VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION
Host-Selection Patterns of Culex tarsalis (Diptera: Culicidae)
Determine the Spatial Heterogeneity of West Nile Virus Enzootic
Activity in Northern California
REBECCA CAMPBELL,1 TARA C. THIEMANN,1,2 DEBRA LEMENAGER,3
1,4
AND WILLIAM K. REISEN
J. Med. Entomol. 50(6): 1303Ð1309 (2013); DOI: http://dx.doi.org/10.1603/ME13089
ABSTRACT The spatial heterogeneity of West Nile virus (WNV) activity in Sutter County, CA,
as measured by mosquito infection rates, was associated with spatial variation in the prevalence
of Culex blood feeding on competent passeriform hosts. Overall, 42 vertebrate host species (31
avian, 11 mammal) were identiÞed from 601 blood-fed Culex tarsalis Coquillett and 151 blood-fed
Culex pipiens L. complex females using sequences of the cytochrome c oxidase I gene and the
Barcode of Life Data Systems database. WNV infection rates were low at sites where the primary
vector, Cx. tarsalis, fed frequently on domestic cattle or incompetent galliform birds and high
when females fed frequently on American Robins, American Crows, and Yellow-billed Magpies.
Opportunistic host selection by Cx. tarsalis in combination with spatial variation in the presence
of highly competent corvid hosts appeared to determine the distribution of WNV activity in rural
Sutter County, CA.
KEY WORDS Culex tarsalis, Culex pipiens, blood-feeding pattern, West Nile virus, transmission
West Nile virus (Flaviviridae, Flavivirus; WNV) is now
well established throughout North America, where it
is maintained by enzootic transmission among a variety of Culex mosquitoes and avian hosts (Kramer et al.
2008, Kilpatrick 2011). Most Culex species are moderately competent vectors (Goddard et al. 2002, Turell
et al. 2005), with infection being dose dependent (Reisen et al. 2005) and following a logistic function (Lord
et al. 2006, Reisen et al. 2008). This doseÐresponse
curve is critical because avian hosts vary widely in
competence (Komar 2003, Kilpatrick et al. 2007,
Wheeler et al. 2009), presenting a broad spectrum of
viremias to blood-feeding mosquitoes. In addition,
most large mammals such as cattle and horses are
considered “dead end” hosts and do not produce sufÞcient viremia to infect Culex mosquitoes. Therefore,
WNV ampliÞcation seems highly contingent upon
vector host selection (Kilpatrick et al. 2006); however,
Culex seem to feed opportunistically on a wide variety
of hosts depending strongly on relative availability
(Thiemann et al. 2012) and female host-seeking behavior (Lothrop and Reisen 2001). In general, WNV
transmission has been most efÞcient in urban envi1 Center for Vectorborne Diseases, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616.
2 Department of Biology, University of the PaciÞc, Stockton, CA
95211.
3 Sutter-Yuba Mosquito and Vector Control District, PO Box 726,
Yuba City, CA 95992.
4 Corresponding author, e-mail: [email protected].
ronments where avian diversity is reduced (Loss et al.
2009) and transmission among competent commensal
passerine species is more efÞcient (Ezenwa et al. 2006,
Swaddle and Calos 2008). These concepts have not
been tested well in rural areas.
In California, Culex tarsalis Coquillett and members
of the Culex pipiens L. complex (Diptera: Culicidae)
feed on a wide variety of birds and mammals, including
maintenance, amplifying, and dead-end hosts (Tempelis et al. 1965, Tempelis and Washino 1967, Molaei
et al. 2010, Thiemann et al. 2012). Collectively, these
studies indicated that host-seeking behavior and associated ßight paths may dictate host contact and
therefore bloodmeal acquisition patterns (Lothrop
and Reisen 2001). Because avian and mammalian species vary widely in their distribution throughout rural
and urban landscapes, host selection patterns also vary
spatially among biomes (Thiemann et al. 2012). However, less is known about Þne-scale spatial variation
within biomes and how this determines variation in
WNV activity.
