1. Vol. 35, no.1 Journal of Vector Ecology 1
Scientific Note
First evidence for presence of Culex pipiens biotype molestus in the Netherlands, and
of hybrid biotype pipiens and molestus in northern Europe
C.B.E.M. Reusken1
, A. de Vries1
, J. Buijs2
, M.A.H. Braks1
, W. den Hartog3
, and E.-J. Scholte1,3
1
Laboratory for Zoonoses and Environmental Microbiology, Centre for Infectious Disease Control Netherlands, Bilthoven,
The Netherlands
2
Public Health Service (GGD) of Amsterdam
3
National Centre for Vector Monitoring, Wageningen, The Netherlands
Mosquitoes (Diptera: Culicidae) are the principal
vectors of West Nile virus (WNV). The likely introduction
pathway of WNV into northwestern Europe is by infected
migratory birds from WNV-endemic regions like Africa and
central Europe. For efficient local enzootic transmission,
mosquito species are required that are capable of sustaining
and transmitting the virus among the indigenous bird
population. Ornithophilic mosquito species are the most
relevant species for WNV establishment in an enzootic cycle
(HubalekandHalouzka1999,Koopmansetal.2008,Rappole
et al. 2000, Rappole and Hubalek 2003). Humans do not
generate enough viremia for mosquitoes to pick up WNV
during blood-feeding. Therefore, humans are considered a
dead-end host. Vectors with a more opportunistic feeding
behavior are required for human WNV epidemics, serving
as a “bridge” for the virus between birds and humans.
Members of the Culex pipiens complex are considered
to be principal vectors for WNV transmission both in
North America and Europe (Hubalek 2008, Hubalek and
Halouzka 1999, Koopmans et al. 2008). In Europe, the Cx.
pipiens complex contains two distinct biotypes; pipiens and
molestus, which are morphologically indistinguishable
but differ in physiology and behavior. Culex pipiens
biotype (b.) pipiens requires a blood meal for each egg
batch (anautogeny), is unable to mate in confined spaces
(eurygamous), and is only seasonally active. In contrast, Cx.
pipiens b. molestus is autogenous, stenogamous, remains
active throughout the year, and mainly feeds on mammals,
especially humans (mammophilic) (Harbach et al. 1984).
In northern Europe, Russia, and the northeastern U.S.A.,
biotype molestus and pipiens occupy different habitats. Cx.
molestus occurs in underground areas in urban settings
while Cx. pipiens lives above ground (Bahnck and Fonseca
2006, Byrne and Nichols 1999, Fonseca et al. 2004). In
southern Europe, sympatric occurrence of both biotypes
has been observed in surface habitats (Chevillon et al.
1995, Gomes et al. 2009). Several studies have shown that
the biotypes represent two distinct genetic entities and that
underground molestus populations in northern Europe are
most likely derived from southern molestus populations
that have dispersed and colonized underground habitats
(Fonseca et al. 2004, Gomes et al. 2009, Weitzel et al. 2009).
While biotype molestus is locally present in northern
Europe, being reported only from Germany and the United
Kingdom from underground breeding sites (Byrne and
Nichols 1999, Fonseca et al. 2004, Medlock and Snow 2008,
Weitzel et al. 2009), biotype pipiens is the ubiquitous species
of the complex (Service 1993).
In the northeastern U.S.A., a large proportion of the
specimens of the Cx. pipiens complex were found to be
hybrids between both biotypes, readily feeding on both
humans and birds (Fonseca et al. 2004, Kilpatrick et al.
2007). Up to now, hybridization has only been observed
in two populations in southernmost Europe (Fonseca et al.
2004, Gomes et al. 2009). Despite the presence of both the
molestusandpipiensbiotypes,noevidenceforhybridization
exists in northern Europe.
Members of the Cx. pipiens complex have different
vector capacities due to their different feeding preferences
with Cx. pipiens being ornithophilic, Cx. molestus being
mammophilic, and hybrids being opportunistic. Local
distinction between these forms in the complex is essential
for understanding local WNV epidemiology and for
assessing risks and for control. To gain insight into the
composition of the Cx. pipiens complex in the Netherlands,
we collected mosquitoes in the underground transit system
of Amsterdam and identified members of the Cx. pipiens
complex at the biotype level. We established the presence
of the three members of the Cx. pipiens complex in the
Netherlands and the epidemiological important presence
of hybrids of biotypes pipiens and molestus in northern
Europe.
Study sites
Potential habitats of biotype molestus were selected
based on the combination of two criteria: 1) reported biting
nuisancetothePublicHealthService(GGD)Amsterdamand
2) the presence of underground larval breeding sites (water
bodies). In the summer of 2009, mosquito biting nuisance
complaints were reported from 11 sites in Amsterdam. Cx.
pipiens was found at three of these sites, but underground
larval breeding sites were present only at one of these: the
Amsterdam metro transit system. Both metro personnel
and travellers had reported a mosquito biting nuisance at
the platforms to the local authorities. The complaints were
not restricted to one station and it was decided to visit three
underground metro stations. The stations were visited on
28 August 2009. Wastewater collection basins were present
at each of the three underground metro stations located at
the southern and northern perimeters of the platforms in a

2. 2 Journal of Vector Ecology June 2010
restricted area accessible to maintenance employees. These
collection basins are closed spaces containing at least 20 cm
of filthy, nutrient-rich water. They are covered with a metal
plate, but adult mosquitoes could enter/exit through at least
one small hole. They are usually treated against mosquito
larvae, but apparently this had not happened for some time,
as huge larval densities consisting of many thousands of
larvae occurred in several of these basins. Sizes of the basins
varied, but most measured approximately 3 m deep, 3 m
long and 2 m wide.
