Eur. J. Entomol. 108: 35–40, 2011
http://www.eje.cz/scripts/viewabstract.php?abstract=1584
ISSN 1210-5759 (print), 1802-8829 (online)
Molecular approach for identification of mosquito species (Diptera: Culicidae)
in Province of Alessandria, Piedmont, Italy
ASGHAR TALBALAGHI 1, 2 and ELENA SHAIKEVICH 3
1
Mosquito Control District of Alessandria
European Mosquito Control Association, Italy; e-mail: zanzare@zanzare.eu
3
Group of Insect Genetics, Department of Comparative Genetics of Animals, N.I. Vavilov Institute of General Genetics,
Russian Academy of Sciences, Gubkin Str. 3, Moscow 119991, Russia; e-mail: elenashaikevich@mail.ru
2
Key words. Mosquitoes, Culicidae, Anopheles maculipennis, Ochlerotatus caspius, Culex pipiens, PCR-RFLP, ITS2, COI
Abstract. The aim of the present work was to identify cryptic species in the Anopheles maculipennis and Culex pipiens complexes
and to study the genetic structure of the dominant mosquito species Ochlerotatus caspius (Diptera: Culicidae) in the Province of
Alessandria close to the vast area untreated rice fields in Piedmont, NW Italy. With the help of PCR-RFLP analysis, four members
of the Anopheles maculipennis complex were identified: A. messeae, A. maculipennis, A. sacharovi and A. atroparvus. Only C.
pipiens f. molestus was identified in 11 habitats studied in Piedmont. Partial sequences of the cytochrome c oxidase subunit 1 (COI)
mitochondrial gene and the second internal transcribed spacer (ITS2) of nuclear ribosomal RNA genes for Italian O. caspius are
reported here for the first time. The results indicate that this species diverged from Iranian representatives of this species about one
million years ago. The great diversity of mosquito species in Piedmont considerably increases the risk of vector-borne dise ases.
INTRODUCTION
Exact identification of mosquitoes is essential for controlling the species that are vectors of human and animal
diseases. It is particularly difficult to identify representatives of species complexes, such as Anopheles maculipennis and Culex pipiens (Diptera: Culicidae). The
morphology of larvae and adults of species within these
complexes are similar or identical. However, they do
show considerable differences in ecological and physiological features, including food preferences. Their epidemiological significance is also different.
Importantly, the causative agents of many diseases are
transferred between hosts by certain species of bloodsucking Culicidae (e.g. Turell et al., 2001; Becker et al.,
2003; McAbee et al., 2008). In a recent study, the following dangerous viruses were detected in pooled mosquito samples from N Italy: West Nile virus in C. pipiens,
two orthobunya viruses and Tahyna virus in Ochlerotatus
caspius and Batai virus in A. maculipennis (Cazolari et
al., 2010). Since different species within these complexes
can sustain arboviral outbreaks (Talbalaghi et al., 2010),
specific diagnosis of the vectors is essential for developing control strategies.
Unfortunately, morphological features are often insufficient for identifying species of bloodsucking mosquitoes
as some are very variable and consequently unreliable
[for example, siphonal index of C. pipiens larvae
(Fedorova & Shaikevich, 2007)], while many diagnostic
features (especially paleolae and chaetae) are not present
on collected material. This problem can be solved by
using molecular-genetic analysis and developing DNA
markers for specifically identifying species and subspecies of these insects.
One of the basic molecular-genetic markers used to
study species composition of mosquitoes is the second
internal transcribed spacer (ITS2) of nuclear ribosomal
ribonucleic acid (rRNA) genes. rRNA genes of mosquitoes are represented in the genome as a set of tandem
repeats. Each transcriptional unit consists of the genes
encoding three ribosomal RNAs (18S, 5.8S, and 28S)
separated by internal transcribed spacers ITSl and ITS2.
The coding 28S gene sequence is followed by an external
transcribed spacer. Transcriptional units are separated by
un-transcribed spacers. The rRNA genes are conserved,
whereas internal transcribed spacers are variable and
useful for comparing species. It is known that the structure of ITS2 varies among different mosquito species
(Porter & Collins, 1991), the length being from 421 bp in
A. maculipennis to 442 bp in A. sacharovi, however A.
messeae, A. melanoon, A. atroparvus, and A. labranchia
have a fragment of similar length (Marinucci et al., 1999).
Recently, a complex of PCR-RFLP methods was developed to identify some cryptic species in the Anopheles
maculipennis complex (Nicolescu et al., 2004; Ejov et al.,
2008).
Another approach is to analyze the 5’ end of the cytochrome c oxidase subunit 1 mitochondrial gene (COI).
