VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION

Host Feeding Patterns of Culex Mosquitoes (Diptera: Culicidae) in

East Baton Rouge Parish, Louisiana
ANDREW J. MACKAY,1 WAYNE L. KRAMER,2 JENNIFER K. MEECE,3 ROBB T. BRUMFIELD,4
2
AND LANE D. FOIL

Department of Entomology, Louisiana State University Agricultural Center, 404 Life Sciences Bldg.,
Baton Rouge, LA 70803

J. Med. Entomol. 47(2): 238Ð248 (2010); DOI: 10.1603/ME09168

ABSTRACT Host feeding patterns were examined for four species of Culex mosquitoes collected
from 18 sites in or adjacent to East Baton Rouge Parish, LA, from November 2002 to October 2004.
Host DNA from 37 bloodfed Culex coronator Dyar and Knab, 67 bloodfed Cx. salinarius Coquillett, 112
bloodfed Cx. nigripalpus Theobald, and 684 bloodfed Cx. quinquefasciatus Say were identiÞed. The
percentages of bloodmeals containing mammalian DNA were 94.6% for Cx. coronator, 82.1% for Cx.
salinarius, 66.1% for Cx. nigripalpus, and 40.1% for Cx. quinquefasciatus. Human DNA was detected in
7% of the bloodmeals from Cx. quinquefasciatus and 2.7% of the bloodmeals from Cx. nigripalpus. The
northern cardinal was the most frequent avian host of Cx. quinquefasciatus and Cx. nigripalpus. In 2003
and 2004, there was no signiÞcant relationship from May through October between the proportion
of Cx. quinquefasciatus feeding on mammalian hosts and the date of collection. Of the six avian species
most frequently fed on by Cx. quinquefasciatus, the northern cardinal, northern mockingbird, common
grackle, and brown thrasher were fed on more frequently than expected based on their abundance.
House sparrows were fed on less frequently than expected based on their abundance. These data
support the conclusions of previous studies that Cx. quinquefasciatus is the most important vector for
both the enzootic ampliÞcation and transmission of West Nile virus to humans in southern Louisiana.

KEY WORDS bloodmeal identiÞcation, cytochrome B, Culex, seasonal host utilization, Louisiana

Seasonal host range studies are required to assess the
role of competent vectors of West Nile virus (Flavi-
viridae, Flavivirus, WNV) in the enzootic transmission
cycle by determining the frequency of blood feeding
on competent reservoir host species. Host range stud-
ies also are important when evaluating the role of
potential bridge vector species in transmission of
pathogens to humans and domestic animals. To serve
as a bridge vector, a mosquito must Þrst feed on a
reservoir host with a sufÞcient viremia for infection
and then feed on a human or domestic animal after
completion of the extrinsic incubation period. In
North America, the most important enzootic vectors
of WNV, and many of the suspected bridge vectors,
are mosquitoes of species in the genus Culex (Turell
et al. 2005).
Trends of seasonal shifts in host feeding, from feed-
ing mostly on avian hosts in the spring and early
1 Dengue Branch, Centers for Disease Control and Prevention, 1324
Calle Cañada, San Juan, Puerto Rico 00920.
2 Department of Entomology, Louisiana State University Agricul-
tural Center, 404 Life Sciences Bldg., Baton Rouge, LA 70803 (e-mail:
wkramer@agcenter.lsu.edu).
3 MarshÞeld Clinic Research Foundation, MarshÞeld, WI 54449.
4 Museum of Natural Science, Louisiana State University, Baton
Rouge, LA 70803.
summer, to increasing mammalian feeding in the late
summer and fall, have been reported for Culex nigri-
palpus Theobald (Edman and Taylor 1968, Edman
1974), Cx. tarsalis Coquillett (Tempelis et al. 1965), Cx.
quinquefasciatus Say (Bertsch and Norment 1983),
and Cx. pipiens L. (Kilpatrick et al. 2006). Changes in
host selection can allow a vector species to circulate
the pathogen among avian reservoir hosts in the spring
and early summer and then transmit the pathogen to
humans and other mammals later in the transmission
season.
From 2002Ð2004, WNV was detected in at least 19
mosquito species collected from East Baton Rouge
(EBR) Parish, including Cx. coronator Dyar and Knab,
Cx. salinarius Coquillett, Cx. nigripalpus, and Cx. quin-
quefasciatus (Mackay et al. 2008). Although the latter
three species have been demonstrated to be compe-
tent vectors of WNV in the laboratory (Sardelis et al.
2001, Goddard et al. 2002), a greater knowledge of
their host range in southern Louisiana is required to
assess their potential as enzootic and/or epidemic
vectors of WNV. A previous study in EBR Parish re-
ported that avian blood was detected in almost one
half of blood engorged Cx. quinquefasciatus collected
from hardwood forests, and that the host range of
Cx. quinquefasciatus varied greatly among habitats