Thousands of adult mosquitoes were present at the
three locations, resting at the walls of the basins. These were
collected using mouth aspirators for biotype identification.
Two adult specimens were collected outside the waterbasins.
It was noted that while collecting, one of the researchers
received numerous mosquito bites.
DNA isolation
After identification, the collected mosquitoes were
stored at –80° C until analysis. Individual specimens were
ground with pestles in liquid N2
and homogenized in 600
µl RLT using a Qiashredder homogenizer according to the
instructions of the manufacturer (Qiagen Inc.). Half of
the homogenate was used for DNA extraction using the
tissue protocol of the QIAamp DNA minikit (Qiagen Inc.)
starting immediately with incubation at 70° C for ten min.
As a quality control for the isolation step, each DNA isolate
was checked by an actin polymerase chain reaction (PCR)
as described by Scholte et al. (2008).
Differentiation between biotypes
Molecular identification of the two biotypes of the
Cx. pipiens complex and hybrid populations was done
using a method based on indels in the flanking region of
microsatellite locus CQ11 (Bahnck and Fonseca 2006).
Specimens of a laboratory mosquito colony of Cx. pipiens
from Bologna (bioype molestus) and Cx. pipiens specimens
collected outdoors in the Netherlands (biotype pipiens)
served as controls.
RESULTS AND DISCUSSION
A total of 29 adult specimens, collected at three different
underground stations in Amsterdam were morphologically
identified as members of the Cx. pipiens complex and were
further typed by PCR. Figure 1 shows a typical result for
typing Cx. pipiens specimens at the biotype level (Bahnck
and Fonseca 2006). Of the analyzed specimens, two were
identified as biotype pipiens, 18 specimens as biotype
molestus, and nine specimens as hybrid forms. These results
wereconfirmedbysequencingofthePCRproductsfromtwo
biotype molestus specimens, one biotype pipiens specimen,
and two hybrid specimens. No other polymorphisms were
observed in the amplified region of the CQ11 gene (data
not shown).
In this study, we provide the first evidence for the
presence of Cx. pipiens b. molestus and b. pipiens hybrids
in northern Europe and Cx. pipiens b. molestus in the
Netherlands. Interestingly, the different biotypes and
hybrids co-existed in the same underground breeding sites.
Sympatric occurrence of biotype molestus and pipiens
in surface habitats has been observed before in southern
Europe and the U.S.A. (Chevillon et al. 1995, Fonseca et al.
2004, Gomes et al. 2009) but never for underground habitats
as observed in this study.
The distinction among the members of the Cx.
pipiens complex is essential for understanding local WNV
epidemiology. The two biotypes and hybrid form of the
Cx. pipiens complex are thought to have different feeding
preferences and, consequently, different vectorial capacities.
Observations on hybrids in the U.S.A. suggest a genetic
basis for feeding preferences of members of the Cx. pipiens
complex. Biotype pipiens females that fed upon mammals,
in particular humans, had a significantly higher proportion
of biotype molestus ancestry than females that fed upon
birds (Huang et al. 2008, Kilpatrick et al. 2007). In the field,
hybrids appeared to bite readily both humans and birds as
an opportunistic feeder (Fonseca et al. 2004). Therefore,
hybrids between the molestus and pipiens biotypes may act
as bridge vectors with a major contribution to human WNV
epidemiology. It has been hypothesized that the presence of
these opportunistic feeders in the Cx. pipiens complex in
1M 1 2 3 4 65 7 8 9 10M
300 bp 
200 bp 
1M 1 2 3 4 65 7 8 9 10M 1M 1 2 3 4 65 7 8 9 10M
300 bp 
200 bp 
Figure 1. Example of DNA fragments amplified from Cx. pipiens specimen collected at underground stations in Amsterdam,
the Netherlands, using the typing method described by Bahnck and Fonseca (2006). Fragments amplified using either a
biotype pipiens-specific PCR (lanes 1, 3, 5, 7, and 9) or a biotype molestus-specific PCR (lanes 2, 4, 6, 8, and 10) were run on
a 2% agarose gel. Biotype molestus (lanes 1 and 2), biotype pipiens (lanes 3 and 4), hybrid (lanes 5 and 6), control Cx. pipiens
b. pipiens from Aalsmeer, the Netherlands (lanes 7 and 8), and control Cx. pipiens b. molestus from the laboratory colony
from Bologna, Italy (lanes 9 and 10). Lane M: 100-bp size marker (Fermentas Inc., St. Leon-Rot, Germany).

3. Vol. 35, no.1 Journal of Vector Ecology 3
combination with a susceptible bird population and highly
concentrated human populations have created conditions
for human epidemics. This is illustrated by the rapid spread
and severity of the WNV epidemic in the U.S.A. since 1999
(Fonseca et al. 2004). By analogy with the circumstances in
North America, the presence of hybrids in northern Europe
might be an important determinant for the dynamics of
WNV in northern Europe. For risk assessment purposes,
information on the geographic distribution, proportion
within the Cx. pipiens complex population, and phenotypic
properties of the European (Dutch) hybrids in more detail
is essential. The public transport company has intensified
the treatments and is improving the basins built in order
to prevent development of larvae and possible entrance or
escape of adults.
Acknowledgments
We are indebted to Anna Medici (Centro Agricoltura
Ambiente Giorgio Nicoli s.r.l., Crevalcore, Bologna, Italy)
for Cx. pipiens mosquitoes that served as controls in the
analysis. We thank employees of the Amsterdam public
transport company (GVB) for their kind cooperation in
visiting the underground metro locations. Joke van der
Giessen is thanked for critical reading of the manuscript.
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