Morphological changes in eggs and embryos of Aedes aegypti (Diptera: Culicidae) exposed to predicted climatic scenarios for the year 2100 in the Central Amazon.

According to the IPCC, by the year 2100, rises in global temperature could reach up to 5 °C above current averages. On a planet-wide scale, this is one of the effects of climate changes that could have repercussions on the biological cycle of Aedes aegypti, the main arbovirus vector in urban environments and a transmitter of the arboviruses that cause dengue, Zika, chikungunya and urban yellow fever. The objective of this study was to evaluate morphological changes in Ae. aegypti eggs and embryos maintained in a climate change simulator. For this, specimens obtained from an insectarium were kept in four chambers that simulated the range of environmental scenarios predicted by the IPCC for the year 2100. The eggs obtained from each room were collected and transported to the laboratory for morphometric and morphological analysis, using confocal and scanning microscopy. Aedes aegypti eggs (n=20) were used to obtain the following variables: total width, total length, length-width ratio and diameter of the micropylar disc. Additionally, 20 embryos were used to obtain the data on head capsule length, width and length-width ratio. The data were subjected to a normality test and the means of each variable were compared using ANOVA and Tukey's post-hoc test, considering (p ≤ 0.05). A significant reduction (p < 0.05) was observed mainly in the mean lengths under the current-extreme scenario (587.5 and 553.6 µm, respectively), as well as in the widths under the current-mild scenario (171 and 158.4 µm, respectively). The length of the cephalic capsule was also affected, showing significant differences in the means under the current-intermediate scenario (189.5 and 208.5 µm, respectively), as well as in the widths between the current-intermediate scenarios (173.7 and 194.9 µm, respectively). The results suggest significant changes in the morphometry of Ae. aegypti eggs and embryos as a result of the climatic influences to which the adults were subjected, which may have an impact on vector population density and, consequently, on arbovirus dynamics in urban environments.

Bioecological Aspects of Species of the Subgenus Mansonia (Mansonia) (Diptera: Culicidae) Prior to the Installation of Hydroelectric Dams on the Madeira River, Rondônia State, Brazil.

The objective of this study was to evaluate ecological aspects of Mansonia species before the construction of hydroelectric plants on the Madeira River, and thus enable the assessment of the impact of these projects on mosquitoes. A total of 199 samplings were carried out between November 2003 and August 2004, using the technique of attraction with protection. Temporal distribution was evaluated from monthly incidence values obtained from the bite index per man/hour. Relative abundance was subsequently calculated to evaluate the spatial distribution of species, according to land use and municipal districts; furthermore, the pattern of hematophagous activity was evaluated from 12-h and 4-h samplings. The data were analyzed according to the negative binomial distribution and generalized linear models to estimate the influence of environmental factors on the presence and abundance of Mansonia. A total of 1479 specimens were collected, distributed among four species-Mansonia titillans (87%), Mansonia humeralis (6.3%), Mansonia amazonensis (6%), and Mansonia indubitans (0.5%), and spatial distribution analysis showed Ma. titillans to be dominant. Hematophagous activity had peaks between 6:00 p.m. and 8:00 p.m. and species incidence was higher during the rainy season and in areas where domestic animals are raised. Therefore, the region studied presented characteristics favorable to the reproduction of Mansonia even before the construction of the hydroelectric plants and after construction, these conditions were enhanced, due to the increase in the availability of breeding sites for immatures and blood sources for females, as a consequence of changes in the environment.

Dirofilaria immitis is endemic in rural areas of the Brazilian Amazonas state capital, Manaus.

The canine filarial parasite Dirofilaria immitis has not been reported in Brazil´s Amazonas state capital, Manaus, for over a century. Here, we report one imported and 27 autochthonous D. immitis infections from a microfilarial survey of 766 domestic dog blood samples collected between 2017 and 2021 in Manaus. An Overall prevalence estimate of 15.44% (23/149) was calculated from our two rural collection sites; a prevalence of 1.22% (4/328) was estimated at our periurban collection site, and an overall prevalence of 0.35% (1/289) was calculated from our two urban clinic collections. Our data suggest that in the urban areas of Manaus, where the parasites are very likely vectored by the same species of mosquito that historically vectored Wuchereria bancrofti (Culex quinquefasciatus), prevalence levels are very low and possibly maintained by an influx from rural areas where sylvatic reservoirs and/or more favorable vector transmission dynamics maintain high prevalences.

