Sand Fly Species Research Articles

Awareness of residents about kala-azar and its related practices in two endemic areas of Bangladesh.

Kala-azar, a worldwide disease, is caused by the parasitic protozoan species of genus Leishmania and transmitted by species of sandflies. Awareness of the population about the disease is essential to run a successful control-strategies program. This cross-sectional study was conducted to assess the awareness of residents about it and related practices in two highly-endemic areas of Bangladesh. In total, 511 household respondents were selected conveniently from two unions (Kushmail and Kanihari) under two sub-districts (Trishal and Fulbaria) of Mymensingh district. Interviewer-administered questionnaires were used for assessing the awareness of the respondents about kala-azar and their practices. Knowledge scores were categorized as poor (<mean -1SD), average (mean ±1SD), good (>mean +1SD). Statistical tests were considered significant at p value of ≤5% (≤0.05). Chi-squared and Student's t-tests were performed for statistical analysis. The mean knowledge score of the respondents of Kanihari union regarding kala-azar was significantly higher than that of the respondents of Kushmail union (mean ±SD, 4.30 ±0.86 versus 4.12 ±0.75, p = 0.002). Of the respondents, 11% and 20% had good (>5.04), 77% and 72% had average (3.43-5.04), 12% and 8% had poor (<3.43) (GAP) knowledge on kala-azar in Kushmail union and Kanihari union respectively. However, the mean knowledge score of the respondents of Kanihari union relating sandfly was significantly lower compared to that of the respondents of Kushmail union (mean ±SD, 2.49 ±0.79 versus 2.65 ±0.85, p = 0.03). Of them, 22% and 19% had good (>3.39), 67% and 64% had average (3.39-1.75), and 11% and 17% had poor (<1.75) knowledge on sandfly in Kushmail union and Kanihari union respectively. More than 70% of the respondents from Kushmail and Kanihari mentioned only injection as the preferred treatment of kala-azar. Eighty-seven percent and 88% of the respondents in the two unions had chosen upazilla health complex for the treatment of their kala-azar. About 50% of the respondents in Kushmail and Kanihari liked to use coil or mat as a preventive measure to avoid sandfly bites. The findings of this study indicate the importance of awareness and related practices of the community people in two endemic areas that will help implement the kala-azar-elimination program successfully in Bangladesh.

Effects of land use type, spatial patterns and host presence on Leishmania tropica vectors activity

BackgroundLeishmaniasis is a vector-borne disease, caused by the infection of Leishmania parasites which are transmitted by the bite of infected female phlebotomine sand flies. Leishmania tropica is transmitted by Phlebotomus sergenti and Phlebotomus arabicus while the main reservoir host is the rock hyrax. A marked increase in the incidence of cutaneous leishmaniasis (CL) caused by L. tropica has been detected in recent years in Israel; it is associated with infections which have emerged in new urban and rural foci. The objective of this study is to contribute to a better understanding of the preferred habitat, spatial activities and host-sand fly relationships of both species of vectors within various types of land use.MethodsUsing CDC-type traps, we investigated the activity levels of sand flies. A field survey was conducted in 2016 at Elifelet, an agricultural village characterized by various types of land use. Movement patterns of P. sergenti between rock-piles were investigated by using colour-marked sugar baits and analyses of recapture patterns. In 2017, a survey was conducted in the hilly Jordan River area, by comparing sand flies and rock hyrax activities in relation to the size of rock-piles and vegetation cover.ResultsBoth sexes of both species were found to have a clear preference for rocky habitats over other land use types in rural landscapes. Movement patterns of P. sergenti were characterized by their high presence close to the rocks and an exponential decrease in their recapture, commensurate with the distance from the rocks. Host-sand fly relationships were found to have a higher correlation between rock hyrax activity levels for females than for males of both species of sand flies. Males exhibited the strongest association with the size of rock-piles.ConclusionsThe results suggest a strong affinity of both phlebotomine vector species to the rocky habitats of the Mediterranean areas. We suggest that rock-piles are associated with populations of rock hyraxes attracting female sand flies seeking blood sources. Rapid human population growth, coupled with intensive land-use changes and the creation of artificial rock-piles, which created potential habitats for both vectors and hosts in the proximity of many settlements, have increased the prevalence of L. tropica among the human population in the region.

