Genetic structure of natural populations of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) from the Brazilian northeastern region

Acta Tropica 98 (2006) 15–24

Genetic structure of natural populations of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) from the Brazilian northeastern region Valdir de Queiroz Balbino a,∗ , Iliano Vieira Coutinho-Abreu a , Ivan Vieira Sonoda a , M´arcia Almeida Melo a , Paulo Paes de Andrade a , Jos´e Adail Fonseca de Castro b , Jos´e Mac´ario Rebˆelo c , S´ılvia Maria Santos Carvalho d , Marcelo Ramalho-Ortig˜ao e a

Laborat´orio de Gen´etica Molecular, Centro de Ciˆencias Biol´ogicas, Departamento de Gen´etica, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil b Universidade Federal do Piau´ı, Teresina, Piau´ı, Brazil c Universidade Federal do Maranh˜ ao, S˜ao Lu´ıs, Maranh˜ao, Brazil d Universidade Estadual de Santa Cruz, Ilh´ eus, Bahia, Brazil e Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA Received 22 July 2005; received in revised form 23 December 2005; accepted 4 January 2006 Available online 14 February 2006

Abstract In Latin America, Lutzomyia longipalpis is the principal vector of Leishmania chagasi, and is associated with the majority of active foci of visceral leishmaniasis. In spite of the fact that this sand fly is spread practically throughout the entire Neotropical Region, its distribution is not uniform due to geographic and environmental barriers. Geographic isolation coupled with reduced flight abilities may contribute to the appearance of cryptic species of Lutzomyia longipalpis, which may differ in their capacity to transmit L. chagasi. In this work, we describe the genetic structuring patterns based on polymorphism analysis of 24 RAPD-PCR loci of 7 natural populations of Lutzomyia longipalpis obtained from Brazil’s northeastern region. The estimated degree of genetic differentiation between populations, based on the population subdivision index θ ST (0.136), suggests a moderate degree of genetic structuring as a result of geographical isolation and restricted gene flow. Genetic distances were found to be compatible with those found between members of a single species, suggesting a taxonomic uniformity of Lutzomyia longipalpis in the region studied. © 2006 Elsevier B.V. All rights reserved. Keywords: Lutzomyia longipalpis; Sand fly; American visceral leishmaniasis; Molecular markers; Polymorphisms; Population genetics

1. Introduction

∗

Corresponding author. Universidade Federal de Pernambuco, Departamento de Gen´etica, Av. Prof. Moraes Rego S/N, Cidade Universit´aria, Recife, Pernambuco 50732-970, Brazil. Tel.: +55 8121268569; fax: +55 8121268569. E-mail address: [email protected] (V. de Queiroz Balbino). 0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2006.01.007

Visceral leishmaniasis (VL) is a grave parasitic illness with a high mortality rate among untreated patients. Approximately, 500 000 new cases are reported worldwide every year, the majority of which occur in poor and suburban areas in Asian and South American developing nations (Desjeux, 2004). The etiological agent of visceral leishmaniasis in the Neotropical Region is Leishmania

