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Mem. Inst. Oswaldo Cruz vol.102 no.1 Rio de Janeiro Feb. 2007
Luiz GSR BauzerI; Nataly A SouzaI; Rhayza DC MaingonII; Alexandre A PeixotoI, +
IDepartamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil
IIInstitute of Science and Technology in Medicine, School of Life Sciences, Keele University, Staffordshire, UK
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.
Key words: visceral leishmaniasis - sand flies - Lutzomyia longipalpis species complex - introgression - Brazil
The primary vector of Leishmania infantum chagasi and hence of visceral leishmaniasis in Latin America, Lutzomyia longipalpis (Lutz & Neiva 1912) (Diptera: Psychodidae: Phlebotominae) is probably the best-studied sand fly species in the Neotropical region (Soares & Turco 2003, Lainson & Rangel 2005). There is a fairly large body of data pointing out to the existence of a species complex (reviewed by Uribe 1999). Despite of this, there is no consensus yet about the number of sibling species of Lu. longipalpis and what are their areas of distribution (Arrivillaga et al. 2003).
In this mini-review, we intend to give a general overview on the Lu. longipalpis species problem and to consider in detail the current data for the Brazilian populations.
Lu. longipalpis, a species complex?
It has been more than 35 years since the first paper suggesting that Lu. longipalpis might be a species complex was published (post mortem) by Mangabeira (1969) (see also Sherlock & Sherlock 1961). Males collected from the state of Ceará (Northeast Brazil) were shown to have a pair of pale patches on the third and fourth abdominal tergites (2-spot phenotype called henceforth 2S), while those from the state of Pará (North Brazil) were shown to have pale patches only on the fourth abdominal tergite (1-spot phenotype called henceforth 1S). Mangabeira also mentioned that the two forms were found in different ecological conditions speculating that they might represent different species or varieties. Finally, considering that Lu. longipalpis could be a highly variable species, he suggested that Lu. gaminarai and Lu. cruzi could be either good species or regional variants of Lu. longipalpis. In this respect, it is worth noting that Lu. cruzi has been established as a bona fide vector for Le. infantum chagasiin the state of Mato Grosso do Sul, Brazil (Santos et al. 1998) and recent data supports the idea that Lu. cruzi might be another species of the Lu. longipalpis complex (Watts et al. 2005).
Mangabeira hypothesis was the starting point for subsequent three and half decades of discussion concerning the real status of the main vector of visceral leishmaniasis in Latin America (see Table). On the one hand, there are some authors that have defended the hypothesis of the existence of Lu. longipalpis as a single species. On the other hand, there are authors that have argued that Lu. longipalpis occurs as a complex of sibling species although, as mentioned before, there is no consensus on the number and distribution range of the different siblings (see Arrivillaga et al. 2003).
Lu. longipalpis may have once occupied a much larger area than at present and climatic changes may have resulted in the current focalised pattern of distribution (Ward et al. 1985). If true, this suggestion is in agreement, for example, with the now classic forest refugee theory of Haffer (1969). This theory proposes that during several dry climatic periods of the Pleistocene and post-Pleistocene much of South America consisted of tracts of open non-forested vegetation, interspersed with " islands " of humid forest in which speciation of many animal groups could have occurred. The isolated forests were re-united during humid climatic periods. This rupturing and rejoining of the various forests was repeated several times during the Quaternary period and led to rapid differentiation of the fauna in geologically and evolutionary relative very recent times. Based on the known habitat preferences of Lu. longipalpis, climatic conditions might have allowed these insects to attain the wide and discontinuous distribution currently seen (Alexander et al. 1998). During the dry period populations of Lu. longipalpis may have spread over a broader area and humid climatic periods may have separated them. Several other hypotheses have been postulated to explain species diversity in the Neotropical region, but no single model can by itself explain evolution in the tropics, (reviewed by Marroig & Cerqueira 1997).
At present, there is considerable geographical isolation among Lu. longipalpis populations. This may be attributed to their low mobility as well as to the extrinsic barriers that exist within their broad but discontinuous areas of distribution (Lanzaro et al. 1993, Young & Duncan 1994, Alexander et al. 1998). Isolation by distance is recognised as one of the conditions that eventually may cause species rising as a consequence of the low gene flow between the populations involved. Due to its importance as the main vector involved in the transmission of visceral leishmaniasis in Central and South America, Lu. longipalpis has been examined in terms of its morphological variability, male pheromones, sexual behaviour and population genetic structure (reviewed by Uribe 1999).
