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BACKGROUND: Reducing mosquito abundance or interfering with its ability to support the parasite cycle can help to interrupt malaria in areas of significant risk of malaria transmission. Fluralaner is a safe and effective drug for veterinary use indicated for the treatment against fleas and ticks which acts as an antagonist of chloride ion channels mediated by γ-aminobutyric acid (GABA), preventing the entry of these ions into the postsynaptic neuron, leading to hyperexcitability of the postsynaptic neuron of the central nervous system of arthropods. Fluralaner demonstrated insecticidal activity against different insect species. METHODS: The study aimed to evaluate the effects of fluralaner on the biology, survival, and reproductive fitness of Anopheles aquasalis. The following lethal concentrations (LC) were determined for An. aquasalis: LC5 = 0.511 µM; LC25 = 1.625 µM; LC50 = 3.237 µM. RESULTS: A significant decrease (P < 0.001) was evident in the number of eggs, larvae, and pupae in the group exposed to a sublethal dose of fluralaner when compared to a control group (without the drug). Using blood from dogs after administration of fluralaner, it was observed that the drug causes 100% mortality in An. aquasalis in less than 24 h after feeding; this effect remains even after 90 days in all samples. DISCUSSION: Fluralaner showed the same result for up to 60 days, and after that, there was a slight reduction in its effect, evidenced by a decrease in the percentage of dead females; however, still significant when compared to the control group. CONCLUSION: Fluralaner affects the biology and reduction of survival in An. aquasalis in a lasting and prolonged period, and its fecundity with lower dosages, is a strong candidate for controlling disease vectors.

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Malaria is a severe public health problem in several developing tropical and subtropical countries. Anopheles aquasalis is the primary coastal malaria vector in Central and South America and the Caribbean Islands, and it has the peculiar feature of living in water with large changes in salinity. Recent research has recognised An. aquasalis as an important model for studying the interactions of murine and human Plasmodium parasites. This study presents the complete genome of An. aquasalis and offers insights into its evolution and physiology. The genome is similar in size and gene content to other Neotropical anophelines, with 162 Mb and 12,446 protein-coding genes. There are 1387 single-copy orthologs at the Diptera level (eg. An. gambiae, An. darlingi and Drosophila melanogaster). An. aquasalis diverged from An. darlingi, the primary malaria vector in inland South America, nearly 20 million years ago. Proteins related to ion transport and metabolism belong to the most abundant gene families with 660 genes. We identified gene families relevant to osmosis control (e.g., aquaporins, vacuolar-ATPases, Na+/K+-ATPases, and carbonic anhydrases). Evolutionary analysis suggests that all osmotic regulation genes are under strong purifying selection. We also observed low copy number variation in insecticide resistance and immunity-related genes for all known classical pathways. The data provided by this study offers candidate genes for further studies of parasite-vector interactions and for studies on how anophelines of brackish water deal with the high fluctuation in water salinity. We also established data and insights supporting An. aquasalis as an emerging Neotropical malaria vector model for genetic and molecular studies.

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Anophelines are vectors of malaria, the deadliest disease worldwide transmitted by mosquitoes. The availability of genomic data from various Anopheles species allowed evolutionary comparisons of the immune response genes in search of alternative vector control of the malarial parasites. Now, with the Anopheles aquasalis genome, it was possible to obtain more information about the evolution of the immune response genes. Anopheles aquasalis has 278 immune genes in 24 families or groups. Comparatively, the American anophelines possess fewer genes than Anopheles gambiae s. s., the most dangerous African vector. The most remarkable differences were found in the pathogen recognition and modulation families like FREPs, CLIP and C-type lectins. Even so, genes related to the modulation of the expression of effectors in response to pathogens and gene families that control the production of reactive oxygen species were more conserved. Overall, the results show a variable pattern of evolution in the immune response genes in the anopheline species. Environmental factors, such as exposure to different pathogens and differences in the microbiota composition, could shape the expression of this group of genes. The results presented here will contribute to a better knowledge of the Neotropical vector and open opportunities for malaria control in the endemic-affected areas of the New World.

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The mosquito larval midgut is responsible for acquiring and storing most of the nutrients that will sustain the events of metamorphosis and the insect's adult life. Despite its importance, the basic biology of this larval organ is poorly understood. To help fill this gap, we carried out a comparative morphophysiological investigation of three larval midgut regions (gastric caeca, anterior midgut, and posterior midgut) of phylogenetically distant mosquitoes: Anopheles gambiae (Anopheles albimanus was occasionally used as an alternate), Aedes aegypti, and Toxorhynchites theobaldi. Larvae of Toxorhynchites mosquitoes are predacious, in contrast to the other two species, that are detritivorous. In this work, we show that the larval gut of the three species shares basic histological characteristics, but differ in other aspects. The lipid and carbohydrate metabolism of the An. gambiae larval midgut is different compared with that of Ae. aegypti and Tx. theobaldi. The gastric caecum is the most variable region, with differences probably related to the chemical composition of the diet. The peritrophic matrix is morphologically similar in the three species, and processes involved in the post-embryonic development of the organ, such as cell differentiation and proliferation, were also similar. FMRF-positive enteroendocrine cells are grouped in the posterior midgut of Tx. theobaldi, but individualized in An. gambiae and Ae. aegypti. We hypothesize that Tx. theobaldi larval predation is an ancestral condition in mosquito evolution.

