Identification of the sibling species of the Anopheles maculipennis complex by heteroduplex analysis.

Abstract

The group of anopheline mosquitoes referred to as 'Anopheles maculipennis complex' includes the most important malaria vectors of the Palearctic Western region. The species belonging to this complex, however, are difficult or impossible to distinguish by morphological characters. To differentiate sibling palearctic species belonging to this complex, interspecific differences in the ITS2 sequences were used to set up a rapid and sensitive diagnostic tool based on heteroduplex analysis. The relative heteroduplex mobility allowed the following seven species to be readily distinguished: An. atroparvus, An. labranchiae, An. maculipennis s.s. , An. martinius, An melanoon, An. messeae and An. sacharovi.

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Insect Molecular Biology (2000) 9(5), 509–513
© 2000 Blackwell Science Ltd
509
Blackwell Science, Ltd
Identification of the sibling species of the Anopheles
maculipennis complex by heteroduplex analysis
R. Romi, D. Boccolini, M. Di Luca, G. La Rosa and
M. Marinucci
Department of Parasitology, Istituto Superiore di Sanità,
Roma, Italy
Abstract
The group of anopheline mosquitoes referred to as
‘Anopheles maculipennis complex’ includes the most
important malaria vectors of the Palearctic Western
region. The species belonging to this complex, how-
ever, are difficult or impossible to distinguish by
morphological characters. To differentiate sibling
palearctic species belonging to this complex, inter-
specific differences in the ITS2 sequences were used
to set up a rapid and sensitive diagnostic tool based
on heteroduplex analysis. The relative heteroduplex
mobility allowed the following seven species to be
readily distinguished: An. atroparvus, An. labranchiae,
An. maculipennis s.s., An. martinius, An melanoon,
An. messeae and An. sacharovi.
Keywords: Anopheles maculipennis complex, malaria
vectors, heteroduplex analysis, ITS2.
Introduction
The group of anopheline mosquitoes referred to as
‘Anopheles maculipennis complex’ (White, 1976, 1978)
includes the most important malaria vectors of the Palearctic
Western region. In the 1970s, following the eradication of
malaria in almost all countries of the European Region of
the World Health Organization (Zahar, 1990), scientific
interest in this complex progressively decreased. However,
there has been renewed interest since the re-emergence
of malaria in Turkey and Armenia (Sabatinelli, 1998),
the epidemics in Azerbaijan and Tajikistan (Sabatinelli &
Joergensen, 1999), and the increasing number of imported
cases in Western Europe, which could result in malaria
transmission being re-established in Europe (Baldari et al.,
1998; Romi, 1999).
The species belonging to the An. maculipennis complex
are impossible to distinguish by adult morphological char-
acters and their identification still largely depends on egg
morphology (Missiroli et al., 1933), larval polythene chro-
mosome banding patterns (Kitzmiller et al., 1967) and
gene-enzyme analysis (Bullini & Coluzzi, 1982). However,
molecular techniques have been shown to be both reliable
and efficient tools in distinguishing cryptic Anopheles
species (Collins & Paskewitz, 1996). For example, for
the An. maculipennis complex, the sequence of the Inter-
nal Transcribed Spacer 2 (ITS2) of ribosomal DNA (rDNA)
was recently determined for seven palearctic species
(Marinucci et al., 1999). Within the taxa examined, the
sequence showed minimal intraspecific and significant
interspecific variations. These are the basic requirements
for the application of heteroduplex analysis (HDA), which
has been shown to be both rapid and sensitive. HDA was
originally developed for clinical diagnoses and geno-
typing (Delwart et al., 1993), yet recently it was successfully
applied to population and phylogenetic studies in medical
entomology (Tang et al., 1995; Tang & Unnasch, 1997). In
HDA, the denaturation–renaturation of homologous DNA
sequences of two species results in the formation of four
products: two products corresponding to the two strands
of the same species re-annealing to themselves (homo-
duplexes) and two chimeric products (heteroduplexes),
each comprising a strand from both species. The differ-
ences in DNA sequences, in altering the conformation of
the heteroduplex products, produce an electrophoretic
retardation (ER) of the heteroduplex products with respect
to the homoduplex products on polyacrylamide gels. On
the basis of molecular data (Marinucci et al., 1999), we
developed an HDA capable of distinguishing seven pale-
arctic taxa belonging to the An. maculipennis complex,
namely An. atroparvus, An. labranchiae, An. maculipennis
s.s., An. martinius, An melanoon, An. messeae and
An. sacharovi.
Results
Figure 1 diagrams the banding patterns of the homoduplexes
and heteroduplexes of the twenty-one possible DNA
Received 4 April 2000; accepted following revision 19 June 2000.
Correspondence: Roberto Romi, Laboratorio di Parassitologia, Istituto
Superiore di Sanità, Viale Regina Elena, 299, 00161 Roma, Italy. Tel.:
(+ 39) 06 49902301; fax: (+ 39) 06 49387065; e-mail: romi@iss.it

