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Medical and Veterinary Entomology (1999) 13, 72±77
Metaphase karyotypes and G-banding in sand¯ies of
the Lutzomyia longipalpis complex
H U A I Z H I Y I N , J . - P . M U T E B I , S . M A R R I O T T and
G. C. LANZARO
Department of Pathology & Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas, U.S.A
Abstract. Abstract. Mitotic metaphase chromosomes (2n = 8) from brain cells of
fourth instar sand¯y larvae of four geographical strains of the Lutzomyia longipaplis
complex were examined microscopically, with bright-®eld illumination, after
staining by a new G-banding technique involving exposure of air-dried
chromosome preparations to quinacrine and ultraviolet light. Differences of
G-banding and/or position of the centromere on chromosome 4 (the smallest
chromosome pair) distinguished four putative sibling species from Costa Rica,
Colombia and Brazil (distinctive populations from Jacobina and Lapinha Caves).
The karyotype of the population from Jacobina, Brazil, showed an apparently
plesiomorphic pattern of G-banding. On the basis of their recognizably different
mitotic karyotypes, cytogenetic identi®cation of separate taxa in the L. longipalpis
complex should be useful for speci®c female vector competence and ecology
studies.
Key words. Lutzomyia longipalpis, leishmaniasis vectors, sibling species,
genetics, cytogenetics, mitotic metaphase karyotype, G-banding, Brazil, Colombia,
Costa Rica.
Introduction
Lutzomyia longipalpis (Lutz & Neiva) sensu lato is an
important sand¯y (Diptera: Psychodidae: Phlebotominae)
vector of visceral (Killick-Kendrick, 1990) and cutaneous
leishmaniasis in the Neotropics (Zeledon et al., 1989; KillickKendrick 1990; Kettle, 1995). L. longipalpis s.l. is regarded as
a species complex (Ward et al., 1983) with three allopatric
sibling species recognized from Brazil, Colombia and Costa
Rica by Lanzaro et al. (1993), based on isozyme analysis and
cross-mating studies. The joint type-localities of L. longipalpis
are in Brazil at Benjamin Constant and SaÄo Paulo (Lutz &
Neiva, 1912). While speciation of L. longipalpis s.l. is
complex, local population structure can be quite simple
(Munstermann et al., 1998)
Chromosomal characters have been widely used in studies
of the taxonomy and population genetics of other haematophagous Diptera, but very few cytogenetic studies have been
Correspondence: Dr G. C. Lanzaro, Department of Pathology,
University of Texas Medical Branch, 301 University Boulevard,
Galveston, Texas 77555-0609, U.S.A.
72
conducted on Phlebotomine sand¯ies (Lanzaro & Warburg,
1995). Polytene chromosomes of L. longipalpis s.l. larval
salivary glands were partly described by White & KillickKendrick (1976).
The mitotic karyotypes (2n = 8) of several sand¯y species
have been described by Kreutzer et al. (1987, 1988) using
a squash method. While this method is technically simple,
it is dif®cult to obtain metaphase chromosomes with a good
spread and clear background. Air-dry methods, such as
those described by Gosden (1994), can be used to obtain
superior chromosome preparations. This method results in
chromosomes well-suited for banding analysis and DNA in
situ hybridization. Chromosome banding is also useful for
systematics and taxonomy, playing a pivotal role in the
construction of genomic maps. Ordering of DNA markers
can be quickly and easily achieved by in situ hybridization
to banded chromosomes (Craig & Bickmore, 1993).
Here we present metaphase karyotypes and chromosome
banding for populations of the L. longipalpis complex from
four geographical areas in three countries, using an improved
air-dry method and G-banding techniques.
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L. longipalpis chromosomes
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Fig. 1. Central nervous system of fourth instar L. longipalpis larva. (Ab Gng, abdominal ganglia; Th Gng, thoracic ganglia; Br, brain).
Materials and Methods
Sources of materials and dissection
Fourth instar larvae from four laboratory strains of L.
longipalpis were used, namely: Jacobina (11°11¢S,
40°31¢W) and Lapinha Caves (20°0¢S, 44°0¢E) in Brazil;
El Callejon in Colombia (4°11¢N, 74°18¢W) and Liberia
(10°38¢N, 85°27¢W) in Costa Rica. The Lapinha Caves, El
Callejon and Liberia populations were interpreted as distinct
species by Lanzaro et al. (1993). The relationship between
the Jacobina strain and the others is uncertain (cf. Dujardin
et al., 1997). All four colonies were maintained by us
following the methods of Modi & Tesh (1983). The brain
(cerebral ganglion) was dissected from fourth instar larvae
using ®ne forceps (Fig. 1) in Ringer's solution for insects.
This dissection is relatively simple. First the integument is
stripped off, starting between the third and fourth
abdominal segments. The digestive system is then removed
to expose abdominal and thoracic ganglia. The brain is
extracted from the head capsule by holding the anterior
portion of the head, between the eyes, with one pair of
forceps and pulling the abdominal ganglia with another.