Table summarises all major research studies reported on the elucidation of the taxonomical status of this putative species complex. These studies collectively sampled populations distributed over a broad geographical range in Central and South America. The table shows the sample collection sites, the different study approaches employed and the main results and conclusion concerning the taxonomic status of the Lu. longipalpis populations (as either single species or species complex). It is interesting to observe that all studies carried out on samples from South America versus Central America have strongly suggested that Lu. longipalpis is a species complex (Lanzaro et al. 1993, Warburg et al. 1994, Morrison et al. 1995, Dujardin et al. 1997, Lanzaro et al. 1998, 1999, Yin et al. 1999, 2000, Soto et al. 2001, Arrivillaga et al. 2002, 2003, Watts et al. 2005). Lutzomyia pseudolongipalpis was formally recognized as the first cryptic species of the complex by virtue of morphological, genetic and behavioural differences when compared to several populations, including those separated by a very long distance (Arrivillaga & Feliciangeli 2001). Equally interesting from the data compiled in this table is that it is only within Brazil where broadly separated Lu. longipalpis populations have been considered either a single species (Mukhopadhyay et al. 1997, 1998a,b, Mutebi et al. 1999, Azevedo et al. 2000, Arrivillaga et al. 2003, de Queiroz Balbino et al. 2006) or a species complex (Ward et al. 1983, 1988, Yin et al. 1999, Souza et al. 2002, 2004, Bauzer et al. 2002a,b, Maingon et al. 2003, Bottecchia et al. 2004, Hamilton et al. 2004, 2005, Watts et al. 2005) depending on the markers used. Therefore the controversy concerning the existence of a complex is centred on data from Brazilian populations.
Lu. longipalpis in Brazil, evidence for a species complex
Following the lead by Mangabeira (1969), Ward et al. (1983, 1988) published results of a number of crossing experiments that provided the first strong evidence that Lu. longipalpis was not a single species in Brazil. Even though this earlier work was somewhat contradicted by subsequent data from isoenzyme studies (Mukhopadhyay et al. 1998b, Mutebi et al. 1999, Azevedo et al. 2000), Ward et al. (1983) was the first study to show clear evidence that Lu. longipalpis was a species complex when reproductive isolation was found in crosses between some allopatric and sympatric populations (see below).
Ward et al. (1983) were, in fact, testing Mangabeira's hypothesis that the 1S and 2S phenotypes might represent different species. Studies on the distribution of Lu. longipalpis showed that males having pale patches only on the fourth abdominal tergite (1S phenotype) are found broadly and discontinuously distributed all over South and Central America whereas males having a pair of pale patches on the third and fourth abdominal tergites (2S phenotype) are more concentrated in the Northeast region of Brazil (Ward et al. 1985). Crossing experiments were performed between sympatric and allopatric Brazilian populations that differed by the number of tergal spots (Ward et al. 1983). Highly reduced insemination rates were observed in crosses between two colonies originated from Sobral, state of Ceará, one with 1S and the other with 2S males, suggesting that they represent different sympatric sibling species. In addition, low insemination rates were also observed between the 2S colony from Morada Nova, Ceará, and 1S sand flies from Lapinha cave and between 1S Marajó Island and 1S Lapinha. Later work on other populations (Ward et al. 1988) came to confirm and extend these earlier results and the conclusion was that Lu. longipalpis was in fact a species complex in Brazil.
The above studies showed that the spot morphology could not be used as a species-specific character. Indeed, an intermediate phenotype (a small spot on the third tergite in addition to the spot on the fourth tergite) was observed in high frequencies in some localities, especially around the Northeast coast, indicating an intraspecific polymorphism (Ward et al. 1988). Crosses between some 1S and 2S populations yielded mainly males carrying an intermediate phenotype strongly suggesting a semi-dominant genetic model of inheritance for this character (Ward et al. 1988). Further evidence regarding spot morphology as a genetic polymorphism was obtained by Mukhopadhyay et al. (1998a) in a visceral leishmaniasis endemic area near the city of Natal, state of Rio Grande do Norte. However, although this character is not species-specific, it may be useful to identify two sympatric species in localities where intermediates are very rare, such as Sobral (Ward et al. 1988, see below).
