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.

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.

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?

Larvicidal Activities against Aedes aegypti of Supernatant and Pellet Fractions from Cultured Bacillus spp. Isolated from Amazonian Microenvironments.

The Aedes aegypti mosquito is the primary vector of Dengue, Chikungunya and Zika causing major problems for public health, which requires new strategies for its control, like the use of entomopathogenic microorganisms. In this study, bacteria from various Amazonian environments were isolated and tested for their pathogenicity to A. aegypti larvae. Following thermal shock to select sporulated Bacillus spp., 77 bacterial strains were isolated. Molecular identification per 16S RNA sequences revealed that the assembled strains contained several species of the genus Bacillus and one species each of Brevibacillus, Klebsiella, Serratia, Achromobacter and Brevundimonas. Among the isolated Bacillus sp. strains, 19 showed larvicidal activity against A. aegypti. Two strains of Brevibacillus halotolerans also displayed larvicidal activity. For the first time, larvicidal activity against A. aegypti was identified for a strain of Brevibacillus halotolerans. Supernatant and pellet fractions of bacterial cultures were tested separately for larvicidal activities. Eight strains contained isolated fractions resulting in at least 50% mortality when tested at a concentration of 5 mg/mL. Further studies are needed to characterize the active larvicidal metabolites produced by these microorganisms and define their mechanisms of action.

Physical Mapping of the Anopheles (Nyssorhynchus) darlingi Genomic Scaffolds.

The genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.

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.

Differentiating Full-Spectrum Hemp Extracts from CBD Isolates: Implications for Policy, Safety and Science.

The passage of the 2018 United States Agriculture Improvement Act removed industrial hemp, defined as Cannabis sativa L. containing less than 0.3% THC content by dry weight, from Schedule I of the Controlled Substances Act and made it an agricultural commodity. Following these changes, the popularity of hemp-derived cannabidiol (CBD) dietary supplements by consumers has greatly exceeded the scientific understanding of purported benefits, safety and composition of these botanical extracts. Further complicating CBD hemp supplement regulation, Food and Drug Administration (FDA) considers CBD to be an approved drug (Epidiolex) in the treatment of severe epilepsy disorders, Dravet and Lennox-Gastaut syndromes. At the same time, hemp-derived CBD supplements can contain a complex phytochemical matrix from the hemp plant, distinguishing the composition of these products from isolated CBD preparations. This work aims to provide clarity on differentiating botanical full-spectrum hemp extracts containing CBD from isolates, from a phytochemical, toxicological and regulatory perspective.

Post Marketing Safety of Plus CBD™ Products, a Full Spectrum Hemp Extract: A 2-Year Experience.

The market for products featuring hemp extracts is large and growing larger. However, safety concerns have been raised by medical and regulatory agencies. Post marketing surveillance of full spectrum hemp extract (FSHE) products manufactured and distributed by CV Sciences (CVSI) and traded under the brand PlusCBD™ was conducted over a 2-year period (2018-2019). The safety of these products was assessed by analyzing adverse events reports. From a total of approximately five million product units sold during the 2-year period, 1,429 (0.03%) adverse events (AE) were reported in 1,151 unique customers. Of those, only two were classified as serious AEs. For orally ingested products, the most common types of AEs reported were gastrointestinal (e.g. abdominal discomfort), while for topically applied products, the most reports mentioned dermatological symptoms (e.g. rashes). There has been no evidence of liver toxicity associated with CVSI products. Based on this longitudinal dataset, the products manufactured using CVSI's proprietary processes are safe and well tolerated at the recommended doses.

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.

nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito, Anopheles stephensi.

Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so.

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.

Transcriptome sequencing and developmental regulation of gene expression in Anopheles aquasalis.

BACKGROUND: Anopheles aquasalis is a major malaria vector in coastal areas of South and Central America where it breeds preferentially in brackish water. This species is very susceptible to Plasmodium vivax and it has been already incriminated as responsible vector in malaria outbreaks. There has been no high-throughput investigation into the sequencing of An. aquasalis genes, transcripts and proteins despite its epidemiological relevance. Here we describe the sequencing, assembly and annotation of the An. aquasalis transcriptome. METHODOLOGY/PRINCIPAL FINDINGS: A total of 419 thousand cDNA sequence reads, encompassing 164 million nucleotides, were assembled in 7544 contigs of ≥ 2 sequences, and 1999 singletons. The majority of the An. aquasalis transcripts encode proteins with their closest counterparts in another neotropical malaria vector, An. darlingi. Several analyses in different protein databases were used to annotate and predict the putative functions of the deduced An. aquasalis proteins. Larval and adult-specific transcripts were represented by 121 and 424 contig sequences, respectively. Fifty-one transcripts were only detected in blood-fed females. The data also reveal a list of transcripts up- or down-regulated in adult females after a blood meal. Transcripts associated with immunity, signaling networks and blood feeding and digestion are discussed. CONCLUSIONS/SIGNIFICANCE: This study represents the first large-scale effort to sequence the transcriptome of An. aquasalis. It provides valuable information that will facilitate studies on the biology of this species and may lead to novel strategies to reduce malaria transmission on the South American continent. The An. aquasalis transcriptome is accessible at http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx.

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.