This region, known as the DNA barcode (Barcode of Life
Initiative, 2003), was used previously to study the Iranian O. caspius (Azari-Hamidian et al., 2009). For O.
caspius in Italy, microsatellite and allozyme polymorphism and mitochondrial COII gene structure are reported
(Porretta et al., 2007). Although the nucleotide structure
of the ITS2 area of C. pipiens is variable it cannot be used
to distinguish between C. pipiens and C. pipiens f. molestus, however, a method based on the analysis of the 5’end
35
TABLE 1. Sampling locations, population number (sample code) and date of collection.
Municipality
Alessandria
Alessandria
Valle S.Bartolomeo
Alluvioni Cambiò
Alluvioni Cambiò
Bosco Marengo
Castellazzo Bormida
Castellazzo Bormida
Frugarolo
Ovada
Ovada
Rivarone
Alessandria
Alessandria
Alluvioni Cambiò
Alessandria
Gamalero
Oviglio
Tortona
Novi Ligure
Tassarolo
Sample
ADULT
O. caspius
O. caspius
O. caspius
C. pipiens
O. caspius
O. caspius
C. pipiens
O. caspius
C. pipiens
C. pipiens
Anopheles spp.
Anopheles spp.
C. pipiens
Anopheles spp.
LARVAE
Anopheles spp.
C. pipiens
C. pipiens
C. pipiens
C. pipiens
C. pipiens
C. pipiens
Sample code
Date of sample
or capture
1
2
3
5
7
10
13
15
18
24
25
33
35
36
08.ix.2009
25.viii.2009
01.ix.2009
26.viii.2009
02.ix.2009
01.vii.2009
17.viii.2009
17.viii.2009
17.vi.2009
29.ix.2009
26.viii.2009
10.viii.2009
01.ix.2009
08.ix.2009
1
1
1
3
1
1
2
1
2
4
1
1
2
2
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Dry
Dry
Dry
Dry
Dry
Dry
Dry
Dry
Dry
67
69
70
71
73
74
75
26.viii.2009
28.viii.2009
24.viii.2009
25.viii.2009
26.viii.2009
24.viii.2009
24.viii.2009
2
1
5
8
5
5
9
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
of the mitochondrial COI gene can be successfully used
to identify mosquitoes of the C. pipiens comlex
(Shaikevich, 2007).
In this study, simple PCR-based assays (amplification
of the ITS2 within nuclear rRNA genes of the A. maculipennis complex and amplification of the COI gene within
mtDNA of C. pipiens complex followed by endonuclease
digestion) were used for the molecular identification of
cryptic species.
MATERIAL AND METHODS
Study area and sampling sites
The adults and larvae of the mosquitoes analyzed were collected in the Province of Alessandria close to the vast area of
untreated rice fields in Piedmont, NW Italy. The Province of
Alessandria is 10 km from the biggest mosquito breeding site in
Europe, approximately 220,000 ha of rice fields, which produce
50% of the rice grown in Europe. These rice fields are unique in
being a major breeding area for O. caspius (Pallas, 1771) from
April to July and later a breeding area for Culex spp. and the A.
maculipennis group.
Samples of insects were collected weekly by a monitoring
network of CO2 traps (see Table 1). The genera and species
complexes were identified morphologically.
DNA isolation and analysis
Mosquito DNA was extracted using DIAtomTM DNA Prep kit
(Isogen, Moscow, Russia). DNA samples were obtained from
individual larvae and adults, either dry or preserved in 96%
ethanol. Prior to DNA isolation from the ethanol-preserved
material, ethanol was evaporated off by heating at 65°C for
30–40 min.
PCR-RFLP assay
The mtDNA gene COI was amplified using primers LCO and
HCO (Folmer et al., 1994) for studying O. caspius and Culex-
36
Number of specimens
Storing conditions
identified
COIF and Culex COIR (Shaikevich, 2007) for studying C. pipiens. The ribosomal ITS2 region was amplified using primers
5.8S and 28S (Porter & Collins, 1991) at research both O. caspius and Anopheles spp.
PCR amplification reactions were run in a final volume of 25
µl with PCR buffer (Isogen), 20 µM of each dNTP, 2.5 mM
MgCl2, one unit of Taq DNA polymerase, 0.2 µM of each primer
and 0.1 µg of the isolated DNA. Thermocycler conditions were
as recommended for each primer pair.
