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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., (© 2023. The Author(s).)

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: LC 5  = 0.511 µM; LC 25  = 1.625 µM; LC 50  = 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., (© 2023. The Author(s).)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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.

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.

The successful spread and maintenance of the dengue virus (DENV) in mosquito vectors depends on their viral infection susceptibility, and parameters related to vector competence are the most valuable for measuring the risk of viral transmission by mosquitoes. These parameters may vary according to the viral serotype in circulation and in accordance with the geographic origin of the mosquito population that is being assessed. In this study, we investigated the effect of DENV serotypes (1-4) with regards to the infection susceptibility of five Brazilian Ae. aegypti populations from Manaus, the capital of the state of Amazonas, Brazil. Mosquitoes were challenged by oral infection with the DENV serotypes and then tested for the presence of the arbovirus using quantitative PCR at 14 days post-infection, which is the time point that corresponds to the extrinsic incubation period of Ae. aegypti when reared at 28 °C. Thus, we were able to determine the infection patterns for DENV-1, -2, -3 and -4 in the mosquito populations. The mosquitoes had both interpopulation and inter-serotype variation in their viral susceptibilities. All DENV serotypes showed a similar tendency to accumulate in the body in a greater amount than in the head/salivary gland (head/SG), which does not occur with other flaviviruses. For DENV-1, DENV-3, and DENV-4, the body viral load varied among populations, but the head/SG viral loads were similar. Differently for DENV-2, both body and head/SG viral loads varied among populations. As the lack of phenotypic homogeneity represents one of the most important reasons for the long-term fight against dengue incidence, we expect that this study will help us to understand the dynamics of the infection patterns that are triggered by the distinct serotypes of DENV in mosquitoes.

Anopheles aquasalis is an important malaria vector in coastal regions of South America and islands of the Caribbean. In its original description, the species was divided into two varieties, based on the scaling patterns of their hind-tarsomere 2. Specimens from our 25-year established colony, used for Plasmodium experimental infections, still exhibit both scaling tarsomere patterns. This study examined the DNA sequence of the nuclear Internal Transcribed Spacer 2 (ITS2) and susceptibility to Plasmodium, looking for differences among the phenotypes 30BS and 50BS. One hundred mosquitoes, 25 males and 25 females of each sex, and phenotype were analyzed. Twenty-seven novel haplotypes were identified. Three were found in both phenotypes (30BS and 50BS) regardless of gender. Among the other 27 genotypes, we observed a male-oriented bias in both phenotypic categories. Evaluation of Plasmodium yoelii N67 infections, based on oocyst counts, showed a higher susceptibility of 30BS compared with 50BS. Future studies need to be conducted to evaluate if these genotype assortments among the phenotypic groups reflect differences in fitness, mating, and their susceptibility to infection by Plasmodium parasites., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: The authors have declared that no competing interests exist.

We investigated by scanning electron microscopy the morphology, distribution, and abundance of antennal sensilla of females Phlebotomus duboscqi sand fly, an important vector of zoonotic cutaneous leishmaniasis at Afrotropical region. Thirteen well-differentiated sensilla were identified, among six types of cuticular sensilla. The probable function of these sensillary types is discussed in relation to their external structure and distribution. Five sensillary types were classified as olfactory sensilla, as they have specific morphological characters of sensilla with this function. Number and distribution of sensilla significantly differed between antennal segments. The results of the present work, besides corroborating in the expansion of the morphological and ultrastructural knowledge of P. duboscqi, can foment future electrophysiological studies for the development of volatile semiochemicals, to be used as attractants in traps for monitoring and selective vector control of this sand fly., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The outbreaks caused by the Aedes aegypti-transmitted dengue virus (DENV), zakat virus (ZIKV), and chikungunya virus (CHIKV) result in a significant impact to the health systems of tropical countries. Furthermore, the occurrence of patients coinfected by at least two of these arboviruses is an aggravating factor in that scenario. On this basis, surveillance tools such as the Rapid Index Survey for Aedes aegypti (LIRAa) are used to estimate vector infestation in order to improve the prediction of human outbreaks. Ae. aegypti eggs were collected in the city of Vitória da Conquista, in Bahia State, Brazil, and subsequently hatched into larvae, which were analyzed in pools or individually for the presence of DENV, ZIKV, and CHIKV by molecular biology methods. The detection data for arboviruses were crossed with the LIRAa obtained in each region of the study city. Thirty larvae pools were analyzed, and fourteen (46.6%) of them were detected positive for DENV, ZIKV, and/or CHIKV. Among the individually analyzed larvae (n = 30), nine (30%) were positive for any of these arboviruses, and four (13.3%) were simultaneously coinfected by DENV and ZIKV. Furthermore, there was a positive correlation between the detection of circulating arboviruses and LIRAa. The simultaneous Ae. aegypti larvae infection by two different arboviruses is an unprecedented finding. This result suggests the occurrence of a vertical arboviruses co-transmission from the female mosquito to its offspring in nature. The occurrence of concomitant circulation of DENV, ZIKV, and CHIKV in Ae. aegypti from a single study region is another finding of this article. Finally, LIRAa seems to not only estimate vector infestation but also to predict circulation of arboviruses., (Copyright © 2021. Published by Elsevier B.V.)

