Individual Sand Flies Research Articles

Trypanosoma infection and bloodmeal analysis in post-feeding sand flies across Thailand

Phlebotomine sand flies are recognized as a primary vector of Leishmania and are also suspected vectors of Trypanosoma. The transmission cycle of these parasites relies on the distribution of sand fly vectors, parasites, and reservoir animals. This study aimed to detect Leishmania and Trypanosoma DNA and identify the sources of bloodmeals in post-feeding sand flies captured across Thailand. A total of 42,911 field female sand flies were collected from 11 provinces across Thailand using CDC light traps. Among these, 253 post-feeding sand flies were selected for analysis. The predominant species in this study was Sergentomyia khawi (33.60 %). The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR), specific to the internal transcribed spacer 1 (ITS1) and the small subunit ribosomal RNA (SSU rRNA) gene regions were used to detect the presence of Leishmania and Trypanosoma DNA, respectively. Additionally, cytochrome c oxidase subunit I (COI) gene region was utilized to identify the sources of host bloodmeals. Leishmania DNA was not detected in any specimens. The analysis of SSU rRNA sequences revealed the presence of Trypanosoma DNA (11.46 %, 29/253) in sand fly samples. Among these samples, T. noyesi (1.58 %, 4/253) was identified in Idiophlebotomus longiforceps and Phlebotomus asperulus, Trypanosoma Anura01+02/Frog2 (1.18 %, 3/253) in Se. khawi, and Trypanosoma Anura04/Frog1 (8.70 %, 22/253) in Se. khawi, Se. hivernus and Grossomyia indica. Bloodmeal analysis utilizing the COI gene revealed a diverse range of vertebrate hosts’ blood, including bird, bat, frog and sun skink. Our findings confirm the presence of Trypanosoma DNA and identify the sources of bloodmeals from vertebrate hosts in various sand fly species, suggesting their potential as possible vectors for Trypanosoma in Thailand. Furthermore, our study is the first to provide molecular evidence using the COI gene to identify frogs as a host blood source for sand flies in Thailand. Further studies focusing on the isolation of live parasites in sand flies to confirm vector potential and examining the role of animal reservoirs will enhance our understanding of the host-parasite relationship and enable more efficient control for disease transmission.

First Detection and Molecular Analysis of Leishmania infantum DNA in Sand Flies of Kosovo.

Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of phleboviruses and Leishmania spp., the causative agents of leishmaniases. The Mediterranean sand fly fauna is diverse, and leishmaniasis, mainly caused by Leishmania infantum, is endemic in the Balkan countries. Despite recent entomological surveys, only some districts of Kosovo have been sampled for sand flies, with no proof/confirmation of L. infantum. This study aimed to gain further insights into the species composition of natural sand fly populations in previously unsampled districts and areas in Kosovo without reports of leishmaniasis and to detect Leishmania DNA in sand flies. A sand fly survey was conducted in 2022 in all seven districts of Kosovo. Collected females were screened for Leishmania DNA by PCR. Positive samples were sequenced and subjected to maximum likelihood analysis with reference sequences for further molecular characterization. The trapping activities at 114 different localities resulted in 3272 caught specimens, comprising seven sand fly species of two genera, namely Phlebotomus neglectus, Ph. perfiliewi, Ph. tobbi, Ph. papatasi, Ph. simici, Ph. balcanicus and Sergentomyia minuta. Leishmania infantum DNA was detected in three individual sand flies of Ph. neglectus and Ph. perfiliewi. This study provides the most extensive sand fly survey in Kosovo and reports the first record of L. infantum DNA in sand flies, indicating autochthonous circulation of L. infantum.

Population Genetic Structure of Phlebotomus sergenti (Diptera: Psychodidae) Collected in Four Regions of Morocco Based on the Analysis of Cyt b and EF-1α Genes.