0022-2585/10/0238Ð0248$04.00/0 䉷 2010 Entomological Society of America

March 2010 MACKAY ET AL.: HOST FEEDING PATTERNS IN Culex MOSQUITOES 239

Table 1. Collection sites sampled for mosquitoes in southern Mosquitoes were collected using a Centers for Dis-
Louisiana ease Control (CDC) miniature UV-light trap (model
512; John W. Hock Co., Gainesville, FL) baited with
Sampling
Parish Latitude Longitude
groupa ⬇3 kg of dry ice, and a CDC gravid trap (model 1712;
John W. Hock Co.), baited with 2 liters of a 1.2% Þsh
East Baton Rouge 30⬚ 25⬘ 58.08⬙ N 91⬚ 10⬘ 13.15⬙ W 1
30⬚ 21⬘ 2.88⬙ N 90⬚ 56⬘ 55.32⬙ W 1
oil emulsion that had been prepared 4 Ð 8 d previously.
30⬚ 24⬘ 49.18⬙ N 91⬚ 0⬘ 6.08⬙ W 1 These methods were chosen to provide concurrent
30⬚ 20⬘ 58.38⬙ N 91⬚ 4⬘ 4.37⬙ W 1 collections of host seeking and gravid females for iso-
30⬚ 27⬘ 24.48⬙ N 90⬚ 59⬘ 58.24⬙ W 1 lation of WNV (Mackay et al. 2008). During weeks in
30⬚ 34⬘ 4.04⬙ N 90⬚ 59⬘ 13.07⬙ W 2
30⬚ 35⬘ 32.04⬙ N 91⬚ 2⬘ 25.00⬙ W 2
which sites were sampled, traps were operated for two
30⬚ 31⬘ 35.04⬙ N 91⬚ 4⬘ 24.01⬙ W 2 consecutive 24 h periods. Collections of insects were
30⬚ 34⬘ 13.04⬙ N 91⬚ 10⬘ 21.01⬙ W 2 transported to the laboratory in a cooler containing
30⬚ 25⬘ 59.66⬙ N 91⬚ 0⬘ 26.57⬙ W 4 dry ice. Bloodfed females were removed from trap
30⬚ 25⬘ 43.64⬙ N 91⬚ 4⬘ 24.13⬙ W 4
30⬚ 29⬘ 35.03⬙ N 91⬚ 5⬘ 1.00⬙ W 4
samples on a chill table and identiÞed to species. All
30⬚ 24⬘ 14.94⬙ N 91⬚ 9⬘ 4.82⬙ W 4 females were stored at ⫺80⬚C until processed. The
30⬚ 23⬘ 8.12⬙ N 91⬚ 12⬘ 15.34⬙ W 4 abdomen of each bloodfed female was removed. The
30⬚ 22⬘ 53.94⬙ N 91⬚ 2⬘ 44.74⬙ W 4 relative size of the bloodmeal was ranked, from a trace
East Feliciana 30⬚ 46⬘ 44.00⬙ N 91⬚ 3⬘ 57.00⬙ W 3
30⬚ 43⬘ 2.00⬙ N 91⬚ 8⬘ 29.00⬙ W 3
bloodmeal, barely visible under low power magniÞ-
Iberville 30⬚ 15⬘ 30.00⬙ N 91⬚ 6⬘ 5.00⬙ W 2 cation (size I) to a full engorgement (size IV).
Host Bloodmeal Identification. DNA was extracted
a
Group 1, sampled every 2 wk from Nov. 2002 to Oct. 2004; group from the abdomens of bloodfed mosquitoes and from
2, sampled every 2 wk (alternate wk to group 1), from Mar. to Nov. tissue samples of reference vertebrate species using
in 2003 and 2004; group 3, sampled every 2 wk (alternate wk to group
1), from Mar. to Nov. in 2003 only; group 4, sampled every wk from the QIAamp DNA Mini Kit (Qiagen, Valencia, CA).
Mar. to Nov. in 2003 and 2004, and sampled every 2 wk from Nov. 2002 The manufacturerÕs protocol for extraction of DNA
to Feb. 2003, and Dec. 2003 to Feb. 2004. from tissue was followed, except that for the mosquito
abdomens, 50 ␮l rather than 200 ␮l of AE buffer was
used per sample to elute the extracted DNA from each
(Niebylski and Meek 1991). However, the authors QIAamp spin column.
used an immunologic assay that did not identify avian For all four mosquito species, two methods were
hosts to species. More recently developed molecular used to identify host DNA extracted from bloodmeals:
based assays have enabled researchers to more accu- a T-restriction fragment-length polymorphism assay
rately identify hosts to the species level based on the and direct sequencing. A majority of the bloodmeals
sequence of host mitochondrial DNA extracted from of Cx. coronator, Cx. nigripalpus, and Cx. quinquefas-
mosquito bloodmeals (Lee et al. 2002, Cupp et al. 2004, ciatus was identiÞed using T-restriction fragment-
Kent and Norris 2005, Meece et al. 2005). In the cur- length polymorphism. This methodology initially was
rent study, we used a terminal restriction fragment chosen as the primary diagnostic tool for identifying
length polymophism (T-restriction fragment-length host DNA to allow us to more easily identify DNA
polymorphism) assay and direct sequencing of verte- from more than one host in the bloodmeal of mosqui-
brate mitochondrial DNA extracted from bloodfed toes that had partially fed on two or more vertebrate
mosquitoes to examine the host ranges of Cx. corona- species. However, because T-restriction fragment-
tor, Cx. salinariusi, Cx. nigripalpus, and Cx. quinque- length polymorphism (TRFLP) was found to be more
fasciatus in southern Louisiana. Seasonal changes in labor intensive than direct sequencing, and patent
host range also was examined for Cx. quinquefasciatus. multiple bloodmeals were rarely detected in the Þrst
three Culex species processed by TRFLP, sequencing
was adopted as the primary method used to identify
Materials and Methods
host DNA extracted from the bloodmeals of Cx. sali-
Mosquito Collections. Bloodfed mosquitoes were narius.
collected from 15 locations in EBR Parish, two sites in The TRFLP assay was performed as described pre-
East Feliciana Parish and one site in Iberville Parish viously (Meece et al. 2005). To expand the preexisting
(Table 1). The sites in EBR parish represented the library of restriction fragment proÞles for known ver-
diversity of habitats in the parish, and were in close tebrate hosts (Meece et al. 2005), tissue samples col-
proximity to the homes of previous human cases of lected from Louisiana and representing 34 avian and
WNV, or to surveillance sites where arbovirus activity 13 mammalian species were obtained from the Loui-
previously had been detected in birds or mosquitoes. siana State University Museum of Natural Science
Trapping sites included urban, suburban, and rural Collection of Genetic Resources and also processed by
habitats; most of the sites (13/18) were located within T-restriction fragment-length polymorphism. Samples
100 m of a residence. A majority of sites were con- from three additional vertebrate species were pro-
sidered ÔwoodedÕ (forest canopy enclosed 50% or vided by East Baton Rouge Mosquito Abatement and
more of the area within a 100 m radius of the trap). Rodent Control. Samples of DNA extracted from ref-
