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Acoustic communication in the form of courtship and mating songs are often involved in reproductive isolation between species of Diptera, such as Drosophila, mosquitoes and sand flies. The patterns of courtship songs in New World sand fly species evolve quickly under sexual selection; and therefore, represent an important trait that can be used as a marker to study the evolution of species complexes and may aid identification of sibling species with a complex. The ability to identify vector species within species complexes is of critical importance for effective and efficient vector control programs. Species-specific song patterns seems to contribute to reproductive isolation in New World sand fly species, suggesting that auditory communication signals may be widespread among these important vectors of leishmaniasis. The main goal of the present study was to characterize the copulatory courtship song of Phlebotomus argentipes, an important vector of visceral leishmaniasis in the Old World. Ph. argentipes males produce acoustic signals during copulation and two types of songs were observed. The one we called primary song is a 'pulse song' with similar length and amplitude to the previously observed 'P1' pattern recorded in Brazilian populations of Lu. longipalpis s.l. The secondary song has 'sine song' characteristics and is quite different from any song produced by New World species. The discovery of this copulation courtship songs in Ph. argentipes supports the possibility that acoustic communication in sandflies might be more widespread than previously thought, including Old World species. Our results highlight the importance of further research on acoustic communication in the Ph. argentipes species complex and other Old World vectors of leishmaniasis. [ABSTRACT FROM AUTHOR]

Phlebotomine sand flies are vectors of Leishmania that are acquired by the female sand fly during blood feeding on an infected mammal. Leishmania parasites develop exclusively in the gut lumen during their residence in the insect before transmission to a suitable host during the next blood feed. Female phlebotomine sand flies are blood feeding insects but their life style of visiting plants as well as animals, and the propensity for larvae to feed on detritus including animal faeces means that the insect host and parasite are exposed to a range of microorganisms. Thus, the sand fly microbiota may interact with the developing Leishmania population in the gut. The aim of the study was to investigate and identify the bacterial diversity associated with wild adult female Lutzomyia sand flies from different geographical locations in the New World. The bacterial phylotypes recovered from 16S rRNA gene clone libraries obtained from wild caught adult female Lutzomyia sand flies were estimated from direct band sequencing after denaturing gradient gel electrophoresis of bacterial 16 rRNA gene fragments. These results confirm that the Lutzomyia sand flies contain a limited array of bacterial phylotypes across several divisions. Several potential plant-related bacterial sequences were detected including Erwinia sp. and putative Ralstonia sp. from two sand fly species sampled from 3 geographically separated regions in Brazil. Identification of putative human pathogens also demonstrated the potential for sand flies to act as vectors of bacterial pathogens of medical importance in addition to their role in Leishmania transmission. [ABSTRACT FROM AUTHOR]

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis. [Copyright &y& Elsevier]

The fine structure of male sex pheromone disseminating structures on abdominal segments of five species of Lutzomyia sandflies (Diptera: Psychodidae) was analyzed. Scanning electron microscopy showed that in Lutzomyia cruzi [Mem. Inst. Oswaldo Cruz 33 (1938) 349] and Lutzomyia longipalpis [Mem. Inst. Oswaldo Cruz 4 (1912) 84], the disseminating structures are located in pale spots on abdominal tergites IV or III and IV and are morphologically similar, appearing as small round cuticular elevations with a central pore. Observation of abdominal tergites of L. longipalpis pupae showed that the spots, but not the structures, are already present in this stage. In Lutzomyia lenti [Mem. Inst. Oswaldo Cruz 33 (1938) 349] and Lutzomyia carmelinoi [Mem. Inst. Oswaldo Cruz 81 (1986) 323] adult males, the disseminating structures are present on tergal segments V and VI, where pale spots could not be observed, and appear as apple-like elevations with a central pore. In Lutzomyia renei, a single disseminating structure is found at the anterior region of tergal segment VI. Transmission electron microscopy was used to analyze the gland cell fine structure in L. cruzi. Several class III gland cells are located side by side in the fourth abdominal segment. Each epidermal secretory cell contains a small reservoir and a short outlet channel through the cuticle. [Copyright &y& Elsevier]

Sauroleishmania spp. comprises one of the four Leishmania subgenera, which has been historically considered a non-pathogenic protozoan of reptiles. However, some strains appear to be transiently infective to mammals, and recent findings have detected these parasites in dogs and humans in areas where leishmaniasis is endemic. Herein, the digestion pattern of PCR–RFLP of the 234 bp-hsp70 fragment was evaluated as a simpler and cheaper tool to distinguish the Sauroleishmania species from the other Leishmania subgenera. As a result, the digestion of the 234 bp-hsp70 fragments with HaeIII produced a banding pattern specific to the four Sauroleishmania strains assessed. This technique could contribute to the identification of Leishmania parasites isolated from sandflies, reptiles, or even mammals in fieldworks as an alternative to the use of laborious and expensive methodologies. [ABSTRACT FROM AUTHOR]

During a field trip to the town of El Carmen in de Department of Santa Cruz we captured 108 specimens of Phlebotominae (Diptera: Psychodidae). Among the specimens examined, we found five species of sand fly comprising four genera. Four species (Lutzomyia cruzi, Lutzomyia forattinii, Evandromyia corumbaensis, Expapillata cerradincola) are new records which increased the number of species known to occur in Bolivia from 117 to 121. [ABSTRACT FROM AUTHOR]

The detection of Leishmania in naturally infected rodents in endemic areas is of fundamental importance for defining these rodents as possible reservoir hosts of infection. The use of polymerase chain reaction in conjunction with molecular hybridization has provided important results that could lead to a better understanding of the natural history of leishmaniasis. [Copyright &y& Elsevier]