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chagasi, which is transmitted through the bite of the sand fly Lutzomyia longipalpis Lutz and Neiva, 1912 (Diptera: Psychodidae) (Ashford, 2000). This insect has proven to be well adapted to different environments, reaching high population densities in rural and peri-urban areas. The presence of Lutzomyia longipalpis in the vicinities of human dwellings enables the establishment of a domestic cycle of VL, where dogs play an important role in transmission (Lainson and Shaw, 1998). Lutzomyia longipalpis can be found from southern Mexico to northern Argentina (Young and Duncan, 1994). However, their distribution in the Neotropical Region is fragmented due to the existence of geographical barriers and unfavorable climatic conditions, limiting their proliferation in some areas (Arrivillaga et al., 2002). The geographic discontinuity among populations, coupled with the sand flies’ reduced flight capability (Morrison et al., 1993) may limit gene flow, resulting in local populations of Lutzomyia longipalpis likely due to mutation and differential selection. Local populations may have different evolutionary patterns that may be reflected in their capacity to transmit L. chagasi (reviewed by Lanzaro and Warburg, 1995). The first report of phenotypic variation in Lutzomyia longipalpis was presented by Mangabeira (1969), who described differences in the number of tergal spots (one or two pairs) found in males from two geographic regions in Brazil. The assumption that the morphological variants occupied distinct ecological niches led the author to suggest that they could represent distinct varieties or species. Subsequently, the sympatric occurrence of the two phenotypes as well as the identification of an intermediary phenotypic form (Ward et al., 1988) resulted in the gradual reduction of taxonomic importance of this characteristic. In the last few years, the number of reports focusing on the genetic characterization of Lutzomyia longipalpis based on various chromosomal, morphological, biochemical and molecular characters has grown considerably (Watts et al., 2005; Souza et al., 2004; Hamilton et al., 2004; Bottecchia et al., 2004; Maingon et al., 2003; Arrivillaga et al., 2003; Yin et al., 1999; Lanzaro et al., 1993). These have indicated the likely existence of a Lutzomyia longipalpis complex, made up of a yet unknown number of species. Possible epidemiological implications of the presence of a Lutzomyia longipalpis complex in regards to the transmission of L. chagasi, have not yet been established. Understanding the complex pattern of geographical structuring of these insects may provide insight on epidemiological importance, if any, of the Lutzomyia longipalpis species complex.

Molecular markers are an invaluable tool in studies of population genetics and evolution, as the levels of genetic variability obtained are usually higher that those produced by morphological and isozyme markers (reviewed by Loxdale and Lushai, 1998). The degree of genetic differentiation between geographic populations can be indirectly estimated using statistical models, which are based on the identification of genotypes within and between evolutionary units (Rousset, 1997). One of the most frequently used methods in this type of inference is the FST index, introduced in population genetics by Wright (1951). FST is an indirect measurement of the level of genetic flow between populations and has shown to be effective in comparative analysis of geographic populations (Neigel, 2002). The random amplified polymorphic DNApolymerase chain reaction (RAPD-PCR) (Williams et al., 1990) enables the investigation of DNA strands without any prior information, and allows for the rapid identification of polymorphic genetic markers (Black, 1993). Although some of these markers have been used in studies directed at various insects (Ram´ırez et al., 2005; Kim and Sappington, 2004; Ocampo and Wesson, 2004; Posso et al., 2003; Skoda et al., 2002) and some species of sand flies (Meneses et al., 2005; de Souza et al., 2004; Margonari et al., 2004; Mukhopadhyay et al., 2000), their use in Lutzomyia longipalpis population studies remain limited. In this work we describe the use of 24 RAPD loci to establish the genetic structure of 7 natural populations of Lutzomyia longipalpis in northeastern (NE) Brazil. We detected levels of genetic variation that were higher than those obtained with the use of isozyme markers, suggesting an advantage in using the former for population genetics studies in Lutzomyia longipalpis. Levels of genetic distance between populations tested in this study were found to be compatible with the existence of a single species of Lutzomyia longipalpis in NE Brazil. 2. Material and methods 2.1. Sand flies Lutzomyia longipalpis were collected from seven locations in NE Brazil (Table 1, Fig. 1). Sand flies were collected using CDC light traps (Sudia and Chamberlain, 1962) set in chicken pens between 18.00 and 05.00 h. Insects were killed by freezing at −20 ◦ C and individually stored at −80 ◦ C prior to utilization. Due to ease of identification, only male specimens were used in this analysis. The identification of the sand flies was made through the observation of their external genitalia and

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Table 1 List of municipalities and areas in NE Brazil where Lutzomyia longipalpis specimens were captured for this study Locality S˜ao Luis Teresina Patos Jo˜ao Pessoa Itamarac´a Calumbi Camac¸ari

Abbreviation SLIS TRSA PATS JPES ITAM CALB CAMI

Sample size

Latitude (S)

Longitude (W)

Altitude (m)

20 20 20 20 20 20 20

02◦ 31

44◦ 18

24.0 72.7 242.0 47.0 36.0 446.0 20.0

05◦ 05 07◦ 01 07◦ 06 07◦ 44 07◦ 56 12◦ 41

42◦ 48 37◦ 16 34◦ 51 34◦ 49 38◦ 09 38◦ 19

Coordinates (latitude, longitude and altitude) were determined with the use of a global positioning system (GPS) (Garmin International Inc, Olathe, KS, USA).

