Phlebotominae (Diptera: Psychodidae) and Biomes in the State of Mato Grosso do Sul, Brazil.

The state of Mato Grosso do Sul, Brazil is an important region of South America regarding leishmaniasis, with the great diversity of sandflies reported since 1938 and wide biodiversity represented by its biomes (Atlantic Forest, Cerrado, and Pantanal). A checklist of sandflies is presented here for the state and respective biomes. The data base was compiled from primary and secondary data. The primary data collection involved automatic light traps at Miranda-Abobral Pantanal subregion and in subregion Baixa Nhecolância of Pantanal. The secondary data were obtained from entomology scientific collections and a literature review of articles from 1938 to 2019, including entomological museum collections. A total of 71 species were reported, belonging to 14 genera and 13 subgenera, in 61 municipalities of the state, including the type locality of 9 species, and the first report of Lutzomyia cruzi (Mangabeira 1938), vector of Leishmania infantum (Nicolle 1937), in the Pantanal region of Miranda-Abobral. Other vector species, Bichromomyia flaviscutellata (Mangabeira 1942), Lutzomyia longipalpis (Lutz & Neiva 1912), Nyssomyia antunesi (Coutinho 1939), Nyssomyia intermedia (Lutz & Neiva 1912), Nyssomyia neivai (Pinto 1926), and Nyssomyia whitmani (Antunes & Coutinho 1939), are reported in the entire area of the state. Maps containing the distribution of sandflies on Mato Grosso do Sul biomes were produced. The eclecticism of the species in relation to their ecotypes was observed, with several species using the three biome types as habitats and breeding sites.

Macrogeographic genetic structure of Lutzomyia longipalpis complex populations using Next Generation Sequencing.

Lutzomyia longipalpis is the main vector of Leishmania infantum, the causative agent of visceral leishmaniasis in the Neotropical realm. Its taxonomic status has been widely discussed once it encompasses a complex of species. The knowledge about the genetic structure of insect vector populations helps the elucidation of components and interactions of the disease ecoepidemiology. Thus, the objective of this study was to genotypically analyze populations of the Lu. longipalpis complex from a macrogeographic perspective using Next Generation Sequencing. Polymorphism analysis of three molecular markers was used to access the levels of population genetic structure among nine different populations of sand flies. Illumina Amplicon Sequencing Protocol® was used to identify possible polymorphic sites. The library was sequenced on paired-end Illumina MiSeq platform. Significant macrogeographical population differentiation was observed among Lu. longipalpis populations via PCA and DAPC analyses. Our results revealed that populations of Lu. longipalpis from the nine municipalities were grouped into three clusters. In addition, it was observed that the levels of Lu. longipalpis population structure could be associated with distance isolation. This new sequencing method allowed us to study different molecular markers after a single sequencing run, and to evaluate population and inter-species differences on a macrogeographic scale.

Synanthropy and diversity of Phlebotominae in an area of intense transmission of visceral leishmaniasis in the South Pantanal floodplain, Midwest Brazil.

Phlebotomines have been recorded from a wide variety of habitats, and some of these vector species have shown preference for human environments, with high levels of adaptation. This study evaluated the degree of preference of these vectors for urban, rural, and forested environments (synanthropic behavior), as well as the diversity of these species, in three areas (forested, rural, and urban, exhibiting different degrees of anthropogenic changes) in a region of intense transmission of visceral leishmaniasis in Corumbá county, Mato Grosso do Sul, Brazil. Using light traps, sand fly specimens were collected from the three environments simultaneously, from May 2015 to April 2017, totaling 7 213 sand flies of 14 species in eight genera. Nuorteva's synanthropy index was determined for the species Lutzomyia cruzi, Brumptomyia brumpti, Micropygomyia peresi, Lu. forattinii, Martinsmyia oliveirai and Evandromyia corumbaensis. Lutzomyia cruzi, the vector of Leishmania infantum in Corumbá, was the most abundant vector species, recorded from all three areas and sampling plots, on all 24 months investigated. This species exhibited the highest synanthropic index (+75.09), indicating a strong preference for urban environments. Brumptomyia brumpti, Micropygomyia peresi, Lu. forattinii, and Martinsmyia oliveirai showed preference, albeit not strong, for urban environments. Overall, males were more abundant than females (W = 490; p < 0.0001). High density, high synanthropic index, and sustained indoor presence were found for Mi. peresi in the rural area. Monitoring changes in the ecological behavior of sand flies is of vital importance, as these changes may indicate an increased likelihood of leishmaniasis emergence or reemergence.

Sandfly fauna (Diptera: Psychodidae) in an urban area, Central-West of Brazil.