Background: Brazilian populations of Lutzomyia longipalpis may constitute a complex of cryptic species, and this report investigates the distribution and number of potential sibling species. One of the main differences observed among Brazilian populations is the type of acoustic signal produced by males during copulation. These copulation song differences seem to be evolving faster than neutral molecular markers and have been suggested to contribute to insemination failure observed in crosses between these sibling species. In previous studies, two main types of copulation songs were found, burst-type and pulse-type. The latter type can, in turn, be further subdivided into five different patterns. Methods: We recorded male song from 13 new populations of the L. longipalpis complex from Brazil and compared the songs with 12 already available. Results: Out of these 25 populations, 16 produce burst-type and 9 produce pulse-type songs. We performed a principal component analysis in these two main groups separately and an additional discriminant analysis in the pulse-type group. The pulse-type populations showed a clear separation between the five known patterns with a high correspondence of individuals to their correct group, confirming the differentiation between them. The distinctiveness of the burst-type subgroups was much lower than that observed among the pulse-type groups and no clear population structure was observed. This suggests that the burst-type populations represent a single species. Conclusion: Overall, our results are consistent with the existence in Brazil of at least six species of the L. longipalpis complex, one with a wide distribution comprising all the populations with burst-type songs, and five more closely related allopatric siblings with different pulse-type song patterns and more restricted distribution ranges. [ABSTRACT FROM AUTHOR]

The sand fly Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), the most important vector of American visceral leishmaniasis, is widely distributed in Latin America. There is currently a consensus that it represents a species complex, however, the number and distribution of the different siblings is still uncertain. Previous analyses have indicated that Brazilian populations of this vector can be divided into two main groups according to the type of courtship song (Burst vs. Pulse) males produce during copulation. Nevertheless, no diagnostic differences have been observed between these two groups with most molecular markers used to date. We analyzed the molecular divergence in a fragment of the paralytic (para) gene, a locus involved in the control of courtship songs in Drosophila, among a number of Lu. longipalpis populations from Brazil producing Burst and Pulse-type songs. Our results revealed a very high level of divergence and fixed differences between populations producing the two types of songs. We also compared Lu. longipalpis with a very closely related species, Lutzomyia cruzi, which produces Burst-type songs. The results indicated a higher number of fixed differences between Lu. cruzi and the Pulse-type populations of Lu. longipalpis than with those producing Burst-type songs. The data confirmed our previous assumptions that the presence of different sibling species of the Lu. longipalpis complex in Brazil can be divided into two main groups, one representing a single species and a second more heterogeneous group that probably represents a number of incipient species. We hypothesize that para might be one of the genes directly involved in the control of the courtship song differences between these two groups or that it is linked to other loci associated with reproductive isolation of the Brazilian species. [ABSTRACT FROM AUTHOR]

Background: The rising incidence of visceral leishmaniasis due to Leishmania infantum requires novel methods to control transmission by the sand fly vector. Indoor residual spraying of insecticide (IRS) against these largely exophilic / exophagic vectors may not be the most effective method. A synthetic copy of the male sex-aggregation pheromone of the key vector species Lutzomyia longipalpis in the Americas, was co-located with residual pyrethroid insecticide, and tested for its effects on vector abundance, hence potential transmission, in a Brazilian community study. Methods: Houses within eight defined semi-urban blocks in an endemic municipality in Brazil were randomised to synthetic pheromone + insecticide or to placebo treatments. A similar number of houses located >100m from each block were placebo treated and considered as "True Controls" (thus, analysed as three trial arms). Insecticide was sprayed on a 2.6m2 surface area of the property boundary or outbuilding wall, co-located within one metre of 50mg synthetic pheromone in controlled-release dispensers. Vector numbers captured in nearby CDC light traps were recorded at monthly intervals over 3 months post intervention. Recruited sentinel houses under True Control and pheromone + insecticide treatments were similarly monitored at 7–9 day intervals. The intervention effects were estimated by mixed effects negative binomial models compared to the True Control group. Results: Dose-response field assays using 50mg of the synthetic pheromone captured a mean 4.8 (95% C.L.: 3.91, 5.80) to 6.3 (95% C.L.: 3.24, 12.11) times more vectors (female Lu. longipalpis) than using 10mg of synthetic pheromone. The intervention reduced household female vector abundance by 59% (C.L.: 48.7, 66.7%) (IRR = 0.41) estimated by the cross-sectional community study, and by 70% (C.L.: 56.7%, 78.8%) estimated by the longitudinal sentinel study. Similar reductions in male Lu. longipalpis were observed. Beneficial spill-over intervention effects were also observed at nearby untreated households with a mean reduction of 24% (95% C.L.: 0.050%, 39.8%) in female vectors. The spill-over effect in untreated houses was 44% (95% C.L.: 29.7%, 56.1%) as effective as the intervention in pheromone-treated houses. Ownership of chickens increased the intervention effects in both treated and untreated houses, attributed to the suspected synergistic attraction of the synthetic pheromone and chicken kairomones. The variation in IRR between study blocks was not associated with inter-household distances, household densities, or coverage (proportion of total households treated). Conclusions: The study confirms the entomological efficacy of the lure-and-kill method to reduce the abundance of this important sand fly vector in treated and untreated homesteads. The outcomes were achieved by low coverage and using only 1–2% of the quantity of insecticide as normally required for IRS, indicating the potential cost-effectiveness of this method. Implications for programmatic deployment of this vector control method are discussed. Author summary: The predominant sand fly vector of the intracellular parasite Leishmania infantum, that causes human and canine visceral leishmaniasis in the Americas, is Lutzomyia longipalpis. Vector control tools to reduce transmission are needed. A sex-aggregation pheromone released by male Lu. longipalpis attracts female conspecifics which facilitates blood-feeding and transmission. This study, conducted in Brazil, quantifies the effects of community deployment of a synthetic version of the sex-aggregation pheromone, in a controlled-release dispenser, and co-located with lethal insecticide applied to a small area of the household compound or outbuilding wall. 50mg synthetic pheromone dispensers were used since they attracted substantially more vectors than 10mg dispensers. Deploying this novel lure-and-kill method to houses in eight replicate study blocks in two suburban endemic areas, demonstrated that it reduces vector numbers at both pheromone + insecticide treated houses, and neighbouring untreated houses. The presence of chickens (a known additional attraction to blood-seeking female Lu. longipalpis) increased the beneficial effects of the intervention. The method used only 1–2% of the quantity of insecticide necessary for IRS for an average sized house. The results demonstrate the efficacy and potential cost-effectiveness of this novel lure-and-kill control method. [ABSTRACT FROM AUTHOR]