The existence of a complex of Lu. longipalpis in Brazil was substantiated by results of analysis of sex pheromones (Ward et al. 1988, Hamilton et al. 1996a,b) and male " lovesongs " that are produced during copulation (Souza et al. 2002, 2004). These two traits can have an important role in the reproductive isolation among closely related species. Interestingly, differences in both traits are strongly correlated in the Brazilian Lu. longipalpis populations studied so far indicating the existence of at least four reproductive isolated populations in Brazil (Figure). Males from Jacobina, Lapinha, Natal, Sobral and Marajó island had both their pheromones (Ward et al. 1988, Hamilton et al. 1996a,b) and copulatory courtship songs (Souza et al. 2002, 2004) analysed. Remarkable differences among these populations were found. Males from Jacobina produce the 3-methyl-a-himachalene pheromone type and song with trains of pulses that resemble those produced by Drosophila (" pulse-song type 1 ") (e.g. Peixoto & Hall 1998) while Lapinha males produce the 9-methyl-germacrene-B pheromone type and songs with trains of essentially monocyclic pulses interrupted by highly polycyclic pulses (" pulse-song type 2 ") (Hamilton et al. 1996a,b, Souza et al. 2002, 2004).
Sobral 1S males also produce 9-methyl-germacrene-B that can be differentiated from Lapinha's pheromone by the amount of specific terpenes (Hamilton et al. 2005). Yet a third type of pulse-song is produced by Sobral 1S, different from Jacobina and Lapinha, in which high and low amplitude pulses alternate almost perfectly (" pulse-song type 3 ") (Souza et al. 2004). These three populations therefore produce different types of pulse-songs and 16C type pheromones (9-methyl-germacrene-B or 3-methyl-a-himachalene) (Figure). Males from Natal, Marajó, and Sobral 2S on the other hand showed a completely different acoustic signal made of highly polycyclic bursts that are modulated in frequency and amplitude (" burst-song "). These three populations also produce the same type of 20C pheromone (cembrene type 1) (Figure). As expected from the crossing experiment results (Ward et al. 1983, 1988), the two forms that coexist in Sobral were shown to be associated with different pheromone types and copulatory courtship songs.
Molecular studies using two sand fly homologues (Peixoto et al. 2001) of Drosophila courtship song genes, period and cacophony were also carried out to address the Lu. longipalpis " species problem " in Brazil. period is involved in the control of circadian and courtship song rhythms and it was used in a number of studies of closely related species of Drosophila (reviewed in Peixoto 2002). Because this gene controls song differences among Drosophila species that are important to their sexual isolation it is considered an example of a " speciation gene " (Coyne 1992). cacophony codes for an a-1 subunit of a voltage-gated calcium channel involved in the control of the Drosophila courtship song (Smith et al. 1998, Peixoto & Hall 1998).
Both genes were used to study the molecular divergence between the allopatric populations of Jacobina, Lapinha, and Natal (Bauzer et al. 2002a, Bottecchia et al. 2004). It was shown that these three broadly distributed populations are highly differentiated and the estimated number of migrants per generation was much smaller, particularly for the period gene (Nem = 0.29), than those estimated using isoenzymes (see below). This suggests that Lu. longipalpis in Brazil might be in an incipient speciation process and that isoenzymes do not evolve fast enough to detect genetic divergence among the Brazilian populations.
Bauzer et al. (2002b) and Bottecchia et al. (2004) also analysed the reproductively isolated sympatric populations of Sobral. Data from both genes confirm that the two sympatric populations were genetically distinct and very high and significant levels of differentiation were found, particularly for period (Fst = 0.395, p < 0.001; Nem = 0.38). Some evidence for introgression was found in the case of cacophony, probably due to the incomplete reproductive isolation observed in mating experiments (Ward et al. 1983, 1988). In fact, this evidence of introgression, associated with the lower evolutionary rates of isoenzymes could easily explain why these markers did not show a significant deviation from HWE in Sobral (Mutebi et al. 1999) when samples from this locality were analysed pooling together 1S and 2S males.
