Volume 23, Issue 2, December 1998

Transcription

Journal
of
Vector
Ecology
Journal of the Society for Vector Ecology
Volume 23, No. 2
December 1998 4/
JVE 23( 2): 107- 201
ISSN 1081- 1710
Printed
on
Acid- Free Paper
Journal of Vector Ecology
Volume 23- Number 2- December 1998
Published by the Society for Vector Ecology
Marc J. Klowden, Editor
Division of Entomology
University of Idaho
Moscow, ID 83844- 2339
Phone: ( 208) 885- 7546
Fax: ( 208) 885- 7760
E-mail: mklowden @uidaho.edu
EDITORIAL BOARD
M. S. Mulla, Chair( 2000)
University
of
C. I. Dahl( 2001)
California
University
of
Uppsala
Riverside, CA, USA
Uppsala, Sweden
R. C. Axtell( 1998)
F. R. Holbrook( 1998)
J. W. Beehler( 2001)
Northwest Mosquito and Vector
Control District, Corona, CA,
USA
North Carolina State
USDA- ARS
M. W. Service( 1998)
Raleigh, NC, USA
Laramie, WY, USA
Liverpool School of Tropical
N. Becker( 2001)
R. S. Lane( 1999)
Liverpool, England
KABS
University of California
Berkeley, CA, USA
E. D. Walker( 1999)
D. R. Barnard( 1998)
G. G. Marten( 1998)
Michigan State University
East Lansing, MI, USA
USDA-ARS
Kwansei Gakuin University
Gainesville, FL, USA
Sanda, Hyogo, Japan
H. Briegel( 1999)
L. S. Mian( 2000)
University
Medicine
Waldsee,
Germany
S. K. Wikel( 1998)
Oklahoma State University
University
of
Zurich
Stillwater, OK, USA
County Vector Control
Zurich, Switzerland
San Bernardino, CA, USA
W. J. Crans( 1998)
R. S. Nasci( 200(3)
Rutgers
CDC- DVBID
S. C. Williams( 1998)
San Francisco State University
University
New Brunswick, NJ, USA
San Francisco, CA, USA
Ft. Collins, CO, USA
The Journal of Vector Ecology is published biannually in June and December. Authors agree to transfer the
copyright for their article to the publisher when the article is accepted for publication. Authorization to photocopy
articles is granted by the Society for Vector Ecology provided the indicated fee is sent to the Copyright Clearance
Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Individuals may make single copies of articles
without charge. Communications relating to editorial matters and manuscripts should be addressed to the Editor.
Communications relating to galley proofs, reprints, subscriptions, SOVE membership, change of address, and
other matters should be addressed to the Business Office.
Publications and Business Office: Society for Vector Ecology, 1966 Compton Avenue, Corona, CA 91719 USA.
Subscription Rates:
Institutional
Membership, including the Journal of Vector Ecology,$ 50.00, Student membership$ 25. 00,
subscription$
50.00,
Sustaining
membership $ 100. 00
SOCIETY FOR VECTOR ECOLOGY
1998 BOARD OF DIRECTORS
OFFICERS
President
Rex E. Thomas
President- Elect
David A. Dame
Vice- President
John D. Edman
Past- President
Michael W. Service
Secretary- Treasurer
Major S. Dhillon
REGIONAL DIRECTORS
Southwestern
Minoo B. Madon
Northwestern
Sammie Dickson
North Central
Thomas R. Wilmot
South Central
Cluff E. Hopla
Northeastern
Wayne J. Crans
Southeastern
Jonathan F. Day
Isabelle Thiery
European
SOVE Journal Editor
Marc J. Klowden
JOURNAL OF VECTOR ECOLOGY
Volume 23
DECEMBER, 1998
Number 2
CONTENTS
Board
of
Directors
ii
Guidelines for Contributors
William Donald
Murray
iv
Memoriam
v
Howard R. Greenfield Memoriam
vii
Submitted Papers
Olfactory Responses of Female Culex quinquefasciatus Say ( Diptera: Culicidae) in a DualChoice Olfactometer
L. E. G. Mboera, B. G. J. Knols, W. Takken,
and
P. W. T. Huisman
107
Antifeedancy of Neem Products Containing Azadirachtin against Culex tarsalis and Culex
quinquefasciatus(
Diptera: Culicidae)
Tianyun Su
and
Mir S. Mulla
114
A New Enzyme Immunoassay to Detect Antibodies to Arboviruses in the Blood of Wild Birds
R. E. Chiles
and
W. K. Reisen
123
Surveillance Studies of Orthopodomyia signifera with Comparisons to Aedes sierrensis
David L. Woodward, Arthur E. Colwell,
and
Norman L. Anderson
136
Effectiveness of Control Measures against Mosquitoes at a Constructed Wetland in Southern
California
William
E. Walton, Parker D. Workman, Louie A. Randall, Joshua A. Jiannino,
and
Yvonne A. Offill
149
Effect of Two Rice Culture Methods on the Seasonal Occurrence of Mosquito Larvae and Other
Aquatic Animals in Rice Fields
of
Southwestern Korea
Dong- Kyu
Lee
161
Evaluation of Attractant-Baited Traps/ Targets for Mosquito Management on Key Island,
Florida, USA
Daniel L. Kline
and
Gene F. Lemire
171
Olfactory Responses and Field Attraction of Mosquitoes to Volatiles from Limburger Cheese and
Human Foot Odor
Daniel L. Kline
186
Egg Maturation in Neotropical Malaria Vectors: One Blood Meal is Usually Enough
L. P. Lounibos, D. Couto Lima, R. Lourenco- de- Oliveira, R. L. Escher,
and
N. Nishimura
195
JOURNAL OF VECTOR ECOLOGY
Guidelines for Contributors
The Journal of Vector Ecology is an international journal published by the Society for
Vector Ecology. It is concerned with all aspects of the biology, ecology, and control of arthropod
vectors and the interrelationships between the vectors and the disease agents they transmit. The
journal publishes original research articles and research notes, as well as comprehensive reviews
of vector biology based on presentations at Society meetings. All papers are reviewed by at least
two referees who are qualified scientists and who recommend their suitability for publication.
Acceptance of manuscripts is based on their scientific merit and is the final decision of the editor,
but these decisions may be appealed to the editorial board.
Manuscripts intended for publication should be sent to Dr. Marc J. Klowden, Editor,
Division of Entomology, University of Idaho, Moscow, Idaho 83844- 2339, U.S. A. Manuscripts
must be double spaced on a single side of bond paper with 25 mm margins. An original and two
clear copies are required. Draft mode dot matrix type should not be used. Submission of text on
a 3- 1/ 2" computer diskette formatted in MS- DOS is encouraged. Microsoft Word, Word Perfect,
or Wordstar formats are acceptable, as well as unformatted text files. Please indicate the type of
format on the diskette label. Papers must be organized under the following headings, each on a
separate page,
in
order:
Title
page, abstract,
text,
acknowledgments ( if appropriate), references
cited, tables, figure legends, and figures. The title page should contain the names of all authors,
their affiliations and the identification and address of the corresponding author. It should also
include a keyword index containing no more than five words that best describe the paper. Pages
should be numbered consecutively starting with the title page. References should conform to the
style in recent volumes. Illustrations that are submitted must be of high quality and remain legible
after reduction.
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SOVE members who are unable to pay page charges may apply for a limited number of waivers.
charges, which
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Communications relating to editorial matters and manuscripts should be addressed to the
Editor. Communications concerning galley proofs reprints, subscriptions, SOVE
membership, and change of address should be addressed to the Business Office.
PUBLICATIONS AND BUSINESS OFFICE
Society for Vector Ecology
1966 Compton Avenue
Corona, CA 91719
Phone: ( 909) 340- 9792; Fax: ( 909) 340- 2515; E- Mail:
nwmvcd
@pe. net
NUMBER 2
DECEMBER, 1998
VOLUME 23
IN MEMORIAM
WILLIAM DONALD MURRAY
1913- 1998
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Dr. W. Donald
it*. 0r.
Murray,
a
Murray
was
His early interest in
i
at
born
Dr.
both
Delong,
University,
influenced him to
received
a
where
who
S;
.
Minnesota.
t
ar
icy
11. t
ill:
.
i#
While there he was awarded a masters
his home in Visalia,
in the research of wasps. He married his wife Frances
August 9, 1913, in
during the time he was in graduate school.
This was the time of the depression years and full-
and colleagues
history was spawned
time employment was
Dr. Donald Borror
and
entomology from Ohio
served as his mentors and
obtain.
Don spent
where he first developed the pants leg count that was
later used to count Aedes mosquitoes in California.
in
After graduate school he taught botany and zoology
he received his BS degree. Don then
at Eveleth Junior College in Eveleth, Minnesota. He
attend
scholarship to
Ohio State
difficult to
summers evaluating grasshopperpopulations and control
operations in the midwestern prairie states.
This is
professors at
Station
entomology,
met
:
1.
by his experience in summer YMCA camp sessions near
Columbus, Ohio. There he
hF
i
degreeandlateraPh. D. Hisgraduateworkwasprimarily
on
natural
s•
in California,
control
Shayside, Ohio, and was known by friends
asDon.
if..}
dedicated leader in the
early development of mosquito
passed away on March 18, 1998,
California.. Dr.
z.
4}
c
+
s•
o
4 tad
ss#
s
ail
tk:
r
l iqi
lib
ra
r+
s
a
attend
the
and
major
University
then went on to teach biology at Bemidji State Teachers
of
v
VOLUME 23
DECEMBER, 1998
College in Bemidji, Minnesota from 1942- 1943.
In November 1943, Don
the U. S.
Pacific
Navy
oil was used
of
developed methods to eliminate larval sources ofMusca
in
domestica using a rotary scraper to spread and dry the
South
feed- manure mixture behind the feed bunkers on large
a malaria
dairy ranches. To control the green blow fly, Phoenicia
received a commission
Lieutenant
island
the
on
control unit.
diesel
as a
to the
and sent
Efate to head- up
In the early phases of the operation, diesel
as a larvicide; DDT powder mixed with
Don
was a
in California)
this approach
of mosquito
Mosquito and Vector Control Association) was hired to
strong early
advocate of
direct the fly control program. John' s effort was very
effective in developing a community fly control
debris
was started on
coconut
hulas
landfill,
and the axil points of the
drained
to
and
eliminate
sp. in the urban areas, John Combs( who later became
District Manager and ExecutiveDirectoroftheCalifornia
His idea
oil gave excellent control.
source reduction (
cans
were
Efate,
disposed
where
in
of
a
education program and working with city officials to
were
employ better methods of refuse storage and collection.
that served as
Don was Past- President( 1956), elected Honorary
Member, and served as Secretary- Treasurer for 16 years
States he talked to Professor
for the California Mosquito and Vector Control
breadfruit trees
water
standing
sources of the malaria vector.
Upon his
Herms
return
U.C.
at
Dick Peters
to the
Berkeley
the
of
who suggested
that
California Department
he
of
contact
Association. He was awarded the" Medal of Honor" for
Public
his service as Treasurer ( 8 years) by the American
Mosquito Control Association. He was a founding
member of the Society for Vector Ecology and a longtime member of the Entomological Society of America.
Health where they were establishing a new program unit
in vector control. Don worked for the newly formed
Bureau
Vector Control
during 1946. In the spring of
job opening for Manager of the Delta
Mosquito Abatement District. He applied and was hired
of
1947, there
for the
He continued to be active as an officer and consultant to
was a
in
position
In his 31
spring of 1947.
1947- 1978) as Manager
the
years (
the U.S. Navy Medical Service Corps., obtaining the
rank of Captain before his retirement from the
of
Commission Officers Corps.
the
District, Don developed many innovative approaches to
mosquito and fly control. He originated the concept of
the section
survey
that
procedure
of each square mile
in
delineating the
his 80s). He was active in the Visalia Ys Men Club, and
the
crop, irrigation
and
served as its President for several years. He was listed
plotting areas of standing water
frequent schedule. This allowed the zone operator
in American Men and Women of Science- Medical and
drainage
to
mapping
Don was very active in his community. He served
on the Tulare County Grand Jury, and was a member of
the Visalia Racquet Club( he was still playing tennis in
involved
each control zone,
field- crop boundaries, type
on a
NUMBER 2
of
patterns, and
become
more
effective
in
locating
source within the proper time sequence.
he
was an
employed personnel and equipment to establish
drain
water systems, and
He is survived by his wife, Frances Murray of
early
Visalia, son David Murray of Arroyo Grande, daughter
that
Lois Feleay ofVisalia, brothers Allen and Robert Murray
of Columbus, Ohio, and grandchildren Kevin Murray,
adopter of an effective source reduction program
channels, return
Health Sciences.
the mosquito
drainage
dairy
drain
Aaron Feleay, and Shara Hendren.
recycle disposal facilities.
In
response
program
during
personnel
to
community pressure for a fly control
1960s, the District, along with
Earl W. Mortenson, Assistant Chief, Retired
Vector Surveillance and Control Branch
the early
from the State Bureau
of
Vector Control,
California Department of Health Services
vi
NUMBER 2
DECEMBER, 1998
VOLUME 23
IN MEMORIAM
HOWARD R. GREENFIELD
1918- 1998
s
dim \
s,,,
it
k
Itt
s
E
4
It
was a sad
colleague,
31, 1998,
had
was
time to hear that our good
Howard Greenfield,
after a year-
long
passed
away
friend
on
town of Prescott. He later moved back to California and
and
lived in Los Altos.
August
struggle with cancer.
I first
College for two years before joining the U.S. Navy.
Most of his Navy experience was in the South Pacific
opportunity to meet Howard in 1950, when he
entomologist for the Merced Mosquito Abatement
an
collecting
soil samples
pastures.
Howard
from
pastures
infested
with
in
other campaigns, where his unit was awarded five
and
helping us
Merced County
Aedes
where he participated in the Leyte, Guadalcanal, and
I had been working on
for Aedes eggs from irrigated
Deed Thurman
District.
was
combat battle stars. After his five year Navy service, he
returned to San Jose State College to complete his BS
to obtain samples
that were
degree.
heavily
Howard' s interest in biology was soon channeled
into courses in entomology. Dr. Carl Duncan, Professor
of Entomology and Dr. Ed Ross of the California
Academy of Science served as his mentors and in the
nigromaculus.
Howard was born in 1918 in Santa Cruz, California.
He
where
his
and grandparents operated a gold mine near
the
spent a portion of
father
his
youth
in Arizona
Howard attended San Jose State
vii
VOLUME 23
senior
DECEMBER, 1998
Howard
year,
Science Student
His first
of
of
was
awarded
the Year.
He was very active and made major contribution to the
Vector Control, California Department
1948 to
evaluate
pretreatment
Merced
to work with
County.
Public
serving as President in 1957 and was on key committees;
Dr. Basil Markos in
Ways and Means, Legislative, and Bylaws. He was one
the effectiveness
application
of
irrigated
on
Ed Smith, Manager
of
DDT
District Entomologist. In 1951, Howard
Merced
of the
designing
Manager- Engineer
the
source
reduction
district to
was
of the
His wife, Betty Greenfield, son Brice Greenfield of
in
Pensacola, Florida, daughter Diane Lott, Rio Vista,
was
grandchildren Nicole Greenfield, Samantha Greenfield,
consultation
program
community organizations.
another
that
and Jennifer Lott survive him.
drainage
help
problems inherent to the noi. h part of Monterey County.
Howard was a Charter Member of the Society for
required
Vector Ecology
Society
in 1971.
Howard was
School Board, Past- President of the Salinas County
Club( a very good golfer), and member of many other
the
Howard in providing
for this
Mosquito Abatement District in 1982.
the
as
active in his community, serving as a member of the
of
Alameda Mosquito Abatement District
to
of the original members of the 1948 Entomology
Committee and was elected Honorary Member in 1983.
Howard retired as manager of the North Salinas Valley
newly formed North
Mosquito Abatement District. The
Valley
legendary Harold Gray,
mentor
a
in
was appointed
Manager- Entomologist
Salinas
as
pastures
Mosquito Abatement District hired Howard
the
California Mosquito and Vector Control Association,
Bureau
professional position was with the
health, designated
Control Association Meritorious Service Award in 1973.
Outstanding
the
NUMBER 2
new
and served as the
He
received
solve the
Earl W. Mortenson, Assistant Chief, Retired
Vector Surveillance and Control Branch
California Department of Health Services
fourth President of the
the
American Mosquito
viii
Journal of Vector
Ecology
23( 2): 107- 113
Olfactory Responses of Female Culex quinquefasciatus Say
Diptera: Culicidae) in a Dual-Choice Olfactometer
L. E. G.
Mboera1"2,
B. G. J.
Kno1s13,
W. Takken', and P. W. T. Huisman'
Laboratory of Entomology, Wageningen Agricultural
University, Wageningen, THE NETHERLANDS.
2 National Institute for Medical Research,
Ubwari Field Station, Muheza, TANZANIA.
3International Centre of Insect Physiology
and Ecology, Nairobi, KENYA.
Received 24 February 1998; Accepted 13 July 1998
ABSTRACT: Olfactory responses of individual female ( n= 1010) Culex quinquefasciatus Say ( Diptera:
Culicidae) to various odor stimuli were studied in a dualchoice olfactometer. Responses( i.e., the number of
mosquitoes entering either of both olfactometer ports) were studied towards clean conditioned air( control),
human foot skin
emanations( collected on worn stockings), carbon
dioxide( 4. 5% in clean air), moistened air,
and various combinations thereof. Skin emanations were significantly more attractive( x2= 23.0, p< 0.001)
than clean stockings( control). The mosquito was also significantly more attracted( x2= 7. 7, p< 0.01) to skin
emanations than to a clean stocking to which water( an equivalent of that absorbed by a worn stocking) was
added. A moistened( 1 g H2O) clean stocking, however, was slightly more attractive than a dry stocking(
x2=
6, p< 0. 025). Carbon dioxide( 4. 5%) did not elicit higher responses than clean air, and no synergistic effect
was observed in combination with skin emanations. With the aim of developing an odor-baited trap, our
results indicate that Cx. quinquefasciatus responds well to human body odors which can be collected on
polyamide materials.
Keyword Index: Culex quinquefasciatus, host- seeking behavior, human skin emanations, carbon dioxide,
olfactometer.
1996) showed a random distribution of bites on a naked
INTRODUCTION
motionless volunteer.
Culex
the most
world.
The
Diptera: Culicidae) is
Host-seeking mosquitoes use visual, physical, and
bancroftian filariasis in the
olfactory cues produced by their host to orient themselves
is widely distributed in tropical and
the world where it has established
to that host( Takken 1991). Of the available stimuli, host
quinquefasciatus
important
Say(
vector of
mosquito
odor is considered to be the most important, especially for
subtropical regions of
itself in towns
to the creation
containing
that
larvae
of favorable
highly
use
nocturnally active species that seek their host when visual
breeding sites, mainly habitats
cues are at a minimum or even nonexistent. Several
polluted water rich
for
Host- seeking
fasciatus
human habitation has led
and areas where
nourishment(
and
constitute
biting
in
behavior
important
filariasis epidemiology. The
aspects
of
transmission
of
the peripheral
1969)
which are
mosquito
dioxide is generally considered to be an important
mosquito attractant( reviews by Clements 1963, Gillies
quinque-
bancroftian
is markedly
White 1971,
behavioral traits
1980, Mboera and Takken 1997), although its specific
role in the host-seeking behavior of different mosquito
species is poorly understood. In addition to carbon dioxide,
favoring the
Wuchereria bancrofti microfilariae from
blood
system
reported a preference
humans but
their role in mosquito host location. Of these, carbon
Cx.
of
endophagic, anthropophilic, and nocturnal(
Beier et al. 1990),
studies have examined organochemical compounds for
organic matter
Subra 1981).
to the vector.
Self
for biting the lower limbs
recent experiments
by
De
Jong
and
other compounds such as
phenols ( Kline et al. 1990), and carboxylic fatty acids
of
Knols
L(+)- lactic acid( Acree et al.
1968), 1- octen- 3- ol( Takken and Kline 1989), butanone,
et al.
(
Knols et al. 1997) have been
shown
to attract mosquitoes.
108
Journal of Vector
Ecology
December, 1998
The majority of these studies, however, focused on the
yellow fever mosquito Aedes aegypti( Acree et al. 1968,
Pumped air ( 1400 ml/ min) was cleaned by activated
charcoal, humidified, and regulated by volume before
Carlson
et al.
1973)
mediated
very little is known for
veterinary importance. The
other
entering the olfactometer. Wind speed, temperature, and
odor-
relative humidity of the air passing the tunnel ports( see
Cx. quinquefasciatus
Fig. 1) were measured before and after each test, and
and
species of medical and
host- seeking behavior
of
has received remarkably little attention despite its
medical
averaged 20± 4 cm/ sec., 26.9± 0.3° C, and 93. 9± 0.4% RH,
importance. In this preliminary study we sought to
investigate the response of Cx. quinquefasciatus toward
respectively.
human foot skin emanations, carbon dioxide, and moisture
Colombo, Sri Lanka( courtesy Prof. C. F. Curtis) and has
been maintained under laboratory conditions for more
first st,; p in the development
for this filariasis vector.
as a
of an effective odor- bait
The Cx. quinquefasciatus strain used originated from
than ten years. Mosquitoes were kept in a climate-
controlled room at 27± 1° C, 80±5% RH and a light regime
MATERIALS AND METHODS
of LD 12: 12. Adult mosquitoes were kept in 30 cm3 cages
and offered a
Dual- Choice Olfactometer, Mosquitoes,
mental
and
Experi-
Procedures
The dualchoice
6% glucose solution. Females were offered
blood from a human arm twice weekly for egg production.
Wet filter paper on glass petri dishes was provided for
Perspex
and was placed
27± 1° C
and
80± 5% RH
Fig.
1)
was made of
oviposition. Larvaewererearedinplastictrayscontaining
a climate- controlled room at
tap water and fed Tetramin® fish food. Females used in
olfactometer(
in
with a
light
intensity of 110 lux.
the experiments were four to eight days old and had not
E-
8 -›<-
8
34
92. 5
g
h3 , 4
OT
r
mir
k
e
;
;
e
16
J
c
a
Figure 1. The dualchoice olfactometer setup( dimensions in cm). Room air was pumped( a) at 1400 ml/min, cleaned
by passing activated charcoal( b), moistened by passing distilled water( c), passed a flow meter( e) for each
port, and entered the olfactometer( f).Baits( worn/ clean stockings,( g) were positioned in compartments and
air that passed over them entered the tunnel through the ports( h). Mosquitoes were released from glass vials
j) at the downwind end of the tunnel, and an imaginary flight path is shown( k). When needed, carbon dioxide
was pumped
into the
circuit
from
a pressurized gas cylinder at(
d).
December, 1998
Journal of Vector
blood meal. They
received a
were
deprived of sugar water
18 h before testing.
Experiments were
carried out
hours
Test mosquitoes
into
glass
the
last
dioxide. Carbon dioxide ( 4. 5% at 300 ml/ min) passed
colony cage and transferred individually
vials( 5 x 1. 5 cm) using a suction tube, which
over foot skin emanations on polyamide stocking and
period.
were
a
sealed with a water- moistened
Individuals
wool.
In a last series of experiments we examined the
during
possibility of synergism between foot odors and carbon
then
were
the test port before the experiment started.
randomly
from
selected
109
six
the dark
of
Ecology
were released at
the
of cotton
plug
downwind
end of
was tested against either foot skin emanations on
polyamide stocking or carbon dioxide ( at a similar
concentration).
the olfactometer and the response( i.e., entering of either
of
the
two
olfactometer
experimental
period)
in
times
worn at all
ports
order
RESULTS
min.
to avoid contamination.
All
Figure 2A shows that when clean conditioned air
hexane
and
was pumped into both ports of the tunnel, no significant
parts of the olfactometer were cleaned with
ethanol(
3-
the
within
Surgical gloves were
recorded.
96%) between test
preference( x2= 0.9, p> 0.05) for either port was observed.
series.
Similar results were obtained when both ports were
Experimental Odor Stimuli
Before running tests in
added
to
baited with clean stockings( x2= 0. 8, p> 0. 05). Mosquitoes
which
Similar tests
from both
dioxide
the
effect of any
the
unknown origin,
Skin
between 06. 00 hr
treatment
response of
and control
tightly
and
bottle
same
cm
until
rolled
up stocking
from the
responded
treatment
A clean
used
for
an
was placed on a
air entrance
ports, and clean air was pumped over
consisted of a clean
being
morning. Throughout the
volunteer ( LEGM) provided the
the
dish 17
stocking. The
it. The
in the tunnel
control odor
number of mosquitoes
to and probed the polyamide
stocking in
was recorded.
polyamide
increase in
stocking onto which water equal
1. 0 g) of a stocking worn for
weight( c
four days was pipetted, was tested against a worn( 4 days)
stocking in
polyamide
on
water
mosquito
order to examine the effect of
responses.
In
another
experiments, clean stockings moistened with
tested against clean
were
dry
ml/ min was
In the
flow
19801) discharged from
The
through the
of water
to the
of those entering the port with the clean stocking.
Worn stockings were also more attractive than moistened
clean
stockings (
x2=
7. 7,
p< 0. 01),
confirming the
attractiveness of body odors other than water.
dioxide
port.
treatment pumping clean air from both test ports. However,
no difference was observed with the treatments using
clean stockings in both ports or those that had water added
to clean stockings.
When the response of Cx. quinquefasciatus to air
with added carbon
dioxide( 4. 5%) was compared to that
0.03%, Fig. 2B), the percentages of
mosquitoes entering the ports did not differ between the
treatments( x2= 0. 9, p> 0.05). These results indicate that
of atmospheric air( c
carbon dioxide at human breath concentration does not
attract this strain of Cx. quinquefasciatus. When both
ports of theolfactometerwerebaitedwithcarbondioxide,
the mosquitoes did not show any preference ( x2= 0. 1,
p> 0.05) for either of the ports showing maintenance of
When carbon dioxide plus a worn polyamide
of air and
When running tests
to flow freely
minutes to
in which foot odors were pumped in the tunnel than in the
stocking was tested versus carbon dioxide only,
significantly more( x2= 24.6, p< 0. 001) mosquitoes were
attracted to the carbon dioxide/worn stocking combination
gas was allowed
for five
ports was significantly higher( p< 0.01) in those treatments
symmetry of the olfactometer.
was compared
flow
more attractive than dry ones( x2= 6, p< 0.025). The total
300
a cylinder at
combined rates of
dioxide, the
conduit
stocking, 87. 7% landed and probed on it as compared
in human
control was conditioned air alone at a
dioxide in the treatment
with carbon
of
initially pumped into both ports of the tunnel.
rate equal
carbon
1 g
concentration
second experiment, carbon
to clean air.
series
polyamide stockings.
Carbon dioxide( 4. 5%, the
breath[ Gillies
worn
to 12. 3%
percentage of mosquitoes responding to either or both test
experiments
glass petri
p< 0.001). Of those mosquitoes entering the port with the
wearing them for 4- 5 days
18. 00 hr at which time they were
after
by
following
The
polyamide stockings than to clean stockings ( x2= 23. 0,
Nevertheless, moistened stockings were significantly
stoppered
skin emanations.
responded significantly more to skin emanations on
every 10 mosquitoes.
from a human foot
the
to the
in
Thereafter, to
biased directional
experiment
each
ports
emanations were collected
on polyamide stockings
that
was run.
from both
system.
positions of the
between ports
were alternated
a
blank tests
tunnel ports)
released
to test the symmetry of the
counteract
were
were also conducted with clean polyamide
stockings or carbon
kept in
stimuli
a clean air stream, a series of routine
clean conditioned air
order
odor
displace
all air
in
than to carbon dioxide alone( Fig 2B). On comparing the
response to carbon dioxide/worn stocking versus worn
stocking alone, it was found that there was no significant
2. 9, p> 0.05),
indicating that the response was more to the worn stocking
difference between the treatments (
x2=
110
Journal of Vector
Treatment 1
A
Ecology
December, 1998
N
Treatment 2
I
I
%
resp.
160
67. 5 a
46
80.4 ab
141
83. 7 b
141
83. 0 b
96
77. 1 ab
1m1 H2O)
1 ml H20)
iiii i4i iiiiiiiiiiiiiii i iiii
I
1
1
1
50
1
1
30
1
10
1
1
10
1
1
30
1
50
1
1
70
choice
N
B
r
w
1
1
1
1
50
%
resp.
181
63. 0 a
54
70.4 ab
163
83. 4 b
68
73. 5 ab
>
1
30
1
1
10
10
1
1
1
1
30
1
50
1
1
70
choice
VA
clean air
clean
stocking
ah,
CO2( 4.5%)
worn stocking+ CO2( 4. 5%)
worn stocking
Figure 2. Results of olfactometer tests with various odor stimuli. Treatments 1 and 2 were tested against each other,
bars indicate the percentage
and
p< 0.01,***:
of
p< 0. 001. N: total
responding
number
mosquitoes
tested.%
flying into either test port(%
choice). *:
p< 0.025;
resp.: mosquitoes that entered either of both test ports.
Percentages with no letters in common are significantly different at p< 0.005.
than to carbon
dioxide,
and that no synergistic effect of
DISCUSSION
skin emanations with carbon dioxide was present. The
overall percentage of mosquitoes
test
ports
in this second
for those treatments in
dioxide
was present
responding to any of the
highest
series of experiments was
which
foot
in the tunnel.
odor and/ or carbon
Our results show that Cx. quinquefasciatus is attracted
to body odors collected on polyamide stockings, that this
effect is partially caused by the uptake of water by the
stocking, that
carbon
dioxide
at
a
human breath
December, 1998
Journal of Vector
is
concentration
not more attractive
that the combination
does
not result
body
odor alone.
by Omer(
a
hand
studies
1979),
was attractive
did
been
also
Cx.
of
quinquefasciatus to
seems that Cx. quinquefasciatus uses water vapor as an
wind tunnel
additional cue to respond to( human) hosts from a distance.
passing over
However, these
As humans have a far greater eccrine sweat gland density
for the presence of water in the
stimulus for anthropophilic mosquitoes such as Cx.
quinquefasciatus and An. gambiae sensu stricto( Takken
et al. 1997). Nevertheless, our studies have indicated that
observed
found that
who
to the mosquitoes.
not compensate
in
air
than other mammals, moisture may be a behavioral
may well have influenced the results. As
for human feet, hands contain high densities(> 300/
emanations
that
cm2)
ofeccrinesweatglands, excretinglargeamountsofwater
Marples 1969). Other workers have
also shown
emanations are attractive to a
Thompson
Price
and
variety
Brown 1955, Khan
1979),
al.
et
that
skin emanations elicit stronger attraction to Cx.
skin
quinquefasciatus than variations in the moisture gradient,
of mosquito species
and that these skin emanations, left as residues in the
and
Maibach 1966,
it has been found that
and
111
with the present finding that moisture is a cofounding
factor in attraction to skin odor. Moisture is naturally
present in the total emanations of the human body and it
dioxide
responses compared with
Responses
have
than clean air, and
odors with carbon
in increased
skin emanations
studies
body
of
Ecology
stockings, are probably a source of human odor that plays
an important role in the host-seeking behavior of this
odors
clothing attract more female Anopheles
Anopheles funestus to an unoccupied hut
collected on worn
species. It should be borne in mind, however, that due to
gambiae and
the size ofthe olfactometer, responses were only observed
than clean
1996)
al. (
clothing( Haddow 1942). Recently, Geier et
collected human skin odors on an ethanoldemonstrated
soaked pad, and
Ae.
attractive to
aegypti as a
over a small distance near the source. It may well be that
over longer distances other kairomones also influence
this behavior. So far, we have failed to attract wild Cx.
quinquefasciatus in Tanzania to unlighted CDC traps
baited with worn stockings( L. E. G. Mboera, unpublished
that this extract was as
human hand
and that the
its activity for months after storage at
low temperatures. Other hematophagous insects have
extract maintained
also
been
even exhibited
on
to
shown
of
the
body.
Simulium damnosum
From these
1996) it
be
odors can
be transferred
results show that Cx. quinquefasciatus is poorly attracted
from
De
Jong
some
Knols
to this compound at a concentration equivalent to that in
respond
human breath. However, the response of the mosquito to
different concentrations of carbon dioxide was not tested
to determine optimum response levels for this species.
and
may
body regions, these
onto an adsorbent cloth( cotton
And due to
lasting
their
that
al. (
1996) found that Cx.
very
above that normally released by one human, it is likely
to note that the addition of small
that at short range carbon dioxide plays a minor role in the
attractiveness of a host. Other workers have made similar
effect
kairomones
are
of
not
amounts of water to clean stockings provides a sufficient
stimulus to attract
et
quinquefasciatus in Burkina Faso responded at doses
a
volatile.
interesting
Although Costantini
the
attractiveness, these specific
It is
Costantini et al. 1996, Dekker and Takken 1998).
Carbon dioxide is considered to be a universal
attractant for many hematophagous Diptera, but our
wool pads, polyamide material, etc.) and still retain
attractiveness.
(
belonging
concluded that mosquitoes
to kairomones originating
although they are readily attracted to human
emanations released in- vivo from a tent or a bed net
complex with worn trousers.
and other studies ( e. g.,
can
worn
Thompson ( 1976) for
instance, in Cameroon, attracted more blackflies
to the
data),
attracted to worn materials and
differential attractiveness to clothing
parts
specific
be
Cx. quinquefasciatus. It
was observed
during all experiments the stockings increased in
by approximately 0. 4 g, presumably as a result of
weight
from the humid airstream. This may have
in intermittent variations of the relative humidity
observations on different mosquito species( Crumb 1922,
Healy and Copland 1995, De Jong and Knols 1995).
Moreover, field studies in Tanzania failed to attract An.
gambiae s. l. to electric nets baited with human breath or
water uptake
carbon dioxide although they were readily caught on nets
resulted
baited with complete human odor from an occupied
passing the tunnel ports, which may have been
responsible for the overall increase of responsiveness of
bedroom ( Knols
the mosquitoes. Like An. gambiae(
ml/ min) attracted
of
the
air
Takken
et al.
1997),
it thus appears that Cx. quinquefasciatus is also extremely
sensitive to small changes
Earlier
in the relative humidity gradient.
that
Cx.
et al.
1998).
Recent work in Tanzania
showed that tents baited with carbon dioxide( pure, at 300
9%
as many An. gambiae s. l.( Mboera
as many Cx. quinquefasciatus as a
tent baited with a human( L. E. G. Mboera, unpublished
et al.
1997)
and
28%
bit
data). As carbon dioxide is not signifying the identity of
body than on
density( De Jong
an upwind host, it is likely that specialized mosquitoes
Knols 1996), suggesting that drier skin is preferred
above a moister surface. These results are not in contrast
compound in their host- seeking behavior than generalist
work
showed
significantly
more on
areas with a
high
and
the
drier
quinquefasciatus
areas of
eccrine sweat gland
the
like Cx. quinquefasciatus show less dependency on this
species; and that, when used as a
kairomone
on
its
own,
Journal of Vector
112
it accounts for onlyaminorpartoftheoverallattractiveness
of a
human( Mboera
Takken 1997).
and
No interaction between
by
observations
workers
other
who
For
for
compounds
Vickery
example,
synergism
that the
various
of carbon
the
of
Lactic
catch of mosquitoes.
dioxide
only in the
1970, Price
et al.
Kline
1990, 1991) have
et al.(
of carbon
odors (
of
presence
dioxide in
namely 1-
showing
has been
the
mosquitoes
dioxide ( Smith
et al.
Kline ( 1989)
and
shown
to
shown
that the
and
presence
conjunction with other mammalian
in
experiments
our
increase
could
octen- 3- ol) resulted
several mosquito species,
Since in
two
1979). Takken
in
particularAe.
the
J. Econ. Entomol. 66: 329- 331.
Clements, A. N. 1963. The Physiology of Mosquitoes.
Pergamon Press, Oxford, 393 pp.
