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.

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.

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.

A re-annotation of the Anopheles darlingi mobilome.

The mobilome, portion of the genome composed of transposable elements (TEs), of Anopheles darlingi was described together with the genome of this species. Here, this mobilome was revised using similarity and de novo search approaches. A total of 5.6% of the A. darlingi genome is derived of TEs. Class I gypsy and copia were the most abundant superfamilies, corresponding to 22.36% of the mobilome. Non-LTR elements of the R1 and Jockey superfamilies account for 11% of the TEs. Among Class II TEs, the mariner superfamily is the most abundant (16.01%). Approximately 87% of the A. darlingi mobilome consist of short, truncated and/or degenerated copies of TEs. Only three retrotransposons, two belonging to gypsy and one to copia superfamilies, are putatively active elements. Only one Class II element, belonging to the mariner superfamily, is putatively active, having 12 copies in the genome. The TE landscape of A. darlingi is formed primarily by degenerated elements and, therefore, somewhat stable. Future applications of TE-based vectors for genetic transformation of A. darlingi should take into consideration mariner and piggyBac transposons, because full length and putatively active copies of these elements are present in its genome.

Collagen-binding protein, Aegyptin, regulates probing time and blood feeding success in the dengue vector mosquito, Aedes aegypti.

Mosquito salivary glands have important roles in blood feeding and pathogen transmission. However, the biological relevance of many salivary components has yet to be determined. Aegyptin, a secreted salivary protein from Aedes aegypti, binds collagen and inhibits platelet aggregation and adhesion. We used a transgenic approach to study the relevance of Aegyptin in mosquito blood feeding. Aedes aegypti manipulated genetically to express gene-specific inverted-repeat RNA sequences exhibited significant reductions in Aegyptin mRNA accumulation (85-87%) and protein levels (>80-fold) in female mosquito salivary glands. Transgenic mosquitoes had longer probing times (78-300 s, P < 0.0001) when feeding on mice compared with controls (15-56 s), feeding success was reduced, and those feeding took smaller blood meals. However, no differences in feeding success or blood meal size were found in membrane feeding experiments using defibrinated human blood. Salivary gland extracts from transgenic mosquitoes failed to inhibit collagen-induced platelet aggregation in vitro. Reductions of Aegyptin did not affect salivary ADP-induced platelet aggregation inhibition or disturb anticlotting activities. Our results demonstrate the relevance of Aegyptin for A. aegypti blood feeding, providing further support for the hypothesis that platelet aggregation inhibition is a vital salivary function in blood feeding arthropods. It has been suggested that the multiple mosquito salivary components mediating platelet aggregation (i.e., Aegyptin, apyrase, D7) represent functional redundancy. Our findings do not support this hypothesis; instead, they indicate that multiple salivary components work synergistically and are necessary to achieve maximum blood feeding efficiency.

Coetzeea brasiliensis gen. nov., sp. nov. isolated from larvae of Anopheles darlingi.

A Gram-stain-negative, rod-shaped strain, Braz8T, isolated from larvae of Anopheles darlingi was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Braz8T was related most closely to species of the genus Thorsellia, with 95.6, 96.5 and 96.6 % similarity to the type strains of Thorsellia anophelis, Thorsellia kandunguensis and Thorsellia kenyensis, respectively, and formed a separate branch in the phylogenetic tree next to the monophyletic cluster of the genus Thorsellia. Chemotaxonomic data supported the allocation of the strain to the family Thorselliaceae. The major fatty acids were C18 : 1ω7c, C16 : 0 and C14 : 0. The quinone system was composed of ubiquinones Q-8 and Q-7 (1 : 0.3), the predominant polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and the polyamine pattern showed the major compound putrescine. However, qualitative and quantitative differences in the major polyamine, polar lipid profile and fatty acid patterns distinguished strain Braz8T from species of the genus Thorsellia. Phylogenetic analysis based on 16S rRNA gene sequences, average nucleotide identity, DNA-DNA hybridization, multilocus sequence analysis as well as physiological and biochemical tests distinguished strain Braz8T both genotypically and phenotypically from the three Thorsellia species but also showed its placement in the family Thorselliaceae. Thus, strain Braz8T is considered to represent a novel species of a new genus most closely related to the genus Thorsellia, for which the name Coetzeea brasiliensis gen. nov., sp. nov. is proposed. The type strain of Coetzeea brasiliensis is Braz8T (=LMG 29552T=CIP 111088T).

Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands.

BACKGROUND: The circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration. METHODS: Peptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system. RESULTS: Transient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum. CONCLUSION: These results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.

The genome of Anopheles darlingi, the main neotropical malaria vector.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

Integrated proteomic and transcriptomic analysis of the Aedes aegypti eggshell.

