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Acta Tropica
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a r t i c l e
i n f o
Article history:
Received 27 March 2015
Received in revised form
29 September 2015
Accepted 5 October 2015
Available online 20 October 2015
Keywords:
Culex pipiens
Mosquito trap
West Nile
Arbovirus surveillance
Physiological age
a b s t r a c t
The selection of the ideal trap for arbovirus surveillance is an issue of primary importance to increase
the sensitivity of virus detection and the cost-effectiveness of the entomological surveillance. During
the summer 2011, the effectiveness of ve types of mosquito traps (CDC gravid trap, CO2- baited trap,
BG-SentinelTM and two experimental prototypes) to attract females potentially infected with West Nile
virus were assessed. The study was carried out in three natural wetland sites located in the EmiliaRomagna Region (Northern Italy), using a Latin square scheme. Single night collections of adult females
were performed and determination of species and physiological state (gravid, nulliparous or parous) was
made upon return to the laboratory. The species most frequently collected in the gravid trap was Culex
pipiens sl. L., being gravid females the large majority of the individuals. Species diversity was much higher
in CO2- baited traps, which may therefore enable a more comprehensive description of the vector species
composition and their role in arboviruses circulation. Our ndings indicate that gravid traps can be a
valid tool and should be integrated in the West Nile virus surveillance system in the Emilia-Romagna
region, mainly based on collections made with CO2 -baited traps.
2015 Elsevier B.V. All rights reserved.
1. Introduction
The increasing international trade and tourism, and the globalization of work are rapidly changing the distribution of arboviruses
worldwide, posing new concerns on public health due to the
increased risk of mosquito-transmitted arboviruses. In Europe,
the most dangerous mosquito-borne viruses are the four dengue
serotypes (DENV) causing the dengue fever, and the West Nile
virus (WNV) that can cause lethal encephalitis, as well as the Usutu
(USUV) and Bagaza viruses (BAGV) (Agero et al., 2011; Roiz et al.,
2012a; Vazquez et al., 2011) all belonging to the Flaviviridae family.
WNV lineage 1 has been responsible for repeated disease outbreaks
in the Mediterranean basin over the past 50 years. In 2004, and in
subsequent years, the WNV lineage 2 appeared to spread throughout Hungary and Austria, and subsequently emerged in Greece in
2010 and in Italy in 2011, involving outbreaks on the Italian mainland and Sardinia. Further spread through the Balkan countries is
also suspected (Hernndez-Triana et al., 2014). WNV is transmitted
in an avian cycle by ornithophilic mosquitoes, chiey of the genus
Corresponding author at: Via Argini Nord, 335140014, Crevalcore (BO) Italy.
E-mail address: bmaccagnani@caa.it (B. Maccagnani).
http://dx.doi.org/10.1016/j.actatropica.2015.10.002
0001-706X/ 2015 Elsevier B.V. All rights reserved.
94
virus detection. WNV can spread very quickly and easily, and may
threaten humans health before its detection (Kramer et al., 2008).
Bustamante and Lord (2010), using a model that simulates the process of mosquito sampling, pooling, and virus testing, found that
mosquito infection rates commonly underestimate the prevalence
of arbovirus infection in a mosquito population. They conclude that
other factors, like mosquito population size, age structure, weather
and historical baseline data have to be considered to assess the risk
of arbovirus transmission. According to Bellini et al. (2014a), the
development of surveillance programs and vector control strategies
in European regions based on data obtained from studies performed in North America is not entirely appropriated (and also
not feasible due to different legislative frameworks). The diversity of the susceptible bird fauna and the vector species involved
in the enzootic and tangential transmission of WNV in Europe,
pose a situation remarkably different to that in US (Bellini et al.,
2014a). The improvement of entomological surveillance methods
and programs can increase the reliability of the risk assessment and
contribute to reduce the gap between infection rate estimates and
the risk of arbovirus transmission to humans and animals (Gu et al.,
2008; Roiz et al., 2012b).
To develop an efcient vector surveillance system it is necessary to gain knowledge about the following issues: (i) which
environmental conditions are necessary for an outbreak to occur;
(ii) biology and ecology of the main vector species in the areas
potentially at risk; (iii) the efciency of the different types of
trap in attracting the vector species; (iv) the physiological age of
the captured females (i.e. nulliparous and parous females) (Hugo
et al., 2008). Recognition of parous females is important because
it implies females had completed at least one gonotrophic cycle
and had taken a blood meal, with the chance to be infected in
case the host is viraemic. In addition, the identication of gravid
females is also important, because having a batch of eggs ready
to be laid implies they already took a blood meal, independently
of the physiological age. In the same way, the presence of blood
in the stomach is an indication of a potentially infectious female.