Background In South America the sand fly Lutzomyia longipalpis is the predominant vector of Leishmania infantum, the parasite that causes canine and human visceral leishmaniasis. Co-location of synthetic male sex-aggregation pheromone with an insecticide provided protection against canine seroconversion, parasite infection, reduced tissue parasite loads, and female sand fly densities at households. Optimising the sex-aggregation pheromone + insecticide intervention requires information on the distance over which female and male Lu. longipalpis would be attracted to the synthetic pheromone in the field. Methodology/Principal findings Wild Lu. longipalpis were collected at two peridomestic study sites in Governador Valadares (Minas Gerais, Brazil). Sand flies were marked with coloured fluorescent powder using an improved protocol and then released into an existing domestic chicken shed at two independent sites, followed by recapture at synthetic-pheromone host-odour baited traps placed up to 30 metres distant from the release point. In total 1704 wild-caught Lu. longipalpis were released into the two chicken sheds. Overall 4.3% of the marked flies were recaptured in the pheromone baited experimental chicken sheds compared to no marked flies recaptured in the control sheds. At the first site, 14 specimens (10.4% of the marked and released specimens) were recaptured at 10m, 36 (14.8%) at 20m, and 15 (3.4%) at 30m. At the second site, lower recapture rates were recorded; 8 marked specimens (1.3%) were recaptured at 5 and 10m and no marked specimens were recaptured at 15m. Approximately 7x more marked males than females were recaptured although males were only 2x as common as females in the released population. 52% of the marked Lu. longipalpis were collected during the first night of sampling, 32% on the second night, and 16% on the third night. Conclusions/Significance The study established that both male and female sand flies can be attracted to the synthetic sex-aggregation pheromone in the presence of host odour over distances up to at least 30m in the field depending on local environmental and meterological conditions. [ABSTRACT FROM AUTHOR]

To evaluate the efficacy of a synthetic sex-aggregation pheromone of the sand fly vector Lu. longipalpis, co-located with residual insecticide, to reduce the infection incidence of Leishmania infantum in the canine reservoir. A stratified cluster randomised trial was designed to detect a 50% reduction in canine incident infection after 24 months in 42 recruited clusters, randomly assigned to one of three intervention arms (14 cluster each): synthetic pheromone + insecticide, insecticide-impregnated dog collars, or placebo control. Infection incidence was measured by seroconversion to anti-Leishmania serum antibody, Leishmania parasite detection and canine tissue parasite loads. Changes in relative Lu. longipalpis abundance within households were measured by setting three CDC light traps per household. A total 1,454 seronegative dogs were followed-up for a median 15.2 (95% C.I.s: 14.6, 16.2) months per cluster. The pheromone + insecticide intervention provided 13% (95% C.I. 0%, 44.0%) protection against anti-Leishmania antibody seroconversion, 52% (95% C.I. 6.2%, 74·9%) against parasite infection, reduced tissue parasite loads by 53% (95% C.I. 5.4%, 76.7%), and reduced household female sand fly abundance by 49% (95% C.I. 8.2%, 71.3%). Variation in the efficacy against seroconversion varied between trial strata. Equivalent protection attributed to the impregnated-collars were 36% (95% C.I. 14.4%, 51.8%), 23% (95% C.I. 0%, 57·5%), 48% (95% C.I. 0%, 73.4%) and 43% (95% C.I. 0%, 67.9%), respectively. Comparison of the two interventions showed no statistically consistent differences in their efficacies; however, the errors were broad for all outcomes. Reductions in sand fly numbers were predominant where insecticide was located (chicken and dog sleeping sites), with no evidence of insecticide-induced repellence onto humans or dogs. The synthetic pheromone co-located with insecticide provides protection particularly against canine L. infantum parasite transmission and sand fly vector abundance. The effect estimates are not dissimilar to those of the insecticide-impregnated collars, which are documented to reduce canine infection incidence, human infection and clinical VL disease incidence, in different global regions. The trialled novel lure-and-kill approach is a low-cost potential vector control tool against ZVL in the Americas. The predominant sand fly vector of the intracellular parasite Leishmania infantum, that causes human and canine visceral leishmaniasis in the Americas, is Lutzomyia longipalpis. Dogs are the proven reservoir. Vector control tools to reduce transmission suited to this predominantly exophilic vector are lacking. Insecticide-impregnated dog collars protect dogs against infectious bites from sand fly vectors, and result in reductions of new infections in both dogs and humans. However, collars are costly for endemic communities, and alternative approaches are needed. Recently the bulk synthesised sex-aggregation pheromone of male Lu. longipalpis was shown to attract large numbers of conspecific females to lethal pyrethroid insecticides, indicating the potential for use in a vector control application. This study, conducted in Brazil, evaluated the efficacy of this novel lure-and-kill approach to reduce seroconversion and infection incidence with L. infantum in the canine reservoir, in addition to measuring its impact on household abundance of Lu. longipalpis. Deployed in 14 stratified clusters, the outcomes were compared to those attributed to insecticide impregnated collars fitted to dogs in another 14 clusters; each intervention was compared to 14 clusters that received placebo treatments. The beneficial effects of the lure-and-kill method were most noticeable on confirmed infection incidence and clinical parasite loads, and in reducing sand fly abundance. The overall effect of the two interventions were not statistically dissimilar, though the confidence intervals were broad. We conclude that the novel low-cost lure-and-kill approach should be added to the vector control toolbox against visceral leishmaniasis in the Americas. [ABSTRACT FROM AUTHOR]