A survey of sand flies (Diptera, Phlebotominae) along recurrent transit routes in Serbia

Serbia was a country endemic for leishmaniasis with rich and abundant sand fly fauna during the middle of the 20th century. After 1968, the disease was considered as eradicated, and as a consequence, all research on vectors and pathogens was neglected. The recent detection of Leishmania infantum in sand flies and confirmed cases of leishmaniasis in humans and dogs indicated the risk of re-emergence in Serbia. Also, for millions of refugees/immigrants, Serbia is a transit route between East-Mediterranean and Middle-East countries, and Central/North Europe, and it is under constant risk of vector and disease introduction.Primary objectives of this research were to determine which sand fly species are present around transit routes, whether they include vectors of the Leishmania spp. moreover, is/are Leishmania spp. present in the vectors.Surveys were conducted at 55 locations, organised in four clusters, which covered main immigrant routes and shelters/camps as well as tourist/trade transit routes. In total, 367 sand fly specimens were collected. Nine species were identified: Phlebotomus papatasi, P. perfiliewi, P. tobbi, P. neglectus, P. sergenti, P. alexandri, P. simici, P. balcanicus and P. mascittii. Detection of P. alexandri represents the first record in Serbia. The diversity of sand fly species increased while the number of collected specimens per night decreased during the period of research neglection. Phlebotomus neglectus, a proven vector of L. infantum, was a predominant species in all surveyed clusters, and in 56,52% of locations, it was the only species present. Although all detected species are regarded either as proven or suspected vectors of Leishmania spp., screening of females for Leishmania presence resulted negative.Our study provides insight into the significant changes of sand fly fauna in Serbia during the end of XX and beginning of XXI century. Diverse sand fly fauna in Serbia suggests that the establishment of new leishmaniasis foci is possible.

Epidemiological aspects of cutaneous leishmaniasis in Southwest of Iran

Background and Aim: Zoonotic rodents (Rodentia: Muridae: Gerbillinae) and Phlebotominae sand flies are the major reservoir hosts and vectors of Leishmania major causing zoonotic cutaneous leishmaniasis, respectively. This study was carried out to evaluate the epidemiological aspects of cutaneous leishmaniasis (CL) in Tangestan County, Southwest Iran. Materials and Methods: This descriptive cross-sectional study was carried out on human cases, probable reservoirs, and vectors of CL at Tangestan County in 2017. The data of patients were taken on health centers and analyzed. To determination faunistic of sand flies, they were collected using sticky paper traps and identified using identification keys. The rodent samples were captured using Sherman live traps and determined for amastigote infection. Results: A total of 436 cases occurred during 2005-2017 and the incidence rate of disease was 0.2-12/100,000 people. Six species of sand flies including three species of Phlebotomus and three species of Sergentomyia including Phlebotomus papatasi, Phlebotomus sergenti, Phlebotomus alexandri, Sergentomyia tiberiadis, Sergentomyia baghdadis, and Sergentomyia clydei were identified. From 185 trapped rodents, 9 species were identified. We founded, 2 out of 132 Tatera indica (1.53%) were positive with Leishmania spp. using the microscopic method. Conclusion: According to the existence of human leishmaniasis cases as a host, T. indica with high abundance and infected to Leishmania as a reservoir, and existence of P. papatasi with high abundance as a suspected vector of disease; there is a potential for transmission of disease in the area. Design of exact control and prevention strategies is highly recommended.

Ecological niche models for sand fly species and predicted distribution of Lutzomyia longipalpis (Diptera: Psychodidae) and visceral leishmaniasis in Bahia state, Brazil.

Visceral leishmaniasis is a public health problem in Brazil. This disease is endemic in most of Bahia state, with increasing reports of cases in new areas. Ecological niche models (ENM) can be used as a tool for predicting potential distribution for disease, vectors, and to identify risk factors associated with their distribution. In this study, ecological niche models (ENMs) were developed for visceral leishmaniasis (VL) cases and 12 sand fly species captured in Bahia state. Sand fly data was collected monthly by CDC light traps from July 2009 to December 2012. MODIS satellite imagery was used to calculate NDVI, NDMI, and NDWI vegetation indices, MODIS day and night land surface temperature (LST), enhanced vegetation index (EVI), and 19 Bioclim variables were used to develop the ENM using the maximum entropy approach (Maxent). Mean diurnal range was the variable that most contributed to all the models for sand flies, followed by precipitation in wettest month. For Lutzomyia longipalpis (L. longipalpis), annual precipitation, precipitation in wettest quarter, precipitation in wettest month, and NDVI were the most contributing variables. For the VL model, the variables that contributed most were precipitation in wettest month, annual precipitation, LST day, and temperature seasonality. L. longipalpis was the species with the widest potential distribution in the state. The identification of risk areas and factors associated with this distribution is fundamental to prioritize resource allocation and to improve the efficacy of the state's program for surveillance and control of VL.