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.

Study of karyotype and constitutive heterochromatin of Mansonia spp. (Diptera: Culicidae) in the area surrounding the Jirau Hydroelectric Dam, Rondônia, Brazil.

BACKGROUND: Mansonia mosquitoes transmit arboviruses to humans. This study describes the karyotypes and C-banding of Mansonia humeralis, Mansonia titillans, Mansonia pseudotitillans, and Mansonia indubitans. METHODS: From the 202 larvae, the brain ganglia were dissected (n=120) for the preparation of slides. Twenty slides with well-distended chromosomes for each species (10 for karyotyping and 10 for C-banding) were selected for further study. RESULTS: The haploid genome and the average lengths of the chromosomal arms differed in relation to the centromere between species, and intraspecific differences also occurred in the distribution of the C-bands. CONCLUSIONS: These results are useful for better understanding of the chromosomal variability of Mansonia mosquitoes.

Study of karyotype and constitutive heterochromatin of Mansonia spp. (Diptera: Culicidae) in the area surrounding the Jirau Hydroelectric Dam, Rondônia, Brazil

ABSTRACT Background: Mansonia mosquitoes transmit arboviruses to humans. This study describes the karyotypes and C-banding of Mansonia humeralis, Mansonia titillans, Mansonia pseudotitillans, and Mansonia indubitans. Methods: From the 202 larvae, the brain ganglia were dissected (n=120) for the preparation of slides. Twenty slides with well-distended chromosomes for each species (10 for karyotyping and 10 for C-banding) were selected for further study. Results: The haploid genome and the average lengths of the chromosomal arms differed in relation to the centromere between species, and intraspecific differences also occurred in the distribution of the C-bands. Conclusions: These results are useful for better understanding of the chromosomal variability of Mansonia mosquitoes.

Endophytic fungus Phomopsis sp. as a source of 3-nitropropionic acid with larvicidal activity against Aedes aegypti (Linnaeus 1762, Diptera: Culicidae).

BACKGROUND: Aedes aegypti is the primary vector of viruses, such as Zika, chikungunya, yellow fever, and dengue. In this context, a biomonitored chemical study was conducted to evaluate the activity of the crude extract of the endophytic fungus Phomopsis sp. against the larvae of Aedes aegypti. METHODS: Crude extract, fractions, and isolated substances were evaluated in in-vitro assays against third-stage larvae of Aedes aegypti. RESULTS: We isolated 3-nitropropionic acid with an LC50 of 15.172 ppm and LC90 of 18.178 ppm after 24 hours of larval exposure. CONCLUSIONS: The results indicated that 3-nitropropionic acid exerted larvicidal activity.

DNA barcoding suggests new species for the Mansonia subgenus (Mansonia, Mansoniini, Culicidae, Diptera) in the area surrounding the Jirau hydroelectric dam, Porto Velho municipality, Rondônia state, Brazil.

Previous studies have linked the construction of hydroelectric dams with increases in the density of mosquitoes, especially Mansonia. In Brazil, Mansonia mosquitoes are still poorly studied at the taxonomic, biological, ecological and epidemiological levels, and nothing is known about the genetic diversity and the cryptic speciation of the group. The current study analyzed the molecular taxonomy of Mansonia species captured in the area surrounding the Jirau hydroelectric dam, Rondônia state, Brazil. Samples were collected from fifteen locations between 2018 and 2019. Genomic DNA of the specimens was extracted, and the DNA barcode region of the Cytochrome Oxidase, subunit I gene was amplified with PCR and both DNA strands were sequenced. The dataset was analyzed using MEGA, Mr. Bayes and DnaSP software. The results provided COI sequences for 100 specimens collected in the area surrounding from Jirau hydroelectric dam. These belonged to five species of the Mansonia subgenus, identified morphologically as Mansonia humeralis, Mansonia amazonensis, Mansonia titillans, Mansonia dyari and Mansonia indubitans. Findings showed that the COI gene is an effective and accessible DNA barcode that provides a high-resolution tool for delimiting species within the subgenus Mansonia, with the tree construction (Bayesian Inference) well supported and non-overlapping intraspecific and interspecific (K2-P) genetic distance values. These findings also indicate the occurrence of cryptic speciation within M. dyari and near of M. titillans. This is the first study to apply molecular tools to the taxonomy of Mansonia species from Brazil.