Brady, and M. Coluzzi. 1996. Mosquito response to
by
carbon
fever mosquitoes:
Costantini, C., G. Gibson, N. Sagnon, A. della Torre, J.
and chicks
acid
and
Yellow
demonstrated
flight behavior in
activate and elicit oriented
the
1973.
Compounds related to lactic acid that attract females.
of mosquitoes.
1966)
al. (
et
dioxide
combination
species
earlier
reported
presence of a synergistic effect of carbon
other
and skin
This is contrary to
observed.
was
emanations
Carlson, D. A., N. Smith, H. K. Gouck, and D. R.
Godwin.
dioxide
carbon
December, 1998
Ecology
synergism
for
taeniorhynchus.
combination
of carbon
carbon dioxide in a West African Sudan savanna
village. Med. Vet. Entomol. 10: 220- 227.
Crumb, S. E. 1922. A mosquito attractant. Science 126:
446- 447.
De Jong, R. and B. G. J. Knols. 1995. Olfactory responses
of host- seeking Anopheles gambiae s. s. Diptera:
Culicidae). Acta Trop. 59: 333- 335.
De Jong, R. and B. G. J. Knols. 1996. Selection of biting
sites by mosquitoes.
Olfaction in mosquito- host
interactions ( G. Cardew,
ed.).
CIBA symposium
200: 89- 99.
significantly
Dekker, T. and W. Takken. 1998. Field studies on the
increase the number of mosquitoes responding compared
to carbon dioxide only, it is likely that there is no
responses of Anopheles arabiensis Patton and An.
dioxide
and
synergistic
skin
emanations
for Cx.
effect
The overall
conclusion of
by
to
this work
which
distance,
attracted over a short
be
quinquefasciatus
by
the
slight variations
and
in
is that the human
that this
relative
effect
may
humidity. The
recently described foot odor- Limburger cheese analogy
and the subsequent identification of attractive carboxylic
acids (
Knols
et
quinquefasciatus
attractiveness of
undertaken
1997)
al.
and
quadriannulatus Theobald to carbon dioxide, a
man and a calf. Med. Vet. Entomol. 12: 136- 140.
Geier, M., H. Sass, and J. Boeckh. 1996. A search for
skin produces compounds
enhanced
not
the two.
combination of
is
could
may
to
studies
these kairomones
to
Cx.
determine
the
also
are
apply
currently
being
in Tanzania.
components in human body odor that attract females
of
Aedes
aegypti.
Olfaction in mosquito- host
interactions ( G. Cardew,
ed.).
CIBA symposium
200: 132- 144.
Gillies, M. T. 1980. The role of carbon dioxide in host-
finding by
mosquitoes
(
Diptera:
Culicidae): a
review. Bull. Ent. Res. 70: 525- 532.
Haddow, A. J. 1942. The mosquito fauna and climate of
native huts at Kisumu, Kenya. Bull. Ent. Res. 33:
91- 142.
Healy, T. P. and M. J. W. Copland. 1995. Activation of
Anopheles gambiae mosquitoes by carbon dioxide
Acknowledgments
and human breath. Med.Vet. Entomol. 9: 331- 336.
Khan, A. A., H. I. Maibach, W. G. Strauss, and W. R.
We
are
grateful
to
Prof. C. F. Curtis for helpful
early draft of the manuscript. BGJK
acknowledges
financial support from the Niels
gratefully
Stensen Foundation, The Netherlands.
comments
on an
Fenley. 1966. Quantitationof effect ofseveral stimuli
on landing and probing by Aedes aegypti. J. Econ.
Entomol. 59: 690- 694.
Kline, D. L., W. Takken, J. R. Wood, and D. A. Carlson.
1990. Field studies on the potential of butanone,
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Electric
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Marples, J. J. 1969. Life on the human
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Tone. 1997. The
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Tanzania. Bull. Ent. Res. 87: 173- 178.
Mboera, L. E. G.
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Rev. Med. Vet. Entomol.
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Exp.
and
of
human hands in
and
female
quadrimaculatus
in
females
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Say)
with
conjunction
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W., B.
G. J. Knols, and H. Otten.
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Journal of Vector
Ecology
23( 2): 114- 122
Antifeedancy of Neem Products Containing Azadirachtin against
Culex tarsalis and Culex quinquefasciatus ( Diptera: Culicidae) 1
Tianyun Su and Mir S. Mulla
Department of Entomology, University of California,
Riverside, CA 92521- 0314, U.S.A.
Received 26 March 1998; Accepted 1 June 1998
ABSTRACT:
In order to develop and utilize new botanical insecticides originating from neem
Azadirachta indica A. Juss) in mosquito control programs as a potential larvicide, the antifeedant activity
of three formulations of neem against the larvae of Culex tarsalis Coquillett and Culex quinquefasciatus
Say was investigated under laboratory conditions. A significant antifeedancy was indicated at 5 ppm and
10 ppm azadirachtin( AZ) for all formulations and both species. Within the test concentration range of AZ
1- 10 ppm), 5 ppm was the minimum effective concentration for antifeedancy in most cases. Some
differences in larval susceptibility in terms of antifeedancy to the test formulations were noted between the
two species. The Cx. tarsalis larvae were more susceptible than Cx. quinquefasciatus larvae to Azad EC
4. 5 at 1 ppm during the first 15- min exposure period and at 5 and 10 ppm during the whole test period. The
Cx. quinquefasciatus larvae were more susceptible than Cx. tarsalis larvae to Azatin WP 4. 5 at 1, 5, and
10 ppm during the first 15- min exposure period or to Neemix EC 4.5 at 1 ppm during the first 45- min
exposure period. The formulation- related differences in antifeedant activity appeared when the concentration
increased from 1 to 10 ppm. In Cx. tarsalis, the Azad EC 4. 5 and Neemix EC 4. 5 were more effective than
Azatin WP 4.5 at 5 ppm during the first 15- min exposure period and at 10 ppm during the whole test period.
In Cx. quinquefasciatus, the Azatin WP 4. 5 and Neemix EC 4. 5 were more effective than Azad EC 4. 5 at
1 ppm during the first 30-min exposure period, and at 5 ppm during the first 60- min exposure period, and
at 10 ppm during the whole test period.
Keyword Index: Neem, azadirachtin, Culex tarsalis, Culex quinquefasciatus, antifeedancy.
INTRODUCTION
that about 2, 000 plant species possess pest control
properties( Ahmed et al. 1984); and among these, 344
Chemical control
of insect pests and
disease vectors
using synthetic insecticides has played an important
role in protecting the food sources and the health of
mankind
during
the past
decades. However,
concerns
impact
species
(
show
some
activities
against
mosquitoes
Sukumar et al. 1991). In these plants producing bioactive
materials, the neem tree, Azadirachta indica A. Juss,
and closely related species A. excelsa Jack, A. siamens
of
Valeton, Melia azedirach L., and M. volkensii Giirke in
search
the family Meliaceae have been well investigated and
years,
proven to possess considerable insecticidal properties.
and
efficacy,
At least 35 components exhibiting some insecticidal
mammalian
toxicity,
activity have been identified from the neem tree ( Rao
acceptability, safety to nontarget and
beneficial biota, and economic viability ( Mulla 1997)
and Parmar 1984), among which is the triterpennoid,
azadirachtin ( AZ). The AZ moiety has structural
have been actively investigated
regarding safety
and
synthetic pesticides
for alternative
biopesticides
specificity
has
strategies
the
environmental
resulted
having attributes
and
in
an
intensified
for pest control. In recent
of
selectivity, low
activity
environmental
in the fields
of
similarity to the molting hormone of insects. It is quite
microbiology, phytochemistry, and entomology. Among
biopesticides, the botanical insecticides constitute a
unique that AZ can act and affect multiple systems in
diverse group of bioactive
of AZ against a variety of phytophagous insects and a
These
compounds.
studies were supported
It has been
by University
noted
insects and exhibit various modes of action. The activities
of California Special Funds for Mosquito Research.
Journal of Vector
December, 1998
few hematophagous insects have been
These
studied.
include antifeedancy, growth regulation,
reproduction suppression, biological fitness alteration,
for 1st, 2nd, 3rd, and 4th- instar larvae, respectively. The
larval food consisted of powdered rabbit pellets
activities
and so
forth. In
neem or experimental
larvicides
Naqvi
by
activity ( Su
et al.
1997,
pans every other day to replenish loss due to evaporation.
Pupae were removed from the pans in a cup containing
1995, Zebitz 1986). The
water and placed in screened cages ( 23 x 23 x 32 cm)
Mulla
et
Mulla 1998a),
and
repellency ( Su
or
attractancy
act as
regulation (
1991, Rao
et al.
ovicidal
al.
Mulla 1998b),
and
where the adults emerged. Adults were provided
oviposition
and
continuously
host- seeking/ blood- feeding repellency ( Sharma and
Ansari 1994, Sharma et al. 1993 a, b) were also studied
in
a
few
to
be
10% sucrose solution in a jar provided
with a restrained one- week-old chick overnight for
blood feeding( Animal Use Protocol No. A- S 95090523, University of California, Riverside). The early 4thinstar larvae from both colonies were used in the feeding
of most mosquito species are considered
omnivorous, which
filter-feed
floating
and particulate materials
sedimented
with
with a cotton wick. On day 5 postemergence, the adults
were deprived of sugar feeding for 12 h, then provided
species.
The larvae
Brookhurst®, Brookhurst Mill, Riverside, CA) and
Brewer' s yeast in the ratio of 3: 1. Water was added to the
AZ formulations primarily
growth
(
of
the crude preparations
mosquitoes,
115
Ecology
to the bottom
on microorganisms
on, suspended
of aquatic
habitats. The
in,
or
test.
mouth
brushes set up the water currents, which carry the
particles
and
toward the mouth.
inert, with suitable
The
both
particles,
Test Materials and Preparation of Stock Pre-
nutritive
indiscriminately ingested
size are
suspensions
experimental
The test materials were three experimental
broadly nutritive materials
such as yeast, wheat flour, dry blood, fishmeal, or algae
formulations of neem products, one wettable powder
Clements
was
However,
1992).
the ingestion
conditions,
faster than that
of the
inert
of
under
kaolin, brick dust, talc,
chalk,
1985, Dadd 1968, 1970a, b; Dadd
and
Mulla 1989). The larval
species,
instars,
factors
by
affected
the
and
dye
or
et al.
feeding
such
water
as
particulates.
As to the
the
effects of
suspensions of the formulated products were prepared
in distilled
among
and is
water at a concentration of
0. 1% (
w/ v)
or
employed in antifeedancy tests, where the needed
food
aliquots
were
added
to
200
ml
of
2% ( WN) yeast
suspension in 250 ml disposable paper cups for feeding
neem products on
feeding
and
1, 000 ppm of AZ. These stock preparations were
temperature,
types of the
behavior in insects, antifeedancy has been
indicated in many species of phytophagous insects
the
Azad EC 4. 5
1982, Rashed
rate varies
phagostimulants, phagodeterrents, and
Azatin WP 4. 5) and two emulsifiable concentrates
(
Neemix EC 4. 5), all supplied by
Thermo- Trilogy Co.( Salt Lake City, UT, USA). Stock
particles (
of starvation,
extent
(
Aly
materials such as charcoal,
(
see
below). For each test, stock suspensions were
freshly prepared.
Ascher 1993, Mordue and Blackwell 1993, Schmutterer
1988, 1990). To date, there
of neem products on
larvae. In
order
neem products
neem
in
gain
products
information
mosquitoes,
on
the
in the
behavior
feeding
and
on
the effects
of mosquito
antifeedancy
of
the current research was
of experimental
effects
feeding
Culex tarsalis Coquillett
Say
are no reports on
investigate the
to
undertaken
to
the
behavior
Culex
of
larvae
of
quinquefasciatus
laboratory.
Antifeedancy Tests
The antifeedancy tests were conducted in a holding
room maintained at
28±
1° C, 35- 45% RH illuminated
by two 40W fluorescent and one 15W incandescent
lamp. The feeding rate was determined according to the
method of Dadd( 1968). Briefly, the larvae were allowed
to feed on contrasting color inert particulates such as
kaolin or charcoal for some time to fill the gut completely.
The inert material acted as color indicator distinguishable
visibly from the color of the test material. Then the
glutted larvae were removed to the test material. The
Mosquito
Colony Rearing
The test
fasciatus
species,
in two
and
Cx.
quinque-
newly ingested test material displaced rearward the
previously ingested inert material column, which was
separate culture rooms
excreted in the form of small pellets. Because the gut
1° C, 50- 60% RH and a 14L: 10D
peristalsis did not mix the gut content( Jones 1960) and
were maintained
under conditions of 26±
h
Cx. tarsalis
1 h dawn
dusk
To
rear
the sharp boundary between indicator particle mass and
5 to 7 egg rafts of each species were placed
in an enamel pan( 40 x 24 x 6 cm) containing 2, 500 ml
distilled water. The larvae were fed dry powder food at
the test material mass was maintained, the ingestion rate
photoperiod with
and
periods.
the colony,
the doses
of
80, 160, 320,
and
240 mg
per pan
every
day
was quantified by counting the segments filled or
displaced by test material within specified time intervals.
If the boundary had reached the posterior margin of the
116
Journal of Vector
thorax, a value of 1
was assigned.
Values
1. 5, 2, 2. 5,
of
Ecology
December, 1998
determined by unpaired, two tailed t-test.
and 3- 6 were given when the boundary was at the
midpoint or posterior margin of the
1st to 5th abdominal
RESULTS
segments, respectively. If the indicator column was
completely displaced but still remained as pellets in the
hindgut, a value of 6. 5 was assigned. When no indicator
in the
mass was seen
Values
of 0 and
occurred or
0. 5
if test
whole gut, a value of
were applied
material
7
was given.
if no displacement had
had only
antifeedancy was indicated at 5 and 10 ppm AZ for all
suspension
formulations and both species. In the cases of Azad EC
4. 5 against Cx. tarsalis or Neemix EC 4. 5 against Cx.
tarsalis and Cx. quinquefasciatus, 10 ppm AZ was more
adding 2 g kaolin( Fisher Scientific, Fair
by
was made
indicator
current research, the
Lawn, NJ) to 1, 000
distilled
ml
was tested at 0( control), 1, 5, and 10 ppm AZ at various
formulations against the two test species. Significant
half way
advanced
through the thorax( Dadd 1968).
In the
Comparisons Among Different Concentrations
The antifeedant activity of the neem formulations
0.2%, WN)
and
effective than 5 ppm AZ in terms of antifeedancy. Five
shaking vigorously to insure the homogeneity of the
prepared suspension. The whitish, amorphous kaolin
ppm AZ was the minimum effective concentration for
singly or aggregated in clusters
2. 5- 37. 5 gm in water( Rodcharoen and
AZ did not affect the feeding rate, except in Azad EC 4. 5
and Neemix EC 4. 5 against Cx. tarsalis( Fig. 1).
water(
particles were suspended
the size
with
of
antifeedancy in most cases. The concentration 1 ppm
Mulla 1995). For feeding, approximately 100 unstarved
early 4th- instars from the rearing pans were transferred
to 200 ml 0.2% kaolin suspension in 250 ml disposable
overnight.
The larvae
transferred
200
to
Biochemical
Corp.,
disposable
ml
different
4. 5,
or
1, 5,
neem
10
0. 2%
cups,
which
inert
this
kaolin
yeast (
Cleveland, OH)
paper
between the two test species, regardless of formulations
then
and concentrations, were indicated. The feeding rates of
United States
4th- instar Cx. tarsalis and Cx. quinquefasciatus on
in 250
yeast suspension under neem- free condition was
were
suspension
treated
essentially the same, with the exception that the former
WP 4. 5, Azad EC
fed slightly faster than the latter during the first 15- min
at the concentrations of 0( control),
feeding period after being transferred to yeast suspension.
The
were
of Azatin
yeast suspension was prepared
previously by adding 2 g yeast powder to 1, 000 ml
distilled water and shaking vigorously. The light brown,
oval or spherical yeast particles were suspended
or aggregated
in clusters( 5- 25 pm)
Mulla 1995). At
Some differences in susceptibility to neem products
material
with
formulations
ppm.
on
engorged with
ml
Neemix EC 4. 5
and
feed
to
paper cups and allowed
Comparisons Between Two Species
singly
in water( Rodcharoen
intervals
The Cx. tarsalis larvae were more susceptible than Cx.
quinquefasciatus larvae to Azad EC 4. 5 at 1 ppm
during the first 15- min exposure period and at 5 and 10
ppm during the whole test period. The Cx. quinquefasciatus larvae were more susceptible than Cx. tarsalis
15, 30,
larvae to Azatin WP 4.5 at 1, 5, and 10 ppm during the
were screened out with
first 15- min of exposure period or to Neemix EC 4. 5 at
by putting them in a 5 ml
plastic dish containing 3 ml of hot water at 75- 85° C. The
numbers of the segments filled or displaced
by yeast
particles were scored within 2 h under
dissecting
microscope( 8x), by noting the different color of kaolin
1 ppm during the first 45- min exposure period( Fig. 2).
and
45, 60,
and
a series of time
90- min, 10 larvae
plastic screener and
killed
The formulation- related differences in antifeedancy
using three
concentrations against two species
Neemix EC 4. 5 were more effective than Azatin WP 4.5
and yeast mass
in the
at
four
was repeated
three times.
at
Comparisons Among Various Formulations
activity occurred when the concentration increased from
1 to 10 ppm. In Cx. tarsalis, the Azad EC 4. 5 and
formulations
filled
of
gut.
The
The
different intervals
experiment
mean values of segment
were
determined.
at 5 ppm during the first 15- min exposure period and at
10 ppm during the whole test period. In Cx.
quinquefasciatus, the Azatin WP 4.5 and Neemix EC
Data Analysis
The
yeast
parameter
in the
4. 5 were more effective than Azad EC 4. 5 at 1 ppm
for
determining feeding
rate
on
aquatic medium treated with neem products
was mean numbers of segments
filled
or
displaced
by
during the first 30- min exposure period and at 5 ppm
during the first 60- min exposure period, and at 10 ppm
during the whole test period( Fig. 3).
yeast particles at various time intervals. At each interval
of
feeding
process,
the comparisons among
DISCUSSION
various
concentrations or formulations were conducted by 1factor
analysis of variance (
while
the
ANOVA Scheffe F test),
difference between two test
species
was
The extracts from the seeds, leaves, and bark of the
neem
tree,
where
the
major pesticidal component
is AZ,
December, 1998
Journal of Vector
Control
III
Azatin WP4.5
vs.
7-
ppm
5
®
117
ppm
10 ppm
:.
Azatin WP4.5 vs. Cx. quinquefasciatus
Cx. tarsalis
as
as
a
1
0
Ecology
a
7-
6-
a
b
a
s
b
as
a a
bb
6-
b
b
a
5-
a
b
a
5-
i
bb
b
4
a
a
ab
f
b
4
4'
b
b
2-
2
05
Azad EC 4. 5
vs.
as
as
a
a
7-
Azad EC 4. 5 vs. Cx. quinquefasciatus
Cx. tarsalis
a
a
b
a
a
7-
a
a
b
ab
6-
a
a
E
b
i:
O
aaaa
aaab
ab
b
::
-
ij:'.
t„,:„„
b
......:•°
b
K.
2_ ::: Ii:
L ....,...
i. iiii
4
I
Neemix EC 4. 5
a
7-
vs.
a
a
Neemix EC 4. 5 vs. Cx. quinquefasciatus
as
s
I
I
I
I
I
Cx. tarsalis
a
as
I
I
f
I
ft;iii
h+
7a
b
a
as
a
s
a
6-
6b
0
b
5-
b
4_
b
5-
b
b
4-
b
c
3-
c
32-
30
60
45
1_ .
fi.
90
tie
iiil: b
I
I
I
1
15
iii
4•
4
Mme,
1 ; i: *.::
c
b
c
2
C
c
b
a
La:*
30
i;
i:
:!:
1.
45
4
I
I
I
I
15
i:: i: + +
t...
60
90
Feeding time( min)
Figure 1.
Antifeedancy of neem formulations, Azatin WP 4. 5, Azad EC 4. 5, and Neemix EC 4.5 against 4th- instar
larvae of Culex tarsalis and Culex quinquefasciatus at various concentrations. Significant differences
of the mean numbers of segments filled among concentrations and control at each feeding time are
indicated
by
different letters
at
0. 05 level using 1- factor ANOVA.
118
Journal of Vector
IllCx. tarsalis
Ecology
December, 1998
Ej Cx. quinquefasciatus
Control
a
6-
54:
Azatin WP4.5 at 1 ppm
a
a
a
a
a
a
iii1;
ri
2:::: ',]
::::::::
1- ::: :: :::::::::
0
a
a
a
a,..
101 : 11
1.
111
i:]:
MI
a
a
a.,
iV
Azatin WP4.5
•::;:
i::;
1:: : : : : : .
M:
I
5
at
Azatin WP4.5 at 10 ppm
ppm
a
7-
a
7-
a
a
3-
b
2-
Azad EC4.5
a
Eli
a
Azad EC4.5 at 5 ppm
ppm
a
a
s.
1
at
iHQ
a
a
a
:
i !lpi
R:
•;
ilF.,
z:I.,
Azad EC4.5
10
at
b
a
s.
a
3_
b
b
2' ,
..... x.,
RIN
1- " :•':":".':
2271
a
a !
!
ip:!
6-
3-
mi.
1g1111111. •
IliCH
11:
Neemix EC4.5
m:::::::::
at
5
a
2
15-;
iii.
15
!
1.: '
I
1111 ;: :
: :::::
:
:.
1•.
i••:::'1:'. ':1: :
i:
11
N:1: i1:1
i:
Ilk
111;1
I
I
Neemix EC4.5 at 10 ppm
7-
45
iri 111
a
i.•. ,.: i.••
kali IT1
:;:::::;::
30
ii
s-
a
a
a
a
5-
ppm
a
5-
a
a
b
i:
in
7-
321-
a'ii:,1,
a
a
a
7-
blhil
i;
TI
Neemix EC4.5 at 1 ppm
b
5-
Illi!I
iitil
7.7
ppm
7-
a b
Ili
ii:
NI:i :
2-
a
:::::::::::
15
60
a a
i.„,
a
'
Tin
.
30
a
a
al
45
60
90
Feeding time( min)
Figure 2.
Species- dependent difference in antifeedancy by neem formulations against Culex tarsalis and Culex
quinquefasciatus. Significant differences of the mean numbers of segments filled between these species
at each
feeding
time
are
indicated
by different
letters
at
0. 05 level using
unpaired
2- tailed t test.
December, 1998
Journal of Vector
E.1
Azatin WP4. 5
Cx. tarsalis
at
1
Azad EC4.5
119
®
Neemix EC4.5
Cx. quinquefasciatus at 1 ppm
ppm
a
aaa
aaa
7-
Ecology
as
b
a
65-
aaa
aaa
ab
b
6a
a
a•
flh1HHiiflH
a
2
1
O
Cx. tarsalis
at
5
1
1
a
5a
a
a
2_
2bb
gi
1-
i: :
a
b
i
ig
a
a
a
1-
b
a
b
a
a
a
b
5
s
4-
b
6-
a
ab
a
b
1177
6-
a3-
I
ppm
7-
a
1
a
a
a
iill.,
.:,:
i:,:
M
I
I
I
Cx. tarsalis
at
10
I
1
I
1
I
ppm
7-
7
6
a
a
5-
b
5
b
a
43_
a
2bb
b
Ilk
b
ti
b
a
b
4
a
b
b
a
6-
1
I
I
a
3
a
b
a
2
1
a
a
Je
a
a
Feeding time( min)
Figure 3.
Antifeedancy of various neem formulations against 4th-instar larvae of Culex tarsalis and Culex
quinquefasciatus.
formulations
Significant differences of the mean numbers of segments filled among various
at each
feeding time are indicated by different letters at 0. 05
level using 1- factor ANOVA.
120
induce
can
by
Journal of Vector
its
these
in
multiple effects
unconventional
effects
fecundity
are
a
variety
pesticidal
growth
antifeedancy,
suppression
insect
of
of
regulation,
and
sterility, oviposition
repellency or attractancy, changes in biological fitness,
or even blocking the development of vector- borne
in
pathogen
1993). Most
of
the
against mosquitoes
frcm different parts
extracts
commercial
formulations
the experimental
WP 4. 5
of the neem
of AZ.
against
Mulla et al. 1997) of the consumed AZ could occur at
these high AZ concentrations of 5 and 10 ppm, which
than
rather
would result in the secondary antifeedancy during the
Azad EC 4. 5
investigated in
of
and
components
and
feeding
as
nucleic
acids,
adenylic
acid,
activity
are powerful phagostimulants to mosquito larvae( Dadd
quinquefasciatus.
1970b, Dadd et al. 1982). The test larvae ingested inert
materials much faster in the presence of yeast extracts
than in suspensions without yeast extract( Dadd 1970b,
antifeedant
Cx.
such
nucleotides, nucleosides, and nucleic acid bases, which
agricultural pest control
investigate the
Cx. tarsalis
Chemical inhibition
well
the detrimental effects through the larvicidal activity
(
process of intoxication. The yeast extracts contain some
to
of
tree
mortality was indicated during antifeedant test period,
in the
were crude
deterrent" cells, or both. Furthermore, even though no
In the current research,
and emulsifiable concentrate
larvae
"
formulations of wettable powder Azatin
Neemix EC 4. 5 developed for
were employed
activity, where the AZ either blocked the input from
phagostimulant chemoreceptors or stimulated specific
Blackwell
and
pesticidal neem products used
investigations
earlier
Mordue
the arthropods (
December, 1998
early as 15- min after the test larvae were transferred to
the yeast suspension containing AZ. Therefore, primary
antifeedancy could be putatively a major form of this
species
Some
properties.
Ecology
behavior has been
insects. The
some phytophagous
primary antifeedancy may function by blocking the
input from chemoreceptors normally responding to
Dadd
et al.
1982). The
present work used
2%
yeast
suspension as test material for feeding rate. Therefore,
by
stimulating the specialized
deterrentreceptors( Chapman 1974, Schoonhoven 1982).
the observed antifeedancy exhibited by neem products
against the test species is probably the result of
The former may have a general effect on all insects, but
the chemicals in the latter category will only be effective
overcoming the phagostimulant activity of yeast
phagostimulants,
or
if the insects have
components by AZ.
The species- and formulation-dependent differences
responding to
them( Chapman 1974). The antifeedancy resulting from
if consumed, is
toxic effects,
dancy (
When
neurons capable of
called
Ascher 1993, Mordue
applied
and
in many different
ways,
AZ
and
AZ
con-
antifeedancy, as well as secondary antifeedancy against
a variety of insects( Mordue and Blackwell 1993).
current studies
using the
Dadd( 1968),
rates of particulates (
found in the
proven as a
for evaluating ingestion
and Mulla 1995). It
current research
This
rate
Rodcharoen
that 5 ppm of AZ
the minimum effective concentration
most cases.
feeding
has been
which
practical and reliable approach
in
antifeedancy
larvae by
for larval
assessment method
by
proposed
was
on
of neem products against mosquito
activity
was
as an antifeedant
effective concentration was
higher
for IGR activity by neem products against test
insect species. For instance, in Cx. quinquefasciatus,
than that
almost
100%
inhibition
and
80%
than
more
1
in the present research. Similar trends were also
secondary antifeeBlackwell 1993).
tainingext ractsfromtheneemtreeshowdistinctprimary
We initiated the
in the magnitudes of antifeedant activity was indicated
adult
emergence
0. 1
(
demonstrated in the larvicidal( Mulla et al. 1997), ovicidal
Su and Mulla 1998a), and ovipositional( Su and Mulla
1998b) tests of these formulations against the same
species. In larvicidal tests, Azad WP 10 was 10 times
more effective than Azad EC 4. 5 against Cx.
quinquefasciatus( Mulla et al. 1997). In ovicidal tests,
the eggs of Cx, quinquefasciatus were more susceptible
than those of Cx. tarsalis to the same neem formulations
at equal AZ concentrations. The Azad WP 10 was more
effective and more persistent than the Azad EC 4. 5 as an
ovicide against Cx. tarsalis and Cx. quinquefasciatus at
the same AZ concentration ( Su and Mulla 1998a). In
ovipositional tests, the Azad WP 10 acted as an
ovipositional attractant at
AZ
concentration?
0. 5 ppm
against Cx. tarsalis, but acted as a repellent at AZ
concentration 10 ppm against Cx. quinquefasciatus( Su
and Mulla 1998b).
AZ,
By applying the neem products to crops, within
respectively, in the formulations of Azad WP 10, Azad
EC 4. 5, and Neemix EC 0. 25 ( Mulla et al. 1997). In
artificial diets, or on simplified feeding discs, or to the
other
ppm
was
attained
at
ppm
and
ppm
test insect species, AZ concentrations of
by injection,
ppm by
0. 14- 4. 5
15- 100%,
and
depending
on
1993). In
our
1. 5- 11. 3
ppm
by
oral application, and
topical application
5- 50%
molt
0.2- 4. 1
yielded
15- 50%,
inhibition, respectively,
Mordue and Blackwell
the test species (
tests the
antifeedancy activity
was noted as
insects as sprays, topical applications, or by injection or
cannulation, both primary and secondary antifeedant
activity of neem products have been indicated in the
members of the following orders: Orthoptera, Isoptera,
Hemiptera, Coleoptera, Lepidoptera, and Diptera
( Mordue
and
Blackwell
1993).
differences, however, regarding
There are clear
the magnitude of this
December, 1998
depending on the concentration
effect,
of
Journal of Vector
in
the active principles, the ways
and
Ecology
121
formulation
Dadd, R. H. 1970a. Relationship between filtering
the neem
products are applied, and species of test
insects. Different
activity and ingestion of solids by larvae of the
mosquito Culex pipiens: a method for assessing
from
larvae inhabit
phagostimulant factor. J. Med. Entomol. 7: 708-
phytophagous
insects,
mosquito
which
an
Therefore, the antifeedancy activity or
only be achieved by treatment of the whole
aquatic medium.
toxicity
can
breeding
medium,
than
rather
larval food
materials
only. Therefore, the stability of insecticidal principles
of neem products in an aquatic environment, as well as
the
to nontarget aquatic organisms
safety
with mosquito
Neem
immatures
should
synthetic organic
insecticides
hazardous to
nontarget
the
be
and
in
and
organisms,
in
1997)
and
some
nontarget aquatic
fish ( Tangtong
source
of
feeds,
and
pest
control (
Koul
environmental
of neem products
for the
of
Culex pipiens( Diptera: Culicidae). Ann. Entomol.
Soc. Am. 75: 605- 612.
Jones, J. C. 1960. The anatomy and rhythmical activity
of the alimentary canal of Anopheles larvae. Ann.
Entomol. Soc. Am. 53: 459- 474.
Koul, O., M. B. Isman, and C. M. Ketkar. 1990. Properties
and uses of neem, Azadirachta indica. Can. J. Bot.
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of
Mordue, A. J., and A. Blackwell. 1993. Azadirachtin: an
furniture, cattle and poultry
for various agricultural crops,
Mulla, M. S. 1997. Nature and scope of biopesticides.
et
the
1990). The
al.
safety,
and public
the
potential
pesticidal
acceptability
control of crop pests
past several years ensure
Entomol. Exp. Appl. 13: 407- 419.
Dadd, R. H., J. E. Kleinjan, and L. D. Merritt. 1982.
the
and
nitrification of soils
efficacy,
vast areas
it is
stimulant effect of water- soluble yeast extract.
used as
pharmaceuticals,
implements
have been
Wattanasirmkit 1997).
and
and subtropical countries, where
agricultural
though
invertebrates ( Stark
Neem trees have been introduced into
tropical
less
are
even
some adverse effects of neem components
noted
than
easier
general
particulate solids by Culex pipiens larvae: Phago-
Phagostimulant effects of simple nutrients on larval
considered.
degrade faster
products
cohabitating
712.
Dadd, R. H. 1970b. Comparison of rates of ingestion of
during the
introduction into
update. J. Insect Physiol. 39: 903- 924.
Proc. 1st Int. Symp. Biopesticides( Rodcharoen, J.,
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and
M. S. Mulla,
eds.)
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Mulla, M. S., J. D. Chaney, and J. Rodcharoen. 1997.
Activity and efficacy of neem products against
mosquito larvae. Proc. 1st Int. Symp. Biopesticides.
mosquito control programs.
Rodcharoen, J., S. Wongsiri, and M. S. Mulla,
eds.).
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Naqvi, S. N. H., S. O. Ahmed, and F. A. Mohammad.
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Ahmed, S., M. Grainge, J. W. Hylin, W. C. Mitchell,
J. A. Litsinger. 1984.
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1985.
Feeding rate of larval Aedes vexans
stimulated by food substances. J. Am. Mosq. Contr.
Assoc. 1: 406- 410.
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Ascher, K. R. S. 1993. Nonconventional insecticidal
effects of pesticides available
Azadirachta indica.
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Biochem.
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Rodcharoen, J. and M. S. Mulla. 1995. Comparative
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Dadd, R. H. 1968. A
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rates in mosquito larvae.
Mosq. News. 28: 226- 230.
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Clements, A. N. 1992. The
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Rashed, S. S. and M. S. Mulla. 1989. Factors influencing
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J. Am.
Effectiveness
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quinquefasciatus (
Culex
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Diptera: Culicidae). Entomol.
R. K. Razdan. 1993a.
Sukumar, K., M. J. Perich, and L. R. Boombar. 1991.
Azadirachta
Botanical derivatives in mosquito control: areview.
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Mosq. Contr. Assoc. 9:
Sharma, V. P., B. N. Nagpal,
of
and
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A. Srivastava. 1993b.
oil
Trans. R. Soc.
359- 360.
mats
Trop.
in repelling
Hyg. 87:
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626.
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nontarget
J. Am. Mosq. Contr. Assoc. 7: 210-237.
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toxicity of neem extract on certain blood parameters
of tilapia, Oreochromis niloticus. Proc. 1st Int.
Symp. Biopesticides.( Rodcharoen, J., S. Wongsiri,
Stark, J. D. 1997. Risk
on
containing azadirachtin on oviposition activity of
Exp. Appl. ( Submitted).
repellent
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Contr. Assoc. 14: 204- 209.
Su, T. and M. S. Mulla. 1998b. Effects of neem products
in kerosene. J. Med. Entomol.
Sharma, V. P., M. A. Ansari,
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1st Int.
Symp.
Biopesticides. ( Rodcharoen, J., S. Wongsiri,
and
and
M. S. Mulla,
eds.). (
Phitsanulok, Thailand
1996). 1: 94- 103.
Zebitz, C. P. W. 1986. Effects of three different neem
seed kernel extracts and azadirachtin on larvae of
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different mosquito species. J. Appl. Entomol. 102:
69- 74.
455- 463.
Journal of Vector
Ecology
23( 2): 123- 135
A New Enzyme Immunoassay to Detect Antibodies
to Arboviruses in the Blood of Wild Birds
R. E. Chiles and W.K. Reisen
Arbovirus Research Unit, Center for Vector-borne Disease Research,
School of Veterinary Medicine, University of California, Davis, CA 95616
Reprint address: Arbovirus Field Station, 4705 Allen Rd, Bakersfield, CA 93312
Received 23 March 1998; Accepted 24 August 1998
ABSTRACT: A new indirect enzyme immunoassay ( EIA) was developed to screen wild bird sera for
antibodies against western equine encephalomyelitis( WEE) and St. Louis encephalitis( SLE) viruses. The
detector antibody was made by immunizing rabbits with serum proteins pooled from single species
representatives of four bird orders and was conjugated with horseradish peroxidase to allow visualization
with the ABTS substrate in an EIA plate reader set at 405 nm. The detector antibody recognized a wide
range of bird species and was more accurate, sensitive, and specific than a hemaglutination inhibition test
when compared to a plaque reduction neutralization test( PRNT).
EIA positive sera frequently could not
be confirmed by PRNT; however, practically all sera positive by PRNT also were positive by EIA. The
new EIA has been incorporated into our field research program and has been used to economically screen
over 10, 000 wild bird sera from 124 species for antibodies against WEE and SLE.