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510R. Romi et al.
© 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 509–513
combinations with the seven examined species. All of the
heteroduplexes originating from different species associations
were characterized by the number of visible heteroduplex
bands (one or two), by the distance between the two
bands and by the distance that the bands migrated from
the origin. The associations sacharovi/melanoon, sacharovi/
martinius, atroparvus/maculipennis, martinius/labranchiae,
maculipennis/messeae and melanoon/messeae showed
a single band (the two heteroduplex bands overlap); all of
the other associations showed distinct double bands.
Figure 2 presents, as an example, a gel showing the
HDA banding pattern of An. labranchiae (labelled ‘LAB’)
and of the associations obtained by mixing DNA of this
species with DNA from each of the remaining species
(labelled with ‘LAB’ and the first three letters of the species
name). As shown at the bottom of the gel, homoduplexes
obtained by coupling DNA from the same species were
recovered as single bands, whereas those obtained by
mixing DNA from two species were recovered as either
double bands (lanes LAB/MAC, LAB/SAC, LAB/MAR) or
single bands, in cases of comigrate products (lanes LAB/
ATR, LAB/MES, LAB/MEL). The slow-migrating hetero-
duplexes were distributed on the gel according to their
electrophoretic mobility. For each species association,
two bands were observed. As expected, no heteroduplex
bands were produced by mixing DNA from the same
species. The associations LAB/MEL, LAB/MES, LAB/MAC
and LAB/MAR migrated to the lower half of the gel and
LAB/ATR to the middle of the gel; the association LAB/
SAC showed the least mobility.
Under the experimental conditions employed, one or
two additional secondary slow-migrating bands were also
observed in the middle of the gel (Fig. 2, at level of the
marker band of 1500 bp). These bands were assumed to be
Figure 1. Schematic representation of
separation of heteroduplexes (black bars) from
homoduplexes (grey bars) by polyacrylamide
gel electrophoresis. Diagnostic patterns of
heteroduplexes are shown for all twenty-one
combinations of the seven species of the
Palearctic Anopheles maculipennis complex.
Relative electrophoretic retardation (ER) of the
heteroduplexes was defined as ER = (Dhm − Dhd)/
Dhm × 100, where Dhm and Dhd are the distances
that homoduplex and heteroduplex products
migrated on gel. In lane 1, a 100 bp ladder
(Takara, Japan). Species abbreviations as in
Table 1.
Figure 2. Separation on polyacrylamide gel of heteroduplex
bands generated from combinations of Anopheles labranchiae
single-stranded rDNA with PCR products from other species of the
Palearctic An. maculipennis complex. Lighter bands in the middle indicate
single-stranded rDNA. In lane 1, a 100 bp ladder (Takara, Japan). The
homoduplex bands of An. maculipennis s.s. (MAC) and An. labranchiae
(LAB) are shown as reference in lanes 2 and 3, respectively. Species
abbreviations as in Table 1.

