West Nile virus vector competency of Culex quinquefasciatus mosquitoes in the Galapagos Islands.

Abstract

The mosquito-transmitted pathogen West Nile virus (WNV) is not yet present in the Galápagos Archipelago of Ecuador. However, concern exists for fragile endemic island fauna after population decreases in several North American bird species and pathology in certain reptiles. We examined WNV vector competency of a Galápagos strain of mosquito (Culex quinquefasciatus Say). Field specimens were tested for their capacity to transmit the WN02-1956 strain of WNV after incubation at 27°C or 30°C. Rates of infection, dissemination, and transmission all increased with days post-exposure to WNV, and the highest rates were observed at 28 days. Infection rates peaked at 59% and transmission rates peaked at 44% (of mosquitoes tested). Vector efficiency increased after day 14. Rates of infection but not of transmission were significantly influence by temperature. No vertical transmission was detectable. We demonstrate that Galápagos Cx. quinquefasciatus are competent WNV vectors, and therefore should be considered an animal and public health risk for the islands and controlled wherever possible.

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Am. J. Trop. Med. Hyg., 85(3), 2011, pp. 426–433
doi:10.4269/ajtmh.2011.10-0739
Copyright © 2011 by The American Society of Tropical Medicine and Hygiene
INTRODUCTION
West Nile virus (WNV; family Flaviviridae, genus Flavivirus )
is a mosquito-borne virus that was originally isolated in Africa
in 1937 but is now established across six continents 1 and con-
sidered as causing an emerging infectious disease. This virus
was successfully introduced to the Americas (New York City)
in 1999, and although it subsequently spread west and south
with unprecedented intensity, and infected wildlife on several
Caribbean islands, 2, 3 its distribution across South America is
largely unknown. There are relatively few reports of WNV
activity in South America, with several hypotheses as to why,
including cross-protection of circulating viruses, lack of sur-
veillance, and dilution effect from increased biodiversity. 1
However, WNV was isolated from horses in Argentina in
2006, and infection was serologically identified in horses in
Colombia in 2004 and birds in Venezuela in 2005, 4– 6 the lat-
ter being the first confirmation of establishment on the South
American mainland. The status of WNV in mainland Ecuador,
and the impact on its island territory six hundred miles west,
the Galápagos Archipelago, has yet to be determined. This
study forms part of a more comprehensive investigation into
the extent of the threat posed by WNV to Galápagos; the dis-
tribution, abundance, and ecology of potential mosquito vec-
tor species in Galápagos, including Culex quinquefasciatus , is
additionally being evaluated.
The wildlife species of the islands of Galápagos ( Figure 1 )
embrace a distinct biodiversity that evolved in isolation over
thousands of years, with high levels of endemism in the flora
and fauna. However, the islands unique ecosystems are faced
with modern pressures from an increasing level of human
population, tourism, invasive species, and disease introduc-
tion. 7 Such pressures led to international conservation con-
cern for the deteriorating quality of the island ecosystems
and, as a United Nations Educational Scientific and Cultural
Organization World Heritage Site, Galápagos was, until
recently, listed in the category ‘under danger’. 8 Disease threats
are one particular concern and several disease agents includ-
ing avian pox virus and Salmonella spp. have already reached
Galápagos with negative impacts on the native wildlife. 9– 11 The
isolation of Galápagos wildlife implies they lack prior expo-
sure to many pathogens (i.e., they are immunologically naive)
and as such may show heightened susceptibility to WNV. 12
West Nile virus is maintained in nature in an avian host–
mosquito vector enzootic cycle. 13 Although the highest vire-
ias generally occur in birds, the main amplification host,
WNV is a multi-host pathogen capable also of infecting
mammals (including humans) and reptiles. Galápagos has
22 endemic reptile species, some already considered endan-
gered. Of the 326 avian species reported with WNV infections
in the United States, approximately 39 have been observed in
Galápagos (e.g., Pelecanus occidentalis) and additional spe-
cies exist as related congenic populations (e.g., mockingbirds
of the family Mimidae ). 14 West Nile virus is linked to popu-
lation decreases of several North American bird species. 15, 16
There fore, this virus represents a substantial threat to the native
species of Galápagos should WNV reach the archipelago. 17
The most likely pathway for a WNV introduction event to
Galápagos is predicted to be human mediated, chiefly by the
transport of infected mosquitoes to the islands by airplane. 18
Invasive insects are known to enter Galápagos by this route
(approximately one-fourth of all insect fauna in Galápagos is
alien 19 ). Recent work detected Culex quinquefasciatus mos-
quitoes inside aircraft arriving to Galápagos (Baltra airport),
and genetic analysis indicates there have been multiple intro-
ductions of this mosquito although pathogen infection status
was not ascertained. 20 Culex spp. draw particular attention for
those monitoring WNV outbreaks because they are the princi-
pal vectors; for example, accounting for more than 98% of all
mosquito pools positive for WNV in surveillance work in the
United States. 21
Three species of mosquito are currently resident in Galápagos.
