Effects of diflubenzuron on associated insect fauna with Anopheles(Diptera: Culicidae) in laboratory, partial-field, and field conditions in the Central Amazon.

This study aimed to analyze the possible effects of diflubenzuron on the associated insect fauna under laboratory, semi-field and field conditions. Laboratory bioassays were performed in aquariums with mortality readings (%) every 24h until 96h, and in semi-fields, insects were kept in basins with readings every 24h for up to 12 days, in triplicates and a control. In the field experiment, a collection was performed before the application of diflubenzuron in ten brick factory pits (25m²) and 15 post-application. The values of LC50 and LC90 for Chironomus were 2.77x10-3g/L and 0.86g/L, respectively, and for Buenoa, they were 0.019g/L and 0.92g/L, a strong relationship was observed between mortality and exposure time (r²>0.8) in all concentrations used. In semi-field, similar mean values of emergency inhibition were observed, except for Euthyplociidae, which was more sensitive. There was no significant difference between the data of richness and diversity of aquatic insects in the field experiment, considering an interval of 15 days (p=0.32). Finally, the data suggest that diflubenzuron may have a negative effect on the associated insect fauna, but in the field experiment the environmental conditions of Anopheles breeding sites may have affected its efficiency.

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.

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).

Anopheles darlingi polytene chromosomes: revised maps including newly described inversions and evidence for population structure in Manaus.

Salivary gland polytene chromosomes of 4th instar Anopheles darlingi Root were examined from multiple locations in the Brazilian Amazon. Minor modifications were made to existing polytene photomaps. These included changes to the breakpoint positions of several previously described paracentric inversions and descriptions of four new paracentric inversions, two on the right arm of chromosome 3 and two on the left arm of chromosome 3 that were found in multiple locations. A total of 18 inversions on the X (n = 1) chromosome, chromosome 2 (n = 7) and 3 (n = 11) were scored for 83 individuals from Manaus, Macapá and Porto Velho municipalities. The frequency of 2Ra inversion karyotypes in Manaus shows significant deficiency of heterozygotes (p < 0.0009). No significant linkage disequilibrium was found between inversions on chromosome 2 and 3. We hypothesize that at least two sympatric subpopulations exist within the An. darlingi population at Manaus based on inversion frequencies.

In vitro and in vivo anti-malarial activity of plants from the Brazilian Amazon.

BACKGROUND: The anti-malarials quinine and artemisinin were isolated from traditionally used plants (Cinchona spp. and Artemisia annua, respectively). The synthetic quinoline anti-malarials (e.g. chloroquine) and semi-synthetic artemisinin derivatives (e.g. artesunate) were developed based on these natural products. Malaria is endemic to the Amazon region where Plasmodium falciparum and Plasmodium vivax drug-resistance is of concern. There is an urgent need for new anti-malarials. Traditionally used Amazonian plants may provide new treatments for drug-resistant P. vivax and P. falciparum. Herein, the in vitro and in vivo antiplasmodial activity and cytotoxicity of medicinal plant extracts were investigated. METHODS: Sixty-nine extracts from 11 plant species were prepared and screened for in vitro activity against P. falciparum K1 strain and for cytotoxicity against human fibroblasts and two melanoma cell lines. Median inhibitory concentrations (IC50) were established against chloroquine-resistant P. falciparum W2 clone using monoclonal anti-HRPII (histidine-rich protein II) antibodies in an enzyme-linked immunosorbent assay. Extracts were evaluated for toxicity against murine macrophages (IC50) and selectivity indices (SI) were determined. Three extracts were also evaluated orally in Plasmodium berghei-infected mice. RESULTS: High in vitro antiplasmodial activity (IC50 = 6.4-9.9 µg/mL) was observed for Andropogon leucostachyus aerial part methanol extracts, Croton cajucara red variety leaf chloroform extracts, Miconia nervosa leaf methanol extracts, and Xylopia amazonica leaf chloroform and branch ethanol extracts. Paullinia cupana branch chloroform extracts and Croton cajucara red variety leaf ethanol extracts were toxic to fibroblasts and or melanoma cells. Xylopia amazonica branch ethanol extracts and Zanthoxylum djalma-batistae branch chloroform extracts were toxic to macrophages (IC50 = 6.9 and 24.7 µg/mL, respectively). Andropogon leucostachyus extracts were the most selective (SI >28.2) and the most active in vivo (at doses of 250 mg/kg, 71% suppression of P. berghei parasitaemia versus untreated controls). CONCLUSIONS: Ethnobotanical or ethnopharmacological reports describe the anti-malarial use of these plants or the antiplasmodial activity of congeneric species. No antiplasmodial activity has been demonstrated previously for the extracts of these plants. Seven plants exhibit in vivo and or in vitro anti-malarial potential. Future work should aim to discover the anti-malarial substances present.

