Am. J. Trop. Med. Hyg., 61(6), 1999, pp. 1004–1009
Copyright q 1999 by The American Society of Tropical Medicine and Hygiene
OCCURRENCE OF SIBLING SPECIES OF LUTZOMYIA LONGIPALPIS
(DIPTERA: PSYCHODIDAE) IN VENEZUELA: FIRST EVIDENCE FROM
REPRODUCTIVELY ISOLATED SYMPATRIC POPULATIONS
MARGARITA LAMPO, DARA TORGERSON, LUIS M. MÁRQUEZ, MILAGRO RINALDI, CARMEN Z. GARCÍA, AND
ALBERTO ARAB
Centro de Ecologı́a, Instituto Venezolano de Investigaciones Cientı́ficas, Caracas, Venezuela; Department of Zoology,
University of Guelph, Guelph, Ontario, Canada; Departamento de Biologia, Universidade Estadual Paulista,
Rio Claro, Sao Paulo, Brazil
Abstract. The delimitation of cryptic species within the main vector of the American visceral leishmaniasis,
Lutzomyia longipalpis, remains a topic of controversy. An analysis of genetic variability based on 8 enzymatic loci
revealed fixed differences in 2 diagnostic loci, adenylate kinase (Ak) and hexokinase (Hk), between sympatric and
allopatric populations at 4 localities in Venezuela. The absence of heterozygotes for these 2 loci within 1 locality
indicates, for the first time, the presence of 2 sympatric reproductively isolated populations or cryptic species within
L. longipalpis. Significant differences were also detected between these cryptic species in the allele frequencies of
glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase, decarboxylating (Me). One species showed mean
heterozygosities that ranged between 6.6% and 6.7%, with 1.6–1.9 alleles detected per locus, while the other had
mean heterozygosities that ranged from 4.3% to 6.3%, with 1.3–1.6 alleles per locus. Comparisons of isozyme profiles
with published data suggests that 1 species is similar to the L. longipalpis described in Colombian and Brazilian
populations, whereas the other has not been previously reported.
Many investigators have suggested that Lutzomyia longipalpis (Lutz and Neiva), the main vector of visceral leishmaniasis in America, is composed of a complex of cryptic
species. The evidence published has been nevertheless controversial. Two forms of L. longipalpis were identified from
Brazilian colonies that differed in the presence of dorsolateral pale spots in the third and fourth abdominal tergites.1
Crossing experiments showed a significant reduction in fertility between sympatric and allopatric strains of both forms,
but a few fertile hybrids from both sexes were also produced.1 Because reduced fertility was also recorded between
similar individuals from different geographic regions, it was
concluded that further field studies should be carried out
before these 2 morphologic forms were assigned to specific
taxa. Further studies on the distribution of these 2 forms
suggested that 1 is found from Mexico to southern Brazil
while the other is restricted from northern Brazil to the border with Paraguay.2
The structure of L. longipalpis as a complex of distinct
reproductively isolated populations became more complex as
new data on isozymes became available. High genetic divergence, decreased fertility, and the presence of several diagnostic loci between laboratory strains from Costa Rica,
Colombia, and Brazil suggested the presence of at least 3
sibling species.3 However, the adequacy of laboratory colonies to study genetic divergence across geographic areas remains debatable since colonies can be subjected to a significant reduction of genetic variability due to the loss of infrequent alleles and decreased heterozygosity.4,5 In fact, the
detection from field samples of several loci intermediate between diagnostic loci from Colombian and Brazilian colonies suggested that a high polymorphism could also explain
the high genetic variability within L. longipalpis throughout
Colombia, Brazil, and Costa Rica.5 Also, recent studies on
the genetic basis for the presence of spots found no evidence
that indicates genetic differentiation at the species level between the 1- and 2-spot phenotypes in Brazilian6 or Bolivian7
populations.
Concise boundaries between cryptic species may be
drawn by identifying field populations composed of sympatric diagnostic phenotypes which show no evidence of
gene exchange. Here, we present isozymatic evidence for the
existence of 2 cryptic species of L. longipalpis within Venezuela, and provide diagnostic loci that allow for the unequivocal separation of both of these cryptic species.