additional morphological characteristics, following the descriptions provided by Young and Duncan (1994). 2.2. DNA extraction and RAPD-PCR analysis DNA was extracted in accordance with Mukhopadhyay et al. (2000), with some modifications. Each insect was individually homogenized in a volume of 50 ␮l of STE buffer (0.1 M NaCl; 10 mM Tris/HCl pH 8.0; 1 mM EDTA pH 8.0), incubated at 95 ◦ C for 10 min and centrifuged at 6000 × g for

1 min. A total of 40 ␮l of supernatant was recovered and diluted with 160 ␮l of H2 O and stored at −80 ◦ C prior to utilization. For RAPD, DNA from each insect was amplified using 20 preselected oligonucleotides from the Operon P series (Operon Technologies Inc., Alameda, CA, USA). PCR reactions were carried out by combining 1 ␮l of the DNA extraction, 5 ␮l of the master mix [2.5 ␮l PCR buffer/MgCl2 (10×); 0.5 ␮l dNTP mix (10 mM); 2 ␮l Taq polymerase (1 U/␮l)]; 2 ␮l of each primer (10 ␮M); and 17 ␮l of H2 O] under the following conditions: a single step at 94 ◦ C for

Fig. 1. Map of the Brazilian territory depicting state lines and the location of cities where Lutzomyia longipalpis were collected as indicated with a dot.

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1 min, followed by 34 cycles at 94 ◦ C for 1 min, 40 ◦ C for 2 min, and 72 ◦ C for 2 min, with a final extension at 72 ◦ C for 10 min. PCR reactions were repeated in three different thermocyclers: an Eppendorf® Mastercycler® Gradient (Eppendorf, Hamburg, Germany); a Biometra TGradiente (Biometra, G¨oettingen, Germany); and a MJ Research PTC-100 (Bio-Rad Laboratories, Inc., Waltham, MA, USA). PCR products were separated on 1.5% agarose gels, stained with ethidium bromide and photographed using a Doc-Print (Vilber Lurmat, Marne-la-Vall´ee, France) digital system. DNA bands were named based on the corresponding primer, and the estimated molecular weight in relation to a 100 bp DNA ladder (Invitrogen, Carlsbad, CA, USA). 2.3. Statistical analysis RAPD-PCR polymorphism was scored based on presence (1) or absence (0) of amplified PCR products. The allelic frequencies, the percentage of polymorphic loci (P%), the expected average heterozygosity (HE) (Nei, 1973), and the effective number of alleles (Ne) (Kimura and Crow, 1964) were estimated using the program POPGEN v. 1.32 (http://cc.oulu.fi/∼jaspi/popgen/popdown.htm). A locus was considered polymorphic only if the most common allele occurred at a frequency of ≤0.95 in the population (a 95% criterion). In accordance to the dominance relationship usually found in RAPD-PCR loci, the statistical analysis followed four general principles described by Apostol et al. (1996): (1) RAPD loci segregate as dominant mendelian markers; (2) genotypic frequencies are in accordance with Hardy–Weinberg equilibrium; (3) recessive alleles are identical and originate from the same mutational events; (4) dominant alleles also originate from the same mutational events. The genetic distance between populations was estimated using Nei’s genetic distance (D) (1978), and was corrected for RAPD markers (Lynch and Milligan, 1994) using the program RAPDDIST (Black, 1995). The genetic distances obtained were used in the construction of a dendrogram based on the unweighted pairgroup method analysis (UPGMA) algorithm (Sneath and Sokal, 1973) with the use of the program Mega v. 2.2 (Kumar et al., 2001). The population subdivision indexes FST (Wright, 1951), FST corrected for dominant markers (Lynch and Milligan, 1994), and θ ST (Weir and Cockerham, 1984) were calculated using the program RAPDFST (Black, 1995), based on the assumption that the populations were in agreement with the Hardy–Weinberg equilibrium, and adopting the dominance condition at each locus. The degree of correlation