Phlebotomus (Ph.) sergenti is the main vector of Leishmania (L.) tropica (Trypanosomatida: Trypanosomatidae), the causative agent of anthroponotic cutaneous leishmaniasis in Morocco. This species has an extended geographical distribution, wider than that of the parasite. The main objective of our study was to analyze the genetic diversity of Ph. sergenti collected in four foci in Morocco: Taza, Foum Jemâa, El Hanchane, and Ouarzazate. We studied a set of diversity and population structure indices by sequencing two markers; nuclear EF-1α and mitochondrial Cyt b from 175 individual sand flies. Our results showed a considerable degree of intraspecific polymorphism with a high number of haplotypes identified in both genes. Many polymorphic sites detected in the Cyt b sequences (SCyt b = 45) indicate that it is the most polymorphic marker showing a distinct distribution of haplotypes according to their geographical origin, whereas the EF-1α marker showed no geographical isolation. Analysis by Tajima's D and Fu's Fs tests revealed a possible recent expansion of the populations, especially with the EF-1α marker, showing significant values in Taza and Ouarzazate sequences. The present study revealed significant genetic diversity within Ph. sergenti populations in Morocco. The results warrant further research using a combination of more than two markers including mitochondrial and non-mitochondrial markers, which may provide more information to clarify the genetic status of Ph. sergenti.

Sandfly Blood-Feeding Habits and Competence in Transmitting Ntepes Virus, a Recently Discovered Member of the Genus Phlebovirus.

Phleboviruses transmitted by sandflies are among emerging public health threats. A novel Phlebovirus named Ntepes virus (NTPV) was recently described and found to infect humans from a wide geographic area in Kenya. However, the entomologic risk factors of this virus such as the potential vectors and the transmission cycles remain poorly defined. This study assessed the ability of the colonized sandfly Phlebotomus duboscqi to transmit NTPV and determined the bloodmeal host sources of field-collected sandflies from the area where NTPV was found in Baringo County, Kenya. Five-day old laboratory-reared P. duboscqi were orally challenged with an infectious dose of NTPV (≈106.0 pfu/ml) and incubated for up to 15 days postinfection. Individual sandflies were dissected into abdomens, legs, and salivary glands and screened for the virus infection by cell culture. Of the 205 virus-exposed sandflies, 19.5% developed non-disseminated infections in the midgut, with no evidence of virus dissemination or transmission in legs and salivary glands, respectively. The midgut infection rates decreased with increasing extrinsic incubation period (Spearman's correlation, ρ = −0.71). Blood-fed specimens analyzed by polymerase chain reaction (PCR) and sequencing of a region of the mitochondrial 12S rRNA, revealed almost exclusive feeding on humans (98%) represented by the sandflies Sergentomyia schwetzi, S. clydei, S. antennata, S. squamipleuris, S. africana, and Phlebotomus martini. One specimen of S. clydei had fed on cattle (2%). These findings suggest P. duboscqi is an incompetent laboratory vector of NTPV. The high human-feeding rate by diverse sandfly species increases the likelihood of human exposure to pathogens associated with these sandflies. Assessment of the susceptibility of Sergentomyia species to NTPV is recommended given their high human-feeding tendency.

A new cluster of rhabdovirus detected in field-caught sand flies (Diptera: Psychodidae: Phlebotominae) collected from southern Thailand

BackgroundThe distribution of phlebotomine sand flies is changing rapidly due to climate change. This issue has implications for the epidemiology of sand fly-borne diseases, especially sand fly-associated viruses. Few studies concerning sand fly-associated viruses have been conducted in Thailand. Therefore, this study aimed to perform a molecular survey of groups of pathogenic RNA viruses belonging to the Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae in sand fly samples collected from southern Thailand.MethodsSand flies were collected at two locations in Trang and Songkhla provinces of southern Thailand, and individual sand fly samples were processed for species identification and virus detection. The Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae molecular determination was performed by RT-PCR, and positive samples were identified by cloning and sequencing, cell culture inoculation, and phylogenetic analysis.ResultsThe results presented in this study were based on the analysis of a total of 331 female sand flies. This molecular study revealed evidence of Rhabdoviridae family virus presence in Phlebotomus papatasi (3/331, 0.9%). The findings demonstrated a new cluster of rhabdovirus that was closely related to Bactrocera dorsalis sigmavirus strain BDSV.abc5 and the lineages of insect-specific Rhabdoviridae. In addition, the Bayesian tree suggested that the common ancestor of this group was the dimarhabdovirus clade. It was assumed that the virus may have switched hosts during its evolution. However, the detection of Orbivirus, Phlebovirus, and Flavivirus genera using specific primers for RT-PCR was negative in the collected sand flies.ConclusionsThere is limited knowledge on the genetic diversity and ecology of Rhabdoviridae in Thailand. This is the first data regarding the circulation of Rhabdoviridae in Ph. papatasi from Thailand. We found a new cluster of rhabdoviruses that was close to the new B. dorsalis sigmavirus. It is possible that there is a great deal of diversity in this family yet to be discovered, and a more extensive survey for new rhabdoviruses may uncover viruses from a wide diversity of host taxa and broaden our understanding of the relationships among the Rhabdoviridae.Graphical abstract