MATERIALS AND METHODS
Study sites. Specimen collections were carried out at 4
localities throughout Venezuela (Figure 1). La Rinconada
(98599N, 698559W; elevation 5 627 meters) is a rural settlement surrounded by xerophytic vegetation, located at the agricultural valley of Curarigua (Lara State). The mean monthly temperature for this area is 25.48C and the mean annual
rainfall 636.6 mm. El Paso (108049N, 698569W; elevation 5
600 meters) is a rural settlement 8 km north of La Rinconada. The climatic characteristics for this locality are similar
to those of La Rinconada. Altagracia (98529N, 668239W; elevation 5 440 meters) is also a small settlement adjacent to
Altagracia de Orituco (Guárico State). Agricultural fields and
patches of deciduous and semideciduous shrub forests surround this locality. Temperature and rainfall records are not
available for this locality. Mapire (78449310N, 648429300W;
elevation 5 54 meters) is a small town located at the northern shore of the Orinoco River (Anzoátegui State). It is surrounded by a seasonally flooded riparian vegetation with a
mean monthly temperature of 26.38C and a mean annual
rainfall of 1,396 mm. Samples at this location were collected
along the shore at the other side of the river.
La Rinconada and El Paso were sampled between September 1997 and January 1998. Additional samples were also
collected from El Paso during September 1998. Altagracia
was sampled during April 1998 and Mapire was sampled
during July 1998. At Mapire, phlebotomine sand flies were
collected using Centers for Disease Control (CDC) light
traps in forest stands and at the mouth of rock crevices. At
1004
1005
L. LONGIPALPIS SPECIES COMPLEX
FIGURE 1. Geographic origin of field collections of Lutzomyia
longipalpis from Venezuela.
all other sites, the CDC light traps were placed inside and
outside houses and within chicken and goat sheds between
6:00 PM and 6:00 AM.
Live adults were cold-anesthetized by keeping them at
approximately 228C for about 10 min. The distal abdominal
tergites and heads were separated and stored in 70% ethanol,
while the rest of the bodies, mainly thorax and the proximal
tergites, were stored in liquid nitrogen. The distal abdominal
tergites and heads of all specimens were cleared and mounted in Berlese fluid. Species identification was based on the
morphology of the spermathecae and the cibaria for females,
and the presence of dorsal curved setae inserted directly on
the paramere for males.8 The presence of tergal spots was
checked only on a subsample from El Paso.
Electrophoretic methods. A total of 148 specimens from
the 4 localities were processed. On each electrophoresis gel,
1 Aedes aegypti (Rockefeller strain) was included as a standard reference.
Standard, vertical, polyacrylamide gel electrophoresis protocols were used.9 Each sample was homogenized in 20 ml
of grinding solution (10% sucrose, 0.1% Triton X-100, 0.02
M Tris-citrate buffer, bromophenol blue), centrifuged for 3
min at 12,000 rpm, and the supernatant was distributed
among 8 gels. Tris-citrate and Tris-borate buffer systems
were used to maximize electrophoretic enzyme separation.
Assays revealed phenotypes for the following enzymes: glucose-6-phosphate isomerase (Gpi, E.C.5.3.1.9), hexokinase
(Hk, E.C.2.7.1.1), and malate dehydrogenase, decarboxylating (Me, E.C.1.1.1.40) for Tris-borate buffer gels; and malate
dehydrogenase (Mdh, E.C.1.1.1.37), isocitrate dehydrogenase (Idh, E.C.1.1.1.42), glycerol-3-phosphate dehydrogenase (Gpd, E.C.1.1.1.8), adenylate kinase (Ak, E.C.2.7.4.3),
and arginine kinase (Ark, E.C.2.7.3.3) for Tris-citrate buffer
gels.
The banding phenotypes of these 8 loci were examined.