between these indexes was estimated using the program “Statistica” (StatSoft Inc., 1999). A matrix containing the straight-line distances between the sampled locations was built based on the geographical coordinates for the locations. This matrix was used in the construction of a UPGMA dendrogram, with the program Mega version 2.2 (Kumar et al., 2001). The matrices containing the geographic and genetic distances, as well as pairwise θ ST estimates between populations, were tested for linear correlation using a Mantel test (Mantel, 1967) for the evaluation of the association level (Z) through 1000 random permutations. Correlation values (r) equal to or greater than 0.5 were considered statistically significant. The mean number of migrants per generation (Nm) was calculated between the populations based on FST values, the corrected FST for RAPD markers and θ ST (Slatkin and Barton, 1989), under the assumptions of the island model of migration (reviewed by Slatkin, 1987). The model was tested through linear regression of the values on the FST /(1 − FST ) relation against the geographical straight-line distance between each of the populations (Rousset, 1997). The regression statistical relevance was tested through the Mantel test (1967) on 1000 permutations. Correlation values equal to or greater than 0.5 were considered statistically relevant. 3. Results The analysis of the amplification profiles of the 20 primers tested showed that primers OPP-04, OPP-06 and OPP-09 produced a satisfactory level of variability and reproducibility. In each case, 10–12 bands with sizes ranging from 200 to 1200 bp were obtained. For the analysis of population variability in Lutzomyia longipalpis, 24 bands were selected based on reproducibility (eight from each primer), with sizes ranging from 260 to 1050 bp. Genetic variability levels for the three primers chosen for the analysis were high. The percentage of polymorphic loci, based on an 85% criterion, varied between 87.5% (OPP-06) and 100.0% (OPP-04 and OPP-09), while the expected mean heterozygosity (HE) varied between 0.386 (OPP-6) and 0.390 (OPP09). Levels of genetic variation for each population are presented in Table 2, including the percentage of polymorphic loci and the expected mean heterozygosity. It was noted that the majority of loci (95.8%) was polymorphic in at least one of the populations, and that 50% of loci were variable in all of them. Diagnostic alleles were not identified among the seven populations of Lutzomyia longipalpis analyzed, and therefore the genetic differences observed were due to variation in allelic frequencies between populations. The lowest percentage of

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Table 2 Genetic variability of 24 RAPD-PCR loci obtained from seven populations of Lutzomyia longipalpis found in NE Brazil Primer