Agriculture within the Sacramento Valley of California in Sutter County is dominated by rice and livestock production, providing a landscape dominated by
rice Þelds interspersed with pasture, riparian corridors, and farmsteads. Each of these semi-isolated farmsteads provides a unique assortment of avian and mammalian hosts allowing us to test the hypothesis that
opportunistic vector host selection in combination
with variation in host availability determines hetero-
0022-2585/13/1303Ð1309$04.00/0 䉷 2013 Entomological Society of America
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JOURNAL OF MEDICAL ENTOMOLOGY
geneity in mosquito infection rates and therefore
WNV activity.
Materials and Methods
Study Sites. Blood feeding patterns and WNV activity were assessed for Cx. tarsalis and Cx. pipiens at
three sites in Sutter County, CA, during 2006 and 2007.
All sites contained shade trees and other ornamental
vegetation that formed ecological “islands” that attracted roosting and nesting birds as well as hostseeking and resting mosquitoes. Site A (39.1172⬚,
⫺121.7567⬚) was a cattle ranch near a wildlife refuge
and water delivery canal. The observed hosts included
a large herd of beef cattle, domestic chickens, and
other domestic mammals, as well as resident and migratory water birds using the adjacent canal and refuge. Site B (38.8194⬚, ⫺121.4969⬚) was a Þrehouse with
an adjacent park and an elementary school ⬇500 m
from the site but otherwise surrounded by pastures.
Passerine birds in scattered trees within the park were
the most visible hosts at this site. Site C (38.8119⬚,
⫺121.5317⬚) was an isolated farm complex that was
surrounded by rice Þelds. A few cattle and a variety of
wild and domestic birds, including turkeys, pheasants,
and peacocks, were observed at this location.
Mosquito Collection. Blood-fed mosquitoes were
aspirated weekly from walk-in red boxes that mimic
natural resting sites for mosquitoes (Meyer 1987).
Mosquitoes were identiÞed to species, sorted by blood
engorgement status, and stored in individual cryovials
at ⫺80⬚C until tested. The Cx. pipiens complex in this
part of California consists of a mixture of form pipiens
(L.) hybridized with Culex quinquefasciatus Say and
form molestus Forskäl (Kothera et al. 2013).
Bloodmeal Identification. Bloodmeals were identiÞed using methods described previously (Thiemann
et al. 2011). In brief, DNA was extracted from bloodfed Culex females using the DNeasy 96 Blood & Tissue
Kit (Qiagen, Valencia, CA). The 658-bp “barcoding”
region of the mitochondrial gene cytochrome c oxidase I (COI) was ampliÞed using a nested polymerase
chain reaction (PCR). First, primers for the tRNAcoding regions ßanking COI were used to amplify
⬇1,900 bp. Second, the barcoding region of COI was
ampliÞed using vertebrate-speciÞc primers (Cooper
et al. 2007, Ivanova et al. 2009). Hosts were identiÞed
from DNA by sequencing and using the “Identify
Specimen” feature of the Barcode of Life Data Systems
(BOLD) database (www.boldsystems.org) (Ratnasingham and Hebert 2007, Kent 2009). To compare host
species richness among study sites, rarefaction was
used to generate the expected number of host species
from a 10 bloodmeal sample for Cx. pipiens and from
a 100 bloodmeal sample for Cx. tarsalis using the rarefy
function in the vegan package version 1.17-D8 of R
(RDC [R Development Core] 2011). The different
base number between species was used because of the
low number of samples available for Cx. pipiens.
WNV Activity. Most female mosquitoes tested for
the virus were collected weekly from red boxes from
June through September concurrent with specimens
Vol. 50, no. 6
for bloodmeal analysis, pooled into groups of ⱕ50,
frozen at ⫺80⬚C, and later tested for WNV RNA by
quantitative reverse transcription (qRT)-PCR using
primers from the envelope region of the genome
(Lanciotti et al. 2000). During 2006 and 2007, 12.5 and
4.7% of mosquito pools, respectively, were derived
from females collected by dry ice-baited Centers for
Disease Control and Prevention (CDC) style traps
(Newhouse et al. 1966). Data were pooled over collection method, weeks, and years to provide statistical
conÞdence and control the variance. Maximum likelihood estimates (MLE) of infection incidence (Biggerstaff 2003) were used to calculate the minimum
number of WNV-infected mosquitoes per 1,000 individuals tested from each study site and from the Sutter
and Yuba County areas as a whole.