Numerical values for the allelic phenotypes (electromorphs)
were based on the relative migration of bands from the gel
origin. For each locus, the Aedes electromorph was designated as the 1.00 electromorph. Following convention, loci
were denoted as polymorphic if the most common allele had
a frequency less than 95% in at least 1 population.
Analyses methods. Electromorphs were separated according to fixed allelic differences found in sympatric populations of L. longipalpis. As a measure genetic variability, the
percentage of polymorphic loci and mean heterozygosity
were determined for allopatric and sympatric populations
and for each electromorph.10 Genotypic frequencies for all
loci were compared with Hardy-Weinberg equilibrium frequencies. Also, allele frequencies among populations for
each electromorph were compared by means of contingency
analyses.10 Means and 95% confidence intervals over all loci
for FIS and FST were calculated using a jacknife method11 to
assess the degree of inbreeding within and the degree of
differentiation between populations. Finally, genetic distances of Nei12 and Roger13 were calculated and a Wagner dendrogram based on the genetic distances of Roger was constructed to determine the genetic relationship between allopatric and sympatric populations, and between electromorphs.10
RESULTS
Marked genetic differences were found between the population at La Rinconada and those from Altagracia and Mapire. Two (25%) of the 8 loci examined showed fixed allelic
differences. Alleles Ak1.08 and Hk0.77, fixed at La Rinconada,
were absent from Altagracia or Mapire (Figure 2). Conversely, Ak1.21 and Hk0.69, found in very high frequencies at Altagracia and Mapire, were absent from La Rinconada (Table
1). These 2 diagnostic loci allowed unambiguous separation
of samples from La Rinconada from those collected at Altagracia or Mapire. Differences in the frequencies of alternative alleles were also detected for Gpi and Me. For Gpi,
the most frequent allele at La Rinconada, Gpi0.38, was absent
from Altagracia and Mapire. In contrast, Gpi0.48, present at
high frequencies at Altagracia and Mapire, was very infrequent at La Rinconada. Similarly, Me0.93, the most frequent
allele at La Rinconada, appeared at very low frequencies at
Altagracia and Mapire, whereas the alternative allele, Me1.00,
which was absent from La Rinconada, was the most frequent
allele at Altagracia and Mapire (Table 1).
Except for a rare allele detected at Altagracia (Hk0.61), all
of the alleles reported at Altagracia, Mapire, and La Rinconada for these 2 diagnostic loci were present at El Paso
(Table 1). However, no heterozygotes between Hk0.69 and
Hk0.77 or between Ak1.08 and Ak1.21 were detected in a sample
of 103 and 98 individuals, respectively (Table 1).
Samples were assigned to distinct electromorphs according to the presence of Ak1.08 and Hk0.77 (Ak 1.08/Hk0.77) or Ak1.21
and Hk0.69 (Ak1.21/Hk0.69) (Table 1). For each of these electromorphs, genotypic frequencies for all loci were not different
from the Hardy-Weinberg expected equilibrium frequencies.
Levels of genetic variability were low for all populations.
One electromorph showed mean heterozygosities that ranged
between 6.6% and 6.7%, with 1.6–1.9 alleles detected per
locus, while the other had mean heterozygosities that ranged
from 4.3% to 6.3% with 1.3–1.6 alleles per locus (Table 2).
A contingency analysis showed no significant heterogeneity between El Paso (Ak1.08/Hk0.77) and La Rinconada
(x2 5 8.14, degrees of freedom [df] 5 9, P 5 0.520), or
1006
LAMPO AND OTHERS
FIGURE 2. Enzymatic profiles showing two diagnostic loci (Ak and Hk) for separating sympatric (A and B) and allopatric (C, D, E, and
F) populations of Lutzomyia longipalpis at 4 localities: El Paso (EP), La Rinconada (LR), Altagracia (A), and Mapire (M). Aedes aegypti
ROCK (Aa) were used as a standard reference. A specimen of L. trinidadensis (Lt) is also shown in F. For definitions of enzyme loci, see
Table 1.