Updates on the distribution and diversity of sand flies (Diptera: Psychodidae) in Romania

BackgroundPhlebotomine sand flies (Diptera: Psychodidae) are haematophagous insects that transmit the protozoan parasite Leishmania infantum (Kinetoplastida: Trypanosomatidae), the main causative agent of both zoonotic visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in the Mediterranean basin. Eight species of sand flies have been previously recorded in Romania: Phlebotomus papatasi, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus perfiliewi, Phlebotomus neglectus, Phlebotomus longiductus, Phlebotomus balcanicus and Sergentomyia minuta. Three of them (P. perfiliewi, P. neglectus and P. balcanicus) were incriminated as vectors of L. infantum. Recent reports of autochthonous CanL in Romania require updates on sand fly distribution and diversity in this country.MethodsBetween 2013–2014 and 2016–2018, CDC light traps and mouth aspirators were used to collect sand flies in 132 locations from Romania, indoors and around various animal species shelters. Species identification of collected specimens was done using morphological keys, genetic tools and MALDI-TOF protein profiling.ResultsSand flies were present in seven localities (5.3%): Eibenthal, Baia Nouă, Gura Văii (south-western Romania, Mehedinţi County); Fundătura, Pâhneşti, Epureni (eastern Romania, Vaslui County); and Schitu (southern Romania, Giurgiu County). Of the total number of collected sand flies (n = 251), 209 (83.27%) were Phlebotomus neglectus, 39 (15.53%) P. perfiliewi, 1 (0.40%) P. papatasi, 1 (0.40%) P. balcanicus and 1 (0.40%) P. sergenti (sensu lato).ConclusionsWe confirmed the presence of five sand fly species previously recorded in Romania. However, their updated distribution differs from historical data. The diversity of sand fly species in Romania and their presence in areas with Mediterranean climatic influences constitutes a threat for the reemergence of vector-borne diseases. In the context of CanL and VL reemergence in Romania, but also due to imported cases of the diseases in both humans and dogs, updates on vector distribution are imperative.

PCR-RFLP analyses of Leishmania species causing cutaneous and mucocutaneous leishmaniasis revealed distribution of genetically complex strains with hybrid and mito-nuclear discordance in Ecuador.

PCR-Restriction Fragment Length Polymorphism (RFLP) analyses targeting multiple nuclear genes were established for the simple and practical identification of Leishmania species without using expensive equipment. This method was applied to 92 clinical samples collected at 33 sites in 14 provinces of Ecuador, which have been identified at the species level by the kinetoplast cytochrome b (cyt b) gene sequence analysis, and the results obtained by the two analyses were compared. Although most results corresponded between the two analyses, PCR-RFLP analyses revealed distribution of hybrid strains between Leishmania (Viannia) guyanensis and L. (V.) braziliensis and between L. (V.) guyanensis and L. (V.) panamensis, of which the latter was firstly identified in Ecuador. Moreover, unexpected parasite strains having the kinetoplast cyt b gene of L. (V.) braziliensis and nuclear genes of L. (V.) guyanensis, L. (V.) panamensis, or a hybrid between L. (V.) guyanensis and L. (V.) panamensis were identified. This is the first report of the distribution of a protozoan parasite having mismatches between kinetoplast and nuclear genes, known as mito-nuclear discordance. The result demonstrated that genetically complex Leishmania strains are present in Ecuador. Since genetic exchanges such as hybrid formation were suggested to cause higher pathogenicity in Leishmania and may be transmitted by more species of sand flies, further country-wide epidemiological studies on clinical symptoms, as well as transmissible vectors, will be necessary.

Experimental Infection of Sand Flies by Massilia Virus and Viral Transmission by Co-Feeding on Sugar Meal.