Antiplasmodial activity of synthetic ellipticine derivatives and an isolated analog.

Ellipticine has been shown previously to exhibit excellent in vitro antiplasmodial activity and in vivo antimalarial properties that are comparable to those of the control drug chloroquine in a mouse malaria model. Ellipticine derivatives and analogs exhibit antimalarial potential however only a few have been studied to date. Herein, ellipticine and a structural analog were isolated from Aspidosperma vargasii bark. A-ring brominated and nitrated ellipticine derivatives exhibit good in vitro inhibition of Plasmodium falciparum K1 and 3D7 strains. Several of the compounds were found not to be toxic to human fetal lung fibroblasts. 9-Nitroellipticine (IC50=0.55µM) exhibits greater antiplasmodial activity than ellipticine. These results are further evidence of the antimalarial potential of ellipticine derivatives.

Experimental Plasmodium vivax infection of key Anopheles species from the Brazilian Amazon.

BACKGROUND: Anopheles darlingi is the major malaria vector in countries located in the Amazon region. Anopheles aquasalis and Anopheles albitarsis s.l. are also proven vectors in this region. Anopheles nuneztovari s.l. and Anopheles triannulatus s.l. were found infected with Plasmodium vivax; however, their status as vectors is not yet well defined. Knowledge of susceptibility of Amazon anopheline populations to Plasmodium infection is necessary to better understand their vector capacity. Laboratory colonization of An. darlingi, the main Amazon vector, has proven to be difficult and presently An. aquasalis is the only available autonomous colony. METHODS: Larvae of An. darlingi, An. albitarsis s.l., An. nuneztovari s.l. and An. triannulatus s.l. were collected in the field and reared until adult stage. Adults of An. aquasalis were obtained from a well-established colony. Mosquitoes were blood-fed using a membrane-feeding device containing infected blood from malarial patients.The infection of the distinct Anopheles species was evaluated by the impact variance of the following parameters: (a) parasitaemia density; (b) blood serum inactivation of the infective bloodmeal; (c) influence of gametocyte number on infection rates and number of oocysts. The goal of this work was to compare the susceptibility to P. vivax of four field-collected Anopheles species with colonized An. aquasalis. RESULTS: All Anopheles species tested were susceptible to P. vivax infection, nevertheless the proportion of infected mosquitoes and the infection intensity measured by oocyst number varied significantly among species. Inactivation of the blood serum prior to mosquito feeding increased infection rates in An. darlingi and An. triannulatus s.l., but was diminished in An. albitarsis s.l. and An. aquasalis. There was a positive correlation between gametocyte density and the infection rate in all tests (Z = -8.37; p < 0.001) but varied among the mosquito species. Anopheles albitarsis s.l., An. aquasalis and An. nuneztovari s.l. had higher infection rates than An. darlingi. CONCLUSION: All field-collected Anopheles species, as well as colonized An. aquasalis are susceptible to experimental P. vivax infections by membrane feeding assays. Anopheles darlingi, An. albitarsis s.l. and An. aquasalis are very susceptible to P. vivax infection. However, colonized An. aquasalis mosquitoes showed the higher infection intensity represented by infection rate and oocyst numbers. This study is the first to characterize experimental development of Plasmodium infections in Amazon Anopheles vectors and also to endorse that P. vivax infection of colonized An. aquasalis is a feasible laboratory model.

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.

Integrated vector management targeting Anopheles darlingi populations decreases malaria incidence in an unstable transmission area, in the rural Brazilian Amazon.