Thus, mosquito traps capturing a higher proportion of potentially
infectious females will enhance the probability to predict a disease
outbreak (Bellini et al., 2014a,b). Concerning the efciency of different models of traps in attracting mosquito females, Kesavaraju
et al. (2011) and Allan and Kline (2004) compared some commercial models of gravid traps with structural differences and found
that several characteristics signicantly affect mosquito collection
efcacy. Commercial gravid traps differ in basic design, color and
size of the tank that contains the infusion, giving different capture results (Allan and Kline, 2004, Dennet, 2007; White et al.,
2009). Moreover, different infusions (aquatic grasses like Juncus
effusus L., Rhyncospora corniculata (Lamarck) and Typha latifolia L.,
cow manure, mix of grass clippings, wheat straw, rabbit chow)
can attract different mosquito species depending on the season
(Burkett and Mullen 2008; Jackson et al., 2005; McPhatter et al.,
2009). According to Williams and Gingrich (2007), the use of gravid
traps could give better results for West Nile virus surveillance over
light traps or resting boxes. Many types of traps use carbon dioxide as the primary attractant. The produced plume of CO2 mimics
human exhalation and thus makes these traps quite specic for
capturing blood-seeking females. CO2 traps allow the collection
of large numbers of mosquitoes and appear to be highly attractive to a wide variety of mosquito species. CO2 traps are widely
used in Italy for vectors monitoring and surveillance (Bellini et al.,
2003; Calzolari et al., 2010). Traps called resting boxes are passive devices that serve as shelters for mosquitoes during the day
(Crans 1995). They have been used to sample mosquito populations
since the time of the malarias control programs, and still they are
successfully used for monitoring many Anopheles species (Kweka
et al., 2010; Pombi et al., 2014). However, they perform very dif-
95
according to the experimental scheme reported in Table 1 (5 collections in total). In the ve stations the traps were simultaneously
activated at 6:00 PM and stopped on the next morning at 9:00
AM. The collected mosquitoes were taken to the laboratory to be
counted and prepared for analyses. Mosquito species determination was performed using the taxonomic keys of Schaffner et al.
(2001) and Becker et al. (2010).
2.4. Potentially infectious females
To the aim of this study, we considered at risk to be infectious
all the females with a hint of having taken a blood meal. 3070 Cx.
pipiens females per collection date per trap were analyzed, and in
instances where collections were less than 30 Cx. pipiens females,
all the specimens were used for the study. The pools were stored
at 20 C until dissection.
Females were classied as nulliparous or parous following
the ovarian tracheation technique described by Detinova (1962).
Mosquito dissections were performed in a drop of saline solution
on a glass microscope slide using a stereomicroscope. With the aid
of an entomological needle, ovaries were separated from the rest of
the surrounding tissues, placed in a drop of clean de-ionized water,
and allowed to air dry to reveal ovary tracheation. Using a compound microscope, females were classied as parous if they had
uncoiled tracheoles (Fig. 3A), as nulliparous if they had coiled tracheoles (skeins) (Fig. 3B). The presence of completely developed
eggs prevented tracheoles visualization, and it was not feasible
to determine the parity status, thus females were classied as
gravid females. When other causes did not allow establishing the
parous condition, females were categorized as non-determined.
In addition, we recorded the presence of blood in the stomach
Table 1
Experimental scheme.
Fig. 2. The ve types of traps. (A) Gravid trap, (B) CAA2004 CO2 -baited trap, (C)
BG-Sentinel trap, (D) Resting trap 1 RT001, (E) Resting trap 3 RT003.
Site
Period
Frequency of
collection
No.
collections
La Rizzaa
Le Vallette
Oasi Val di Sole
June 24July 8
July 15August 5
August
19September 16
5
5
5
96
Fig. 3. Ovaries of Culex pipiens s.l.. (A) parous females ovary, (B) nulliparous females ovary. 200X.
transformation was necessary to satisfy the variances homogeneity requirements for 2-way ANOVA. Study sites and types of trap
were set as the main factors. The signicance level for statistics was
stated at alpha = 0.05. Analysis were performed using Statistica 7.0.
3. Results
3.1. Species composition in the three study sites
The total number of mosquitoes collected in the three sites was
18,760, 18,036 of which were females and 724 males. Considering
all the mosquito species, 6,204 mosquito females were captured in
La Rizza, 5,796 in Le Vallette, 6,036 in Oasi Val di Sole. The mosquito
species were those typically found in the Po plain rural areas: Cx.
pipiens, Cx. modestus Ficalbi, Aedes caspius Pallas, Ae. vexans Meigen,
Ae. cinereus Meigen, Ae. albopictus Skuse and Anopheles maculipennis
s.l. Meigen. The large majority of the specimens (85.0% of the males
and 86.7% of the females) belonged to the species Cx. pipiens. In
Table 2, for each site of study and for each trap type, the mean number of females per species per sampling day is reported. In La Rizza
Cx. pipiens accounted for 96.7% of the total number of catches, while
2.1% were Ae. vexans; being present the other species cited above
with percentages well below 1%. This was the only site in which
Table 2
Collection capacity of different trap types to different mosquito species: mean number of females per site per trap.