Keyword Index: Enzyme immuno assay, birds, arboviruses, antibodies, WEE, SLE.
INTRODUCTION
Wild birds
several
are the principal vertebrate
North American
encephalitis viruses,
western equine encephalomyelitis(
and
St. Louis
Reisen
and
Species in
they detect antibody in a variety of bird species and do
not require laboratory containment procedures.
encephalitis (
hosts for
However, the HI test is labor intensive, tedious to
including
perform, and notorious for producing nonspecific results
WEE, Togaviridae)
SLE, Flaviviridae)
Monath 1989, Tsai
and
viruses
Mitchell 1989).
variety of avian taxa( mostly Passeriformes
and Columbiformes) become infected and are important
in
virus
enzootic
McLean
Milby
a
and
maintenance
Bowen 1980,
Reeves
and
1990).
and
amplification
Hardy and Reeves 1990,
A key component in
encephalitis virus surveillance programs
monitoring
of enzootic
seroconversion
Reeves
et
al.
transmission
has been the
by measuring either
rates
in
sentinels,
1990)
or
seroprevalence
such
as
chickens
rates
among
field- collected birds, especially house sparrows and
house finches( McLean et al. 1988, Gruwell et al. 1988).
Monitoring
rates
in
wild
temporal changes
birds in
by
sensitive,
effective
and
cost
species (
Chiles
inhibition
and
assays (
seroprevalence
arbovirus surveillance programs
has been complicated
antibodies against a
in
the absence
of a rapid, specific,
assay that can detect
in different bird
of viruses
variety
Reisen 1997).
HI) have been
Hemagglutination
used
widely because
at
low antibody titers (
e.
g.,
Hall
et
al.
1995).
Neutralization tests, such as the plaque reduction
neutralization
test (
PRNT), do not require species-
specific reagents and are considered to be the" standard"
for serological testing. However, PRNTs are expensive,
require up to two weeks to perform, and must be
conducted within adequate containment facilities
because of the test requirement for live virus culture.
Several enzyme immunoassays ( EIA) have been
developed.
A blocking or competitive enzyme
immunoassay( EIA) was developed for Murray Valley
encephalitis and several other flaviviruses ( Hall et al.
1995).
One of the monoclonal antibodies( 3H6) cross
reacts with several of the Flaviviridae, including SLE;
however, similar competitive monoclonals currently
are not available
for the Togaviridae.
Indirect EIAs
have been developed to detect antibodies in specific bird
species, such as sentinel chickens( Oprandy et al. 1988,
Reisen et al. 1994); however, these EIAs rely on host
specific reagents and are not suited for a surveillance
program that tests a
variety
of
bird
species.
Calisher
et
Journal of Vector
124
al. (
1986)
IgG
and
EIA using
antibodies
commercial
reactivity
bird
several
both
were
in
previously elicited a strong antibody response in a
variety of bird species ( Hardy and Reeves 1990) and
However, in
an
our
and
chickens(
Renewed interest in using temporal
avian seroprevalence as an
California
by
stimulated research to
a
WEE
species.
induce
Reisen
et al.
1994). Both viral antigens and
SLE in
a
variety
of
Procedures and test parameters are summarized in
Figure 1. Briefly, viral antigen was allowed to bind to
be
cost
Immulon 1 ( Dynatek Laboratories Inc., Winooski, VT)
and
96 well plates overnight at 4° C. Nonspecific binding
used
was
and
field
antibodies
most
bird
species
(
with
2%
a
casein/ PBS- T ( phosphate
at 37°C. After blocking, 0. 1 ml of unknown bird sera
diluted 1: 50; e. i., 1: 10 field dilution x 1: 5 laboratory
dilution in physiological saline) was added to each well
California bird
and allowed to react with the antigen coated surface. A
EIA may be able
for pathogens that
positive reaction was detected by the subsequent binding
of the rabbit anti- bird detector antibody that was
in birds.
antibodies
blocked
buffered saline— Tween detergent) solution for 30 min
describes the
slight modification, this
from
to screen sera
current paper
indirect EIA that detects
and
With
and could
development, and laboratory
evaluations of an
against
Both viral strains
coating buffer( pH 9. 6)
agencies
assay that was
to both WEE
an
species,
The
without containment.
control
antibodies
bird
of
variety
rationale, reagent
develop
in
changes
of arbovirus
early warning
mosquito
detect
to
effective, able
SLE in
controls.
the control antigen were diluted in carbonate- bicarbonate
Reisen 1997).
activity
negative
to WEE and SLE in humans ( Reisen et al. 1996) and
several
Passeriformes ( Chiles
as
used
have been used as antigens in EIAs to detect antibodies
antibodies
birds from
positive
including
orders
against
species
anti- chicken
goat
known
react with
different
cross
from
goat anti- chicken reagents.
laboratory
failed to
broad
reported
IgM
December, 1998
Ecology
conjugated with horseradish peroxidase to the unknown
bird sera, now bound to the virus antigen.
Rationale
Assay
and
Procedures
Our assay is based upon the hypothesis that a
mammalian host immunized with the IgG antibody
fraction from normal sera pooled from bird species
different
representative of several
antibodies
most
is
that
birds were tested per plate for each virus using the map
from
shown
related
to
were
Montgomery,
immunized
formes),
rabbits
from the same bird taxa that is being tested, it was
from
difficult to get large amounts of high titered positive
sparrows
sera from small birds such as sparrows, finches, and
order
( Streptopelia
domestic
Galliformes),
and
Anseriformes).
based
on
these
bird
availability
fraction
pooled
Our
Passeriformes),
order
risoria,
chickens (
Muscovy
order
degree
Texas, in domestic
within
produced
white- crowned
Zonotrichia leucophrys,
turtle- doves
4, 7, and 10) and known positive ( B 1- 3) and negative
( A1- 3) bird sera. Although it is best to use known sera
proteins
was
the IgG
with
from
collected
sera
the
Bethyl
of
Our antibody
Laboratories,
Gallus
bird
orders with enzootic virus
anti sera production could
being
In practice, wild bird sera
tested.
presumptively positive with a mean optical density> 2X
species was
the negative controls were retested by a PRNT. If used
transmission.
reactivity may be
bird taxa
order
moschata,
The
enhanced
be directed specifically
as a stand alone assay, we recommend using the mean
of
by the
addition of sera from additional orders. Alternatively,
of cross-
Therefore, known positive and negative
warblers.
chicken sera were utilized at dilutions to simulate the
Columbi-
gallus,
ducks( Cairina
choice of
ringed
the historical involvement
and
Controls on each plate included
by
diversity
the
in Figure 2.
mock antigen( uninfected Vero cell extract, columns 1,
bird taxa. The degree of cross- reactivity presumably
immunogen.
that
Instruments, Winooski, VT). In practice, sera from 30
orders will produce
serum proteins
will " recognize"
The latter
reaction was detected by color development after the
addition of the ABTS substrate( Kirkegaard and Perry,
Gaithersberg, MD) with plates quantitatively assessed
at 405 nm using a standard EIA plate reader( Dynatek
against
a single
taxon such as the order Passeriformes to provide
a more
focussed
reagent.
+
3 STD of the negative control wells per plate as the
cutoff for positive sera.
Evaluation
The specificity and sensitivity of the new detector
antibody conjugate was evaluated in five ways.
Inactivated viral antigens were derived from Vero
cell cultures
or
infected
the BFS 1750
were
with
strain of
the
BFS 1703
SLE. Both
isolated from Culex
strain of WEE
viruses
tarsalis
originally
collected
near
and were passaged twice in suckling
Vero cell infection. Uninfected Vero cells
1.
Species Recognition
The ability of the rabbit anti- bird sera to detect
proteins from a variety of avian taxa was evaluated by
Bakersfield in 1953
a sandwich EIA at Bethyl Laboratories
mice prior to
Arbovirus
Research
Laboratory (
ARL).
and the
In
the
December, 1998
Journal of Vector
ABTS SUBSTRATE
Ecology
125
READ AT 405 NM
WASH
CONJ
POLYCLONAL
ANTI- BIRD ABY
HORSERADISH PEROXIDASE
LABELED ANTI- BIRD ANTIBODY
WASH
BIRD SERA
UNKNOWN SERA DILUTED 1: 50
WASH & BLOCK
BLOCK FOR 30 MIN WITH 2% CASEIN
ITT
---- ormsrem
ilvER
INACTIVATED VIRAL ANTIGEN
GE N
FROM VERO CELL CULTURE
IMMULON® 1 96 WELL PLATE
Figure 1.
A diagram showing the components of the indirect EIA.
1
A
-
3
-
4
5
-
6
7
8
9
10
11
7
15
23
+
8
16
24
C
1
9
17
25
D
2
10
18
26
E
3
11
19
27
F
4
12
20
28
G
5
13
21
29
H
6
14
22
30
B
Figure 2.
2
+
+
12
Protocol for specimen positioning on the 96 well plates. A1- 3 negative chicken sera. B1- 3 positive
chicken sera. Columns 1, 4, 7, and 10 are negative antigen control wells and 2, 3, 5, 6, 8, 9, 11, and 12
contain positive antigen. Numbers refer to sera tested in duplicate adjacent wells.
sandwich
EIA, affinity
purified
rabbit anti-
bird IgG
dilution that produced an absorbance of 1. 0 was> 1: 100.
was used as a capture antibody and was adsor-bed
to
96
well
dilutions
added,
of
plates,
bird
rinsed,
sera
incubated,
and
blocked. Ten- fold
from different taxa
and
rinsed,
after
Two groups of six adult ( after hatching year by
plumage) house finches ( Carpodacus mexicanus) of
ABTS
mixed sexes each were inoculated subcutaneously in the
cervical region with either 4. 0log1oPFU( plaque forming
units)/ 0. 1 ml of the BFS 1703 strain of WEE or 3. 0
log10
substrate then was added and absorbance read at
The
Experimental Infections
the
which
conjugated rabbit anti- bird sera was added.
nm.
2.
then were
405
reaction was considered positive when the
Journal of Vector
126
PFU/ 0. 1
BFS 1750
ml of the
hatching
after
domesticus)
Zenaida
year
and two after
SLE.
A
single
encephalitis
virus
surveillance
program.
Selected
Passer
positive and negative sera then were shipped to the ARL
hatching year mourning doves
where they were tested blindly by EIA and PRNT( at
male
also
macroura)
strain of
house
December, 1998
Ecology
sparrow (
inoculated
were
with
SLE.
dilutions of 1: 20 and 1: 40).
Birds were bled by jugular puncture( 0.2 cc diluted in
0. 8 cc physiological
saline) on
days 2 and 3 post infection
RESULTS
PI) for acute antibody samples for WEE and SLE, and
days 13, 17, 20, 22, 24,
then on
and
27 PI. Sera
Species Recognition
were
by the new EIA and by PRNTs in
cultures.
Confirmatory PRNTs were done at
1: 20 except on day 27 when end point titers
Sera from 13 of 16 bird species in 7 of 10 orders
tested for antibody
Vero
cell
a titer of
cross- reacted with the rabbit anti- bird antibody and
produced an absorbance value of 1 at a dilution of
1: 100 in the
determined.
were
sandwich
EIA ( TABLE 1).
Strongest
reactions were observed for sera from species that
3.
on
the EIA
PRNT
Comparative
Sensitivity
Sera
from experimentally infected birds
collected
of
and
day 27 were diluted in a doubling sequence and then
by EIA. EIA formula values then were plotted as
tested
a
function
Species with end points< 100 included the least bittern
(
PRNT titers to determine EIA
of calculated
test results on sera of
comprised the immunogen, but strong reactions also
were obtained for domestic turkey, emu, and ostrich.
known but varying PRNT titers.
Ixobrychus exilis, order Ardeiformes), sora( Porzana
order
carolina,
(
Calidris
Ralliformes), and least sandpiper
minutilla,
order
Charadriiformes).
Experimental Infections
Comparison Between EIA
4.
PRNT in Wild
and
Birds
Wild birds
were collected
in Riverside, Kern,
traps
California,
and sex,
and
by
mist nets or ground
Sacra- mento
counties,
immediately identified to species, age,
by jugular puncture( 0. 1 cc blood in 0. 9 cc
and
bled
Blood
saline), and released.
separate at ambient
sera stored atand
samples were allowed
to
temperature, centrifuged, and the
70° C. Sera from Riverside( September
October 1996)
Sacramento ( 1997)
and
counties
for antibody to either WEE or SLE using
both the new EIA and a PRNT in Vero cells. Positive
tested
were
EIAs
exhibited
negative
of>
80%
controls at a serum
to
analyzed
post-infection( PI), respectively( TABLE 2, Fig. 3). On
day 13 PI, antibody to WEE was detected at low levels
in three of five house finches, but results for the two
assays were only in agreement for sera from two birds.
Antibody to SLE was detected first on day 17 PI in five
of five house finches and two of two mourning doves;
the single house sparrow remained negative. By day 22
PI all birds were positive for antibodies against both
by both tests. Variable test
results during days 13 to 20 reflected the low concenWEE
and
SLE
viruses
2 times the
trations of antibodies present before the rise in titer at 22
Positive PRNTs inhibited the
days post- infection( Fig. 3). On day 27 PI, the reciprocal
of the geometric mean titer by PRNT was 101 for house
absorbance
controls.
formation
values
of plaques
dilution
estimate
the
of >
developed in
of>_
1:
negative
20. These data
were
and
accuracy, sensitivity,
specificity of the EIA in comparison to the PRNT.
Additional sera from Riverside County( March 1996September 1997) and Kern County ( 1997)
were
screened by EIA and presumptive positives confirmed
by PRNT. These demonstrated the extent of cross
reactivity by the detector antibody and the effectiveness
of
EIA and PRNT tests on house finch sera were
negative for antibody to WEE and SLE on days 2 and 3
the EIA as a screening assay.
finches infected with WEE, and 60 for house finches
and 631 for mourning doves infected with SLE.
Comparative Sensitivity of the EIA and PRNT
EIA test results ( expressed by formula as mean
optical density of the two test wells divided by the
negative control well) were plotted as a function of the
doubling dilution of sera collected from experimentally
infected birds on day 27 PI( Fig. 4). EIA formula values
did not vary proportionally with PRNT titers, even for
5.
Comparison Between HI and EIA in Wild Birds
the undiluted sera. For example, house finch 2113 with
Birds ( house finches
a PRNT titer of 1: 320 had an EIA value of 3. 5 that was
in
collected
jugular
Blood
puncture(
traps
0. 1
cc
house
in Orange
in 0.9
cc
SLE
County
by
sparrows)
were
by
lower than the remaining birds with a titer of 1: 80.
and released.
However, there was complete congruence of test
County,
diluent),
bled
at
results; i.e., all undiluted sera( points on Fig. 4 furthest
for antibody against either WEE
HI( Clarke and Casals 1958) at the Orange
to the right) had PRNT titers >_1: 20 and EIA formula
samples were centrifuged, and
4° C. Sera
or
crow
and
the sera stored
were tested
Vector Control District
as
part
of
their
values > 2. The EIA for WEE ( Fig. 4a) seemed less
sensitive
than the EIA for SLE(
Fig.
4b), because EIA
December, 1998
Journal of Vector
PRNT titers
values at
but> 2 for PRNT titers
1: 10
of
1: 5
of
were all <
2 for WEE
9),
1: 10 for SLE. In fact,
and
A total
were tested
EIA
and
898 birds comprising 70
in Riverside
for antibodies
WEE
and
PRNT( TABLE 3). Twelve birds
for WEE
EIA,
by
including
PRNT,
against
a
black- crowned
night
false
were no
these, 47 ( 35%)
and
Columbiformes,
were positive
by
heron (
order
by
EIA;
negatives
0.997 ( 886/ 889).
For SLE,
WEE
and
and/ or
SLE ( TABLE 5).
Of
14 ( 20%)
were confirmed by
PRNT, respectively. Confirmed positives included 15
species in four orders ( Gallliformes, Ardeiformes,
SLE by both
of which eight were confirmed
Ardeiformes). There
antibodies against
and
1997
was
in Riverside and Kern counties were screened by EIA,
of which 136 and 69 birds were positive by EIA for
species was
September
specificity
7), and specificity was 0.997( 889/ 891).
Overall, 10,077 sera from 124 bird species collected
PRNT in Wild Birds
County during
1996 and Sacramento County during
collected
October
of
and
and
127
accuracy was 0.996( 894/ 898), sensitivity was 0.714( 5/
only birds 2121 and 2118 gave negative values for SLE
at PRNT titers of 1: 20 and 1: 40,
respectively( Fig. 4b).
Comparison Between EIA
Ecology
and
Passeriformes).
Higher confir-
mation rates for WEE than SLE reflected the greater
number of birds with WEE titers? 1: 40 collected inKern
County.
i. e., sera negative for antibodies to WEE by EIA also
were negative
song
confirmed
by
EIA
these
by
PRNT. Four house finches
for SLE
sparrow positive
by
were
quail
and>_1:
antibodies
PRNT, but two Gambel'
again
positive,
Comparisons Among HI, EIA, and PRNT Assays
were
A total of 141 sera was collected in Orange County,
s quail negative
tested for WEE and SLE antibody by HI, and then
shipped to the ARL where they were retested by EIA
and PRNT. Overall, 47 were positive for SLE by HI; 3
PRNT. Repeat PRNTs
by
positive
were
by
and one
EIA
with
titers
of
on
1: 20
40.
had titers of 1: 40, 15 of 1: 20, and an additional 29 were
ElAdataforbothviruseswerecomparedstatistica lly
PRNT
determine accuracy,
sensitivity and specificity ( TABLE 4). For WEE,
accuracy was 0. 997( 895/ 898), sensitivity was 1. 000( 9/
against
the
listed
to
standard
TABLE 1.
as "
borderline."
By EIA, 17 were positive by
formula ( i. e., mean of positive wells divided by the
negative well was> 2) for SLE and six were nonspecific
(
i. e., noticeable color development in both positive and
Ability of rabbit anti- bird sera to recognize wild bird sera in a
sandwich EIA at Bethyl Laboratories and the Arbovirus Research
Laboratory ( ARL). Values are the reciprocal titers from dilution
series starting at 1: 100 that gave an optical density of 1. 0 at 405 nm.
Bird
Domestic
species
chicken*
Ringed turtle dove*
White- crowned
Muscovy
sparrow*
duck*
Ostrich
Emu
Domestic turkey
Gambel' s quail
Marsh
Bethyl
AR
100, 000
100, 000
100, 000
1, 600
65, 000
nd
100, 000
nd
6, 000
nd
10, 000
nd
100, 000
nd
nd
100
nd
100< 200
wren
Audobon'
s warbler
nd
100
nd
100< 800
nd
100
nd
100
Least bittern
nd
100
Sora
nd
100
nd
100
Northern
Bullock'
rough- winged swallow
s oriole
Bonaparte'
Least
s gull
sandpiper
Species sera used in the rabbit immunogen.
Nd,
not
done
Journal of Vector
128
TABLE 2.
December, 1998
Ecology
Ability of the new EIA( rabbit anti- bird conjugated
antibody) and PRNT to detect antibodies to WEE
and SLE in experimentally infected after hatching
year
Virus(
house finches(
strain,
innoculum)
6 per virus).
n=
No. positive*
Days Post
infection
n
WEE( BFS 1703, 4.O log
10
EIA
PRNT
PFU/ 0. 1 ml)
2
6
0
13
5
3
3
17
5
2
2
20
5
1
5
22
5
5
5
24
5
5
5
27
5
5
5
SLE( BFS 1750, 3. O log
io
nd
PFU/0. 1 ml)
3
6
0
nd
13
6
0
0
17
5
4
3
20
5
4
3
22
5
5
5
24
5
5
5
27
5
5
5
n, sample size (
1 bird died in
each
group);
EIA, positive by
formula where mean optical density of antigen positive wells/
control well>
negative antigen wells,
were positive
but
value<
2; PRNT, titer>_ 1: 20.
2).
By
PRNT, 19
for SLE; 12 at a titer of 1: 20 and 7
at?.
1: 40.
Two birds strongly positive for WEE by PRNT ( titer
1: 40) and EIA( formula>_4. 0) were reported as being
negative
by
When
missed and 16 PRNT negative birds would have been
reported as positive. In contrast, the EIA detected all
seven PRNT positive birds, with formula values
As indicated earlier, the 10 false EIA positives were not
considered to be a problem because these would be
HI.
all positives were
included
and compared
discarded after the confirmatory PRNT.
against the PRNT results, the HI was less accurate,
sensitive, and specific
than the
EIA in
detecting
SLE
DISCUSSION
antibody in bird sera ( TABLE 6).
Considering that
birds with a PRNT titer of 1: 20 may represent old or
Rabbits immunized with sera pooled from single
borderline positive infections, the analyses were repeated
species representatives of four orders of birds produced
using more conservative criteria of PRNT titer? 1: 40,
HI titer>_1: 20, and EIA>_2. 0 by formula. This analysis
antibodies that reacted with serum proteins from 29
markedly improved the
PRNT, but decreased
PRNT.
have
agreement
Reporting only HI titers?
resulted
results;
agreement
in only
five PRNT
species of birds in seven orders. This extensive cross
between the EIA
and
reactivity was exploited in a simple three- step indirect
between the HI
and
EIA that detected antibodies in wild birds against WEE
and SLE viruses. When compared against the PRNT,
1: 20 as
two tests
in
positive
birds
positive would
agreement with
would
PRNT
have been
the new EIA was acceptably accurate and sensitive to
function
as a rapid,
inexpensive, screening assay for
December, 1998
Journal of Vector
Ecology
129
OD
2. 5A. WEE
2-
HOUSE FINCH
1. 5-
1—
0. 5r
8
9
CUTOFF
0
2
13
17
20
22
24
27
DAYS POST INFECTION
OD
2. 5
B.
SLE
2
HOUSE FINCH
HOUSE SPARROW
1. 5-
+
MOURNING DOVE
1—
0. 5
cfi
CUTOFF
0
3
13
17
20
22
24
27
DAYS POST INFECTION
Figure 3.
EIA test results expressed as mean optical density of two test wells per serum sample plotted as a function
of days after infection of adult birds with either( A) WEE or( B) SLE. Cut off values above which values
are positive were expressed as the mean optical density plus two standard deviations( solid line) or two
times the mean of the negative control wells.
processing the large numbers
for surveillance programs and
Although currently in
minor anomalies with
the
use
bird
sera needed
vigilance may be necessary to ensure that false negatives
epidemiological studies.
are not recorded for taxa not adequately cross reacting
of wild
by our research program,
new
EIA
were
bothersome.
First, the
sandwich EIA used to screen for cross
reactivity
failed to react strongly( titer< 1: 100 at an optical density
of 1. 0) with sera from a least bittern, sora, and least
sandpiper.
However, the final
antibody reacted positively at
from least bitterns and black
confirmed as positive
some
Chadriiformes
EIA, these
a
by
by
night
PRNT.
Ralliformes
were not confirmed
detector
1: 50 dilution to
crowned
for WEE
and
conjugated
with our detector antibody. Future studies will evaluate
the reactivity of detector antibody prepared by combining
sera from rabbits immunized with serum from individual
bird taxa.
Secondly, two Gambel' s quail and two house finches
were positive for SLE antibodies by PRNT, but negative
sera
by EIA. The quail were collected on the same day and
herons
were tested initially in adjacent wells. Repeat tests on
the original sera again were positive by PRNT, but ne-
Although
were positive
by
PRNT. Continued
gative by EIA. Interestingly, the two finches missed by
the
EIA
were
marginally
positive
by HI.
At
present, our
130
Journal of Vector
12
December, 1998
Ecology
A. WEE
2110
10r
1—
A
2111
42112
8
2113
F
6
O
R
M
U
L
A
2115
4
n
2L
0
1
_ -
80
40
20
10
160
320
PRNT TITER
14
r
B. SLE
19. 3
12i
2116
-
2117
A10
2118
8-
F
2120
O
2121
R
M
64
A
r
2
0
5
10
20
80
40
160
320
PRNT TITER
Figure 4.
EIA results expressed by formula( mean optical density of two test wells divided by the control well) and
plotted as a function of PRNT titer of sera collected on day 27 post- infection from adult house finches
experimentally infected with( A) WEE or( B) SLE. Sera from each bird were doubly diluted and then
tested by EIA; initial concentrations furthest to the right.
only
or
explanation was
very
body
old
infections
titers to
that these
and
did
birds had
not
have
produce positive reactions
in both tests.
Similar problems of assay agreement were observed
for birds infected experimentally with SLE and tested
earlier than 17 d PI.
Overall, testing California and
Gambel'
SLE
s quail sera
by
EIA detected 10 WEE
positives, of which
PRNT, respectively.
3
and
4
35%
of
WEE
and
20% of SLE EIA positives were
by PRNT. Hall et al. ( 1995) attributed
comparable assay disparity for several flaviviruses to
reduced sensitivity by a comparable neutralization
confirmed
procedure. In contrast, Calisher et al.( 1986) and Olson
and
12
were confirmed
by
1991) documented good agreement among their
EIAs and HI and neutralization assays. Comparing the
house finches
performance of our new EIA on sera from experi-
Similarly, 29 and 5
by both EIA
Thirdly, when used to screen field samples, only
either recent
sufficient anti-
PRNT for WEE
et al.(
SLE,
mentally infected birds indicated that EIA formula
respectively. Therefore, the detector antibody readily
detects the serum proteins of these two bird species, and
values did not vary quantitatively as a function of
PRNT titer, although there was congruence among
were positive
we
currently have no
results.
and
explanation
for these
and
negative
EIA
test results among undiluted samples. The WEE EIA
seemed
less
sensitive than the
SLE EIA, especially
at
December, 1998
TABLE 3.
Journal of Vector
Ecology
131
Birds collected in Riverside and Sacramento counties and tested for antibodies to WEE and SLE
viruses by both EIA and PRNT assays.
No.
COMMON NAME
SCIENTIFIC NAME
GAMBEL' S QUAIL
Callipepla
LEAST BI1"1' ERN
Ixobyrchus
GREAT EGRET
Casmerodius
GREEN- BACKED HERON
Butorides
BLACK- CROWNED NIGHT HERON
Nycticorax
gambelii
exilis
albus
striatus
nycticorax
WEE
SLE
Tested EIA PRNT EIA PRNT
30
0
0
0
2
3
0
0
0
0
1
0
0
0
0
4
0
0
0
0
3
1
1
0
0
2
0
0
0
0
1
0
0
0
0
1
0
0
0
0
11
0
0
0
0
34
0
0
0
0
1
0
0
0
0
SORA
Porzana
COMMON MOORHEN
Gallinula
AMERICAN COOT
Fulica
MOURNING DOVE
Zenaida
COMMON GROUND DOVE
Columbina
RED- NAPED SAPSUCKER
Sphyrapicus
NUTTALL' S WOODPECKER
Picoides
nuttallii
6
1
0
0
0
SAY' S PHOEBE
Sayornis
saya
1
0
0
0
0
BLACK PHOEBE
Sayornis
nigricans
11
0
0
0
0
UNIDENTIFIED FLYCATCHERS
34
0
0
0
0
ASH- THROATED FLYCATCHER
Empidonax spp.
Myiarchus cinerascens
1
0
0
0
0
BROWN- HEADED COWBIRD
Molothrus
1
0
0
1
0
RED- WINGED BLACKBIRD
Agelaius
4
0
0
0
0
GREAT-TAILED GRACKLE
Quiscalus
1
0
0
0
0
CASSIN' S FINCH
Carpodacus
cassinii
1
0
0
0
0
HOUSE FINCH
Carpodacus
mexicanus
93
5
5
5
4
LESSER GOLDFINCH
Carduelis
6
0
0
0
0
LAWRENCE' S GOLDFINCH
Carduelis lawrencei
1
0
0
0
0
24
1
0
0
0
3
0
0
0
0
13
0
0
0
0
1
0
0
0
0
6
0
0
0
0
14
0
0
0
0
133
1
0
0
0
2
0
0
0
0
6
0
0
0
0
0
carolina
chloropus
americana
macroura
passerina
nuchalis
ater
phoeniceus
mexicanus
psaltria
AMERICAN GOLDFINCH
Carduelis
tristis
VESPER SPARROW
Pooecetes
gramineus
SAVANNAH SPARROW
Passericulus
GRASSHOPPER SPARROW
Ammodramus
LARK SPARROW
Chondestes
grammacus
GOLDEN- CROWNED SPARROW
Zonotrichia
atricapilla
WHITE-CROWNED SPARROW
Zonotrichia leucophrys
WHITE- THROATED SPARROW
Zonotrichia
BREWER' S SPARROW
Spezella breweri
SAGE SPARROW
Amphispiza belli
SONG SPARROW
Melospiza
LINCOLN' S SPARROW
Melospiza lincolnii
FOX SPARROW
Passerella iliaca
SPOTTED TOWHEE
Pipilo
CALIFORNIA TOWHEE
sandwichensis
savannarum
albicollis
melodia
5
0
0
0
62
1
1
1
1
89
0
0
0
0
8
0
0
0
0
20
0
0
0
0
Pipilo fuscus
3
0
0
0
0
GREEN- TAILED TOWHEE
Pipilo
1
0
0
0
0
LAZULI BUNTING
Passerina
5
0
0
0
0
BLACK-HEADED GROSBEAK
Pheucticus
4
0
0
0
0
WESTERN TANAGER
Piranga ludoviciana
2
0
0
0
0
BARN SWALLOW
Hirundo
13
0
0
0
0
NO. ROUGH- WINGED SWALLOW
Stelgidopteryx
1
0
0
0
0
TABLE 3
continued on next page
erythrophthalmus
chlorurus
amoena
melanocephalus
rustica
serripennis
132
Journal of Vector
Ecology
December, 1998
TABLE 3 continued.
No.
SCIENTIFIC NAME
COMMON NAME
WEE
SLE
Tested EIA PRNT EIA PRNT
LOGGERHEAD SHRIKE
Lanius ludovicianus
1
0
0
0
0
WARBLING VIREO
Vireo
6
0
0
0
0
ORANGE-CROWNED WARBLER
Vermivora
celata
19
0
0
0
0
NASH\ ILLE WARBLER
Vermivora
ruficapilla
3
0
0
0
0
YELLOW WARBLER
Dendroica
petechia
28
0
0
0
0
YELLOW- RUMPED WARBLER
Dendroica
coronata
10
0
0
0
0
BLACK- THROATED GRAY W.
Dendroica
nigrescens
3
0
0
0
0
HERMIT WARBLER
Dendroica
occidentalis
1
0
0
0
0
NORTHERN WATERTHRUSH
Seiurus
noveboracensis
1
0
0
0
0
COMMON YELLOWTHROAT
Geothlypis trichas
27
0
0
0
0
WILSON' S WARBLER
Wilsonia
19
0
0
0
0
MACGILLIVRAY' S WARBLER
Oporornis tolmiei
4
0
0
0
0
AMERICAN REDSTART
Setophaga
1
0
0
0
0
HOUSE SPARROW
Passer domesticus
56
0
0
0
0
NORTHERN MOCKING BIRD
Mimus
3
0
0
0
0
SCRUB JAY
Aphelocoma
4
0
0
0
0
BEWICK' S WREN
Thryomanes bewickii
7
1
1
0
0
MARSH WREN
Cistothorus
15
0
0
0
0
HOUSE WREN
Troglodytes
5
0
0
0
0
WHITE- BREASTED NUTHATCH
Sitta
2
0
0
0
0
OAK TITMOUSE
Parus inornatus
2
1
1
0
0
WRENTIT
Chamea fasciata
2
0
0
0
0
VERDIN
Auriparus flaviceps
2
0
0
0
0
BLUE- GRAY GNATCATCHER
Polioptila
2
0
0
0
0
HERMIT THRUSH
Catharus
guttatus
8
0
0
0
0
AMERICAN ROBIN
Turdus
migratorius
1
0
0
0
0
898
12
9
7
7
gilvus
pusilla
ruticilla
polyglottos
coerulescens
palustris
aedon
carolinensis
caerulea
TOTAL
TABLE 4.
Comparison of the EIA with the PRNT standard for detecting
WEE and SLE antibodies in wild birds collected in Riverside and
Sacramento counties.
PRNT
Virus
EIA
Positive
WEE
Positive
9
3
12
Negative
0
886
886
Total
9
889
898
SLE
Negative
Total
Positive
5
2
7
Negative
2
889
891
Total
7
891
89
December, 1998
TABLE 5.
Journal of Vector
Ecology
133
Birds collected in Riverside( March 1996— September 1997) and Kern( March—October
1997) counties, screened by EIA, and confirmed by PRNT assays for WEE and SLE
antibodies.
NO.
COMMON NAME
WEE
TESTED
EIA
612
69
EARED GREBE
MALLARD
GAMBEL' S QUAIL
CALIFORNIA QUAIL
LEAST BI'1- FERN
SNOWY EGRET
GREEN-BACKED HERON
BLACK- CROWNED NIGHT HERON
WESTERN SANDPIPER
KILLDEER
SLE
PRNT
EIA
PRNT
9
3
12
4
1
0
0
0
8
1
0
0
0
1
1
0
0
0
68
5
1
3
0
5
1
0
0
0
17
0
0
1
0
8
1
1
0
0
123
2
0
0
0
9
0
0
1
0
45
0
0
1
0
203
1
1
4
2
LESSER NIGHTHAWK
21
1
0
0
0
BLACK PHOEBE
72
0
0
1
0
EUROPEAN STARLING
84
1
0
1
0
MOURNING DOVE
COMMON GROUND DOVE
BROWN- HEADED COWBIRD
145
2
1
1
0
RED- WINGED BLACKBIRD
263
10
3
7
0
NORTHERN ORIOLE
19
2
0
1
0
BULLOCK' S ORIOLE
151
4
2
0
0
0
GREAT-TAILED GRACKLE
17
2
0
0
HOUSE FINCH
1, 256
65
29
10
5
WHITE- CROWNED SPARROW
1, 577
7
0
7
0
GOLDEN- CROWNED SPARROW
51
2
0
0
0
1, 133
1
0
3
0
ABERT' S TOWHEE
55
1
0
2
0
BLACK-HEADED GROSBEAK
18
1
1
0
0
0
SONG SPARROW
VIOLET-GREEN SWALLOW
1
1
0
0
NORTHERN ROUGH- WINGED SWALLOW
66
0
0
1
0
LOGGERHEAD SHRIKE
25
1
0
0
0
WARBLING VIREO
79
1
1
0
0
NASHVILLE WARBLER
27
0
0
1
0
693
1
0
1
0
27
0
0
1
0
186
1
0
2
0
14
0
0
1
0
291
1
0
3
1
NORTHERN MOCKING BIRD
39
0
0
1
0
CALIFORNIA THRASHER
19
2
2
0
0
7
0
0
1
1
211
1
0
2
1
39
6
2
0
0
TOTAL POSITIVE( 41 SPECIES)
7, 754
136
47
69
14
TOTAL NEGATIVE( 83 SPECIES)
2, 323
0
0
0
0
10, 077
136
47
69
14
YELLOW- RUMPED WARBLER
MACGILLIVRAY' S WARBLER
COMMON YELLOWTHROAT
YELLOW- BREASTED CHAT
HOUSE SPARROW
CACTUS WREN
MARSH WREN
AMERICAN ROBIN
TOTAL( 124 SPECIES)
Journal of Vector
134
December, 1998
Ecology
TABLE. 6. Comparison of HI, EIA, and PRNT on bird sera collected in Orange County.
Standard
Pos.
Comparison'
Neg.