For the Anopheles maculipennis complex, the improved PCRRFLP method was used (Ejov et al., 2008). The PCR-RFLP was
run using CfoI (Promega, Madison, WI, USA) and BctACI
(Sibenzyme, Moscow, Russia) endonucleases. Restriction volumes were 20 µl. The digestion mixtures consisted of (i) 3 µl of
the ITS2 PCR-product, 0.5 µl (5 U) of CfoI, 2 µl of the manufacturer’s incubation buffer, 0.2 µl BSA (Promega) and 14.3 µl
ddH2O, and (ii) 3 µl of the ITS2 PCR-product, 0.5 µl (5 U) of
BctACI, 2 µl of the manufacturers incubation buffer and 14.5 µl
ddH2O (BSA is included in all Sibenzyme’s incubation buffers).
Incubation time was not less then 2 h at 37°C.
For the precise identification of C. pipiens mosquitoes using
the PCR-RFLP assay amplicons were digested with HaeIII
(Promega) restriction endonuclease. Restriction enzyme digestions were performed in 20 µl volume. Digest master mix consisted of 5 µl of the COI PCR product, 0.5 µl (5 U) of HaeIII, 2
µl buffer C (Promega), 0.2 µl BSA (Promega) and 12.3 µl
ddH2O. The HaeIII restriction reaction was incubated for 1 h at
37°C. The digested products were visualized on a 2% agarose
gel.
Sequence analysis
PCR products were identified by electrophoresis, using a 1%
agarose gel (Sigma, St. Louis, MO, USA). Amplified DNA
fragments were isolated from the gel using JetQuiCk Gel
Extraction Spin Kit (Genomed, Loehne, Germany) and then
directly sequenced on an ABI PRISM 310 using the ABI
PRISM BigDye Terminator Cycle Sequencing kit (Applied Bio-
Fig. 2. Fragments resulting from restriction with HaeIII of
603 bp amplificates of DNA of COI gene of C. pipiens from
Piedmont: 5-1, 13-1, 18-1, 24-1 (the sample codes correspond to
the numbers in Table 1); of anautogenous mosquitoes from open
rural habitats in the region of Moscow (pip-1) and Krasnodar
(pip-2); of autogenous mosquitoes from basement habitats in
Moscow (mol-1) and Krasnodar (mol-2); M100 – 100 bp ladder
(Isogen) 1000, 900, 800, 700, 600, 500 (bright), 400, 300, 200,
100 bp.
Fig. 1. Fragments resulting from restriction with CfoI of ITS2
amplificates of A. maculipennis complex mosquitoes from Piedmont; M50 – 50 bp ladder (Isogen, Moscow, Russia): 500, 450,
400, 350, 300, 250, 200, 150, 100, 50 bp. The sample codes
above lanes correspond to those in Table 1.
systems, Foster City, CA, USA) according to the instructions of
the manufacturer. The obtained sequences of O. caspius DNA
are available in GenBank under accession numbers
HM140413–HM140418 for COI and HM140419–HM140424
for ITS2. The DNA sequences were aligned using ClustalW and
analyzed using MEGA version 4 (Tamura et al., 2007).
RESULTS AND DISCUSSION
Anopheles spp.
The PCR with primers 5.8S and 28S produced amplicons with lengths typical for mosquitoes of the complex
A. maculipennis (420–440 bp). An improved PCR-RFLP
method was used to identify species of the A. maculipennis complex (Ejov et al., 2008). Identification was
based on species-specific restriction sites in the ITS2
locus. The recognition site for CfoI is the sequence
GCGC. After digestion with CfoI, variable fragments
were obtained (Fig. 1). Two fragments (56 and 42 bp) are
characteristic for all members of the A. maculipennis
complex. Because of their low molecular weight these
fragments merged into one another on agarose gels (about
50 bp).
For individuals from population 36, 389-bp and 50-bp
fragments, common for A. atroparvus, were obtained.
DNA of the mosquito from population 33 was cut into
fragments of 207, 111, 78, and 50 bp, corresponding to A.
sacharovi. 50 bp is almost not visible in lane 33-1 on Fig.
1, but other visible fragments are specific only for A.
sacharovi (Nicolescu et al., 2004). For the individuals
from populations 25 and 67 (sample 67-2) restriction
fragments of 272, 102, and 50 bp were obtained. These
fragments correspond to A. maculipennis. DNA from
other individual from population 67 (sample 67-1) was
cut into fragments of 141, 135, 111 and 50 bp, as in both
A. messeae and A. melanoon. For exact identification of
the latter one an additional restriction reaction with
BstACI was run, which indicated fragments of 292 and
150 bp, common for ITS2 of A. messeae (not shown).
(There is no site for BctACI in the ITS2 of the DNA of A.
melanoon).