BACKGROUND: Studies on vector behaviour should be conducted in order to evaluate the effectiveness of vector control measures on malaria protection in endemic areas of Latin America, where P. vivax predominates. This work aims to investigate the fauna of anopheline mosquitoes and verify the impact of integrated vector management in two colonization projects in the Careiro Municipality, Western Brazilian Amazon. METHODS: Four mosquitoes' captures were carried out from August 2008 to March 2010, with an interval of six months between each collection. Since September 2009 a large programme to reduce the burden of malaria has started in the two communities by distribution of insecticide-treated bed nets (ITN) and intensification of indoor residual spraying (IRS). Human biting rates (HBRs), entomological inoculation rates (EIRs), malaria incidence rate (MIR) and Plasmodium carrier's prevalence were used as outcomes to estimate the impact of the control measures. RESULTS: A total of 3,189 anophelines were collected, belonging to 13 species. Anopheles darlingi was the predominant species in the period (42.6%), followed by Anopheles albitarsis (38.4%). An. darlingi HBRs showed a notable decreasing trend from the start to the end of the study. Conversely, An. albitarsis increased its contribution to overall HBRs throughout the study. For An. darlingi there was a significant positive correlation between HBRs and MIR (p = 0.002). Anopheles albitarsis HBRs showed a significant negative correlation with the corresponding MIR (p = 0.045). EIR from total anophelines and from An. darlingi and An. albitarsis presented decreasing patterns in the successive collections. Four species of anophelines (An. darlingi, An. albitarsis, Anopheles braziliensis and Anopheles nuneztovari) were naturally infected with Plasmodium, albeit at very low infection rates. There were a decrease in the MIR for both vivax and falciparum malaria and in the prevalence of Plasmodium vivax and Plasmodium falciparum carriers during the period of study. CONCLUSIONS: There is strong evidence of association between the density of An. darlingi and the incidence of malaria in the studies sites, further highlighting the importance of this vector in malaria transmission in this region. An. darlingi susceptibility to control using ITN and IRS is likely to be high in the rural settlements studied.

Cyt1Aa toxin gene frequency in Bacillus thuringiensis isolates and its relation with pathogenicity for vector mosquitoes.

Bacillus thuringiensis produces several virulence factors, the main ones being the Cry and Cyt toxins, present in the parasporal body produced during sporulation. The Cyt toxins have mechanisms specific for mosquitoes and Cyt1Aa, the most studied cytolytic toxin, is effective for mosquito control by acting in synergism with Cry toxins. The goal of the present work was to study the frequency of the codifying gene for Cyt1Aa in B. thuringiensis native isolates acquired from samples of soil, insect and water, as well as to verify any possible genetic polymorphism. 1,448 B thuringiensis strains were used for DNA extraction and PCR technique, all with the use of a primer that amplifies a fragment of 300 pairs of the cyt1Aa gene. The strains that showed amplification in the PCR reaction were sequenced and compared to each other and to the sequences available at Genbank. 32 (2.3%) strains of B. thuringiensis showed positive amplification for the cyt1Aa gene. The highest frequency of isolates with cyt1Aa gene was acquired from samples coming from the Cerrado biome, both isolates from soil and from insects, equally with 3.4%. The cyt1Aa gene sequencing highlighted that, for that 300 bp region, the gene is conserved and there is no single-base polymorphism.

Essential oil of Piper purusanum C.DC (Piperaceae) and its main sesquiterpenes: biodefensives against malaria and dengue vectors, without lethal effect on non-target aquatic fauna.

The mosquito vectors of the genera Aedes and Anopheles present resistance to several commercial insecticides, which are also toxic to non-predator targets. On the other hand, essential oils are a promising source of insecticides. Thus, in this work, the essential oil from the leaves of Piper purusanum was characterized by gas chromatography-based approaches and evaluated as biodefensive against malaria and dengue vectors. The main compounds of P. purusanum essential oil were ß-caryophyllene (57.05%), α-humulene (14.50%), and germacrene D (8.20%). The essential oil inhibited egg hatching (7.6 ± 1.5 to 95.6 ± 4.5%), caused larval death (LC50 from 49.84 to 51.60 ppm), and inhibited the action of acetylcholinesterase (IC50 of 2.29 µg/mL), which can be related to the mechanisms of action. On the other hand, the biological activities of ß-caryophyllene, α-humulene, and germacrene D were higher than that of essential oil. In addition, these sesquiterpenes and essential oil did not show a lethal effect on Toxorhynchites splendens, Anisops bouvieri, Gambusia affinis, and Diplonychus indicus (LC50 from 2098.80 to 7707.13 ppm), although D. indicus is more sensitive (SI/PSF from 48.56 to 252.02 ppm) to essential oil, representing a natural alternative against these relevant vectors.

Identification of a fibrinogen-related protein (FBN9) gene in neotropical anopheline mosquitoes.