Total
Statistic'
PRNT(? 1: 20)
15
32
47
ACC
0. 745
4
90
94
SEN
0. 789
Total
19
122
141
SPE
0. 738
Positive
17
6
23
ACC
0. 943
2
116
118
SEN
0. 895
19
122
141
SPE
0. 951
Positive
HI
Negative
all)
EIA
Negative
all)
Total
PRNT(>_1: 40)
HI
1: 20)
EIA
2.0)
Positive
2
16
18
ACC
0. 851
Negative
5
118
123
SEN
0.286
Total
7
134
141
SPE
0. 881
Positive
7
10
17
ACC
0.929
Negative
0
124
124
SEN
1. 000
Total
7
134
141
SPE
0.925
Comparison: HI,
all=
1: 20, 1: 40
and
borderline
pooled;
EIA,
all=>
2. 0 and
nonspecific
Statistic: ACC, accuracy, SEN, sensitivity, SPE, specificity
and this may have accounted for the
higher confirmation rate among sera from field- collected
Lothrop, and Vincent Martinez, University of
birds.
Mosquito and Vector Control District for collecting the
low PRNT titers,
The
new
First, the
birds in Kern, Riverside, and Sacramento counties.
relatively inexpensive to produce
David Gutierrez, University of California, Davis,
assisted in screening the bird sera
by EIA. Carrie
Fogarty, Orange County Vector Control District,
performed HI tests on the Orange County birds. We
EIA has
detector antibody
was
several
advantages.
in large volumes. Immunization,
and conjugation of rabbit sera
required
bleeding, purification,
by the Bethyl Corporation
approximately three
months and<$
3, 000 for
antibody to test thousands of birds when diluted
1: 1500. Production would be more cost- effective using
a goat as the host animal to produce the antisera.
enough
the three step EIA is rapid and inexpensive to
We routinely screen 240 bird sera for WEE
SLE by this EIA in a single day at a cost of about
Secondly,
perform.
and
1. 50
California, Davis, and Stan Wright, Sacramento/ Yolo
Third,
thank Jame P. Webb, Jr. for permission to cite this data.
Laura D. Kramer, University of California, Davis,
critically read the manuscript. We especially thank two
anonymous reviewers of the Journal of Vector Ecology
whose comments markedly improved an earlier version
of this manuscript.
detector antibody
This research was funded, in part, by grants from
variety of similar assays to
test any bird taxa for infection with any pathogen
providing a suitable antigen is available and will bind to
the Mosquito and Vector Control Association of
Immulon® plates.
Division of Agriculture and Natural Resources,
per
theoretically
sera.
can
be
used
in
the
a
Acknowledgments.
California, the Coachella Valley Mosquito Abatement
District, the Mosquito Research Program of the
University of California, and the US NIH ( Grant R21
AI39483- 01A1). Logistical support was provided by
the Kern Mosquito and Vector Control District and the
We especially thank Robert Cusack, Hugh
Coachella
Valley
Mosquito Abatement District.
December, 1998
Journal of Vector
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California, 1983- 1987 ( W. C. Reeves,
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Reisen, W. K. and T. P. Monath. 1989. Western equine
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Reisen, W. K., S. B. Presser, J. Lin, B. Enge, J. L.
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with western equine encephalomyelitis and St. Louis
encephalitis viruses. J. Am. Mosq. Contr. Assoc.
10: 549- 555.
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St. Louis encephalitis viruses in sentinel chickens.
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Journal of Vector
23( 2): 136- 148
Ecology
Surveillance Studies of Orthopodomyia signifera
with Comparisons to Aedes sierrensis
David L. Woodward, Arthur E. Colwell, and Norman L. Anderson
Lake County Vector Control District
P. O. Box 310, Lakeport, CA 95453
Received 26 March 1998; Accepted 13 May 1998
The oviposition behaviors of Orthopodomyia signifera and Aedes sierrensis were
ABSTRACT:
examined with ovitraps and larval surveys during a five-year field study in northern California.
Ovipositional periodicity was found to be an important factor affecting the segregation of the species into
tree holes that were temporarily ( Ae. sierrensis only) or permanently ( both species) filled with water.
Orthopodomyia signifera females also used cues associated with the permanence of the habitat when
selecting oviposition sites. The distributions of the eggs of each species along horizontal and vertical
did
transects
indicate that interspecific
not
competition affected oviposition
site selection.
The data
indicate that temporal partitioning of larval development may reduce interspecific competition. Ovitraps
were determined to be more sensitive tools for surveillance of Or. signifera than larval surveys, CO2- baited
Fay traps and CDC light traps.
Keyword Index: Tree hole, mosquito, Orthopodomyia, oviposition.
because females are economically important biting
INTRODUCTION
pests of humans( Papineau 1984) and known vectors of
Seasonal
flooding
places
an
mental constraint on mosquitoes
breeding
This
sites
region
is
in
important
utilizing tree
California
northern
characterized
by
a
Dirofilaria immitis (
environ-
holes
as
oak woodlands.
Mediterranean
(
Leidy), the canine heartworm
Weinmann and Garcia 1974, Walters and Lavoipierre
1982).
Comparatively, much less is known about the
climate
dry
adaptive traits which allow Or. signifera populations to
completely
Hartmann 1992), making
survive the annual perturbation of summer drought.
habitats seasonally unsuitable for larval
development. As a result, the mosquito fauna lacks
shown Or. signifera eggs hatch upon completion of
diversity
The lack of a resting state in the egg stage coupled with
an unusually long larval development period ( Bohart
Steinhauser 1979)
with mild, wet winters and
More than 90%
summers.
each summer(
Washburn
of
tree
and
holes
hot,
dry
most of these
relative
Orthopodomyia
sierrensis (
Culicidae
to other regions of
largely
Coquillett)
signifera (
Ludlow)
are
the
restricted
North America.
only
and
northern
to tree
Aedes
California
highly
for
the western tree
survival
oviposit
hole
through the
conditions that characterize most
Females
is
signifera larvae do not pupate in California until high
dry- summer
outdoor temperatures have been reached. This trait may
delay adult activity until temporary rainfilled tree holes
have dried, thereby restricting oviposition to permanent
mosquito,
California forests.
drought- resistant
eggs
above
the
in tree holes
during spring and summer. The eggs
do not hatch until flooded by rain, usually in fall and
winter( Reisen and Reeves 1990). The life
history( e. g.,
Peyton 1956, Hawley 1985) and seasonal abundance
water
Garcia
al.
et al.
1996)
1950) restricts successful populations to the low
percentage of tree holes that hold water year- round.
Grant ( 1953)
sierrensis,
adapted
embryonic development, a period of four days at 30° C.
and
holes ( Bohart
Washino 1978).
Aedes
Previous studies ( Chapman 1964, Baerg 1968) have
1989, Washburn
ofAe. sierrensis
et al.
1989, Woodward
have been extensively
et
studied
habitats.
and
Baerg ( 1968) observed that Or.
Due to a lack of effective surveillance
techniques, this hypothesis has not been directly
investigated.
Or.
Further study of the ecology and vector potential of
signifera is needed.
This species is widely
distributed throughout temperate
regions
of eastern
December, 1998
Journal of Vector
North America.
includes
The known distribution in
lower
the
California
central
Ward 1981).
by human
the west
elevation mountains and valleys of
and
Oregon ( Darsie
southern
Although females have been
J. O. Washburn,
sentinels(
Ecology
137
Weather Data
Daily precipitation and maximum temperature data
and
for each year of the seasonal study were obtained from
collected
a United States National Weather Service station
pers. commun.),
located 10 km north of the study site.
they are not known to bite people ( Zavortink 1968).
may, however,
They
indirectly
because they readily
Chapman 1964,
females
signifera
al. (
et
and
in
Prior to the study, the volumes of the 10 tree holes
in the woodland were determined when they were
both EEE
WEE(
laboratory
western
volumes. The distance from the water surface to the
ground was measured for each tree hole. Polyethylene
Or.
cally important
Mexico. Further evaluation
eastern
in
signifera
arbovirus
maintenance of medi-
in
cycles
has been hampered
by
a
lack
In the
present
the
seasonal
the
analyze
flooding
of
oviposition.
of ecological
monthly ( December to March), with the exceptions of
January and December 1991 ( no data were collected).
compared.
traps(
Fay
to
natural
pipette were identified ( Bohart and Washino 1978) in
The temporal
and
for
water samples( ca. 45 ml each) by pipette( 5 mm mouth
diameter).
among climate, seasonal
tree holes, and the timing of
and spatial relationships of
Prince 1970), CDC light traps( Sudia
Chamberlain 1962),
methods
designed
was
On each sampling date, the water in each tree hole
was gently agitated prior to removal of each of three
relationships
by Or. signifera and Ae. sierrensis were also
Finally, larval surveys, CO2- baited Fay
oviposition
year of the seasonal study, water depths were monitored
weekly ( April to November), or one to four times
study, ovitraps were utilized to
distribution of oviposition by Or.
females. The investigation
signifera
rulers were mounted in each tree hole and during each
bird
sylvan
data.
monitor
naturally full of water. All standing water was removed,
measured, and replaced to determine maximum
transmission
of the role of
populations
Immature Mosquitoes
Vargas ( 1960) isolated EEE from field-
females in
collected
Surveillance of Natural Tree Holes for Water and
1954) determined Or.
were effective vectors of
encephalitis virus)
studies,
from birds
For
eastern equine encephalitis virus) and
equine
health
public
meals
1965, Zavortink 1968).
Baerg
Chamberlain
example,
affect
blood
take
and
and
ovitraps were compared
surveillance of
Or.
as
Immature mosquitoes collected by the
the field ( Ae. sierrensis) or the laboratory ( all other
species) and returned to the tree holes. Tree holes were
considered to be dry when no water could be removed
with the pipette. All ten tree holes were surveyed for
water and immature mosquitoes on a total of 36 dates in
1991, 43 dates in 1993, 42 dates in 1995, and 50 dates in
signifera.
1997. In addition, on July 14, 1993, a water sample( 50
METHODS AND MATERIALS
ml) was removed from each of the five deepest tree
holes and replaced with deionized water. The pH of
Study
Site
each sample was measured in the laboratory with a
The study
forested
was
northern
conducted
oak
in
woodland (
Potter Valley, Mendocino
by
densely
Munz 1965)
near
County, California( Latitude
39° 14' N, Longitude 123° 06' W,
dominated
1. 2 ha
a
interior live
oak (
elevation
Quercus
332
m)
wislizenii
meter. The samples were then centrifuged for 8 minutes
at 2200 rpm to remove suspended particles. The true
color( platinum- cobalt units) of the supernatant of each
sample was determined by spectrophotometer
according to Greenberg ( 1985).
Candolle) and Pacific madrone ( Arbutus menziessii
Pursh). Maximum height
m.
Winter searches of the
holes( 0- 5
of
holes
Pacific
on
were
foot
was ca.
16
for water- filled tree
and with an extension
located
madrones ( n=
year of
the tree canopy
m above ground) were conducted each year
the study
tree
of
woodland
the study.
on
2),
The
interior live
all of which
ladder. Ten
woodland was
8)
A translucent polyethylene cup( 473 ml) was filled
with 380 ml ofattractant water. Each cup was lined with
a Teri- wiper® towel strip( 10 x 27 cm), the oviposition
and
substrate, and placed in a plywood box( 29. 2 cm height,
water each
15. 2 cm width, and 17. 8 cm depth) that was painted
gloss black on all surfaces. An 11. 4 x 11. 4 cm vertical
entrance hole ( backed with 2. 5 cm mesh hardware
cloth) was centered on the front of each box at a point 7. 7
oaks( n=
held
Ovitrap Design
bordered
by
a
highway to the south and in the other directions by less
densely forested foothill woodland dominated by blue
oak( Q. douglassii Hooker and Arnott).
Study periods
cm below the top. Each lid was hinged and hasped to
included the
allow access( see Woodward et al. 1996).
seasonal
vertical
years
1991,
surveillance
distribution
of
1993,
1995,
and
1997
oviposition)
and
1994
of oviposition).
Blue oak tree hole water was used in the ovitraps as
an ovipositional attractant
from 1991
to
1995.
During
Journal of Vector
138
these
tree
during
volume
85 liters)
ca.
by
periods
study
evaporated
1997
during
16 liters)
from
75% blue
and
March 19, 1997
1996).
of
least
Baited Fay Traps and CDC Light Traps
Two stations, one near the middle of each ovitrap
or
Similar
transect, were established as locations for the operation
of Fay traps. These suction traps present a black-and-
water.
hole,
hole
tree
oak
white visual pattern without light and were baited with
2. 3
volume ca.
two
dates
July
on
14, 1993
4140 Pt- Co
and
as per
Garcia
et al.
1989). Traps were
following day at ca. 1000 hr. Each was operated once
during each year
during 1991 to
1991, on 34 dates between March 25 and November 16,
5773 Pt- Co
the attractant water measured
ice(
per week on 32 dates from April 3 to November 18,
1995, and on 34 dates between March 4 and October 21,
1995. In 1997 the pH ranged from 7. 78 to 7. 93. The true
color of
dry
according
pH of
from 7. 56 to 7. 83
the study) ranged
of
the attractant
and stored
The
kg
placed in the field at ca. 1400 hr and retrieved the
leaf infusion. The infusion
Woodward
water( measured on at
Response of Or. signifera Adults to Carbon Dioxide-
removed
25% blue
was prepared on
et al. (
same
kept full
except the attractant
a second tree
oak
was
replacing
water used was a mixture of
water( all collected
which
deionized
with
water
methods were used
to
from the
years attractant water was collected
hole (
December, 1998
Ecology
July
units on
1997. Live mosquitoes were anesthetized with carbon
dioxide,
units
24, 1997.
(
counted (
10X
magnification),
identified
Bohart and Washino 1978), and released in the field.
Dead adults were returned to the laboratory for
Seasonal Distribution
Seasonal
eight ovitraps
and
woodland
transition to
study
on
standard CDC light traps were operated at stations
two east- west transects of the
transecting the center of the woodland east to west in
foothill
site.
5- 8
stations
Using
using
woodland at
Each ovitrap
0.2
were
dioxide once per week on 15 dates between June 26 and
northern edge of
September 14, 1997. Other methods conformed to those
was staked
25- 50
m
to the
each year,
the
of
from
ground on
m apart).
north.
ovitraps were
continuously from April 2 to December 4,
1991, from April 5 to December 16, 1993, from April 11
operated
to
December 11, 1995,
1, 1997. Tree hole
Mosquito
per week.
and
from March 26 to December
water and
liners
were replaced once
eggs oviposited onto
These traps were operated without carbon
described for operation of the Fay traps.
Ovitrap
faced
m above ground and
locations
the same
1997.
the
center
15
the
a
the
were <
the north side of a tree trunk(
entrances
enumeration and identification. In addition, three
total of
oviposition was studied
located
Oviposition
signifera
Stations 1- 4 transected the
woodland.
the
Or.
of
liners
were
Statistical Analyses
Statistical analyses were performed according to
described by Zar ( 1980). Variables used in
statistical analyses were tested for normality with the
methods
Shapiro- Wilks'
W test.
Analyses which included
variables with a significant W( P< 0.05) were completed
with nonparametric methods.
identified using methods described by Woodward et al.
1996). Species determinations were confirmed by
Daily precipitation from January 1 to June 30 and
daily maximum temperatures from June 1 to September
identification ( Bohart
of reared
30 were compared between years with a Kruskal- Wallis
in
ANOVA by ranks followed by a multiple range test.
Using a Spearman rank order correlation ( R), total
monthly collections of Or. signifera eggs were
fourth- instar larvae
and
Washino 1978)
Eggs
and adults.
were counted
dissecting microscope ( 10X
magnification).
Rarely, Or. signifera eggs hatched
before the liners were returned to the laboratory. All Or.
the
laboratory
signifera eggs,
with
a
hatched
or unhatched, were
included in
the counts.
regressed with corresponding mean monthly maximum
temperatures, total monthly precipitation, the number
of natural tree holes holding water and mean tree hole
water depth. Seasonal totals of Or. signifera and Ae.
Vertical Distribution
The
with
six
of
Or.
signifera
Oviposition
sierrensis oviposition at each of the eight ovitrap
vertical pattern of oviposition was examined
ovitraps
mounted
the north side
on
of
the
primary stem of an interior live oak with the entrances
0. 2, 1. 0, 3. 0, 5. 0, 7. 5, and 10. 0 m above ground. The tree
was near
the
2
of
station
center of
were serviced with a
pulley
using the
study.
site and corresponded
Higher level
7. 5
ladder( 3. 0
10. 0
and
5. 0 m)
These
to
ovitraps
or a rope and
a Spearman
rank order correlation
analysis.
Oviposition site selection by Or. signifera and Ae.
sierrensis females was further investigated by Chisquare analysis. The frequencies of ovitrap liners with
and without the eggs of each species were analyzed for
independence using
a
2
x
2 contingency table.
The
ovitraps were
vertical ovipositional distributions of each species were
continuously from May 9 to November 2, 1994
analyzed using Kruskal- Wallis ANOVA by ranks
system (
operated
the study
the seasonal study.
stations were compared for each year of the study with
and
same methods
m).
described for
other years of
the
followed by multiple range tests. The numbers of eggs
collected at each
height
were compared
for the
entire
December, 1998
Journal of Vector
Ecology
139
during the entire year( Fig. 2). Throughout the study, the
seasonal ovipositional periods of each species.
five deepest tree holes (
RESULTS
data are
and maximum temperature
in Figure lA ( 1991, 1993,
1995)
and
and
shown
ranged
Figure 2A
Daily precipitation totals from January
1997).
depth = 23. 9 cm) held
tree holes had an exposed, horizontal opening. On July
14, 1993 the pH of the water in these deep tree holes
Weather Data
Rainfall
mean
water longest into the summer months. Each of these
from 6. 19 to 7. 26(
mean± std.
dev.= 6. 84± 0. 42)
and true color measured between 1144 and 2356 Pt- Co
1 to June
units( mean±
std.
dev.=
1732± 454 Pt- Co units).
30 were significantly( P< 0.05) lower in 1991 ( 57. 9 cm)
in 1993 ( 88. 9
than
106. 7
the
dry
for the
30. 1,
and
1997
and
Temporal Distribution of Or. signifera Oviposition
during
Orthopodomyia signifera females oviposited totals
of 4, 247, 6, 083, and 4, 135 eggs into the eight ovitraps
From June 1 to September 30, 1991
during 1991, 1993, and 1995, respectively( Fig. 1B). An
32. 1° C. Means
additional 750 Or. signifera eggs were found in the
1997
ovitraps during 1997( Fig. 2C). A single, peak period of
ovipositional activity occurred during August or
temperatures
in 1993, 1995,
same months
peaked
but
temperatures averaged
maximum
cm),
highest
air
summer months each year,
P< 0. 05) in 1991.
daily
1995 ( 136. 8
cm),
Maximum
cm).
and
were
30. 1,
were
29. 9° C, respectively. The months of August
September lacked rain each year except 1997.
and
September
of
each
year.
Orthopodomyia signifera
oviposition was detected in the woodland from June 19
Surveillance
Natural Tree Holes for Water
of
Maximum
volumes
from0.4to 12. 21iters(
and maximum water
cm( mean± std.
tree
holes
of
shown
holes, but
onset of summer
from
July
and
between 6. 4
cm).
water
kept
30.0
The oviposition periods of Or. signifera coincided
m
with the hottest and driest months during each year
Figs. 1
(
2 indicate the
and
was
rainfall-
levels high in
with
the tree
the
holes
Fig.
1B).
September
TABLE 1.
of
from August 24
Following
to
unusual rains
2).
More than 97%
of the oviposition
maximum
temperatures >
30° C.
The cessation of
all of
oviposition also showed close coincidence with the
annual
onset of cooler weather. The last detectable oviposition
dry
of each year occurred within two weeks of the last trap
were
24 to October 31, 1991, from August 30 to
and
and
coincided with trap periods that completely lacked
rainfall and 77% occurred during periods with mean
m).
water
of
and
0. 07 to 1. 31
depths declined
drought. All
duration in 1993( July 8 to October 5), 1995( July 26 to
October 5), and 1997( July 17 to September 4).
The height of the
from
in Figures 1
rain
November 29, 1993,
11, 1995(
8. 2
to October 9, 1991. Oviposition was more restricted in
ranged
dev.= 4. 5± 4. 3 liters)
in the tree holes
water
dependent. Winter
the tree
were
dev.= 17. 1±
dev.= 0. 34± 0. 38
The data
presence
std.
above ground ranged
std.
mean±
mean±
depths
holes
the ten tree
of
periods
with
mean
maximum
temperatures > 30° C.
December
Regression analysis( TABLE 1) showed mean monthly
in August
maximum temperatures were positively correlated with
1997, three tree holes held
total monthly Or. signifera oviposition during each year
water
Spearman rank correlation coefficients ( R) for the total monthly ovitrap collections of
Orthopodomyia signifera eggs and the mean monthly maximum temperature, total monthly
rainfall, mean number of tree holes holding water, and mean monthly tree hole water depth.
Data for the months from April through November are included for each year.
Spearman R
Eggs
Mean
Monthly
Total
for
Maximum
Monthly
Year
Temperature
Rainfall
Number
of
Tree Holes
Holding
Water
Mean Monthly
Tree Hole
Water Depth
1991
0. 88 **
0. 76 *
0. 90 **
0.90 **
1993
0. 80 *
0. 91 **
0. 76 *
0.74 *
1995
0. 86 **
0. 63
0. 54
0.53
1997
0. 85 **
0. 46
0. 82 *
0. 85 **
P< 0. 05 **
P< 0. 01
140
Journal of Vector
Ecology
December, 1998
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December, 1998
Journal of Vector
Ecology
141
35 -
A
MI Rainfall
36
30 -
Max. Temp.
U
25 -
E
30 °
g.
20 •
cu
24
F"
15 18
10
5-
1
10 -
1.k.*..1*..
k
k. A•*
k..A..,.*..
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12
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frk..,..+..,.A.
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k•+
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ct
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'
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16
kr .'
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6
sz
A
o
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l
t
i
ln
it'
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0—
Mean Water Depth
with
Water
6
c
4
Z
2
0•
0
60
k
C
Ae. sierrensis
-
5
50
0—
Or. signifera
4
i
c,
Q
40 '
a
i
i
0.
on
cn
W
L.
2
i
20 '
ti
4
10 '
1
A
i
i
0
A
h'.+•
Jan
Feb
Mar
Apr
+.+..
A
May
Jun
Jul
Aug
Sep
0
Oct
Nov
Dec
Figure 2. A. Mean maximum temperatures and total precipitation recorded at a United States Weather service at
Potter Valley, CA during weekly sampling periods in 1997.
B. Numbers of natural tree holes holding water and mean tree hole water depth during 1997.
C. Mean
numbers of Or. signifera and Ae. sierrensis eggs per ovitrap day during weekly trap periods from
March 26 to December
1,
1997.
Journal of Vector
142
of
Oviposition
the study.
with
total
monthly
Orthopodomyia
monitored tree
holes
oviposition was
had dried, but
after all of
In 1997
held
depth
of the
the oviposition(
78%)
patterns
Or.
by
each
38, 725(
May
79%
of
eggs
tree
of
holes in the
1993,
in
Ae.
tree holes
During
were
these
years all
partially filled
all of the tree
September 24, totaling 27, 761
reached
August 14.
four
year
study
period,
presence of mosquito eggs.
A total
of
36. 3%
of the
liners was positive for Or. signifera eggs and 87. 5%
were positive for Ae. sierrensis eggs. The frequencies
their mean
4
and
of
1. 83, P> 0. 17)
on
liners
with(
38. 8%)
or
28. 6%) the presence of Ae. sierrensis eggs.
between June 6 and October 4.
All of the eggs were
oviposited between 3. 0 and 10.0 m above ground( Fig.
4),
and most(
74%) were oviposited in the 5. 0 and 7. 5 m
3. 0, 5. 0, 7. 5, and 10. 0 m ovitraps had six, seven, six, and
five Or. signifera positive liners, respectively, over the
course
of
the season.
Although the number of Or.
between
signifera eggs found at middle( 3. 0 and 5. 0 m) and high
Fig.
( 7. 5 and 10.0 m) heights above ground were not
significantly different from each other, all of the
elevated ovitraps collected significantly( P< 0. 05) more
eggs(
laid
Or. signifera
During
without(
x2=
evenly distributed among the higher level ovitraps. The
minimum water
84%
different(
ten of the
occurred
August 7.
Ae.
ovitrap liners. During the four yearly Or. signifera
oviposition periods 394 liners were examined for the
ovitraps. The frequencies of positive liners were more
sierrensis eggs were
Conversely,>
oviposition occurred after
analysis of the distributions of eggs on individual
to
first detected
and
before the tree holes
May
holes had dried. In
oviposition
theAe.
were
with water
April 16
of
0. 18), or 1997( R= 0. 00, P= 1. 00), indicating females of
each species chose oviposition sites independently of
the other species. This conclusion was supported by an
In the 1994 vertical distribution study, Or.signifera
ovitraps ( with
sierrensis
2C). More than 97%
for 1991 ( R= - 0.43, P=
females laid a total of 1, 293 eggs into the ovitraps
parenthesis)
sierrensis oviposition was
before
not a significant correlation
0.29), 1993 ( R= 0. 14, P= 0. 74), 1995 ( R= - 0. 52, P=
females
65, 054 ( April 25
and
species were compared for all of the stations there was
sierrensis
sierrensis
Ae.
on
into the
17 to October 23) in 1991, 25, 358(
occurred
depth
temporal
oviposited
eight
occurrence
of
in
However, when yearly ovipositional totals of the two
of liners with Or. signifera eggs were not significantly
all
1997
occurred
oviposition
Yearly
from the
totals
with
Ae.
and
the study.
November 9) in 1995.
Ae.
holes
differences in the
signifera
October 20) in
monitored
1995,
tree
2). Mean monthly tree hole
holes holding
Fig.
marked
year of
inclusive dates
and
dried completely( Fig. 1).
on August 14, coinciding
fewest number
numbers
ovipositional
when
also
the mean number of tree
were
and
ovitraps
after
negatively correlated
and 1997( TABLE 1).
1991, 1993,
There
the
water(
and
water were
to
30%
the tree holes had
date when
woodland
during
most of
In 1991, 1993,
dry.
were
first detected
oviposition peaked
the
water
was
oviposition
signifera
restricted to periods when most, or all, of the
largely
with
was negatively correlated
in 1991 and 1993.
rainfall
December, 1998
Ecology
the entire
sierrensis oviposition was
21 days
eggs than ovitraps at low ( 0.2 and 1. 0 m) levels above
ground.
In the same study, Ae. sierrensis females
oviposited a total of 21, 538 eggs into the ovitraps
after
between May 24 and October 25. There was relatively
The
little variation in the percentage of Ae. sierrensis
magnitude ofAe. sierrensis oviposition
into the ovitraps
oviposition that occurred at each height. Ovitraps at 0. 2,
10 times that of Or. signifera. No
other species of
1. 0, 3. 0, 7. 5, and 10. 0 m above ground each had between
detected
the
an average of
was>
73 days before
oviposition periods
yearly
mosquito oviposited
into the
of
and
Or.
ovitraps
signifera.
during the
study.
16%
and
20%
percentage(
Spatial Distributions
All
of
the Or.
of
Oviposition
signifera eggs
by Or. signifera
found in
the ovitraps
adhering to the ovitrap liners.
The external membranous sheath( Chapman 1964) was
were above
nearly
eggs.
eggs.
oriented away from the oviposition
Positive liners ( n= 168) held between 1 and
Most( 54%) had totals
signifera
signifera
30
and
150
all
of
the
ovitraps
were
during each year of the
location.
ovipositional
8( 35%
Station 8
total of
utilized
study(
Fig. 3),
The lowest
no significant difference in the number of Ae. sierrensis
eggs oviposited at any height( P> 0. 65).
Larval Surveys of Natural Tree Holes
A total of 28, 129 immature mosquitoes
was
collected from the ten natural tree holes surveyed and
identified to species during the four year seasonal study.
vast
majority
of
the
larvae (> 96%)
were Ae.
sierrensis, the only species identified during surveys
females
conducted from August until April. Eggs of this species
by
any
had the lowest
any location for Ae.
oviposition.
Or.
of the total) than at
also
the total
Kruskal- Wallis ANOVA by ranks indicated there was
The
egg.
more eggs at station
other
of between
More than 27%, however, had< 30 eggs and 4 held
only 1 Or.
While
laid
waterline
always
substrate.
758
the
of
10%) occurred at 5. 0 m above ground. A
sierrensis.
hatched in all of the tree holes as soon as they were
flooded by rain each fall. Nearly all overwintering Ae.
sierrensis immatures had emerged by the end of May
December, 1998
Journal of Vector
Ecology
143
oo
1r;
e•'
C.'
pa
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CT
1
1 ..
C7,
S:
r,-,-
CT
CT
g
B
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Q
4
Ell
I
•::::'.:::::::;:.:
1 - •'..........:. . . ....-.. ....... ..:.. .-.::..... ...:
a'
E.5
o
o
i.
vs
N
1
7)
o
51)
o;
c)
c)
1
1
s,
4:
Ri
0
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N
00
d-
0
N
0
0
0
00
0
VD
0
ytt
0
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4
Journal of Vector
144
December, 1998
Ecology
40
Or. signifera
A Ae. sierrensis
30
0
0
0
-
ao 20
to
0
1. 0
0. 2
5. 0
3. 0
10. 0
7. 5
Height above Ground( m)
Figure 4. Percentages of Or. signifera ( n= 1, 293 eggs) and Ae. sierrensis( n= 21, 538 eggs) oviposition that occurred
at each of six heights above ground during weekly trap periods between May 16 and November 2, 1994.
each
but
year
following
July 18), three
small
rainfall
and
in
tree
occurred
northeast of the study site. This tree hole had a large
tree holes in 1991 ( June 30-
volume( 9. 3 liters) and maximum depth( 52. 1 cm) with
20- August 9),
a restricted, vertical opening. Intermittent observations
summer
seven
holes in 1995 (
17- September 4).
were
July
August
indicated rain filled the tree hole each winter, but the
Culiseta incidens Thomson
small opening reduced evaporation, which prevented
five tree holes in 1997(
immatures
generations
May
28- July 4
found in five tree holes in 1993 (
19- July 14), four tree holes in 1995(
five tree holes in 1997 ( April 23
stigmatosoma
and
Dyar immatures
holes in both 1993(
July
1-
July
May 23- July 5), and
to July 16).
Culex
21)
and
Or. signifera. However, Or.
were never
found in any
of the tree
1997( June 12-
identified from the tree hole during each year of the
signifera
holes
larvae
during
the
study.
Response of Or. signifera Adults to Carbon Dioxide-
Baited Fay Traps and CDC Light Traps
Carbon dioxide- baited Fay traps have been
determined to be an accurate method for surveillance of
study.
The
closest natural
tree
signifera
immatures
located 1. 58
on a
pH= 9. 03, true color= 288, 000 Pt- Co units on July 24,
1997). Larvae of Or. signifera and Ae. sierrensis were
species of
July
mosquitoes utilized the tree holes as breeding sites
during summer months that overlapped the oviposition
periods of
tree hole held opaque, black water at each observation
(
in four tree
occurred
These data indicate that three
31).
the habitat from drying completely during summer. The
May
Pacific
was
madrone
hole known to hold Or.
in the foothill
m above ground
woodland ca.
90
m
adult Ae. sierrensis( Garcia et al. 1989, Washburn et al.
1992).
Although they collected large numbers of Ae.
sierrensis,
they
were
not attractive
to
Or.
signifera
December, 1998
during
adults
Journal of Vector
three
years
TABLE 2).
of operation (
Standard CDC light traps failed to detect the presence
Or.
adult
during
signifera or
Ae.
in the
sierrensis
Ecology
145
possibility that females may have used photoperiod as a
of
cue affecting the cessation of oviposition cannot be
woodland
ruled out since eggs were last found during early
1997.
October in four of the five studied years. It is less likely
that minimal rainfall or the absence of rain directly
DISCUSSION
affected ovipositional
periodicity.
Although signifi-
cantly lower rainfall totals preceded the early
Unlike Ae.
a
for
signifera
holes temporarily filled
that
with
Or. signifera lack
oviposition period in 1991, the ends of the oviposition
dry- summer
periods did not coincide with the onset of rain in 1995 or
through
survival
Female Or.
conditions.
the eggs of
sierrensis,
state
resting
that oviposit
their
progeny will
development before the habitat dries
chances
shown
1968)
of
in Figures 1
largely
not
complete
The data
out.
Zavortink'
support
s
that in
California the activity periods
females occur after most rainfilled tree
conclusion
Or. signifera
2
and
into tree
adaptive
For
example,
that Or.
Leon
in
Florida,
the tree
of
majority
through the
after
detected
1953)
and
in
until
the observed
larvae do
a
holes
Baerg(
1968)
concluded
reached.
factors reported in TABLE 1,
was also
the
most
six
likely
to
each
year
of
the study;
and
occurred ca. one month earlier
TABLE 2.
that
Or.
than
signifera
high
until
still
held
water.
Despite this
the
outdoor
physical
and tree holes were used by female mosquitoes selecting
oviposition
sites.
Bradshaw and Holzapfel ( 1988)
previously reported that water from permanent rainfilled tree holes at their Florida study site tended to have
higher optical density, pH, conductivity, and tanninlignin concentration than water from intermittently
flooded tree holes.
They demonstrated that Or.
signifera females avoided oviposition into jars holding
water from tree holes fated to dry out, but they did lay
temperature
eggs in jars with water from permanent tree holes. In the
a cause- and- effect
during
present study, Or. signifera females exhibited similar
behavior. They consistently avoided ovipositing into
temporary tree holes which had exposed, horizontal
significantly
openings and held light-colored water with acidic to
oviposition period
slightly basic pH. However they readily laid eggs in the
in
ovitraps
oviposition.
oviposition
following
higher temperatures in 1991, the
weeks
in Florida. Grant
maximum air
relationship with the periodicity of
factor was positively correlated with
oviposition
to twelve
Among
have had
water
dry- summer
California
July,
in California
have been
hold
to
Under the
onset of ovipostion
not pupate
temperatures
where rain caused the
examined
or
holes
These data suggest that cues associated with the ovitraps
northern
June
tree
began in late April in
location
summer months.
conditions present
was not
and
oviposition
signifera
County,
North America.
some other areas of
Bradshaw
occurred during periods when one to seven of the
monitored
Holzapfel ( 1984) found
trait for Or. signifera females in California that
not occur
Although most Or. signifera oviposition occurred
after all of the monitored tree holes had dried > 20%
overlap Or. signifera females avoided ovipositing into
the temporary tree holes; conversely, Culiseta incidens
and Culex stigmatosoma females utilized many of the
temporary tree holes for oviposition. These generalists
are attracted to a wide variety of habitats for breeding
( Colwell et al. 1995), but both species completely
avoided use of the ovitraps for deposition of egg rafts.
holes have dried, thereby excluding these temporary
habitats as oviposition sites. This periodicity is an
does
1997.
may increase the
water
other years.
This
The
apparently after receiving cues
which
Seasonal totals of tree hole mosquitoes captured in two carbon dioxide- baited Fay traps and three CDC
light traps operated at the study site.
Total Number of Adults Captured
Trap
Or.
Trap
Period
Days
4/ 03-
11/ 18
64
0
CO2- baited Fay
3/ 25-
11/ 16
68
CO2- baited
4/ 04-
10/ 21
68
6/ 26-
10/ 08
45
Type
1991
CO2- baited Fay
1995
1997
1997
CDC light trap
Year
Total
Trap
Fay
9
signifera
d'
Ae. sierrensis
9
d'
0
1, 543
2, 081
0
0
4, 554
5, 695
0
0
2, 132
5, 242
0
0
0
0
146
Journal of Vector
by permanent rainfilled
mimicked those presented
Ecology
December, 1998
tree
sierrensis larval development does not begin until fall
Each ovitrap provided a darkened, sheltered
cavity and held dark- colored, basic water. A similar
association of Or. signifera with tree holes
holding
rains inundate resting eggs. Data from the present study
holes.
highly
basic
has been
indicate Or. signifera larvae had a one to four month
window for development each year during periods
many times( e. g.,
Bohart 1950, Chapman 1964, Mitchell and Rockett
water
1981, Woodward
In
oviposited
later in the
located
Ae.
both into tree holes that dried completely
summer and into the permanent tree hole
near the
the end
after
study
Most
site.
eggs at
by
filled
2).