Loci

SLIS

TRSA

PATS

JPES

ITAM

CALB

CAMI

Total

OPP 04

920 850 780 680 550 430 380 300

0.6384 0.4015 0.5558 0.3351 0.0000 0.5558 0.3351 0.5558

0.6384 0.5096 0.7116 0.2537 0.4015 0.5558 0.3351 0.5558

0.5558 0.4015 0.9493 0.0000 0.0000 0.6760 0.3351 0.7454

0.6384 0.3351 0.9493 0.3351 0.3351 0.4587 0.0000 1.0000

0.5986 0.4587 0.8958 0.0000 0.3351 0.4587 0.0000 1.0000

0.6760 0.5096 0.8678 0.0000 0.0000 0.5986 0.5558 1.0000

0.5986 0.5986 1.0000 0.3351 0.2537 0.4587 0.5558 0.4015

0.6162 0.4567 0.8580 0.2275 0.2426 0.5357 0.3608 0.7842

OPP 06

1050 950 900 700 650 550 300 260

0.3351 0.5986 0.5558 0.5986 0.0000 0.5558 0.5096 0.0000

0.4015 0.5986 0.5558 0.7116 0.2537 0.5558 0.5558 0.0000

0.5096 0.5558 0.5096 0.6760 0.0000 0.5986 0.6760 0.0000

0.4587 0.5096 0.4587 0.6760 0.0000 0.6384 0.5558 0.0000

0.4587 0.7116 0.5558 0.2537 0.0000 0.7116 0.4015 0.0000

0.4015 0.5558 0.5096 0.7454 0.0000 0.5986 0.5986 0.0000

0.5558 0.6384 0.4015 0.5558 0.4587 0.5558 0.2537 0.0000

0.4408 0.5926 0.5013 0.6162 0.1937 0.5986 0.5154 0.0000

OPP 09

950 880 750 650 550 450 350 300

1.0000 0.6384 0.0000 0.4015 0.5986 0.5558 0.4587 0.0000

1.0000 0.4587 0.3351 0.2537 0.4587 0.5096 0.5096 0.0000

0.0000 0.4587 0.0000 0.0000 0.5096 0.5096 0.2537 0.4587

0.0000 0.4587 0.5096 0.2537 0.4587 0.4015 0.0000 0.0000

0.0000 0.5096 0.0000 0.4015 0.5096 0.4587 0.0000 0.0000

0.2537 0.8678 0.0000 0.0000 0.4015 0.5986 0.4015 0.4587

0.0000 0.7116 0.0000 0.4587 0.4587 0.3351 0.4587 0.0000

0.5423 0.5986 0.2275 0.2956 0.4795 0.4795 0.3507 0.2426

P% HE Ne

75.0 0.369 1.58

87.5 0.415 1.71

70.8 0.323 1.48

70.8 0.326 1.48

62.5 0.289 1.41

70.8 0.321 1.47

79.2 0.380 1.61

95.8 0.420 1.72

Abbreviations are as indicated in Table 1. P% = percentage of polymorphic loci (95% criterion); HE = genetic diversity (Nei, 1973); Ne = effective number of alleles (Kimura and Crow, 1964).

polymorphic loci was observed in ITAM (P = 62.5%), while the highest percentage was observed in TRSA (P = 87.5%). The mean heterozygosity was 0.346 and varied between 0.289 (ITAM) and 0.415 (TRSA). Nei (1978) genetic distance between all populations was 0.087 and varied between 0.016 (PATS/CALB and SLIS/TRSA) and 0.136 (SLIS/JPES). The UPGMA dendrogram (Fig. 2), generated from the genetic distances in Table 3, showed two main clades. One of the

clades includes five populations (JPES, PATS, ITAM, CALB and CAMI), which are subdivided into two smaller clades, formed by the clustering of populations JPES/ITAM and PATS/CALB, with CAMI appearing isolated from the others. In the second clade, there is a clustering of populations TRSA and SLIS. The clustering pattern observed in Fig. 2 is consistent with clustering based on geographic distance between the samples locations (Fig. 3). The correlation between genetic and

Fig. 2. UPGMA dendrogram of Nei’s genetic distance (1978) between Lutzomyia longipalpis populations in NE Brazil. Abbreviations are as indicated in Table 1.

Fig. 3. UPGMA dendrogram of geographic distance (in km) of Lutzomyia longipalpis populations in NE Brazil. Abbreviations are as indicated in Table 1.

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Table 3 Genetic (gray) and geographical distances (white) between seven populations of Lutzomyia longipalpis from NE Brazil SLIS

TRSA

PATS

JPES

ITAM

CALB

CAMI

SLIS TRSA

–

329.50

924.10

1162.50

1198.10

908.20

1307.30

0.016

–

646.90

905.50

928.90

603.50

977.80

PATS JPES ITAM CALB CAMI

0.110 0.136 0.123 0.088 0.112

0.115 0.190 0.130 0.100 0.103

266.70 – 0.030 0.089 0.099

282.20 70.40 – 0.084 0.081

139.90 373.30 366.60 – 0.091

640.70 726.80 669.90 528.80 –

– 0.057 0.055 0.016 0.088

Abbreviations are as indicated in Table 1. Genetic distances were calculated according to Nei (1978).

geographic distances of Lutzomyia longipalpis populations could also be confirmed through a Mantel (1967) test, (r = 0.835) (Fig. 4). Patterns of genetic differentiation between populations of Lutzomyia longipalpis were also evaluated using measures of a fixation index FST (Wright, 1951), FST corrected for dominant markers (Lynch and Milligan, 1994), and θ ST (Weir and Cockerham, 1984). The most significant genetic differentiation was observed in loci OPP-09.950, OPP-09.300, and OPP-09.750 (Table 4). The average FST and FST corrected values were identical (0.162), and were slightly above θ ST (0.136). All three fixation indexes were positively correlated with each other. The correlation coefficient between FST and FST corrected was 0.990, while the values obtained between FST and θ ST , and between corrected FST and θ ST were 0.958 and 0.951, respectively. As a result of the positive correlation between the three indexes, and the fact that the θ ST index does not incorporate restrictions regarding the number of alleles per locus, sample size and the number of populations analyzed (Weir and Cockerham, 1984), the use of this estimator was chosen for the remainder of the analysis on the genetic structure of Lutzomyia longipalpis populations.

