Oral and Intragastric: New Routes of Infection by Leishmania braziliensis and Leishmania infantum?

Although Leishmania transmission in nature is associated with the bite of an infected sandfly vector, other possible transmission routes are speculated to occur, such as the oral route. We evaluated the possibility of infection by this route in golden hamsters (Mesocricetus auratus) using Leishmania braziliensis (Lb) and Leishmania infantum (Li). Hamsters were exposed to experimental oral or intragastrical infection with axenic promastigotes, besides oral ingestion of a suspension of cultivated macrophages infected with amastigotes, lesion-fed Lutzomyia longipalpis, skin lesion or infective spleen fragment. The parasite's isolation, besides a positive PCR and IFAT, confirmed the intragastric infection by promastigote parasites. The oral ingestion of macrophages infected with L. braziliensis amastigotes was also infective. These results confirmed that Leishmania parasites could infect mammals by the intragastric route through the ingestion of promastigote forms (what can happen after a sandfly ingestion) and by the oral ingestion of infected macrophages (what can happen in nature in a predator-prey interaction). The better understanding of these alternative routes is essential to understand their transmission dynamics in nature. As far as we know, this is the first time that oral and intragastric Leishmania transmission has been experimentally demonstrated, constituting new infection routes, at least for L. infantum and L. braziliensis.

Community deployment of a synthetic pheromone of the sand fly Lutzomyia longipalpis co-located with insecticide reduces vector abundance in treated and neighbouring untreated houses: Implications for control of Leishmania infantum.

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.

Insecticide-impregnated netting: A surface treatment for killing Lutzomyia longipalpis (Diptera: Psychodidae), the vector of Leishmania infantum.

The sand fly Lutzomyia longipalpis is the main vector of Leishmania infantum in Brazil. Synthetic male-produced sex/aggregation pheromone co-located with micro-encapsulated λ-cyhalothrin in chicken sheds can significantly reduce canine infection and sand fly densities in a lure-and-kill strategy. In this study, we determined if insecticide-impregnated netting (IN) could replace insecticide residual spraying (IRS). We compared numbers of Lu. longipalpis attracted and killed in experimental and real chicken sheds baited with pheromone and treated with a 1 m2 area of either insecticide spray or netting. First, we compared both treatments in experimental sheds to control mortality established from light trap captures. We then compared the long-term killing effect of insecticide spray and netting, without renewal, in experimental sheds over a period of 16 weeks. Finally, a longitudinal intervention study in real chicken sheds compared the numbers and proportions of Lu. longipalpis collected and killed before and after application of both treatments. In Experiment 1, a higher proportion of males and females captured in IRS- and IN-treated sheds were dead at 24 h compared to controls (P < 0.05). No difference was found in the proportion of females killed in sheds treated with IN or IRS (P = 0.15). A slightly higher proportion of males were killed by IRS (100%) compared to IN (98.6%; P < 0.05). In Experiment 2, IN- and IRS-treated traps were equally effective at killing females (P = 0.21) and males (P = 0.08). However, IRS killed a significantly higher proportion of females and males after 8 (P < 0.05) and 16 (P < 0.05) weeks. In Experiment 3, there was no significant difference between treatments in the proportion of females killed before (P = 0.88) or after (P = 0.29) or males killed before (P = 0.76) or after (P = 0.73) intervention. Overall, initially the IN was as effective as IRS at killing female and male Lu. longipalpis in both experimental and real chicken sheds. However, the relative lethal effect of the IN deteriorated over time when stored under prevailing environmental conditions.

Synthetic sex-aggregation pheromone of Lutzomyia longipalpis, the South American sand fly vector of Leishmania infantum, attracts males and females over long-distance.

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.

Dinner and a show: The role of male copulatory courtship song and female blood-feeding in the reproductive success of Lutzomyia longipalpis from Lapinha, Brazil.

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.

Characterization of copulatory courtship song in the Old World sand fly species Phlebotomus argentipes.

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.

Sand fly synthetic sex-aggregation pheromone co-located with insecticide reduces the incidence of infection in the canine reservoir of visceral leishmaniasis: A stratified cluster randomised trial.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

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.

Attraction of Lutzomyia longipalpis to synthetic sex-aggregation pheromone: Effect of release rate and proximity of adjacent pheromone sources.

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.

A temporal comparison of sex-aggregation pheromone gland content and dynamics of release in three members of the Lutzomyia longipalpis (Diptera: Psychodidae) species complex.

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.

Spotted Fever: Epidemiology and Vector-Rickettsia-Host Relationship in Rio de Janeiro State.