Site
Species
SD
Mean
La
Rizza
Ae. albopictus
Ae.caspius
Ae.cinereus
Ae.vexans
An. maculipennis
Cx. modestus
Cx. pipiens
0.0
6.6
4.8
27.8
0.8
0.8
536.0
0.0
3.1
10.7
36.7
1.8
1.3
368.3
0.5
0.0
0.0
0.0
0.0
0.0
724.0
Le
Vallette
Ae. albopictus
Ae.caspius
Ae.cinereus
Ae.vexans
An. maculipennis
Cx. modestus
Cx. pipiens
1.0
145.8
0.0
3.6
2.2
4.2
468.4
1.0
238.2
0.0
3.4
2.4
4.1
336.2
1.6
0.0
0.0
0.6
2.6
0.0
456.4
Oasi
Val
di
Sole
Ae. albopictus
Ae.caspius
Ae.cinereus
Ae.vexans
An. maculipennis
Cx. modestus
Cx. pipiens
0.0
59.4
0.0
0.8
0.8
191.8
344.6
0.0
46.2
0.0
1.3
1.3
98.4
129.1
0.0
0.0
0.0
0.4
1.6
0.8
589.2
BG-S
SD
RT001
Mean
SD
0.2
0.0
0.0
0.0
0.2
0.2
78.2
0.5
0.0
0.0
0.0
0.5
0.5
120.2
0.0
0.2
0.0
0.0
0.0
0.0
1.4
0.0
0.5
0.0
0.0
0.0
0.0
2.61
0.0
0.0
0.0
0.0
0.2
0.0
5.0
0.0
0.0
0.0
0.0
0.5
0.0
8.6
1.67
0.0
0.0
0.89
2.3
0.0
327.8
3.8
0.0
0.0
0.0
3.8
0.4
20.8
3.8
0.0
0.0
0.0
6.9
0.9
10.8
0.0
0.2
0.0
0.0
6.2
0.0
15.6
0.0
0.45
0.0
0.0
10.5
0.0
5.7
0.4
0.0
0.0
0.0
1.2
0.2
20.4
0.9
0.0
0.0
0.0
0.8
0.5
11.5
0.0
0.0
0.0
0.5
1.5
1.10
404.1
0.2
0.2
0.0
0.0
1.2
1.6
6.4
0.5
0.5
0.0
0.0
1.6
1.1
4.6
0.0
0.4
0.0
0.0
0.8
0.0
2.0
0.0
0.0
0.0
0.0
1.4
0.0
3.6
0.0
0.0
0.0
0.0
2.1
0.0
4.3
1.0
0.0
0.0
0.0
0.0
0.0
1,244.3
Mean
RT003
SD
0.0
0.5
0.0
0.0
1.1
0.0
2.0
Mean
SD
4. Discussion
The entomological surveillance plays an important role in
vector-borne disease surveillance thanks to the capability to provide early detection of arboviruses circulation (Gu et al., 2008;
Almeida et al., 2008; Bellini 2014a,b). Many different mosquito
2000
No. individuals
2400
1600
1200
800
400
0
BG S CO2 RT001 RT003 Gr T
Median
25%-75%
Non-Outlier Range
No. individuals
600
400
200
0
-200
No. individuals
Ae. cinereus was captured, on 29/06/2011 with the CO2- baited trap
CAA2004 (0.39%). In Le Vallette 84.7% of the females were Cx. pipiens, while Ae. caspius accounted for 12.6%, and An. maculipennis s.l.
for 1.4%; Ae. cinereus was not present, while the other species were
below 1%. In Oasi Val di Sole 78.4% of the collected females were
Cx. pipiens, 5.0% were Ae. caspius and 16.1% were Cx. modestus; all
the other species were present at percentages below 1%.
97
800
600
400
200
0
BG S CO2 RT001 RT003 Gr T
Median
25%-75%
Non-Outlier Range
Fig. 4. AC. Median number of Cx. pipiens females per trap per night in the three
study sites. (A) La Rizza, (B) Le Vallette, (C) Oasi Val di Sole. (Software: Statistica 7.0).
98
Le Vallette
140
120
100
80
60
40
20
0
-20
Gravid trap
CAA2004
Fig. 5. Percentage of potentially infectious females in the pools formed from the
captures of the Gravid trap and of the CAA2004 CO2 -baited trap in two study areas.
The following females categories were considered potentially infectious: all the
parous females, nulliparous females and females of undetermined parity status with
blood in the stomach, gravid females. (A) Le Vallette, (B) Oasi Val di Sole. (Software:
Statistica 7.0).
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