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.

Discovery and genome sequencing of a new virus related to members of the family Tymoviridae, isolated from mosquitoes of the genus Mansonia in Brazil.

A new virus, named Mutum virus, related to members of the family Tymoviridae, was isolated from mosquitoes (Mansonia spp.) in clone C6/36 cells, and its complete genome was sequenced. Its genome is 6494 nt in size with an organization resembling that of tymovirids. The isolated virus is phylogenetically related to two viruses isolated from Culex spp. mosquitoes: Ek Balam virus, reported in Mexico, and Culex-originated Tymoviridae-like virus, isolated in China. The results of this study suggest that this virus is a new member of the family Tymoviridae.

Oxidative stress induction by crude extract of Xylaria sp. triggers lethality in the larvae of Aedes aegypti (Diptera: Culicidae).

BACKGROUND: Aedes aegypti is currently controlled with synthetic larvicides; however, mosquitoes have become highly resistant to these larvicides and difficult to eradicate. Studies have shown that insecticides derived from fungal extracts have various mechanisms of action that reduce the risk of resistance in these mosquitoes. One possible mechanism is uncontrolled production of reactive oxygen species (ROS) in the larvae, which can cause changes at the cellular level. Thus, the crude extract of Xylaria sp. was evaluated to investigate the oxidative effect of this extract in A. aegypti larvae by quantifying the oxidative damage to proteins and lipids. METHODS: The larvicidal potential of the crude extract of Xylaria sp. Was evaluated, and the extract was subsequently tested in human lung fibroblasts for cytotoxicity and ROS production. ROS level was quantified in the larvae that were killed following exposure to the extract in the larvicide test. RESULTS: The crude extract of Xylaria sp. Caused cytotoxicity and induced ROS production in human lung fibroblasts and A. aegypti larvae, respectively. In the larvicide trial, the extract showed an LC50 of 264.456 ppm and an LC90 of 364.307 ppm, and was thus considered active. The extract showed greater oxidative damage to lipids and proteins, with LC90 values of 24.7 µmol MDA/L and 14.6278 ×10-3 nmol carbonyl/ mg protein, respectively. CONCLUSIONS: Crude extracts of Xylaria sp. induced oxidative stress that may have caused the mortality of A. aegypti larvae.

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.

Oxidative stress induction by crude extract of Xylaria sp. triggers lethality in the larvae of Aedes aegypti (Diptera: Culicidae)

ABSTRACT Background: Aedes aegypti is currently controlled with synthetic larvicides; however, mosquitoes have become highly resistant to these larvicides and difficult to eradicate. Studies have shown that insecticides derived from fungal extracts have various mechanisms of action that reduce the risk of resistance in these mosquitoes. One possible mechanism is uncontrolled production of reactive oxygen species (ROS) in the larvae, which can cause changes at the cellular level. Thus, the crude extract of Xylaria sp. was evaluated to investigate the oxidative effect of this extract in A. aegypti larvae by quantifying the oxidative damage to proteins and lipids. Methods: The larvicidal potential of the crude extract of Xylaria sp. Was evaluated, and the extract was subsequently tested in human lung fibroblasts for cytotoxicity and ROS production. ROS level was quantified in the larvae that were killed following exposure to the extract in the larvicide test. Results: The crude extract of Xylaria sp. Caused cytotoxicity and induced ROS production in human lung fibroblasts and A. aegypti larvae, respectively. In the larvicide trial, the extract showed an LC50 of 264.456 ppm and an LC90 of 364.307 ppm, and was thus considered active. The extract showed greater oxidative damage to lipids and proteins, with LC90 values of 24.7 µmol MDA/L and 14.6278 ×10-3 nmol carbonyl/ mg protein, respectively. Conclusions: Crude extracts of Xylaria sp. induced oxidative stress that may have caused the mortality of A. aegypti larvae.