During
females may prefer to oviposit at locations above
ground. However since Ae. sierrensis females showed
Since
holes
tree
levels declined
this
they
sites and
locations above the
rains.
into
the oviposition
water
Fig.
triseriatus showed preference for oviposition near
ground level, while Ae. hendersoni preferred to oviposit
at above- ground locations in the same eastern
woodlands.
Figure 4 indicates that Or. signifera
access to the maximum number of
developmental
future
of
hole
the rainy season (
of
females have
available
Several researchers ( Scholl and DeFoliart 1977,
Sinsko and Grimstad 1977) have reported that Ae.
females readily
sierrensis
the ovitraps occurred as tree
period
minimum.
1988).
et al.
contrast,
when Ae. sierrensis larval populations were at their
reported
waterline
their
no ovipositional preference relative to height above
be flooded
ground, Or. signifera females would not avoid
can place
that
will
levels fluctuate in
water
regardless
of
interspecific competition by depositing their eggs at
rain-
they dry
whether
specific
during summer, Ae. sierrensis eggs hatch
and
develop in both temporary and permanent habitats
Zavortink 1985). During the late summer overlap of
the
oviposition
signifera,
periods
most
the
of
Ae.
of
eggs
sierrensis
both
of
and
species
of the aquatic
North America, the
Bradshaw
predation (
interspecific
are
habitat( Bradshaw
oviposition sites might also prefer locations high in
Or.
trees. It is possible that most permanent rainfilled tree
holes in the region occur well above ground. Copeland
were
that Ae.
Copeland
Craig (
oviposition
based
with
on
et
1988)
al.
for limited food
hole
tree
Ae.
tree
In summary, this analysis indicates the oviposition
used
periods of both northern California tree hole mosquitoes
quality
are adapted to weather patterns associated with the
Say).
Mediterranean climate. Peak oviposition periods of Or.
predatory trophic
but intraspecific
signifera occurred during August and September,
water
triseriatus (
a
is
Colwell
susceptible habitats. Most Ae. sierrensis oviposition
force regulating larval populations of Ae.
( Hawley 1985, Washburn et al. 1991,
occurred from late spring to mid- summer when the
et
al.
limitations in
1995).
Despite the known
regional tree
holes, data from
were available as oviposition sites. The segregation of
the present
the species into temporary ( Ae. sierrensis only) and
ovipositing
locations that
Instead, the
in
the species.
the
modulation of
between tree hole
cues used by Or. signifera females for oviposition site
of each species,
selection. The study did not produce any evidence that
interspecific competition affected the spatial
larval
distribution of oviposition by Or. signifera females, but
interspecific larval competition may be modulated
reduce
competition
between
shown that temporal
interspecific
mosquitoes.
compe-
Orthopodomyia
oviposited
during
summer
within
a
eggs
periodicity of the oviposition of each species, as well as
sierrensis.
begin larval development
signifera
permanent ( both species) habitats results from the
characteristics of the eggs,
Barrera ( 1996) has
can
to Ae.
periodicity
hatching
habitat partitioning
tition
unattractive
ovipositional
coupled with
result
were
largest numbers of tree holes with declining water levels
resource
study did not indicate that Or. signifera females avoided
interspecific competition by
their eggs at
may
temporally excluding most of the eggs from drought-
considered to
resources
a major
sierrensis
(
1992)
hendersoni ( Cockerell)
competition
level ( Woodward
Craig
individual
and
Northern California tree holes lack
be
and
that occur within
concluded
competition
to the bases of tree branches. In a northern California
and
survey, all of the rainfilled tree holes positive for Or.
complexes
avoid
1984)
signifera occurred at above ground locations
Woodward et al. 1988).
example,
to
Holzapfel
Copeland
be important factors affecting the
holes. For
differential
permanence
1990)
competition (
considered to
culicid
and
and Craig ( 1992) positively associated Or. signifera
populations with " elevated
deep rotholes" at their
Indiana study site and concluded these types of tree
holes only occurred above ground at the sites of wounds
Holzapfel 1988),
and
Although host- seeking female Or.
the ground, it is less apparent why females seeking
probably laid into tree holes that held water all year.
In the more diverse tree hole mosquito
communities of eastern
heights.
signifera might find greater numbers of birds well above
completely
hatch
and
few days; Aedes
temporally.
The data also indicate that California
populations of Or. signifera possess a number of traits
that may cause their distributions and densities to be
underestimated.
Adults
are nocturnal and not
known
to
December, 1998
Journal of Vector
bite humans ( Zavortink 1968).
Although
Ecology
147
are
Culicidae) and Chaoborids in relations to weather
occasionally collected in light traps ( Grant 1953:
Zavortink 1968), in the present study they failed to
respond to traps using either light or CO2 as attractants.
Bradshaw, W. E. and C. M. Holzapfel. 1988. Drought
larval habitat in
The
nature of
tree
holes with restricted openings)
the
occur at above ground, cryptic
larval
oak woodlands(
deep
often causes them to
locations. For
four isolated
surveys of
adults
example,
oak woodlands
in Lake
County, California failed to locate any tree holes
holding Or. signifera larvae. The ovitraps described in
this
all
paper
later detected the
four locations ( D.L. W.
Similar
in
in
woodlands
J. O. Washburn,
indicate Or.
and
southern
widely
signifera
associated
also
Ae.
Tree hole
sierrensis.
Mendocino
in Fresno
County
County,
reports
may be more
is widely believed. The
permanent
have implications for the
do
surveys which
rain-
control of
not
locate
may be missing large numbers of Ae.
that co- habit permanent tree holes with Or.
signifera
sierrensis
1954. Studies of the North American
arthropod- borne encephalides.
VI. Quantitative
determination of virus- vector relationships. Amer.
J. Hyg. 63: 278- 85.
ecology of Orthopodomyia californica Bohart.
Or.
of
presence
locating
with
filled tree holes
D. Sudia.
Chapman, H. C. 1964. Observations on the biology and
populations
than
communities. Oecologia 74: 507- 514.
Chamberlain, R. W., R. K. Sikes, D. B. Nelson, and W.
unpublished).
in California, these
used
and the organization of treehole mosquito
at
Tucker 1978). Since" tree hole"
widespread and abundant
difficulties
Or. signifera
A.E.C.,
pers. comm.) and
California( Lewis
ovitraps are not
and
have detected the
ovitraps
signifera
Or.
presence of
and predation. J. Med. Entomol. 21: 366- 378.
Mosq. News 24: 432- 439.
Colwell, A. E., D. L. Woodward, and N. L. Anderson.
1995. Environmental factors affecting the western
treehole mosquito ( Aedes sierrensis).
Northwest
Sci. 69: 151- 162.
Copeland, R. S.
G. B.
Craig, Jr. 1990. Habitat
segregation among treehole mosquitoes ( Diptera:
and
Culicidae) in the Great Lakes region of the United
States. Ann. Entomol. Soc. Am. 83: 1063- 1073.
Copeland, R. S. and G. B. Craig, Jr. 1992. Differential
oviposition by Aedes hendersoni and Aedes
triseriatus ( Diptera: Culicidae) in response to
chemical cues associated with treehole water.
signifera.
J.
Med. Entomol. 29: 33- 36.
Darsie, R. F., Jr. and R. A. Ward. 1981. Identification
Acknowledgments
and geographical distribution of the mosquitoes of
We thank Dr. Jan Washburn
Colwell for reviewing
earlier
The technical
manuscript.
Haberthur
an
Dr. Robert
version
assistance
Sanderson
Terry
and
and
is
this
of
of
Joann
gratefully
North America, north of Mexico. Am. Mosq. Cont.
Assoc., Fresno, CA., 313 pp.
Fay, R. W. and W. H. Prince. 1970. A modified visual
trap for Aedes aegypti. Mosq. News 30: 20- 23.
Garcia, R., A. E. Colwell, W. G. Voigt, and D. L.
acknowledged.
Woodward. 1989. Fay- Prince trap baited with CO2
for monitoring adult abundance ofA edes sierrensis
REFERENCES CITED
Diptera: Culicidae). J. Med. Entomol. 26: 327-
Baerg,
of
D. C. 1965. A study
the treehole
Thesis,
Baerg,
D.
of
1968.
Orthopodomyia
Culicidae).
Mosq.
community
biology
of
and
ecology
California.
MS
California, Davis, 66 pp.
Overwintering habits
californica
of
Bohart ( Diptera:
California.
pp.
Bradshaw, W. E.
Proc. Calif.
Mosq. Cont. Assoc. 22: 73.
Greenberg, A. E.( ed.). 1985. Standard Methods for the
Public Health Assoc., Washington, D.C., 1268 pp.
new species of
Orthopodomyia
Ann.
Entomology Laboratory, Vero Beach, FL., 579 pp.
R. K. Washino. 1978. Mosquitoes
University
Bohart.
Hawley, W. A. 1985. Population dynamics of Aedes
sierrensis. Pp. 167- 184 in Ecology of Mosquitoes:
Proceedings of a Workshop( L. P. Lounibos, J. R.
Rey and J. H. Frank, eds.) Florida Medical
Entomol. Soc. Am. 43: 399- 404.
and
Notes on the occurrence of
californica
insects in tree holes. J.
from California ( Diptera: Culicidae).
Bohart, R. M.
1953.
Examination of Water and Wastewater. American
Vector Ecol. 21: 66- 80.
Bohart, R. M. 1950. A
Grant, C. D.
structure
concurrence and
of aquatic
the
331.
Orthopodomyia
News 28: 234- 235.
Barrera, R. 1996. Species
of a
the
mosquitoes
University
C.
of
of California,
Berkeley,
of
153
Lewis, L. F. and T. W. Tucker. 1978. Fabrication of
artificial tree holes and their performance in field
tests with Aedes sierrensis and Orthopodomyia
and
development
of
C. M. Holzapfel. 1984. Seasonal
treehole
mosquitoes (
Diptera:
signifera. Mosq. News 38: 132- 135.
Mitchell, L.
and
C. L. Rockett. 1981. An investigation
148
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larval habitat
the
on
breeding
five
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species of treehole
mosquitoes ( Diptera:
Press,
1965.
tree holes with particular reference to parasite
loads. J. Med. Entomol. 26: 173- 182.
A California Flora. Univ. Calif.
Washburn, J. O. and E. U. Hartmann. 1992. Could
1, 681 pp.
Berkeley,
Papineau, E. A. 1984.
Aedes
Controlling Aedes
sierrensis
in
County, a year- round program. Proc.
Calif. Mosq. Contr. Assoc. 52: 63- 64.
Peyton, E. L. 1956.
Biology of the Pacific Coast
treet. ole mosquito A edes varipalpus( Coq.). Mosq.
1991.
Regulatory role of parasites: Impact on
host population shifts with resource availability.
Science 253: 185- 188.
and
324- 325 in
Epidemiology
and
J. R. Anderson.
Control
1943- 1987
by
W. C. Reeves, Calif.
Mosq.
Weinmann, C. J.
150
Journal of Vector
host- seeking
predominant
thorax
1996)
Dyar,
flooding
after
significant
role
mosquito,
during
collected
the
Culex
the first two
wetland
in St. Louis
erythro-
1995-
years(
is thought
not to
encephalitis (
play
SLE)
transmission in southern California( Reisen et al. 1992b).
Large
populations
Coquillett
were
host- seeking Culex
of
collected
the wetland
tarsalis
during
1992.
Correlations of Aedes
sierrensis captures at human sentinels with CO2baited Fay- Prince and duplex cone traps.
J.
Amer. Mosq. Contr. Assoc. 8: 389- 393.
Vect.
Contr. Assoc., Sacramento, 508 pp.
a
Washburn, J. 0.,D. L. Woodward, A. E. Colwell, and
Mosquito- borne Arboviruses in California,
of
J. Med.
Washburn, J. 0., D. R. Mercer, and J. R. Anderson.
of Culex tarsalis and other potential vector
Pp
Culicidae) become
in California tree holes?
Entomol. 29: 995- 1005.
News 16: 220- 224.
Reisen, W. K. and W. C. Reeves. 1990. Bionomics
species.
albopictus ( Diptera:
established
Jackson
ecology
December, 1998
sierrensis( Diptera: Culicidae) from California
Culicidae). Great
Lakes Entomologist 14: 123- 129.
Munz, P. A.
Ecology
and
R. Garcia.
Ecology
1974.
Canine
December, 1998
californicus ( Meyer) Sojak and S. acutus ( Muhl. ex.
Bigel.) Love and Love. In 1997, the wetland was in the
third year of operation and the surface area was
approximately 70%
al.
1998).
covered by vegetation ( Thullen et
The inlet and outlet marshes contained
bands of vegetation separated by open water. The
the
water depth within the inlet and outlet marshes was
approximately 1, 000 females per trap night in
1996: WEW, unpublished data). Culex tarsalis is the
maintained between approximately 0.5 and 0. 6 m during
at
summer(
vector of
primary
myelitis(
central
SLE
and western equine encephalo-
WEE) to humans in
California( Reeves
rural areas of southern and
and
Hammon 1962, Reeves
the summer. Water temperatures at the middle of the
water column of the open water regions in the inlet and
outlet marshes ranged between 21. 5° C and 26.5° C
during the summer.
1990).
We
report
here
on the
efficacy
of control efforts
against mosquitoes associated with a 10 ha multipurpose,
constructed
surveys,
wetland
trapping
in
southern
California.
Larval
of emerging adults, and collections of
host- seeking females
by
carbon
dioxide- baited
traps
were used to evaluate the effectiveness of larvicides and
adulticides applied to the entire wetland.
Treatment of the wetland was carried out using a
helicopter by a local pest control company under contract
with the water district. Pelletized Bacillus thuringiensis
var. israelensis ( Bti, Bactimos® pellets) or granular
Bacillus sphaericus( Vectolex®CG) formulations were
applied using a hopper- spreader apparatus suspended
below the helicopter. The two Bti treatments were made
SITE DESCRIPTION
in
The 10 ha demonstration
wetland
is located
at
Eastern Municipal Water District'
s Hemet- San Jacinto
Regional Wastewater Reclamation Facility( HSJRWRF)
in San Jacinto, California. The demonstration
consisted of five treatment wetlands(
wetland
conjunction
pyrethrins,
quarts/
30
with an
adulticide (
Pyrenone®; 6. 0%
60% piperonyl butoxide; application rate: 3
acres=
0.014 kg AI/ha) during mid-August.
The adulticide was applied by cold aspiration through
an hydraulic spray rig suspended below the helicopter.
inlet marshes 1- 5),
These treatments were followed by approximately
a central pond, and two outlet wetlands( outlet marshes
biweekly applications ofB. sphaericus until mid-October
A and B)( USBR, NB S,
1997). The
gallons
species
EMWD 1994, Walton
approximately one million
of
from the HSJRWRF.
effluent
secondary
wetland was planted
of
et al.
wetland received
daily
The
and
in
autumn
1994
bulrush, Schoenoplectus (=
with
two
Scirpus}
and a final application in early November( TABLE 1).
Treatment rate for the bacterial larvicides was either 19
kg/ha or 23. 6 kg/ ha. Because of restricted after- hours
access to the site and safety concerns, treatments were
carried out
during daylight hours between approximately
Journal of Vector
Ecology
23( 2): 149- 160
Effectiveness of Control Measures against Mosquitoes at a
Constructed Wetland in Southern California
William E. Walton, Parker D. Workman, Louie A. Randall,
Joshua A. Jiannino, and Yvonne A. Offill
Department of Entomology, University of California, Riverside CA 92521 USA
Received 20 April 1998; Accepted 18 May 1998
ABSTRACT: The effectiveness of larvicide and adulticide treatments against mosquitoes at a constructed
wetland in San Jacinto, California was assessed with larval surveys, trapping of emerging adults, and
collections of host- seeking females by carbon dioxide-baited traps. Bacillus thuringiensis var. israelensis
Bti, Bactimos®pellets) applied at a rate of 19 kg/ ha did not demonstrably affect Culex larval and emergent
adult populations. Larval populations in the seven marshes of the wetland decreased from approximately
one third- fourth instar larva/ dip to undetectable levels following two applications of Bacillus sphaericus
Vectolex®CG) at a rate of either 19 or 23. 6 kg/ ha. The largest decline in the number of adult mosquitoes
emerging per day from vegetated regions of the wetland occurred after B. sphaericus treatments. The Culex
erythrothorax host-seeking population declined about 80- fold during September beginning three weeks
after the first treatment with B. sphaericus; however, the Culex tarsalis host-seeking population did not
decline abruptly until mid-October 1997. This result suggests that immigration of females from other
developmental sites might be an important factor influencing the Cx. tarsalis host- seeking population at
the wetlands. Safety concerns required that insecticide applications were carried out during daylight hours,
and two daytime applications ofadulticide( Pyrenone®) in early August were ineffective against mosquitoes
resting in the thick vegetation.
Keyword Index: Culex, constructed wetlands, Bacillus, bacterial larvicides, bulrush.
INTRODUCTION
Rapid growth of vegetation in constructed wetlands
quickly creates ideal conditions for mosquito
The incorporation
facilities,
such
as
of alternative water reclamation
constructed
resource management programs
control efforts.
In
order to
fulfill
into
wetlands,
is likely to
the
such
as
southern
development( Walton et al. 1996, Walton and Workman
1998).
Mosquito production enhanced by constructed
affect vector
wetlands is likely to require increased mosquito
increasing demand
abatement efforts, particularly where human populations
for water and wastewater treatment in densely
arid regions,
water
California,
populated
alternative
continue to encroach on wetlands situated in previously
agriculture- dominated regions.
be needed. California
In 1997, encephalitis virus activity in western
has approximately 200 water reclamation facilities that
recycle about 450, 000 acre- feet annually (
McCarthy
Riverside and San Bernardino counties and large host-
1997).
facilities is
constructed wetland in San Jacinto, California prompted
approximately double by 2000( McCarthy
increase is projected to occur in
the undertaking of control efforts against Culex
populations. Seroconversion to SLE in sentinel chicken
water management strategies will
The
projected to
1997);
most of the
southern
are
number of water reclamation
California. Multipurpose,
expected
reclamation
to
play
programs.
an
constructed wetlands
important
In
addition
in early summer 1997 indicated a comparatively early
processing
onset of annual virus activity in the region( cf. Emmons
et al. 1990, Kramer et al. 1996). Marked increases in the
secondary- or tertiary- treated effluent, these wetlands
provide habitat for wildlife and a site for public education
on
issues
related
to
water and
flocks in western San Bernardino and Riverside counties
water
role
to
in
seeking adult mosquito populations at a multipurpose,
wildlife conservation.
host- seeking and larval mosquito populations were also
observed at the wetland
during 1997. Although the
Journal of Vector
150
host- seeking
predominant
Dyar, collected
thorax
1996)
a
flooding
after
Large
first two
the
is thought
wetland
erythro-
californicus ( Meyer) Sojak and S. acutus ( Muhl. ex.
1995-
Bigel.) Love and Love. In 1997, the wetland was in the
years(
not to
play
SLE)
third year of operation
and the
surface area
was
in southern California( Reisen et al. 1992b).
approximately 70% covered by vegetation( Thullen et
al.
1998).
The inlet and outlet marshes contained
host- seeking Culex tarsalis
bands of vegetation separated by open water. The
significant
transmission
during
the
Culex
mosquito,
December, 1998
Ecology
in St. Louis
role
of
populations
encephalitis (
the
water depth within the inlet and outlet marshes was
approximately 1, 000 females per trap night in
1996: WEW, unpublished data). Culex tarsalis is the
maintained between approximately 0. 5 and 0.6 m during
Coquillett
were
at
collected
the
wetland
during
summer(
primary
vector of
myelitis(
central
SLE
and western equine encephalo-
WEE) to humans in
California( Reeves
rural areas of southern and
and
Hammon 1962, Reeves
the summer. Water temperatures at the middle of the
water column of the open water regions in the inlet and
outlet marshes ranged between 21. 5° C and 26. 5° C
during the summer.
1990).
We
report
here
the
on
efficacy
of control efforts
against mosquitoes associated with a 10 ha multipurpose,
Larval
Treatment of the wetland was carried out using a
trapping
emerging
host- seeking females by carbon dioxide- baited traps
helicopter by alocal pest control company under contract
constructed
wetland
in
southern
of
surveys,
were used
California.
adults, and collections of
with the water district. Pelletized Bacillus thuringiensis
to evaluate the effectiveness of larvicides and
adulticides applied
to the
var. israelensis ( Bti, Bactimos® pellets) or granular
Bacillus sphaericus( Vectolex®CG) formulations were
entire wetland.
applied using a hopper- spreader apparatus suspended
below the helicopter. The two Bti treatments were made
SITE DESCRIPTION
in
The
10 ha demonstration
wetland
Eastern Municipal Water District'
s
is located
at
Hemet- San Jacinto
consisted of five
treatment wetlands(
and
B)( USBR, NB S,
and
wetland
inlet marshes 1- 5),
EMWD 1994, Walton
et al.
species
wetland was planted
of
in
autumn
1994
bulrush, Schoenoplectus {=
TABLE 1.
with
Pyrenone®; 6. 0%
Treatment rate for the bacterial larvicides was either 19
1997). The wetland received approximately one million
gallons daily of secondary effluent from the HSJRWRF.
The
an adulticide (
30 acres= 0.014 kg AI/ha) during mid- August.
The adulticide was applied by cold aspiration through
an hydraulic spray rig suspended below the helicopter.
These treatments were followed by approximately
biweekly applications ofB. sphaericus until mid-October
and a final application in early November( TABLE 1).
a central pond, and two outlet wetlands( outlet marshes
A
with
60% piperonyl butoxide; application rate: 3
quarts/
Regional Wastewater Reclamation Facility( HSJRWRF)
in San Jacinto, California. The demonstration
conjunction
pyrethrins,
kg/ ha or 23. 6 kg/ ha. Because of restricted after- hours
two
access to the site and safety concerns, treatments were
Scirpus}
carried out during daylight hours between approximately
Larvicide and adulticide applications to the 10 ha demonstration wetlands
at the Hemet- San Jacinto Regional Water Reclamation Facility during
1997.
Date
Larvicide
(
Quantity
kg/ ha)
Adulticide
(
Quantity
kg AI/ha)
August 13
Bactimos
19. 0
Pyrenone
0.014
August 20
Bactimos
19. 0
Pyrenone
0.014
August 29
Vectolex CG
19. 0
September 12
Vectolex CG
23. 6
September 26
Vectolex CG
19. 0
October 10
Vectolex CG
23. 6
November 3
Vectolex CG
19.0
Journal of Vector
December, 1998
10: 00 A.M.
In
demonstrable
a
for emerging
CO2-
baited traps
each marsh of
in
transects were positioned
the
At
each station,
in
95%
determined
dissecting
25X
at
Bohart
and
The
0. 25
m2
were divided into two subpopulations: early instar larvae
were
The abundance of larvae and emergent adults was
opening:
Develop-
under- represented in larval surveys ( Walton and
mosquitoes
Workman 1998) and, therefore, the efficacy of control
were
and
measures against this species was assessed using adults
using a
IV larvae
collected in emergence traps. Because the entire marsh
was treated, both untreated areas and replicated
and
experimental units were lacking.
wetlands
Chemical and physical parameters related to water
using eighty- four,
traps. Collapsible emergence traps
quality were routinely measured at the wetland.
number of adults
emergence
N+1)- ln( N)/t, where N is
and t is the interval between samples in days. Larvae
Washino( 1978).
determined from
was
ln(
along
using Loomis ( 1959)
species
The rate of
number of emergent adults/ m2/ week, x is sample date,
magnification
Stage III
microscope.
identified to
were
50X
to
cycle.
( stages I and II) and late instar larvae( stages III and IV).
dips
ethanol.
immature
in the life
averaged for each of the seven marshes in the wetland by
sample date. The predominant host- seeking mosquito,
Cx. erythrothorax, found at the wetlands was typically
ml
mesh
cup(
stage
inlet
the
Samples
400
particular
the average number of larvae per dip or the average
along
The three
either
a
adults was calculated as:
by dipping
wetland.
three
mental stage and abundance of
stereo
of
population change( per day) for larvae and for emergent
host- seeking
spaced stations
a concentrator
m), and preserved with
were
mosquito
each marsh at successive
five equally
at
taken, combined
200µ
the
southernmost outlet weirs.
biweekly
transect.
each
on
were examined.
interfaces from
open water- vegetation
taken
effect
mosquitoes were sampled
three transects in
weir or
and adulticide
adult mosquitoes, and
in
adult abundance
Immature
larvicide
assess whether
larval abundance in dip samples, population
populations,
trends
to
had
treatments
populations was indicated by a marked reduction in the
abundance, or by an abrupt change in population growth,
and noon.
order
151
Ecology
emerging from the
collections
the results presented here: residual chlorine concentration
frame. The gently sloping sides of
the trap concentrated emerging insects into a widemouth( 16 oz.) Mason jar fitted with a removable plastic
and bacterial density. Residual chlorine in the water
entering the wetland was measured using the iodometric
method( APHA 1995). Coliform bacteria density in the
influent water was measured using the fermentation
to a hinged
wooden
Quadrats (
funnel.
shoots/
by
above
mean
number
SD: 265± 98)
m2±
seven marshes of
just
affixing fiberglass
However, two parameters are particularly relevant to
window screen
were constructed
the
Schoenoplectus
of
wetland and
the water surface on
bulrush
the
tube test and calculated as the Most Probable Number
the
were established within
was cut
June 26- 27. Emergence
(
MPN) using the number of positive reactions in the
dilution series( APHA 1995).
traps were placed into the wetlands on July 1. Collections
were made
weekly from
8 through September 26,
July
RESULTS
except for the period between July 15 and July 22 when
no
jars
At
the traps.
were placed on
collection,
the
jar
Larval Populations
the funnel in
Larval samples contained predominantly Cx.
were returned
tarsalis. Late instar larvae of Cx. quinquefasciatus Say,
laboratory where the collections were killed by
freezing and then enumerated under a stereo dissecting
Cx. stigmatosoma Dyar, and Cx. erythrothorax were
was removed
from the
apex of
the
each jar was plugged with cotton.
trap
and
The jars
to the
Because Schoenoplectus
microscope.
and could upset emergence
shoots was carried out
were
mosquitoes at
collected
dioxide- baited traps. Mosquitoes
May
February through
December
1997.
approximately
identified to
April
using
and
Traps
15: 00
species (
of emergent
until
a stereo
San Jacinto
using three
carbon
and
weekly
biweekly from
from November through
were
run
08: 00.
Bohart
the
were collected
2 through October 30
from
counted
clipping
rapidly
twice each week.
Host- seeking female
wetlands complex
traps,
grows
and
dissecting
overnight
from
Mosquitoes
were
Washino 1978)
and
microscope at
12X
magnification.
Effectiveness
of
the treatments against mosquito
less frequent in dip samples. The abundance of third and
fourth instar larvae in dip samples increased from fewer
than one larva per dip in April to an average of four
larvae per dip in May ( Fig. 1). The abundance of late
instar larvae in the inlet marshes decreased in mid- June;
in four of the five inlet marshes, larval abundance
declined
by
nearly two
orders of magnitude.
Larval
abundance in four of the inlet marshes on the next
sampling date increased to levels observed in late May.
For most dates during the summer, the average number
of late instar larvae per marsh ranged between one and
larvae
dip. In contrast to older larvae, the
abundance of young larvae was comparatively stable
from April through July, averaging between three and
seven larvae per
dip( Fig. 1).
eight
per
152
Journal of Vector
Early instar
100 .
larvae ( L1 &
0— Late instar larvae ( L3 &
Ecology
December, 1998
4/ 4 4
L2)
j
r/
L4)
Q.
10 -
ii•
Z
co<
0. 1
w
0.01
I
I
31- Mar
I
I
30- Apr
I
I
30- May
I
I
29- Jun
I
I
29- Jul
I
•
28- Aug
27- Sep
Date
11— Early instar larvae ( L1 &
0.2 —
L2)
0 —Late instar larvae ( L3 &
L4)
0. 15 —
0. 1 —
d
A
31.- ---
c
mi\
0. 05 —
I
cd
U
0
ao
v
s - 0. 05 —
0
n.
c
-0. 1 —
0
0. 15 —
111.
0.2 —
0.25 —
0. 3
I
I
31- Mar
30- Apr
I
I
I
30- May
I
I
29- Jun
I
I
29- Jul
I
28- Aug
1
27- Sep
Date
Figure 1.
Population trends of Culex larval subpopulations in dip samples from the HSJRWRF demonstration
wetland
during
1997.
Upper
panel: abundance of
larvae ( mean
±
SE) in dip samples. The arrows
indicate treatments of bacterial insecticides. Open arrows are Bacillus thuringiensis var. israelensis
treatments. Closed arrows are Bacillus sphaericus treatments. Lower panel: the rate of population
change( mean±
SE) for two larval
mosquito subpopulations.
Journal of Vector
December, 1998
The
abundance
declined
during
collected
from
both larval
of
August
in late September(
all marshes
mid- August, larval
subpopulations
late instar larvae
and
abundance
declined
ten- fold
only 0. 3 larva
marshes
were lower
counts in the inlet
and averaged
the end
marshes at
abundance of
of
August.
levels in inlet marshes 2
in the last
By
late instar larvae
1).
Fig.
by
dip.
than
more
Larval
per
than
In
in the
outlet
mid- September,
was
of
population change(<-
from late September.
On
from
were absent
all
comparatively large negative
0. 1/ d) were observed in the late
August(- 0. 176/ d),
of
These large
1997.
during
rate
growth
population occurred
the
late instar larval
during early June(- 0. 122/ d), early
during September(<- 0. 118/ d:
and
The early instar larval subpopulation exhibited
comparatively large declines in growth rate in early
Fig. 2).
August(- 0. 229/ d)
0. 211/ d).
older
during
and
However, the
larvae
observed
September(- 0.084/ d, growth rate
negative
in early June
the
of
was not observed
for
young larval subpopulation; the subpopulation of
1st and 2nd larval instars declined at 3%/ d during the
the
in early June when the
instar larvae declined appreciably.
two week period
older
abundance of
meter
of vegetated
Culex spp. emerging per square
surface declined gradually in all
8
August 15 (
2).
On
marshes
between
average,
the number of mosquitoes emerging per unit
in early
July
The
marshes
July
and
Fig.
week
from approximately 1, 500 adults/
to 300 adults/
week in early August.
m2/
m2/
Culex emerging from the inlet
declined during the third week of August( Fig.
number
of
2). Mosquito production from the inlet marshes declined
from 250 to 300
during
the third
from inlet
m2/
adults/
week of
marshes
week
outlet
mosquitoes/
The
m2/
1, 2, 4,
and
most
emergence
during
marked
occurred
mosquito production
80
adults/
5 increased
m2/
week
emergence
during
early
marshes, adult production
was
marshes
week
to
August. Mosquito
September. Unlike the inlet
from the
emergence declined approximately 10- fold between
July 14 and July 28. Production for the second Cx.
during the summer( Fig. 3). An abrupt decline
in adult emergence was not observed in early September;
however, adult production was already very low at the
of
7%/ d
end of
August(<
10 females/ m2/ week).
Host-Seeking Populations
In 1997, host- seeking populations of Cx.
erythrothorax and Cx. tarsalis ( Fig. 4) increased
throughout April and May and reached annual maxima
in June. The maximum number of host- seeking Cx.
erythrothorax collected was nearly 33, 000 individuals/
trap night. The maximum number of host- seeking Cx.
tarsalis collected was 4, 560 individuals/ trap night, about
one- tenth of Cx. erythrothorax.
4). Two periods of stasis in population numbers occurred
of
number
area declined
Culex erythrothorax
samples.
The Cx. erythrothorax host- seeking population
declined approximately 80- fold during September( Fig.
Adult Emergence
The
in
400 individuals/ m2/ week from late July until early
September, and then declined about 10- fold during
September( Fig. 3).
The number of Cx. tarsalis females produced weekly
level
subpopulation
decreases in
rarely
encountered
from vegetated quadrats in the wetland declined at a rate
periods
instar larval
Culex stigmatosoma adults were
quinquefasciatus.
erythrothorax generation in 1997 was between 200 to
seven marshes.
Three
and more than one hundred times greater than Cx.
the
and remained at
September 26, late instar larvae
per quadrat was about ten times greater than Cx. tarsalis
below detectable
this
5
and
of samples
set
were not
153
Ecology
nearly
constant
at
500
August.
declines in
after
adult
mosquito
mid- September.
decreased to about 25
Adult
mosquitoes/
Fig. 4). Catches of Cx. erythrothorax hostseeking females fluctuated around a mean abundance of
23, 700 individuals/ trap night from June 12 until the last
week of August.
After declining for a three- week
period, the host- seeking population stabilized at an
in 1997 (
average of 475 females/ trap night for four weeks
( September 18 through October 16).
The Cx. tarsalis host- seeking population attained a
maximum in June and declined slowly throughout the
summer and early autumn( Fig. 4). The rate of decline
for
the
host- seeking
population
in 1997
was
2. 6%/ d.
Unlike the Cx. erythrothorax host- seeking population
which declined abruptly in early September, Cx. tarsalis
collections in carbon dioxide- baited traps fluctuated
between 100 and 300 females/ trap night during
September and then declined abruptly in mid- October.
Host- seeking females of both species disappeared in
early November.
m2/ week between the second and third week of
September.
This
was
largest
the
negative change
in
Water Quality
nearly twice the largest
average decrease observed earlier in the summer(- 0. 14/
limit of detection( 0. 2 mg/ L) for the iodometric method
d:
on five dates between April and September 1997.
population
Fig
size (-
0. 24/ d),
2).
The
number of adult
Cx.
erythrothorax produced
Residual chlorine concentration was above the
Residual
chlorine concentrations
were greater
than 1
154
Journal of Vector
Ecology
December, 1998
1
10000
III-- 2
6- 3
11000
ia
0-5
r-.
art—
o
i_
i
A
B
100
Q
in-
10
E
w
1
I
I
1- Jul
I
1
16- Jul
I
1
31- Jul
I
I
15- Aug
1
30- Aug
I
I
14- Sep
I
29- Sep
Date
0. 15 —
b
0. 1 —
0.05 —
et
A
0
0
o-
1
0.05 —
0. 1 —
a
0 -
0. 15 —
a-
0.2 —
N
0.25 -
cl -
0. 3
I
i
1- Jul
I
i
16- Jul
I
i
31- Jul
I
I
15- Aug
I
30- Aug
I
I
i
14- Sep
29- Sep
Date
Figure 2.
Number of Culex spp. emerging from the five inlet marshes( 1- 5) and two outlet marshes( A and B) in
the HSJRWRF demonstration wetland during summer 1997. Upper panel: number of individuals
emerging
per
week.
Lower
panel:
the rate
of population
change ( mean ±
SE) for the emerging
mosquitoes.
mg/ L
on
three dates: the
first
week of
May,
a
two
DISCUSSION
week
period in June- July, and at the end of August( Fig. 5).
Increases in
inversely
residual
related
to
chlorine
bacterial
bacteria densities declined
by
were
Wetlands constructed for wastewater treatment
Coliform
present a significant challenge for vector control agencies
or more orders of
in the arid southwestern United States. The nutrients
concentration
abundance.
three
magnitude when residual chlorine concentration was
greater
than 1 mg/ L(
Fig.
5).
supplied by wastewater and the nearly year-round
favorable growing
conditions create
dense
stands of
December, 1998
Journal of Vector
Ecology
155
10000 y
erythrothorax
X-- Cx. tarsalis
1000 —
a1
100 —
E
W
10 —
1=
0. 1
1- Jul
16- Jul
31- Jul
15- Aug
30- Aug
14- Sep
29- Sep
Date
The
Figure 3.
average number(±
SE) of Culex erythrothorax and Culex tarsalis females emerging from the seven
marshes in the demonstration wetland, San Jacinto, California during 1997.