Two of the trapping sites were located in equestrian erence vertebrate tissue and from Þeld-collected mos-
parks, and two sites were located adjacent (within quitoes were ampliÞed using the primer BM1 (5⬘-CCC
20 m) to a permanent, freshwater swamp. CTC AGA ATG ATA TTT GTC CTC A), labeled at the
240 JOURNAL OF MEDICAL ENTOMOLOGY
Table 2. Identification of vertebrate host DNA from blood-fed Culex mosquitoes
No. bloodmeals
% samples identiÞed (% of
Species
ampliÞeda total bloodmeals
processed)
Cx. coronator 76.3 37 (62.7)
Cx. salinarius 81.4 67 (77.9)
Cx. nigripalpus 68.0 112 (56.0)
Cx. quinquefasciatus 63.8 684 (62.4)
a
Bloodmeals processed for host identiÞcation that were sufÞciently ampliÞed by PCR to produce a clear and unambiguous terminal
restriction fragment proÞle and/or sequence proÞle.
b
DNA identiÞed from more than one host in same bloodfed mosquito.
5⬘ end with 6-carboxyßourescein (6-FAM), and the
primer BM2 (5⬘-CCA TCC AAC ATC TCA GCA TGA
TGA AA), labeled at the 5⬘ end with 4,7,2⬘,4⬘,5⬘,7⬘-
hexachloro-6-carboxyßuorescein (HEX) (Integrated
DNA Technologies, Coralville, IA). Control DNA, for
the restriction digest and terminal fragment length
measurement, was ampliÞed using unlabeled BM1 and
N,N,N⬘,N⬘-tetramethyl-6-carboxyrhodamine (TAMRA)
labeled BM2.
Polymerase chain reaction (PCR) products from
mosquito bloodmeals and reference vertebrate tissue
samples were digested using four restriction enzymes;
AciI, AluI, HaeIII, and RsaI (New England Biolabs,
Beverly, MA). A chromosomal DNA preparation from
the common grackle (Quiscalus quiscala) was used as
a positive control. Digestion products were puriÞed
using the DyeEx 96 removal kit (Qiagen) according to
the manufacturerÕs instructions. Restriction fragment
lengths were measured in a model 3100 capillary DNA
sequencing instrument using GeneMapper software
(Applied Biosystems, Foster City, CA). The fragment
length proÞles for each digest were compared with a
database of fragment proÞles for known host DNA
samples (CYTBD 2009). Sequencing was performed
on host DNA samples from bloodfed Cx. coronator, Cx.
nigripalpus, and Cx. quinquefasciatus that failed to
match a TRF proÞle in the database, lacked a diag-
nostic TRF proÞle (no recognition sites for any of the
four restriction enzymes), or possessed a TRF proÞle
that was shared by more than one host species in the
TRF database. Sequencing also was used as a conÞr-
matory assay for some of the species identiÞcations
obtained by TRFLP.
For sequencing, unlabeled BM1 and BM2 primers
were used to amplify host DNA by PCR as described
previously. Products were puriÞed by polyethylene
glycol precipitation. Sequence reactions were ana-
lyzed using a model 3100 capillary DNA sequencing
instrument, and compared with nucleic acid databases
(GenBank) using the basic local alignment search tool
(BLAST) service to Þnd matches to host species. Host
DNA extracted from Cx. salinarius that were success-
fully ampliÞed but produced poor quality sequence
data were reprocessed by the TRFLP assay.
Seasonal Host Utilization Patterns of Cx. quinque-
fasciatus. To test for signiÞcant seasonal trends in the
utilization of host species or class, logistic regression
(SPSS, Chicago, IL) was used to model host identities
Vol. 47, no. 2
% identiÞed samples
Single host species Multiple host speciesb
Both Single
Aves Mammalia
classes class
5.4 94.6 0 0
16.4 80.6 0 3.0
33.9 64.3 1.8 0
59.9 39.2 0.3 0.6
of bloodfed Cx. quinquefasciatus by ordinal date of
collection for each year, from May to October. Pro-
portions of Cx. quinquefasciatus bloodmeals from spe-
ciÞc host species or groups were compared between
years or months using the Fisher Exact Test (Graph-
Pad, Software, San Diego, CA).
Avian Host Preferences of Cx. quinquefasciatus.
The utilization of avian hosts, relative to their density
in the Parish, was examined for Cx. quinquefasciatus
using the forage ratio (FR) method (Hess et al. 1968).
Values were calculated by dividing the percentage of
bloodmeals obtained by Cx. quinquefaciatus from each
host species by the percentage that the host species
represents in the total avian community. A FR value
signiÞcantly greater than one was considered to indi-
cate selective preference by Cx. quinquefasciatus for
that host species. A FR value signiÞcantly less than one
was considered to indicate that the mosquito was
exhibiting selective avoidance of the host. The relative
abundances of avian hosts were calculated using data
obtained from the North American Breeding Bird Sur-
vey (NABBS) for the Baton Rouge route from 2003
and 2004 (USGS 2007). Because the NABBS only col-
lects data on wild bird species during the peak breed-
ing season, domestic species (Gallus gallus and Anser
anser) and winter resident species were excluded
from the numerator (percentage of avian host usage)
and denominator (relative avian host abundance) of
the FR calculation. FR values only were calculated for
wild avian hosts identiÞed from ⬎1% of the bloodfed
Cx. quinquefasciatus. Ninety-Þve percent conÞdence
intervals were computed as described by Lardeux et
al. (2007).
Results
Host Bloodmeal Identification. Blood meals from 59
Cx. coronator, 86 Cx. salinarius, 200 Cx. nigripalpus, and
1097 Cx. quinquefasciatus were processed for host
identiÞcation. Among the four species, sufÞcient am-
pliÞcation of host DNA to produce an unambiguous
TFR proÞle and/or sequence was achieved in 64 Ð 81%
of all bloodmeals (Table 2). Ranked bloodmeal size
signiÞcantly affected ampliÞcation success in Cx. ni-
gripalpus (␹2 ⫽ 35.4, df ⫽ 3, P ⱕ 0.001) and Cx.
quinquefasciatus (␹2 ⫽ 29.6, df ⫽ 3, P ⱕ 0.001). The
percentages of identiÞed bloodmeals that were ranked
as fully engorged were 22% for Cx. coronator, 16% for
March 2010 MACKAY ET AL.: HOST FEEDING PATTERNS IN Culex MOSQUITOES 241