Fig. 4. Positive correlation between genetic and geographic distance between populations of Lutzomyia longipalpis from NE Brazil using a Mantel test with 1000 permutations (r = 0.835).

θ ST estimates vary between 0.0252 (TRSA/SLIS) and 0.1937 (ITAM/SLIS) (Table 5). The majority of comparisons (85.71%) confirmed the existence of moderate levels of genetic divergence (0.10 > θ ST < 0.20) between Lutzomyia longipalpis populations. The comparison of matrices containing the geographic distances (Table 3) and the θ ST estimates between paired populations (Table 5) also confirmed a positive correlation between them (r = 0.731; p = 0.0001). The effective number of migrants (Nm), resulting from the simultaneous analysis of all loci was 1.6. The comparison of gene flow levels between paired populations (Table 5) resulted in values between 1.04 (JPES/SLIS and ITAM/SLIS) and 9.67 (TRSN/SLIS). Based on these results, we suggest that the genetic differentiation between populations of Lutzomyia longipalpis are compatible with the principle of isolation through distance. 4. Discussion The use of molecular markers in studies of Lutzomyia longipalpis population biology has increased over recent years, and in general has contributed towards a greater understanding of the genetic variation and patterns of evolution in this species. Analyses of polymorphism in mitochondrial genes (Soto et al., 2001; Arrivillaga et al., 2003) and microsatellites (Maingon et al., 2003) have confirmed the existence of a Lutzomyia longipalpis species complex in the Neotropical Region, formed by an as yet unknown number of species, as proposed by Lanzaro et al. (1993). In addition, Lutzomyia pseudolongipalpis (Arrivillaga and Feliciangeli, 2001) was formally recognized as the first cryptic species of the complex by virtue of genetic techniques (Arrivillaga et al., 2003), and behavioral differences (Feliciangeli et al., 2004; Agrela et al., 2002) from Lutzomyia longipalpis sensu stricto. The taxonomic status of Lutzomyia longipalpis in Brazil has been a recurring subject in the literature (Soares and Turco,

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Table 4 Estimated fixation indexes for FST (Wright, 1951), corrected FST * (Lynch and Milligan, 1994), θ ST (Weir and Cockerham, 1984) and for the number of migrants (Nm) found in seven populations of Lutzomyia longipalpis from NE Brazil FST

Nm

FST *

Nm

θ ST

Nm

920 850 780 680 550 430 380 300

0.006 0.029 0.170 0.157 0.175 0.026 0.209 0.302

42.7 8.3 1.2 1.3 1.2 9.5 0.9 0.6

−0.043 −0.013 0.195 0.170 0.192 −0.011 0.241 0.313

−6.1 −19.0 1.0 1.2 1.1 −22.0 0.8 0.5

−0.019 0.009 0.172 0.158 0.178 0.005 0.216 0.319

−13.6 26.7 1.2 1.3 1.2 50.2 0.9 0.5

OPP-06

1050 950 900 700 650 550 300 260

0.021 0.016 0.013 0.110 0.306 0.012 0.075 0.000

11.7 15.3 19.4 2.0 0.6 20.1 3.1 0.0

−0.030 −0.025 −0.038 0.124 0.304 −0.032 0.065 0.000

−8.6 −10.4 −6.8 1.8 0.6 −8.2 3.6 0.0

−0.001 −0.007 −0.010 0.010 0.032 −0.011 0.063 0.000

−329.7 −38.6 −24.2 2.2 0.5 −23.0 3.7 0.0

OPP-09

950 880 750 650 550 450 350 300

0.886 0.094 0.353 0.159 0.014 0.031 0.193 0.366

0.0 2.4 0.5 1.3 17.3 7.9 1.0 0.4

0.854 0.103 0.354 0.178 −0.037 −0.009 0.221 0.360

0.0 2.2 0.5 1.2 −7.0 −28.5 0.9 0.4

0.898 0.085 0.374 0.160 −0.009 0.011 0.198 0.387

0.0 2.7 0.4 1.3 −29.2 22.5 1.0 0.4

0.162 0.196

1.3

0.162 0.207

1.3

0.136 0.209

1.6

Primers

Loci

OPP-04

Average S.D.