Aedes taeniorhynchus is ubiquitous and naturally colonized the
archipelago approximately 200,000 years ago 22 (Eastwood G,
unpublished data). Aedes aegypti is a recently introduced
anthropophilic mosquito that because of its associations
with dengue viruses has been largely controlled. Culex
West Nile Virus Vector Competency of Culex quinquefasciatus Mosquitoes in the
Galápagos Islands
Gillian Eastwood ,* Laura D. Kramer , Simon J. Goodman , and Andrew A. Cunningham
Institute of Zoology, Zoological Society of London, London, United Kingdom; Institute of Integrative and Comparative Biology,
University of Leeds, Leeds, United Kingdom; Galápagos Genetics, Epidemiology and Pathology Laboratory, Puerto Ayora, Santa Cruz,
Galápagos Islands, Ecuador; Wadsworth Center, New York State Department of Health, Slingerlands, New York
Abstract. The mosquito-transmitted pathogen West Nile virus (WNV) is not yet present in the Galápagos Archipelago
of Ecuador. However, concern exists for fragile endemic island fauna after population decreases in several North American
bird species and pathology in certain reptiles. We examined WNV vector competency of a Galápagos strain of mosquito
( Culex quinquefasciatus Say). Field specimens were tested for their capacity to transmit the WN02-1956 strain of WNV
after incubation at 27°C or 30°C. Rates of infection, dissemination, and transmission all increased with days post-exposure
to WNV, and the highest rates were observed at 28 days. Infection rates peaked at 59% and transmission rates peaked at
44% (of mosquitoes tested). Vector efficiency increased after day 14. Rates of infection but not of transmission were sig-
nificantly influence by temperature. No vertical transmission was detectable. We demonstrate that Galápagos Cx. quin-
quefasciatus are competent WNV vectors, and therefore should be considered an animal and public health risk for the
islands and controlled wherever possible.
* Address correspondence to Gillian Eastwood, Wildlife Epidemiol-
ogy, Institute of Zoology, Zoological Society of London, Wellcome
Building, Room 29, Outer Circle Regents Park, London, NW1 4RY,
United Kingdom. E-mail: gillian.eastwood@ioz.ac.uk

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WNV VECTOR COMPETENCY OF CX. QUINQUEFASCIATUS IN GALÁPAGOS
quinquefasciatus , a wide-ranging semi-tropical/tropical species
associated with fresh water, had been introduced to Galápagos
by 1985 and is now established around areas of human habita-
tion. 20, 23 Culex quinquefasciatus also has been recently found
in uninhabited agricultural or tortoise reserve zones away
from human habitation, in the highlands of Santa Cruz, San
Cristobal, Isabela, and Floreana islands (Eastwood G, unpub-
lished data). The invasive nature of Cx. quinquefasciatus was
demonstrated by a study at the Miami, Florida, airport, which
examined 102 arriving aircraft and found live specimens on
28 of these. 24 In Hawaii, an oceanic island ecosystem similar
to Galápagos, Cx. quinquefasciatus is an introduced vector for
the agents of avian malaria and avian pox, co-introduced dis-
eases associated with catastrophic decreases and extinctions
of endemic avifauna. 25, 26
Culex quinquefasciatus is of key importance to the risk of
WNV introduction and establishment in Galápagos for sev-
eral reasons. First, it is a primary enzootic vector for WNV
in North America 1, 13 (expanded details below). Second, it
is associated with outbreaks of related arboviruses. 27 Third,
its feeding behavior shows preferences for bird and mam-
mal blood. 28, 29 Although local feeding patterns in Galápagos
require further investigation, the broad diet of Cx. quinque-
fasciatus known from elsewhere indicates that this mosquito
would act as a bridge vector for WNV between bird and mam-
mals in Galápagos. 23, 30, 31
North American Cx. quinquefasciatus populations are
reported as competent vectors of WNV, and in southern
United States this is the principal mosquito species associ-
ated with WNV. 28, 30 Infection rates of up to 100% have been
observed (Florida and Texas), and high WNV transmission
rates of up to 52% (southern California), calculated as a pro-
portion of the number of mosquitoes tested. 30, 32– 35 There are
noticeable influences on mosquito susceptibility to WNV
infection including extrinsic (e.g., initial viral dose, temper-
ature, and length of incubation) and intrinsic (e.g., genetic
strain, midgut physiology) factors. 32, 36– 38 Generally, longer incu-
bation periods, higher viral doses, and higher temperatures
are expected to yield higher rates of infection. 30, 32, 39 However,
Cx. quinquefasciatus in Australia was shown to be a highly
competent vectors when exposed to a dose of only 10 4.0±0.3
plaque-forming units (PFU)/mL of WNV (NY99 strain). 40
Extensive geographic variation can arise in the rates of basic
infection, dissemination beyond the midgut, and salivary
transmission of WNV, even within the same state in the United
States. For example, in California under comparable test con-
ditions, one population of Cx. quinquefasciatus was refractory
to infection, whilst populations from adjacent counties reached
rates of 43% infection and 30% transmission. 32 Similarly,
WNV vector competence of the sibling species Cx. pipiens
Linnaeus, was shown to vary spatially and temporally in New
York and Massachusetts. 41 If one considers vertical transmis-
sion of WNV from parent to progeny, minimum filial infection
rates of approximately 3% are reported for Cx. quinquefascia-
tus in the United States. 42
Little is known about South American Cx. quinquefasciatus ,
including its potential to play a significant role as a vector in
WNV epidemiology in Galápagos. The available literature sug-
gests that we cannot make assumptions about the WNV vector
competency of Galápagos Cx. quinquefasciatus on the basis
of studies conducted on the species elsewhere in the world. If
we are to accurately model how Galápagos populations would
interact with WNV, it is imperative to determine the unique
vector characteristics specific to this strain.