1007
L. LONGIPALPIS SPECIES COMPLEX
TABLE 1
Allelic frequencies for 8 enzyme loci from the sand fly Lutzomyia
longipalpis from 5 populations from Venezuela*
Altagracia
Ak1.21/Hk0.69
La Rinconada
Ak1.08/Hk0.77
Ak
1.33
1.21
1.08
n
Ho
He
0
1.000
0
10
0
0
0
0
1.000
18
0
0
Ark
0.89
n
Ho
He
1.000
16
0
0
Gpd
1.51
1.34
1.17
n
Ho
He
Mapire
Ak1.21/Hk0.69
El Paso
Ak1.08/Hk0.77
El Paso
Ak1.21/Hk0.69
0
1.000
0
7
0
0
0
0
1.000
77
0
0
0.048
0.952
0
21
0.095
0.093
1.000
24
0
0
1.000
7
0
0
1.000
84
0
0
1.000
21
0
0
0
1.000
0
14
0
0
0
0.987
0.022
23
0.043
0.043
0.071
0.929
0
7
0.143
0.143
0
1.000
0
56
0
0
0
1.000
0
14
0
0
Gpi
0.48
0.38
0.26
n
Ho
He
0.950
0
0.050
10
0.100
0.100
0.056
0.889
0.056
18
0.222
0.210
0.929
0
0.071
7
0.143
0.143
0.063
0.861
0.076
72
0.250
0.250
0.941
0.059
0
17
0.118
0.114
Hk
0.94
0.77
0.69
0.61
n
Ho
He
0
0
0.884
0.156
16
0.188
0.272
0
1.000
0
0
24
0
0
0
0
1.000
0
7
0
0
0.012
0.988
0
0
83
0.024
0.024
0
0
1.000
0
20
0
0
Idh-2
0.77
0.64
0.44
n
Ho
He
0
1.000
0
10
0
0
0
1.000
0
17
0
0
0
1.000
0
7
0
0
0.016
0.984
0
62
0.032
0.032
0.031
0.938
0.031
16
0.125
0.123
Mdh
0.69
0.58
0.43
n
Ho
He
0.050
0.950
0
10
0.100
0.100
0.059
0.941
0
17
0.118
0.114
0
1.000
0
7
0
0
0.110
0.884
0.006
82
0.183
0.207
0
1.000
0
21
0
0
Me
1.00
0.93
0.88
n
Ho
He
0.993
0.067
0
15
0.133
0.129
0
0.891
0.109
23
0.217
0.198
0.857
0.143
0
7
0.286
0.264
0
0.958
0.042
83
0.084
0.084
0.976
0.024
0
21
0.048
0.048
* Ak 5 adenylate kinase; Ark 5 arginine kinase; Gpd 5 glycerol-3-phosphate dehydrogenase; Gpi 5 glucose-6-phosphate dehydrogenase; Hk 5 hexokinase; Idh-2 5 isocitrate
dehydrogenase-2; Mdh 5 malate dehydrogenase; Me 5 malate dehydrogenase; n 5 number
of single diploid genomes; Ho 5 observed heterozygotes for all alleles at a locus; He 5
expected heterozygotes (Hardy-Weinberg equilibrium). Bold numbers indicate the most
common alleles.
FIGURE 3. Wagner dendrogram based on Roger’s genetic distance.10 Five populations of Lutzomyia longipalpis from field collections are shown. For definitions of enzyme loci, see Table 1.
among El Paso (Ak1.21/Hk0.69), Altagracia, and Mapire (x2 5
26.62, df 5 18, P 5 0.0864). Also, the mean FST values
between El Paso (Ak1.08/Hk0.77) and La Rinconada or between
El Paso (Ak1.21/Hk0.69), Altagracia, and Mapire were very low
(20.0045 and 0.0280, respectively) compared with that obtained when all 5 populations were considered (0.8091). As
for the Altagracia and Mapire populations, the El Paso
(Ak1.21/Hk0.69) population showed a very high frequencies of
Gpi0.48 and Me1.00, while the El Paso (Ak1.08/Hk0.77) population
showed high frequencies of the alternative alleles Gpi0.38 and
Me0.93, as reported in La Rinconada (Table 1).