100000
X
Cx.
tarsalis
y
erythrothorax
10000
oq
l000
Ippp
loo
to
1
i
1- Mar
I
31- Mar
I
30- Apr
30- May
29- Jun
29- Jul
28- Aug
27- Sep
27- Oct
Date
Figure 4.
Culex tarsalis and Culex erythrothorax host- seeking populations collected by carbon dioxide- baited
trapping at the demonstration wetland, San Jacinto, California during 1997. The arrows indicate
treatments of bacterial insecticides. Open arrows are Bacillus thuringiensis var. israelensis treatments.
Closed
arrows are
Bacillus
sphaericus
treatments.
156
Journal of Vector
December, 1998
Ecology
4
100, 000, 000
chlorine
Q— bacteria
10, 000, 000
t
3
1, 000, 000
0 2
U
a
0
100, 000
a
10, 000
y
N
•
1, 000
i
10
0
1
1
1- Apr
1-
Figure 5.
wetland
1
i
31-
May
1
1
May
I
1
30- Jul
29- Aug
1
30- Jun
1
1
i
28- Sep
Residual chlorine concentration and coliform bacterial density in influent water to the HSJRWRF
demonstration wetland, San Jacinto, CA during 1997.
Mosquito
vegetation.
concomitantly( Walton,
densities increased
lateral
and
increased
populations
unpublished
data)
as vegetation
growth
of
bulrush,
mounted application
devices.
Environmental factors
such as temperature, salinity, and suspended solids
influence the effectiveness and persistence of bacterial
particularly S. californicus, reduced the proportion of
open water habitat at the San Jacinto wetland
during the
toxins (
initial three
effluent, Bti is less effective in organically enriched
1997, the
years of operation(
Thullen
et al.
1998). In
reviewed
in Walton
and
Mulla 1992).
For
constructed wetlands receiving secondary- treated
host- seeking Culex spp. population
averaged nearly 30,000 females/ trap night during the
summer. The large populations of resident and migratory
emergent adult populations at the San Jacinto wetland.
birds that utilize constructed
wetlands serve as potential
Although larval and emergent adult populations declined
Reeves 1990). Rapid human
appreciably in the inlet marshes after application of Bti,
adult
reservoirs of arboviruses(
development in
water than is B. sphaericus( Mulla et al. 1990).
Bti did not have a demonstrable effect on larval and
surrounding constructed
important public health concern
larval and emergent adult mosquito populations in the
especially when mosquitoes capable of vectoring disease,
such as Cx. tarsalis, can
readily move from develop-
Because the entire wetland was treated with Bti, some
regions
wetlands can create an
mental sites
into human
chemical
provide
an
other factor was presumably associated with the decline
in the larval and emergent adult populations in the inlet
neighborhoods.
Bacterial larvicides
outlet marshes were not affected by the treatments.
alternative
to
insecticides ormosquitocidaloilsformosquito
marshes observed in August. The comparatively high
levels of residual chlorine (> 1 mg/ L) observed in the
control at constructed
wetlands where effluent water
influent water during late August were directly related
quality is
consideration.
However, thick
to marked declines in both the older larval subpopulation
an
important
stands of vegetation and
other environmental
factors
and the number of adults emerging from the inlet marshes.
limit the
bacterial larvicides ( Walton
A similar concurrent decline of mosquito populations in
the inlet marshes with high levels of residual chlorine in
and
effectiveness of
Mulla 1992).
effective
formulations
wetlands.
Jacinto
Thick
application
of the
The
of
stands
of
bulrush limit
pelletized
and
bacterial larvicides to
The declines in larval abundance and larval
in the San
population growth rate during September were associated
nearly 83 m in places) precludes
larvicides by backpack and truck-
wetland( e. g.,
the influent water was observed in June.
constructed
width of vegetated regions
the application of
the
granular
with
B.
declined
sphaericus
by
80% in
treatments.
samples
Larval abundance
taken two weeks after
December, 1998
treatment
Journal of Vector
the
of
Vectolex.
wetland with
weeks
failed to disperse farther than 2 km from the wetland,
and were concentrated only at the wetland and not at
greatest negative
other developmental sites within a 3 km radius of our
were
levels throughout the
The
population change
for
the B.
treatments.
sphaericus
chlorine was measured
larvae
mosquito
only
residual
The difference in the dispersal tendencies of the
August 29, high
two dominant Culex may have a very important
in the influent water during
consequence for control programs that utilize B.
September indicated that residual
decline in
the
chlorine
did
sphaericus was effective against
prevalent species
in dipper
Bacillus
Cx.
host- seeking
Culex
in
dip
Emergent
the
samples
effectiveness
erythrothorax
best indirect
the
adults provided
bacterial larvicides
of
magnitude
studies( Georghiou et al. 1992, Rodcharoen and Mulla
treatment
of
the
orders of
the
at
from
wetland
vegetated quadrats
host- seeking
the
m2/
8 generations)
after
decline naturally
decline for female
site, and if males exhibit similar dispersal tendencies,
this mosquito has a greater potential to evolve resistance
the
of
B.
sphaericus
toxins than
does Cx.
tarsalis.
Out-
crossing of Cx. erythrothorax individuals from treated
host- seeking
areas with susceptible individuals from untreated sites
was
adult
already low
in late August,
is less
likely
than
for Cx.
tarsalis.
Because of their
greater tendencies for dispersal, Cx. tarsalis populations
from Bacillus-treated wetlands are likely to mix to a
greater extent with susceptible populations from other
larvicide treatments,
developmental sites that either are untreated or where an
decline abruptly
alternative larval control is utilized. Cross resistance to
not
female
populations
The timing of the
at the San Jacinto wetland
autumn.
abundance
to
important
an
and
tarsalis adult
during
be
Sinegre et al. 1994, Rao et al. 1995, Silva- Filha et al.
tarsalis
tarsalis adults
did
have also been demonstrated in several field studies
(
emerging
week)
Cx.
population
October. Culex
until
rapid (<
the wetland
Culex tarsalis
wetlands.
declined
of
maintenance of
though the number of
from the
versus
high levels(> 10, 000- fold) of B. sphaericus
1995). Because host- seeking Cx. erythrothorax females
do not move very far from their natal developmental
with
that immigration
peripheral sources might
approximately 5 females/
even
treatment
suggests
factor contributing to the
production
selection)
evolution of
B.
wetland
decline in the Cx.
of a marked
populations
laboratory
Cx.
the
erythrothorax adult
declined nearly two
population after
females from
30- fold resistance after 30 or more generations of
measure of
sphaericus.
with
of multiple mechanisms in low- level( e. g., approximately
wetland.
on
Nielsen- LeRoux et al. 1997) indicated the involvement
of resistance in Cx. quinquefasciatus to B. sphaericus
after
The lack
(
resistance in the Cx. pipiens complex. Different levels
population
host- seeking
1994) to increase the risk for resistance. A recent study
were
followed the Vectolex
Furthermore, the Cx.
host- seeking
B.
larval population. The largest declines in
the emergent adult population
treatments.
1997),
et al. 1991) which have been demonstrated in laboratory
adjacent
from the demonstration
Wirth
the
Workman 1998)
and
and
wetland,
larvae
from
surveys
Walton
wetlands (
in the
Georghiou
mosquitoes (
sphaericus has a pair of mosquitocidal toxins( Baumann
tarsalis.
erythrothorax
in larval
under- represented
experimental
mosquito
in
the
sphaericus was also effective against
erythrothorax.
and
Cx.
and does not readily promote the evolution of resistance
surveys
indicated that B.
samples,
sphaericus. Unlike Bti which contains multiple toxins
not cause
Larval
mosquito populations.
most prevalent
study site( Walton et al. 1998).
occurred after
Even though
once after
coliform bacterial abundance
157
below detectable
later, third and fourth instar larvae
wetland.
Two
Ecology
other compounds used for mosquito abatement ( e. g.,
Bti, methoprene, etc.) is unlikely( Nielsen- LeRoux et al.
1997).
in New
Our analyses suggest that B. sphaericus was
Jersey light trap samples from the cooler, more northern
Central Valley and from the hotter, agricultural valleys
effective against Culex at this wetland; however, the
in
interpreted cautiously. The timing of the declines for
was
intermediate to the
southern
phenologies observed
California( Reisen and Reeves 1990). Mark-
recapture studies carried out
Cx.
host- seeking females
tarsalis
more
kilometers in
large
numbers at
WEW,
moved
estimated
Bailey et al.
In
three sites
contrast
data).
the larval populations,
for emergent adult Cx.
two or
erythrothorax, and for host- seeking adult Cx.
in
erythrothorax are suggestive that bacterial larvicides
surrounding the wetland
The maximum distances
affected the mosquitoes. The results might be viewed as
could move
populations occurred towards the end of the season and
between approximately 10 and 40 km
1965, Dow et al. 1965, Reisen et al. I992a).
there were no untreated populations against which to
to
female Cx.
equivocal because the declines in the mosquito
have been
marked
at
1995 indicated that
one night and were also produced
unpublished
by
during
effectiveness of the larvicide treatments should be
Cx. tarsalis, Cx.
seeking females
tarsalis
erythrothorax
moved an average of
0. 5 km
host-
per night,
compare the effects of the larvicides. Clearly, replicated
plots, untreated controls, and posttreatment samples
closer
to application
dates(
e. g., within
48 hours for B.
158
Journal of Vector
treatments)
sphaericus
interannual
detect
not
larval
lived
facilitate a less ambiguous
in
the
wetlands operations procedures( e. g., changing access
intended for
policies so that adulticiding can take place after dusk,
of
larval
efficacy
Biweekly larval
wetland.
comparisons
a short- lived
by
abundance
surveys,
( i. e., <
2
larvae
has been
observed
Bacillus
sphaericus reduced
by
establishment
1997). There is a need to reevaluate
of effective
vegetation
management
Short-
humans residing near man- made wetlands are fulfilled.
Our results suggest that B. sphaericus may provide
Culex larval
populations
for
one
in
to three
some measure of mosquito control in large- scale
multipurpose
constructed
wetlands
that
receive
organically enriched wastewater.
in Schoenoplectus{= Scirpus} - Typha wetlands
Mulla
et
1988,
al.
and
in
and
1997). Bti
and
longevity
because
for three
catch
e. g.,
Mulla
sites
weeks or
et al.
This study was supported by Special Funds for
and
Mosquito Research from the Division of Agriculture
1997).
and Natural Resources of the University of California
had
precipitation)
in
Acknowledgments
Bacillus
basins ( Siegel
of control
a
highly polluted
in Thailand( Mulla
and by a grant from the Academic Senate at U.C.Riverside. We appreciate the continued cooperation of
Eastern Municipal Water District and the U. S.
only partially
Culex at treatment wetlands in Arizona
Geological Survey. We thank Dr. G. Smith( University
sphaericus
1997).
Levy
have
proven
inhibited
thick vegetation
larvicides (
1996).
et al.
B.
effective against
in
factors (
developmental
mosquito
wastewater lagoons
al.
polluted water(
environmental
marked effect on
et
effective
against mosquitoes
Novak 1997)
in dairy
Binding
however
sphaericus was
Yet,
Levy
strategies) so that the public health and water needs of
reduction
in other organically enriched habitats.
Yoshimura et al. 1996)
longer
wetlands(
in
weeks)
treated plots relative to untreated plots
weeks
at
populations, might
bacterial larvicides.
the
control of mosquito
December, 1998
of
would
the
statement of
Ecology
penetration of the
Nevertheless, trends for the
emergent adult populations at the
San Jacinto
indicated that significant, short- lived
wetland
reductions
between sampling dates did
The projected increase in the use
abundance
in larval
of Wisconsin- Whitewater) for clarification of bulrush
classification
and
Stella Denison ( EMWD)
for
providing additional information regarding mosquito
abatement operations and water quality data collected
by the EMWD. We benefited from discussions with Dr.
not occur.
M. C. Wirth and P. DeChant. We thank Dr. P. Walton
of constructed
for review of the manuscript.
wetlands in regions of rapid human population growth
is
likely
to cause a greater need
for intensified
vector
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Reisen, W. K., M. M. Milby, and R. P. Meyer. 1992a.
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Surveillance for mosquito- borne
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encephalitis virus
human disease in California, 1995.
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Levy,
1997. Mosquitoes
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and
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Mosquitoes in California.
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Mulla, M. S., H. Axelrod, H. A. Darwazeh,
mosquito
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Mosq.
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wastewater
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and
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of some environmental
Bacillus
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Rodcharoen, J.
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Resistance
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Efficacy
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of California, 1987- 1990. J. Med. Entomol. 29:
Newsletter 28( 3): 13- 14.
Loomis, E. C.(
Population dynamics of adult Culex mosquitoes
to the microbial
agent Bacillus
sphaericus. J. Econ. Entomol. 87: 1133- 1140.
Siegel, J. P.
and
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1997.
Field trials of
Vectolex CG, a Bacillus sphaericus lorvicide, in
Bull.
Illinois waste tires and catch basins. J. Am. Mosq.
Mulla, M. S., J. Rodcharoen, W. Ngamsuk, A. Tawatsin,
Silva- Filha, M.- H., L. Regis, C. Neilsen- LeRoux and J.-
thuringiensis ( H- 14)
against
mosquitoes.
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Soc. Vector Ecol. 15: 166- 175.
P. Pan- Urai,
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and
Bacillus
U. Thavara.
polluted water mosquitoes
Bangkok, Thailand.
1997.
Field trials
formulations
sphaericus
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against
a suburban area of
Mosq.
Contr. Assoc.
Sinegre, B. Gaven,
Bacillus
mechanisms
sphaericus in a field- treated population of Culex
quinquefasciatus (
Diptera: Culicidae). J. Econ.
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Sinegre, G., M. Babinot, J.- M. Quermel, and B. Gaven.
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Nielsen- LeRoux, C., F. Pasquier, J.- F. Charles, G.
to
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First field occurrence of Culex pipiens
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Expansion
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Randall. 1997. Survival of two larvivorous fishes
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1994.
Walton, W. E.
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P. D. Workman.
1998.
Effect of
Multipurpose Wetlands: Phase II/III Report. Final
marsh design on the abundance of mosquitoes in
Design
experimental constructed wetlands in southern
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Ongoing
Research Investigations. San
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Walton, W. E.
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California. J. Amer. Mosq. Contr. Assoc. 14: 95-
M. S. Mulla. 1992. Impacts
microbial
and
in the
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Pp. 205- 237
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Walton, W. E., P. D. Workman, and C. H. Tempelis.
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Organisms into Aquatic Ecosystems( A. Rosenfield
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Walton, W. E., M. C. Wirth, P. D. Workman
Randall. 1996.
mosquitoes
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Yoshimura, G., S. Wright,
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1996.
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Mosquito Control Research Annual Report 1996.
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Calif. 64: 124- 129.
Journal of Vector Ecology 23( 2): 161- 170
Effect of Two Rice Culture Methods on the Seasonal Occurrence
of Mosquito Larvae and Other Aquatic Animals in
Rice Fields of Southwestern Korea
Dong- Kyu Lee
Department of Biological Sciences,
Kosin University, Pusan 606- 701, KOREA
Received 19 May 1998; Accepted 14 September 1998
ABSTRACT: An ecological study has been performed on the community structures of aquatic animals and
on abiotic factors in organically and conventionallyfarmed rice fields at Bulkyo, Bosong- gun, Chollanamdo during the rice growing periods between May 1995 and October 1996. The total numbers of aquatic
insect taxa in these fields were 25 species, 22 families in 10 orders. In 1995 and 1996, three and then four
more species of aquatic insects were collected in the organicallyfarmed rice fields than in conventionallyfanned rice fields. The common dominant species in both rice fields during the rice cultivation period was
Chironomus sp. The abundance oftwo vector mosquitoes, Anopheles sinensis and Culex tritaeniorhynchus,
was lower in the organicallyfarmed rice fields as compared to the conventionallyfarmed rice fields. The
populations of mosquito larvae in the organicallyfarmed rice fields might have been influenced by Chinese
muddy loaches, Misgurnus mizolepis. Coefficients of correlation between Chinese muddy loaches and
abundance of mosquito larvae showed negative correlations in An. sinensis(- 0. 66) and Cx. tritaeniorhynchus
The average of the species diversity index for organicallyfarmed rice fields was almost twice
0. 47).
0. 62)
is
as
much
as
conventionallyfarmed
of
rice
fields ( 0. 35).
The average community similarity
between both types of fields was very low( 0. 33).
Keyword Index: Culex, organic farming, rice field, Anopheles, larvivorous fish.
enemies and various abiotic factors in ecosystems, and
INTRODUCTION
Culex
sinensis
Wiedemann
and are vectors of
malaria
Dyar
tritaeniorhynchus
and
mosquitoes
Anopheles
and
distributed widely in Korea
are
Japanese
encephalitis and of
both
the ecology of these mosquitoes.
Traditional mosquito control strategies
in
conventionallyfarmed rice fields used mainly
adulticides which include fogging, aerosol sprays, and
These
inland filariasis, respectively.
are particularly abundant in riceland
larval control from agro- pesticides in Korea( Ree et al.
1981, Shim
et al.
1995a
and
b). Each of these methods
fields
involves the application of relatively large amounts of
sinensis and
insecticides into the environment of the rice ecosystems.
Cx. tritaeniorhynchus are the predominant blood- seeking
These methods also may deposit insecticidal residues in
agroecosystems where
mosquitoes
in
species
animal
areas.
and, when present
present
a
health through
disease. Because
of
serious
is
a need
to
human
and
as
and
animal
well
as
farmers.
Therefore, environmental concerns
and
have stimulated other farming methods to reduce the use
of
of insecticides for controlling pests in rice fields. One
approach that has recently gained popularity is organic
vectors
sinensis and
rice that can be taken inadvertently by consumers as
Cx.
health,
farming.
In Korea, 0. 08% of farmers cultivate several
crops using organic farming, which utilizes organic
these species at acceptably
fertilizers instead of chemical fertilizers and which does
The development
environmental
numbers,
that
such programs require a detailed
between
et al.
management programs
will maintain populations of
low levels.
Shim
in large
the threat that An.
develop
rice
threat to human
annoyance
tritaeniorhynchus pose to
there
Anopheles
July and August, respectively(
1987, 1990, 1997);
these
they breed in irrigated
lowland
and associated
and
implementation
of the
of
relationship
study
factors, including natural
not use pesticides(
Paek 1992).
The objectives of this
study were to establish the population abundance patterns
of vector mosquitoes and other aquatic animals and
to
162
Journal of Vector
evaluate the associations
factors and the
rice versus
between several environmental
Hitachi) and an atomic absorption spectrophotometer
Model Smith-Hieftje 12, Thermo Jarrell Ash).
Sampling
Four paddies each from organicallyfarmed and
conventionallyfarmed rice fields were randomly
Study Areas
The study
was carried out
which was
in the
field( 150 ha)
rice
less than 2m
estuary,
located in Bulkyo, Chullanam- do,
southwestern
In
used
this area, most
culture
in June
but three
Korea.
conventional
employing chemical fertilizers
The insecticides were each applied
and
times
August,
July,
a
names of
and
in other months because the water of the rice paddies
one
was
July, and three times in August of 1995;
in early July, and each one time in late
September of 1996. The chemical
in the field
used
each
from
a road and an
watering
might
the two
from
of
have
1, 730
and
m2
rectangle,
organicallyfarmed
irrigation ditch connected
draining
habitat disruption in
either
fields
rice
to a main
There
canal.
fields
rice
the
canal and a
evidence
m2
The conventionallyfarmed
were separated
by
1, 860
was a
paddy
was
one,
no
which
created an effective ecological change
in
different farming conditions. Water was supplied
a reservoir and an underwater
fields. A
network of
individual
allowed
permitted quick
irrigation
flooding
and
The
of
Water
and
spring near the rice
drainage channels
for each rice paddy and
water control
efficient network of parallel
Analysis
and
draining by
ditches
means of an
connected to them.
Soil
here
were
based
on
in Field and Laboratory Methods for General
Brower and Zar 1977). The
and pH of
those given
water
salinity
in the two
portable
salinity
field types
rice
Once
or twice
meter(
every
were measured
using a
NS- 3P, Merbabu
Trading. Co.)
and a portable pH meter (
Model TS- 1, Suntex Ins.).
month, water
paddies were measured at
ignition
determined by
methods
water.
were
To
the
used
analyze
USDA
for
using
analysis(
proportion of soil particles( e.
g.,
was
depths
clay
spots.
a method of
by 32 sweeps with an aquatic net of 32 mesh/ cm and 33
cm in diameter. Fish samplings in the rice paddies were
(
performed by using an aquarium- type vinyl fish trap
16cm dia. x 20cm long)( Yu et al. 1981), placed at two
sites in each rice paddy.
The samples of three different types from the rice
paddies were put into a small plastic concentrator with
a fine mesh on the bottom. They were then transferred
to 500 ml plastic bottles with water and transported back
to
the
laboratory
in two ice
chests.
To minimize
physical damage and prevent predators from killing or
eating other animals during transportation, the chests
were chilled with ice packs. The aquatic animals were
identified under a dissecting microscope and a stereo
microscope, according to characters described in each
specific key of the corresponding taxa ( Merritt and
Cummins 1984, Stehr 1987a and b, Usinger 1956,
Peterson 1982, Fitzpatrick 1983, Yoo 1986, Yoon 1988,
Yoon and Ahn 1988a and b, Yoon and Kong 1990).
method.
examinations
rice
Also,
of
50
The physical and chemical characteristics of the
soils exposed to the two farming techniques were
statistically
compared
by
Student'
a
s
t- test.
In the
present study, dominant species, dominance indices,
and species diversity indices of the organicallyfarmed
rice field were compared with those of the
conventionallyfarmed rice field in order to analyze the
The
aquatic
loss
dominance index is a method measuring the simplicity
Cox 1976). The
and silt<
water contained
sampled water with soils
the
of
10 to 16 different
soil organic matter was analyzed
of organic carbon on
The
Data Analysis
analytical methods given
Ecology(
in late September.
taking 16 samples of water with a 355 ml dipper. Other
aquatic animals and benthic samplings were also taken
w. s.
respectively.
out
paddy. Larval mosquito abundance was monitored by
were cartab
p. 50%, fenitrothion E.C. 50%, BPMC G. 2%, and
Quratel G. 3%. However, one farmer has cultivated rice
since 1980 in the rice field of 3 ha,
using an organic
culturing method. Each of the four organically- and
conventionallyfarmed rice paddies for the study were
randomly selected from 16 and 850 rice paddies, and
completely drained
aquatic animals were randomly collected in each rice
and
insecticides
the
selected as study plots. The survey was carried out once
or twice a month from June to September in both 1995
and 1996 when the rice fields were flooded with
agricultural water. The collections were not carried out
level,
above sea
method
insecticides.
time
farmers have
December, 1998
with an UVNIS spectrophotometer ( Model U 3210,
in organicallyfarmed
conventionallyfarmed rice.
vector mosquitoes
near an
Ecology
pm)
standard
ions in the
main ions, the
in each rice field were examined
animal
community
and
its dynamics.
The
of each community and was here calculated with
McNaughton'
s
dominance index ( DI) ( McNaughton
1967).
This index is as follows: DI=(n1+ n2)/ N( N: the
total number of individuals in all the species; n1 and n2:
the numbers of individuals of the 1St and 2nd dominant
species,
respectively).
The
species
diversity
was
December, 1998
calculated
1969)
pi
Journal of Vector
using the Shannon- Wiener index( H')( Pielou
H'=- Sigma pi log pi, where, pi= n/ N. The
proportion of
the total number of
163
depending on rice conditions, pesticides were sprayed
five to six times during the growing season from June to
September.
The pesticides used were cartap and
such as
is the
Ecology
individuals
occurring in species i.
carbofuran for the control of agricultural pests such as
was calculated
the rice stemborers, the brown planthoppers, the rice
green leafhoppers, the smaller brown leafhoppers, and
Brower
and
Finally, the community similarity
using the Sorensen Coefficient ( CCs)
Zar 1977).
the grass
Rice culturing practices
rence of An. sinensis and
as other aquatic animals.
cultivation
changed
in the
little
affected
the seasonal occur-
Cx. tritaeniorhynchus
as well
The observed schedule for rice
during the year.
during the study period, except for minor
by the making of a road near the rice
The physical characteristics of the soil and the ricefield water of two rice fields is shown in TABLE 1.
adjustments caused
fields in
In the organicallyfarmed rice
However, no pesticide was applied in the rice field
fields
conventional and organic rice
leafrollers.
field, an organic fertilizer was supplied once in late May
before transplanting rice seedlings. The organic fertilizer
was made mainly from a mixture of chicken dung, rice
bran, and sawdust at the ratio of 6: 3: 1, respectively.
RESULTS AND DISCUSSION
1996. In both 1995 and 1996, plowing
both fields in early May, and water was introduced between the end of May and the beginning of
depth of water, various water ions, organic matter, and
June. The
from transplanting
the organicallyfarmed and the conventionallyfarmed
The seedling transplantation was corna week in early June of both 1995 and
rice fields, except for the concentration of P2O5 in the
In the conventionallyfarmed rice field, chemical
were distributed in late
May of both years to
appeared somewhat higher than those of other rice
began
May,
rice
rice seedlings.
pleted
Some abiotic factors, such as water pH, water salinity,
at
within
plants
were grown
texture of soil were not significantly different between
water.
The water salinity of both the organicallyfarmed and the conventionallyfarmed rice fields
1996.
fertilizers
supply
phosphate,
potassium,
herbicide before transplanting
and
rice
TABLE 1.
nitrogen
seedlings.
fields:
0.4° I
and
1. 1%. ,
respectively.
might be that the rice fields are located near an estuary.
Also,
The concentration of phosphoric acid ( P2O5) in the
Physical and chemical factors of soil and water in the
conventionally- and organicallyfarmed rice fields, 4- 16 replicates.
Rice Fields( Mean±
Factor
Conventional
S. D.)
Organic
Water
pH
5. 6a'±
0. 10
5. 4a ±
0. 10
Salinity M.)
1. 1a ±
1. 20
0. 4a ±
0. 10
Depth(
7. 2a ±
2. 10
6. 8a ±
1. 43
cm)
Soil
Soil
Texture2(%)
59. 9a -
73. 0a
-
Organic Matter(%)
4. 1a ±
0. 20
4. 4a ±
pH
6. 0a ±
0. 60
6. 3a ±
1. 00
Na+ ( ppm)
47. 5a ±
16. 57
52. 4a ±
17. 62
K+ (ppm)
27. 0a ±
23. 37
27. 8a ±
25. 97
3. 6a ±
1. 49
8. 1a ±
13. 63
0. la ±
0. 10
0. 1a ±
0. 08
ppm)
35. 7a ±
6. 46
32. 6a ±
7. 93
ppm)
1. 9a ±
0. 41
2. 3a ±
0. 37
ppm)
65. 5b ±
10. 61
173. 0a ±
32. 53
Mgt,(
Cat+(
S042-(
P042-(
P2O5(
ppm)
ppm)
0. 20
Means within a row followed by the same letters were not significantly
different( P> 0.05; t- test).
2
Soil
percentage
indicates
The reason
a
with
particles<
50
pm
in diameter.
164
Journal of Vector
organicallyfarmed rice field water ( 173. 0 ppm) was
significantly higher than that of the conventionallyfarmed rice fields( 65. 5 ppm), due to a farmer' s use of
dung
chicken
as
a
fertilizer.
natural
components of the environment
abundance and
1977).
distribution
However, there
was
no
larvae
of mosquito
and
between the
correlation
number
concentration
of
NH4- H
characteristics
of
water
such
quantity, pH, DO content,
There are few reports
invertebrates
of aquatic
in the
that
the
insecticide
application
in
rice
fields.
Service ( 1977)
Ikemoto
aquatic insects in rice fields in Kenya before and after
spraying Dimecron [ 0, 0- dimethyl- 0-( diethylanido- 1-
was a positive
water
NH4 H
other than mosquitoes, as particularly related to
studied mortalities of An. gambiae larvae and other
the
chloro- crotnyl) phosphate] applied for the control of
among the
rice stem borer. Whereas before spraying there was a
very rich and numerous aquatic fauna, exceedingly few
live invertebrates remained after spraying. The larval
densities of An. gambiae 14 days after spraying were
significantly larger than pre- spray densities, whereas
and
water
concentration.
on the population
other
Zar
temperature,
as
and
and
distribution
larvae
of
the pesticide application acted as one of the main
on the population dynamics of aquatic invertebrates
evidence
that there
reported
observed that in the conventionallyfarmed rice field,
mortality factors. Other reports showed similar results
or other aquatic organisms.
Sakai(: ( 1979)
such as fish and insect predators, however, it was
affect the
Brower
phosphoric acid affected the abundance and
December, 1998
Chemical
importantly
of species(
Ecology
dynamics
than mosquitoes and the
in Korea,
other aquatic invertebrates were not. In spite of the lack
related
to the various aquatic animals
of quantitative data, it was clear that spraying with
farmed
rice
relationships
them
among
fields,
as well as
in conventionallyfarmed
as
particularly
in organicallythe insecticide application
rice
fields. TABLE 2
Dimecron drastically reduced the numbers of aquatic
insects whereas recolonization with An. gambiae was
shows
from organicallyand
farmed
fields
in 1995 and 1996.
rice
conventionallythe
number of the aquatic animal
The
aquatic animal
22 Families
taxa showed a total of 25
10 Orders
and
taxa
or
species
Subclasses,
such
rapid.
Simpson ( 1949)
in
Branchiopoda,
Mesogastropoda,
Ostracoda,
total that occurs in each species. A collection of species
Copepoda,
with
Pisces, from the organically- or
farmed
rice fields of Bulkyo from June
conventionally-
24
during
in those
tritaeniorhynchus.
were collected
this period.
rice
fields
were
The
An.
More species( 24)
Shim
a
et al.(
high
a,
b)
between the rice fields. Chironomus sp. was frequently
the dominant species in both rice fields during the
period of rice cultivation. In the conventionallyfarmed
rice field, individual numbers ofAn. sinensis in July and
Cx. tritaeniorhynchus in September were very large.
Anopheles sinensis and Chironomus sp. appeared to be
dominant species at both rice fields, and were especially
of aquatic animals
rice
concluded that
both
prevalence was
In the
TABLE 2.
field
field.
species,
Cx. tritaeniorhynchus, had developed
conventionallyfarmed
generally
case of
not
rice
fields
influenced
the predators
The
field that the range of dominance was not so different
had been
resistance to most of the pesticides that
applied to
cides.
1995
have low dominance.
Cx.
sinensis and
from the organicallyfarmed
sinensis and
will
vector mosquito
than( 20) from the conventionallyfarmed rice
An.
diversity
fluctuation in 1996( TABLE 3). The values of dominance
index ranged between 0. 48 and 0.96 in the organic rice
field, and between 0. 52 and 0.93 in the conventional rice
1996( TABLE 2). The monthly occurrence
numbers at the rice fields ranged from 14 to
species
species
high
dominance indices of the rice fields showed a little
and
to September,
of species
McNaughton ( 1967) con-
sidered not only the number of species and the total
number of individuals, but also the proportion of the
as
Ephemeroptera, Odonata, Hemiptera, Coleoptera,
Diptera,
and
that their
abundant in early July, 1996. Also, An. sinensis was a
the pesti-
dominant mosquito species in the conventionallyfarmed
larvae,
rice field during July. The populations of Cx. tritaenior-
so
by
of mosquito
Number of the aquatic animal taxa from the organicallyfarmed and conventionallyfarmed rice
fields of Bulkyo from June through September, 1995 and 1996.
1995
Rice fields
1996
Order
Family
Genus
Species
Order
Family
Genus
Species
Organic
7
12
14
14
10
22
23
24
Conventional
6
9
10
11
10
17
19
20
Total
7
12
15
16
10
22
24
25
December, 1998
Journal of Vector
hynchus increased
both fields in September
at
these mosquito larvae
The
were not
smaller
Chinese muddy loaches, Misgurnus
Gunther)( Cypriniformes: Cobitidae) showed
fluctuations than did the
organicallyfarmed
dominant
rice
field.
only in June
both
twice as much as that of the conventional farm ( 0. 35).
The majority of living aquatic animals probably less
in the
mosquitoes
Yet, this fish
was
a
165
caused by a heavy rainy season. The average of the
species diversity at the organic farm( 0.62) was almost
although
the dominant species.
abundance of
mizolepis(
Ecology
tolerate the chemicals than mosquito larvae in rice fields
(
Ree et al. 1981, Shim et al. 1985, 1995a,b). Furthermore,
Sigara sp.
the effect of pesticides might have been evident on the
in the organicallyearly July they did
predator populations because development rates of the
not appear to
be a dominant species in the conventionally-
from the comparatively rapidly developing and fast
farmed
rice
field. Also, Cercion hieroglyphicum
Hebrus
nipponicus were
was
species
frequently
farmed
and
farm
and
Aquatic
rice
after
dominant
September.
have been impacted
organic
farmed
species
fields in 1996, but
rice
fields in August
might
dominant
a
of
by
species at
The
the
insecticides in the
were more
both
values of species
and
community similarities were the highest ( 0. 42) in
September before harvest but the lowest( 0.28) in early
in the
July. These results might be caused by polyphagous
Chinese muddy loaches and King mud snails in the
conventional
farm.
the
organicallydiverse than those in the
to
organic rice
0. 39 in June
field
were
the
TABLE 3.
lowest,
rice
and
which might
organically farmed
rice paddies.
The average of the
similarities ( 0. 33) showed dissimilar communities
between the rice field ecosystems during the year.
The results ofthe monthly or biweekly prevalencies
of An. sinensis, Cx. tritaeniorhynchus, and Chinese
muddy loaches are summarized in Figures 1, 2, and 3,
respectively.
The population of An. sinensis larvae
1996
appeared from early July, which was much earlier than
of
that of Cx. tritaeniorhynchus, and kept a rather constant
have been
seasonal prevalence, showing an unusual peak in the
during
In late July, the species diversity indices
TABLE 4).
both fields
July
conventional
mosquitoes (
insects
diversity index ranged from
from 0.24 in late
August in the
In 1996, the
colonizing
rice
aquatic
0. 15 in late July to 0. 86 in September in the
field
Service 1977).
and
conventionallyfarmed rice fields, possibly because of
various insecticide applications in the conventional rice
field. The
predators are slower and life histories differ appreciably
predators
in
animal communities
fields
years.
Dominant species and their dominance indicies( DI) of the aquatic animal communities
at the organically- and conventionallyfarmed rice fields in Bulkyo from June through
September, 1996.
Date
Rice Field
Dominance
Jun. 19
Organic
Sigara sp. ( Hemiptera: Corixidae)
species
DI
0. 51
Misgurnus mizolepis( Cypriniformes: Cobitidae)
Convent.
Chironomus sp. ( Diptera: Chironomidae)
0. 85
Jul. 10
Organic
0. 83
Anopheles sinensis( Diptera: Culicidae)
Convent.
0. 52
Jul. 25
Organic
0.96
Convent.
0. 93
Aug.
21
Organic
Cercion hieroglyphicum( Odonata: Coenagrionidae)
0. 57
Convent.
0. 87
Hebrus nipponicus( Hemiptera: Hebridae)
Sep.
12
Organic
Hebrus
nipponicus(
Hemiptera: Hebridae)
0.48
Convent.
Hebrus
nipponicus(
Hemiptera: Hebridae)
0. 87
166
Journal of Vector
TABLE 4.
Ecology
December, 1998
Species diversity( H') and community similarity( CCs)
of the aquatic animal communities in the organicallyfarmed and conventionallyfarmed rice fields in Bulkyo,
1996.
Month
TABLE 5.
Species
diversity(
H')
Community
Similarity( CCs)
Date
Organic
Conventional
Jun. 19
0. 74
0. 39
0.29
Jul. 10
0. 53
0.36
0.28
Jul. 25
0. 15
0.24
0. 32
Aug. 21
Sep. 12
0. 81
0. 39
0. 35
0. 86
0. 37
0.42
Average
0. 62
0. 35
0. 33
Association between the aquatic predators and the population of mosquito larvae in the organicallyfarmed rice fields of Bulkyo.