BACKGROUND: Malaria has a devastating impact on worldwide public health in many tropical areas. Studies on vector immunity are important for the overall understanding of the parasite-vector interaction and for the design of novel strategies to control malaria. A member of the fibrinogen-related protein family, fbn9, has been well studied in Anopheles gambiae and has been shown to be an important component of the mosquito immune system. However, little is known about this gene in neotropical anopheline species. METHODS: This article describes the identification and characterization of the fbn9 gene partial sequences from four species of neotropical anopheline primary and secondary vectors: Anopheles darlingi, Anopheles nuneztovari, Anopheles aquasalis, and Anopheles albitarsis (namely Anopheles marajoara). Degenerate primers were designed based on comparative analysis of publicly available Aedes aegypti and An. gambiae gene sequences and used to clone putative homologs in the neotropical species. Sequence comparisons and Bayesian phylogenetic analyses were then performed to better understand the molecular diversity of this gene in evolutionary distant anopheline species, belonging to different subgenera. RESULTS: Comparisons of the fbn9 gene sequences of the neotropical anophelines and their homologs in the An. gambiae complex (Gambiae complex) showed high conservation at the nucleotide and amino acid levels, although some sites show significant differentiation (non-synonymous substitutions). Furthermore, phylogenetic analysis of fbn9 nucleotide sequences showed that neotropical anophelines and African mosquitoes form two well-supported clades, mirroring their separation into two different subgenera. CONCLUSIONS: The present work adds new insights into the conserved role of fbn9 in insect immunity in a broader range of anopheline species and reinforces the possibility of manipulating mosquito immunity to design novel pathogen control strategies.

Chemical composition and larvicidal activity of the essential oil from the leaves of Onychopetalum periquino (Rusby) D.M. Johnson & N.A. Murray.

The essential oil (EO) from the leaves of Onychopetalum periquino, obtained by hydrodistillation, was analyzed by gas chromatography coupled with mass spectrometry (GC-MS), and also was investigated for its larvicidal activity against Aedes aegypti larvae. Thirteen compounds, representing 91.31% of the crude oil, were identified. Major compounds were sesquiterpenes, including ß-elemene (53.16%), spathulenol (11.94%) and ß-selinene (9.25%). The EO showed high larvicidal activity with a lethal concentration (LC50) of 63.75 µg/mL and 100% mortality at 200 µg/mL. These results represent the first report about the chemical composition of O. periquino and the first larvicidal evaluation with Onychopetalum species.[Figure: see text].

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.

Impact on cholinesterase-inhibition and in silico investigations of sesquiterpenoids from Amazonian Siparuna guianensis Aubl.

The plant popularly known as "negramina" (Siparuna guianensis Aubl.), member of the family Siparunaceae produces an essential oil that presents several biological activities reported in literature. Here, the essential oil was obtained by hydrodistillation from fresh leaves collected in the state of Roraima, far north of the Amazon. Chemical composition of the essential oil was characterized by gas chromatography coupled to mass spectrometry (GC-MS) and flame ionization detector (GC-FID). The sesquiterpenoid shyobunone and its derivatives were identified as major compounds in the oil (>40%). The effect of S. guianensis essential oil on the acetylcholinesterase (AChE) activity from Crassostrea rhizophorae, Litopenaeus vannamei and Electrophorus electricus was tested by spectrophotometric assays. The essential oil has been identified as an AChE inhibitor. The mechanism of inhibition was investigated as well as spectrofluorimetric interactions between the essential oil and the enzyme. 1H NMR titration and molecular docking were also investigated. The spectrophotometric results revealed that shyobunone and its derivatives strongly interact with AChE with a kind of non-competitive inhibition. Interaction studies support the results of enzyme inhibition. Molecular coupling predicted that iso-shyobunone is the strongest ligand, corroborated by fluorescence suppression and 1H NMR titration results. In conclusion, Siparuna guianensis essential oil can be a new source of shyobunone and derivatives capable to reversibly inhibit AChE showing potential neuroprotective properties to be applied in the treatment of Alzheimer's disease.

Piper capitarianum essential oil: a promising insecticidal agent for the management of Aedes aegypti and Aedes albopictus.