MATERIALS AND METHODS
Mosquitoes. Field populations of Cx. quinquefasciatus were
collected from highland and coastal areas of Santa Cruz and
Isabela islands of Galápagos by using mainly gravid traps
(CO 2 -baited light traps were also used). The resulting egg
rafts deposited by gravid females and first-instar larvae were
transported under U.S. Department of Agriculture (no. 47279)
and Centers for Disease Control and Prevention (no. 2009-
06-182) permits to the Wadsworth Center Arbovirus Labora-
tory (New York State Department of Health) during 2009.
Mosquitoes were reared to adult stage in a walk-in chamber in
a BioSafety Level 2 quarantine insectary main tained at 28°C
with a 12 hour:12 hour (light:dark) photoperiod and a relative
humidity of 85% to simulate natural conditions in Galápagos.
Experiments to evaluate WNV vector competence took place
under BioSafety Level 3 conditions.
Infection with WNV. Once maturity and mating were
achieved (allowing 96 hours after emergence of adult females),
mosquitoes were fed a viral preparation by using the WNV
strain WNV02-1956 isolated from an American crow. The virus
had been passaged once in African green monkey kidney (Vero)
cells and amplified once in Ae. albopictus (C6/36) cells to yield
a stock concentration of 9 log 10 PFU/mL, which was stored
at –80°C in multiple aliquots. To encourage feeding, blood
meals consisting of diluted (1:20) virus suspended in 9 mL of
defibrinated goose blood containing 50% sucrose and 100 μL
(0.2 mM) ATP were presented to mosquitoes overnight in a
sausage skin membrane and also soaked onto gauze pledgets.
Prior experience showed this combined technique to yield
higher feeding success in field mosquitoes. The virus level was
determined both pre-feeding and post-feeding to ensure titers
had remained constant during overnight feeding. Infected
blood meals had a median WNV dosage of 7.7 log 10 PFU/mL.
The titer used was relatively high; comparative studies showed
a titer range from 4.1 log 10 PFU/mL 40 to > 8.0 log 10 PFU/mL. 32
However, such virus levels realistically occur in nature in
passerine birds. 43
After feeding, mosquitoes were anaesthetized with CO 2 ,
and engorged female mosquitoes were separated from unfed
F igure 1. Map of the Galápagos Islands showing proximity to
mainland Ecuador and South America.

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EASTWOOD AND OTHERS
or male mosquitoes and sorted randomly into groups incu-
bated at either 27°C or 30°C. Mosquitoes were held at the
relevant temperature in mesh-topped 0.47-liter cartons with
access to 10% sucrose solution ad libitum until transmission
testing. Light cycling was 12 hour:12 hour (light:dark) at 85%
relative humidity for the duration of each incubation period.
An ovicup was included in each carton to harvest F 1 mosqui-
toes for vertical transmission testing.
Because viral development changes over time and is lia-
ble to interact non-linearly with temperature, 36, 38, 44 we chose
to examine the status of mosquito infection and transmis-
sion ability and consider the effect of (i)incubation time
post-exposure and (ii)temperature variation. The Galápagos
ecosystem experiences seasonal variation with a hot, humid,
rainy season with temperatures exceeding 30°C in January–
April. In contrast, June–September are cooler months with
temperatures less than 26°C. Variation in climate influences
host–pathogen interactions and, plausibly, disease dynamics,
varying host-contact rates or the duration in which infections
develop. 45 It is therefore reasonable to hypothesize that the
WNV vector competency of Galápagos Cx. quinquefasciatus
may vary over the course of a year.