Results
Bloodmeal Identification. Using the BOLD database, 42 vertebrate host species (31 avian, 11 mammal)
were identiÞed from 601 blood-fed Cx. tarsalis and 151
blood-fed Cx. pipiens of the 799 mosquitoes tested
(Table 1). The numbers of each species tested reßected their relative abundance, with a subsample of
Cx. tarsalis and all Cx. pipiens tested. In rural Sutter
County, Cx. tarsalis females were usually 30 times
more abundant than Cx. pipiens females in the New
Jersey light trap samples (unpublished surveillance
data), and the red box collections in our study areas
reßected this disparity. Overall, 20 Cx. tarsalis and 27
Cx. pipiens bloodmeals (6% in total) were not identiÞed, likely because of degradation of the sample or
errors in extraction and PCR. The higher percentage
of Cx. pipiens not identiÞed was the result of broadening our selection criteria for blood-fed females in an
attempt to increase our sample sizes for this less common species. All bloodmeals for which a clean DNA
sequence was obtained were identiÞed to host species
using the BOLD system, and none of the data seemed
to represent sequences from more than one host species. This apparent absence of multiple bloodmeals
agreed with our previous bloodmeal identiÞcation
studies in California (Thiemann et al. 2012) and with
the low proportion of partially blood-fed females collected during host seeking at dry ice-baited traps
(Mitchell et al. 1981, Reisen et al. 1995).
The number of bloodmeals apportioned among the
four taxonomic groups in Table 1 varied signiÞcantly
between the two mosquito species when tested by
contingency chi-square (␹2 ⫽ 54.6; P ⬍ 0.001). Overall, Cx. tarsalis fed on 30 different avian and 11 mammalian host species, whereas Cx. pipiens fed on 17
avian and 1 mammalian host. Cx. tarsalis fed most often
on American Robins (21.8%), domestic cattle (19.5%),
and Yellow-billed Magpies (14.1%), whereas Cx. pipiens fed almost entirely on birds, including American
Robins (19.9%), American Crows (17.2%), Yellowbilled Magpies (16.6%), and domestic chickens
(14.6%). Few Culex fed on smaller-sized peridomestic
passerines such as House Finch and House Sparrow that
were the dominant hosts of Cx. quinquefasciatus in urban
November 2013
Table 1.
CAMPBELL ET AL.: HOST-SELECTION PATTERNS OF Cx. tarsalis
Bloodmeals from Culex listed by number that are positive within each order or group
Cx. pipiens
Order
Species
ScientiÞc name
Passeriformes
American Robin
Yellow-billed Magpie
American Crow
House Finch
House Sparrow
European Starling
BrewerÕs Blackbird
Western Kingbird
Red-winged Blackbird
Western Scrub-jay
Brown-headed Cowbird
Barn Swallow
Cliff Swallow
Lark Sparrow
Northern Mockingbird
Tricolored Blackbird
Western Meadlowlark
Chicken
Indian Peafowl
Wild Turkey
Ring-necked Pheasant
Mourning Dove
American Kestrel
Common Barn Owl
Mallard
Great Blue Heron
American Bittern
CooperÕs Hawk
Rock Dove
Common Ground Dove
White-faced Ibis
Domestic cattle
Black-tailed jackrabbit
Horse
Sheep
Cat
Human
Domestic pig
Raccoon
Black rat
Dog
North American river otter
UnidentiÞed
Turdus migratorius
Pica nuttalli
Corvus brachyrhynchos
Haemorhous mexicanus
Passer domesticus
Sturnus vulgaris
Euphagus cyanocephalus
Tyrannus verticalis
Agelaius phoeniceus
Aphelocoma californica
Molothrus ater
Hirundo rustica
Petrochelidon pyrrhonota
Chondestes grammacus
Mimus polyglottos
Agelaius tricolor
Sturnella neglecta
Gallus gallus
Pavo cristatus
Meleagris gallopavo
Phasianus colchicus
Zenaida macroura
Falco sparverius
Tyto alba
Anas platyrhynchos
Ardea herodias
Botaurus lentiginosus
Accipiter cooperii
Columba livia
Columbina passerina
Plegadis chihi
Bos taurus
Lepus californicus
Equus caballus
Ovis aries
Felis catus
Homo sapiens
Sus scrofa
Procyon lotor
Rattus rattus
Canis familiaris
Lontra canadensis
Galliformes
Other
Mammals
1305
Totals
Los Angeles (Thiemann et al. 2012) and Cx. tarsalis in
Kern County (Tempelis et al. 1976) where American
Robins were scarce. In agreement with previous surveys,
only three human bloodmeals were identiÞed, two from
Cx. pipiens and one from Cx. tarsalis.