Mosquitoes are responsible for serious public health problems worldwide, and as such, Aedes aegypti and Aedes albopictus are important vectors in the transmission of dengue, chikungunya, and Zika in Brazil and other countries of the world. Due to growing resistance to chemical insecticides among populations of vectors, environmentally friendly strategies for vector management are receiving ever more attention. Essential oils (EOs) extracted from plants have activities against insects with multiple mechanisms of action. These mechanisms hinder the development of resistance, and have the advantages of being less toxicity and biodegradable. Thus, the present study aimed to evaluate the chemical composition of the EOs obtained from Piper capitarianum Yunck, as well as evaluating their insecticidal potential against Aedes aegypti and A. albopictus, and their toxicity in relation to Artemia salina. The yields of the EOs extracted from the leaves, stems, and inflorescences of P. capitarianum were 1.2%, 0.9%, and 0.6%, respectively, and their main constituents were trans-caryophyllene (20.0%), α-humulene (10.2%), ß-myrcene (10.5%), α-selinene (7.2%), and linalool (6.0%). The EO from the inflorescences was the most active against A. aegypti and A. albopictus, and exhibited the respective larvicidal (LC50 = 87.6 µg/mL and 76.1 µg/mL) and adulticide activities (LC50 = 126.2 µg/mL and 124.5 µg/mL). This EO was also the most active in the inhibition of AChE, since it presented an IC50 value of 14.2 µg/mL. Its larvicidal effect was observed under optical and scanning electron microscopy. Additionally, non-toxic effects against A. salina were observed. Docking modeling of trans-caryophyllene and α-humulene on sterol carrier protein-2 (SCP-2) suggests that both molecules have affinity with the active site of the enzyme, which indicates a possible mechanism of action. Therefore, the essential oil of P. capitarianum may be used in the development of new insecticide targets for the control of A. aegypti and A. albopictus in the Amazonian environment.

Complete mtDNA genomes of Anopheles darlingi and an approach to anopheline divergence time.

BACKGROUND: The complete sequences of the mitochondrial genomes (mtDNA) of members of the northern and southern genotypes of Anopheles (Nyssorhynchus) darlingi were used for comparative studies to estimate the time to the most recent common ancestor for modern anophelines, to evaluate differentiation within this taxon, and to seek evidence of incipient speciation. METHODS: The mtDNAs were sequenced from mosquitoes from Belize and Brazil and comparative analyses of structure and base composition, among others, were performed. A maximum likelihood approach linked with phylogenetic information was employed to detect evidence of selection and a Bayesian approach was used to date the split between the subgenus Nyssorhynchus and other Anopheles subgenera. RESULTS: The comparison of mtDNA sequences within the Anopheles darlingi taxon does not provide sufficient resolution to establish different units of speciation within the species. In addition, no evidence of positive selection in any protein-coding gene of the mtDNA was detected, and purifying selection likely is the basis for this lack of diversity. Bayesian analysis supports the conclusion that the most recent ancestor of Nyssorhynchus and Anopheles+Cellia was extant ~94 million years ago. CONCLUSION: Analyses of mtDNA genomes of Anopheles darlingi do not provide support for speciation in the taxon. The dates estimated for divergence among the anopheline groups tested is in agreement with the geological split of western Gondwana (95 mya), and provides additional support for explaining the absence of Cellia in the New World, and Nyssorhynchus in the Afro-Eurasian continents.

Simulated climate change, but not predation risk, accelerates Aedes aegypti emergence in a microcosm experiment in western Amazonia.

Climate change affects individual life-history characteristics and species interactions, including predator-prey interactions. While effects of warming on Aedes aegypti adults are well known, clarity the interactive effects of climate change (temperature and CO2 concentration) and predation risk on the larval stage remains unexplored. In this study, we performed a microcosm experiment simulating temperature and CO2 changes in Manaus, Amazonas, Brazil, for the year 2100. Simulated climate change scenarios (SCCS) were in accordance with the Fourth Assessment Report of Intergovernmental Panel on Climate Change (IPCC). Used SCCS were: Control (real-time current conditions in Manaus: average temperature is ~25.76°C ± 0.71°C and ~477.26 ± 9.38 parts per million by volume (ppmv) CO2); Light: increase of ~1,7°C and ~218 ppmv CO2; Intermediate: increase of ~2.4°C and ~446 ppmv CO2; and Extreme: increase of ~4.5°C and ~861 ppmv CO2, all increases were relative to a Control SCCS. Light, Intermediate and Extreme SCCS reproduced, respectively, the B1, A1B, and A2 climatic scenarios predicted by IPCC (2007). We analyzed Aedes aegypti larval survivorship and adult emergence pattern with a factorial design combining predation risk (control and predator presence-Toxorhynchites haemorrhoidalis larvae) and SCCS. Neither SCCS nor predation risk affected Aedes aegypti larval survivorship, but adult emergence pattern was affected by SCCS. Accordingly, our results did not indicate interactive effects of SCCS and predation risk on larval survivorship and emergence pattern of Aedes aegypti reared in SCCS in western Amazonia. Aedes aegypti is resistant to SCCS conditions tested, mainly due to high larval survivorship, even under Extreme SCCS, and warmer scenarios increase adult Aedes aegypti emergence. Considering that Aedes aegypti is a health problem in western Amazonia, an implication of our findings is that the use of predation cues as biocontrol strategies will not provide a viable means of controlling the accelerated adult emergence expected under the IPCC climatic scenarios.