Table 3. Number and percentage of bloodmeals obtained from avian hosts by Culex mosquitoes

Cx. quinquefasciatus Cx. nigripalpus Cx. salinarius

Host species
No. % avian % total No. % avian % total No. % avian % total
Northern cardinal, Cardinalis cardinalis 102 24.8 14.9 9 22.5 8.0 2 16.7 3.0
Northern mockingbird, Mimus polyglottos 62 15.0 9.1 1 8.3 1.5
Common grackle, Quiscalus quiscula 43 10.4 6.3 8 20.0 7.1
Mourning dove, Zenaida macroura 33 8.0 4.8
Domestic chicken, Gallus gallus 28 6.8 4.1 6 15.0 5.4 3 25.0 4.5
House sparrow, Passer domesticus 22 5.3 3.2
Brown thrasher, Toxostoma rufum 16 3.9 2.3 1 2.5 0.9
Blue jay, Cyanocitta cristata 13 3.2 1.9 2 5.0 1.8
Tufted titmouse, Baeolophus bicolor 9 2.2 1.3
Fish crow, Corvus ossifragus 8 1.9 1.2 1 2.5 0.9
Carolina chickadee, Poecile carolinensis 7 1.7 1.0 1 2.5 0.9
Yellow-billed cuckoo, Coccyzus americanus 7 1.7 1.0
Barred owl, Strix varia 5 1.2 0.7 1 8.3 1.5
Gray catbird, Dumetella carolinensis 5 1.2 0.7 2 5.0 1.8
Purple martin, Progne subis 5 1.2 0.7
Green heron, Butorides virescens 4 1.0 0.6
Mallard duck, Anas platyrhynchos 4 1.0 0.6
Summer tanager, Piranga rubra 4 1.0 0.6 2 5.0 1.8
Eastern bluebird, Sialia sialis 3 0.7 0.4
European starling, Sturnus vulgaris 3 0.7 0.4 1 2.5 0.9
Red-eyed vireo, Vireo olivaceus 3 0.7 0.4 1 2.5 0.9
Red shouldered hawk, Buteo lineatus 3 0.7 0.4
Blue-gray gnatcatcher, Polioptila caerulea 2 0.5 0.3
Great blue heron, Ardea herodias 2 0.5 0.3 1 8.3 1.5
White-eyed vireo, Vireo griseus 2 0.5 0.3 1 2.5 0.9
Wood thrush, Hylocichla mustelina 2 0.5 0.3
Cedar waxwing, Bombycilla cedrorum 2 16.7 3.0
Wild turkey, Meleagris gallopavo 1 0.2 0.1 1 0.2 0.1
Red-winged blackbird, Agelaius phoeniceus 1 0.2 0.1 1 2.5 0.9
Other 13a 2.9 1.8 3b 7.5 2.7 2c 16.7 3.0
Total 412 60.2 40 35.7 12d 17.9

a
Includes single specimens containing DNA from American redstart (Setophaga ruticilla), brewerÕs blackbird (Euphagus cyanocephalus),
Carolina wren (Thryothorus ludovicianus), downey woodpecker (Picoides pubescens), eastern meadowlark (Sturnella magna), grasshopper
sparrow (Ammodramus savannarum), great horned owl (Bubo virginianus), greyleg goose (Anser anser), little blue heron (Egretta caerulea),
red-bellied woodpecker (Melanerpes carolinus), song sparrow (Melospiza melodia), swamp sparrow (Melospiza georgiana), and yellow-breasted
chat (Icteria virens).
b
Includes single specimens containing DNA from American goldÞnch (Carduelis tristis), black vulture (Coragyps atratus), and mandarin
duck (Aix galericulata).
c
Includes single specimens containing DNA from Þeld sparrow (Spizella pusilla) and yellow-crowned night-heron (Nyctanassa violacea).
d
Total includes single bloodmeal containing DNA from multiple avian species (cedar waxwing and Þeld sparrow).

Cx. salinarius, 30% for Cx. nigripalpus, and 22% for Cx.
quinquefasciatus.
The percentage of bloodmeals identiÞed as avian or
mammalian origin varied among the mosquito species
(Table 2). The majority of identiÞed Cx. coronator, Cx.
salinarius, and Cx. nigripalpus bloodmeals were from
a mammalian host, whereas only 40% of identiÞed Cx.
quinquefasciatus bloodmeals were from a mammalian
host, signiÞcantly lower by ␹2 analysis (P ⬍ 0.001) than
the other three species. Host utilization by Cx. quin-
quefasciatus varied by location. The percentages of
identiÞed bloodmeals containing avian DNA ranged
from 40 to 83% among sites where the host DNA of at
least 10 Cx. quinquefasciatus bloodmeals were identi-
Þed. The proportions of Cx. quinquefasciatus blood-
meals identiÞed as avian derived did not signiÞcantly
differ among the four bloodmeal size ranks (␹2 ⫽ 4.96,
df ⫽ 3, P ⫽ 0.175). Host DNA from more than one
vertebrate species was detected in a small percentage
of bloodfed Cx. salinarius, Cx. quinquefasciatus, and Cx.
nigripalpus.
A total of 47 avian species, mostly permanent resi-
dent species in EBR Parish, were identiÞed from DNA
extracted from bloodfed Culex species mosquitoes
(Table 3). Over 20% of the avian-derived bloodmeals
of Cx. quinquefasciatus and Cx. nigripalpus were ob-
tained from northern cardinals. Other relatively com-
mon (⬎3% of avian derived bloodmeals) avian hosts
of Cx. quinquefasciatus included the northern mock-
ingbird, the common grackle, the mourning dove, the
domestic chicken, the house sparrow, the brown
thrasher, and the blue jay. Common grackle and do-
mestic chicken DNA frequently were detected in
bloodfed Cx. nigripalpus. The domestic chicken was
the most common avian host of Cx. salinarius. Avian
DNA was only detected in two of the 37 identiÞed
bloodmeals of Cx. coronator (Carolina chickadee and
tufted titmouse; data not shown).
Sixteen species of mammals were identiÞed from
DNA extracted from bloodfed Culex mosquitoes (Ta-
ble 4). The most common mammalian host of C. quin-
quefasciatus and Cx. nigripalpus was the northern rac-
coon. The most common mammalian host of Cx.
salinarius and Cx. coronator was the white-tailed deer.
Human DNA was detected in 7% of the identiÞed
bloodmeals of C. quinquefasciatus and almost 3% of the
242 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 2

