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    Sampling sites of Lutzomyia longipalpis populations comprising a transect across eastern Brazil.

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    Distribution of haplotypes among Lutzomyia longipalpis populations and identification of segregating sites within a 261-basepair fragment of cytochrome b. JU = Juazeiro; Na = Natal; Pa = Pancas; Mo = Monte Santo; It = Itamaraca; Fe = Feira de Santana. GenBank accession numbers Ll-22 to Ll-27, AF480170-AF480175; Ll-29 to Ll-33, AF480177-AF480181. Accession numbers for Ll-1 to Ll-21 were reported previously.30

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    Minimum spanning network of haplotypes from Lutzomyia longipalpis populations. Each population is represented by a distinct color. Each node (pie diagram) corresponds to a unique haplotype indicated in Figure 2. The nodes are proportional in size to the haplotype frequency. Each cross bar represents one nucleotide substitution between two observed haplotypes. Narrow, curved lines represent alternative branching between haplotypes. The overlap among Itamaraca, Juazeiro, and Natal is particularly evident in the left hand side of the figure, whereas the differentiated Monte Santo (red), Pancas (gold), and Feira de Santana (blue) extend in tight groups from the most common haplotype L1–2.

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MITOCHONDRIAL CYTOCHROME B VARIATION IN POPULATIONS OF THE VISCERAL LEISHMANIASIS VECTOR LUTZOMYIA LONGIPALPIS ACROSS EASTERN BRAZIL

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  • 1 Biology Department, Fairfield University, Fairfield, Connecticut; Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut; United States Army Center for Health Promotion and Preventative Medicine, Fort Lewis, Washington; Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil

A population analysis of peridomestic, light-trapped, field specimens of the phlebotomine sand fly Lutzomyia longipalpis was targeted to six locations representing a geographic transect across eastern Brazil. Mitochondrial cytochrome b gene sequences established the pattern of genetic variation among the populations. Alignment of a 261-basepair region at the 3′end of cytochrome b identified 30 haplotypes and 21 segregating sites from 78 sand flies. Pairwise comparisons indicated statistically significant population structuring between northern and southern populations, as well as structuring among the southern populations. Prominent spatial clustering was evident for two of the populations in a minimum spanning network of the haplotypes, but sequence divergence was not sufficient to indicate cryptic species.

INTRODUCTION

Lutzomyia longipalpis (Lutz and Neiva) is the primary vector for Leishmania chagasi, the causative agent of New World visceral leishmaniasis and occurs in Mexico and Central and South America.1 In Central and South America, 16,000 new cases of visceral leishmaniasis were reported per year.2 In Brazil, incidences of visceral leishmaniasis have been increasing steadily since 1989. In 1993–1994, 3,000 new cases were identified in the two northeastern states of Piauí and Maranhão,3 and new foci continue to be reported across eastern Brazil.4 Despite continuous documentation of the increasing disease incidence and distribution, corresponding data on the vector with respect to population structure and distribution is relatively sparse.

Population genetic studies of geographically dispersed phlebotomine concentrations are critical both for delineating the genetically distinct groups and for the eventual design of effective vector control programs. Basing population genetic analyses on field-collected specimens eliminates the inherent problems associated with population studies based on colony specimens.5–7 Secondino and others8 further emphasized this, demonstrating that colony flies are more susceptible to infection by leishmanias than are field specimens. In the past decade, isoenzymes provided the primary insights into population genetic structure and biochemical taxonomy of phleboto-mine sand flies. For the species L. longipalpis, an initial analysis of three laboratory colonies suggested it to be a complex of at least three species in Brazil, Colombia, and Costa Rica.5,9 The extension of a morphologic study of larval characteristics and isoenzymes10 to 26 field populations and five laboratory strains documented characteristics that are distinctive to populations from Brazil, Central America, and Colombia.11 Isoenzymes identified two sympatric species in Venezuela.12 Questions have emerged with respect to the conspecific status of Brazilian populations. Within Brazil, abdominal tergal spot patterns (one-spot, two-spot, and intermediate), first identified by Mangabeira,13 were the basis for the designation of species complexes,14 but population genetic studies where two forms co-occur did not support this conclusion.15,16 In addition, isoenzyme analyses of field populations have indicated a single species status in Brazil.16–18 Nonetheless, the genetic divergence of outlying populations suggested a degree of allopatric population differentiation.18 Cytogenetic studies identified two distinct karyotypic forms in Brazil.19 Recently, molecular sequence data of the mitochondrial ND4 gene20 and the song genes, cacophony and period21–23 also question the conspecific nature of the Brazilian populations. Conversely, sequences of the mitochondrial cytochrome c oxidase I gene are consistent with a single species status, with high genetic differentiation among local Brazilian populations.24