In South America, the Protist parasite that causes visceral leishmaniasis, a potentially fatal human disease, is transmitted by blood-feeding female Lutzomyia longipalpis sand flies. A synthetic copy of the male produced sex-aggregation pheromone offers new opportunities for vector control applications. We have previously shown that the pheromone placed in plastic sachets (lures) can attract both females and males to insecticide treated sites for up to 3 months. To use the pheromone lure in a control program we need to understand how the application of lures in the field can be optimised. In this study we investigated the effect of increasing the number of lures and their proximity to each other on their ability to attract Lu. longipalpis. Also for the first time we applied a Bayesian log-linear model rather than a classic simple (deterministic) log-linear model to fully exploit the field-collected data. We found that sand fly response to pheromone is significantly related to the quantity of pheromone and is not influenced by the proximity of other pheromone sources. Thus sand flies are attracted to the pheromone source at a non-linear rate determined by the amount of pheromone being released. This rate is independent of the proximity of other pheromone releasing traps and indicates the role of the pheromone in aggregation formation. These results have important implications for optimisation of the pheromone as a vector control tool and indicate that multiple lures placed in relatively close proximity to each other (5 m apart) are unlikely to interfere with one another. [ABSTRACT FROM AUTHOR]

Background: Lutzomyia longipalpis is the South American vector of Leishmania infantum, the etiologic agent of visceral leishmaniasis (VL). Male L. longipalpis produce a sex-aggregation pheromone that is critical in mating, yet very little is known about its accumulation over time or factors involved in release. This laboratory study aimed to compare accumulation of pheromone over time and determine factors that might influence release in three members of the L. longipalpis species complex. Methodology/Principal findings: We investigated male sex-aggregation pheromone gland content at different ages and the release rate of pheromone in the presence or absence of females under different light conditions by gas chromatography-mass spectrometry (GC-MS). Pheromone gland content was determined by extraction of whole males and pheromone release rate was determined by collection of headspace volatiles. Pheromone gland content appeared age-related and pheromone began to accumulate between 6 to 12 h post eclosion and gradually increased until males were 7–9 days old. The greatest amount was detected in 9-day old Campo Grande males ((S)-9-methylgermacrene-B; X ± SE: 203.5 ± 57.4 ng/male) followed by Sobral 2S males (diterpene; 199.9 ± 34.3) and Jacobina males ((1S,3S,7R)-3-methyl-α-himachalene; 128.8 ± 30.3) at 7 days old. Pheromone release was not continuous over time. During a 4-hour period, the greatest quantities of pheromone were released during the first hour, when wing beating activity was most intense. It was then substantially diminished for the remainder of the time. During a 24 h period, 4–5 day old male sand flies released approximately 63 ± 11% of the pheromone content of their glands, depending on the chemotype. The presence of females significantly increased pheromone release rate. The light regime under which the sand flies were held had little influence on pheromone release except on Sobral 2S chemotype. Conclusions/Significance: Accumulation of pheromone appears to occur at different rates in the different chemotypes examined and results in differing amounts being present in glands over time. Release of accumulated pheromone is not passive, but depends on biotic (presence of females) and abiotic (light) circumstances. There are marked differences in content and release between the members of the complex suggesting important behavioural, biosynthetic and ecological differences between them. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article regarding the correlation of Lutzomyia Nyssomyia intermedia and Lutzomyia Nyssomyia whitmani in the transmission of Leishmania Viannia.

In this paper we review the natural history of pheromone communication and the current diversity of aggregation-sex pheromones in the sand fly Lutzomyia longipalpis. This species complex is the main vector of Leishmania infantum, the agent of visceral leishmaniasis in the Americas. The identification of variation in pheromone chemotypes combined with molecular and sound analyses have all contributed to our understanding of the extent of divergence among cryptic members of this complex. The importance of chemical signals as pre-mating barriers and drivers of speciation is discussed. Moreover, the importance of aggregation-sex pheromones as sexually selected signals is highlighted with evidence from the literature suggesting their potential role in species and mate recognition as well as mate assessment. The distinct evolutionary forces possibly involved are briefly reviewed and discussed in the context of this intriguing insect. [ABSTRACT FROM AUTHOR]