The ultrastructure of the mouthparts of Haematobia irritans (L.) was investigated by scanning electron microscopy. The morphological characteristics of the maxillary palps, labium (prementum and postmentum), labrum, hypopharynx, haustellum, and labellar lobes are described, as well as of the sensilla evidenced on all the surface of the mouthparts, and the set of different positions assumed by the mouth apparatus of this fly. Based on their morphology, 12 well-differentiated sensilla were identified, among three types of cuticular sensilla: trichoidea, coeloconica, and campaniformia. A slight sexual dimorphism in the sensilla patterns found in the mouthparts of H. irritans was evidenced. These observations are discussed with reference to the current literature on the functional morphology of sense organs of Insecta. These results could facilitate the recognition of the chemosensory sensilla by electrophysiological techniques, and foment future taxonomic and phylogenetic studies to better elucidate the evolution of Diptera, Muscomorpha., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Several tropical cities are permissive to Aedes aegypti and dengue virus (DENV) endemicity and have allowed for invasion and circulation of Zika virus (ZIKV) in the same areas. People living in arbovirus-endemic regions have been simultaneously infected with ≥2 arboviruses., Methods: A. aegypti mosquitoes from Manaus, the capital city of Amazonas State in Brazil, were coinfected with circulating strains of DENV and ZIKV. The coinfected vectors were allowed to bite BALB/c mice., Results: A. aegypti from Manaus is highly permissive to monoinfection and coinfection with DENV and ZIKV and is capable of cotransmitting both pathogens by bite. Coinfection strongly influences vector competence, favoring transmission of ZIKV to the vertebrate host., Conclusions: This finding suggests that A. aegypti is an efficient vector of ZIKV and that ZIKV would be preferentially transmitted by coinfected A. aegypti. Coinfection in the vector population should be considered a new critical epidemiological factor and may represent a major public health challenge.

Background: The leishmaniases are a group of diseases caused by protozoans of the genus Leishmania, which are transmitted by the bite of phlebotomine sand flies. In the New World, Lutzomyia longipalpis is the most important vector of visceral leishmaniasis and is a proven vector for Leishmania infantum chagasi in Brazil. During development within the vector, Leishmania can interact with a variety of microorganisms such as fungi and bacteria. The presence of bacteria in the midgut of sand flies can influence the development and survival of the parasite., Results: The bacteria-targeted metagenomic analysis revealed different community compositions between the distinct physiological stages of those tested. The amplicon-oriented metagenomic profiling revealed 64 bacterial genera and 46 families. By crossing the taxa indices from each experimental condition a core composed of 6 genera was identified (Enterobacter, Serratia, Stenotrophomonas, Enhydrobacter, Pseudomonas and Chryseobacterium)., Conclusions: The observed dynamic nature of the bacterial community expands the knowledge pertaining to the tripartite host-microbiota-pathogen interactions. Further studies addressing how laboratory and field collected communities differ are critical to successfully develop control strategies based on bacterial symbionts and paratransgenesis, as already tested in other arthropod vectors.

Background: Transovarial transmission of dengue virus in Aedes spp. mosquitoes is considered an important mechanism for the maintenance of the virus in nature and may be implicated in the occurrence of outbreaks and epidemics of the disease. However, there are few studies involving transovarial transmission and viral vector monitoring as a surveillance tool and control strategy. The present study evaluated transovarial transmission of dengue virus in Aedes aegypti populations as a xenomonitoring strategy in municipalities of the Amazonas state., Results: Aedes sp. eggs (13.164) were collected, with 30% viability of third- and fourth-instar larvae. Transovarial transmission of DENV was detected in all municipalities. The transovarial infection rate (TOR) in the municipalities was 46% of the DENV positive samples. The minimum infection rate (MIR) was 17.7 in the state, varying from 11.4 to 24.1 per 1,000 larvae tested in the respective municipalities. Four DENV serotypes were identified, with DENV I and IV being present in all municipalities investigated. The number of reported dengue fever cases varied during this period., Conclusions: Our results suggest that transovarial transmission may be an important mechanism for the maintenance and spreading of the disease in Amazonas municipalities. Using qRT-PCR, it was possible to identify the four DENV serotypes in larval samples. The methodology used in the present study proved suitable as a DENV xenomonitoring model in immature mosquitoes, contributing to the development of systems for early detection of viral circulation and predictive models for the occurrence of outbreaks and epidemics of this disease., Trial Registration: CAAE34025414200005015 .