Correlation
Species
An.
n
Cx. tritaenior.
sinensis
P
r
n
r
P
Cercion hieroglyphicum( Odonata: Coenagrionidae)
8
0. 25
0. 274
4
0.04
0. 478
Symprtrum darwinianum( Odonata: Libellulidae)
8
0. 21
0. 305
4
0. 63
0. 186
Hydaticus grammicus( Coleoptera: Dytiscidae)
8
0. 01
0. 490
4
0. 84
0.082
8
-
0. 38
0. 177
4
0. 44
0. 279
Helochares
8
-
0. 10
0. 404
4
0. 88
0.059
Ampullarius insularus( Mesogastropoda)
8
-
0. 26
0. 267
4
0. 13
0.437
Misgurnus
5
-
0. 66
0. 111
5
0.47
0. 211
middle
striatus(
Coleoptera: Hydrophilidae)
September (
of
tritaeniorhynchus were
until
Cypriniformes: Cobitidae)
mizolepis(
the middle of
drainage
1).
The larvae
-
-
-
Cx.
for the relative density for the field comparison because
found from the middle of August
the fish are active in night time, yet they were collected
using fish traps in daytime. No mortality was observed
Fig.
September. This
resulted
from both
of
from the
fields. As
for the Chinese muddy loaches even during the draining
population of this species
The
out of the organicallyfarmed rice paddies and over the
winter. On the other hand, some mortality was observed
Cx.
for both young and adult Chinese muddy loaches in the
organicallyfarmed rice field
were
generally much lower than those in the conventional
conventionallyfarmed rice paddies just after insecticides
complete
shown
increased in the
mosquito
larval
fields
rice
in
Fig.
3). The
itself is low
fish
rice paddies
much smaller
The
The population of this species in the
conventional rice field seemed to be deeply affected by
insecticide applications showing only 0. 3, 0. 0, 0. 0, 0. 0,
and 1. 0, compared to 3. 3, 3. 0, 4. 5, 4. 0, and 4. 0 in the
difficult to
density
organicallyfarmed rice field in June, early July, late
July, August, and September of 1996, respectively.
Four insecticides were used in the conventionallyfarmed
method
is inadequate for
rice field to control mainly the Pyralid moth larvae and
this
species appeared
the Delphacid planthoppers. The results showed that
number of
than the actual
were applied.
the
survey
dynamics of those fish,
not clear whether
the collection
populations.
during
the mosquitoes, were
accuracy. It is
or
The Chinese muddy
were collected mostly in
population
who were predators of
assess with
sinensis and
period.
mizolepis,
the organicallyfarmed
period(
An.
the
field throughout the
loaches, Misgurnus
of
rice
September.
during
populations
tritaeniorhynchus
rice
of the water
in the Figure 2, the
the
density, and is only reliable
the
monthly
populations of
Misgurnus
mizolepis
in the
Journal of Vector
December, 1998
167
Ecology
55
Organic F
45
c—
Conven. F.
35
a)
a)
n 25
E
z
g
15
a)
t•16.
5
0
Jun., 95
Jul.
Aug.
Jun., 96
Sep.
E. Jul.
L. Jul.
Aug.'
Sep.'
Month
Figure 1. Seasonal prevalence ofAnopheles sinensis larvae in the organically- and conventionallyfarmed rice fields
in 1995 and 1996.
34
0—Organic F
28
Conven. F.
CS.
22
I)
S
16
l0
4
0
Jun., 95
Jul.
Aug.
Jun., 96
Sep.
E. Jul.
L. Jul.
Aug.'
Sep.'
Month
Figure 2. Seasonal prevalence of Culex tritaeniorhynchus larvae in the organically- and conventionallyfarmed rice
fields in 1995
and
1996.
168
Journal of Vector
Ecology
December, 1998
6. 5
0– Organic F
5. 5
a– Conven. F.
c 4. 5
F.
cl
H
t..N
a.
3. 5
E
2 2.5
01
1. 5
0. 5
n
a-----___
e
Jun., 95
Jul.
Aug.
Sep.
'
Jun., 96
E. Jul.
A
L. Jul.
Aug.'
Sep.'
Month
Figure 3. Collection of Chinese muddy loaches, Misgurnus mizolepis in the organically- and conventionallyfarmed rice fields in 1995 and 1996.
fields
conventional rice
insecticide
were
application,
and
by
Sympet: um darwinianum against the vector mosquito
their recovery was not
species were 0.21 and 0. 63, respectively. Also, Sigara
seriously
suppressed
observed.
Hydaticus grammicus( Coleoptera: Dytiscidae)
collected
in
adults and
the rice
larvae
insecticide
to the
decreased
fields
very
rice
insecticides
fields.
aquatic animals were not
The
Both
susceptible
application, as abundance
whenever the
conventional
during the study period.
of this species seemed
was
markedly
were applied to the
populations
of
other
significantly different between
farming of rice fields,
the organic and the conventional
except
for Cloeon dipterum( Ephemeroptera: Baetidae)
Cercion hieroglyphicum ( Odonata: Coenagrio-
and
nidae).
However, it is
pressure or
populations
hieroglyphicum
in the
of
and
rice
related to the mosquito
not
closely
correlation coefficients of
0. 25
An.
and
nymphs
(
sinensis and
larval
The
Cercion
Sympetrum darwinianum)
The
against
fields ( TABLE 5).
Odonata
larvae in the rice fields might have been influenced
largely by oligophagous Chinese muddy loaches,
Misgurnus mizolepis. The coefficients of correlation
It has shown that there are not many effective
control methods against adult mosquitoes because of
their outdoor feeding, resting habits, and the sociogeographical condition ( Ree et al. 1981, Shim et al.
1987).
Therefore, studies on population dynamics of
immature stages of the vectors are urgently needed and
populations.
considered to be essential for effective control. It is
Cx. tritaeniorhynchus
The
in An. sinensis and- 0.47 in Cx. tritaeniorhynchus.
were
Cercion hieroglyphicum
0. 04, respectively.
inversely to the mosquito populations, as the correlation
coefficients were- 0. 38 with An. sinensis and- 0. 44 with
Cx. tritaeniorhynchus. The population of mosquito
mosquito larvae showed the highest correlation: - 0. 66
methods.
populations
mosquito populations. On the other hand, Hydaticus
grammicus ( Coleoptera: Dytiscidae) was related
between Chinese muddy loaches and the population of
field. Most predatory insect species were not affected
by the decrease ofAn. sinensis and Cx. tritaeniorhynchus
larval
Ampullarius insularus did not significantly reduce the
fluctuation
It probably resulted from the
insecticide treatments in the conventionallyfarmed rice
sampling
sp. ( Hemiptera: Corixidae), Helochares striatus
Coleoptera: Hydrophilidae), and King snails,
due to inadequate
not clear whether the
due to insecticide
was
(
were -
coefficients
of
important to understand the factors regulating natural
populations, particularly agricultural pesticides and
predators
such
as
fish,
adults,
and/ or
nymphs
of
Journal of Vector
December, 1998
Coleoptera, Hemiptera,
which are
found in
that some
natural
regulating the
Odonata
and
enemies
population
carried out
showing that
Odonata
Cx.
the
nymphs, or
the
of
are
important in reducing
Service ( 1977)
large
numbers of potential aquatic predators
from
and
invertebrate predators
has been
diversity
and
of
ofmosquitolarvaeintheirhabitats
in the literature ( Jenkins 1964,
reported
1974, Collins
A broad
habits.
feeding
their predatory
techniques
serological
Washino 1985).
Among
crustacea. Wm. C. Brown. Dubuque, 227 pp.
Ikemoto, T.
and
I. Sakaki. 1979.
Physico- chemical
characters of the water in rice fields in relation to
their suitability for breeding of the mosquito larvae
A. sinensis. Jap. J. Saint. Zool. 30: 87- 92.
Bay
of medically important arthropods. Bull. W.H. O.
for nearly 100 years in the U.S. ( Meisch
Yu et al. ( 1981, 1982, 1983) and Yu and Lee
Kim, H. C., M. S. Kim, and H. S. Yu. 1994. Biological
proved
Coleoptera
that
experimentally
and
rice
According
control agents.
et al. (
larvae
1994), the introduction
to
until
fish
control of vector mosquitoes by the use of fish
predators, Moroco oxycephalus and Misgurnus
1985,
anguillicaudatus in the laboratory and semi- field
rice paddy. Korean J. Entomol. 24( 4): 269- 284.
McNaughton, S. J. 1967. Relationship among functional
and potential
the report of
of other species of
muddy loaches, Misgurnus anguillicaudatus, at 2 fish/
produced a 47. 7% reduction of An. sinensis larvae in
m2,
simulated rice
five
over
paddy
weeks.
reduction of mosquito abundance was
organicallyfarmed
rice
fields
and
A
marked
observed at all
might
have been
by the Chinese muddy loaches. These results
indicated that Misgurnus mizolepis is a promising
caused
biological control
for use against
agent
breeding
species
in the
rice
several mosquito
fields
of
30( Suppl.): 1- 150.
which
native
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biological
the
Aplocheilus latipes,
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Fitzpatrick Jr., J. F. 1983. How to know the freshwater
Jenkins, D. W. 1 964. Pathogens, parasites and predators
Aphyocypris
a
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6, 218 pp.
Cox, G. W. 1976. Laboratory manual of General ecology.
3rd Ed. Wm. C. Brown. Dubuque, 232 pp.
ed.).
them, the
abatement
Kim
C. Chapman,
Hem iptera.
Larvivorous fish have been
1985)
Daily
a biological tool for mosquito
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1985).
1996.
Pp. 25- 42. in Biological Control of Mosquitoes( H.
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Collins, F. H. and R. K. Washino. 1985. Insect predators.
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in
role
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tritaeniorhynchus
Central
mosquito
the experimental
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others,
apparent
important
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play
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Meisch, M. V. 1985. Gambusia
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Acknowledgments
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The
author would
Hong for assistance in the field, and
Dae In Kang, for information on the
and
Ms. Hye- kyung
the
farmer, Mr.
organicallyfarmed
This study
Science
and
like to thank Ms. Sun- hee Choi
rice
cultivation
of
his
rice
field.
financially supported by the Korea
Engineering Foundation( 94- 0402- 03- 01-
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H. Wharton,
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Evaluation of Attractant-Baited Traps/ Targets for Mosquito
Management on Key Island, Florida, USA
Daniel L. Kline' and Gene F. Lemire2
Centerfor Medical, Agricultural and Veterinary Entomology, Agricultural Research
Service, U.S. Department of Agriculture, Gainesville, Florida 32604 USA.
2Collier MCD, 600 North Road, Naples, FL 34104- 3464 USA.
Received 24 August 1998; Accepted 28 September 1998
ABSTRACT: A three- year research project was conducted on Key Island, Collier County, Florida, USA,
to evaluate an innovative attractant-based mosquito management technique. In the first year, species
composition, relative abundance, and spatial distribution were determined. Although 16 species were
collected, the dominant species was the black salt marsh mosquito, Aedes taeniorhynchus, which was the
primary pest species. Efficacy of a single line barrier, consisting of 52 carbon dioxide ( 200 cc/ min) +
octenol( ca. 4 mg/ h) baited traps( 1994) or insecticide( lambdacyhalothrin) impregnated shade cloth targets
1995), spaced ca. 16. 5 m apart, to reduce mosquito abundance in a resort area, was evaluated on the
northern end of the island. Success was evaluated by means of baited surveillance traps located on both
sides of the barrier. Traps and targets performed equally well. Though not statistically significant( p>
0.05), there was a reduction in mosquito abundance in the resort area when the barrier was functional. These
data indicate that with refinement this mosquito management technique may be practical in certain
situations.
Keyword Index:
Aedes taeniorhynchus, Anopheles atropos, Culex nigripalpus, mosquito, attractant,
carbon dioxide, octenol, barrier, traps, target, control.
INTRODUCTION
impact of a perimeter barrier of attractant- baited traps
on mosquito abundance in a resort area( 1994), and the
Development
strategies
are
new
mosquito
that reduce reliance
needed
to
environmental
the
counter
concerns,
approved chemical
and an
of
a
on
management
impact of a similar barrier of insecticide- impregnated
chemical pesticides
cloth targets on mosquito abundance, i. e., how would
by
targets perform compared to traps to reduce mosquito
problems
dwindling
insecticides for
posed
selection
of
mosquito control,
increasing number of mosquito species that have
abundance
in
a
designated
area (
1995).
This paper
summarizes the data obtained for the first phase of this
project.
developed resistance to these approved insecticides.
One
possible alternative is to use strategically
attractant- baited traps and/ or targets.
Research towards the development of such a strategy
for
the management of the
black
salt marsh mosquito,
Aedes taeniorhynchus( Wiedemann),
and other species
associated with estuarine ecosystems
was
initiated in 1993. The
project
Florida,
its abundant salt marsh mosquito populations and limited
access ( either by boat or helicopter), that reduced the
this research
likelihood of vandalism. This ca. 14. 5 km long island,
the largest remaining undeveloped barrier island in
friendly, attractant- based operational mosquito
Florida, combines with a number of smaller islands to
management program.
of
south
Study Site. Key Island, located in Collier Co., FL,
immediately south of Naples, was selected because of
of a cost- effective, environ-
is the development
mentally
in
placed
ultimate goal of
Specific
goals
this research program were the
species composition and spatial
for the first phase
determination
of:
distribution( 1993), the
provide an essential physical barrier between the 3644
ha Rookery Bay National Estuarine Research Preserve
and
the Gulf
of
Mexico. These islands
contain critical
Journal of Vector
172
habitats,
wildlife
sea
is
turtles
including nesting areas for endangered
The
and shorebirds.
a mixed` jungle" of red and
thickets,
vegetation on
Fauna includes bobcats,
This combination of
attractants and release rates was based on studies
black mangroves, palmetto
Australian
December, 1998
found on the pressure regulators.
Key Island
arboreal orchids," slash" pine, and
Ecology
previously conducted with Everglades mosquito species
(
Kline et al. 1990, Kline et al. 1991b, Takken and Kline
gopher
1989). The traps were hung from metal poles so that the
tortoises, raccoons, deer, as
well as
nesting of the Great
Pileated Woodpeckers, and
top of the trap was approximately 1. 8 m above ground
Horned Owls,
level.
pine.
Osprey,
armadillos,
In 1993, 42 traps were placed throughout the north
Towhees.
Because
its location
of
island has been the
this
environmentalists
decades. In 1993,
island
was
relatively
center
pristine state,
between
of conflict
land developers for
and
all
and
but the
northernmost
several
24. 3 ha of the
by the state of Florida and
Rookery Bay Preserve. A resort
purchased
incorporated into the
in the
which remained
The land
corporation.
incorporation into the
seasonal incidence and relative abundance, these traps
the
also provided information on spatial distribution. From
possession of a private
July 16 through August 18 only six of the traps, i.e., 1, 5,
with small rustic guest cottages was established on
24. 3 ha,
end of Key Island within the resort area and along the
various nature trails established by the proprietors of the
resort( Fig. 1).
All traps were baited as described above
and operated continuously from June 21 until July 16. In
addition to providing data on species composition,
purchase
and
has had
preserve
an
impact
6, 33, 35, and 43 were used to obtain additional baseline
on
data. Data obtained in 1993 determined placement of
Collier
ten surveillance traps in 1994. These 10 surveillance
personnel consider
traps were positioned so that five were located on each
important resting stop for salt marsh
mosquitoes believed to be involved in massive migrations
side of a protective barrier, i. e., outside and within the
Everglades into
These surveillance traps were operated from May 19
Naples, CMCD has
through July 20. In 1995, surveillance trap locations
that might
mosquito control options
Mosquito Control District( CMCD)
this
island
and the
Naples. To prevent their invasion
aerial
on
utilized.
as an
from Ten Thousand Islands
relied
be
subsequent
sprays
of
Baytex ( fenthion).
of
Since
Rookery Bay Preserve allows no aerial sprays of chemical
its lands,
pesticides over
to
methods need
and
estuarine
receptive
preserve
to the
personnel
resort owners
have become very
proposed research project.
Surveillance:
dance,
alternative mosquito control
be developed. Thus, the
species composition, relative abun-
seasonal, and spatial
distribution
The
studies.
512 CDC type trap( John Hock Co., Gainesville,
FL) was used without a light source. Each trap was
model
baited
wick(
this
a
3- ol
microreaction
PA) fitted
with
octen-
with plastic
Dills 15-
Octenol
Kline
et
to the
1991a).
al.
stage
pressure
was monitored
This provided the same defined time interval for all
weekend work and servicing of traps during inclement
folded into
a
using a
V- shape
With
weather.
Collections were made into 568 ml ( pint) Mason
trap
illustrated in
agent. All collections were weighed and then stored in
from
double-
a freezer until further processing. In 1993, the entire
sample was frozen after weighing. Therefore, upon
Victor Model VTS 453B;
removal from the freezer, the entire sample was
Denton, TX). Gas flow
reweighed and a 0. 5 gm subsample was taken, identified
neoprene septum.
4 mg/ h
CO2
CO2 was
200
Company,
metered
ml/ min
using
a
the
a
over
9kg
using a compact# 12 flowmeter( Gilmont
delivered to its
to species, and an estimate of the total trap collection
release
extrapolated from this data. In 1994 and 1995, the total
5 cm from the top trap entrance via polyethylene
sample was weighed and a 1 gm subsample removed
tubing.
metering
were
Bottle Rotator- Model 1512, John W. Hock Company,
Gainesville, FL) was used with each surveillance trap.
jars containing a small piece ( ca. 2. 5 x 5. 0 cm) of
Revenge®Bug Strip""( Roxide International, Inc., New
Rochelle, NY) with dichlorvos ( 18. 6%) as the killing
the
Instruments, Barrington, IL)
point ca.
1995 an interval rotator device with timer ( Collector
vicing of traps. This also helped to reduce the need for
release point(
regulator (
Victoria Equipment
Trapping intervals for surveillance traps were
approximately the same each day and were serviced
daily in the same sequence between 8: 00- 10: 00 AM
EDT. The only major change to this routine was that in
Supelco, Bellefonte,
vials were affixed near
compressed gas cylinder at
were operated
locations and also provided flexibility in the daily ser-
octenol was released at ca
entrance adjacent
1994 and
continuously from May 15 through July 6.
was released
and neoprene septa
contact with
technology
period.
lids
ml;
cm pipe cleaner)
its point in
24 h
5
vials (
remained the same as
dioxide
( octenol) and carbon
Octenol( Aldrich, Milwaukee, WI)
CO2).
from
1-
with
area designated for protection from mosquito invasions.
To
assure
accurate
valves ( series
used
in
place of
and
flow
calibration,
M, Nupro,
the
standard
micro-
immediately before placement into the freezer. Only
OH)
the subsample was identified to species. An estimate of
Willoughby,
open- shut valves
the total collection size
was made
by
extrapolation.
If
December, 1998
Journal of Vector
Ecology
173
Naples
r__,
42
l
7
n
Ci
0rdo.'
41
26•_
24
27
a\.-
23
28
t
limmit
22
29
X7
21
40
3 1•
30
20
1•
18
32•
16
I•
1
3•
4
1.
12
34•
Q
I•
10
36•
g
1
1J `
1J
7
3•
31•
36•
5.
l(_
C
•
2
50
0
I
N
A
E
W- • •
Scale I in. = 725 ft.
2. 54
Figure 1.
1993 distribution
of
Model 512
cm. =
S
22 I m.)
surveillance traps(
black dots), baited
with
CO2(
200
cc/ min)+
octenol
ca. 4 mg/ h), on the northern end of Key Island, Naples, Collier Co., FL
the total
sample was
counted and
1
gm or
identified to
less the
entire sample was
species.
to CO2 or calf bait( 15 m for CO2 alone and 9- 18 m for
Barrier studies— baited traps. Based on 1993 data,
a single
line
baited the
barrier
protective
same as
along New Trail
to a small cove [
the
and
its
Fig.
of
of attraction for some species of West Africa mosquitoes
52 Model 512 traps,
surveillance
traps,
one calf) and partially to logistical restraints, i. e., the
fact that this spacing yielded the maximum number of
was placed
traps that we felt we could supply with CO2. Barrier
extensions( extended northeast
traps were operated 24 h/ day from June 14 through July
2]
and southwest
Mexico). The trap barrier
was
the entire northern resort area
These barrier traps
to the
strategically
Gulf
of
placed so
with cottages would
16. 5
be
11.
The ten surveillance traps were operated as a pre-
treatment from May 19 through June 13, and as a post-
treatment from July 12 through July 20.
m
Barrier studies— baited targets. Since our ultimate
This spacing was based partially on the data
reported by Gillies and Wilkes( 1972) on effective range
objective was to kill the mosquitoes rather than retain
protected.
apart.
were placed ca.
them
for
surveillance purposes,
it
was
decided that
a
174
Journal of Vector
Ecology
December, 1998
15
i
IIMININ
i
20
AM
C
25 •
5•
10
30
1
9
35
h
r
40
45
Z
50
5
60
8
\
7•
Figure 2.
Illustration of placement of barrier of traps( 1994)/ targets( 1995) on Key Island and distribution of the
surveillance traps. The resort area( area to be protected) is cross hatched.
barrier of targets,
be
which require
less
more economical than traps.
maintenance, would
Therefore, in 1995 the
barrier traps were replaced with cloth targets impregnated
with
an
insecticide ( lambdacyhalothrin), baited
the same
the traps.
objective
same
The ten surveillance traps were
operated during this time and as a pretreatment from
May 16 through June 3; and as a posttreatment from
June 29 through July 6.
combination and release rates of attractants as
The collapsible targets consisted of cylinders ( ca.
Each trapping site remained the same. The
was to determine if targets would have the
60.25 cm length x 53. 21 cm diameter)( Fig. 3), of 60%
polypropylene black shade cloth ( DeWitt Company,
impact
within
with
June 4 through 28.
as
baited traps
the resort area.
Targets
on
mosquito abundance
were placed on site
from
Sikeston, MO) treated with an EC formulation( 120 gni/
1)
of
lambdacyhalothrin
at
0.2 g A.I./
m2.
The targets
December, 1998
Journal of Vector
Ecology
175
F-'
M! w''
4.
p114..
M*.
YYIIiiiLE+.
d+.
x'"§+
y.M
9• y`....
o
E
ka.
3.,. ,
iii94e7...
4„, -..„ .
1
m
3" 41
A§`.
4104 . .
et
41 '
SRC
'".... .,°
a
t:
1 M,
fir,
j,
04,,'
F
ro.. ;.
at
4
i
re
f
a y
'..
r
4._
x'
1' 1'
i
1
4
ai,
1
4;-),,,...:
4 ,
msµ„
4,0(
4
v
r4
t-
y^,
f
big
Figure 3.
were
cloth
to upper and
lower 5. 13
1. 56 mm thickness)
The targets
were constructed
The
covered with
bottom
was
rest on the
suspended
traps
to allow
be
upper surface of
left
from
bands
for
removed
of
lexan
a
for
10. 26
cm
bioassay
the cylinder was also
insecticide impregnated
shade cloth.
The
open to allow mosquitoes to enter and
inner
in 1994
cm wide
by means of six sheet metal screws.
of cloth, which could
purposes.
4,
T
r
Photograph of cylindrical shade cloth target with stake holding CO2 release tube and octenol vial.
fabricated by attaching the insecticide impregnated
overlap
4)
surface of the target.
poles
with
that
had been
used
string tied to the sheet
Targets
were
for the barrier
metal screws
of the target' s upper ring. Each target was suspended so
the
bottom
was
ca.
15
cm above ground
level.
The
octenol vial and CO2 release tube were taped( duct tape)
to a short stake located centrally under each target so
odors were dispensed ca. 67 cm above ground level.
Our goal for 1995 was to utilize a target impregnated
with an insecticide formulation that would be effective
for at least 30 days before retreatment was necessary.
Lambdacyhalothrin was selected as the insecticide of
choice based on preliminary screening of seven candidate
insecticides (
unpublished
data)
against
a
laboratory
Journal of Vector
176
to treat the shade cloth
mixture required
by
first
determining
the
was
Psorophora ciliata( Fabricius), Aedes infirmatus Dyar
determined
and Knab, Aedes atlanticus Dyar and Knab, and Aedes
to
amount of water required
mitchellae (
0. 2 g AU
was obtained by mixing2.4mlofLambdacyhalothrin
saturate
m2)
the
fabric. The desired treatment
E. C.( 119 g AI/1)
the
The amount of insecticide
ofAe. tacniorhynchus.
colony
195
with
cloth required
to
ml of water.
make
the target
were placed
distribution
and
into
varied
fence
hung from a
dry. This treat-
30
to
patterns (
from
week
<
in
week
to
94. 2%.
1. 0%
Culex nigripalpus started out in week 1 as
37. 7%
stable
Effective field
declined to 4.4%.
longevity
by bioassaying
current
study
from five randomly
each
sampling
hynchus were
minute.
Two
beginning
exposed
to
of
exposing
bioassay. Mortality
hour
All
post- exposure.
targets
Data Analysis. In 1994
the
inside
mean outside/ mean
determined
was
treatment(
Data
daily,
analyzed
and
for
each
by
comparison procedures(
catch
surveillance
the
trial
were
for
would
an
functional)
barrier traps/ targets.
the
increased
the treatment period
during
that mosquitoes
were
being"
by
on June 28.
Several smaller peaks occurred after the
In 1995, the same three species again dominated
indication
collections (
TABLE 1),
but their relative abundance
was different compared with the previous two years.
perimeter
Aedes
taeniorhynchus comprised only slightly> 54% of
the collections. Aedes taeniorhynchus populations were
highest in
May ( Fig.
declined for the
Surveillance:
abundance.
species
In 1993,
were captured,
composition
an estimated
of
16
and
consisting
dominated by three species: Aedes
taeniorhynchus(
84.7%), Culex
and Anopheles atropos
species
Psorophora
sollicitans (
in
order of
columbiae
nigripalpus
decreasing
(
Theobald
Dyar and Knab( 1. 4%).
Dyar
Walker), Anopheles
abundance were:
and
of
(
Fig. 5), except that after reaching a spike on July 6, this
species virtually disappeared from the trap collections
for the remainder of the study. Anopheles atropos was
the second most abundant species collected, composing
ca.
28%
of
the collections compared to
only 1- 2% the
Knab), Aedes
previous two years. The An. atropos population was
Walker,
relatively high throughout most of the evaluation period
in 1995( Fig. 6) until the end of June when it crashed and
crucians
Mansonia spp., Psorophora howardii Coquillett,
Psorophora ferox ( Von Humboldt), Coquillettidia
perturbans (
after which they gradually
remainder
nigripalpus comprised
6, 592, 164 mosquitoes
species/ species groups.
4),
this study.
Culex
16%
of
the
collections.
slightly>
Culex nigripalpus followed a similar pattern as in 1994
relative
collections were
Other
Aedes taeniorhynchus had several
and no Ps. ciliata were caught.
the
RESULTS
13. 8%),
and
the most part of 1994 this species was relatively low in
the trap collections. The only differences in species
composition were that some An. quadrimaculatus Say
of mean
barrier.
Trap
8. 4%),
6) populations had two spikes, June 24 and June 28. For
ratio that
the
nigripalpus (
barrier of traps was turned off. Anopheles atropos( Fig.
means
of
atropos(
until the third week in June. The largest peak occurred
of
trial, trial,
A
was an
filtered out"
of
periods.
and
indication
effectiveness of
Cx.
peaks of abundance, the largest occurring June 27- 30
Fig. 4). After this major peak, the population crashed
and never rebounded during our evaluation period.
Culex nigripalpus ( Fig. 5) populations were very low
they
and post-
pre-
89. 6%),
1. 8%).
time
collections
pre-
inside traps
give
taeniorhynchus (
comparisons
trap
SAS GLM
the three major species as was found in 1993, i. e., Ae.
1
An.
SAS 1988). This ratio
outside/ mean catch
post-
year,
barrier traps/ targets
were
entire
by
4% in week 8.
operated
five inside traps. A ratio
outside and
to 1.
with each
min. and
1995, the efficacy
and
barrier traps/ targets was determined
between the five
increased to 12. 9% in week 7 at which time it declined
In 1994, an estimated 3, 045, 312 mosquitoes were
were
continuously( i. e., 24 hours/ day) for the
were in the field.
29. 5%, respectively. In week 8 it
Anopheles atropos composed< 1%
collected, with a similar relative abundance pattern for
30
at
and
one
to untreated pieces of shade cloth were run
determined
32. 0
mosquitoes
each piece of cloth
consisting
was
for
in week 5; weeks 6 and 7 remained relatively
at
of the collections during weeks 1 through 3, and gradually
of
Ten field- collected Ae. taenior-
week.
checks
the
pieces of cloth removed
targets at the
selected
during
monitored
was
Although Ae.
of the total collection, and gradually peaked at
effective specifica-
day
Relative abundance
TABLE 1).
1 to 57. 6% in week 7. In week 8 it rebounded
tion when exposed to natural environmental conditions.
ment combination met the
Figs. 7- 9).
to week(
collected, its degree of dominance fluctuated from 99. 1%
treatment,
with clothespins, and allowed
Each of the three most abundant
taeniorhynchus was always the dominant species
an
approximately 8 liter resealable freezer bag. The bag
was sealed and inverted daily for four days. The cloth
was removed one week after
Dyar).
species had a different temporal( Figs. 4- 6) and spatial
rate(
This mixture
December, 1998
Ecology
Walker), Culex ( Melanoconium) spp.,
never rebounded.
Psorophora
ciliata
No Ae. mitchellae were collected.
specimens
were
again
found in
December, 1998
Journal of Vector
Ecology
177
INa.
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Journal of Vector
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December, 1998
Ecology
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December, 1998
Journal of Vector
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179
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180
Journal of Vector
December, 1998
Ecology
42
260
041
250
024
027
23
028
I
22
029
040
021
0 30
31•
043
020
19•
I
018
320
170
16
154
0 12
330
13
34•
10
9•
08
35•
30
2
0
•
,/
Average #
Aedes taeniorhynchus
per trap/ night
20, 000
7
37
m > 10, 000 - 20, 000
36
5, 000 - 10,000
61>
0 >
1, 000 -
I 1<
1, 001
5, 000
5
Figure 7.
Relative spatial distribution of Aedes taeniorhynchus on the northern end of Key Island, June 22- July
16, 1993.
some collections.
This
was
the
first
year
that some
Wyeomyia spp. were found in the collections.
Spatial distribution. Mean trap collection and
relative spatial distribution data for the three major
species are
illustrated in Figures 7- 9. Asa generalization
forAe. taeniorhynchus(
trap
was placed,
The majority
Fig. 7), the further
between 5, 000 and 10, 000 Ae. taeniorhynchus per trap
night. Culex nigripalpus was generally most abundant
along New Trail, the eventual location of the perimeter
barrier. Anopheles atropos was most abundant in traps
located
south and east of the resort area.
Very few
southeast the
specimens of this species were collected in the traps
the greater the mean collection size.
located along New Trail. Trap# 38 and Trap# 2 located
of traps
located along New Trail
averaged
within
and
just
outside the resort area,
respectively,
December, 1998
Journal of Vector
Ecology
181
42
260
041
25
024
J027
23
28
22
029
/
40
021
31•
013
X30
020
190
I8
32•
170
16
150
331>
El 14
13•
I2
34•
I
1 O 10
9•
35
30
o
36 ,/-
3740
/
Average #
oCulex
s
i
per trap/ night
j
7
nigripalpus
• 2000
>
1500- 2000
2
0 >
60
i<
N
1000 - 1500
500 - 1000
501
4
5•
Figure 8.
0
Relative spatial distribution of Culex nigripalpus on the northern end of Key Island, June 22- July 16,
1993.
caught a moderate number of this species.
was functional. Similar trends were not obtained forAn.
Effectiveness of trap/ target barrier. No statistical
differences ( p > . 05) among the outside/ inside ratios
atropos for both years. No filtering was indicated in
during
any
period were observed either year.
the trends ( TABLE 2)
nigripalpus
were
indicated for
filtering
for Ae.
similar
these
taeniorhynchus and
for both
species
out" some of
both
However,
was
years.
that the
species
during
Cx.
The trend
barrier
was
the times
it
1994 and only slight filtering in 1995.
Results of
bioassays from targets indicated 100% mortality 30 min
post-exposure for the first three weeks. By the beginning
of the fourth week some of the targets were showing ca.
90% mortality 30 min. post- exposure, but by one hour
post- exposure all the target species tested resulted
in
182
Journal of Vector
December, 1998
Ecology
042
041
26
250
24
27
1
1
23
028
i
El 22
1
i
029
E4
02
3
03
20
19
I8
32
17
IMINIIIIMINIIMMINIMIN
WIIIIIIIIIIIMINIIIMINIIIIIMI__
16
1
337
1
11014
13012
34
INIIMI
0.
1
Anopheles atropos
9o
35
08
v
3 '
Average #
10
370
300
m > 200 - 300
1
36
per trap/ night
K •>
7
100 - 200
6•
o >
50 - 100
11 <
51
N\
4/
r
Figure 9.
l
5m
Relative spatial distribution of Anopheles atropos on the northern end of Key Island, June 22- July 16,
1993.
100% mortality.
No
check
either post- exposure period
mortality
any
was observed at
insecticide applied, allows the choice of where and
when the spot treatments will be made, and greatly
week.
reduces the impact of insecticides on nontarget
DISCUSSION
organisms.
This study, to the best of our knowledge, is the first
The use of attractant baited traps/ targets for removal
attempt to use a barrier of baited traps/ targets for
trapping
of
mosquitoes
conventional
insecticides
the
approach
over
advantage
offers
of
large tracts
of
an
broadcasting
of
to the
population management of any species of mosquito. In
chemical
Zimbabwe( Torr 1994), tsetse flies( Glossina spp.) are
has
controlled using insecticide- impregnated targets baited
alternative
land. This
approach
greatly reducing the
amount
of
with a
blend
of acetone, octenol, and several phenolic
December, 1998
Journal of Vector
v
v
4
00
v
o
N
o
compounds. While we are disappointed that our barrier
of traps/ targets did not result in a more dramatic reduction
in mosquito abundance in the resort area, we remain
-.
TN
encouraged by the fact that there was any reduction. In
`°
O
a
0
in
rn
o
CA
probably have to be made in trap/ target type, spacing,
strategic placement, as well as the quality and quantity
of attractants used. Among the refinements under
M
N
consideration are reduction in distance between traps/
5
R °°
targets( e. g. utilize a spacing of 6- 8 m), increased CO2
o
4
complete encirclement of the area designated for
U
o
M
O
levels, a double and staggered line of traps/ targets, or
o
v:s
definitely toward reduction of mosquito abundance
From the outset we knew that adjustments would
r
00 4
R °
'"
both years when the barrier was present, the trend was
within the area designated for protection.
o
cr
N
00
183
o
rn ° v
d
Ecology
protection.
Therefore, further studies on mosquito
00
o
o
o
behavior need to be conducted, including movement
N
v
a
N
3
n
06 r-
"'
n
o
0
patterns( horizontal, vertical and diel) and their responses
to different kinds and heights of traps/ targets. Do these
o
4
o
M 'r
N
three major species respond to different combinations
o
of chemical and physical cues? Do they fly low or high?
One of us( DLK) observed early morning movements of
An. atropos along the sandy beach parallel to the island' s
o =
coastline. This type of movement pattern allowed at
06
o
s
co
en
6
'r
14
o
o
N
3
acis
a.
vs
i
N
VD
rn
m -
protective barrier was least effective for this species.
cNV
Another important reason why the barrier may have
been least effective for this species may be its spatial
distribution. Assuming that spatial distribution for this
o
b
Qz
least a portion of this species to completely circumvent
00
D
O
"'
00
M
i
o
N
o
o
o
the barrier. Indeed the data( TABLE 2) indicate that the
o
species was the same in 1994 and 1995, as it was in 1993
Fig. 9), then very few An. atropos would have been
aN
attracted to the traps/ targets making up the barrier along
M °
7
N
G
aN
o en
o °`
r
o
o
en
N
rn
a-
r
N
v
0
two major species. In contrast, Cx. nigripalpus should
have been the most impacted of the three major species
since its spatial distribution pattern indicated that its
ppulations were most concentrated along New Trail.