Background: American visceral leishmaniasis (AVL) is an emerging disease in the state of São Paulo, Brazil. Its geographical expansion and the increase in the number of human cases has been linked to dispersion of Lutzomyia longipalpis into urban areas. To produce more accurate risk maps we investigated the geographic distribution and routes of expansion of the disease as well as chemotype populations of the vector. Methodology/Principal Findings: A database, containing the annual records of municipalities which had notified human and canine AVL cases as well as the presence of the vector, was compiled. The chemotypes of L. longipalpis populations from municipalities in different regions of São Paulo State were determined by Coupled Gas Chromatography – Mass Spectrometry. From 1997 to June 2014, L. longipalpis has been reported in 166 municipalities, 148 of them in the Western region. A total of 106 municipalities were identified with transmission and 99 were located in the Western region, where all 2,204 autochthonous human cases occurred. Both the vector and the occurrence of human cases have expanded in a South-easterly direction, from the Western to central region, and from there, a further expansion to the North and the South. The (S)-9-methylgermacrene-B population of L. longipalpis is widely distributed in the Western region and the cembrene-1 population is restricted to the Eastern region. Conclusion/Significance: The maps in the present study show that there are two distinct epidemiological patterns of AVL in São Paulo State and that the expansion of human and canine AVL cases through the Western region has followed the same dispersion route of only one of the two species of the L. longipalpis complex, (S)-9-methylgermacrene-B. Entomological vigilance based on the routes of dispersion and identification of the chemotype population could be used to identify at-risk areas and consequently define the priorities for control measures. Author Summary: Information on the geographical distribution, dispersal mechanisms and dispersion route of insect-borne diseases can help to identify ongoing transmission areas, new risk areas and guide surveillance and control activities. Lutzomyia longipalpis, the principal vector of American visceral leishmaniasis disease in the Americas, is a group of closely related species that can be separated according to the type of pheromone produced by male individuals. It is still unclear how many members there are in this complex, how they are related and if some are more important vectors than others. In the present study, we show that the expansion of human visceral leishmaniasis in the state of São Paulo, Brazil, has followed the same dispersion route of only one of the two related species of L. longipalpis found in the state. The maps that we produced in the present study will allow us to determine risk areas for the occurrence of human visceral leishmaniasis, and reinforces our hypothesis that within São Paulo state these chemotype populations could have different biological capacities to act as a vector. [ABSTRACT FROM AUTHOR]

Background: American visceral leishmaniasis (AVL) is an emerging disease in the state of São Paulo, Brazil. Its geographical expansion and the increase in the number of human cases has been linked to dispersion of Lutzomyia longipalpis into urban areas. To produce more accurate risk maps we investigated the geographic distribution and routes of expansion of the disease as well as chemotype populations of the vector. Methodology/Principal Findings: A database, containing the annual records of municipalities which had notified human and canine AVL cases as well as the presence of the vector, was compiled. The chemotypes of L. longipalpis populations from municipalities in different regions of São Paulo State were determined by Coupled Gas Chromatography – Mass Spectrometry. From 1997 to June 2014, L. longipalpis has been reported in 166 municipalities, 148 of them in the Western region. A total of 106 municipalities were identified with transmission and 99 were located in the Western region, where all 2,204 autochthonous human cases occurred. Both the vector and the occurrence of human cases have expanded in a South-easterly direction, from the Western to central region, and from there, a further expansion to the North and the South. The (S)-9-methylgermacrene-B population of L. longipalpis is widely distributed in the Western region and the cembrene-1 population is restricted to the Eastern region. Conclusion/Significance: The maps in the present study show that there are two distinct epidemiological patterns of AVL in São Paulo State and that the expansion of human and canine AVL cases through the Western region has followed the same dispersion route of only one of the two species of the L. longipalpis complex, (S)-9-methylgermacrene-B. Entomological vigilance based on the routes of dispersion and identification of the chemotype population could be used to identify at-risk areas and consequently define the priorities for control measures. [ABSTRACT FROM AUTHOR]

Background: Notifications concerning American Cutaneous Leishmaniasis have increased in recent years in the state of Acre, Brazil. Despite identification of distinct Leishmania species isolated from cutaneous lesions, there are still no records of visceral leishmaniasis in the state. However, studies on the sand fly fauna in this region are still limited. Findings: Insects were collected from April 2011 to April 2012, using HP light traps distributed in four residential areas and one recreational area in Rio Branco, Capital of the State of Acre in the Amazon region of Brazil. A total of 456 sand flies were collected, comprising 256 females and 200 males. Taxonomic identification revealed 16 Phlebotominae genera and 23 species, as follows: Trichophoromyia auraensis, Nyssomyia whitmani, Nyssomyia antunesi, Pressatia choti, Evandromyia saulensis, Evandromyia walkeri, Evandromyia begonae, Migonemyia migonei, Pintomyia serrana, Psychodopygus paraensis, Sciopemyia sordelii, Migonemyia pusilla, Pintomyia nevesi, Brumptomyia avellari, Micropygomyia acanthopharynx, Micropygomyia micropyga, Pintomyia odax, Lutzomyia sherlocki, Pressatia calcarata, Pressatia duncanae, Bichromomyia flaviscutellata, Evandromyia bourrouli and Evandromyia bacula. From this group, Tr. auraensis and Ny. whitmani were the most abundant species in both forested areas and the peridomiciliary environment. Conclusions: We find that the sand fly fauna in the urban and peri urban areas of Rio Branco is very diverse comprising 23 species, as diverse as that in areas of primary forest. Some species, such as Nyssomyia whitmani, Ny. antunesi and Bichromomyia flaviscutellata are known vectors of parasites responsible for cutaneous leishmaniasis, and Trichophoromyia auraensis is a putative vector in this Amazonian region. [ABSTRACT FROM AUTHOR]