First molecular identification of Leishmania major in Phlebotomus papatasi in an outbreak cutaneous leishmaniasis area in Iraq

Cutaneous leishmaniasis (CL) is transmitted by Phlebotomine sand fly vectors, among which Phlebotomus papatasi is prevalent in Western Asia, Northern Africa and Southern Europe, and it is known as a vector for Leishmania major parasite in the world. However, in Iraq, morphological studies showed that P. papatasi is a predominant sand fly species and hypothesised to transmit CL causing Leishmania species including L. major and L. tropica. Few studies have found Leishmania species in sand flies in mixed pools of samples in this country. Accurate identification of sand flies as vectors of Leishmania species is required in Iraq.The current study aims to identify sand fly species, using both morphological and molecular phylogenetic analyses, in a region where CL tends to be endemic. Furthermore, molecular phylogenetic analysis has also used to confirm Leishmania species in the sand fly samples collected in 11 villages between Diyala and Sulaymaniyah Provinces. For the first time, we have found L. major in three individual sand flies, one engorged (with fresh blood meal) and two non-engorged (without visible fresh blood meal) P. papatasi females in an area of CL outbreaks since 2014-till now due to civil wars and internal conflicts happen in the region. Further study should be performed on sand fly population and Leishmania reservoirs in this region.

DNA extraction from individual Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) specimens: Which is the method with better results?

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are a group of small insects of great concern for Public Health. These dipterous are intensely studied worldwide due to their involvement in the transmission of several pathogens, mainly Leishmania spp. parasites. Nowadays, the molecular tools have been included in Phlebotomine sand flies studies and has shown to be powerful tools in bioecology studies of these dipterous. Thereby, when molecular approaches are employed, there is a great concern regarding the amount and quality of the DNA obtained for analysis. Here, seven methods of DNA extraction, between commercial kits and in house extraction protocols were evaluated. We considered measure of DNA concentration and purity ratios using a spectrophotometer to check the performance of each protocol. In addition, the quality evaluation of the DNA extracted was performed by endogenous gene PCR on samples. The results of the seven evaluated DNA extraction protocols and their implications are discussed.

Establishment of a Colony of Phlebotomus argentipes under Laboratory Conditions and Morphometric Variation between Wild-Caught and Laboratory-Reared Populations.

The field-based studies on sand flies are not adequate to uncover information required for the control of the leishmaniasis through reduction of vector populations. Therefore, establishment and maintenance of laboratory colonies of sand flies is an essential step in leishmaniasis research. In the current study, a colony of P. argentipes was established from wild-caught sand flies following standard procedures from the published literature. Morphological measurements of laboratory-reared and wild-caught individual sand flies were compared to assess the difference between two groups. The colony was successfully established under confined laboratory conditions. The comparison of morphometric parameters revealed that the laboratory-reared sand flies are significantly larger than those caught from wild, suggesting a possibility of increased fitness of sand flies under favorable environmental conditions which may cause higher prevalence in the disease. The current study reports the first successful attempt in colonizing sand flies under laboratory conditions. However, the colony data suggest that the conditions extracted from the published literature need to be optimized to suit local settings in order to achieve maximum population sizes within the available amount of resources.