The eco-epidemiological scenario of spotted fever (SF), a tick-borne disease that affects humans and other animals in several countries around the world, was analyzed in Rio de Janeiro (RJ) State, Brazil. During the last 34 years, 990 SF cases were reported in RJ (the Brazilian state with the highest population density), including 116 cases confirmed by serology (RIFI) or PCR, among 42.39% of the municipalities with reported cases of SF. The epidemiologic dynamics of SF in RJ State are very heterogeneous in time and space, with outbreaks, high mortality rates and periods of epidemiological silence (no SF cases reported). Furthermore, it exhibited a changing epidemiological profile from being rural to becoming an urban disease. This study identified arthropods infected with Rickettsia felis, R. bellii and R. rickettsii, and found that the abundance of ectoparasites was associated with specific hosts. The R. rickettsii-vector-host relationship was most evident in species-specific parasitism. This suggests that the association between dogs, cattle, horses, capybaras and their main ectoparasites, Rhipicephalus sanguineus and Ctenocephalides felis, Rhipicephalus microplus, Dermacentor nitens, and Amblyomma dubitatum, respectively, has a key role in the dynamics of R. rickettsii transmission in enzootic cycles and the maintenance of carrier ectoparasites, thus facilitating the existence of endemic areas with the ability to produce epidemic outbreaks of SF in RJ. This study found confirmed human infections for only the R. rickettsii carrier Amblyomma sculptum, which reinforces the importance of this species as a vector of the pathogen in Brazil. This study can be adapted to different eco-epidemiological scenarios of spotted fever throughout the Americas.

The current status of the Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) species complex

Lutzomyia longipalpis s.l. is a complex of sibling species and is the principal vector of American visceral leishmaniasis. The present review summarises the diversity of efforts that have been undertaken to elucidate the number of unnamed species in this species complex and the phylogenetic relationships among them. A wide variety of evidence, including chemical, behavioral and molecular traits, suggests very recent speciation events and complex population structure in this group. Although significant advances have been achieved to date, differential vector capacity and the correlation between structure of parasite and vector populations have yet to be elucidated. Furthermore, increased knowledge about recent epidemiological changes, such as urbanisation, is essential for pursuing effective strategies for sandfly control in the New World.

The current status of the Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) species complex.

Lutzomyia longipalpis s.l. is a complex of sibling species and is the principal vector of American visceral leishmaniasis. The present review summarises the diversity of efforts that have been undertaken to elucidate the number of unnamed species in this species complex and the phylogenetic relationships among them. A wide variety of evidence, including chemical, behavioral and molecular traits, suggests very recent speciation events and complex population structure in this group. Although significant advances have been achieved to date, differential vector capacity and the correlation between structure of parasite and vector populations have yet to be elucidated. Furthermore, increased knowledge about recent epidemiological changes, such as urbanisation, is essential for pursuing effective strategies for sandfly control in the New World.

The Lutzomyia longipalpis complex: a brief natural history of aggregation-sex pheromone communication.

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.

Morphometric Analysis of Longipalpis (Diptera: Psychodidae) Complex Populations in Mato Grosso do Sul, Brazil.

Owing to the existence of cryptic species that are difficult to distinguish morphologically, the search for new taxonomic characters and methods for identifying and classifying sand flies continues. Lutzomyia longipalpis (Lutz & Neiva, 1912) and Lutzomyia cruzi (Mangabeira, 1938) (Diptera: Psychodidae) are two such species that occur in sympatry in some regions of Mato Grosso do Sul State (MS). Twenty females and twenty males from each of the five populations of Lu. longipalpis and one population of Lu. cruzi from MS were examined. An outlying population of Lu. longipalpis from Estrela de Alagoas, State of Alagoas, was used to compare the degree of divergence among the groups in MS. Specimens were cleared, mounted on slides, identified, and measured using LAS-Leica. The principal component analysis of morphometric characters showed a high degree of variation among females, while males varied to a lower degree. The populations of Alagoas and Miranda demonstrated the greatest variation. The first region, Alagoas, is geographically distant from the others and occurs under distinctly different ecological conditions, which likely accounts for the variation. Further studies should be made to elucidate the factors that contribute to the differences found between the populations of MS.

Phenotypic differentiation in love song traits among sibling species of the Lutzomyia longipalpis complex in Brazil.

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.

Distribution of Lutzomyia longipalpis chemotype populations in São Paulo state, Brazil.

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.

Sand fly fauna (Diptera: Psychodidae: Phlebotominae) in an area of leishmaniasis transmission in the municipality of Rio Branco, state of Acre, Brazil.

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.

Synthetic sex pheromone in a long-lasting lure attracts the visceral leishmaniasis vector, Lutzomyia longipalpis, for up to 12 weeks in Brazil.

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.

Expression of the mevalonate pathway enzymes in the Lutzomyia longipalpis (Diptera: Psychodidae) sex pheromone gland demonstrated by an integrated proteomic approach.

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.