Biological and ecological relations among vectors and their pathogens are important to understand the epidemiology of vector-borne diseases. Camapuã is an endemic area for visceral and tegumentary leishmaniasis. The aim of this study was to characterize the sandfly fauna present in Camapuã , MS, Brazil. Sand flies were collected every fortnight from May 2014 to April 2015 using automatic light traps in the domicile and peridomicile of twelve neighborhoods and forest. The collected specimens were identified based on morphology according to the valid identification keys. In total, 2005 sandflies of five genera and nine species were collected. Nyssomyia whitmani and Lutzomyia cruzi were the most abundant species. Males were more abundant, with a male-to-female ratio of 2.14. The highest diversity was observed in peripheral neighborhood, with abundant plant cover. The peridomicile presented greater abundance of sandflies, with the predominance of Ny. whitmani . No significant correlation between the absolute frequencies of the most abundant species and the precipitation variable was observed; however, there was a predominance of Lu. cruzi in the rainy season. We observed a high frequency of sandflies in urban area, especially vector species. The presence of Nyssomyia whitmani and Lutzomyia cruzi indicate the necessity for health surveillance in the municipality. Additional method of collection such as sticky trap is also recommended for appropriate faunestic study.

Standardization of DNA extraction from sand flies: Application to genotyping by next generation sequencing.

Standardization of the methods for extraction of DNA from sand flies is essential for obtaining high efficiency during subsequent molecular analyses, such as the new sequencing methods. Information obtained using these methods may contribute substantially to taxonomic, evolutionary, and eco-epidemiological studies. The aim of the present study was to standardize and compare two methods for the extraction of genomic DNA from sand flies for obtaining DNA in sufficient quantities for next-generation sequencing. Sand flies were collected from the municipalities of Campo Grande, Camapuã, Corumbá and Miranda, state of Mato Grosso do Sul, Brazil. Three protocols using a silica column-based commercial kit (ReliaPrep™ Blood gDNA Miniprep System kit, Promega®), and three protocols based on the classical phenol-chloroform extraction method (Uliana et al., 1991), were compared with respect to the yield and quality of the extracted DNA. DNA was quantified using a Qubit 2.0 fluorometer. The presence of sand fly DNA was confirmed by PCR amplification of the IVS6 region (constitutive gene), followed by electrophoresis on a 1.5% agarose gel. A total of 144 male specimens were analyzed, 72 per method. Significant differences were observed between the two methods tested. Protocols 2 and 3 of phenol-chloroform extraction presented significantly better performance than all commercial kit extraction protocols tested. For phenol-chloroform extraction, protocol 3 presented significantly better performance than protocols 1 and 2. The IVS6 region was detected in 70 of 72 (97.22%) samples extracted with phenol, including all samples for protocols 2 and 3. This is the first study on the standardization of methods for the extraction of DNA from sand flies for application to next-generation sequencing, which is a promising tool for entomological and molecular studies of sand flies.

SAND FLIES (DIPTERA: PSYCHODIDAE) IN AN ENDEMIC AREA OF LEISHMANIASIS IN AQUIDAUANA MUNICIPALITY, PANTANAL OF MATO GROSSO DO SUL , BRAZIL.

The Aquidauana municipality is considered an endemic area of leishmaniasis and an important tourist site in Mato Grosso do Sul State. The aim of this study was to investigate the sand fly fauna in the city of Aquidauana. Captures were carried out twice a month, from April 2012 to March 2014 with automatic light traps and active aspiration, in the peridomicile and domicile of six residences. A total of 9,338 specimens were collected, 3,179 and 6,159 using light traps and active aspiration, respectively. The fauna consisted of: Brumptomyia brumpti, Evandromyia aldafalcaoae, Ev. evandroi, Ev. lenti, Ev. orcyi, Ev. sallesi, Ev. termitophila, Ev. walkeri, Lutzomyia longipalpis and Psathyromyia bigeniculata. The most abundant species captured was Lutzomyia longipalpis, present in all the ecotopes, predominantly in peridomicile areas, and mainly males. Leishmania DNA was not detected in the insects. It was observed the abundance of the sand fly fauna in the region, as well as the high frequency of Lu. longipalpis, the main vector of L. infantum. The results of this study show the need to increase the monitoring and more effective control measures. It is noteworthy that the studied region presents several activities related to tourism and recreation, increasing the risk of transmission of leishmaniasis to this particular human population.

Monthly Distribution of Phlebotomine Sand Flies, and Biotic and Abiotic Factors Related to Their Abundance, in an Urban Area to Which Visceral Leishmaniasis Is Endemic in Corumbá, Brazil.

The monthly distribution and abundance of sand flies are influenced by both biotic and abiotic factors. The present study aimed to evaluate the seasonal distribution of sand flies and the relation between their abundance and environmental parameters, including vegetation and climate. This study was conducted over a 2-year period (April 2012 to March 2014). Monthly distribution was evaluated through the weekly deployment of CDC light traps in the peridomicile area of 5 residences in an urban area of the municipality of Corumbá in the State of Mato Grosso do Sul, Brazil. Meteorological data were obtained from the Mato Grosso do Sul Center for Weather, Climate, and Water Resources. The spectral indices were calculated based on spatial resolution images (GeoEye) and the percentage of vegetal coverage. Differences in the abundance of sand flies among the collection sites were assessed using the Kruskal-Wallis test, and the strength of correlations between environmental variables was determined by calculating Spearman's correlation coefficients. Lutzomyia cruzi, Lu. forattinii, and Evandromyia corumbaensis were the most frequently found species. Although no significant association was found among these sand fly species and the tested environmental variables (vegetation and climate), high population peaks were found during the rainy season, whereas low peaks were observed in the dry season. The monthly distribution of sand flies was primarily determined by Lu. cruzi, which accounted for 93.94% of the specimens collected each month throughout the experimental period. The fact that sand flies were detected year-round indicates a continuous risk of infection to humans, demonstrating the need for targeted management and education programs.