Biological activity of neosergeolide and isobrucein B (and two semi-synthetic derivatives) isolated from the Amazonian medicinal plant Picrolemma sprucei (Simaroubaceae).

In the present study, in vitro techniques were used to investigate a range of biological activities of known natural quassinoids isobrucein B (1) and neosergeolide (2), known semi-synthetic derivative 1,12-diacetylisobrucein B (3), and a new semi-synthetic derivative, 12-acetylneosergeolide (4). These compounds were evaluated for general toxicity toward the brine shrimp species Artemia franciscana, cytotoxicity toward human tumour cells, larvicidal activity toward the dengue fever mosquito vector Aedes aegypti, haemolytic activity in mouse erythrocytes and antimalarial activity against the human malaria parasite Plasmodium falciparum. Compounds 1 and 2 exhibited the greatest cytotoxicity against all the tumor cells tested (IC50 = 5-27 microg/L) and against multidrug-resistant P. falciparum K1 strain (IC50 = 1.0-4.0 g/L) and 3 was only cytotoxic toward the leukaemia HL-60 strain (IC50 = 11.8 microg/L). Quassinoids 1 and 2 (LC50 = 3.2-4.4 mg/L) displayed greater lethality than derivative 4 (LC50 = 75.0 mg/L) toward A. aegypti larvae, while derivative 3 was inactive. These results suggest a novel application for these natural quassinoids as larvicides. The toxicity toward A. franciscana could be correlated with the activity in several biological models, a finding that is in agreement with the literature. Importantly, none of the studied compounds exhibited in vitro haemolytic activity, suggesting specificity of the observed cytotoxic effects. This study reveals the biological potential of quassinoids 1 and 2 and to a lesser extent their semi-synthetic derivatives for their in vitro antimalarial and cytotoxic activities.

High proportions of asymptomatic and submicroscopic Plasmodium vivax infections in a peri-urban area of low transmission in the Brazilian Amazon.

BACKGROUND: Population-based studies conducted in Latin America have shown a high proportion of asymptomatic and submicroscopic malarial infections. Considering efforts aiming at regional elimination, it is important to investigate the role of this asymptomatic reservoir in malaria transmission in peri-urban areas. This study aimed to estimate the prevalence of Plasmodium spp. and gametocyte burden on symptomatic and asymptomatic infections in the Brazilian Amazon. RESULTS: Two cross-sectional household surveys (CS) were conducted including all inhabitants in a peri-urban area of Manaus, western Amazonas State, Brazil. Malaria parasites were detected by light microscopy (LM) and qPCR. Sexual stages of Plasmodium spp. were detected by LM and RT-qPCR. A total of 4083 participants were enrolled during the two surveys. In CS1, the prevalence of Plasmodium vivax infections was 4.3% (86/2010) by qPCR and 1.6% (32/2010) by LM. Fifty percent (43/86) of P. vivax infected individuals (qPCR) carried P. vivax gametocytes. In CS2, 3.4% (70/2073) of participants had qPCR-detectable P. vivax infections, of which 42.9% (30/70) of infections were gametocyte positive. The P. vivax parasite density was associated with gametocyte carriage (P < 0.001). Sixty-seven percent of P. vivax infected individuals and 53.4% of P. vivax gametocyte carriers were asymptomatic. CONCLUSIONS: This study confirms a substantial proportion of asymptomatic and submicroscopic P. vivax infections in the study area. Most asymptomatic individuals carried gametocytes and presented low asexual parasitemia. This reservoir actively contributes to malaria transmission in the Brazilian Amazon, underscoring a need to implement more efficient control and elimination strategies.