Highly variable microsatellite marker loci have been isolated from Lu. longipalpis by Watts et al. (2002). Maingon et al. (2003) used five of these microsatellite markers to genotype 190 field specimens captured at Sobral, in two separate years at different seasons. As mentioned, at Sobral, a 1S, 9-methyl-germacrene-B, pulse-song type 3 (with alternate high and low pulses) overlaps with a 2S, cembrene type 1 isomer, burst-song population. No temporal genetic differences were found within each of these populations. In contrast, divergent allelic frequencies (mainly at two of the five loci) indicated significant genetic differentiation (q = 0.221; P < 0.05) for comparisons between the two populations. Genetic differentiation remained high, (q = 0.229; P < 0.001), when temporal collections were pooled according to their spot/pheromone/song type. Estimated gene flow between the two sympatric populations was relatively low (Nem = 0.84), and comparable to that obtained from the analysis of polymorphisms in the period gene (Nem = 0.38), by Bauzer et al. (2002b).
Analyses of polymorphism in period and cacophony genes (Bauzer et al. 2002a,b, Bottecchia et al. 2004) also indicate that Natal and Sobral 2S are highly similar to each other and distinct from the other three populations. This is in agreement with the fact that these two populations produce the same type of pheromone and copulatory courtship song. Furthermore, a correlation seems to exist between the level of divergence in the period gene amongst Natal, Sobral 2S, Marajó, Lapinha, Jacobina, and Sobral 1S, and the level of phenotypic differences in the copulation songs of these six populations (Souza et al. 2004).
Watts et al. (2005) have extended their studies on the allele frequency distribution of the microsatellite markers used for Sobral to nine other Brazilian and Venezuelan populations. Temporal genetic differences were non-significant within populations collected at the same location. Geographic separation, however, influenced genetic differentiation, although the effect was relatively weak (r2 = 0.095; P = 0.0014) when samples were pooled together irrespective of their spot/pheromone type. The correlation between genetic and geographic distances between samples increased when populations with different pheromone types were separately analysed (r2 = 0.541, P = 0.0028 for 9-methyl-germacrene-B populations; and r2 = 0.965, P = 0.039 for cembrene type 1 populations). A cluster analysis revealed, for the first time, a highly distinct group of Brazilian cembrene-1 populations mapping to the Northeast of the country, plus a geographically dispersed non-cembrene (9-methyl-germacrene-B and 3-methyl-a-himachalene) cluster comprising Brazilian and Venezuelan populations. Interestingly, this separation between cembrene and non-cembrene samples matchs that observed between populations with burst and pulse songs (Souza et al. 2004).
Evidence against a complex in Brazil
The technique most used, so far, to estimate genetic divergence among Brazilian Lu. longipalpis populations is the variation in isoenzyme loci. According to Mukhopadhyay et al. (1998b), Mutebi et al. (1999), and Azevedo et al. (2000), isoenzyme analysis failed to show a genetic distance large enough to indicate the existence of two or more species in Brazil. Nevertheless, the comparison of allele frequencies among Brazilian populations indicated a certain degree of genetic substructure. The estimated number of migrants per generation ranged from 2.0 to 3.6 in these three different studies (Mukhopadhyay et al. 1998b, Mutebi et al. 1999, Azevedo et al. 2000). As stated by the authors, these numbers suggested a level of gene flow between the Brazilian populations that would not have allowed a speciation process to occur. Although the isoenzyme data did not seem to support the existence of different sibling species in Brazil, on closer examination, the results are not inconsistent with the molecular studies mentioned above. For example, Azevedo et al. (2000) showed that Natal and Salvaterra (Marajó island) were far more similar to each other than either is to Lapinha even though the geographical distance between the two Northern populations, Salvaterra and Natal (1550 km), is about the same as the distance between Natal and Lapinha (1700 km). These results confirmed similar observations of two other broader population analyses in Brazil (Mukhopadhyay et al. 1998b, Mutebi et al. 1999). In both studies, the populations occurring along the Northeast coast (down to the southeastern locality of Pancas, state of Espírito Santo) were shown to be genetically more homogeneous and distinct from the populations of Jacobina and Lapinha, even though the distance that separates some of the Northeastern populations is about the same as the distance that separates them from Jacobina or Lapinha. Therefore, the isoenzyme data does not agree with a simple isolation by distance model of genetic differentiation and the patterns observed are consistent with the results obtained with period, cacophony, and microsatellite loci (Bauzer et al. 2002a,b, Bottecchia et al. 2004, Maingon et al. 2003, Watts et al. 2005).