Identification of Blood Meal of Sand Flies in a Cutaneous Leishmaniasis Endemic Area, Volta Region-Ghana

<i>Background</i>: Vector incrimination is of utmost importance in the transmission of <i>Leishmania</i> species identified in the Volta region of Ghana. The feeding preference of sand flies provides valuable information about the vector-host interactions, including reservoir host. <i>Objective</i>: The main aim of this study was to identify the blood meal in collected sand flies and its source from three villages in a cutaneous leishmaniasis endemic area in the Ho Municipality. <i>Method</i>: Sand flies was collected using CDC light straps and sticky paper traps from Lume Atsiame, Dodome Dogblome and Dodome Awuiasu. They were morphologically identified by taxonomic keys. DNA was extracted from the individual sand flies using the potassium acetate extraction method. Polymerase chain reaction (PCR) amplification of cytochrome b gene (<i>cyt b</i>) fragment was carried out and subsequently DNA sequenced. <i>Results</i>: Of a total of three hundred and sixty-three (363) female sand flies that were analysed, eighty-four (84) representing 23.14% were positive for blood meal presence with an amplified DNA of the expected size, 359bp. Out of the number that was sequenced, the <i>cyt b</i> sequencing revealed that four (4) blood fed females fed on bloodmeal from human (<i>Homo sapiens</i>) and house mouse (<i>Mus musculus</i>). <i>Conclusion</i>: The blood meal presence was more predominant in <i>Sergentomyia ghesquierei</i> and least in <i>Sergentomyia schwetzi</i>. This may aid in the development of effective strategies to control cutaneous leishmaniasis in the endemic areas.

Genetic Structure of Phlebotomus orientalis (Diptera: Psychodidae) in Leishmaniasis Endemic Foci of Sudan.

Visceral leishmaniasis (VL) remains a major concern in many parts of Sudan. The disease is transmitted by Phlebotomus orientalis. The objective of this study was to determine genetic structure of Phlebotomus orientalis population from 5 geographical regions in Sudan. A total of 194 individual sand flies were collected from 5 geographic regions in Sudan. The field collected sand flies were analyzed by Random Amplified Polymorphic DNA (RAPD) using 30 primers. Eight hundred and 65 bands from 4 RAPD primers were analyzed for genetic variation. A higher level of intrapopulational variability was detected in populations of P. orientalis from eastern Sudan compared to those populations from central and northern Sudan. Diagnostic bands were detected in populations of P. orientalis central Sudan. Hieratical clustering analysis showed clear clustering into 2 main populations with 1 population subdivided into 4 subpopulations. However, these populations did not show any correlation with their geographical origins. Furthermore, the low genetic differentiation among subpopulations was supported by fixation index (FST) estimated by analysis of molecular variance (AMOVA). It is concluded that the populations of P. orientalis from the selected areas in Sudan have a low genetic differentiation. However, assessment of genetic structure of P. orientalis populations is important for understanding the patterns of transmission of VL in different endemic areas.

Detection of Leishmania and Trypanosoma DNA in Field-Caught Sand Flies from Endemic and Non-Endemic Areas of Leishmaniasis in Southern Thailand.

Phlebotomine sand flies are tiny, hairy, blood-sucking nematoceran insects that feed on a wide range of hosts. They are known as a principal vector of parasites, responsible for human and animal leishmaniasis worldwide. In Thailand, human autochthonous leishmaniasis and trypanosomiasis have been reported. However, information on the vectors for Leishmania and Trypanosoma in the country is still limited. Therefore, this study aims to detect Leishmania and Trypanosoma DNA in field-caught sand flies from endemic areas (Songkhla and Phatthalung Provinces) and non-endemic area (Chumphon Province) of leishmaniasis. A total of 439 sand flies (220 females and 219 males) were collected. Head and genitalia dissection of female sandflies were done for morphology identification, and the remaining parts of those sand flies were then used for the detection of Leishmania and Trypanosoma parasites. The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR) anneal, specific to the ITS1 and SSU rRNA gene regions, was used to detect Leishmania and Trypanosoma DNA, respectively. The positive PCR products were cloned and sequenced. The results showed that the female sand fly species in this study consisted of Sergentomyia khawi (35.9%); Se. anodontis (23.6%); Phlebotomus betisi (18.6%); Ph. kiangsuensis (9.5%); Ph. asperulus (6.4%); Se. barraudi (2.3%); 0.9% of each Se. indica, Ph. stantoni, and Ph. major major; and 0.5% of each Se. sylvatica and Ph. mascomai. The PCR and sequence analysis were able to detect Leishmania and Trypanosoma DNA in sand fly samples, which were identified as L. martiniquensis, 1/220 (0.45%) in Se. khawi, 3/220 (1.36%) of T. noyesi in Se. anodontis, and Ph. asperulus. Fourteen (6.36%) of the unidentified trypanosome species in Se. khawi, Se. indica, Se. anodontis, Ph. asperulus, and Ph. betisi were found in all of the areas of this study. Interestingly, we found a 1/220 (0.45%) co-infection sample of L. martiniquensis and Trypanosoma in Se. khawi from Songkhla Province. These data indicate that several species of sand flies might be potential vectors of Leishmania and Trypanosoma parasites in southern Thailand. However, more extensive study for potential vectors using a larger number of sand flies should be conducted to prove whether these sand flies can be natural vectors of leishmaniasis and trypanosomiasis in both humans and animals. In addition, our study could be useful for the future study of infection prevention, including effective vector control for leishmaniasis and trypanosomiasis in Thailand.

