Background Lutzomyia longipalpis is the primary vector of American visceral leishmaniasis. There is strong evidence that L. longipalpis is a species complex, but until recently the existence of sibling species among Brazilian populations was considered a controversial issue. In addition, there is still no consensus regarding the number of species occurring in this complex.Methodology/Principal FindingsUsing period, a gene that controls circadian rhythms and affects interpulse interval periodicity of the male courtship songs in Drosophila melanogaster and close relatives, we analyzed the molecular polymorphism in a number of L. longipalpis samples from different regions in Brazil and compared the results with our previously published data using the same marker. We also studied the male copulation songs and pheromones from some of these populations. The results obtained so far suggest the existence of two main groups of populations in Brazil, one group representing a single species with males producing Burst-type copulation songs and cembrene-1 pheromones; and a second group that is more heterogeneous and probably represents a number of incipient species producing different combinations of Pulse-type songs and pheromones.Conclusions/SignificanceOur results reveal a high level of complexity in the divergence and gene-flow among Brazilian populations of the L. longipalpis species complex. This raises important questions concerning the epidemiological consequences of this incipient speciation process.

The sand fly Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae: Phlebotominae), the main vector of Leishmania infantum in the Americas, is believed to be a species complex, although the status of different Brazilian populations is still somewhat unclear. Preliminary analysis of the acoustic signals that are produced during copulation by L. longipalpis males has suggested the existence of three sibling species in Brazil. In the current report, we analyze in more detail a number of parameters of the copulatory courtship songs of L. longipalpis males from four allopatric populations from different parts of the country (Marajó Island, Natal, Jacobina, and Lapinha Cave) and from two sympatric populations from the locality of Sobral, where two types of males can be differentiated by the number of pale spots (one or two pairs) found on the abdomen. We show that males from the localities of Natal, Marajó, and Sobral (two-spot morph) have very similar songs composed of successive bursts, which are modulated in frequency and amplitude. No significant differences were found in the song parameters of these three populations. In contrast, one-spot males from Sobral and males from Jacobina and Lapinha produce songs that are made of pulses but with distinct patterns for each population and significant differences in all song parameters studied. The results suggest that the L. longipalpis complex in Brazil is composed of four sibling species and that the differences in song patterns between the populations are consistent with the level of divergence found in the period gene.

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Lutzomyia longipalpis, a sibling complex, is the main vector of Leishmania chagasi/infantum. Discriminating between siblings is important as they may differ in vectorial capacity. Lutzomyia longipalpis populations display distinct male sex pheromone chemotypes. We investigated the phylogeographic pattern of variation at microsatellite loci from 11 populations from Brazil and Venezuela related to their male pheromone. Temporal genetic differentiation was mostly not significant at the same site. Spatial genetic differentiation was, however, strong, although there was only a weak relationship between genetic differentiation and the geographic distance separating the samples (r2 < 0.10); geographic separation explained a much greater (54-97%) percentage of the genetic differences among populations when samples with the same pheromone type were analyzed separately. A cluster analysis showed five groups: Lu. cruzi (Brazil) and Lu. pseudolongipalpis (Venezuela) as separate species, two (mostly 9-methyl-germacrene-B) Venezuelan and Brazilian groups, and a very distinct cluster of Brazilian cembrene populations.

Despite the importance of circadian rhythms in vector-borne disease transmission, very little is known about its molecular control in hematophagous insect vectors. In Drosophila melanogaster, a negative feedback loop of gene expression has been shown to contribute to the clock mechanism. Here, we describe some features of the circadian clock of the sandfly Lutzomyia longipalpis, a vector of visceral leishmaniasis. Compared to D. melanogaster, sandfly period and timeless, two negative elements of the feedback loop, show similar peaks of mRNA abundance. On the other hand, the expression of Clock (a positive transcription factor) differs between the two species, raising the possibility that the different phases of Clock expression could be associated with the observed differences in circadian activity rhythms. In addition, we show a reduction in locomotor activity after a blood meal, which is correlated with downregulation of period and timeless expression levels. Our results suggest that the circadian pacemaker and its control over the activity rhythms in this hematophagous insect are modulated by blood intake.

In a focus of cutaneous leishmaniasis in Jacarepaguá, Rio de Janeiro, one specimen of Lutzomyia intermedia was found naturally infected with Leishmania braziliensis.

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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 (theta = 0.018; RST = -0.005), and genetic differentiation was low between the cembrene collections (theta = 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 (theta = 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 (theta = 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 (theta = 0.229-0.268; P < 0.0001 for the 2001 and theta = 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.