Background: Notifications concerning American Cutaneous Leishmaniasis have increased in recent years in the state of Acre, Brazil. Despite identification of distinct Leishmania species isolated from cutaneous lesions, there are still no records of visceral leishmaniasis in the state. However, studies on the sand fly fauna in this region are still limited. Findings: Insects were collected from April 2011 to April 2012, using HP light traps distributed in four residential areas and one recreational area in Rio Branco, Capital of the State of Acre in the Amazon region of Brazil. A total of 456 sand flies were collected, comprising 256 females and 200 males. Taxonomic identification revealed 16 Phlebotominae genera and 23 species, as follows: Trichophoromyia auraensis, Nyssomyia whitmani, Nyssomyia antunesi, Pressatia choti, Evandromyia saulensis, Evandromyia walkeri, Evandromyia begonae, Migonemyia migonei, Pintomyia serrana, Psychodopygus paraensis, Sciopemyia sordelii, Migonemyia pusilla, Pintomyia nevesi, Brumptomyia avellari, Micropygomyia acanthopharynx, Micropygomyia micropyga, Pintomyia odax, Lutzomyia sherlocki, Pressatia calcarata, Pressatia duncanae, Bichromomyia flaviscutellata, Evandromyia bourrouli and Evandromyia bacula. From this group, Tr. auraensis and Ny. whitmani were the most abundant species in both forested areas and the peridomiciliary environment. Conclusions: We find that the sand fly fauna in the urban and peri urban areas of Rio Branco is very diverse comprising 23 species, as diverse as that in areas of primary forest. Some species, such as Nyssomyia whitmani, Ny. antunesi and Bichromomyia flaviscutellata are known vectors of parasites responsible for cutaneous leishmaniasis, and Trichophoromyia auraensis is a putative vector in this Amazonian region. [ABSTRACT FROM AUTHOR]

Current control methodologies have not prevented the spread of visceral leishmaniasis (VL) across Brazil. Here, we describe the development of a new tool for controlling the sand fly vector of the disease: a long-lasting lure, which releases a synthetic male sex pheromone, attractive to both sexes of Lutzomyia longipalpis. This device could be used to improve the effectiveness of residual insecticide spraying as a means of sand fly control, attracting L. longipalpis to insecticide-treated animal houses, where they could be killed in potentially large numbers over a number of weeks. Different lure designs releasing the synthetic pheromone (±)-9-methylgermacrene-B (CAS 183158-38-5) were field-tested in Araçatuba, São Paulo (SP). Experiments compared numbers of sand flies caught overnight in experimental chicken sheds with pheromone lures, to numbers caught in control sheds without pheromone. Prototype lures, designed to last one night, were first used to confirm the attractiveness of the pheromone in SP, and shown to attract significantly more flies to test sheds than controls. Longer-lasting lures were tested when new, and at fortnightly intervals. Lures loaded with 1 mg of pheromone did not attract sand flies for more than two weeks. However, lures loaded with 10 mg of pheromone, with a releasing surface of 15 cm2 or 7.5 cm2, attracted female L. longipalpis for up to ten weeks, and males for up to twelve weeks. Approximately five times more sand flies were caught with 7.5 cm2 10 mg lures when first used than occurred naturally in non-experimental chicken resting sites. These results demonstrate that these lures are suitably long-lasting and attractive for use in sand fly control programmes in SP. To our knowledge, this is the first sex pheromone-based technology targeting an insect vector of a neglected human disease. Further studies should explore the general applicability of this approach for combating other insect-borne diseases. Author Summary: Visceral leishmaniasis is a neglected disease of increasing worldwide importance. In Brazil, the protozoan parasites which cause visceral leishmaniasis are transmitted through blood-feeding of the female sand fly Lutzomyia longipalpis. As no human vaccine is available against VL, new tools for targeting sand flies are urgently needed to reduce transmission. Here, we develop and test a new tool for controlling L. longipalpis: a long-lasting lure, which releases a synthetic sex pheromone that attracts both male and female L. longipalpis. Through a series of field experiments conducted in São Paulo state, we show that these lures can attract female L. longipalpis for up to 10 weeks from first use, and males for up to 12 weeks. This technology could be applied by disease control agencies to attract sand flies away from feeding on people and their animals, and towards insecticide treated surfaces where they can be killed. To our knowledge, this is the first example of a feasible insect sex pheromone technology that could be applied as part of a programme designed to protect against a human disease. [ABSTRACT FROM AUTHOR]

Current control methodologies have not prevented the spread of visceral leishmaniasis (VL) across Brazil. Here, we describe the development of a new tool for controlling the sand fly vector of the disease: a long-lasting lure, which releases a synthetic male sex pheromone, attractive to both sexes of Lutzomyia longipalpis. This device could be used to improve the effectiveness of residual insecticide spraying as a means of sand fly control, attracting L. longipalpis to insecticide-treated animal houses, where they could be killed in potentially large numbers over a number of weeks. Different lure designs releasing the synthetic pheromone (±)-9-methylgermacrene-B (CAS 183158-38-5) were field-tested in Araçatuba, São Paulo (SP). Experiments compared numbers of sand flies caught overnight in experimental chicken sheds with pheromone lures, to numbers caught in control sheds without pheromone. Prototype lures, designed to last one night, were first used to confirm the attractiveness of the pheromone in SP, and shown to attract significantly more flies to test sheds than controls. Longer-lasting lures were tested when new, and at fortnightly intervals. Lures loaded with 1 mg of pheromone did not attract sand flies for more than two weeks. However, lures loaded with 10 mg of pheromone, with a releasing surface of 15 cm2 or 7.5 cm2, attracted female L. longipalpis for up to ten weeks, and males for up to twelve weeks. Approximately five times more sand flies were caught with 7.5 cm2 10 mg lures when first used than occurred naturally in non-experimental chicken resting sites. These results demonstrate that these lures are suitably long-lasting and attractive for use in sand fly control programmes in SP. To our knowledge, this is the first sex pheromone-based technology targeting an insect vector of a neglected human disease. Further studies should explore the general applicability of this approach for combating other insect-borne diseases. [ABSTRACT FROM AUTHOR]