Mitochondrial DNA (mtDNA) has been widely used in population genetic and evolutionary studies of vector species.20,25,26 Although mitochondrial genes are linked, sequence evolution varies between as well as within genes.27,28 For population genetic studies, a suitable marker is one that is sufficiently variable to detect differences between individuals of the same population. The 3′-end of the mitochondrial cytochrome b (cyt b) gene has proven suitably variable in the sand fly species L. whitmani29 and L. longipalpis.30

In the present study, mtDNA variation of the 3′-end of the cyt b gene in L. longipalpis was characterized in a geographic transect of field populations across eastern Brazil. Population structure was assessed by evaluating the pattern of genetic variation in DNA sequences within this selected geographic range.

MATERIALS AND METHODS

Specimen collection.

Field specimens of L. longipalpis were collected in November and December 1998 from six peridomestic populations using Centers for Disease Control light traps (Table 1). The sites were distributed along a 1,600-km transect through eastern Brazil within an elevation range of 58–567 meters (Figure 1). Sites were selected to sample zones with demonstrated differences in isozyme frequency18 and with a range of altitude and rainfall (Table 1). Specimens were frozen immediately in liquid nitrogen and later transferred to 80% ethanol and stored at −20°C.

Isolation of DNA.

Prior to isolation of DNA, male genitalia were removed for species verification and deposited as voucher specimens in the Division of Entomology at the Yale Peabody Museum (New Haven, CT). The DNA was isolated from individual specimens following extraction in lysis buffer (0.5% sodium dodecyl sulfate, 0.1 M NaCl, 0.2 M sucrose, 0.1 M Tris, pH 9.2, and 0.05 M EDTA) and incubation at 65°C for 30 minutes. Protein was precipitated by addition of 8 M potassium acetate (to a final concentration of 1 M) to the warm homogenate, followed by overnight incubation on wet ice. Protein was removed the next day by centrifugation at 22°C. Genomic DNA was precipitated from the supernatant and washed three times in ethanol. The pellet of 80–240 ng was dried and then resuspended in 20 μL of buffer (10 mM Tris, 1 mM EDTA, pH 7.6) for 10 minutes at 60°C (entire method from Bender and others,31 modified by Romans P, unpublished data). DNA extracts were stored at −20°C.

Polymerase chain reaction (PCR) amplification and sequencing.

Double-stranded amplifications of the 3′-end of the cyt b gene were accomplished using the following conditions: an initial denaturation at 98°C for five minutes; followed by 10 cycles at 95°C for one minute, 38°C for one minute, and 72°C for 1.5 minutes; 35 cycles at 95°C for one minute, 40°C for one minute, and 72°C for 1.5 minutes; and a final extension at 72°C for three minutes. The PCR was performed in a 50-μL reaction volume containing 10 mM Tris, 50 mM KCl, 3.2 mM MgCl2, 0.1% Triton X-100, pH 8.8, 10–20 ng of total genomic DNA, 200 μM of each dNTP, 0.15 μM of each primer; and 0.4 units of Taq DNA polymerase (Roche Molecular Biochemicals [formerly Boehringer-Mannheim], Indianapolis, IN). Primers 11226F (5′-GAATGATATTTTT TATTTGC-3′) and 11587R (5′-CTTATGTTTTCAAGA CATATGC-3′) were used as forward and reverse primers, respectively.30 The primer number corresponds to the nucleotide position of the 5′-end of the primer based on the Anopheles gambiae Giles mitochondrial genome.32 A negative control containing all reactants but no DNA was always included. The PCR products were stored at −20°C following purification with a kit according to supplier’s specifications (QIAquickTM PCR Purification Kit; Qiagen, Valencia, CA). The same PCR primers were used for automated sequencing (Howard Hughes Medical Institute, Biopolymer/W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, CT).