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification.

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

Quantifying Leishmania Metacyclic Promastigotes from Individual Sandfly Bites Reveals the Efficiency of Vector Transmission

Predicting how Leishmania will respond to control efforts requires an understanding of their transmission strategy. Using real-time quantitative PCR to quantify infectious metacyclic and non-metacyclic forms in mouse skin from single sandfly bites we show that most transmissions were highly enriched for infectious parasites. However, a quarter of sandflies were capable of transmitting high doses containing more non-infectious promastigotes from the vector’s midgut. Mouse infections replicating “high” to “low” quality, low-dose transmissions confirmed clear differences in the pathology of the infection and their onward transmissibility back to sandflies. Borrowing methods originally developed to account for exposure heterogeneity among hosts, we show how these high-dose, low-quality transmitters act as super-spreading vectors, capable of inflating Leishmania transmission potential by as much as six-fold. These results highlight the hidden potential of transmission of mixed Leishmania promastigote stages on disease prevalence and the role of dose heterogeneity as an underlying strategy for efficient transmission.

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.

First detection of Leishmania donovani in sand flies from Cameroon and its epidemiological implications.

A sound knowledge of the vector-host-parasite transmission dynamics is a prerequisite for adequate control measures of vector-borne diseases. To achieve this, an entomological investigation was conducted in the cutaneous leishmaniasis (CL) endemic focus of Mokolo District, northern Cameroon to identify the insect vector(s) of the disease. Phlebotomine sand flies were collected in and around Mokolo using New Standard CDC Miniature Light Traps. Individual sand flies were used for morphological species identification, and the remainder of the body for DNA analysis. Sand flies were demonstrated to harbour Leishmania spp. parasites using ITS1 PCR. Mitochondrial vertebrate-specific Cytochrome b -PCR was used to identify blood meals ingested by female sand flies. PCR amplicons were sequenced for Leishmania and blood sources discrimination. This study revealed the presence of Leishmania donovani complex DNA (n = 1) in Phlebotomus duboscqi and of lizard-borne Leishmania tarentolae-like DNA (n = 3) in Sergentomyia spp. in 79 sand fly specimens from Mokolo district. The causative agent of CL could not be detected in potential vectors. Instead, we found evidence for visceral leishmaniasis (VL) parasites in Phlebotomus duboscqi as well as enzootic reptile parasites in the Mokolo area. We recommend that an epidemiological survey be carried out in the area to evaluate the prevalence and eventually describe the clinical manifestations of VL in the human population. Political instability in neighbouring countries and the resulting refugee migration are likely explanations for the emergence of VL in Mokolo.

Population genetics analysis of Phlebotomus papatasi sand flies from Egypt and Jordan based on mitochondrial cytochrome b haplotypes