Leishmania amazonensis DNA in wild females ofLutzomyia cruzi (Diptera: Psychodidae) in the state of Mato Grosso do Sul, Brazil

Studies on natural infection by Leishmania spp of sandflies collected in endemic and nonendemic areas can provide important information on the distribution and intensity of the transmission of these parasites. This study sought to investigate the natural infection by Leishmaniain wild female sandflies. The specimens were caught in the city of Corumbá, state of Mato Grosso do Sul (Brazil) between October 2012-March 2014, and dissected to investigate flagellates and/or submitted to molecular analysis to detect Leishmania DNA. A total of 1,164 females (77.56% of which were Lutzomyia cruzi) representing 11 species were investigated using molecular analysis; 126 specimens of Lu. cruziwere dissected and also submitted to molecular analysis. The infection rate based on the presence of Leishmania DNA considering all the sandfly species analysed was 0.69%; only Leishmania (Leishmania) amazonensis was identified in Lu. cruzi by the molecular analysis. The dissections were negative for flagellates. This is the first record of the presence of L. (L.) amazonensis DNA in Lu. cruzi, and the first record of this parasite in this area. These findings point to the need for further investigation into the possible role of this sandfly as vector of this parasite.

Leishmania amazonensis DNA in wild females of Lutzomyia cruzi (Diptera: Psychodidae) in the state of Mato Grosso do Sul, Brazil.

Studies on natural infection by Leishmania spp of sandflies collected in endemic and nonendemic areas can provide important information on the distribution and intensity of the transmission of these parasites. This study sought to investigate the natural infection by Leishmaniain wild female sandflies. The specimens were caught in the city of Corumbá, state of Mato Grosso do Sul (Brazil) between October 2012-March 2014, and dissected to investigate flagellates and/or submitted to molecular analysis to detect Leishmania DNA. A total of 1,164 females (77.56% of which were Lutzomyia cruzi) representing 11 species were investigated using molecular analysis; 126 specimens of Lu. cruziwere dissected and also submitted to molecular analysis. The infection rate based on the presence of Leishmania DNA considering all the sandfly species analysed was 0.69%; only Leishmania (Leishmania) amazonensis was identified in Lu. cruzi by the molecular analysis. The dissections were negative for flagellates. This is the first record of the presence of L. (L.) amazonensis DNA in Lu. cruzi, and the first record of this parasite in this area. These findings point to the need for further investigation into the possible role of this sandfly as vector of this parasite.

Spatiotemporal analysis of sandfly fauna (Diptera: Psychodidae) in an endemic area of visceral leishmaniasis at Pantanal, central South America.

BACKGROUND: Environmental changes caused by urbanization can cause alterations in the ecology and behavior of sandflies and in the epidemiology of leishmaniasis. Geotechnological tools allow the analysis and recognition of spatiotemporal patterns by monitoring and mapping risk areas of this vector-borne disease. This study aims to describe the sandfly fauna in the municipality of Corumbá and to compare it with the data described in a three-year period from 1984 to 1986 by Galati. A further aim was to analyze the influence of environmental changes on the composition of the fauna. METHODS: Captures were conducted weekly from April 2012 to March 2013, in intra and peridomicile areas with automatic light traps, from 6:00 pm to 6:00 am. The following indices were calculated for both periods analyzed: Standardized Index of Species Abundance (SISA), Shannon's diversity index (H) and Pielou's index (J). The Normalized Difference Vegetation Index (NDVI) was extracted from a remote sensing LANDSAT-5 image. RESULTS: In total, 7,370 specimens (6,169 males and 1,201 females) were collected, distributed among 12 species. Lutzomyia cruzi was the most frequent species (93,79%) and the first in the ranking of standardized species abundance index in both studies. The dominance of the species Lu. cruzi in the neighborhoods of Maria Leite and Centro was demonstrated by the low equitability index. The neighborhood of Cristo Redentor had the greatest diversity of sandflies in the present study and the second greatest in the study performed by Galati et al. (Rev Saúde Pública 31:378-390, 1997). Analyzing the satellite images and the NDVI from 1984 and 2010, the largest amount of dense vegetation was found in the neighborhood of Cristo Redentor. CONCLUSIONS: It was, therefore, possible to show how changes caused due to urbanization have affected the density and distribution of Lu. cruzi and other species over time. Moreover, the data suggest that different populations of sandflies adapt in different ways according to environmental conditions and the adaptation does not necessarily depends on the presence of high vegetation cover.