Data analysis.

DNA sequences were aligned, first with the program CAP sequence assembly (software by Huang X., available at http://gcg.tigem.it/) and then manually with the program SeqApp.33 Sequences were translated into their amino acid counterparts according to the Drosophila mtDNA genetic code using the program MacClade 3.08a.34 The software POPSTR (supplied by H. Siegismund, University of Copenhagen, Copenhagen, Denmark) was used for the following analyses: 1) to determine the number of haplotypes, segregating sites, and number of substitutions between haplotypes; 2) to estimate the nucleotide diversity (Nei,35 equation 10.5) as a measure of population variability; 3) to calculate the number of net nucleotide substitutions (genetic distance) between populations (Nei,35 equation 10.21); and 4) to estimate the extent of population differentiation (population subdivision) using the test statistic, K*ST.36 The sequence statistic, K*ST proved more appropriate than FST since it accommodated for variation in sample size among populations. The statistical significance of the observed K*ST values was estimated by 1,000 Monte Carlo permutations, where individuals were sampled without replacement and randomly assigned to populations.

Phylogenetic relationships between haplotypes were illustrated by the software Arlequin.37 The program constructed the most probable minimum spanning network, using Kimura two-parameter distance estimates38 as well as alternative minimum spanning trees. A minimum spanning network portrayed the evolution of sequences or haplotypes as nodes in the network rather than terminal tips of a tree. Networks can only be used in intraspecific relationships because they portray the relationships among sequences for populations in which many sequences may be derived from the same ancestral genotype. Spanning trees also assume that the direct common ancestor of all observed haplotypes is present in the sample.39 The tree was drawn manually with the size of the nodes scaled to represent the haplotype frequency.

RESULTS

A 261-basepair region of the 3′-end of the mtDNA cyt b gene was aligned for 78 specimens; 30 haplotypes were identified with 21 segregating sites (Figure 2). Sequences of the haplotypes were deposited in GenBank (see Figure 2 for accession numbers). No more than two different nucleotides were identified at each segregating site, indicating minimal homoplasy. No ambiguities or insertions/deletions were found during sequence alignment. No stop codons were found within the amino acid alignment. Among the haplotypes, transitions outnumbered transversions 21:1. The most frequent haplotype (Ll-2) was identified in all populations except the two with the smallest sample size, Itamaraca (It) and Feira de Santana (Fe) (Table 1 and Figure 2). Unique and shared haplotypes were present in each population.

The nucleotide diversity within the total sample was relatively high (1.1%). The genetic diversity varied within populations, with the lowest in Monte Santo (Mo, 0.5%) and the highest in Juazeiro (Ju, 1.5%). The genetic distance was lowest between Juazeiro and Natal (−0.02%) and highest between Itamaraca or Monte Santo and Pancas (0.43% and 0.42%, respectively). Additional details of the latter two parameters are shown in Table 2.

Since some haplotypes occurred in relatively high frequencies (total haplotype diversity HT = 0.942, average within population diversity HS = 0.858), the haplotype statistic was of limited value for population analysis. Analysis of population structure was based on the sequence statistic, K*ST 34. Initial analysis revealed homogeneity between Juazeiro and Natal (K*ST = −0.001, P ≤ 0.466; Table 2). The two populations were subsequently combined and found to be homogeneous with Itamaraca (K*ST = 0.010, P ≤ 0.148). These populations were pooled in homogeneity tests and will be referenced subsequently as northern populations. Significant structuring was indicated between the northern populations and the remaining populations south of 10°S latitude (Mo, Fe, Pa) (K*ST = 0.023–0.077, P ≤ 0.023) as well as among the southern populations (Mo, Fe, Pa) (K*ST = 0.064–0.095, P ≤ 0.020; Table 2). The sequence divergence between the northern and southern populations was 0.18–0.27%.