Standard deviation (S.D.) was calculated for all the fixation indexes.

2003), and the cause of some degree of contradiction. Data based on insect morphology, isozymes, and certain molecular markers (Arrivillaga et al., 2003; Hodgkinson et al., 2003; Azevedo et al., 2000; Mutebi et al., 1999; Mukhopadhyay et al., 1997) are compatible with a single species theory. However, this taxonomic uniformity has been questioned by a number of authors who consider the occurrence of at least three Lutzomyia longi-

palpis allopatric species (Watts et al., 2005; Hamilton et al., 2004; Bottecchia et al., 2004; Souza et al., 2004). Additionally, even more potentially cryptic species of Lutzomyia longipalpis have been suggested (Hamilton et al., 2005; Maingon et al., 2003; Bauzer et al., 2002). Dias et al. (1998) applied RAPD to study polymorphism in four laboratory-reared colonies of Lutzomyia longipalpis generated from populations from Lapinha

Table 5 Estimated fixation index θ ST (Weir and Cockerham, 1984) (gray), and the number of migrants (Nm) (white) from seven populations of Lutzomyia longipalpis from NE Brazil SLIS

TRSA

PATS

JPES

ITAM

CALB

CAMI

SLIS TRSA PATS JPES ITAM

– 9.67 1.26 1.04 1.04

0.0252 – 1.32 1.40 1.08

0.1653 0.1596 – 2.16 2.04

0.1933 0.1516 0.1038 – 3.72

0.1937 0.1879 0.1090 0.0629 –

0.1502 0.1297 0.1253 0.1382 0.1370

CALB CAMI

1.56 1.41

1.48 1.68

7.56 1.75

1.40 1.56

1.36 1.57

0.1384 0.1441 0.0320 0.1513 0.1556 – 1.53

Abbreviations are as indicated in Table 1.

0.1401 –

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(Brazil), Melgar (Colombia) and Liberia (Costa Rica). The RAPD profiles obtained by the authors did not differ significantly for the specimens used, despite an earlier suggestion that they probably constituted cryptic species (Lanzaro et al., 1993). The apparent inefficacy of RAPD in the identification of the genetic differences between these populations could be attributed, among other factors, to the limited number of loci included in the study. In this work we investigated the genetic structure of natural populations of Lutzomyia longipalpis in NE Brazil by means of RAPD markers. A total of 140 specimens from seven populations, representing a longitudinal transect of approximately 1300 km of this region were analyzed (Fig. 1). The levels of genetic variation detected in our study are compatible with those obtained through the analysis of RAPD loci of other insect species (Ram´ırez et al., 2005; Duncan et al., 2004; Ocampo and Wesson, 2004; Kim and Sappington, 2004). The expected mean heterozygosity found using RAPD loci is at least twice as high as values commonly obtained through the use of isozymic markers (M´arquez et al., 2001; Azevedo et al., 2000; Mutebi et al., 1999), and is similar to that of some microsatellites systems used in the study of natural populations of Lutzomyia longipalpis (Watts et al., 2002, 2005). Higher levels of polymorphism, combined with a reduced cost and ease of use, demonstrate the viability of RAPD markers as an alternative tool in studies of population genetics and the evolution of Lutzomyia longipalpis. The degree of genetic differentiation between seven geographic populations of this species was initially estimated based on Nei’s (1978) genetic distances. This method has been frequently used as the tool of choice in population biology, due to its statistical simplicity and for its suitability for analysis that involve a small sample size. Nei’s genetic distances between populations in this study were relatively low (Table 3), and are compatible with those expected among members of the same species (Thorpe and Sol´e-Cava, 1994). The analysis of geographic structural patterns using θ ST (Weir and Cockerham, 1984) also demonstrated moderate levels of genetic differentiation (0.10 < θ ST < 0.20), and are also compatible with those observed among members of the same species. The high level of correlation between matrices containing geographic distance and θ ST (Fig. 4) demonstrate that the differences observed between populations are compatible with the principle of isolation by distance. This hypothesis was evaluated through determining rates of gene flow (Nm) between populations based on considerations by Slatkin and Barton (1989) in the island model of migration (Wright, 1940, 1931). Genetic differentiation can be explained at least to some