This procedure results in a preparation free of extraneous
tissue and exoskeleton, suitable for stained nerve cells with
minimal background staining.
Karyotype preparation
Metaphase karyotypes were prepared from brain tissue
using established techniques (Stock et al., 1972; Lakhotia &
Kumar, 1978) with some modi®cation. The dissected brain
tissue was placed in a drop of hypotonic solution (0.075 M
KCl) on a clean slide. The liquid was withdrawn after
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3±5 min. A drop of 50% acetic±methanol ®xative (1:2:3,
glacial acetic acid: absolute methanol: distilled water) was
added and the preparation incubated at room temperature
for 4±5 min. The ®xative was removed and a small drop of
45±60% acetic acid was added to the tissue. Excess
solution was withdrawn after the brain tissue became clear.
A drop of freshly mixed absolute acetic±methanol ®xative
(1:3, glacial acetic acid: methanol) was dropped onto the
clear tissue from a height of 10±20 cm and cells were
spread by immediately blowing on the preparation (this
must be done quickly before the ®xative evaporates). The
slide was allowed to dry then stained with 5% Giemsa
(Sigma, St. Louis, MO) solution for 20 min. Metaphase
chromosomes were examined microscopically with bright®eld illumination and photographic records were made, as
described below.
G-banding preparation
The G-banding technique stains euchromatin and is
usually achieved using the ASG (acid/saline/Giemsa)
method of Sumner et al. (1971). However, it is dif®cult
to obtain G-bands using the ASG procedure in most
invertebrates. We therefore developed a modi®ed G-banding
method using quinacrine stain, following Bregmen (1975).
Air-dried slides were kept at 37°C for at least 2 weeks and
then stained with 0.5% quinacrine (Sigma) for 20 min. Each
slide was then rinsed with distilled water and the tissue
covered with a thin layer of 2x SSC buffer (0.3 M sodium
chloride, 30 mM sodium citrate). The slide was then
irradiated with ultraviolet light (254 nm) in a UV
Stratalinker (Stratagene model no. 1800) for 30 min. This
method has been used by Daga et al. (1996) for studying
replication banding in zebra®sh. Following UV irradiation,
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74 H. Yin et al.
Fig. 2. Karyotypes of metaphase chromosomes (c. 4000 3) from the brain tissue of four populations of L. longipalpis s.l. from: A: Jacobina,
Brazil; B: Lapinha Caves, Brazil; C: El Callejon, Colombia; D: Liberia, Costa Rica.
the preparation was incubated in 0.2x SSC buffer (0.03 M
sodium chloride, 3 mM sodium citrate) at 60°C for 60 min,
then rinsed brie¯y with distilled water. The slide was then
stained with 3% PBS (pH 7.2) Giemsa solution for 30 min,
and mounted in Euparal medium, dried and kept for further
analysis.
Observation and photography
Slide preparations were examined through a 100 3
objective with an Olympus (BX 50) microscope system
and photographed with ILFORD PANF 50 ®lm. Karyotypes
were described from metaphase preparations of at least ten
individuals from each population. Likewise, ideograms
illustrating G-band patterns were based on observation of
chromosomes from a minimum of ten individuals. Karyotypes were described according to the International System
for Human Cytogenetic Nomenclature (ISCN), as de®ned
by Bergsma (1978). We feel that it is appropriate to follow
this system which differs from the terminology employed
for sand¯y chromosomes in earlier studies ± Kreutzer et al.
(1988) described sand¯y karyotypes by numbering them in
ascending order of chromosome size so that the smallest
chromosome was designated no. 1. The ISCN protocol calls
for numbering chromosomes by descending order of size
and we followed this convention.
Results
The mitotic karyotype formula for L. longipalpis of our
strain from Jacobina, Brazil is 2n = 6M + 2SM (M = meta# 1999
centric, SM = submetacentric). Chromosomes 1, 2 and 3 are
metacentric, whereas chromosome 4 is submetacentric
Fig. 2A). The G-banded karyotype of the Jacobina strain
is shown in Fig. 3(A). Chromosome 1 has ®ve bands per
arm. Chromosome 2 has four bands on one arm and three
on the other. Chromosome 3 has three bands on one arm
and two on the other, one occupying nearly half of that
arm. Chromosome 4 has two bands on both short and long
arms, the latter with a broad band resembling that of
chromosome 3.
The karyotype formula of L. longipalpis from Lapinha
Caves, Brazil is 2n = 6M + 2ST (ST = subtelocentric). As
previously described by White & Killick-Kendrick (1976),
chromosomes 1, 2 and 3 are metacentric while chromosome
4 is subacrocentric = subtelocentric (Fig. 2B). The G-banded
karyotype of the Lapinha Cave strain is illustrated in
Fig. 3(B). Chromosome 1 has four bands on one arm and
three on the other. Chromosome 2 has three bands on one
arm and two on the other, with a large band occupying
two-thirds of this arm. Bands on chromosome 3 resemble
those on chromosome 2, but the large band is more distal.