The genetic distance between allopatric populations of different electromorphs was very large (D 5 0.43) compared
with those between allopatric populations of similar electromorphs (D , 0.01) (Table 3). Similarly, the genetic distance
between both electromorphs from El Paso, where they occurred in sympatry, was also very large (D 5 0.545). A
Wagner dendrogram based on Roger’s genetic distances
showed 2 distinct groups: 1 including La Rinconada and El
Paso (Ak1.08/Hk0.77) and the other including Altagracia, Mapire, and El Paso (Ak1.21/Hk0.69) (Figure 3).
DISCUSSION
The presence of fixed allelic differences for Ak and Hk
for the 2 sympatric populations found at El Paso suggests
that L. longipalpis consists of 2 distinct reproductively isolated cryptic species. The absence of heterozygotes between
Ak1.08 and Ak1.21 and between Hk0.69 and Hk0.77 at this locality
is extremely improbable under the 1 species hypothesis.
Species separation between L. longipalpis and L. cruzi
may be misleading because females from both species appear to be structurally indistinguishable.14 However, males
may be separated by comparing the setal tufts at the base of
the coxites.14 Lutzomyia cruzi has 2 long superior and 2 short
basal setae, whereas in L. longipalpis, all 4 setae are equally
long. Comparisons of all collected males with reference
specimens of L. cruzi and L. longipalpis provided by the
Centro Nacional de Referencia de Flebótomos (Universidad
de Carabobo, Maracay, Venezuela) indicated that all males
collected were L. longipalpis. Isozymatic comparisons with
L. cruzi were not possible because there are no records of
L. cruzi in Venezuela.8,15
Nei’s genetic distances between both electromorphs (D .
0.517) were larger than those between presumed cryptic species of L. longipalpis from Colombia, Costa Rica, and Brazil
(0.121–0.333).3 For each electromorph, genetic distances between localities (D # 0.002) were as low as those reported
between some field populations from Brazil.16 Even though
1008
LAMPO AND OTHERS
TABLE 2
Genetic variability within 5 populations of Lutzomyia longipalpis from Venezuela
Population*
Mean sample
per locus
Mean number of
alleles per locus
Percentage of loci
polymorphic
Mean
heterozygosity
13.0
20.0
7.0
72.2
17.6
1.4
1.6
1.3
1.9
1.6
44.4
33.3
33.3
22.2
22.2
0.058
0.067
0.063
0.066
0.043
Altagracia (Ak1.21/Hk0.69)
La Rinconada (Ak1.08/Hk0.77)
Mapire (Ak1.21/Hk0.69)
El Paso (Ak1.08/Hk0.77)
El Paso (Ak1.21/Hk0.69)
* For definitions of enzyme loci, see Table 1.
this actually occurs in insects. Moreover, this explanation
seems even more unlikely considering that such differences
occurred in more than 1 locus.
The delimitation of cryptic species within L. longipalpis
has a significant relevance with respect to the epidemiology
of leishmaniasis. Life histories and vectorial capacities of
phlebotomines may have direct effect on the incidence of
leishmaniasis.19–21 In Lutzomyia, vectorial capacity is highly
species specific;8,22,23 thus, it may vary significantly between
cryptic species. Furthermore, clinical manifestation of leishmaniasis has also been found to vary between geographic
strains.24,25 The high incidence of visceral leishmaniasis at
La Rinconada, where only the newly described electromorph
was present, suggests that this cryptic species may also be
involved in the transmission of visceral leishmaniasis. Nonetheless, further studies should be conducted to compare the
ecologic requirements and vectorial capacity of both of these
cryptic species. Morphologic studies are now being carried
out to identify diagnostic characters that allow us to separate
these 2 cryptic species (Arrivillaga J and others, unpublished
data).
all samples were collected in the field, the average heterozygosities reported here appear to be lower than those reported for field samples from Colombia17,18 and Brazil.16
However, this may also be attributed to the smaller number
of loci analyzed here.