Abstract: In Latin America, Lutzomyia longipalpis is the main vector of the protozoan parasite Leishmania infantum, which is the causal agent of American Visceral Leishmaniasis. This insect uses male-produced pheromones for mate recognition. Elucidation of pheromone biogenesis or its regulation may enable molecular strategies for mating disruption and, consequently, the vector's population management. Motivated by our recent results of the transcriptomic characterization of the L. longipalpis pheromone gland, we performed a proteomic analysis of this tissue combining SDS-PAGE, and mass spectrometry followed by an integrative data analysis. Considering that annotated genome sequences of this sand fly are not available, we designed an alternative workflow searching MS/MS data against two customized databases using three search engines: Mascot, OMSSA and ProLuCID. A total of 542 proteins were confidently characterized, 445 of them using a Uniref100-insect protein database, and 97 using a transcript translated database. In addition, use of PEAKS for de novo peptide sequencing of MS/MS data confirmed ~90% identifications made with the combination of the three search engines. Our results include the identification of six of the seven enzymes of the mevalonate-pathway, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis. Biological significance: L. longipalpis is the main vector of the protozoan parasite L. infantum, which is the causal agent of American Visceral Leishmaniasis. One of the control measures of such disease is focused on vector population control. As this insect uses male-produced pheromones for mate recognition, the elucidation of pheromone biogenesis or its regulating process may enable molecular strategies for mating disruption and, consequently, this vector's population management. On this regard, in this manuscript we report expression evidence, at the protein level, of several molecules potentially involved in the pheromone production of L. longipalpis. Our results include the identification of the mevalonate-pathway enzymes, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis. In addition, considering that the annotated genome sequences of this sand fly are not yet available, we designed an alternative workflow searching MS/MS data against proteomic and transcript translated customized databases, using three search engines: Mascot, OMSSA, and ProLuCID. In addition, a de novo peptide sequencing software (PEAKS) was used to further analyze the MS/MS data. This approach made it possible to identify and annotate 542 proteins for the pheromone gland of L. longipalpis. Importantly, all annotated protein sequences and raw data are available for the research community in protein repositories that provide free access to the data. [Copyright &y& Elsevier]

Background: Molecules involved in pheromone biosynthesis may represent alternative targets for insect population control. This may be particularly useful in managing the reproduction of Lutzomyia longipalpis, the main vector of the protozoan parasite Leishmania infantum in Latin America. Besides the chemical identity of the major components of the L. longipalpis sex pheromone, there is no information regarding the molecular biology behind its production. To understand this process, obtaining information on which genes are expressed in the pheromone gland is essential. Methods: In this study we used a transcriptomic approach to explore the pheromone gland and adjacent abdominal tergites in order to obtain substantial general sequence information. We used a laboratory-reared L. longipalpis (one spot, 9-Methyl GermacreneB) population, captured in Lapinha Cave, state of Minas Gerais, Brazil for this analysis. Results: From a total of 3,547 cDNA clones, 2,502 high quality sequences from the pheromone gland and adjacent tissues were obtained and assembled into 1,387 contigs. Through blast searches of public databases, a group of transcripts encoding proteins potentially involved in the production of terpenoid precursors were identified in the 4th abdominal tergite, the segment containing the pheromone gland. Among them, protein-coding transcripts for four enzymes of the mevalonate pathway such as 3-hydroxyl-3-methyl glutaryl CoA reductase, phosphomevalonate kinase, diphosphomevalonate descarboxylase, and isopentenyl pyrophosphate isomerase were identified. Moreover, transcripts coding for farnesyl diphosphate synthase and NADP+ dependent farnesol dehydrogenase were also found in the same tergite. Additionally, genes potentially involved in pheromone transportation were identified from the three abdominal tergites analyzed. Conclusion: This study constitutes the first transcriptomic analysis exploring the repertoire of genes expressed in the tissue containing the L longipalpis pheromone gland as well as the flanking tissues. Using a comparative approach, a set of molecules potentially present in the mevalonate pathway emerge as interesting subjects for further study regarding their association to pheromone biosynthesis. The sequences presented here may be used as a reference set for future research on pheromone production or other characteristics of pheromone communication in this insect. Moreover, some matches for transcripts of unknown function may provide fertile ground of an in-depth study of pheromone-gland specific molecules. [ABSTRACT FROM AUTHOR]

Abstract: The sand fly Lutzomyia cruzi (Mangabeira, 1938) is implicated as a vector of American visceral leishmaniasis (AVL) in some areas of Brazil. Lutzomyia cruzi is closely related to Lutzomyia longipalpis s.l. (Lutz and Neiva, 1912) the main Latin American vector of AVL and a species complex. Although females of the two species are identical, the males can be distinguished by differences in the genitalia. Nevertheless, pheromone analysis shows that Lu. cruzi males produce 9-methyl-germacrene-B, which has also been found in a number of Latin American populations of Lu. longipalpis s.l. In addition, analysis of microsatellite loci shows that the level of divergence between Lu. cruzi and Lu. longipalpis s.l. is similar to that observed among the Lu. longipalpis s.l. sibling species. Here we present the lovesongs of Lu. cruzi males which are similar to the Burst-type songs produced by one of the Lu. longipalpis s.l. sibling species. We also present data on the molecular polymorphisms of the period gene of Lu. cruzi that indicate this species as another sibling within the Lu. longipalpis complex. The results highlight the importance of an integrative approach to understand the patterns of genetic and phenotypic divergence among very closely related vector species. [Copyright &y& Elsevier]

Abstract: In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37°C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28°C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365. [Copyright &y& Elsevier]

Abstract: Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi. [Copyright &y& Elsevier]