Most engorged females were collected from June
through August of both years, except for Site B, where
104 females collected from September and early October of 2006 had imbibed 41 American Robin and 33
Yellow-billed Magpie bloodmeals. Therefore, most
mosquitoes were collected during the late nesting and
ßedging periods. Host selection varied not only between species but also among sites (Table 2; Fig. 1).
Bloodmeal diversity estimated as the number of species per bloodmeal was consistently the lowest at Site
B and highest at Site C. Similarly, species richness of
hosts used for bloodmeals, which were estimated using rarefaction, was the lowest at Site B and highest at
Site C, although Sites A and C were not statistically
different. Because of differences in sample sizes, cal-
Total
Cx. tarsalis
No.
% total
No.
% total
No.
%
30
25
26
7
11
9
5
1
0
3
1
1
0
0
0
0
0
22
0
0
0
1
4
1
0
0
1
1
0
0
0
0
0
0
0
0
2
0
0
0
0
0
27
178
19.9
16.6
17.2
4.6
7.3
6.0
3.3
0.7
0.0
2.0
0.7
0.7
0.0
0.0
0.0
0.0
0.0
14.6
0.0
0.0
0.0
0.7
2.6
0.7
0.0
0.0
0.7
0.7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1.3
0.0
0.0
0.0
0.0
0.0
131
85
49
25
17
11
9
4
3
0
1
0
1
1
1
1
1
22
13
7
5
16
6
6
6
5
2
1
2
1
1
117
19
13
6
5
1
2
2
1
1
1
20
621
21.8
14.1
8.2
4.2
2.8
1.8
1.5
0.7
0.5
0.0
0.2
0.0
0.2
0.2
0.2
0.2
0.2
3.7
2.2
1.2
0.8
2.7
1.0
1.0
1.0
0.8
0.3
0.2
0.3
0.2
0.2
19.5
3.2
2.2
1.0
0.8
0.2
0.3
0.3
0.2
0.2
0.2
161
110
75
32
28
20
14
5
3
3
2
1
1
1
1
1
1
44
13
7
5
17
10
7
6
5
3
2
2
1
1
117
19
13
6
4
3
2
2
1
1
1
47
799
21.4
14.6
10.0
4.3
3.7
2.7
1.9
0.7
0.4
0.4
0.3
0.1
0.1
0.1
0.1
0.1
0.1
5.9
1.7
0.9
0.7
2.3
1.3
0.9
0.8
0.7
0.4
0.3
0.3
0.1
0.1
15.6
2.5
1.7
0.8
0.7
0.4
0.3
0.3
0.1
0.1
0.1
Table 2. Bloodmeal host diversity depicted as the number of
host species identified per bloodmeal and rarefaction analysis per
10 meals for Cx. pipiens and 100 meals for Cx. tarsalis for sites
A–C, Sutter County, 2006 –2007
Site
Cx. pipiens
Site A
Site B
Site C
Total
Cx. Tarsalis
Site A
Site B
Site C
Total
a
No. host
Bloodmeals
species
Host species
per
Richness SEa
bloodmeal
11
11
8
18
37
96
18
151
0.297
0.115
0.444
0.119
5.39
5.02
6.16
1.13
1.04
0.87
23
20
25
40
152
306
143
601
0.151
0.065
0.175
0.067
19.28
13.95
22.65
1.53
1.62
1.26
SE of species richness estimated by rarefaction analysis.