Table 4. Number and percentage of bloodmeals obtained from mammalian hosts by Culex mosquitoes

Cx.
Cx. quinquefasciatus Cx. nigripalpus Cx. salinarius
Host species coronator
No. % mammalian % total No. % mammalian % total No. % mammalian % total No. % total
Northern raccoon, Procyon lotor 79 28.8 11.5 41 55.4 36.6 12 21.8 17.9 2 5.4
Domestic dog, Canis lupus familiaris 57 20.8 8.3 2 2.7 1.8 8 14.5 11.9 1 2.7
Human, Homo sapiens 48 17.5 7.0 3 4.1 2.7
Virginia opossum, Didelphis virginiana 41 15.0 6.0 8 10.8 7.1 1 1.8 1.5 1 2.7
Domestic horse, Equus caballus 19 6.9 2.8 2 2.7 1.8 2 3.6 3.0 2 5.4
Domestic cat, Felis silvestris catus 11 4.0 1.6 2 2.7 1.8 6 10.9 9.0 1 2.7
Domestic cow, Bos taurus 5 1.8 0.7 2 2.7 1.8 4 7.3 6.0
Nine-banded armadillo, Dasypus 5 1.8 0.7
novemcinctus
White-tailed deer, Odocoileus 4 1.5 0.6 13 17.6 11.6 17 30.9 25.4 27 73.0
virginianus
Domestic pig, Sus scrofa domestica 3 1.1 0.4
Hispid cotton rat, Sigmodon hispidus 2 0.7 0.3 1 1.8 1.5
Fox squirrel, Sciurus niger 1 0.4 0.1
River otter, Lontra canadensis 1 0.4 0.1 1 2.7
Roof rat, Rattus rattus 1 0.4 0.1 1 1.8 1.5
Nutria, Myocastor coypus 1 1.4 0.9 1 1.8 1.5
Domestic rabbit, Oryctolagus 2 3.6 3.0
cuniculus
Golden mouse, Ochrotomys nuttalli 1 1.8 1.5
Total 274a 40.1 74 66.1 55b 82.1 35 94.6

a
Total includes four bloodmeals containing DNA from multiple mammalian species (domestic dog and northern raccoon 关2兴, domestic dog
and Virginia opossum 关1兴, and domestic dog and unidentiÞed mammalian host 关1兴).
b
Total includes single bloodmeal containing DNA from multiple mammalian species (white-tailed deer and domestic dog).

identiÞed bloodmeals of Cx. nigripalpus. Domestic dog
DNA was frequently detected in bloodfed C. quinque-
fasciatus and Cx. salinarius. The proportion of blood-
meals obtained from domestic and wild mammalian
hosts differed signiÞcantly among the four Culex spe-
cies (␹2 ⫽ 46.6, df ⫽ 3, P ⬍ 0.001). Domestic species
represented 40% of the mammalian derived blood-
meals of Cx. salinarius and 52% of the mammalian
derived bloodmeals of Cx. quinquefaciatus. In contrast,
most of the mammalian bloodmeals of Cx. coronator
(87%) and Cx. nigripalpus (85%) were obtained from
wild species. DNA from small mammalian hosts (ro-
dent species, excluding Nutria) were detected in only
6% of mammalian derived bloodmeals of Cx. salinarius,
2% of mammalian derived bloodmeals of Cx. quinque-
faciatus, and none of the bloodfed Cx. coronator or Cx.
nigripalpus.
Seasonal Host Utilization Patterns of Cx. quinque-
fasciatus. From May though October, the percentages
of Cx. quinquefasciatus feeding on avian hosts ranged
from 76 to 52% in 2003, and from 83 to 52% in 2004 (Fig.
1). In both years, the highest percentages of Cx. quin-
quefasciatus that obtained blood from a mammalian
host were observed in August. The highest monthly
percentage of Cx. quinquefasciatus containing human
blood was 5% in 2003 (May), and 15% in 2004 (Sep-
tember). Logistic models for mammalian and human
feeding by ordinal date were not signiÞcant for either
year. However, the proportion of identiÞed blood-
meals that were from humans was signiÞcantly higher
by Fisher Exact Test (P ⫽ 0.001) in 2004 (9%) than in
2003 (3%). In both years, there were no signiÞcant
differences in the percentage of human feeds detected
between trap sites ⱕ100 m from a residence (n ⫽ 13),
and trap sites ⬎100 m from a residence (n ⫽ 5).
From May through November, the northern cardi-
nal and the northern mockingbird, together ac-
counted for between 15Ð31% of all identiÞed blood-
meals from Cx. quinquefasciatus (Table 5) and were
most important as hosts during the period. The per-
centage of identiÞed bloodmeals of Cx. quinquefascia-
tus represented by mourning doves was greatest in the
spring. In 2004, the proportion of bloodmeals obtained
from northern cardinals increased signiÞcantly from
May through October (Fig. 1; logistic regression: in-
tercept ⫾ SE ⫽ ⫺4.274 ⫾ 0.763, slope ⫾ SE ⫽ 0.012 ⫾
0.003, n ⫽ 394, P ⬍ 0.001), while the proportion of
bloodmeals obtained by all other avian hosts declined
(logistic regression: intercept ⫾ SE ⫽ 1.666 ⫾ 0.530,
slope ⫾ SE ⫽ ⫺0.009 ⫾ 0.002, n ⫽ 394, P ⬍ 0.001). In
2004, northern cardinal DNA was identiÞed in a sig-
niÞcantly greater proportion of avian-derived blood-
meals collected in September and October (P ⫽
0.017), than during the same months in 2003.
Avian Host Preferences of Cx. quinquefasciatus. FR
values calculated for the northern cardinal, the north-
ern mockingbird, the common grackle, and the brown
thrasher were signiÞcantly greater than one, indicat-
ing that these species were fed on more frequently
than expected based on their abundance measured by
the BBS (Table 6). The FR value for the house sparrow
was signiÞcantly less than one, indicating that this
species was fed less frequently than expected based on
its abundance. FR values calculated for the European
starling, the Carolina wren and the red-bellied wood-
pecker also were signiÞcantly less than one (data not
shown). Host DNA from 30 permanent and summer
resident species, representing ⬇26% of all permanent
and summer residents counts recorded during the
March 2010 MACKAY ET AL.: HOST FEEDING PATTERNS IN Culex MOSQUITOES 243

Fig. 1. Seasonal host utilization by Cx. quinquefasciatus collected from sites in and adjacent to EBR Parish, from November
2002 to November 2004.