Abstract: The technique of polymerase chain reaction (PCR) associated to hybridization was used to screen 123 samples collected from wild and synanthropic rodents captured in a cutaneous and visceral leishmaniasis endemic area in the State of Minas Gerais, Brazil. The detection of Leishmania spp in naturally infected rodents is of fundamental importance for incriminating them as possible reservoir hosts of the diseases in Minas Gerais. A total of 62 specimens belonging to wild (Thrichomys apereoides, Oryzomys subflavus, Galea spixii, Bolomys lasiurus and Wiedomys pyrrhorhinos) and synanthropic (R. rattus) rodent species were captured in different ecotopes. Blood and skin samples were submitted for PCR analyses followed by molecular hybridization with specific probes for the three Leishmania-species complexes. Fifteen samples were found positive after PCR-hybridization and identified as follows: nine belonging to the L. mexicana complex, three to the L. braziliensis complex and three to the L. donovani complex. Positive PCR results were found in 11 out of the 61 (18%) blood samples and in four out of the 62 (6.4%) skin fragments screened. R. rattus and T. apereoides were the most abundant species in the area also presenting high prevalence of natural infection. The presence of parasite DNA belonging to L. braziliensis, L. mexicana and L. donovani complexes was confirmed in several individuals of a rodent species, R. rattus. This work is the first report of the detection of L. (L.) chagasi in a naturally infected T. apereoides. The utility of filter paper as a substrate for PCR analyses and the efficacy of the procedure associated to the hybridization is emphasized. [Copyright &y& Elsevier]

Background: The direct agglutination test (DAT) has proved to be a very important serodiagnostic tool combining high levels of intrinsic validity and ease of performance. Otherwise, fast agglutination screening test (FAST) utilises only one serum dilution making the test very suitable for the screening of large populations. Results: We have tested FAST and DAT for the detection anti-Leishmania antibodies in serum samples from patients with American visceral (AVL) and cutaneous leishmaniases (ACL) in Minas Gerais State, Brazil. The DAT on serum and blood samples of confirmed AVL patients found all samples positive at a serum dilution of ≥ 1:800. This dilution was subsequently used as cut off value in the study. The blood and serum samples of these confirmed patients could also be clearly read in FAST using a 1:100 dilution with the same high sensitivity. DAT and FAST were not able to detect significant amounts of antibodies in samples from ACL patients and are not suitable for the diagnosis of this manifestation of the disease. Conclusion: We suggest that both DAT and FAST are very practical diagnostic tools for the serodiagnosis of AVL under rural conditions as both serological tests do not require sophisticated equipment, a cold chain and are very simple to perform. [ABSTRACT FROM AUTHOR]

Lutzomyia longipalpis is the main vector of visceral Leishmaniasis in the Americas and is composed of a species complex. Males of this sand-fly produce acoustic signals during copulation and different patterns are observed among Brazilian populations. Such acoustic signals are commonly involved in species recognition. However, since the song is only produced during copulation it is not clear how it affects mating success or contributes to sexual isolation. Another aspect that may affect reproductive success is the presence of food. Since hematophagy is such an important aspect of L. longipalpis biology, we wanted to test if blood-feeding can influence the reproductive behaviour of this insect. We performed crossing experiments removing males' wings (silencing them) and playing back either the homo-specific or the hetero-specific song to either unfed or blood-fed females. Our results showed that both songs and blood-feeding affect insemination success, but not the frequency of copulation. In trials where females were not blood-fed song clearly affected insemination; males with wings, and males with homo-specific song playback had a higher insemination success than wingless males (no song) and trials with hetero-specific song. Blood-feeding females prior to the trials increased insemination in all groups including the control group which suggests that mating happens simultaneously with, or immediately after, the blood meal. Blood-fed females also seemed to discriminate less against the wrong song or the lack of song (wingless) one day after feeding, however trials with the correct song still had higher insemination rates. Altogether, our results show that both the male copulatory courtship songs and female blood-feeding are important for reproductive success and as such are important components of the sexual behaviour of L. longipalpis. • Male copulatory courtship song and female blood-feeding affect insemination success in Lutzomyia longipalpis. • Homo-specific song increased insemination in comparison to hetero-specific song. • Blood-feeding L. longipalpis females prior to mating increased insemination. [ABSTRACT FROM AUTHOR]

Background: In 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. Methods: During 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. Results: We 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. Conclusions: Our 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. [ABSTRACT FROM AUTHOR]

Background:Lutzomyia longipalpis (Diptera: Psychodidae) is the major vector of Leishmania (Leishmania) infantum and thus plays a crucial role in the epidemiology of American visceral leishmaniasis (AVL). This vector is the best studied species of sand fly in the Neotropical region. Many studies claim that this vector is in fact a species complex; however there is still no consensus regarding the number of species that belong into this complex or the geographical distribution of sibling species. The aim of the present study was to analyze the genetic relationships within Lu. longipalpis populations in the state of Mato Grosso do Sul (MS), Brazil. Methodology/Principal Findings:We collected 30 Lu. longipalpis (15 females and 15 males) from five localities (Campo Grande, Três Lagoas, Aquidauana, Miranda and Bonito) and 30 Lu. Cruzi from Corumbá, totaling 180 sandflies from MS, and 30 Lu. longipalpis from Estrela de Alagoas, state of Alagoas (AL), Northeast Brazil. We show that eight previously described microsatellite loci were sufficient in distinguishing Lu. longipalpis from Lu. Cruzi, which is a closely related species, and in differentiating between Lu. longipalpis collected in MS versus Estrela de Alagoas. Analyses of the genotypes revealed introgression between sympatric Lu. longipalpis and Lu. Cruzi. Conclusions/Significance:Our findings support the hypothesis of cryptic species within the Lu. longipalpis complex. Furthermore, our data revealed introgression between Lu. longipalpis and Lu. cruzi. This phenomenon should be further investigated to determine the level and incidence of hybridization between these two species. We also demonstrated that microsatellite markers are a powerful tool for differentiating sand fly populations and species. The present study has elucidated the population structure of Lu. longipalpis in MS and, by extension, the Neotropical Lu. longipalpis complex itself. [ABSTRACT FROM AUTHOR]