1306
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 50, no. 6
Fig. 1. Proportion of Cx. pipiens and Cx. tarsalis feeding on different hosts at Sites AÐC. Hosts comprising ⬍5% of the total
bloodmeals were grouped as “other.”
culations were done using base 10 and 100 for Cx.
pipiens and Cx. tarsalis, respectively, and therefore
were not comparable. Host species utilization varied
among sites.
Site A. Overall, 73% of 152 Cx. tarsalis (Fig. 1)
bloodmeals were from mammals, with 77% of these
from cattle. In marked contrast, only 2.7% of 37 Cx.
pipiens feedings were from mammals, and both of
these were from humans. No Cx. pipiens fed on cattle
despite their abundance and frequent feeding by Cx.
tarsalis collected concurrently from the same walk-in
red box. Cx. pipiens fed frequently (31.1%) on domestic chickens, a poorly competent host for WNV. Passeriformes made up 21.2% of Cx. tarsalis and 44.4% of
Cx. pipiens bloodmeals.
Site B. In marked contrast to Site A, passeriforms
made up 83.1% of the 306 Cx. tarsalis and 74.8% of the
96 Cx. pipiens bloodmeals identiÞed at Site B (Fig. 1).
Most of the passerine bloodmeals were from moderately competent American Robins and highly compe-
tent American Crows and Yellow-billed Magpies
(Komar et al. 2003). Most mosquitoes that fed on
American Crows were collected during MayÐJune,
followed by Yellow-billed Magpies from June to September and American Robins from August to September after the nesting American Crows departed from
this site (Fig. 2). The seasonal pattern changed somewhat between years, with virus activity and transition
in bloodmeal hosts occurring earlier in 2007 than 2006,
showing the dynamics of host availability and utilization at this isolated study area (Fig. 2).
Site C. This site supported a variety of domestic and
natural hosts as reßected in the high diversity of bloodmeals, especially for Cx. tarsalis (Table 2). Cx. tarsalis
fed most often on American Robins and cattle and
infrequently on a variety of other hosts (each ⬍5% of
the total) that cumulatively accounted for 48% of the
total feeds. The few Cx. pipiens that were collected fed
mostly on American Robins, chickens, and Yellowbilled Magpies (Fig. 1).
November 2013
CAMPBELL ET AL.: HOST-SELECTION PATTERNS OF Cx. tarsalis
1307
Fig. 2. Proportion of total bloodmeals from American Robin, Yellow-billed Magpie, and American Crow hosts (total
sample size for each month above columns) and MLE of infection incidence calculated as the number of WNV infected per
1,000 Cx. tarsalis females tested from Site B.
and bridge vectors. Previous studies on Cx. tarsalis
from Northern California also found that females frequently fed on passeriform and bovine hosts (Tempelis and Washino 1967, Thiemann et al. 2012),
whereas in Southern California, Cx. tarsalis fed on a
variety of hosts in rural areas (Molaei et al. 2010,
Thiemann et al. 2012). In all studies, Cx. tarsalis accepted an assortment of hosts, ranging from large
mammals such as cattle and horse to small passerines
such as House Finch and European Starling. Herein,
mosquitoes at Site A most frequently fed on hosts with
low WNV competence: Cx. tarsalis on cattle and Cx.
pipiens on chicken. In contrast at Site B, Culex fed on
a low diversity of competent passeriform hosts, including American Robin, Yellow-billed Magpie, and
American Crow. At Site C, mosquitoes fed on a wide
variety of host species, many of which have low WNV
competence, including members of the order Galliformes and domestic cattle.