Background: Massilia virus (MASV) is a phlebovirus isolated from Phlebotomus perniciosus in various regions of southwestern Europe. It is closely related to human pathogens such as Toscana virus and sandfly fever Naples virus. The natural cycle of phleboviruses is poorly understood. Indeed, experimental studies demonstrate that transovarial and sexual transmission are not efficient enough for the maintenance of the virus in nature and to date there is no convincing evidence that a species of vertebrates is the reservoir of the virus. Here, we studied various transmission routes of MASV taking advantage of experimental colonies representing different species of sand flies. Methodology/Principal findings: In P. perniciosus, four sources of infection were compared: (i) Virus-seeded larval food to the first instar larvae (L1), or (ii) to the fourth instar larvae (L4), (iii) virus-seeded blood meal to adult females, and (iv) virus-seeded sugar meal to adults of both sexes. From 875 adults emerged from infected L1 and L4, only three were positive. In females infected by bloodmeal the infection rate was high before defecation, then it decreased drastically; MASV RNA was detected in only 5 out of 27 post-defecation. Surprisingly, the most efficient route of infection was observed after intake of virus-seeded sugar meal: 72% of females (79/110) and 52% of males (51/99) were found to be MASV RNA-positive. In addition, MASV-infected sandflies regurgitated virus particules into the sugar drop and MASV RNA was detectable in this drop for at least 24 h after regurgitation. MASV RNA was detected in about one third of the P. perniciosus exposed to this sugar drop contaminated by regurgitation. Sugar meal infection was also tested with six other species of sand flies. In males, there were no significant differences in infection rates when compared to P. perniciosus. In females, most species tested showed high infection rate at the beginning but then significant gradual decrease in infection rate during the experiment. Conclusions/Significance: We present the first description of arboviral infection of a dipteran vector using sugar meal. In all seven sand fly species tested, MASV was detected for two weeks post-infection. Our results showed that MASV can be transmitted between P. perniciosus either through co-feeding or via an infected sugar source such as plant sap. These newly described routes of horizontal transmission may play an important role in the circulation of phleboviruses in nature.

Inventory and taxonomy of phlebotomine sand flies of the Mokolo leishmaniasis focus, northern Cameroon, with description of new Sergentomyia taxa (Diptera: Psychodidae)

Leishmaniasis is endemic in northern Cameroon. However, the sand fly vectors have not been incriminated. A sand fly species inventory was generated by integrating a number of techniques. Miniature light traps were used for collecting sand flies in a variety of ecotopes found across the area, and a morphological and molecular identification approach for taxonomic confirmation was undertaken. In a pilot survey conducted in September 2012, we captured 687 sand flies, 259 of which were morphologically identified to species level. They represent 14 species of the genera Sergentomyia and Grassomyia. No Phlebotomus spp. were found. A second series of collections was carried out during 2013 in five different environmental setups: two urban, two peri-urban/rural and one sylvatic; 14,036 sand flies (6665 males and 7371 females) were collected. A total of 5926 females and 98 males were morphologically identified to species level, representing 19 species of the genera Sergentomyia, Grassomyia and Phlebotomus, including Ph. duboscqi, a known vector of cutaneous leishmaniasis in the region. Two new taxa were found and are described: Sergentomyia (Sintonius) thomsoni mandarai ssp. nov. and Se. coronula sp. nov. Our study is the first to report the following species in Cameroon: Se. (Sin.) thomsoni (as ssp. nov. mandarai), Se. (Ser.) cincta, Se. (Sin.) affinis ssp. vorax, Se. (Sin.) adami, Se. (Sin.) herollandi, and Se. (Sin.) christophersi. In addition, some morphologically atypical Sergentomyia specimens (combination of Ser. x Sin. traits) were recorded. A checklist of 32 species reports from Cameroon is presented.

Leishmania spp. Infection Rate and Feeding Patterns of Sand Flies (Diptera: Psychodidae) from a Hyperendemic Cutaneous Leishmaniasis Community in Panamá.

American cutaneous leishmaniasis (ACL) is a common and important vector-borne parasitic zoonosis in Panamá. Here, we study Leishmania spp. infection rates and blood-feeding patterns among common sand flies in Trinidad de Las Minas, a rural community with hyperendemic ACL transmission, and where a deltamethrin fogging trial was performed. Sand flies were collected from April 2010 to June 2011 with light traps installed inside and in the peridomicile of 24 houses. We restricted our analysis to the most abundant species at the study site: Lutzomyia trapidoi, Lutzomyia gomezi, Lutzomyia panamensis, Lutzomyia triramula, and Lutzomyia dysponeta. We detected Leishmania spp. infection in sand flies by polymerase chain reaction (PCR) amplification of the internal transcribed spacer region 1 (ITS-1) in pooled females (1-10 females per pool). Host species of engorged sand flies were identified using a cytochrome b PCR. From 455 sand fly pools analyzed, 255 pools were positive for Leishmania spp., with an estimated infection rate (confidence interval) of 0.096 [0.080-0.115] before the deltamethrin fogging which slightly, but not significantly (P > 0.05), increased to 0.116 [0.098-0.136] after the deltamethrin fogging. Blood meal analysis suggested that pigs, goats, and birds were the most common sand fly blood sources, followed by humans and domestic dogs. DNA sequencing from a subsample of ITS-1 positive pools suggests that Leishmania panamensis, Leishmania naiffi, and other Leishmania spp. were the parasite species infecting the most common vectors at the study site. Our data confirm an association between sand fly species, humans, domestic dogs, and pigs and Leishmania spp. parasites in rural Panamá.