Chromosome 4 has one band on the short arm and three on
the long arm.
L. longipaplis of the Colombia and Costa Rica populations,
previously recognized as different species by Lanzaro et al.
(1993), have the same karyotype formula, 2n = 8M (Fig. 2C, D)
with four pairs of metacentric chromosomes. However, the Gbanding patterns differ between them. In the Colombian
species (Fig. 3C), chromosomes 1 and 2 appear the same, with
3 bands on each arm, whereas in the Costa Rica species
(Fig. 3D), chromosome 1 has four bands per arm and
chromosome 2 has three bands on each arm. Chromosome 3
has two bands on one arm and three on the other in both
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L. longipalpis chromosomes
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Fig. 3. G-Banding on metaphase chromosomes (c. 3800 3) of four populations of L. longipalpis s.l. and their diagrammatic representations, from:
A: Jacobina, Brazil; B: Lapinha Caves, Brazil; C: El Callejon, Colombia with large band; D: Liberia, Costa Rica.
Table 1. Karyotypes of Lutzomyia spp., as described by Kreutzer
et al. (1987, 1988).
Group
2n
Karyotype formula
Species
1
2
6
8
4M, 2SM
6M, 2ST
3
8
6M, 2SM
4
8
4M, 2SM, 2ST
trapidoi
gomezi
longipalpis
carmelinoi
columbiana
erwindonaldoi
walkeri
spinicrassa
species (Fig. 3C, D). Both arms of chromosome 3 have one
large band occupying half the length in the Colombian species.
In contrast, the Costa Rican chromosome 3 has three subequal
bands on one arm, whilst the other arm has a broader band
distally. The banding pattern of chromosome 4 is similar to
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that of chromosome 3 in the Colombian species, whereas
chromosome 4 has two bands on each arm in the Costa Rican
species.
Discussion
Four different karyotype formulae of Lutzomyia spp. were
reported by Kreutzer et al. (1987, 1988), with chromosome
numbers varying from 2n = 6 in L. trapidoi to 2n = 8 in other
species (Table 1). All our population samples of the L.
longipalpis complex had 2n = 8, but different con®gurations
in the four populations sampled. Our ®ndings are inconsistent
with the observation of Kreutzer et al. (1987) that karyotypes
of Colombian and Brazilian samples of L. longipaplis s.l. were
identical, although they did not report the exact locality or
source of the populations used in their study. Our population
from Lapinha Caves in Brazil had the same karyotype as that
described for L. longipalpis by Kreutzer (Group 2) and by
White & Killick-Kendrick (1975) from the same locality, but
our three other geographical populations were unique. We
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76 H. Yin et al.
Fig. 4. Hybrid karyotype of an F1 individual from the cross: Costa Rica X 3 Y Lapinha Caves.
Fig. 5. Possible sequence of karyological changes in chromosome 4 of L. longipalpis from Jacobina (A); Lapinha Caves (B); Colombia (C) and
Costa Rica (D). Idiograms A¢ and A² are hypothetical. Dotted lines indicate a deletion of euchromatin (A®A¢), broken lines indicate a
duplication (A¢®C, A²®D) and solid lines indicate a pericentric or paracentric inversion (A¢®B, A¢®A²).
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L. longipalpis chromosomes
examined chromosomes of hybrids from the cross Lapinha
Á
Cave X3 Y Liberia. Hybrid progeny of this cross are
dysfunctional, males being sexually sterile (Lanzaro et al.,
1993). The chromosome 4 homologues were heteromorphic in
these hybrids (Fig. 4).
G-banding was used to construct a theoretical phylogeny of
chromosome 4 polymorphism (Fig. 5). The Jacobina chromosome 4 (designated as type A) appears to be plesiomorphic. A
deletion in the long arm would produce a hypothetical type A¢
chromosome from which extant forms could be derived via
appropriate pericentric inversions and duplications, as suggested in Fig. 5. Another hypothetical type A² chromosome
would be needed as a step towards the Costa Rican
chromosome 4 (type D).
The air-dry method for the preparation of insect chromosomes (Stock et al., 1972; Imai et al., 1977; Lakhotia & Kumar
1978) is laborious and time consuming. We describe a superior
method, resulting in a larger number of readable mitotic
complements per preparation and with less background
staining. We also report the ®rst application of quinacrine
treatment and UV irradiation for G-banding in the preparation
of insect chromosomes. This method may be useful with other
insects and invertebrates to obtain G-banding in mitotic
chromosome preparations.
Acknowledgments
This research was supported by grant AI39540 from the
National Institutes of Health, U.S.A. to G.C.L.
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