Transmission testing. Mosquito groups at each temperature
(27°C or 30°C) were held for an extrinsic incubation period
(EIP) of 7, 11, 14 or 21 days post-feeding to determine the
influence of time on the extent of infection and/or transmission
of WNV. A single group held at 27°C only was included 28 days
post-exposure; survival rates of Cx. quinquefasciatus at 30°C
restricted testing beyond 21 days for the higher temperature.
At each time point, approximately 30 mosquitoes from each
temperature group were immobilized by using triethylamine
(Sigma, St. Louis, MO). To investigate WNV infection status,
bodies of mosquitoes were examined for midgut infections.
Legs were used to check for disseminated infections spread-
ing beyond the midgut as described. 46 Briefly, mosquito legs
were first removed from bodies and separately triturated
in a microfuge tube containing 1 mL of mosquito diluent
(phosphate-buffered saline, 20% fetal bovine serum [FBS],
and antibiotics) and a ball-bearing. To check for transmission
ability, salivary secretions were obtained by using a modi-
fied in vitro capillary transmission assay 36, 47 in which the mos-
quito proboscis is inserted into a capillary tube containing a
1:1 mixture of FBS and 50% sucrose. This procedure invokes
a reflex action by the live mosquito to secrete its saliva into
the solution and after 30 minutes the contents of the capillary
are dispensed into 300 μL of mosquito diluent. All samples
were stored at –80°C. After homogenization by using a grinder
mill at 24 cycles/seconds for 30 seconds and centrifugation at
12,000 rpm, the resultant suspensions were screened for virus.
Virus assay. A plaque assay was used to screen mosquito
body, legs, and salivary sample for infectious virus by using six-
well Vero cell culture plates (Costar, Corning, NY) basically
as described by Payne and others. 48 Briefly, cell monolayers
were inoculated with 100 μL of each sample, then incubated at
37°C in an atmosphere of 5% CO 2 for 1 hour to enable virus
to enter cells. An overlay of 10% FBS Eagles-Agar containing
fungizone (amphotericin B) to limit contamination, was then
added. After an additional incubation for 48 hours at 37°C,
a second overlay of 2% FBS containing 2% neutral red was
applied. Following a further 12 hours of incubation plates
were examined for development of plaques to determine the
presence of virus in each sample.
Vertical transmission. The F 1 offspring obtained from field-
collected mosquitoes that had been infected with WNV in the
above experiments were reared to adulthood and tested in
8 pools of up to 30 mosquitoes in each pool for WNV by using
the plaque assay described.
Statistical analysis. Infection rates were calculated as the
percentage of mosquitoes tested in each group that were
found to contain WNV in their body. An infection limited
to the midgut was determined to have taken place when the
mosquito body sample showed a positive plaque assay result
but the corresponding leg sample showed a negative result.
Dissemination rates were calculated as the percentage of all
mosquitoes tested with virus-positive leg samples. Recovery
of virus in the salivary secretion of mosquitoes indicated that
the virus could be transmitted by bite. Thus, transmission rates
were calculated as this proportion, of all mosquitoes, that
would be competent vectors for WNV. Confidence intervals
at the α = 0.05 level of significance were calculated for rates
of vector competency or disseminated infection in accordance
with the Wald interval (
zp
a
ˆ
2/
±
Generalized linear regression (GLM) models based on esti-
mation of binomial parameters were constructed to compare
infection, dissemination, and transmission rates across EIP
and temperature to determine if these parameters resulted
in any significant differences. The fitted GLM models were
used to fit predicted future rates according to varying temper-
ature and EIP.
np
/ ) ˆ
p
1 ( ˆ
-
).
RESULTS
Groups of Galápagos Cx. quinquefasciatus F 0 mosquitoes
collected in Galápagos were tested for WNV vector compe-
tency at two temperatures (27°C or 30°C) and various time
periods (7, 11, 14, 21, or 28 days). Each group showed some
degree of midgut infection and further dissemination of WNV
( Table 1 ). Overall WNV infection rates of exposed mosqui-
toes averaged 35.5% across all time points from 7 to 28 days
post-feeding.
To test the hypothesis that the rates of infection, dissemina-
tion, and transmission increased as a function of temperature
and days post-feeding, a regression model was constructed.
This model also generated predictive rates for Galápagos Cx.
quinquefasciatus WNV vector competency on the basis of
influences found to be significant. Binomial data, i.e., a posi-
tive or negative result for the viral screening of each tissue
suspension, were weighted to eliminate any effects of size
variation. Overdispersion of generalized linear models was
avoided by fitting a quasi-binomial model (logit function)
where necessary.
Effect of incubation period. An increase in the rates of
infection, virus dissemination, and transmission were evident
with increasing period post-feeding ( Table 1 ). Longer EIPs,
which enabled WNV to replicate further within mosquitoes,
significantly increased detection for percentage infected (F =
8.01, degrees of freedom [df] = 7, P < 0.05), percentage with
disseminated infections (F = 20.4, df = 7, P < 0.01), and the
percentage transmitting (F = 20.3, df = 7, P < 0.01).