Functional and structural similarities of D7 proteins in the independently-evolved salivary secretions of sand flies and mosquitoes

The habit of blood feeding evolved independently in many insect orders of families. Sand flies and mosquitoes belong to separate lineages of blood-feeding Diptera and are thus considered to have evolved the trait independently. Because of this, sand fly salivary proteins differ structurally from those of mosquitoes, and orthologous groups are nearly impossible to define. An exception is the long-form D7-like proteins that show conservation with their mosquito counterparts of numerous residues associated with the N-terminal domain binding pocket. In mosquitoes, this pocket is responsible for the scavenging of proinflammatory cysteinyl leukotrienes and thromboxanes at the feeding site. Here we show that long-form D7 proteins AGE83092 and ABI15936 from the sand fly species, Phlebotomus papatasi and P. duboscqi, respectively, inhibit the activation of platelets by collagen and the thromboxane A2 analog U46619. Using isothermal titration calorimetry, we also demonstrate direct binding of U46619 and cysteinyl leukotrienes C4, D4 and E4 to the P. papatasi protein. The crystal structure of P. duboscqi ABI15936 was determined and found to contain two domains oriented similarly to those of the mosquito proteins. The N-terminal domain contains an apparent eicosanoid binding pocket. The C-terminal domain is smaller in overall size than in the mosquito D7s and is missing some helical elements. Consequently, it does not contain an obvious internal binding pocket for small-molecule ligands that bind to many mosquito D7s. Structural similarities indicate that mosquito and sand fly D7 proteins have evolved from similar progenitors, but phylogenetics and differences in intron/exon structure suggest that they may have acquired the ability to bind vertebrate eicosanoids independently, indicating a convergent evolution scenario.

Antibody response to Phlebotomus perniciosus saliva in cats naturally exposed to phlebotomine sand flies is positively associated with Leishmania infection

BackgroundZoonotic leishmaniosis, caused by the protozoan Leishmania infantum, is a public and animal health problem in Asia, Central and South America, the Middle East and the Mediterranean Basin. Several phlebotomine sand fly species from the subgenus Larroussius are vectors of L. infantum. Data from dogs living in endemic areas of leishmaniosis advocate the use of antibody response to phlebotomine sand fly saliva as an epidemiological biomarker for monitoring vector exposure. The aim of this study was to analyse the exposure of cats to phlebotomine sand flies using detection of IgG antibodies to Phlebotomus perniciosus saliva. The association between phlebotomine sand fly exposure and the presence of Leishmania infection was also investigated.ResultsIgG antibodies to P. perniciosus saliva were detected in 167 (47.7%) out of 350 cats; higher antibody levels were present in sera collected during the period of phlebotomine sand fly seasonal activity (OR = 19.44, 95% CI: 9.84–38.41). Cats of 12–35 months had higher antibody levels than younger ones (OR = 3.56, 95% CI: 1.39–9.16); this difference was also significant with older cats (for 36–95 months-old, OR = 9.43, 95% CI: 3.62–24.48; for older than 95 months, OR = 9.68, 95% CI: 3.92–23.91). Leishmania spp. DNA was detected in the blood of 24 (6.9%) cats, while antibodies to L. infantum were detected in three (0.9%). Only one cat was positive to Leishmania by both techniques. Cats presenting IgG antibodies to P. perniciosus had a significantly higher risk of being positive for Leishmania infection.ConclusionsTo our knowledge, this is the first study demonstrating anti-sand fly saliva antibodies in cats. The evaluation of the contact of this animal species with the vector is important to the development of prophylactic measures directed to cats, with the aim of reducing the prevalence of infection in an endemic area. Therefore, studies evaluating whether the use of imidacloprid/flumethrin collars reduces the frequency of P. perniciosus bites in cats are needed. It is also important to evaluate if there is a correlation between the number of phlebotomine sand fly bites and IgG antibody levels.