The relationships among haplotypes/populations and the frequency of each haplotype are portrayed simultaneously in the minimum spanning network of 30 haplotypes (Figure 3). Alternative minimum spanning trees, indicated by the thinner lines, suggest alternate but equivalent relationships between haplotypes. Theoretical and empirical expectations indicate that mtDNA genotypes sharing a single recent ancestor show a strong tendency for the ancestral (often frequent) haplotype to be immediately interior and followed by more distantly related haplotypes in decreasing frequency.40 The most widely distributed and frequent haplotype (Ll-2) forms an internal node with several derivatives. A tendency towards structuring is evident from the prominent spatial clustering of the Pancas and Monte Santo populations in contrast to the more distant association of the Feira de Santana population. The network does not coincide with the geographic location of the populations as revealed by the scattered distribution of haplotypes.

Male specimens from five of the six populations were evaluated for abdominal tergal spot morphology. All male specimens from Monte Santo possessed two tergal spots. In each of the remaining populations a mix of one-spot and two-spot morphotypes were identified. The two morphotypes were both associated with the same haplotype (Ll-15) in Natal and the same haplotype in different geographic locations (Ll-1, Ll-9). However, when the two morphotypes were combined across the five sampling areas and analyzed as two separate populations, a pairwise comparison using K*ST indicated them to be significantly different (P ≤ 0.05) with respect to mitochondrial genotype.

DISCUSSION

Analysis of the mtDNA cyt b gene in L. longipalpis appeared to identify a geographic transition at 10°S latitude that separated the populations in eastern Brazil. Populations north of 10°S latitude (Na, It, and Ju) were sufficiently homogeneous to indicate a high probability of gene flow among them. Although several studies have focused on L. longipalpis populations in this region,16–18 the zone of transition has not been well defined. The phylogeographic pattern of the sampled populations cannot be attributed simply to isolation by distance, since Monte Santo and Juazeiro, 168 km apart, show significant structuring, whereas Natal, Itamaraca and Juazeiro, separated by 650–700 km, each indicate no structuring. Our findings are consistent with other gene sequence reports21,23 that differentiate the Natal population from sites south of 10°S latitude, Jacobina (approximately 160 km from Monte Santo) and Lapinha (approximately 320 km from Pancas). Genetic and morphologic differentiation among sand flies has been associated with adaptations to climate, latitude or altitude.17,41,42 In the present transect study, differentiation between Monte Santo and Juazeiro was associated with latitude, but not climatic conditions (both zones are arid) or elevation (512 and 566 meters, respectively). In spite of a northern versus southern haplotype clustering, the relatively small sequence divergence (≤ 0.27%) between these clades points to within-species, geographic differentiation.

The genetic structuring of Pancas and Monte Santo paralleled data indicating genetic differentiation reported by others10,18,19,23,24 for Lapinha and Jacobina. Pairwise comparisons of Pancas, Monte Santo, and Feira de Santana supported differentiation of these populations from each other as well as between each of these three and the northern populations. This indicated limited gene flow between populations. However, the degree of discontinuity between populations has varied through evolutionary time, as revealed in the minimum spanning network (Figure 3). In Pancas, the tendency towards a close clustering of haplotypes in the network provided evidence of a past fragmentation event.43–45 The current distribution of L. longipalpis is widespread and the populations closely juxtaposed.1 However, in the present study potential transitional populations north of Pancas were not represented. A possible alternative explanation of the Pancas haplotype structure (as seen in Figure 3) may be attributed simply to isolation by distance.