The analysis of Anopheles spp. from various biotopes
using PCR-RFLP revealed four members of the A. maculipennis complex: A. messeae, A. maculipennis, A. sacharovi, and A. atroparvus.
Five species of the A. maculipennis complex are
common in Europe: A. maculipennis, A. messeae, A.
melanoon, A. sacharovi, and A. atroparvus. All are zooand anthropophilic and attack humans and animals both
indoors and outdoors. After feeding on blood, A. maculipennis, A. sacharovi, A. messea, and A. atroparvus rest
indoors (are endophilic), while A. melanoon rests outdoors (is exophilic). A. messeae hibernates, while A.
maculipennis, A. melanoon, A. sacharovi, and A.
atroparvus can suck blood all year round, but do not lay
eggs in winter. Therefore, a correct identification of the
vectors is essential for the development of control strategies. Since the mtDNA of A. maculipennis mosquitoes is
more polymorphic than the ITS regions of the rRNA transcriptional units, using the ITS2 sequences for the PCRRFLP analysis for identifying the species within this complex is likely to prove more accurate. It has been possible
to identify species using these methods and these markers
are very promising for use in future studies.
Culex pipiens
The C. pipiens complex in Europe is represented by
Culex pipiens (Linnaeus) and C. pipiens f. molestus
(Forskal). Although very similar in morphology these two
37
Fig. 3. Variable nucleotide and amino acid sites of the 563 bp
5’region of COI (Barcode fragment) and variable nucleotide
sites of the ITS2 fragment of O. caspius from Piedmont. The
sample codes correspond to those in Table 1. Vertical numbers
in square bracklets indicate positions of variable sites. V –
valine; I – isoleucine.
forms are quite different in their physiology and behaviour. C. pipiens is rather ornithophilic while C. pipiens f.
molestus is mostly anthropophilic. C. pipiens is anautogenous (unable to lay a batch of eggs until it has had blood
meal) and eurygamous (unable to mate in confined
spaces). In contrast, the molestus form is autogenous (can
produce a first batch of eggs prior to feeding on a blood
meal) and eugamous (mates in confined spaces). C.
pipiens can hibernate, but not C. pipiens f. molestus.
Although both forms are vectors there is no data on their
relative abilities as vectors of disease.
Earlier, the DNA polymorphism in a 603-bp fragment
of the 5’end of the mitochondrial COI gene of two forms
of C. pipiens from Russia was used to develop PCRRFLP assays using HaeIII restriction endonuclease to distinguish between anautogenous (pipiens) and autogenous
(molestus) forms (Shaikevich, 2007). After restriction of
amplificates of DNA of COI gene using endonuclease
HaeIII two fragments (206 and 397 bp) were recorded in
anautogenous C. pipiens, whereas the 603 amplicon from
autogenous C. pipiens remained unchanged (Fig. 2). This
method was used in the analysis of mosquitoes from Piedmont. Some examples are presented in Fig. 2. After
amplification and digestion with HaeIII only the uncut
603-bp fragment was obtained for all the specimens of C.
pipiens studied. Altogether 48 individuals of the molestus form were identified in Piedmont using restrictase
HaeIII. Almost all the C. pipiens studied were collected
from cans, water containers and sewage water in an urban
area. Homogeneous populations of the anthropophilic
form molestus were recorded in 11 populations (see Table
1).
Mainly anthropophilic populations of C. pipiens are
recorded in Egypt (Gad et al., 1999). In regions of
southern Russia in summer, mosquitoes of the molestus
form may temporarily establish homogeneous populations
or coexist with the anautogenous form of pipiens in
above-ground water bodies (barrels) (Vinogradova et al.,
38
Fig. 4. Dendrogram based on 563 bp COI gene mtDNA
sequence data of the relationship of the haplotypes within O.
caspius. Och1, Och2, Och3, Och7, Och10, and Och15 – O. caspius from Piedmont; the sample numbers correspond to those in
Table 1. The tree was constructed using neighbour-joining
method with Kimura 2-parameter distances. Numbers are bootstrap percentages (1000 replicates).
2007). Heterogeneous populations are also recorded in
Germany, where some populations in water barrels, buckets, and garden ponds consist of up to 20% of the autogenous form (Becker et al., 1999).
These results confirm that molestus mosquitoes can survive if the temperature conditions are favourable in open
habitats, although in northern zones these mosquitoes
occur mostly underground and biotopically separated
from the form pipiens.