Influence of Deforestation on the Community Structure of Sand Flies (Diptera: Psychodidae) in Eastern Amazonia.

Variation in the structure of phlebotomine (sand fly) communities in forest fragments with different degrees of preservation and human occupation (peridomicile) in eastern Amazonia was studied. We identified 43 species of sand flies in our study, of which 38 occurred in both preserved forest areas and in the peridomiciles of short-term settlements, while another 28 species occurred in altered forest fragments and long-term settlements. The composition of the community at each site changed with the type of environment (forest or peridomicile), with the species Lutzomyia evandroi, L. whitmani, L. choti, L. serrana, L. triacantha, L. migonei, L. hirsuta, L. shannoni, and L. brachyphylla accounting for more than 54% of the differences among environments. The quality of the environment exerted a significant influence on the structure of phlebotomine communities, and affected their species composition, richness, and abundance.

Susceptibility of wild-caught Lutzomyia longipalpis (Diptera: Psychodidae) sand flies to insecticide after an extended period of exposure in western S\xe3o Paulo, Brazil

BackgroundIn Brazil, members of the sand fly species complex Lutzomyia longipalpis transmit Leishmania infantum, a protist parasite that causes visceral leishmaniasis. Male Lu. longipalpis produce a sex pheromone that is attractive to both females and males. During a cluster randomised trial, to determine the combined effect of synthetic sex-aggregation pheromone and insecticide on Le. infantum transmission Lu. longipalpis had been continuously exposed to insecticide for 30 months. The objective of this study was to determine the effect of continuous exposure to the insecticides used in the trial on the susceptibility of Lu. longipalpis population.MethodsDuring the trial the sand flies had been exposed to either lambda-cyhalothrin [pheromone + residual insecticide spray (PI)], deltamethrin [dog collars (DC)] or no insecticide [control (C)], for 30 months (November 2012 to April 2015). The insecticide treatment regime was kept in place for an additional 12 months (May 2015-April 2016) during this susceptibility study. Sand flies collected from the field were exposed to WHO insecticide-impregnated papers cyhalothrin (0.05%), deltamethrin (0.5%) and control (silicone oil) in a modified WHO insecticide exposure trial to determine their susceptibility.ResultsWe collected 788 Lu. longipalpis using CDC-light traps in 31 municipalities across the three trial arms. Probit analysis showed that the knockdown times (KDTs) of Lu. longipalpis collected from the lambda-cyhalothrin exposed PI-arm [KDT50: 31.1 min, confidence interval (CI): 29.6–32.6 and KDT90: 44.2 min, CI: 42.1–46.7] were longer than the KDTs from the non-insecticide-treated C-arm (KDT50: 26.3 min, CI: 25.1–27.6 and KDT90: 38.2, CI: 36.5–40.2) (no-overlapping 95% CIs). KDTs of Lu. longipalpis collected from the deltamethrin exposed DC-arm had similar values (KDT50: 13.7 min, CI: 10.1–16.2 and KDT90: 26.7 min, CI: 21.8–30.6) to those for the C-arm (KDT50: 13.5 min; CI: 12.2–14.8 and KDT90: 23.2 min, CI: 21.4–25.4) (overlapping CIs). The wild-caught unexposed Lu. longipalpis (C-arm), took approximately twice as long to knock down as laboratory-colonised specimens for both insecticides.ConclusionsOur study reveals slight changes in KDT, in sand flies after prolonged exposure to lambda-cyhalothrin in the presence of pheromone. These changes are not considered to have reached the reference levels indicative of resistance in sand flies suggesting that pheromone and insecticide treatment at the level indicated in this study do not constitute a significant risk of increased insecticide resistance. Prolonged exposure to deltamethrin in dog collars did not result in changes to KDT.

Identification of phlebotomine sand flies through MALDI-TOF mass spectrometry and in-house reference database

Phlebotomine sand flies are vectors for many pathogens responsible for human and animal diseases worldwide. Their identification at species level is of importance in epidemiological studies and control programmes. MALDI-TOF MS has been increasingly investigated as an alternative approach to the conventional identification of arthropods species. To establish an in-house protein spectra database for a quick and reliable species identification of phlebotomine sand flies, 166 field-caught sand fly specimens, morphologically identified as Phlebotomus perniciosus (no = 56; 26 males and 30 females), Phlebotomus neglectus (no = 4 males), Phlebotomus sergenti (no = 6; 4 males and 2 females) and Sergentomyia minuta (no = 100; 45 males and 55 females), were subjected to MALDI-TOF MS analyses. Out of 166, 149 specimens (89.8%) produced consistent species-specific protein spectra. Good quality database for P. perniciosus and S. minuta were generated; no databases have yet constructed for P. neglectus and P. sergenti due to the low number of specimens examined. The identification of 80 sand flies (no = 20 P. perniciosus; no = 60 S. minuta) were confirmed using the new generated SuperSpectra as validation test. The results reported support the use of MALDI-TOF MS for rapid, simple and reliable phlebotomine sand fly species identification suggesting its usefulness in accurate survey studies, ultimately improving biological and epidemiological knowledge on these important vectors of pathogens.