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Identification of Anopheles maculipennis by HDA511
© 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 509–513
single-stranded DNA fragments, in that they were present
when running single-species denaturated-renaturated DNA
and absent when running single-species nondenaturated
DNA (data not shown).
Discussion
Seven Palearctic members of the An. maculipennis species
complex were subjected to HDA of the PCR amplified
ITS2 fragment. All of the combinations of species pro-
duced a specific heteroduplex pattern, demonstrating that
HDA is a useful tool for identifying species.
In general, the mobility of heteroduplexes is related to the
number and type of mismatched nucleotides between the
species-specific sequences compared by HDA (Zimmerman
et al., 1993). However, when noncoding sequences are
involved (e.g. rDNA ITSs), the ER does not necessarily
respect the genetic distance between taxa, because the
presence of deletions or insertions may decrease the
heteroduplex mobility due to the formation of bends (Tang
& Unnasch, 1997). Nevertheless, comparing the relative
ER of heteroduplexes with the genetic divergences of
the species, calculated based on the nucleotide differenti-
ation of ITS2 (Table 1) (Marinucci et al., 1999), a very
significant correlation (R2 = 0.84; P < 0.001) was found.
HDA revealed three main groups of heteroduplexes
(Fig. 1 and Table 1): group I, which had the highest ER
(average ER = 15.2) (i.e. greatest distance between
heteroduplexes and homoduplexes), due to hybridization
of An. sacharovi with the other species; group II, inter-
mediate ER (average ER = 8.3), due to An. atroparvus
hybridization with each of the other An. maculipennis
species (excluding An. sacharovi); and group III, lowest
ER (average ER = 5.5), due to hybridization among
An. labranchiae, An. martinius, An. maculipennis,
An. melanoon and An. messeae.
Identifying the species within unknown samples would
thus require that a relatively large number (seven) of hetero-
duplexes be allowed to form. Thus, a screening assay
was developed that minimizes the number of molecular
combinations to accurately and simply diagnose the spe-
cies of all of the An. maculipennis complex specimens
tested. Anopheles messeae from Lazio was selected as
the reference species for the preparation of the marker. A
mix that incorporates all of the possible heteroduplexes
derived from HDA of An. messeae rDNA was produced
and used as a marker to distinguish the seven species
of the complex (Fig. 3, lane 7). The heteroduplexes
from unknown samples, derived from their hybridization
with An. messeae rDNA, were identified by their relative
Figure 3. Diagnostic separation of
heteroduplexes from different populations
of Anopheles labranchiae (lanes 1–5),
An. maculipennis s.s. (lanes 6, 8, 9) and
An. messeae (lanes 10–13) vs. an An. messeae
reference marker (lane 7). This marker
incorporates all of the possible heteroduplexes
derived from the HDA of An. messeae rDNA.
The species producing the different heteroduplex
bands in the marker (lane 7) are illustrated to
the left of the figure. In lane 14 a 100 bp ladder
(Takara, Japan). Species and geographical origin
abbreviations as in Table 2.
Table 1. Electrophoretic retardation values of the heteroduplexes (bold)
and rate of divergence in the ITS2 region within seven species belonging
to the Anopheles maculipennis complex
MAC MEL MESLABMARATR SAC
MAC
MEL
MES
LAB
MAR
ATR
SAC
– 19.8
–
4.6
6.4
5.7
9.1
15.1
29.4
16.7
–
5.8
5.5
8.6
15
30
17.6
22.7
–
6.2
7
15.9
30.2
33.5
40
37.8
–
9
14.2
64.4
60.5
64
53.4
65.2
–
16.7
85.8
86
84.2
76.1
84.8
89.1
–
5
4
5.3
6.8
7.8
14
Species abbreviations: An. atroparvus (ATR); An. labranchiae (LAB);
An. maculipennis (MAC); An. martinius (MAR); An. melanoon (MEL);
An. messeae (MES); An. sacharovi (SAC).