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Zika virus (ZIKV) is transmitted to humans by the infectious bite of mosquitoes such as Aedes aegypti. In a city, the population control of mosquitoes is carried out according to alerts generated by different districts via the analysis of the mosquito index. However, we do not know whether, besides mosquito abundance, the susceptibility of mosquitoes could also diverge among districts and thus impact the dissemination and transmission of arboviruses. After a viremic blood meal, the virus must infect the midgut, disseminate to tissues, and reach the salivary gland to be transmitted to a vertebrate host. This study evaluated the patterns of ZIKV infection in the Ae. aegypti field populations of a city. The disseminated infection rate, viral transmission rate, and transmission efficiency were measured using quantitative PCR at 14 days post-infection. The results showed that all Ae. aegypti populations had individuals susceptible to ZIKV infection and able to transmit the virus. The infection parameters showed that the geographical area of origin of the Ae. aegypti influences its vector competence for ZIKV transmission.

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BACKGROUND: Arthropod-borne viruses have recently emerged and are pathogens of various human diseases, including dengue, zika, and chikungunya viruses. METHODS: We collectedAedes aegyptilarvae (N = 20) from Brumado, Bahia, Brazil, and treated and individually preserved the specimens. We analyzed the samples for dengue, zika, and chikungunya viruses using molecular biology methods. RESULTS: We found that 25% (N = 5) and 15% (N = 3) were positive exclusively for dengue and chikungunya viruses, respectively; 15% (N = 3) were coinfected with both. CONCLUSIONS: This is the first report of dengue and chikungunya virus coinfection in A. aegypti larvae.

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BACKGROUND: Emerging and re-emerging vector-borne diseases (VBDs) pose a recurring threat to tropical countries, mainly due to the abundance and distribution of the Aedes aegypti mosquito, which is a vector of the Zika, dengue, chikungunya, and yellow fever arboviruses. METHODS: Female 3-5 day-old Ae. aegypti were distributed into two experimental groups: group I-survey of cultivable bacteria; sucrose group: fed only on sucrose, i.e., non-blood-fed (UF); blood-fed group: (i) fed with non-infected blood (BF); (ii) fed with blood infected with the Zika virus (BZIKV); (iii) pretreated with penicillin/streptomycin (pen/strep), and fed with non-infected blood (TBF); (iv) pretreated with pen/strep and fed blood infected with ZIKV, i.e., gravid with developed ovaries, (TGZIKV); group II-experimental co-infections: bacteria genera isolated from the group fed on sucrose, i.e., non-blood-fed (UF). RESULTS: Using the cultivable method and the same mosquito colony and ZIKV strain described by in a previous work, our results reveled 11 isolates (Acinetobacter, Aeromonas, Cedecea, Cellulosimicrobium, Elizabethkingia, Enterobacter, Lysinibacillus, Pantoea, Pseudomonas, Serratia, and Staphylococcus). Enterobacter was present in all evaluated groups (i.e., UF, BF, BZIKV, TBF, and TGZIKV), whereas Elizabethkingia was present in the UF, BZIKV, and TBF groups. Pseudomonas was present in the BZIKV and TBF groups, whereas Staphylococcus was present in the TBF and TGZIKV groups. The only genera of bacteria that were found to be present in only one group were Aeromonas, Lysinibacillus, and Serratia (UF); Cedacea, Pantoea and Acinetobacter (BF); and Cellulosimicrobium (BZIKV). The mosquitoes co-infected with ZIKV plus the isolates group fed on sucrose (UF) showed interference in the outcome of infection. CONCLUSIONS: We demonstrate that the distinct feeding aspects assessed herein influence the composition of bacterial diversity. In the co-infection, among ZIKV, Ae. aegypti and the bacterial isolates, the ZIKV/Lysinibacillus-Ae. aegypti had the lowest number of viral copies in the head-SG, which means that it negatively affects vector competence. However, when the saliva was analyzed after forced feeding, no virus was detected in the mosquito groups ZIKV/Lysinibacillus-Lu. longipalpis and Ae. aegypti; the combination of ZIKV/Serratia may interfere in salivation. This indicates that the combinations do not produce viable viruses and may have great potential as a method of biological control.

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ABSTRACT Background: Arthropod-borne viruses have recently emerged and are pathogens of various human diseases, including dengue, zika, and chikungunya viruses. Methods: We collectedAedes aegyptilarvae (N = 20) from Brumado, Bahia, Brazil, and treated and individually preserved the specimens. We analyzed the samples for dengue, zika, and chikungunya viruses using molecular biology methods. Results: We found that 25% (N = 5) and 15% (N = 3) were positive exclusively for dengue and chikungunya viruses, respectively; 15% (N = 3) were coinfected with both. Conclusions: This is the first report of dengue and chikungunya virus coinfection in A. aegypti larvae.

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Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti. After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae. aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae. aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections.

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