Effect of temperature. Although there appeared to be
increased WNV development at the higher of the two
temperatures tested ( Figure 2 ), this effect was only found
to be significant for basic infection rates. As confirmed by
the associated models, rates of disseminated infection and

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WNV VECTOR COMPETENCY OF CX. QUINQUEFASCIATUS IN GALÁPAGOS
subsequent transmission by Galápagos Cx. quinquefasciatus
showed no significant relationship to temperature even when
taking possible interaction effects with time into consideration.
Infection rates showed a significant relationship with both
temperature (c 2 = 9.6, P < 0.05) and time post-feeding (c 2 =
14.1, P < 0.01), although no interaction between these two
factors occurred. The fitted model (Proportion Infected =
0.08 * EIP + 0.16 * Temperature, AIC = 51.4, P < 0.05) based
on incubation period and temperature produced the following
likelihoods for midguts of Galápagos Cx. quinquefasciat to
contain detectable WNV post-feeding: 21.1%/30.4% infected
(by day 7) at 27˚C/30˚C respectively, 26.9%/37.6% (day 11),
31.9%/43.4% (day 14), 45.2%/57.4% (day 21), and 59.1%
(day 28).
Dissemination of virus showed no relationship with tem-
perature. However, incubation time post-feeding significantly
influenced the extent that WNV moves beyond the midgut
epithelial cells of the mosquito (F = 20.4, df = 7 P < 0.01). The
fitted model produced the following likelihoods for WNV
dissemination rates in Galápagos Cx. quinquefasciatus : 9.4%
(day 7), 15.5% (day 11) and 22.0% (day 14), 43.4% (day 21),
and 67.6% (day 28).
No viral transmission was observed until day 11 at either
temper ature, and even then the rate of transmission was low:
3.2% at 27°C, 5.5% at 30°C. However, by day 14, 22.6% of mos-
quitoes tested could transmit WNV, and significantly higher
rates of transmission occurred over time (F = 20.3, df = 7, P <
0.01), 31.3% by day 21 (27°C), and 44% by day 28 (27°C). No
significant difference between temperatures was discernable.
Predicted WNV transmission rates obtained by our model
for Galápagos Cx. quinquefasciatus were 3.3% (day 7), 6.3%
(day 11), 10.0% (day 14), 26.9% (day 21), and 54.9% (day 28).
Lack of vertical transmission. No virus was detected in
any of the 8 pools (comprising a total of 200 mosquitoes) of
F 1 adult progeny tested to investigate vertical passage of WNV
in Galápagos Cx. quinquefasciatus .
Increased efficiency with time. If one considers those
mosquitoes already showing detectable infections, the
corresponding rate of WNV dissemination beyond the midgut
also increased significantly with incubation period (c 2 = 9.6,
df = 7, P < 0.001) and showed up to 100% efficiency, Also,
consequent rates of transmission increased (c 2 = 15.9, df = 7,
P < 0. 0001) and an efficiency up to 75% was observed ( Table 1 ).
Once the virus disseminated beyond the midgut of Galápagos
Cx. quinquefasciatus , transmission was highly likely, i.e., up to
80% of mosquitoes with disseminated-infections could then
transmit WNV.
DISCUSSION
We demonstrated WNV vector competency for a unique
population of Cx. quinquefasciatus mosquitoes within South
America. West Nile virus has yet to reach Galápagos, but with
a presence in South America, this pathogen has the potential to
reach these islands in the near future. 18 The research we report
forms part of a larger risk framework evaluating the threat
posed to the islands from this flavivirus. Knowledge of how
Galápagos strains of mosquito would interact with WNV is an
essential component of this framework. The concern underly-
ing the study was that Cx. quinquefasciatus would be a highly
competent and efficient vector for WNV, able to circulate the
virus rapidly among rare fauna, and perpetuate harmful conse-
quences of an introduction event to Galápagos. The results of
our study do not alleviate concerns because they suggest that
the strain of Cx. quinquefasciatus in Galápagos could have a
role in WNV maintenance should the pathogen be introduced
into the islands. This mosquito does show susceptibility to viral
infection from feeding on an infected blood meal, and subse-
quently approximately 50% would be expected to able to trans-
mit WNV to future hosts with an optimal timeframe of 28 days.
