Rhayza D. C. Maingon

Lutzomyia longipalpis in Brazil: a complex or a single species? A mini-review

Lutzomyia longipalpis is the main vector of Leishmania infantum chagasi, the causative agent of American visceral leishmaniasis (AVL). Although there is strong evidence that Lu. longipalpis is a species complex, not all data concerning populations from Brazil support this hypothesis. The issue is still somewhat controversial for this large part of Lu. longipalpis distribution range even though that it is the Latin American region contributing to most of the cases of AVL. In this mini-review we consider in detail the current data for the Brazilian populations and conclude that Lu. longipalpis is a complex of incipient vector species with a complexity similar to Anopheles gambiae s.s. in Africa.

Analysis of the sex pheromone extract of individual male Lutzomyia longipalpis sandflies from six regions in Brazil.

Abstract.  Although the phlebotomine sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) is generally accepted to be a species complex, it is unclear how many members there are, how they are related and which are the main vectors of leishmaniasis. The vectorial capacity of each sibling species is likely to differ, thus a means of identifying the most important vector species is of critical importance to the epidemiology and control of this debilitating disease in South and Central America. In Brazil four chemotypes have been distinguished by sex pheromone analysis. In this study the sex pheromone extracts of L. longipalpis from six regions of Brazil were analysed in detail. Samples included the sympatric 1‐spot, 2‐spot and intermediate spot morphotypes from Sobral, Ceará State. The results strongly suggest that members of the complex that produce different sex pheromones are reproductively isolated, thus strengthening the argument that the different chemotypes represent true sibling species. The study also found significant differences in morphology and the amounts of sex pheromone produced by members of each chemotype from different parts of Brazil, which suggests population substructuring that has not previously been recognized. Evidence of a fifth chemotype in Brazil is also presented.

Genetic identification of two sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) that produce distinct male sex pheromones in Sobral, Ceará State, Brazil.

Lutzomyia longipalpis, the main sandfly vector for New World visceral leishmaniasis is a complex of an as yet undefined number of sibling species. At present, there is no consensus on the status (single species vs. species complex) of Brazilian populations. We applied five microsatellite loci to test the hypothesis that L. longipalpis occurs as two sympatric cryptic species in Sobral, Ceará State, Brazil as predicted by male sex pheromone chemotypes described previously for field specimens from this site [S‐9‐methyl‐germacrene‐B (9MGB) and a cembrene compound]. Abdominal spot morphology corresponds with pheromone type at this locality (9MGB in ‘1 spot’ males and cembrene in ‘2 spot’ males). Genotype data from 190 wild‐caught L. longipalpis specimens collected in October 1999 and April 2001 were used to estimate genetic differentiation between the two sex pheromone populations and sampling dates. No significant (P > 0.05) genetic differences were found between the 1999 and 2001 9MGB samples (θ = 0.018; RST = −0.005), and genetic differentiation was low between the cembrene collections (θ = 0.037, P < 0.05; RST = −0.043, P > 0.05). By contrast, highly divergent allelic frequencies (largely at two microsatellite loci) corresponded to significant (P > 0.05) genetic differentiation (θ = 0.221; RST = 0.215) for all comparisons between samples with different pheromones. When pheromone samples were pooled across sample date, genetic differentiation was high (θ = 0.229; P < 0.001; Nem = 0.84). The allele frequency distribution at each of the five microsatellite loci was similar for males and females from the two collection years. Two of these loci showed highly divergent allele frequencies in the two sex pheromone populations. This was reflected in the highly significant genetic differentiation obtained from the male genotypes, between populations producing different pheromones (θ = 0.229–0.268; P < 0.0001 for the 2001 and θ = 0.254–0.558; P < 0.0001 for the 1999 collections, respectively). Similar results were obtained when the females, assigned to a pheromone type, were included in the analysis. Both a Bayesian analysis of the data set and a population assignment test provided strong evidence for two distinct populations corresponding to pheromone type. Given its genotype, the probability of assigning a 9MGB male to the original 9MGB population was 100% once the two years’ collections were pooled. For cembrene‐producing ‘2 spot’ males this probability although still high, was lower than for 9MGB males, at 86%. This microsatellite data together with previously reported reproductive isolation between the two Sobral populations confirm that premating barriers are important in speciation of L. longipalpis.

A real-time PCR assay to estimate Leishmania chagasi load in its natural sand fly vector Lutzomyia longipalpis.