BACKGROUND: Mosquito eggshells show remarkable diversity in physical properties and structure consistent with adaptations to the wide variety of environments exploited by these insects. We applied proteomic, transcriptomic, and hybridization in situ techniques to identify gene products and pathways that participate in the assembly of the Aedes aegypti eggshell. Aedes aegypti population density is low during cold and dry seasons and increases immediately after rainfall. The survival of embryos through unfavorable periods is a key factor in the persistence of their populations. The work described here supports integrated vector control approaches that target eggshell formation and result in Ae. aegypti drought-intolerant phenotypes for public health initiatives directed to reduce mosquito-borne diseases. RESULTS: A total of 130 proteins were identified from the combined mass spectrometric analyses of eggshell preparations. CONCLUSIONS: Classification of proteins according to their known and putative functions revealed the complexity of the eggshell structure. Three novel Ae. aegypti vitelline membrane proteins were discovered. Odorant-binding and cysteine-rich proteins that may be structural components of the eggshell were identified. Enzymes with peroxidase, laccase and phenoloxidase activities also were identified, and their likely involvements in cross-linking reactions that stabilize the eggshell structure are discussed.

Probing functional polymorphisms in the dengue vector, Aedes aegypti.

BACKGROUND: Dengue is the most prevalent arboviral disease world-wide and its primary vector is the mosquito Aedes aegypti. The current lack of commercially-available vaccines makes control of vector populations the only effective strategy to prevent dengue transmission. Aedes aegypti geographic populations exhibit great variability in insecticide resistance and susceptibility to dengue infection. The characterization of single nucleotide polymorphisms (SNPs) as molecular markers to study quantitatively this variation is needed greatly because this species has a low abundance of microsatellite markers and limited known restriction fragments length polymorphisms (RFLPs) and single-strand conformation polymorphism (SSCP) markers. RESULTS: We used RNA-seq to characterize SNPs in three Ae. aegypti strains, including the Liverpool (LVP) strain, from which the current genome annotation is derived. We identified 131,764 unique genome locations with at least one alternative nucleotide to what is reported in the reference annotation. These comprised changes in both open-reading frames (ORFs) and untranslated regions (UTRs) of transcripts. An in depth-look at sequence variation in immunity genes revealed that those associated with autophagy, MD2-like receptors and Peptidoglycan Recognition Proteins had more sequence variation in their 3'UTRs than mutations associated with non-synonymous changes. This supports the conclusion that these genes had maintained their functional specificity while being adapted to different regulatory domains. In contrast, a number of peroxidases, serpins and Clip-domain serine proteases exhibited conservation of putative UTR regulatory sequences while displaying diversification of the ORFs. Transcriptome evidence also was found for ~2500 novel transcriptional units (NTUs) not annotated in the reference genome. CONCLUSIONS: The transcriptome-wide assessment of within and inter-strain polymorphisms in Ae. aegypti adds considerably to the number of molecular markers available for genetic studies in this mosquito. Additionally, data supporting NTU discovery emphasizes the need for continuous amendments of the reference genome annotation.

Engineered resistance to Plasmodium falciparum development in transgenic Anopheles stephensi.

Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs) designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.

Development of a population suppression strain of the human malaria vector mosquito, Anopheles stephensi.

BACKGROUND: Transgenic mosquito strains are being developed to contribute to the control of dengue and malaria transmission. One approach uses genetic manipulation to confer conditional, female-specific dominant lethality phenotypes. Engineering of a female-specific flightless phenotype provides a sexing mechanism essential for male-only mosquito, release approaches that result in population suppression of target vector species. METHODS: An approach that uses a female-specific gene promoter and antibiotic-repressible lethal factor to produce a sex-specific flightless phenotype was adapted to the human malaria vector, Anopheles stephensi. Transposon- and site-specific recombination-mediated technologies were used to generate a number of transgenic An. stephensi lines that when combined through mating produced the phenotype of flight-inhibited females and flight-capable males. RESULTS: The data shown here demonstrate the successful engineering of a female-specific flightless phenotype in a malaria vector. The flightless phenotype was repressible by the addition of tetracycline to the larval diet. This conditional phenotype allows the rearing of the strains under routine laboratory conditions. The minimal level of tetracycline that rescues the flightless phenotype is higher than that found as an environmental contaminant in circumstances where there is intensive use of antibiotics. CONCLUSIONS: These studies support the further development of flightless female technology for applications in malaria control programmes that target the vectors.

Female-specific flightless phenotype for mosquito control.

Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50-100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A "sterile-male-release" strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release ("genetic sexing"), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.

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.

Comparative genomics allows the discovery of cis-regulatory elements in mosquitoes.