Unlike Pancas, the genetically differentiated populations, Monte Santo and Feira de Santana, were separated by more than one mutation within the same population (Figure 3). The scattering of haplotypes in a structured population, as observed in Feira de Santana, indicates recurrent genetic contact with all northern populations.43,44 In contrast, and under the assumption that the intermediate haplotypes along the branch (2–4 mutations from the ancestral haplotype) are extinct, the long branch lengths within the Monte Santo population support a past fragmentation event.43–45 Generally, the coexistence of large genetic differences within a single locality is evidence of either unidentified sympatric sibling species or previously allopatric populations that have undergone secondary contact.45 To substantiate this level of within population differentiation based on mtDNA variation, larger sample sizes will be required.

The present day geographic distribution of fauna and flora has been influenced strongly by dramatic effects of paleoclimatic changes.46,47 During the late Pleistocene, the Atlantic type forest covered most of central Bahia, leaving arid regions of only limited extension.48 The beginning of desertification characterized the early Holocene and resulted in the predominant caatinga (thorn scrub and cactus) of today. Whitmore and Prance47 contended that vegetation expanded during humid periods and survived in refugia during dry periods. During these climatic cycles, the phlebotomine populations of Monte Santo and Feira de Santana may have survived in allopatric refugia. Monte Santo is located at the edge of one of the refuges identified in eastern Bahia.49 Because the majority of Monte Santo haplotypes are present in the ancestral node (Figures 2 and 3), Monte Santo may represent a relict population. Although the refuge theory has been challenged by more recent observations,50 it continues to be strongly defended by the original proponents.51

High genetic variation within a geographic locale is a phenomenon commonly found in populations that have not undergone drastic size reductions over evolutionary time. Where the population size has been large and expanding, the effects of genetic drift are reduced and the retention of ancestral polymorphisms is promoted.52 Because the majority of the Monte Santo sand flies possess the ancestral haplotype (Figure 3), the refugium hypothesis seems applicable.

The arid climatic condition common to Monte Santo, Feira de Santana, and Juazeiro does not define the haplotype distributions, however. Whereas Juazeiro haplotypes are consistent with those of the northern populations, Monte Santo and Feira de Santana are distinctive. A plausible explanation is that variation in the onset age of aridity (associated with latitude) may not have been synchronous between sites.53 Studies on additional sand fly or other insect species with similar habitat requirements, behavior, and congruent distribution may provide further evidence of the effect of cyclical changes in the arid Bahia region.

The relationship between tergal spot morphology and genetic differentiation is not clear. In the current study, when the male morphotype data were combined from five locations, a marginally significant K*ST value was obtained, indicating a degree of mitochondrial association with the spot morphology. In Sobral, researchers have found additional diagnostic nucleotide characters in the period gene that are associated with the tergal spot morphology. However, a direct relationship between spot morphology and differentiated populations has not been consistently supported by other studies in Jacobina, Lapinha, or Natal sand fly populations.22

This current report represents the first use of mtDNA cyt b gene sequence analysis in a geographic transect of populations of L. longipalpis from eastern Brazil. Furthermore, sand fly populations spanning the Juazeiro, Monte Santo, and Feira de Santana zone have not been included in previous analyses of genetic variation, although this region has been identified as significant foci of leishmaniasis transmission.4 Therefore, the analysis of cyt b gene variation in transect across this region has not only provided insights into the present day population structure but also has elicited inferences concerning its population history.

Table 1

Geographic characteristics of transect sites in eastern Brazil sampled for the phlebotomine sand fly Lutzomyia longipalpis

Locality, StateLocationAltitude (meters)Rainfall (mm)54
Natal (Na), Rio Grande do Norte5.77°S, 35.30°W581,500–2,000
Itamaraca (It), Pernambuco7.75°S, 34.83°W911,500–2,000
Juazeiro (Ju), Bahia9.47°S, 40.49°W567<500
Monte Santo (Mo), Bahia10.44°S, 39.34°W512<500
Feira de Santana (Fe), Bahia12.20°S, 39.02°W226500–1,500
Pancas (Pa), Espirito Santo19.20°S, 40.83°W1431,500–2,000
Table 2