Prior to 14 days post-exposure to WNV, Galápagos popula-
tions of Cx. quinquefasciatus demonstrated low rates of WNV
infection and dissemination in comparison to North American
Table 1
West Nile virus Vector competence of Culex quinquefasciatus mosquitoes of Galápagos *
Temperature (°C) No. tested Days post-feedingInfection, % (95% CI)
Dissemination, % of
no. tested (95% CI)
Transmission, % of
no. tested (95% CI)
Efficiency of dissemination,
% of no. infected
Efficiency of transmission,
% of no. infected
27
27
27
27
27
30
30
30
30
57
62
31
32
27
54
55
31
20
721.1 (10–32)
21.0 (11–31)
38.7 (22–56)
50.0 (33–67)
59.3 (41–78)
38.9 (26–52)
25.5 (14–37)
54.8 (37–72)
50.0 (28–72)
5.3 (0–11)
8.1 (1–15)
32.3 (16–49)
50.0 (33–67)
55.6 (37–74)
9.3 (2–17)
12.7 (4–22)
45.2 (28–63)
40.0 (19–61)
0 25
38.5
83.3
100
93.8
23.8
50
82.4
80
0
11
14
21
28
3.2 (0–8)
22.6 (8–37)
34.4 (18–47)
44.4 (26–63)
0
5.5 (0–11)
22.6 (8–37)
20.0 (2–38)
15.4
58.3
68.8
75
0
21.4
41.2
40
7
11
14
21
* Values are percentages of mosquitoes in each group with tissues positive for West Nile virus or with virus in salivary secretion. CI = confidence interval. All groups were exposed to the WNV02-
1956 strain of West Nile virus at a median titer of 7.65 log 10 plaque-forming units/mL of blood.
F igure 2. Influence of temperature upon rates of West Nile virus
infection, dissemination (of those tested), and transmission (of those
tested) in Galápagos Culex quinquefasciatus . Dashed lines indicate
upper and lower 25% quartiles.

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EASTWOOD AND OTHERS
strains. Studies in Florida showed much higher infection and
dissemination rates at one-week post-exposure at compa-
rable or lower doses, even at lower temperatures (transmis-
sion rates were not mentioned). 30 Similarly, although studies
in California (which did cite transmission rates) showed
variability in results on the basis of comparable viral titers,
mosquitoes there tended to have higher vector competency
than those of Galápagos. 32, 34 In our own study, transmission
ability in the first two weeks of infection was less than 10%,
which suggests a lower WNV risk for Galápagos fauna if other
more-competent mosquito vectors are not present. However,
increasing rates of WNV infection, dissemination, and trans-
mission in Galápagos Cx. quinquefasciatus were observed
from 14 days onwards (until the end of the study at day 28).
Given that mosquitoes have a limited lifespan, survival issues
become more relevant with longer incubation periods, i.e., if
a longer period of time is required for virus to develop in the
mosquito body, there is reduced opportunity for vector-host
contact while the mosquito is in an infectious state. It becomes
increasingly likely that the mosquito will die before it reaches
the period of optimal transmission. Nevertheless, even at 30°C,
Cx. quinquefasciatus generally survive for longer than 28 days
(Eastwood G, unpublished data). 49 Thus, virus transmission
would be expected to occur and maintain pathogen cycling in
Galápagos, assuming susceptible vertebrate hosts are present
that support sufficiently high virus titers to infect mosquitoes.
The dosage of virus used must be taken into account for vec-
tor competency studies and should represent the range of
titers realistically found in nature. A WNV titer of 7.3–8.3 log 10
PFU/mL of blood was used in blood meal preparations and
produced infection in 39–55% of Galápagos Cx. quinquefas-
ciatus at day 14. This titer is consistent with those found in
nature and those used in other studies. However, studies else-
where have used lower doses to yield higher rates of WNV
infection in populations of Cx. quinquefasciatus . 39, 40
Contrary to evidence of vertical transmission in North
American Cx. quinquefasciatus , 42, 50 Galápagos populations
showed no transmission to progeny, which suggests that ver-
tical transmission may have a limited role in maintenance
of WNV in Galápagos. However, the number of progeny we
tested was limited and only progeny from the first oviposition
cycle, 14 days after feeding on the infectious blood meal, could
be tested. Because the mosquitoes in our study did not live
long enough to complete the second and third ovarian cycles,
it is difficult to draw firm conclusions.
South America has a unique climate, geology, avifauna, and
genetically distinct mosquitoes. 21, 51 We simulated seasonal cli-
mate variation by testing WNV vector competency at two
temperatures, and hypothesized that temperature would influ-
ence viral transmission in Galápagos Cx. quinquefasciatus .
The impact that changes in global climate would have on dis-
ease dynamics is a hotly debated topic, with some predicting
increased disease emergence and serious implications for pub-
lic health control. 38, 52 We demonstrated an environment with
higher temperatures to influence mosquito infection rates.