Summary Leishmania chagasi, transmitted mainly by Lutzomyia longipalpis sand flies, causes visceral leishmaniasis and atypical cutaneous leishmaniasis in Latin America. Successful vector control depends upon determining vectorial capacity and understanding Leishmania transmission by sand flies. As microscopic detection of Leishmania in dissected sand fly guts is laborious and time-consuming, highly specific, sensitive, rapid and robust Leishmania PCR assays have attracted epidemiologists’ attention. Real-time PCR is faster than qualitative PCR and yields quantitative data amenable to statistical analyses. A highly reproducible Leishmania DNA polymerase gene-based TaqMan real-time PCR assay was adapted to quantify Leishmania in sand flies, showing intra-assay and inter-assay coefficient variations lower than 1 and 1.7%, respectively, and sensitivity to 10 pg Leishmania DNA (∼120 parasites) in as much as 100 ng sand fly DNA. Data obtained for experimentally infected sand flies yielded parasite loads within the range of counts obtained by microscopy for the same sand fly cohort or that were around five times higher than microscopy counts, depending on the method used for data analysis. These results highlight the potential of quantitative PCR for Leishmania transmission studies, and the need to understand factors affecting its sensitivity and specificity.

A Fourth Chemotype of Lutzomyia longipalpis (Diptera: Psychodidae) from Jaíbas, Minas Gerais State, Brazil

Abstract Lutzomyia longipalpis (Lutz and Neiva) is a species complex of Lutzomyia pseudolongipalpis (Arrivillaga and Feliciangeli) and at least three other as yet undefined siblings. Isozyme and mitochondrial studies of allopatric populations across Central and South America have suggested the presence of four “clades” that have been hypothesized to have arisen mainly because of geographical isolation mechanisms. Parallel studies of sexual behavior as well as cross-mating and genetic analysis, of both allopatric and sympatric populations, suggest at least four sibling species that do not seem to correspond to the defined four “clades.” In an effort to understand this apparent discrepancy, sympatric populations of L. longipalpis from a single South American country, Brazil, are being studied. In Brazil, three putative species can be identified by their male-produced sex pheromones: (S)-9-methylgermacrene-B, 3-methyl-α-himachalene, and a cembrene. We report here that analysis by coupled gas chromatography–mass spectrometry shows that L. longipalpis from Jaíbas, Minas Gerais State, Brazil, occurs as two sympatric sex pheromone chemotypes. One chemotype is the cembrene type previously recorded in a L. longipalpis population from Sobral, Ceará State, Brazil, and the other is a new cembrene isomer not previously observed in L. longipalpis. The finding of this new chemotype strongly suggests that the L. longipalpis species complex in Brazil consists of four members rather than the three previously recognized and confirms previous analysis of genetic variation that had suggested the presence of a complex in Brazil.

Fixed differences in the paralytic gene define two lineages within the Lutzomyia longipalpis complex producing different types of courtship songs

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.

A possible role for Phlebotomus (Anaphlebotomus) rodhaini (Parrot, 1930) in transmission of Leishmania donovani

BackgroundVisceral leishmaniasis (VL, kala azar), caused by Leishmania donovani is a major health problem in Sudan and other East African countries. In this region the only proven vectors of L. donovani are Phlebotomus orientalis in eastern Sudan, Ethiopia and Upper Nile areas of Southern Sudan and Phlebotomus martini in Ethiopia, Kenya and Southern Sudan. In this report, we present the first evidence that Phlebotomus rodhaini may also play a role in maintaining transmission of L. donovani between animal reservoir hosts in eastern Sudan. The study was conducted in a zoonotic focus of visceral leishmaniasis in Dinder National Park, eastern Sudan, where previous work showed high infection rates of L. donovani in P. orientalis. Sand flies, captured by CDC traps were dissected and examined for infection with Leishmania parasites. Parasite isolates were subjected to L. donovani specific PCR. Field experiments were also carried out to compare efficiency of rodent baited and un-baited CDC traps in collection of P. rodhaini and determine its man-biting rate.ResultsThree female P. rodhain i were found infected with Leishmania parasites in an astonishingly small number of flies captured in three separate field trips. Two of these isolates were typed by molecular methods as L. donovani, while the third isolate was inoculated into a hamster that was subsequently lost. Although P. rodhaini is generally considered a rare species, results obtained in this study indicate that it can readily be captured by rodent-baited traps. Results of human landing collection showed that it rarely bites humans in the area.ConclusionIt is concluded that P. rodhaini is a possible vector of L. donovani between animal reservoir hosts but is not responsible for infecting humans. It is suggested that the role of P. rodhaini in transmission of L. donovani in other zoonotic foci of visceral leishmaniasis in Africa should be re-examined.

Aluminium: a natural adjuvant in Leishmania transmission via sand flies?

Genetically identical Leishmania chagasi/infantum parasites cause both atypical cutaneous leishmaniasis and visceral leishmaniasis. In this report we have tested the first part of a hypothesis that states that the form of this disease that is manifested depends upon the adjuvant-like activity of aluminium of dietary origin accumulated in the salivary gland of the sand fly vector. In sand flies fed aluminium-supplemented sucrose we have used histochemistry to qualitatively identify aluminium in their salivary glands and graphite furnace atomic absorption spectrometry to quantify the aluminium content of dissected salivary glands. Aluminium may be acting as a natural adjuvant in some forms of leishmaniasis.