Genetic relationships among Lutzomyia longipalpis populations, based on 261 basepairs of the mitochondrial cytochrome b gene*

SitesJuazeiroNatalItamaracaPancasFeira de SantanaMonte Santo
* Values in the upper right half of the table, above the bold diagonal, are net interpopulational distance (%); the bold diagonal values are Nei’s nucleotide diversity; values in the lower left half of the table, below the bold diagonal, are significance levels of pair wise tests for population differentiation (K*ST).
Juazeiro0.015−0.018−0.0050.2870.1850.284
Natal0.4660.0110.0960.1980.1170.192
Itamaraca0.4390.0640.0090.4270.3580.422
Pancas<0.001<0.001<0.0010.0060.1920.057
Feira de Santana0.0400.0650.0040.0040.0100.075
Monte Santo0.001<0.001<0.0010.0200.0110.005
Figure 1.
Figure 1.

Sampling sites of Lutzomyia longipalpis populations comprising a transect across eastern Brazil.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.386

Figure 2.
Figure 2.

Distribution of haplotypes among Lutzomyia longipalpis populations and identification of segregating sites within a 261-basepair fragment of cytochrome b. JU = Juazeiro; Na = Natal; Pa = Pancas; Mo = Monte Santo; It = Itamaraca; Fe = Feira de Santana. GenBank accession numbers Ll-22 to Ll-27, AF480170-AF480175; Ll-29 to Ll-33, AF480177-AF480181. Accession numbers for Ll-1 to Ll-21 were reported previously.30

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.386

Figure 3.
Figure 3.

Minimum spanning network of haplotypes from Lutzomyia longipalpis populations. Each population is represented by a distinct color. Each node (pie diagram) corresponds to a unique haplotype indicated in Figure 2. The nodes are proportional in size to the haplotype frequency. Each cross bar represents one nucleotide substitution between two observed haplotypes. Narrow, curved lines represent alternative branching between haplotypes. The overlap among Itamaraca, Juazeiro, and Natal is particularly evident in the left hand side of the figure, whereas the differentiated Monte Santo (red), Pancas (gold), and Feira de Santana (blue) extend in tight groups from the most common haplotype L1–2.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.386

Authors’ addresses: Virginia H. Hodgkinson, Biology Department, Fairfield University, Fairfield, CT 06824, Telephone: 203-254-4000, extension 2744, Fax: 203-254-4253, E-mail: vhodgkinson@mail.fairfield.edu. Josephine Birungi, Uganda Virus Research Institute, HIV Vaccine Program, PO Box 49, Entebbe, Uganda, Telephone: 256-41-321-091, Fax: 256-41-321-124, E-mail: Jbirungi@iavi.org. Miguel Quintana, United States Army Center for Health Promotion and Preventative Medicine, Fort Lewis, WA, Telephone: 253-966-3771, Fax: 253-966-0163, E-mail: Michael.Quintana@nw.amedd.army.mil. Reynaldo Dietze, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil, Fax: 55-27-335-7206, E-mail: rdietze@ndi.ufes.br. Leonard E. Munstermann, Department of Epidemiology and Public Health, Yale University School of Medicine, PO Box 208034, 60 College Street, 706 LEPH, New Haven, CT 06520-8034, Telephone: 203-785-5533, Fax: 203-787-4782, E-mail: leonard.munstermann@yale.edu.

Acknowledgments: Technical assistance was provided by Michelle Haghpanah and Swati Joshi. We give special thanks to the field team provided by the Núcleo de Doenças Infecciosas/Universidade Federal do Espírito Santo, Brazil, and to Professor William C. Black IV for the DNA extraction protocols.

Financial support: This work was supported by grants AI-34521, AI-44793, and AI-56254 from the National Institutes of Health.

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Author Notes

Reprint requests: Leonard E. Munstermann, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, New Haven, CT 06520-8034.
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