Detection of multiple circulating Leishmania species in Lutzomyia longipalpis in the city of Governador Valadares, southeastern Brazil.

Leishmaniasis encompasses a group of diverse clinical diseases caused by protozoan parasites of the Leishmania genus. This disease is a major public health problem in the New World affecting people exposed in endemic regions. The city of Governador Valadares (Minas Gerais/Brazil) is a re-emerging area for visceral leishmaniasis, with 191 human cases reported from 2008 to 2017 and a lethality rate of 14.7%. The transmission of the parasite occurs intensely in this region with up to 22% of domestic dogs with positive serology for the visceral form. Lu. longipalpis is one of the most abundant sand fly species in this area. Despite this scenario, so far there is no information regarding the circulating Leishmania species in the insect vector Lutzomyia longipalpis in this focus. We collected 616 female Lutzomyia longipalpis sand flies between January and September 2015 in the Vila Parque Ibituruna neighborhood (Governador Valadares/MG), which is located on a transitional area between the sylvatic and urban environments with residences built near a preserved area. After DNA extraction of individual sand flies, the natural Leishmania infections in Lu. longipalpis were detected by conventional PCR, using primers derived from kDNA sequences, specific for L. (Leishmania) or L. (Viannia) subgenus. The sensitivity of these PCR reactions was 0.1 pg of DNA for each Leishmania subgenus and the total infection rate of 16.2% (100 positive specimens). Species-specific PCR detected the presence of multiple Leishmania species in infected Lu. longipalpis specimens in Governador Valadares, including L. amazonensis (n = 3), L. infantum (n = 28), L. (Viannia) spp. (n = 20), coinfections with L. infantum and L. (Viannia) spp. (n = 5), and L. (Leishmania) spp (n = 44). Our results demonstrate that multiple Leishmania species circulate in Lu. longipalpis in Governador Valadares and reveal a potential increasing risk of transmission of the different circulating parasite species. This information reinforces the need for epidemiological and entomological surveillance in this endemic focus, and the development of effective control strategies against leishmaniasis.

Identification of French Guiana sand flies using MALDI-TOF mass spectrometry with a new mass spectra library.

Phlebotomine sand flies are insects that are highly relevant in medicine, particularly as the sole proven vectors of leishmaniasis. Accurate identification of sand fly species is an essential prerequisite for eco-epidemiological studies aiming to better understand the disease. Traditional morphological identification is painstaking and time-consuming, and molecular methods for extensive screening remain expensive. Recent studies have shown that matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for rapid and cost-effective identification of arthropod vectors, including sand flies. The aim of this study was to validate the use of MALDI-TOF MS for the identification of Northern Amazonian sand flies. We constituted a MALDI-TOF MS reference database comprising 29 species of sand flies that were field-collected in French Guiana, which are expected to cover many of the more common species of the Northern Amazonian region, including known vectors of leishmaniasis. Carrying out a blind test, all the sand flies tested (n = 157) with a log (score) threshold greater than 1.7 were correctly identified at the species level. We confirmed that MALDI-TOF MS protein profiling is a useful tool for the study of sand flies, including neotropical species, known for their great diversity. An application that includes the spectra generated here will be available to the scientific community in the near future via an online platform.