The discovery and mapping of cis-regulatory elements is important for understanding regulation of gene transcription in mosquito vectors of human diseases. Genome sequence data are available for 3 species, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus (Diptera: Culicidae), representing 2 subfamilies (Culicinae and Anophelinae) that are estimated to have diverged 145 to 200 million years ago. Comparative genomics tools were used to screen genomic DNA fragments located in the 5'-end flanking regions of orthologous genes. These analyses resulted in the identification of 137 sequences, designated "mosquito motifs," 7 to 9 nucleotides in length, representing 18 families of putative cis-regulatory elements conserved significantly among the 3 species when compared to the fruit fly, Drosophila melanogaster. Forty-one of the motifs were implicated previously in experiments as sites for binding transcription factors or functioning in the regulation of mosquito gene expression. Further analyses revealed associations between specific motifs and expression profiles, particularly in those genes that show increased or decreased mRNA abundance in females following a blood meal, and those accumulating transcription products exclusively or preferentially in the midgut, fat bodies, or ovaries. These results validate the methodology and support a relationship between the discovered motifs and the conservation of hematophagy in mosquitoes.

RNA-seq analyses of blood-induced changes in gene expression in the mosquito vector species, Aedes aegypti.

BACKGROUND: Hematophagy is a common trait of insect vectors of disease. Extensive genome-wide transcriptional changes occur in mosquitoes after blood meals, and these are related to digestive and reproductive processes, among others. Studies of these changes are expected to reveal molecular targets for novel vector control and pathogen transmission-blocking strategies. The mosquito Aedes aegypti (Diptera, Culicidae), a vector of Dengue viruses, Yellow Fever Virus (YFV) and Chikungunya virus (CV), is the subject of this study to look at genome-wide changes in gene expression following a blood meal. RESULTS: Transcriptional changes that follow a blood meal in Ae. aegypti females were explored using RNA-seq technology. Over 30% of more than 18,000 investigated transcripts accumulate differentially in mosquitoes at five hours after a blood meal when compared to those fed only on sugar. Forty transcripts accumulate only in blood-fed mosquitoes. The list of regulated transcripts correlates with an enhancement of digestive activity and a suppression of environmental stimuli perception and innate immunity. The alignment of more than 65 million high-quality short reads to the Ae. aegypti reference genome permitted the refinement of the current annotation of transcript boundaries, as well as the discovery of novel transcripts, exons and splicing variants. Cis-regulatory elements (CRE) and cis-regulatory modules (CRM) enriched significantly at the 5'end flanking sequences of blood meal-regulated genes were identified. CONCLUSIONS: This study provides the first global view of the changes in transcript accumulation elicited by a blood meal in Ae. aegypti females. This information permitted the identification of classes of potentially co-regulated genes and a description of biochemical and physiological events that occur immediately after blood feeding. The data presented here serve as a basis for novel vector control and pathogen transmission-blocking strategies including those in which the vectors are modified genetically to express anti-pathogen effector molecules.

What is in a name? Anopheles darlingi versus Nyssorhynchus darlingi.

Taxonomic reassignments were suggested for Neotropical anopheline malaria vectors, elevating four monophyletic groups Kerteszia, Lophopodomyia, Nyssorhynchus, and Stethomyia to the genus level, upending their conventional status as subgenera of the genus Anopheles. Two questions are proposed. Do the advantages of reclassification outweigh its disadvantages? Is the reclassification generally accepted and/or scientifically imperative?

Extracts of Amazonian Fungi With Larvicidal Activities Against Aedes aegypti.

The global increase in diseases transmitted by the vector Aedes aegypti, new and re-emerging, underscores the need for alternative and more effective methods of controlling mosquitoes. Our aim was to identify fungal strains from the Amazon rain forest that produce metabolites with larvicidal activity against Aedes aegypti. Thirty-six fungal strains belonging to 23 different genera of fungi, isolated from water samples collected in the state of Amazonas, Brazil were cultivated. The liquid medium was separated from the mycelium by filtration. Medium fractions were extracted with ethyl acetate and isopropanol 9:1 volume:volume, and the mycelia with ethyl acetate and methanol 1:1. The extracts were vacuum dried and the larvicidal activity was evaluated in selective bioassays containing 500 µg/ml of the dried fungal extracts. Larval mortality was evaluated up to 72 h. None of the mycelium extracts showed larvicidal activity greater than 50% at 72 h. In contrast, 15 culture medium extracts had larvicidal activity equal to or greater than 50% and eight killed more than 90% of the larvae within 72 h. These eight extracts from fungi belonging to seven different genera (Aspergillus, Cladosporium, Trichoderma, Diaporthe, Albifimbria, Emmia, and Sarocladium) were selected for the determination of LC50 and LC90. Albifimbria lateralis (1160) medium extracts presented the lowest LC50 value (0.268 µg/ml) after 24 h exposure. Diaporthe ueckerae (1203) medium extracts presented the lowest value of LC90 (2.928 µg/ml) at 24 h, the lowest values of LC50 (0.108 µg/ml) and LC90 (0.894 µg/ml) at 48 h and also at 72 h (LC50 = 0.062 µg/ml and LC90 = 0.476 µg/ml). Extracts from Al. lateralis (1160) and D. ueckerae (1203) showed potential for developing new, naturally derived products, to be applied in integrated vector management programs against Ae. aegypti.