NABBS in EBR Parish in 2003 and 2004, were not
identiÞed in any bloodfed Cx. quinquefasciatus.
Discussion
In the current study, over 60% of identiÞed Cx.
quinquefasciatus bloodmeals contained avian DNA,
mostly from Passeriformes species, providing further
evidence for the importance of Cx. quinquefasciatus as
an enzootic vector of WNV in southern Louisiana
(Gleiser et al. 2007). The frequency of avian feeding
may have been inßuenced by the types of habitats
where the majority of Cx. quinquefasciatus were col-
lected in our study. The NABBS route for Baton Rouge
and most of the sites sampled for bloodfed mosquitoes
in the current study are located in areas of low to
moderate urban development interspersed with
patches of hardwood forest. More than half of the
bloodfed Cx. quinquefasciatus were collected from
sites classiÞed as wooded. In a previous host range
study of Cx. quinquefasciatus in EBR Parish, avian
blood was detected in ⬍20% of females collected di-
rectly adjacent to human residences, but in over 45%
of females collected from hardwood forests (Niebylski
and Meek 1992). In Florida, only 33% of bloodfed Cx.
quinquefasciatus collected from a sewage pond adja-
cent to a residential area were shown to have fed on
an avian host, compared with 73% of females collected
from a swamp adjacent to an agricultural area (Edman
1974). It was recently reported that avian DNA was
identiÞed in only 47% of bloodfed Cx. quinquefasciatus
collected from Harris County, TX (39% contained
only avian DNA, 8% contained avian and mammalian
DNA; Molaei et al. 2007). An earlier study in Harris
County had detected avian feeding by Cx. quinque-
fasciatus in 58% of bloodfed females collected from
storm drains, and 66% of bloodfed females collected
resting in abandoned houses and chicken houses

