Elerson Matos Rocha1, Osvaldo Marinotti2, Deidre Machado Serrão3, Laura Viana Correa3, Ricardo de Melo Katak1, Juan Campos de Oliveira1, Veranilce Alves Muniz3, Marta Rodrigues de Oliveira4, Joaquim Ferreira do Nascimento Neto5, Marcos Cézar Fernandes Pessoa1, Rosemary Aparecida Roque5, Adolfo Jose da Mota1, Piero Onorati6, Jayme A Souza-Neto7, Olle Terenius8,9, Wanderli Pedro Tadei10. 1. Programa de Pós-Graduação em Biotecnologia, Universidade Federal do Amazonas - PPGBIOTEC / UFAM, Manaus, Brazil. 2. MTEKPrime, Aliso Viejo, CA, USA. 3. Universidade Estadual do Amazonas - MBT, UEA, Manaus, Brazil. 4. Universidade Estadual do Amazonas - BIONORTE, UEA, Manaus, Brazil. 5. Laboratório de Malária E Dengue, Instituto Nacional de Pesquisas da Amazônia, INPA, Manaus, Brazil. 6. Department of Ecology, Swedish University of Agricultural Sciences (SLU), Box 7044, 750 07, Uppsala, Sweden. 7. School of Agricultural Sciences, Department of Bioprocesses and Biotechnology, Central Multi User Laboratory, São Paulo State University (UNESP), Botucatu, Brazil. 8. Department of Ecology, Swedish University of Agricultural Sciences (SLU), Box 7044, 750 07, Uppsala, Sweden. olle.terenius@icm.uu.se. 9. Department of Cell and Molecular Biology, Microbiology, Uppsala University, Box 596, 751 24, Uppsala, Sweden. olle.terenius@icm.uu.se. 10. Laboratório de Malária E Dengue, Instituto Nacional de Pesquisas da Amazônia, INPA, Manaus, Brazil. wptadei@gmail.com.
Abstract
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.
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.
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