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n=24) and asymptomatic (n=14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p=0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p=0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p=0.027) and was directly correlated with antibody levels (p=0.0379). Age and gender did not influence the transmissibility. Our data show that asymptomatic dogs are highly infective and competent for establishing sand fly infection, indicating their role in maintaining L. (L.) infantum chagasi cycle as well as their involvement in VL spreading in endemic areas., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The effectiveness of a polyester mesh cover (evidengue), aimed at preventing the access of female Aedes aegypti mosquitoes to flowerpot saucers, was evaluated in laboratory. Two saucers of flowerpot with water were individually wrapped with the cover was placed with their respective pots in two entomological cages. One identical set of flowerpot and saucer was placed in a third cage. In each cage, 20 gravid females, fed on mouse blood, were released. Results show that the cover was effective to prevent access of females. Further tests are necessary to assess cover effectiveness as a device to prevent saucer oviposition.

In order to compare the saliva effect from wild-caught and lab-reared L. longipalpis on the development of experimental cutaneous leishmaniasis, C57BL/6 mice were inoculated subcutaneously into the hind footpads with promastigotes of L. (L.) amazonensis plus salivary gland lysate from wild-caught (SGL-W) and lab-colonized (SGL-C) vectors. Lesion sizes were significantly larger in the mice infected with both saliva compared to mice infected with parasites alone; moreover, the lesions caused by parasite+SGL-C were significantly larger than the lesions caused by parasite+SGL-W. Histopathological morphometric studies regarding the acute phase of infections showed lower numbers of polymorphonuclear cells, greater numbers of mononuclear cells and parasites in SGL-C infected mice compared to SGL-W infected mice. In the chronic phase of infection, the number of mononuclear cells was lower and the number of parasites was greater in SGL-C infected mice than SGL-W infected mice. In vitro studies showed increased infection index of macrophages infected with parasites plus saliva compared to infection with parasites alone, with no difference between the saliva infection indices. SDS-PAGE gel for SGL-C and SGL-W showed differences in the composition and quantity of protein bands, determined by densitometry. These results call attention to the experimental saliva model, which shows exacerbation of infection caused by sandfly saliva.

The specific aims of this work were to examine the antennal sensilla of Lutzomyia longipalpis (Lutz & Neiva 1912) (Diptera: Psychodidae) adults and to characterize their typology and topography, with special attention to olfactory sensilla. The surfaces of the antennal segments of Lu. longipalpis males and females were studied by scanning electron microscopy (SEM). Lu. longipalpis used in the current study were obtained from a colony originating from Lapinha Cave, Minas Gerais state, Brazil. Microtrichiae and 11 subtypes of sensilla were observed and characterized according to the following categories: five subtypes of trichoid sensilla (short, medium, long blunt-tipped, long pointed-tipped, and apical), two coeloconic sensilla (grooved and praying hands), and campaniform, chaetic, basiconic, and squamiform sensilla. SEM analyses showed few differences between males and females in the typology, topography, and quantity of antennal sensilla described. The current study is the first to identify several categories of antennal sensilla of the genus Lutzomyia and their distribution patterns. The identification of these sensillar types may be important in planning future electrophysiological studies to develop alternative measures of control and monitoring of Lu. longipalpis.

Egg hatching has been studied in Aedes aegypti (L.) through scanning electron microscopy. The first sign of egg hatching is a small protrusion on the eggshell in the anterior pole. The larval movement provokes a crack in the eggshell with the egg buster located in the dorsal head. The egg buster provokes a small transverse fissure in the eggshell that gradually increases in the chorion. Then, the rupture is completed around the eggshell. The separation of the anterior pole occurs, showing the dorsal region of the larva head with the egg buster and the cap. After sequential movements, the larva looses the cap. Finally, the first instar is ready to be free showing details of its body with the egg buster over its head. This structure is a cuticular formation, similar to a cone structure that ends in a very fine tip and emerges from a pear-like depression with high rounded borders. Our results describe the anatomy of the egg hatching process in Ae. aegypti, showing details of the participation of the egg buster.