Table 5. Seasonal utilization of common avian hosts by Cx. quinquefasciatus

No. bloodmeals identiÞed from avian host species (% all identiÞed bloodmeals)
Month Northern Northern Common Mourning Domestic House Brown
Blue jay
cardinal mocking bird grackle dove chicken sparrow thrasher
Feb. 1 (33) 1 (33)
Mar. 1 (33) 1 (33)
April 3 (25) 4 (33)
May 8 (13) 8 (13) 8 (13) 7 (11) 1 (2) 2 (3) 2 (3) 1 (2)
June 12 (9) 8 (6) 8 (6) 7 (6) 4 (3) 3 (2) 7 (6) 2 (2)
July 23 (14) 17 (11) 4 (2) 6 (4) 8 (5) 7 (4) 2 (1) 3 (2)
Aug. 17 (13) 15 (11) 5 (4) 5 (4) 4 (3) 6 (5) 2 (2) 1 (1)
Sept. 19 (19) 11 (11) 4 (4) 3 (3) 8 (8) 3 (3) 1 (1) 1 (1)
Oct. 19 (29) 1 (2) 6 (9) 3 (5) 1 (2) 2 (3) 1 (2)
Nov. 1 (7) 3 (21) 3 (21) 1 (7)
Dec. 2 (33) 1 (17) 2 (33)
244 JOURNAL OF MEDICAL ENTOMOLOGY
Table 6. Forage ratios for permanent and summer resident,
wild avian species representing >1% of identified Cx. quinquefas-
ciatus bloodmeals
% bloodmeals
% NABBS Forage ratio
Species from wild
counts (95% CI)
avian hosts
Northern cardinal 27.2 9.4 2.9 (2.5, 3.3)
Northern mockingbird 16.5 9.3 1.8 (1.4, 2.1)
Common grackle 11.5 7.3 1.6 (1.2, 2.0)
Mourning dove 8.8 6.2 1.4 (1.0, 1.8)
House sparrow 5.9 12.1 0.5 (0.3, 0.6)
Brown thrasher 4.3 1.7 2.5 (1.5, 3.5)
Blue jay 3.5 4.0 0.9 (0.5, 1.3)
Tufted titmouse 2.4 1.2 2.0 (0.9, 3.1)
Fish crow 2.1 3.2 0.7 (0.3, 1.1)
(Kokernot et al. 1974). Our data support previously
published observations that Cx. quinquefasciatus is an
opportunistic bloodfeeder, and that signiÞcant host
range variability can be observed at relatively Þne
spatial scales, likely a result of the relative availability
of mammalian and avian hosts among habitats.
In the current study, about half of all mammalian
bloodmeals were obtained by Cx. quinquefasciatus fe-
males from domestic species. This domestic associa-
tion is supported by the higher degree of anthropo-
phagy observed in this species, relative to the other
Culex species examined, even though the percentage
of bloodmeals obtained from mammalian hosts was
signiÞcantly lower in Cx. quinquefasciatus. The overall
percentage of Cx. quinquefasciatus feeding on humans
observed in our study (7%) was less than previously
reported in EBR Parish. Niebylski and Meek (1992)
found that between 11 and 15% of bloodfed females
collected from residential areas and between 16 and
23% of bloodfed Cx. quinquefasciatus females collected
from adjacent hardwood forests had fed on a human
host. However, human feedings were detected in ⬍2%
of Cx. quinquefasciatus collected from urban and sub-
urban locations Harris County, TX (Kokernot et al.
1974, Molaei et al. 2007), and rural habitats in Missis-
sippi (Bertsch and Norment 1983). Molaei et al.
(2007) suggested that the low rate of anthropophagy
observed in Harris County, TX, may have been be-
cause of high temperatures inhibiting human outdoor
activities during the summer months, although much
higher human feeding rates (⬇50%) have been re-
ported in Cx. quinquefasaciatus collected from urban
habitats in Arizona (Zinser et al. 2004).
In Harris County, TX, Molaei et al. (2007) reported
observing a signiÞcant increase in Cx. quinquefasciatus
mammalian feeding after August. However, previous
host range studies of Cx. quinquefasciatus in the south-
ern U.S. have failed to demonstrate signiÞcant sea-
sonal changes in the mammalian:avian feeding ratio. In
Oktibbeha County, MS, Bertsch and Norment (1983)
observed an apparent peak in the proportion of mam-
malian bloodmeals obtained during the summer
months, but the trend was not statistically signiÞcant.
Similarly, Niebylski and Meek (1992) failed to detect
a signiÞcant seasonal increase in the rate of mamma-
lian feeding in EBR parish. In the current study, we did
Vol. 47, no. 2
not observe a signiÞcant seasonal change in the pro-
portion of bloodmeals obtained from mammalian
hosts in either 2003 or 2004. However, a seasonal shift
in the feeding behavior of Cx. quinquefasciatus may not
be a requirement for epidemic transmission of WNV
in southern Louisiana, as has been suggested for Culex
vectors of WNV in Shelby County, TN, and Chicago,
IL (Savage et al. 2007, Hamer et al. 2009). During the
summer and early fall months when the majority of
human cases of WNV are reported, Cx. quinquefascia-
tus is often very abundant in urban areas, exhibits a
high rate of blood feeding on competent passerine
hosts and a relatively high rate of WNV infection
(Mackay et al. 2008), and consistently feeds on human
hosts.
Although we did not detect a seasonal shift in the
proportion of Cx. quinquefasciatus feeding on avian
versus mammalian hosts within either year, speciÞc
differences in the bloodfeeding patterns of Cx. quin-
quefasciatus may have contributed to increased rates
of WNV infection in the vector and higher incidence
of human disease in 2004. From 2003Ð2004, we de-
tected a threefold increase in the proportion of Cx.
quinquefasciatus feeding on humans. In 2004, the high-
est rates of human feeding were observed from July
through October. From May to October 2004, we also
observed a signiÞcant seasonal increase in the Cx.
quinquefasciatus feeding on northern cardinals, which
has been demonstrated to be a competent host of
WNV in the laboratory (Komar et al. 2005), with a
concurrent decline in the rate of feeding on all other
avian hosts. While the density of Cx. quinquefasciatus
in EBR parish was similar in both years from July to
October, in 2004 WNV was isolated from a greater
proportion of pools tested and the number of human
cases of WNV associated illnesses was ⬎7⫻ higher
than in 2003 (Mackay et al. 2008).
In Florida, Cx. nigripalpus is considered to be one
of the most important epizootic vectors of WNV
(Rutledge et al. 2003, Godsey et al. 2005), and the
primary enzootic and epidemic vector of St. Louis
Encephalitis virus in central and southern part of
the state (Dow et al. 1964, Shroyer 1991). This was
the Þrst study to examine the host range of Cx.
nigripalpus in Louisiana. Our data are consistent
with previous studies documenting opportunistic
feeding by this species (Edman 1974, Gomes et al.
2003). Approximately two-thirds of the bloodmeals
identiÞed were from mammalian hosts common to
EBR Parish; primarily the northern raccoon and the
Virginia opossum. Although the host competence of
these two species for WNV is unknown, they have
been shown to be very poor hosts of St. Louis En-
cephalitis virus (McLean et al. 1985). The remaining
hosts included a diverse range of avian species,
primarily passeriformes. Previous studies in rural
habitats in Florida have reported a very low per-
centage feeding on humans (Provost 1969). In the
current study, a relatively low rate of anthropoph-
agy was observed. In both 2003 and 2004, host-
seeking females were not collected in light traps
before July in EBR Parish and were not abundant
March 2010 MACKAY ET AL.: HOST FEEDING PATTERNS IN Culex MOSQUITOES
until September (data not shown). Although WNV
activity was detected in several pools of Cx. nigri-
palpus collected concurrently from EBR parish
(Mackay et al. 2008), the seasonal pattern of host-
seeking activity, and frequent use of mammals as
hosts, may limit the importance of this mosquito
species in enzootic ampliÞcation of WNV in south-
ern Louisiana.
The majority of identiÞed Cx. salinarius blood-
meals were from a wide diversity of mammalian
hosts, indicating an opportunistic feeding strategy.
The most frequent host detected was the white-
tailed deer, followed by the northern raccoon and
domestic cats and dogs. A signiÞcant proportion of
bloodmeals were derived from domestic mammalian
hosts, and ⬇9% of identiÞed bloodmeals were from
passeriform birds. This is consistent with previous
host range studies reporting that this species typi-
cally obtains most of its blood meals from mammals,
particularly white-tailed deer and canines (Suyemoto et
al. 1973, Irby and Apperson 1988, Apperson et al. 2004,
Molaei et al. 2006), although the proportion can vary