Endophytic fungus Phomopsis sp. as a source of 3-nitropropionic acid with larvicidal activity against Aedes aegypti (Linnaeus 1762, Diptera: Culicidae)

ABSTRACT Background: Aedes aegypti is the primary vector of viruses, such as Zika, chikungunya, yellow fever, and dengue. In this context, a biomonitored chemical study was conducted to evaluate the activity of the crude extract of the endophytic fungus Phomopsis sp. against the larvae of Aedes aegypti. Methods: Crude extract, fractions, and isolated substances were evaluated in in-vitro assays against third-stage larvae of Aedes aegypti. Results: We isolated 3-nitropropionic acid with an LC50 of 15.172 ppm and LC90 of 18.178 ppm after 24 hours of larval exposure. Conclusions: The results indicated that 3-nitropropionic acid exerted larvicidal activity.

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.

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.

Seasonality modulates the direct and indirect influences of forest cover on larval anopheline assemblages in western Amazônia.

Serious concerns have arisen regarding urbanization processes in western Amazônia, which result in the creation of artificial habitats, promoting the colonization of malaria vectors. We used structural equation modelling to investigate direct and indirect effects of forest cover on larval habitats and anopheline assemblages in different seasons. We found 3474 larvae in the dry season and 6603 in the rainy season, totalling ten species and confirming the presence of malaria vectors across all sites. Forest cover had direct and indirect (through limnological variables) effects on the composition of larval anopheline assemblages in the rainy season. However, during the dry season, forest cover directly affected larval distribution and habitat variables (with no indirect affects). Additionally, artificial larval habitats promote ideal conditions for malaria vectors in Amazonia, mainly during the rainy season, with positive consequences for anopheline assemblages. Therefore, the application of integrated management can be carried out during both seasons. However, we suggest that the dry season is the optimal time because larval habitats are more limited, smaller in volume and more accessible for applying vector control techniques.

New traps for the capture of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) (Diptera: Culicidae) eggs and adults.

The control of arboviruses carried by Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) can be performed with tools that monitor and reduce the circulation of these vectors. Therefore, the efficiency of four types of traps in capturing A. aegypti and A. albopictus eggs and adults, with the biological product Vectobac WG, was evaluated in the field. For this, 20 traps were installed in two locations, which were in the South (Londrina, Paraná) and North (Manaus, Amazonas) Regions of Brazil, from March to April 2017 and January to February 2018, respectively. The UELtrap-E (standard trap) and UELtrap-EA traps captured A. aegypti and A. albopictus eggs: 1703/1866 eggs in Londrina, and 10268/2149 eggs in Manaus, respectively, and presented high ovitraps positivity index (OPI) values (averages: 100%/100% in Londrina, and 100%/96% in Manaus, respectively); and high egg density index (EDI) values (averages: 68/75 in Londrina, and 411/89 in Manaus, respectively), so they had statistically superior efficiency to that of the CRtrap-E and CRtrap-EA traps in both regions, that captured less eggs and adults: 96/69 eggs in Londrina, and 1091/510 eggs in Manaus, respectively. Also presented lower OPI values (averages: 28%/4% in Londrina, and 88%/60% in Manaus, respectively); and lower EDI values (averages: 10.5/9 in Londrina, and 47/30 in Manaus, respectively). The capture ratios of Aedes adults in the UELtrap-EA and CRtrap-EA traps in Londrina and Manaus were 53.3%/29.5% and 0%/9.8%, respectively. UELtrap-EA can be adopted as efficient tool for Aedes monitoring due to their high sensitivity, low cost and ease of use.

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.