BackgroundPhlebotomus papatasi sand flies are major vectors of Leishmania major and phlebovirus infection in North Africa and across the Middle East to the Indian subcontinent. Population genetics is a valuable tool in understanding the level of genetic variability present in vector populations, vector competence, and the development of novel control strategies. This study investigated the genetic differentiation between P. papatasi populations in Egypt and Jordan that inhabit distinct ecotopes and compared this structure to P. papatasi populations from a broader geographical range.MethodsA 461 base pair (bp) fragment from the mtDNA cytochrome b (cyt b) gene was PCR amplified and sequenced from 116 individual female sand flies from Aswan and North Sinai, Egypt, as well as Swaimeh and Malka, Jordan. Haplotypes were identified and used to generate a median-joining network, FST values and isolation-by-distance were also evaluated. Additional sand fly individuals from Afghanistan, Iran, Israel, Jordan, Libya, Tunisia and Turkey were included as well as previously published haplotypes to provide a geographically broad genetic variation analysis.ResultsThirteen haplotypes displaying nine variant sites were identified from P. papatasi collected in Egypt and Jordan. No private haplotypes were identified from samples in North Sinai, Egypt, two were observed in Aswan, Egypt, four from Swaimeh, Jordan and two in Malka, Jordan. The Jordan populations clustered separately from the Egypt populations and produced more private haplotypes than those from Egypt. Pairwise FST values fall in the range 0.024–0.648.ConclusionThe clustering patterns and pairwise FST values indicate a strong differentiation between Egyptian and Jordanian populations, although this population structure is not due to isolation-by-distance. Other factors, such as environmental influences and the genetic variability in the circulating Le. major parasites, could possibly contribute to this heterogeneity. The present study aligns with previous reports in that pockets of genetic differentiation exists between populations of this widely dispersed species but, overall, the species remains relatively homogeneous.

Ecology, feeding and natural infection by Leishmania spp. of phlebotomine sand flies in an area of high incidence of American tegumentary leishmaniasis in the municipality of Rio Branco, Acre, Brazil

BackgroundPhlebotomine sand flies (Diptera: Psychodidae) are insects of medical importance due to their involvement in the zoonotic transmission of Leishmania spp. to vertebrates. The aim of this work was to study the ecology of the sand fly fauna of two types of environments, a rural environment (the Transacreana Road) and an urban park (Horto Florestal Park), both located in the municipality of Rio Branco in the state of Acre, Brazil. Additionally, this study intended to investigate Leishmania infection and blood meal sources of these sand flies using molecular techniques.MethodsThe sand fly fauna was studied in different environments (i.e. forest and peridomestic environments in a rural area, and an urban forest) using Shannon traps and HP light traps to collect sand fly specimens over 13 consecutive months (December 2014 to January 2016). For investigating natural infection by Leishmania and the source of sand fly blood meals, DNA samples were extracted from female sand flies and subjected to polymerase chain reaction targeting ITS1 and cytb genes. DNA sequencing was subsequently used to identify species of Leishmania and the source of blood meals.ResultsA total of 2515 individual sand flies of 43 species were collected and identified, Trichophoromyia auraensis (839; 33.35%), Trichophoromyia spp. (537; 21.35%) and Evandromyia saulensis (187; 7.43%) were more abundant in the rural area (S = 41 species) than in the urban forest. No significant differences were found in species richness between forest and peridomestic environments in the rural area (H = 0.04; P > 0.05), but a larger number of species was found in the forest. Leishmania DNA was sequenced in 13 samples, confirming the presence of L. (V.) braziliensis in Th. auraensis (n = 1), Ev. saulensis (n = 2), Ev. walkeri (n = 1), Ps. llanosmartinsi (n = 1), Pi. nevesi (n = 2), Ps. davisi (n = 1), Ps. ayrozai (n = 1), Pa. aragaoi (n = 1), Ny. antunesi (n = 1) and Ev. infraspinosa (n = 1). Only Ps. ayrozai possessed a sequence similar to that of L. (V.) guyanensis (99%). Through microscopic analysis, five specimens of Ev. saulensis were found to possess flagellate forms in the hindgut, with an infection rate of 2.4%. Samples from 33 fed females were submitted to cytb gene amplification, for which sequencing determined that all were similar to the sequence deposited on GenBank for Gallus gallus (domestic chicken).ConclusionsThe high abundance of Trichophoromyia auraensis and Ev. saulensis, and the detection of L. (V.) braziliensis DNA, suggests that both species may be vectors of American tegumentary leishmaniasis. Psychodopygus ayrozai was found to be infected by L. (V) braziliesnsis and L. (V.) guyanensis, and although collected in low abundance, it may be a potential vector in the region. The sand fly fauna was found to be rich and diverse with predominance of the genus Psychodopygus. Identification of food sources of fed females showed that 100% amplified a gene region compatible with the domestic chicken, which although considered refractory in the disease transmission cycle, may have an influence on the population dynamics of sand flies.