extent by restricted gene flow under geographic isolation, and would not necessarily be associated with an incipient speciation process. Our results are compatible with the existence of a single species of Lutzomyia longipalpis in the NE Brazilian, as previously suggested for other populations (Azevedo et al., 2000; Mukhopadhyay et al., 1998; Arrivillaga et al., 2003). Simultaneous analysis of different classes of molecular markers may provide answers towards the real taxonomic situation of those populations of Lutzomyia longipalpis in NE Brazil. References Agrela, I., Sanchez, E., Gomez, B., Feliciangeli, M.D., 2002. Feeding behavior of Lutzomyia pseudolongipalpis (Diptera: Psychodidae), a putative vector of visceral leishmaniasis in Venezuela. J. Med. Entomol. 39, 440–445. Apostol, B.L., Black IV, W.C., Reiter, P., Miller, B.R., 1996. Population genetics with RAPD-PCR markers: the breeding structure of Aedes aegypti in Puerto Rico. Heredity 76, 325–334. Arrivillaga, J.C., Feliciangeli, M.D., 2001. Lutzomyia pseudolongipalpis: the first new species within the longipalpis (Diptera: Psychodidae: Phlebotominae) complex from La Rinconada, Curarigua, Lara State, Venezuela. J. Med. Entomol. 38, 783–790. Arrivillaga, J., Mutebi, J.-P., Pin˜ango, H., Norris, D., Alexander, B., Feliciangeli, M.D., Lanzaro, G.C., 2003. The taxonomic status of genetically divergent populations of Lutzomyia longipalpis (Diptera: Psychodidae) based on the distribution of mitochondrial and isozyme variation. J. Med. Entomol. 40, 615–627. Arrivillaga, J.C., Norris, D.E., Feliciangeli, M.D., Lanzaro, G.C., 2002. Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences. Infect. Genet. Evol. 2, 83–95. Ashford, R.W., 2000. The leishmaniases as emerging and re-emerging zoonoses. Int. J. Parasitol. 30, 1269–1281. Azevedo, A.C.R., Monteiro, F.A., Cabello, P.H., Souza, N.A., RosaFreitas, M.G., Rangel, E.F., 2000. Studies on populations of Lutzomyia longipalpis (Lutz and Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Brazil. Mem. Inst. Oswaldo Cruz 95, 305–322. Bauzer, L.G.S.R., Souza, N., Ward, R.D., Kyriacou, C., Peixoto, A.A., 2002. The period gene and genetic differentiation between three Brazilian populations of L. longipalpis. Insect. Mol. Biol. 11, 315–323. Black IV, W.C., 1995. FORTRAN Programs for the Analysis of RAPD-PCR Markers in Populations. Colorado State University, Ft. Collins, CO 80523. Black IV, W.C., 1993. PCR with arbitrary primers: approach with care. Insect. Mol. Biol. 2, 1–6. Bottecchia, M., Oliveira, S.G., Bauzer, L.G., Souza, N.A., Ward, R.D., Garner, K.J., Kyriacou, C.P., Peixoto, A.A., 2004. Genetic divergence in the cacophony IVS6 intron among five Brazilian populations of Lutzomyia longipalpis. J. Mol. Evol. 58, 754–761. de Souza, C.M., Fortes-Dias, C.L., Linardi, P.M., Dias, E.S., 2004. Estudos fen´eticos de variabilidade de polimorfismos de DNA amplificados ao acaso pela reac¸a˜ o em cadeia da polimerase em quatro populac¸o˜ es geogr´aficas de Lutzomyia whitmani (Diptera: Psychodidade) no Brasil. Rev. Soc. Bras. Med. Trop. 37, 148– 153.

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