While the authors of the isoenzyme studies mentioned above were unanimous in their conclusion concerning the single species status of the Brazilian population of Lu. longipalpis, analysis of mitochondrial DNA in three different studies (Soto et al. 2001, Arrivillaga et al. 2002, Hodgkinson et al. 2003) gave somewhat ambiguous results. According to Soto et al. (2001), their results question the conspecific status of Brazilian populations. Arrivillaga et al. (2002) and Hodgkinson et al. (2003) reach a different conclusion, even though the latter study did find a significant differentiation between Northern and Southern populations in their analysis of mitochondrial cytochrome b gene sequences in six locations representing a geographic transect across Eastern Brazil.
Recently the genetic structure of seven populations from Brazil Northeastern region was described based on polymorphism analysis of 24 RAPD-PCR loci (de Queiroz Balbino et al. 2006). Although the detected levels of genetic variation were higher than those obtained with the use of isoenzyme markers, genetic distances were considered to be compatible with those found between members of a single species. However, inspection of the data indicates a few loci with very large, even fixed, differences in some comparisons. This suggests that a different conclusion would be reached if a more detailed analysis was carried out on populations where evidence for a complex was found with other molecular markers (Bauzer et al. 2002a,b, Bottecchia et al. 2004, Maingon et al. 2003, Watts et al. 2005).
Lu. longipalpis in Brazil, a complex of incipient species?
It is becoming evident that gene flow and differential introgression among loci between closely related or incipient vector species is far more common than previously thought, as the studies on the An. gambiae complex and An. gambiae s.s. are showing (Black & Lanzaro 2001, della Torre et al. 2002, Besansky et al. 2003, Donnelly et al. 2004, Tripet et al. 2005, Slotman et al. 2005, Stump et al. 2005, Turner et al. 2005).
For Lu. longipalpis, it is fair to say that the available evidence for the occurrence of a complex in Brazil is overwhelming, particularly from the studies carried out in Sobral where two populations with distinct phenotypes overlap. Crossing experiments, pheromone and copulatory courtship song analysis, studies on the " lovesong " genes period and cacophony, and variation at microsatellite loci, all indicate the existence of at least two sympatric sibling species in this Brazilian locality (Ward et al. 1983, 1988, Bauzer et al. 2002b, Maingon et al. 2003, Bottecchia et al. 2004, Souza et al. 2004, Hamilton et al. 2005). The data for other Brazilian allopatric populations based on the same markers and types of analysis used in Sobral are also very convincing in our view (Ward et al. 1988, Bauzer et al. 2002a, Souza et al. 2002, 2004, Bottecchia et al. 2004, Hamilton et al. 2005, Watts et al. 2005). However, it is also fair to say that the differentiation among Brazilian populations, based on isoenzymes and mitochondrial loci, in general, is not as large as the divergence between the Brazilian and other South and Central American populations (Soto et al. 2001, Arrivillaga et al. 2003).
These inconsistencies between different markers may probably be attributed to a number of factors such as: (a) maintenance of ancestral polymorphisms caused by recent speciation events, (b) slow rates of evolution in some markers, such as isoenzymes, due to negative selection, and (c) the occurrence of introgression among these populations caused by incomplete reproductive isolation. Moreover, the levels of introgression are very likely quite different among the various loci, probably affecting much less those closely linked or directly involved in reproductive isolation (Stump et al. 2005, Turner et al. 2005).
There are a number of difficulties associated with the study of recently diverged species and populations in an incipient speciation process (Hey 2001, Coyne & Orr 2004) such as the members of the Lu. longipalpis complex within Brazil. However, in our view, the data reviewed in this paper is strong enough to indicate without any doubt, that the Brazilian populations do not belong to a single panmictic species given that there are strong reproductive barriers even among some sympatric populations. We conclude that Lu. longipalpis in Brazil is one example of a complex of incipient vector species showing perhaps a similar level of complexity presented by An. gambiae s.s. in Africa.
To Richard Ward and Gordon Hamilton for comments on the manuscript and Gabriel Melim Ferreira for drawing the figure.
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Received 9 June 2006
Accepted 20 September 2006
Financial support: Howard Hughes Medical Institute, CNPq, Faperj, Fiocruz-Papes4