of malaria-control programs, particularly in remote or difficult-to-access areas and in regions where existing interventions have been unable to eliminate disease transmission. However, much effort still has to be put into research expanding the knowledge of neotropical malaria vectors to set the steppingstones for the optimization of conventional and development of innovative vector-control tools.

Characterization of Bacterial Communities in Breeding Waters of Anopheles darlingi in Manaus in the Amazon Basin Malaria-Endemic Area.

The microbiota in mosquito breeding waters can affect ovipositing mosquitoes, have effects on larval development, and can modify adult mosquito-gut bacterial composition. This, in turn, can affect transmission of human pathogens such as malaria parasites. Here, we explore the microbiota of four breeding sites for Anopheles darlingi, the most important malaria vector in Latin America. The sites are located in Manaus in the Amazon basin in Brazil, an area of active malaria transmission. Using 16S rRNA gene sequencing by MiSeq, we found that all sites were dominated by Proteobacteria and Firmicutes and that 94% of the total number of reads belonged to 36 operational taxonomic units (OTUs) identified in all sites. Of these, the most common OTUs belonged to Escherichia/Shigella, Staphylococcus, and Pseudomonas. Of the remaining 6% of the reads, the OTUs found to differentiate between the four sites belonged to the orders Burkholderiales, Actinomycetales, and Clostridiales. We conclude that An. darlingi can develop in breeding waters with different surface-water bacteria, but that the common microbiota found in all breeding sites might indicate or contribute to a suitable habitat for this important malaria vector.