Ochlerotatus caspius
The 563-bp sequences of the mitochondrial gene COI
(The Barcode fragments) of O. caspius were obtained for
specimens from six populations 1, 2, 3, 7 10 and 15
(Table 1). Four unique haplotypes were identified. The
individuals studied varied by less than 1%. The mosquitoes from populations 1, 2 and 3 were identical in this
563-bp DNA fragment. DNA from a mosquito of population 7 differed by only one nucleotide. The highest polymorphism was shown by a mosquito from population 10:
six nucleotide substitutions. Most substitutions were at
the third codon positions and did not affect the amino
acid sequence. Only the transition G o A at position 37
at the first codon position results in the amino acid substitution valine-isoleucine (Val o Ile); this substitution was
detected in two of the mosquitoes studied from populations 10 and 15 (Fig. 3).
GenBank search revealed that the COI sequences of O.
caspius from Piedmont share a 98% similarity with specimens from Iran (Azari-Hamidian et al., 2009). The
neighbour-joining dendrogram shows two clusters: the
first is formed by Italian and the second – Iranian O. caspius (Fig. 4).
In addition, sequences of the variable ITS2 region were
analyzed in the ribosomal RNA of O. caspius. Samples
from the same populations: 1, 2, 3, 7, 10, and 15 (Table
1) were studied. The size of the obtained PCR fragments
of the ITS2 region of O. caspius was about 370 bp (not
shown). Primers were selected so that the ITS2 sequences
were flanked with a 88-bp fragment of the 5.8S and 45-bp
fragment of the 28S rRNA genes. Therefore, the size of
the actual ITS2 of O. caspius is about 254 bp.
The ITS2 of the five individuals investigated was
almost identical. Only one mosquito differed from the
others by two nucleotide substitutions and one deletion.
Four variable sites were detected in six sequences (Fig.
3). Pair-wise distances between samples from different
populations varied from 0 to 1.1%. In comparison with all
ITS2 sequences of O. caspius from Spain and Tunis
available in GenBank, the overall average pair-wise distance is below 0.2%.
Unfortunately, only the mtDNA of Iranian mosquitoes
was studied and only ITS2 of Tunisian and Spanish, mosquitoes. Consequently, it is difficult to compare the
genetic structure of geographically remote populations of
O. caspius using the same marker. The polymorphism in
mtDNA of Italian and Iranian O. caspius is 10 times more
than that of the ITS2 sequences of Italian, Spanish, and
Tunisian O. caspius. O. caspius is thought to have originated in central Asia and later spread to Western Europe
and North America (Ross, 1964; Minar, 1976 cited from
Milankov et al., 2009). The nucleotide sequence divergence in insect mtDNA is thought to be approximately
2% for pair-wise divergence per million years (Guillemaud et al., 1997). If so, the differences in COI suggest
that it is about one million years since the divergence of
the geographically distinct populations in Iran and Italy.
During this period, the spread in the distribution of
insects was strongly affected by the availability of their
habitats, and probably, the distinctions operated at the
subspecies level. It would be interesting to compare the
DNA of O. caspius from Italy and central Asia with other
populations of this species using the same set of mitochondrial and nuclear markers.
CONCLUSIONS
By using molecular analysis it was possible to confirm
the presence in the Province of Alessandria of several
species of mosquito that are capable of transmitting infections.
The DNA analysis revealed that the Anopheles maculipennis complex in the area of Piedmont includes A.
messeae, A. maculipennis, A. sacharovi, and A. atroparvus, and the Culex pipiens complex is represented by
the antropophilic form molestus. All these species are of
high epidemiological significance in Mediterranean countries.
It is suggested that the level of genetic homogeneity in
O. caspius in the region of Piedmont is high, even though
only six individuals were studied. Our data accord with
the conclusions reached using other markers such as allozymes and the gene COII, which are not significantly
genetically diverse in O. caspius from the plain of the Po
River in the north of Italy (Porretta et al., 2004).
Probably, there are no genetic barriers preventing
crossing between populations of O. caspius in northern
Italy; this could increase the risk of rapid population
growth in this favourable rice field habitat.
A change in the climate in the Piedmont region,
together with the development of insecticide resistance,
could result in a rapid increase in the abundance and geographic range of mosquito populations. This could complicate the development of an effective control of
mosquitoes. The use of a DNA-based method to confirm
the identity of mosquito species is likely to greatly reduce
the risk of misidentification and results in a more efficient
monitoring of control programs.
ACKNOWLEDGEMENTS. The authors are grateful to I.
Zakharov and E. Gupalo for comments and advice. We thank N.
Oyun for technical help and two anonymous reviewers for their
corrections and recommendations. This work was supported by
the Russian Foundation for Basic Research (grant 08-0401511-a) and by the Program of Basic Research of the Russian
Academy of Sciences “Gene pool and the genetic diversity”.
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Received June 8, 2010; revised and accepted July 28, 2010
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