Leishmania proteophosphoglycans regurgitated from infected sand flies accelerate dermal wound repair and exacerbate leishmaniasis via insulin-like growth factor 1-dependent signalling.

Leishmania parasites are transmitted to vertebrate hosts by female phlebotomine sand flies as they bloodfeed by lacerating the upper capillaries of the dermis with their barbed mouthparts. In the sand fly midgut secreted proteophosphoglycans from Leishmania form a biological plug known as the promastigote secretory gel (PSG), which blocks the gut and facilitates the regurgitation of infective parasites. The interaction between the wound created by the sand fly bite and PSG is not known. Here we nanoinjected a sand fly egested dose of PSG into BALB/c mouse skin that lead to the differential expression of 7,907 transcripts. These transcripts were transiently up-regulated during the first 6 hours post-wound and enriched for pathways involved in inflammation, cell proliferation, fibrosis, epithelial cell differentiation and wound remodelling. We found that PSG significantly accelerated wound healing in vitro and in mice; which was associated with an early up-regulation of transcripts involved in inflammation (IL-1β, IL-6, IL-10, TNFα) and inflammatory cell recruitment (CCL2, CCL3, CCL4, CXCL2), followed 6 days later by enhanced expression of transcripts associated with epithelial cell proliferation, fibroplasia and fibrosis (FGFR2, EGF, EGFR, IGF1). Dermal expression of IGF1 was enhanced following an infected sand fly bite and was acutely responsive to the deposition of PSG but not the inoculation of parasites or sand fly saliva. Antibody blockade of IGF1 ablated the gel’s ability to promote wound closure in mouse ears and significantly reduced the virulence of Leishmania mexicana infection delivered by an individual sand fly bite. Dermal macrophages recruited to air-pouches on the backs of mice revealed that IGF1 was pivotal to the PSG’s ability to promote macrophage alternative activation and Leishmania infection. Our data demonstrate that through the regurgitation of PSG Leishmania exploit the wound healing response of the host to the vector bite by promoting the action of IGF1 to drive the alternative activation of macrophages.

First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru.

The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area.

An ecological study of sand flies (Diptera: Psychodidae) in the vicinity of Lençóis Maranhenses National Park, Maranhão, Brazil.

BackgroundThe Lençóis Maranhenses National Park, located in Maranhão, Brazil, is a region of exceptional beauty and a popular tourist destination. The adjoining area has suffered from the impact of human activity and, consequently, has experienced outbreaks of leishmaniasis. This study aimed to evaluate the composition, abundance, species richness and seasonal distribution of sand flies in the region and to determine the constancy of the insect population.MethodsThe survey was conducted at three sites located in the municipalities of Barreirinhas and Santo Amaro between September 2012 and August 2013. Sampling was performed monthly using automatic light traps installed 1.5 m above the soil adjacent to 13 randomly selected rural dwellings. At each site, one trap was placed in the peridomicile near to animal enclosures and another (extradomicile) at 500 m from the peridomicile.ResultsA total of 4,474 individual sand flies were collected over the year with the highest abundance recorded during the rainy season (December to June). Nine species were collected: L. whitmani, L. longipalpis, L. lenti, L. sordellii, L. evandroi, L. flaviscutellata, L. wellcomei, L. termitophila and L. intermedia. Although peridomiciliary and extradomiciliary environments presented similar species richness, the Shannon diversity index was significantly lower in the former (H’ = 2.4) compared with the latter (H’ = 4.98). Lutzomyia whitmani and L. longipalpis were the most abundant species and were classified as constant (constancy index, CI = 100 %) along with L. lenti (CI = 58.3), L. evandroi (CI = 58.3) and L. sordellii (CI = 66.7). The remaining four species presented CI values between 25 and 50 % and were considered accessory.ConclusionsThe present results confirm the present of L. whitmani and L. longipalpis in the peridomicile of houses in Lençóis National Park. The abundance of these species could explain, respectively, the endemicity of cutaneous leishmaniasis and sporadic cases of visceral leishmaniasis in the study area. However, in the case of cutaneous leishmaniasis, the presence of other sand fly vectors (in addition to L. whitmani) cannot be neglected. Finally, this study emphasizes the need for a more effective and permanent supervision to control the expansion of these vectors and leishmaniasis outbreaks.