This suggests that if WNV emerges in Galapagos during the
hotter months of rainy season, more mosquitoes are likely to
become infected. However, there was no significant indication
in our study for transmission being influenced by temperature
(despite adequately sized samples). Therefore, despite studies
using other Culex species demonstrate even small temperature
gradients to influence vector competency, 36 it is unlikely that
seasonal temperature fluctuations in Galápagos would alter
the epidemiology of an outbreak on the islands. One excep-
tion to that conclusion is the possible transfer of WNV via the
consumption of infected mosquitoes by vertebrates, a pathway
which has been shown to produce host infection. 43 Although
this mechanism for WNV maintenance is not well docu-
mented, regular vector ingestion during hotter periods when
we predict more vectors to be a) infected, and b) increased in
abundance (Eastwood, unpublished data), could have impli-
cations for virus epidemiology. It is hence important to under-
stand vector-pathogen-host- environment interactions. 36
Although we report infection and virus dissemination rates,
it is the transmission rate that is key to determining the extent
to which a mosquito species is a competent vector. For exam-
ple, Cx. quinquefasciatus from Florida is considered to be a
competent but only a moderately efficient vector: despite high
infection rates, the virus often does not disseminate beyond
the midgut (22% being the greatest rate). 33 However, when
exposed to intrathoracic inoculation of WNV (mimicking
virus dissemination), 94% of the Florida population could
transmit WNV. 33 In the case of Galápagos Cx. quinquefascia-
tus, we were more interested in what could occur in nature on
the islands, and although only a proportion of infected mosqui-
toes showed dissemination beyond the midgut with a smaller
proportion further able to transmit virus, the vector efficiency
greatly increases over time ( Table 1 ). Our results indicate that
the Galápagos strain of Cx. quinquefasciatus , although less
competent than strains from the United States, is a competent
and efficient vector for WNV that is capable of transmission
once viral infection extends beyond the midgut.
It should be noted that even when a mosquito is capable
of transmitting virus, an infection may still not develop in the
vertebrate host on which the mosquito feeds, depending on
the viral dose transmitted and the susceptibility of that host.
Ability to induce or develop infection may be restricted until
viral titers reach a certain threshold. To clarify the WNV titer
that could be inoculated by Galápagos Cx. quinquefasciatus , a
subset of our samples were tested beyond the basic assay for
presence of virus, and the range deduced was comparable with
those of other studies (1–5 log 10 PFU/mL). 53 Therefore, a vari-
able amount of WNV could be inoculated by the vector while
they feed on live hosts.
In addition to the presence of susceptible hosts, establish-
ment of WNV in Galápagos would require hosts that also
amplify WNV. There may be reduced risk posed to systems such
as Galápagos when WNV amplification cycles are restricted
to involving only species circulating a high dose of virus. For
example, West Nile virus titers less than 4 log 10 PFU/mL rarely
appear to induce infection in Cx. quinquefasciatus (although
further experimentation with Galápagos Cx. quinquefascia-
tus to establish a 50% infectious dose is recommended). Bird
species such as chickens or psittacines rarely produce viremias
exceeding 3 log 10 PFU/mL. Thus, some species encountered in
Galápagos, for example Tyto alba (peak viremia 3.6 log 10 PFU/
mL when tested in the United States), would have a limited
role in WNV amplification by being unlikely to develop titers
high enough to infect mosquitoes. 54 However, passerine birds
have viremic profiles with the highest magnitude of titers; cer-
tain species peak at 11 log 10 PFU/mL. 43
A study to determine the host susceptibility and likely mor-
bidity of Galápagos native vertebrates is required. Galápagos
has an abundance of passerines that could be important for

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WNV VECTOR COMPETENCY OF CX. QUINQUEFASCIATUS IN GALÁPAGOS
the epidemiology of WNV, including 13 unique species of finch
famed by Charles Darwin. Finch species in United States show
high susceptibility to WNV infection, high mortality rates, and
reservoir competence with viremias greater than 5 log 10 PFU/
mL sustained for 6 days. 43, 55 The susceptibility of Galápagos
finches and other key wildlife species, and the likelihood they
would support WNV viremias high enough to infect a feed-
ing mosquito, needs to be determined. Although such testing
is notably politically sensitive, it would ascertain the vulner-
ability and potential role of Galápagos fauna to understand
the threat presented by WNV. Should Galápagos wildlife
prove susceptible or reach viremias high enough to enable the
virus to become established, the island fauna face a serious
threat. There is particular concern for endemic Galápagos spe-
cies, which given their evolution in isolation from flavivirus
are likely to have limited resistance to WNV. The generally
small population sizes exacerbate vulnerability from stochas-
tic events such as disease outbreaks.
Our finding that Galápagos Cx. quinquefasciatus has the
ability to infect its hosts with WNV prompts the research
question: what exactly is the feeding behavior of this species in
Galápagos? Although it has been suggested that Cx. quinque-
fasciatus can feed on mammals and birds, the relative propor-
tion of preferred host order varies greatly among studies and
vertebrate availability. 28, 31, 56, 57 Knowledge of local diet prefer-
ences specific to Galápagos is critical to determine which ver-
tebrate species would most likely be affected.