Differences in host feeding patterns among sites
correlated well with virus activity. At Site B, where
bloodmealÐ host diversity was the lowest and most
bloodmeals were from highly competent corvids, Cx.
tarsalis had the highest WNV infection incidence in
West Nile Virus Activity. In total, 712 pools of Cx.
tarsalis and 24 pools of Cx. pipiens were tested for
WNV RNA during 2006 Ð2007, of which 86 Cx. tarsalis
and no Cx. pipiens were positive (Table 3). Overall,
51% of the positive pools from Sutter and Yuba Counties were collected at Site B, and the infection rate per
1,000 here was signiÞcantly greater (95% CL) than
either Site A or C, or all samples combined from Sutter
and Yuba Counties. Overall, 88% of positive pools
were collected during July and August.
Discussion
Culex blood feeding patterns were extremely variable among our three study sites and seemed determined by host availability rather than strong host preference. Although both Culex species were collected
concurrently in the same red boxes at the same time,
few Cx. pipiens fed on mammals throughout, supporting the general ornithophagic patterns of this species
complex throughout California (Molaei et al. 2010,
Montgomery et al. 2011, Thiemann et al. 2012). In
contrast, Cx. tarsalis fed frequently on mammals when
available, establishing their role as both amplifying
Table 3. WNV activity at Sites A–C and at all sites in Sutter–Yuba Counties (SUYA) measured by the MLE of the infection rate in Cx.
tarsalis and Cx. pipiens tested during 2006 and 2007
Sites
Culex species
MLE
All SUYA
Site A
Site Ba
Site C
All SUYA
tarsalis
tarsalis
tarsalis
tarsalis
pipiens
2.94
1.09
5.77
0.72
0.00
a
95% limits
Lower
Upper
2.37
0.41
4.26
0.13
0.00
3.61
2.42
7.69
2.36
0.00
MLE signiÞcantly greater than remaining values by inspection of 95% CL.
The lower and upper 95% CIs of the MLE are shown.
Pools
WNV positive
Females tested
712
110
183
66
24
86
5
44
2
0
31,176
4,665
8,664
2,815
359
1308
JOURNAL OF MEDICAL ENTOMOLOGY
Sutter and Yuba Counties during the 2006 Ð2007 study
period. In contrast, at Site A, Culex females fed frequently on dead-end hosts such as cattle and chicken,
whereas at Site C, they fed on a diversity of hosts,
including a high proportion of low competent galliform species. Presumably, the dilution of bloodmeals
among a wide variety of dead-end and low competent
hosts contributed to the low MLE values at Sites A and
C, whereas blood feeding concentrated on a few competent host species resulted in the high MLE values
recorded at Site B (Swaddle and Calos 2008).
In summary, the spatial heterogeneity of WNV activity among study sites in rural Sutter County seemed
to be related to host availability and broad opportunistic host selection patterns by the principal vector,
Cx. tarsalis. At sites with low WNV activity, females
fed frequently on noncompetent hosts, whereas at the
site with the highest WNV activity, over 76% bloodmeals were from competent hosts. Our data indicated
that frequent blood feeding on highly competent corvid hosts may be necessary for efÞcient WNV ampliÞcation.
Acknowledgments
We thank Holly Ernest, University of California Davis, for
supplies and equipment to run gels. Christopher Barker,
Center for Vectorborne Diseases, University of California,
Davis, assisted with the rarefaction analysis. Mosquito pools
were tested for WNV by Ying Fang and the Arbovirus Laboratory, Center for Vectorborne Diseases. This research was
supported, in part, by funds from the California Mosquito and
Vector Control Association (CMVCA) Research Foundation
and from grant number R01-AI65507 from the National Institute of Allergy and Infectious Diseases, National Institutes
of Health. Logistical support was provided by the SutterÐ
Yuba Mosquito and Vector Control District.
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Received 5 May 2013; accepted 7 September 2013.