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512R. Romi et al.
© 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 509–513
mobility in comparison with the heteroduplex banding
pattern of the reference species (Fig. 3).
The possibility that different geographical populations of
the same species produce different heteroduplex bands was
also investigated. Figure 3 illustrates an electrophoretic run
in which five populations of An. labranchiae (lanes 1–5),
three of An. maculipennis s.s. (lanes 6, 8, 9) and three
of An. messeae (lanes 10–12) were compared to the
An. messeae reference species. The banding pattern of
the reference species is shown in lane 13. As expected
(Marinucci et al., 1999), no differences were observed in
the ER of the heteroduplexes within An. labranchiae and
An. maculipennis populations. In An. messeae populations,
the two bands observed (lanes 10–12) represented the
homoduplexes that did not completely overlap. These data
are in accordance with the fact that within populations of
An. labranchiae and An. maculipennis, no nucleotide indels
in the ITS2 sequence have been previously observed
(Marinucci et al., 1999). In An. messeae, although negligible
levels of intraspecific polymorphism have been reported
(Marinucci et al., 1999), they are not able to generate
heteroduplexes with different electrophoretic mobility.
HDA combines the advantages of all PCR-based methods
and constitutes a simple and rapid tool for conducting
entomological investigations through the rapid screening
of multiple individuals on a single gel. HDA may be applied
to samples consisting of small portions of an individual
and to all life stages of anopheline mosquitoes, stored in
various ways (fresh, dried, frozen or in fixative solutions).
In conclusion, this procedure was shown to be a sensit-
ive, reliable and inexpensive method for approaching the
problems in the identification of the seven palearctic taxa
of the An. maculipennis complex, and, possibly, of cryptic
mosquito species belonging to other medically important
complexes.
Experimental procedures
Field collection and specimen identification
Blood-fed or gravid females were individually held in Petri dishes
for laying. Egg morphology was used to identify the specimens
according to Missiroli et al. (1933). The identified specimens
were either frozen at −80 °C or stored in silica-gel until molecu-
lar analysis was carried out. The species, name abbreviations,
geographical origins and the number of specimens examined of
the mosquito field populations are listed in Table 2.
DNA extraction and polymerase chain reaction
Genomic DNA was extracted from individual adult mosquitoes
according to Coen et al. (1982). The ITS2 rDNA region was
amplified by polymerase chain reaction (PCR) according to Porter
& Collins (1991) and Collins & Paskewitz (1996). PCR amplifica-
tion was performed using the Promega® kit (Madison, WI, USA),
following the manufacturer’s instructions.
Heteroduplex analysis
Approximately 0.5 µg of PCR-amplified ITS2 sequences from
different couples of species were mixed, adding water to a final
volume of 50 µl and denatured at 98 °C for 5 min. The reaction
tubes were then placed on ice for 5 min to allow heteroduplex
and homoduplex formation. The same procedure was followed
mixing two aliquotes of DNA from the same species as control.
Heteroduplexes and homoduplexes were separated by electro-
phoresis in 5% polyacrylamide gels according to the method of
Tang & Unnasch (1997). Relative electrophoretic retardation
(ER) of the heteroduplexes was defined as ER = (Dhm − Dhd)/
Dhm × 100, where Dhm and Dhd are the distances that homo-
duplex and heteroduplex products migrated on gel. The HDA was
standardized using the seven species of the An. maculipennis
complex previously identified by egg morphology (marked with
an asterisk in Table 2). The method was then tested on unknown
individuals from other localities. The identification obtained by
HDA was confirmed by sequence analysis.
Species and name
abbreviation
No.
populations
No.
individuals Geographical origin
An. atroparvus (ATR)
An. labranchiae (LAB)
1
5
10
10
3
5
3
3
10
5
10
10
10
10
Toscana* (TOS) – Italy
Toscana* (TOS) – Italy
Sardegna (SAR) – Italy
Puglia (PUG) – Italy
Calabria (CAL) – Italy
Sicilia (SIC) – Italy
Toscana* (TOS) – Italy
Veneto (VEN) – Italy
Masis (ARM) – Armenia
Samarcanda* (UZB) – Uzbekistan
Lazio* (LAZ) – Italy
Lazio* (LAZ) – Italy
Piemonte (PIE) – Italy
Tomsk (RUS) – Russia
Pavlodar (KAZ) – Kazakhstan
Adana* – Turkey
An. maculipennis (MAC)3
An. martinius (MAR)
An. melanoon (MEL)
An. messeae (MES)
1
1
4
An. sacharovi (SAC)110
*Populations identified by egg morphology and used for the standardization of the HAD.
Table 2. Mosquito populations belonging to the
Anopheles maculipennis complex and subjected
to heteroduplex analysis

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Identification of Anopheles maculipennis by HDA513
© 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 509–513
Statistical analysis
Linear regression was used to evaluate the relationship between
relative ER of the heteroduplexes and genetic divergence among
the species. ER was measured according to Tang & Unnasch
(1997). Divergence among taxa was calculated as the number
of mismatched nucleotides divided by the number of aligned
nucleotides. Statistical analysis was processed by BMPD software.
Acknowledgements
We thank Dr Antonino Bella (Istituto Superiore di Sanità)
for performing the statistical analyses.
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