There are other potential vectors of WNV in addition to Cx.
quinquefasciatus in Galápagos. In particular, given the prolific
presence of a Galápagos strain of Ae. taeniorhynchus , determin-
ing the WNV vector competency of this mosquito is necessary
to understand the potential dynamics of WNV in Galápagos.
Aedes taeniorhynchus is far more widely distributed through-
out the archipelago than Cx. quinquefasciatus (Eastwood G,
unpublished data), has a broad host preference, and is another
potential WNV vector. 21 Aedes taeniorhynchus has been found
in WNV-positive surveillance pools in the United States since
2002, which reflects the southerly spread of WNV to the hot-
ter areas where this and other species including Cx. quinque-
fasciatus are found. 58 A study using Ae. taeniorhynchus from
the United States found that although partially refractory to
WNV infection (infection rates of only 12% occurred even
when exposed to titers exceeding 7 log 10 PFU/mL), mosqui-
toes when inoculated showed transmission rates of 93%. 59
Because there is a potential for WNV to arrive in Galápagos
in the near future without appropriate mitigation measures, 18
we recommend that determination of the WNV vector compe-
tency of Galápagos Ae. taeniorhynchus is a priority to inform
control measures to alleviate the future impact of WNV in the
Galápagos Islands.
The results of our study indicate that on-going transport
of Cx. quinquefasciatus from the South American mainland 20
could be effective in co-introducing WNV to Galápagos. The
established presence of Cx. quinquefasciatus on the main
islands and evidence of vector capability suggest that the
Galápagos strain of this mosquito species could have a sig-
nificant role in the maintenance and spread of WNV once
the virus has reached the islands. Increased movement of
passengers and freight to and around the islands (servicing
both the local population and the tourist industry) perpetu-
ates the chance of pathogen dispersal and widespread dis-
ease. Pathogens reaching Galápagos are most likely to derive
penultimately from continental South America (currently all
flights pass through Guayaquil or Quito, Ecuador), and the
risk assessment of WNV reaching Galápagos performed by
Kilpatrick and others 18 was based on an assumption of WNV
being established in mosquitoes at the ports of origin for air-
craft and vessels traveling to the islands. Therefore, determin-
ing the status of WNV activity on mainland Ecuador is a key
requirement for determining the risk of an introduction event
into Galápagos itself.
We also recommend that current measures to minimize the
incursion of live mosquitoes from the mainland (e.g., aircraft
disinfection) are rigorously enforced and additional measures
(e.g., disinfection of sea vessels traveling to Galápagos) are
introduced. With increasing human populations and visitor
numbers, management authorities overseeing the Galápagos
Islands are already faced with the challenge of conserv-
ing the unique range of wildlife that in addition to invalu-
able natural history value, provide considerable economic
benefit, through ecotourism, to Ecuador. West Nile virus has
shown unprecedented activity on arriving in the Americas
and placed substantial pressure on resources in the United
States, also identifying flaws in the existing public health sys-
tem (e.g., recording issues). 60 In addressing the WNV threat
to Galápagos ecosystems, it is advised that identification of
the principal risks from this pathogen is fully explored. This
includes greater knowledge of the ecological characteristics
of Galápagos mosquitoes, how they interact with both native
species, and the increasing influx of invasive species.
Received December 31, 2010. Accepted for publication May 9, 2011.
Acknowledgments: We thank Marilyn Cruz, Pamela Martinez,
Alberto Velez, and Macarena Piaz for support at the Galápagos field
site; Yongjing Jia, Pam Chin, Joe Maffei, Mary Franke, and Alex Ciota
for laboratory support in the United States; and Virna Cedeño for sci-
entific and logistical support in Ecuador. We also thank the Galápagos
National Park for cooperation and support of the overall project
(PNG09-21) and for granting access for sample collection.
Financial support: This study was supported by a Natural Environ-
mental Research Council Doctoral Training Grant to the Faculty of
Biological Sciences, University of Leeds, with additional support from
Darwin Initiative grant EIDPO15.
Authors’ addresses: Gillian Eastwood, Institute of Zoology, Zoological
Society of London, London, United Kingdom and University of Leeds,
Leeds, United Kingdom, E-mail: gillian.eastwood@ioz.ac.uk . Laura
D. Kramer, New York State Department of Health, Slingerlands,
NY, E-mail: ldk02@health.state.ny.us . Simon J. Goodman, Institute
of Integrative and Comparative Biology, University of Leeds, Leeds,
United Kingdom, E-mail: s.j.goodman@leeds.ac.uk . Andrew A.
Cunningham, Wildlife Epidemiology, Institute of Zoology, Zoological
Society of London, London, United Kingdom, E-mail: a.cunningham@
ioz.ac.uk .
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