Aerobic midgut microbiota of sand fly vectors of zoonotic visceral leishmaniasis from northern Iran, a step toward finding potential paratransgenic candidates

BackgroundLeishmaniasis is caused by Leishmania parasites and is transmitted to humans through the bite of infected sand flies. Development of Leishmania to infective metacyclic promastigotes occurs within the sand fly gut where the gut microbiota influences development of the parasite. Paratransgenesis is a new control method in which symbiotic bacteria are isolated, transformed and reintroduced into the gut through their diet to express anti-parasitic molecules. In the present study, the midgut microbiota of three sand fly species from a steppe and a mountainous region of northern Iran, where zoonotic visceral leishmaniasis (ZVL) is endemic, was investigated.MethodsBriefly, adult female sand flies was collected during summer 2015 and, after dissection, the bacterial composition of the guts were analyzed using a culture-dependent method. Bacterial DNA from purified colonies was extracted to amplify the 16S rRNA gene which was then sequenced.ResultsThree ZVL sand fly vectors including Phlebotomus major, P. kandelakii and P. halepensis were found in the highlighted regions. In total, 39 distinct aerobic bacterial species were found in the sand fly midguts. The sand fly microbiota was dominated by Proteobacteria (56.4%) and Firmicutes (43.6%). Bacterial richness was significantly higher in the steppe region than in the mountainous region (32 vs 7 species). Phlebotomus kandelakii, the most important ZVL vector in the study area, had the highest bacterial richness among the three species. Bacillus subtilis and Pantoea agglomerans were isolated from the guts of the sand flies; these are already used for the paratransgenesis of sand flies and mosquitoes, respectively.ConclusionsThe existence of B. subtilis and P. agglomerans in the ZVL vectors and other sand fly species studied so far suggests that these two bacterial species are potential candidates for paratransgenic approach to prevent ZVL transmission. Further research needs to test the possible relationship between the gut microbiome richness and the vector competence of the ZVL vectors.

Spatial distribution and infection rate of leishmaniasis vectors (Diptera: Psychodidae) in Ardabil Province, Northwest of Iran

Objective: To determine the spatial distribution and infection rate of sand flies as vectors of Leishmania parasite in Ardabil province, northwest of Iran. Methods: This was a descriptive cross-sectional study. The sand flies were collected from 30 areas in all 10 districts of Ardabil province during 2017. The specimens were caught using the sticky traps. The head and genitalia of sand flies were separated and mounted in Berlese solution for microscopic identification. The Geographical Information System ArcMap10.4.1 software was used to provide the spatial maps. Results: A total of 2 794 sand flies specimens were collected and 22 species of sand flies were identified from the two genera: Phlebotomus and Sergentomyia from Ardabil province. The highest frequency was found in Phlebotomus papatasi (23.7%) followed by Phlebotomus kandelakii (13.0%). The promastigote form of Leishmania infantum parasite has been reported from the three main vectors of visceral leishmaniasis (Phlebotomus kandelakii, Phlebotomus perfiliewi and Phlebotomus tobbi) from Ardabil province, where the spatial distribution map of these visceral leishmaniasis vectors was prepared. Some important species of sand flies such as Phlebotomus kandelakii, Phlebotomus perfiliewi and Phlebotomus tobbi were reported and identified as main and probable vectors of visceral leishmaniasis in Ardabil. Conclusions: According to the Geographic Information System based maps, the frequency of the sand flies as leishmaniasis vectors, the leishmania parasite infection rate and the prevalence of the disease in the central areas of Ardabil province are higher than in other areas in Ardabil province.

Diversity, natural infection and blood meal sources of phlebotomine sandflies (Diptera, Psychodidae) in the western Brazilian Amazon.

BACKGROUND The state of Rondônia (RO) is a hot spot for human cases of cutaneous leishmaniasis. Many sandfly species in RO are putative vectors of leishmaniasis.OBJECTIVES This study examines the diversity patterns and the presence of Leishmania DNA and blood meal sources of sandflies in RO.METHODS A sandfly survey was performed between 2016 and 2018 in 10 municipalities categorised into three different environment types: (i) Conservation Unit (CUN) - comprised of preserved ombrophilous forests; (ii) Forest Edge (FE) - small forest fragments; and (iii) Peridomicile (PE) - areas around dwellings.FINDINGS A total of 73 species were identified from 9,535 sandflies. The most abundant species were Psychodopygus davisi (1,741 individuals), Nyssomyia antunesi (1,397), Trichophoromyia auraensis (1,295) and Trichophoromyia ubiquitalis (1,043). Diversity was the highest in CUN, followed by the FE and PE environments. One pool of Ps. davisi tested positive for Leishmania braziliensis, reinforcing the possibility that Ps. davisi acts as a vector. The cytochrome b (cytb) sequences were used to identify three blood meal sources: Bos taurus, Homo sapiens and Tamandua tetradactyla.MAIN CONCLUSIONS Our results demonstrated that sandflies can switch between blood meal sources in differing environments. This study enhances the knowledge of the vector life cycle in RO and provides information relevant to leishmaniasis surveillance.