Further genetic comparisons must be carried out before
we can elucidate how these 2 electromorphs relate to other
populations from Central and South America. Colombian,
Costa Rican, and Brazilian strains were considered to be
sibling species based on fixed differences in aldolase (Ald),
Me, and phosphoglucomutase (Pgm). Although we have no
data on Ald and Pgm, we found no fixed differences in Me
between both Venezuelan cryptic species. Most recently,
fixed differences in mannose-6-phosphate isomerase (Mpi)
were found between 2 Colombian populations (Mukhopadhyay J and others, unpublished data). However, the 2 diagnostic loci (Ak and Hk) separating both species at El Paso
appear to be very conservative among all Brazilian and Colombian populations.5 Thus, the fixed differences detected between the 2 Venezuelan electromorphs appear to be distinct
from differences reported earlier for Colombian and Brazilian populations. Whether both cryptic species described
herein occur in Brazil, Colombia, or Costa Ricas remains to
be determined. However, comparisons of Rf values and alleles frequencies for Ak and Hk between Venezuelan and
other South American populations previously studied5 suggest that the Colombian and Brazilian populations are more
similar to the electromorph (Ak1.21/Hk0.69) found at El Paso,
Mapire, and Altagracia than to the electromorph (Ak1.08/
Hk0.77) found at El Paso and La Rinconada. The latter electromorph has not been described previously, although no
data on Hk or Ak has been published for Costa Rican populations.
Although the presence of fixed differences in sympatric
populations strongly indicates the presence of cryptic species, it is at least theoretically possible to attribute the absence of heterozygotes to a strong selection against this genotype. However, we know of no examples that indicate that
Acknowledgments: We are grateful to Maria Dora Feliciangeli (Centro Nacional de Referencia de Flebótomos, Universidad de Carabobo, Maracay, Venezuela) for assisting with the species identification, and Milena Mazzarri for supplying specimens of Ae. aegypti
ROCK strain for reference standards.
Financial support: This study was supported by Consejo Nacional
de Investigaciones Cientı́ficas y Tecnológicas (CONICIT) (No. 96–
0001370) and the World Health Organization–World Bank (No.
021–007).
Authors’ addresses: Margarita Lampo, Milagro Rinaldi, and Carmen
Z. Garcı́a, Centro de Ecologı́a, Instituto Venezolano de Investigaciones Cientı́ficas, Apartado 21827, Caracas 1020-A, Venezuela.
Dara Torgerson, Department of Zoology, University of Guelph,
Guelph, Ontario NIG 2W1, Canada. Luis M. Márquez, Department
of Biochemistry and Molecular Biology, James Cook University,
Townsville, Queensland 4811, Australia. Alberto Arab, Departamento de Biologia, Universidade Estadual Paulista, CEP 13506–900, Rio
Claro, Sao Paulo, Brazil.
TABLE 3
Nei’s unbiased genetic distance12 (above diagonal) and modified as Roger’s13 distance (below diagonal)10 between 5 populations of Lutzomyia
longipalpis from Venezuela*
Altagracia (Ak1.21/Hk0.69)
La Rinconada (Ak1.08/Hk0.77)
Mapire (Ak1.21/Hk0.69)
El Paso (Ak1.08/Hk0.77)
El Paso (Ak1.21/Hk0.69)
Altagracia
Ak1.21/Hk0.77
La Rinconada
Ak1.08/Hk0.69
Mapire
Ak1.21/Hk0.77
El Paso
Ak1.08/Hk0.69
El Paso
Ak1.21/Hk0.77
–
0.619
0.065
0.620
0.064
0.523
–
0.618
0.032
0.629
0.001
0.517
–
0.619
0.057
0.529
,0.001
0.523
–
0.631
0.002
0.537
,0.001
0.545
–
* For definitions of enzyme loci, see Table 1. Bold numbers indicate genetic distances between 2 different electromorphs.
L. LONGIPALPIS SPECIES COMPLEX
Reprint requests: Centro de Ecologı́a, Instituto Venezolano de Investigaciones Cientı́ficas, Apartado 21827, Caracas 1020-A, Venezuela.
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