greatly depending on the local host fauna (Edman
1974). This species is considered a highly competent
vector of WNV (Sardelis et al. 2001), and the results
of the current study suggest that it may play a role
as a bridge vector of WNV in EBR Parish.
Several recent reports have documented an ap-
parent range expansion of Cx. coronator in the south-
eastern United States (Debboun et al. 2005, God-
dard et al. 2006, Smith et al. 2006, Gray et al. 2008,
Moulis et al. 2008). The ecology of Cx. coronator in
this region has not been well described. In the
current study, almost three-quarters of the identi-
Þed bloodmeals from Cx. coronator were from
white-tailed deer, suggesting a preference for large
mammals. This pattern is consistent with previous
observations where large numbers of host-seeking
females have been collected from equine hosts in
Mexico, Texas, and New Mexico (Jones et al. 1977,
Reyes-Villanueva et al. 2006), and human hosts in
Belize, Brazil, and Mexico (Bertram 1971, Roberts et
al. 1981, Reyes-Villanueva et al. 2006). The public
health importance of Cx. coronator is unclear. Al-
though WNV has been detected in Cx. coronator
collected from EBR Parish (Mackay et al. 2008), the
vector competence of this species for WNV has not
been evaluated. The host range described by our
data and previous studies indicate that Cx. coronator
is unlikely to contribute signiÞcantly to the enzootic
ampliÞcation cycle of WNV, but is a potential bridge
vector of WNV to equines and possibly humans.
The high rate of feeding by Culex mosquitoes on
raccoons and opossums is consistent with WNV se-
ropositive rates observed for these species in south-
ern Louisiana. Approximately 75% of Virginia opos-
sums and 60% of northern raccoons collected in 2002
from Slidell, a community in the southeastern cor-
ner of St. Tammany Parish, were positive for WNV-
speciÞc antibodies (Dietrich et al. 2005). Although
oral infection by WNV of mesopredator species
feeding on infected prey may increase their expo-
245
sure (Root et al. 2005), the results of the current
study indicated that vector-borne transmission of
WNV alone could support the high seropositive
rates observed in northern raccoons and Virginia
opossums in southern Louisiana.
Collectively the northern cardinal and northern
mockingbird represented about one-quarter of all
identiÞed bloodmeals of Cx. quinquefasciatus. Both
avian species are well adapted to residential habitats
(Blair 1996, Brennan and Schnell 2005). Experimen-
tally infected northern cardinals can develop a peak
viremia ⬎108 plaque forming units (PFU)/ml of
serum (Komar et al. 2005), sufÞcient to infect most
susceptible vector species, including Cx. quinque-
fasciatus (Sardelis et al. 2001). Experimental infec-
tions in northern mockingbirds indicated that this
species is a moderately competent host of WNV,
developing an average peak viremia of ⬇106 PFU/ml
of serum (Komar et al. 2005). Based on the abun-
dance of these species, their close associations with
residential development, their importance as hosts
of Culex vectors throughout the spring and summer
months, and their susceptibility to WNV infection,
the northern cardinal and the northern mockingbird
may be two of the most important amplifying hosts
of WNV in urban habitats of southern Louisiana.
The common grackle was the third most common
avian host of Cx. quinquefasciatus, and second most
common avian host of Cx. nigripalpus. This species is
abundant throughout most parts of Louisiana, nesting
and foraging in large groups (Yang and Selander
1968). This species is considered a highly competent
host for WNV (Komar et al. 2003), and was a frequent
source of blood for Cx. quinquefasciatus in the spring.
The common grackle may be an important amplifying
host of WNV during the early season in southern
Louisiana.
The blue jay is one of the most common corvid
species in urban areas of EBR Parish and has been
demonstrated in the laboratory to be an extremely
competent reservoir host for WNV (Komar et al.
2003). Our data suggest that this species may be a
frequent host of Cx. quinquefasciatus during the
winter months. This is consistent with the Þndings
of a dead bird surveillance program in Harris
County, TX, where dead blue jays yielded the great-
est number of WNV positive samples from January
2003 to March 2004 (Tesh et al. 2004). The blue jay
may be an overwintering host for WNV in southern
Louisiana.
For many avian species, the relative rates of ex-
posure to blood feeding Cx. quinquefasciatus ob-
served in our study were consistent with the results
of a concurrent study of WNV neutralizing anti-
bodies in wild birds conducted in EBR Parish
(Gruszynski 2006). From 2002Ð2004, the overall
percentages of birds positive for neutralizing anti-
bodies for WNV were 26% in northern cardinals,
40% in northern mockingbirds, 35% in mourning
doves, 20% in house sparrows, 19% in brown thrash-
ers, and 14% in blue jays. Similarly high WNV se-
roprevalence rates in northern cardinals, northern
246 JOURNAL OF MEDICAL ENTOMOLOGY
mockingbirds, mourning doves, and blue jays were
reported from St. Tammany Parish, LA, in 2002 and
from Harris County, TX, in 2005 (Komar et al. 2005,
Molaei et al. 2007). In contrast the Carolina wren
was fed on by Cx. quinquefasciatus less frequently
than expected based on concurrent measures of
WNV seroprevalence (17%; Gruszynski 2006) and
relative abundance (5% of the total NABBS counts).
Similarly, Molaei et al. (2007) detected a relatively
high WNV seroprevalence (19%) in Carolina wrens
in Harris County, TX, but identiÞed DNA from this
species in only 0.3% of Cx. quinquefasciatus blood-
meals. These data suggest that exposure of Carolina
wrens to WNV is occurring via a different route,
such as a vector other than Cx. quinquefasciatus, or
orally after feeding on infected mosquitoes. Alter-
natively, Cx. quinquefasciatus may be feeding on
Carolina wrens in a habitat not sampled in the cur-
rent study.
In the current study, we examined the host-uti-
lization patterns of four species of Culex mosquitoes
common in EBR Parish. Cx. quinquefasciatus fed
more frequently on host species closely associated
with human habitation, as opposed to less synan-
thropic hosts, than did the other Culex species ex-
amined. In contrast, Cx. nigripalpus and Cx. corona-
tor fed frequently on host species associated with
undeveloped areas. The host-feeding pattern of Cx.
salinarius was intermediate. Based on host selection
patterns, Cx. quinquefasciatus likely serves as the
primary enzootic vector as well as the primary ep-
idemic vector of WNV in southern Louisiana. Cx.
nigripalpus may be important as a late summer
bridge vector, whereas Cx. salinarius may serve as a
bridge vector to equines and other domestic ani-
mals. The vector competence of Cx. coronator is
unknown, but our host range data indicate that it
may be a bridge vector to large mammals, such as
equines.
Acknowledgments
We thank Alex Folsom, Rod Wells, Gerardo Boquin, Fran-
cis Currin, Lana Gallegos, and Laura Latil for their assistance
with the collection and sorting of mosquito samples. We
thank Fred Augustine and Randy Vaeth of East Baton Rouge
Mosquito Abatement and Rodent Control, and the Louisiana
State University Museum of Natural Science Collection of
Genetic Resources, for providing the samples of vertebrate
tissue. Cory Reynolds of the MarshÞeld Research Foun-
dation, and Susan Murray of the Louisiana State University
Museum of Natural Science Collection of Genetic Re-
sources, provided advice and assistance with sequencing
and fragment length measurements. We also acknowledge
NSF Grant DBI-400797 to R.T.B. that was used to purchase
the sequencing/fragment analysis instrument. This paper
is published with approval of the Director of the Louisiana
Agricultural Experiment Station, as manuscript number
2009-234-3641.
Vol. 47, no. 2
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Received 2 July 2009; accepted 11 November 2009.