Indeed the barrier' s impact was greatest on this species
oo
or
New Trail. Perhaps that is why the barrier was not as
effective in" filtering out" this species as it was the other
r,
o
00
kn
en
00
0
0
E
N
Q
.--,
o
4 n o
00
7:). — —N
N
a_
both years.
Therefore, we remain optimistic that this technology
will be developed where it can be used to effectively
reduce mosquito annoyance in areas designated for
r''
protection.
This optimism is based partly on the
concurrent cooperative research projects that we initiated
with academia, government, and private industry
0
C
Ct
o
a
VI
6,
Gs
° ...
U
0
4..
a
t
0°
f'
C.)
o
personnel at the outset of phase one of this project.
o
These cooperative research projects have focused on the
z
development of better traps/ targets, attractants, and the
h
use of spatial analysis
to
strategically locate the traps/•
y o
targets for maximum impact on the target species.
Q
These
Q
efforts
have already
resulted
in the development
184
Journal of Vector
TABLE 2.
Ecology
December, 1998
Effectiveness of a single line barrier of traps/ targets to prevent mosquito invasions into
designated
a
area;
z =mean number mosquitoes captured/ trap night and standard
error of mean( S. E.)
1994
Period
Area
1995
T (S. E.)
0/I
z (S. E.)
0/I
Aedes taeniorhynchus
Inside
Pretreatment
2964. 86 ( 458. 01)
Outside
2. 57
752. 64 ( 138. 58)
7634. 50 ( 919. 00)
Inside
Treatment
2572. 48 ( 539. 79)
Outside
3. 21
325. 87 ( 37. 27)
8249. 58 ( 122. 30)
Inside
Posttreatment
466. 47 ( 88. 22)
Outside
2. 54
1910.01 ( 312. 73)
3. 19
1040.08 ( 225. 47)
1. 69
141. 04
789. 73 ( 122. 3)
( 28. 48)
4. 11
580. 16 ( 75. 69)
Anopheles atropos
Inside
Pretreatment
Outside
Inside
Treatment
Outside
Inside
Posttreatment
21. 88
(
44. 24
( 16. 35)
100. 22
( 28. 89)
160. 79
( 42. 72)
9. 19)
55. 73 ( 16. 07)
Outside
2. 02
392.91 ( 88. 72)
1. 58
621. 54 ( 144. 87)
1. 60
1. 05
58. 39 ( 22. 13)
374. 40
( 51. 96)
687. 02
( 93. 18)
19. 46
(
5. 37)
42. 12
(
9. 73)
3. 01
(
0. 64)
3. 64
(
0. 97)
1. 83
2. 16
Culex nigripalpus
Inside
Pretreatment
Outside
Inside
Treatment
2. 64
(
0.56)
10. 70
(
2. 69)
190. 62
Outside
( 59. 42)
4. 06
5. 28
1007. 41 ( 280.51)
Inside
Posttreatment
566.04 ( 258. 44)
Outside
3. 61
2045. 92 ( 586. 31)
140. 54
( 38. 82)
353. 99
( 58. 83)
238. 08
( 84. 93)
1. 21
2. 52
2. 35
560. 19 ( 130. 33)
All Mosquito Species Combined
Inside
Pretreatment
2990.72 ( 462. 65)
Outside
Inside
Treatment
2872. 90 ( 565. 08)
Outside
Posttreatment
1089. 68 ( 303. 89)
Outside
new
trap
types,
have
greater
than those obtained
resulted
attractant
unpublished
heat,
and
electricity to
in
which
in
by
new
operate
the
with
the
2. 21
849. 24
( 82. 95)
2. 51
2134. 12 ( 263. 65)
2. 67
400. 41 ( 107. 85)
systems
3. 04
1218. 18 ( 162. 69)
are
now
commercially
available.
New host
odors have been isolated, identified, and successfully
tested in the laboratory ( unpublished data). Another
one
reason for optimism is that the control of tsetse in
trapping technology has
Zimbabwe, which is the best example of the use of
used
which even produces
vapor)
in
field
5- 10 times
the traps/ targets
combination
water
small scale
captures which are
data). One
been developed,
CO2,
3. 28
2907.03 ( 620.04)
studies
same
1149.03 ( 207. 69)
2536. 88 ( 427. 85)
9437. 29( 1650. 46)
Inside
of several
2. 57
7698. 23 ( 925. 49)
and
phase
its own
attractants
generates
its
own
trap. This trap is already
commercially available. Progress has also been made
in the attractants area. Several different octenol delivery
kairomones to control a medical/ veterinary entomology
pest, was not an overnight success ( Vale 1993, Torr
1994).
Success was achieved as the result of a long,
dynamic process over many decades through close
interdisciplinary collaboration of many dedicated
December, 1998
Journal of Vector
Our three- year research
scientists.
first step towards
the
project
development
is simply the
of attractants/
Ecology
185
Africa. Bull. Entomol. Res. 61: 389- 404.
traps/
Kline, D. L., W. Takken, J. R. Wood, and D. A. Carlson.
1990. Field studies on the potential of butanone,
carbon dioxide, honey extract, 1- octen- 3- ol, Llactic acid and phenols as attractants for mosquitoes.
personnel
Kline, D. L., D. A. Dame, and M. V. Meisch. 1991a.
Evaluation of 1- octen- 3- ol and carbon dioxide as
targets for mosquito management.
Acknowledgments
Med.Vet. Entomol. 4: 383- 391.
This
by
project was a cooperative effort
of the United States Department of Agriculture( USDA),
Agricultural Research Service ( ARS), Center for
Medical, Agricultural
Veterinary Entomology
and
CMAVE), Gainesville, FL,
and
Collier Mosquito
Control District ( CMCD), Naples, FL. The
authors
like to thank H.T. McKeithen ( USDA, ARS,
CMAVE) for his technical help in setting up and
servicing
would
the
field traps/ targets, identification
conducting
the
statistical
the graphics
preparing
field assistance
Salinas
and
of
analyses
used
of
of
appreciated.
E.
the
data,
and
illustrate the data. The
to
E. Lavagnino( USDA, CMAVE), A.
C. Laidlaw- Bell ( CMCD)
identification
mosquitoes,
Lavagnino
also
was also much
assisted
in
the
of mosquitoes.
attractants for mosquitoes associated with irrigated
rice fields in Arkansas. J. Am. Mosq. Contr. Assoc.
7: 165- 169.
Kline, D. L., J. R. Wood,
and
J. A. Cornell.
1991b.
Interactive effects of 1- octen- 3- ol and carbon
dioxide
on
surveillance
mosquito (
and
control.
Diptera:
Culicidae)
J. Med. Entomol. 28:
254- 258.
SAS. 1988. SAS/ STAT user' s guide release 6. 03. SAS
Institute, Cary, NC, 1, 028 pp.
1- octen- 3- ol as mosquito attractants. J. Am. Mosq.
Contr. Assoc. 5: 311- 316.
Torr, S. J.
REFERENCES CITED
1994.
The tsetse ( Diptera: Glossinidae)
story: implications for mosquitoes. J. Am. Mosq.
Contr. Assoc. 10: 258- 265.
Gillies, M. T.
and
T. J. Wilkes.
attraction of animal
mosquitoes.
baits
Studies in
a
1972.
The
and carbon
freshwater
range of
Vale, G. A. 1993. Development of baits for tsetse flies
dioxide for
Diptera: Glossinidae)
West
Entomol. 30: 831- 842.
area of
in Zimbabwe.
J.
Med.
Olfactory Responses and Field Attraction of Mosquitoes to
Volatiles from Limburger Cheese and Human Foot Odor
Daniel L. Kline
USDA, ARS, CMAVE, P. 0. Box 14565, Gainesville, FL 32604 USA
Received 26 August 1998; Accepted 24 September 1998
ABSTRACT: Olfactory responses of femaleAedes aegypti( Linnaeus) to various odor stimuli were studied
in a dualport olfactometer. Responses( i. e., the percent of ca. 75 available female mosquitoes in flight
chamber entering each olfactometer port) were studied toward clean conditioned air( control), human foot
skin emanations( collected on socks by wearing them for three days), human hand, and Limburger cheese.
Mean
percent response was
66. 1%),
Limburger
cheese(
greatest
6. 4%),
to the
human hand ( 80. 1%),
and control(<
0. 1%).
followed by the human worn sock
In field studies the worn sock alone attracted
very few mosquitoes but a synergistic response occurred to the sock+ carbon dioxide baited traps for most
species of mosquitoes in six genera( Aedes, Anopheles, Coquillettidia, Culex, Culiseta, and Psorophora).
This synergistic effect persisted even when the socks were exposed to environmental conditions for eight
consecutive days. Limburger cheese alone did not attract mosquitoes to traps compared to unbaited traps,
and there appeared to be a slight repellent effect for most mosquito species when used in combination with
carbon dioxide.
Keyword Index:
Foot odor, olfactometer, Limburger cheese, mosquitoes, kairomones, host-seeking.
INTRODUCTION
between octenol and CO2 has been reported for several
mosquito species( Takken and Kline 1989).
A
major
research
is the development
tally friendly
research
is
methods
management
Recently, Limburger cheese has been identified as
of selective, environmen-
an effective attractant for Anopheles gambiae s. s. Giles
of current
emphasis
pest
of control.
One
no exception.
Mosquito
control
such mosquito manage-
(
Knols
(
1996) implied that Limburger cheese is an effective
and
De
1996, Knols
Jong
et al.
1997).
Knols
of
attractant for this species because the blend of volatiles
attractant- baited traps/ targets to capture/ kill nuisance
from this cheese is very similar to foot odor. The use of
analytical chemistry confirmed the similarity of fatty
acids found emanating from Limburger cheese and foot
ment concept
mosquitoes(
depends
the
the
subsequently,
for
success of
development
discovery
traps/ targets
scrutiny is the
under
Kline 1994). The
upon
technology,
and
currently
use
this approach
of efficient
trapping
scrapings (
of new, effective attractants,
strategic placement of these
maximum
impact
on
the
baited
study
Meijerink 1997).
The foot is a
Knols 1995). The research reported herein was conducted
to evaluate the potential use of Limburger cheese and
The present
the
and
preferred biting site for An. gambiae s. s. ( De Jong and
target
population.
on
Knols
work was undertaken as part of a
behavioral
The
semiochemicals.
research program
responses
larger
of mosquitoes
ultimate goal of
this
is to isolate and identify
phase of
to
the
new attractants.
human foot volatiles as attractants for Aedes aegypti
(
Linnaeus)( olfactometer studies) and natural populations
of mosquitoes associated with wooded wetlands( field
studies).
To date, few chemicals other than carbon dioxide have
been isolated
and
identified
as
behaviorally
active
for
host- seeking mosquitoes. 1- Octen- 3- ol ( octenol),
originally identified from ox emanations ( Hall et al.
1984) as an attractant for tsetse flies has been shown to
elicit positive
Takken
and
behavioral
responses
from
Kline 1989, Kline 1994).
mosquitoes
Synergism
Attractants
Limburger cheese.
Amish Country Limburger
cheese( Amish Country Cheese, Linwood, MI 48634)
was purchased from
a
local
supermarket.
It was used for
Journal of Vector
December, 1998
both
laboratory
field
and
One hour before initiation of tests, ca. 75 femaleAe.
fiber
aegypti were aspirated into the olfactometer chamber
consisting of 82% cotton and 18% polyester
in both olfactometer and field studies. Black
and allowed to acclimatize for ca. one hour before
crew socks (
content
filters and then heated and humidified, if necessary.
studies.
in these trials.
Socks. Two types
White
of socks were used
AthletechTM,
MI)
Troy,
with
Farah®
crew
casual
Greensboro, NC)
24%
the
wore
lay
Lycra® spandex
1%
each
Corp.,
were used
human
a
subject
floor
the
by
bag
(
the traps and
olfactometer
chamber.
A
1= top; 2= middle cage; 3= bottom chamber) and port
or B).
For each test, one port contained a candidate
A
human arm, sock worn by human, or
Limburger cheese) and the other remained empty
attractant (
were
were
of
completely randomized design was used for cage position
(
consecutive
1800, they
the shoes, i. e., they
socks were removed at
on
upwind
75% Hi- Bulk
content of
study
from 0600- 1800 for three
socks
to
fiber
For
studies.
days. When the
allowed
with
nylon, and
only in field
Kayser- Roth
socks (
Treatments were placed into two test ports
testing.
were used
acrylic,
187
Ecology
(
control).
Treatments( worn folded sock or Limburger
the
cheese) were presented in glass Petri dishes that were
a resealable plastic
cleaned and sterilized in a vacuum oven and then handled
following
only with gloves to minimize any chance of
morning. Two subjects, the author of this paper( DLK)
and his son( JDK), contributed worn socks for the field
contamination. A small block( ca. 12. 4 gm) of Limburger
not placed
day
third
bag
special
they
when
White
studies.
in
laboratory
on
by
socks were worn
the
both
of
cheese was placed on the sterilized glass petri dish. A
fresh piece of cheese was used for each test. Treatments
subjects and
and
JDKWS for these
Black socks were worn only
by DLK( designated
exposure period, the relative numbers caught in the
the
white socks.
treatment and control traps upwind of each port were
the
author was also
recorded.
designated
were
the evening
until
were placed
for transport to the
studies.
as
in any
DLKWS
as
DLKBS) in the
same manner as
Human hand. The left hand
of
were alternated
between
ports.
After a three minute
used as a treatment in the olfactometer studies.
Substances
to
be
obtained
attractive
to
from human hands have been
several mosquito species
Schreck
olfactometer studies(
Carbon dioxide.
from 9
kg(
for
rate used
was
with
20 lb)
treatments that
of
flow rate
CO2
supplied
was
compressed gas cylinders.
500 ml/ min. Control
Field Trapping Studies
Field trapping experiments were conducted from
May through June 1997 against natural populations of
shown
previous
1981, 1990).
Carbon dioxide
trap- bait
all
et al.
in
woodland
The flow
included
CO2
is
which
comprised of a pressure regulator
ml/ min
fixed
fittings.
Olfactometer Studies
worn
sock
volatiles
Limburger
and
emanating from
cheese
orientation response, six to eight
host- seeking Ae.
reared,
port olfactometer,
Posey
choose
is
illustrated
1998). This
et al(
old,
constructed
sleeves,
in
of clear
acrylic,
tested
filtered
allows
temperature (±
precise
humidity (±
was used
entry
for
2%)
external air
control.
assays.
through the
in
a
dual-
Only
Outside
ground level.
Paired comparisons were made between CO2- baited
PJ traps with and without the addition of a human worn
three test
sock. Treatments were alternated on a daily basis between
two sites. Two studies were conducted between May
14- 22, 1997. In the first study a sock worn by the author
paired removable
supply
0. 5 ° C)
and
is
that
system
time
air was conditioned prior
the mosquito
was taped near the bottom of one of the PJ traps. In the
relative
one chamber at a
choice ports,
the attractant is released, was ca. 50 cm( 19. 69 in) above
by
olfactometer
traps on each chamber, and
equipped with a
defined sampling stations. At each station the trap was
hung from a pole so that the bottom tube, through which
to
comprises
has
connected in series were used. Traps were placed at well
laboratory-
system allows mosquitoes
a tiered configuration,
and mosquito
human
upwind
described in detail
between two different stimuli. The
chambers
to
day
aegypti were
and
a
elicited an
with wooded
counterflow concept for capturing
mosquitoes. The trap is constructed from a clear PVC
container( ca. 11. 4 liter volume).
The trap design and
operating principle have been described in detail by
Kline( 1998). The trap requires 12 VDC; in these field
studies two Panasonic 6 volt, 10 amp- hr gel- cells,
at
To determine if
associated
pending,
15 prig, a 10 micron line filter, a 500
flow control orifice, and quick- connect luer
with output
species
Gainesville, FL. ABC' s Pickle Jar( PJ) trap was used for
all the field trials. This new trap design, which has not
yet been made commercially, utilizes a novel, patent
was achieved
American Biophysics( ABC, East Greenwich, RI)
FLOWSET1,
mosquito
wetlands located near a water management area in
trap,
and
theolfactometerbypassingthroughaseriesofcharcoal
(
second study a white crew sock worn by the author' s son
JDK) was taped in the same way to another PJ trap.
Each day the mosquitoes were identified and counted.
Statistical analysis of the trap collection data consisted
of
the
paired
difference
t- test (
SAS
PROC
188
Journal of Vector
UNIVARIATE [ SAS Institute 1985]).
each
were analyzed
study
Three
studies
resulted
were
December, 1998
compared to
separately.
field
additional
The data from
Ecology
in
0. 1%
for the control; and the human hand
80. 1%
an
mean response compared to 0. 1%
for the control( TABLE 1).
conducted
using a 4 x 4 Latin square experimental design. Four
traps
were
located
provided with a
18
ca.
m
Each trap
apart.
different bait
for
daily
ca.
Field Studies. Species Composition
was
16 hours
Twenty- two species of mosquitoes were collected
during these experiments. In order of decreasing
abundance they were: Aedes atlanticus Dyar and Knab,
1800- 0600 Eastern Daylight Savings Time[ EDST]).
In
four bait
each experiment the
In the first Latin
a single nigh..
26- 30, 1997) the treatments
sock,
CO2
second
only,
treatments were no
and
CO2 +
12. 4
A
the
(
CO2 only,
fresh block( ca.
(
in
bottom
bag,
a small sandwich
of the
PJ trap
In the third
treatments
only,
were no
June 08- 12,
bait, DLK
CO2 +
the mosquitoes
(
DLK
the
(
and
only,
Each day
Each
counted.
salinarius
Coquillett,
Culex
Melanoconion) spp. Theobald, Aedes fulvus-pallens
Wiedemann),
Psorophora howardii Coquillett,
ciliata (
Fabricius), Anopheles quadri-
maculatus Say, Aedes triseriatus( Say), Aedes sticticus
Meigen), Culex quinquefasciatus Say, Psorophora
columbiae ( Dyar and Knab), Anopheles punctipennis
white crew sock
white crew sock.
identified
were
1997)
Culex
Psorophora
which was taped to the
so that the volatiles could escape.
experiment (
and
It was
day.
Coquillettidia perturbans( Walker), Culiseta melanura
Coquillett), Aedes vexans( Meigen), Culexnigripalpus
Theobald,
cheese,
small
gm) of Limburger cheese was used each
placed
CO2
cheese.
1997)
Theobald), Aedes dupreei ( Coquillett),
canadensis (
In the
sock.
30- June 02,
bait, Limburger
Limburger
Anopheles crucians Wiedemann, Aedes infirmatus Dyar
and Knab, Psorophora ferox( Von Humboldt), Aedes
May
bait, DLK black
were no
May
experiment (
square experiment(
DLK black
CO2+
and
combinations were
bait occupied each trap station for
alternated so that each
( Say), and Uranotaenia sapphirina ( Osten Sacken)
(
TABLES 2- 6).
experiment was statistically analyzed separately. In all
these
Latin
square
transformed to
transformed
log(
data
the
experiments
n+
1) for
were
data
were
analysis of variance.
analysed
with
The
Statistical
Analysis System ( SAS) PROC ANOVA
and
Means/
REGWQ for
and
means
comparisons(
the
analysis
of variance
Paired Tests
DLKWS+
CO2 vs CO2 only. In this experiment 18
of the species mentioned above were collected( TABLE
2).
No Cx. quinquefasciatus or Ur. sapphirina were
collected in either trap. Overall, 2. 09x more mosquitoes
were collected when the trap was baited with both the
SAS Institute 1985).
worn sock and CO2 compared with CO2 only baited
traps. However, the effectiveness of the addition of the
RESULTS
worn sock varied with species. FewerAe. triseriatus( 4
vs 5) and only one more specimen of Cx. salinarius( 21
Olfactometer Studies
In the olfactometer trials utilizing femaleAe.
Limburger cheese resulted in
of
6. 4%
the
aegypti,
a mean attractive response
0.0% for the simultaneously run control;
sock resulted in a 66. 1%
mean response
versus
worn
TABLE 1.
vs
20, index=
1. 05) were collected when the sock was
combined with the CO2 compared to CO2 alone. The
index for the other 15 species ranged from 1. 35x more
mosquitoes collected with the addition of the sock for
Mean1,2 percent attraction ( SE) of Aedes aegypti to human
hand, human worn sock, and Limburger cheese odors in dualport olfactometer.
Treatment
n=
Mean%
Responding( S. E.)
Hand
80. 12
2. 38)
Sock
66.08
3. 21)
B
Cheese
6. 44
1. 47)
C
Control
0. 04
0. 04)
D
A
10 for hand, sock and cheese treatments and 30 for control.
Means with the same letter are not significantly different; SAS PROC
GLM/ means
REGWQ
multiple range test(
SAS Institute, 1985).
December, 1998
TABLE 2.
Journal of Vector
Effect
of DLKWS worn sock on the mean number(±
CO2 ( 500
with
All
species
Ae.
atlanticus
Index3
0. 0001
2. 09
0. 0002
2. 17
48. 88 ( 10. 44)
77. 63 ( 14. 07)
3. 88
0. 0060
1. 59
40. 25 ( 7. 86)
66. 25 ( 7. 92)
2. 92
0. 0224
1. 65
1. 13)
1. 55
0. 1643
3. 76
98. 13 ( 9. 45)
233. 38 ( 27. 98)
6. 42
0. 0004
2. 37
0. 13 ( 0. 13)
0. 75 ( 0. 25)
2. 38
0. 0492
5. 77
0. 63
( 0. 26)
0. 50
( 0. 38)
0. 23
0. 8264
0. 79
10. 38
( 2. 05)
14. 00
( 2. 82)
1. 45
0. 1892
1. 35
173. 25 ( 27. 60)
3. 39
0. 0116
1. 64
sticticus
vexans
( 0. 33)
105. 50 ( 14. 97)
perturbans
1. 88 (
-
0. 25
( 0. 16)
0. 13 ( 0. 13)
0. 55
0. 5983
0. 52
0. 50
( 0. 27)
0.75
( 0. 31)
0. 68
0. 5165
1. 50
10. 50
( 1. 87)
16. 00
( 2. 81)
1. 54
0. 1663
1. 52
9. 38 ( 2. 22)
2. 17
0. 0663
2. 50
punctipennis
quadrimaculatus
Coquillettidia
1T12
6. 86
0. 50
crucians
Pr>
7. 93
Ae. triseriatus
An
ITI'
486. 75 ( 51. 43)
Ae. infirmatus
An.
Sock
1285. 75( 112. 93)
canadensis
Anopheles
No Sock
615. 88 ( 44. 42)
Ae. fulvus-pallens
Ae.
SE) of mosquitoes collected by PJ traps baited
224. 50 ( 22. 85)
Ae. dupreei
Ae.
189
8 nights).
ml/ min)( n=
Species
Aedes
Ecology
-
Culex( Melanoconion) spp.
Cx. nigripalpus
0. 63
( 0. 42)
1. 75
( 0. 65)
2. 35
0. 0514
2. 78
Cx.
2. 50
( 0. 65)
2. 63 ( 0. 42)
0. 20
0. 8436
1. 05
4. 88 ( 0.77)
9. 38 ( 2. 22)
2. 17
0. 0663
1. 92
0.50
( 0. 38)
1. 63 ( 0. 98)
1. 76
0. 1218
3. 26
65. 13 ( 8. 00)
193. 00 ( 26. 08)
5. 51
0. 0009
2. 96
3. 38 ( 0. 82)
2. 92
0. 0222
5. 37
4. 88 ( 0. 77)
salinarius
Culiseta
melanura
Psorophora
ciliata
Ps. ferox
Ps. howardii
0. 63
( 0. 26)
T test statistic for paired- difference t- test determined by SAS Institute Proc Univariate.
2Pr> ITI values less than 0.05 indicate the average difference is significantly different from zero.
3Index= #
Ae.
of
vexans
the
to
the worn
JDKWS+
Cx.
sock+
5. 37x for Ps. howardii. The collection
Psorophora spp.
addition of
in
CO2- baited trap
mosquitoes in CO2- only baited trap
mosquitoes
CO2
was
to
sock
vs
quinquefasciatus,
CO2
Ps.
increased the
size
by
the
CO2.
Ps.
columbiae, or
TABLE 3).
Ur.
Overall 2. 45x
more mosquitoes were collected when the sock was
used
in
alone.
combination with the
Only
a
few
CO2
specimens
of
compared to
Ae.
triseriatus, and An. quadrimaculatus
and
these were
Although
in the
baited
with
had
at
the
worn socks,
least twice the
CO2
least one of the treatments( TABLE 4). The trap baited
were
worn
sock +
CO2 caught 16 species
followed by 13 species for the CO2 only baited trap, 12
species for the sock only baited trap and 7 species for the
unbaited trap. Total mosquitoes collected in the worn
sock only trap was 3. 71x greater than that collected in
the unbaited
trap.
Individual species varied in their
relative collection size between sock only baited and
collected
unbaited trap from 1. 6x ( Ae. canadensis) to 10.5x
the sock+
the sock
DLKBS
Ae.
sticticus,
all the other species were collected
with and without
always
traps
mosquito species were caught by traps baited with at
with
only. No An. punctipennis,
ciliata,
sapphirina were collected(
most
CO2.
by
( Ae. infirmatus) greater for the sock baited trap. The
traps
sock only baited trap caught three species considered
baited traps
to be major annoyance pests ( Ae. atlanticus, Ae.
collection size as the
CO2
only baited traps. The index for these other species
ranged from 2. 11x for Ae. dupreei to 6. 46x for Ae.
vexans.
infirmatus and Cq. perturbans) at 0. 18x, 0. 25x and
0. 14x, respectively, their collection level in CO2 only
baited traps. In traps baited with worn socks+ CO2 these
same three species were caught at 2. 39x, 2. 84x and
2. 48x greater, respectively, than in traps baited with
Latin Square Experiments
1: DLKBS and
CO2. In this experiment, 17 different
CO2 only.
2: Limburger cheese
and
CO 2. Eighteen mosquito
Journal of Vector
190
TABLE 3.
Effect
with
of
JDKWS
500
CO2 (
worn sock on
378. 67
Aedes
Ae.
150. 67
atlanticus
49. 00
canadensis
(
88. 05)
928. 00
(
38. 92)
362. 67
(
(
14. 57)
115. 67
( 4. 04)
57. 00
1. 20)
4.67
14. 72)
127. 33
( 0. 00)
0.67
27. 00
Ae. dupreei
1. 67
Ae. fulvus-pallens
51. 67
Ae. infirmatus
(
(
ITI'
Sock
No Sock
species
SE) of mosquitoes collected by PJ traps baited
number(±
3 nights).
ml/ min)( n=
Species
All
the mean
December, 1998
Ecology
(
Pr>
ITI2
Index3
129. 33)
11. 66
0.0073
2. 45
45. 48)
7. 22
0.0187
2. 41
( 21. 88)
6. 64
0. 0219
2. 36
(
9. 00)
5. 12
0. 0361
2. 11
(
2. 40)
1. 44
0. 2863
2. 80
16. 02)
5. 51
0. 0314
2. 46
(
0. 33)
2. 00
0. 1835
(
Ae.
sticticus
0. 00
Ae.
triseriatus
0. 00
( 0.00)
1. 00
(
0. 58)
1. 73
0. 2254
vexans
0. 67
(
0.67)
4. 33
(
1. 20)
3. 05
0. 0927
6. 46
21. 67
( 7. 84)
56. 00
(
6. 66)
2. 71
0. 1136
2. 58
0. 00
( 0. 00)
0. 33
(
0. 33)
1. 00
0.4226
Ae.
Anopheles
An
crucians
quadrimaculatus
3. 33
(
0. 88)
7. 67
(
1. 33)
4.91
0. 0390
2. 30
1. 00
( 0. 58)
5. 33
(
2. 96)
1. 23
0. 3442
5. 33
Cx.
nigripalpus
0. 67
(
0. 33)
2. 67
(
1. 45)
1. 15
0.3675
3. 99
Cx.
salinarius
2. 33
(
0. 33)
3. 00
(
1. 73)
0.46
0.6914
1. 29
2. 67
( 0. 88)
8. 33
(
2. 91)
2. 43
0. 1358
3. 12
66. 33 (
21. 99)
171. 00
( 44. 64)
4. 35
0.0490
2. 58
0. 00
( 0. 00)
0. 33
0. 33)
1. 00
0.4226
Coquillettidia
Culiseta
perturbans
melanura
Psorophora ferox
Ps. howardii
(
T test statistic for paired- difference t-test determined by SAS Institute Proc Univariate.
ITI values less than 0.05 indicate the average difference is significantly different from zero.
2Pr>
3lndex= #
in
CO2- baited trap
mosquitoes in CO2- only baited trap
mosquitoes
species were collected
in
sock+
traps
during
maculatus and Ae. sticticus were collected only in the
this experiment:
17 by
CO2 only baited traps, 13 by traps baited with
Limburger cheese+ CO2 and 8 each in the Limburger
cheese
and unbaited
only
traps(
TABLE 5).
specimen of
Only
was collected and
in the
and
the sock+ CO2 compared to CO2 only baited traps. With
this
the exception of Ae. infirmatus species, which in the
burger
previous field experiments had their collection size
unbaited
unbaited
traps.
cheese+
CO2
baited
than traps
Overall, traps baited
caught
with
CO2
fewer( 0. 84x)
alone.
Only
with
Lim-
mosquitoes
five
CO2 baited trap. Overall, mosquito collections
in this experiment were only slightly better( 1. 33x) for
one
trap. A total of only 38 mosquitoes
were collected in each the Limburger cheese only baited
was
sock+
species,
>
1. 5x in
baited
CO2
compared to CO2 alone, still maintained the same type
traps
with
the
combination sock+
increase between the two baited trap types. Different
species dominated during this last experiment.
Ae. infirmatus ( 1. 05x), Ae. fulvus-pallens( 2. 73x), Cs.
melanura (
DISCUSSION
1. 22x), Cx. ( Melanoconion) spp. ( 1. 56x),
and An. quadrimaculatus( 4. 0x) were caught in greater
numbers
in
traps
baited
with
Limburger
cheese +
These data show that there are chemical compounds
produced by the human foot that actively attract
CO2.
3: DLKWS awl CO2.
Twenty species ofmosquitoes
were collected during this experiment: six by unbaited,
seven by sock only, 17 each by
CO2 only and sock+ CO2
mosquitoes without the need for CO2. This was most
baited traps ( TABLE 6).
available Ae. aegypti mosquitoes.
Two
specimens
of
Ur.
only in the sock alone baited
Ps.
columbiae were collected
Ae.
triseriatus
and
trap;
the
alone
baited
in
trap; and An. quadrionly
CO2
sapphirina were collected
obvious in the olfactometer studies where a human sock
worn
for three days consistently
attracted ca.
50% of the
The results of the
field studies were also encouraging in that the sock
alone baited trap attracted significantly( p< 0. 05) more
mosquitoes overall
than
did the
unbaited
traps
in
one of
December, 1998
TABLE 4.
Journal of Vector
Ecology
191
Mean' catch( standard error)( raw data) of mosquitoes associated with woodlands in PJ traps baited with
CO2 and/ or black human worn socks( DLKBS) near Gainesville, FL( May 26- 30, 1997).
Bait
Species
All
species
Aedes
Ae.
atlanticus
None
DLKBS
7. 75 ( 2. 56) D
28. 75 ( 7. 89) C
5. 00 ( 1. 87) C
17. 25 ( 6. 26) B
1. 25 ( 0. 48)
canadensis
B
2. 00 ( 0. 91)
CO2
2
DLKBS+
223. 00 ( 23. 16)
B
Index'
CO2
B
537. 75 ( 73. 39) A
2. 41
95. 00 ( 17. 87) A
227. 50 ( 38. 96) A
2. 39
31. 25 ( 5. 02) A
58. 75 ( 7. 82) A
1. 88
21. 25 ( 0.48)
A
2. 43
9. 00 ( 3. 03)
A
5. 14
Ae. dupreei
0.00 ( 0. 00) C
0. 50 ( 0.50) C
Ae. fulvus-pallens
0. 00 ( 0. 00) B
0. 00 ( 0. 00) B
Ae. infirmatus
0. 50 ( 0. 29) D
5. 25 ( 1. 38)
C
20. 75 ( 7. 32) B
59. 00 ( 11. 05) A
2. 84
Ae. triseriatus
0.00 ( 0.00) A
0. 25 ( 0. 25)
A
0. 25 ( 0. 25) A
0. 50 ( 0. 29) A
2. 00
Ae.
0. 00 ( 0. 00) A
0. 00 ( 0. 00) A
0. 25 ( 0. 25) C
0. 25 ( 0. 25)
C
0. 00 ( 0.00) A
0. 00 ( 0. 00)
A
0. 25 ( 0. 25)
A
0. 75 ( 0. 48) A
3. 00
0. 25 ( 0.25) B
0. 75 ( 0. 48)
B
5. 25 ( 2. 14)
B
12. 75 ( 3. 40) A
2. 48
0. 00 ( 0.00) B
0. 25 ( 0. 25)
B
2. 50 ( 0. 87) AB
5. 75 ( 2. 14) A
2. 30
0. 00 ( 0.00) A
0. 00 ( 0. 00) A
0.00 ( 0. 00) A
0. 25 ( 0.25) A
0.00 ( 0. 00) A
vexans
Anopheles
An.
crucians
quadrimaculatus
Coquillettidia
perturbans
Cx. nigripalpus
8. 75 (
1. 65) B
1. 75 ( 0. 48) B
0. 00 ( 0. 00) A
23. 25 ( 5. 89) B
2. 25 (
1. 11)
A
66. 50 ( 24. 97) A
2. 86
Cx.
quinquefasciatus
0. 25 ( 0.25) A
0. 25 ( 0.25)
A
0. 00 ( 0.00) A
Cx.
salinarius
0. 00 ( 0. 00) B
0. 75 ( 0. 75)
B
7. 00 ( 3. 67) AB
8. 75 ( 0. 63) A
1. 25
0. 00 ( 0. 00) B
0. 50 ( 0.50) B
6. 25 ( 0. 48) A
3. 00 ( 1. 50) A
0. 88
0. 00 ( 0. 00) A
0.00 ( 0. 00) A
0. 25 ( 0. 25) C
0. 75 ( 0. 48) C
Culiseta
melanura
Psorophora
ciliata
Ps. ferox
4
n=
nights;
means
in the
same row
followed
by
the same
0. 00 ( 0. 00) A
20. 75 ( 4. 61)
letter
are not
B
0. 25 ( 0. 25) A
59. 00 ( 8. 46) A
2. 84
significantly different( P> 0. 05);
REGWQ multiple range test( SAS Institute 1985) applied to log- transformed data.
2CO2
500 ml/ min.
release rate=
Index=
mean#
the Latin
Latin
square sock studies(
to
TABLE 4). In the
study( TABLE 5),
had changed from those
square sock
composition
to the
in DLKBS+
CO2 baited trap
mosquitoes in CO2 baited trap
mosquitoes
mean#
to those
sock volatiles
sock volatiles.
Overall
with
other
Also encouraging is the persistence of the synergistic
relative mosquito
effect observed with the worn socks over time observed
attracted
in
little
or no
strongly
attractancy
density
the
first
paired
difference study.
This effect was
relatively consistent over the eight day period that this
was also
study was conducted, which indicates that the attractant
In the first study Ae. atlanticus was the
most abundant species, but in the last study An. crucians
was ca. 3. 5x more abundant than Ae. atlanticus. Aedes
substances were probably bound up in the sock fibers
mosquito
much reduced.
atlanticus
alone
to
be
appeared
be
baited trap than An.
more
attracted
crucians.
a synergistic response
the combination of
of
to
CO2 +
There
to the
sock
also seemed
by most mosquito species
to
worn sock.
same
basic
The overall response of mosquitoes
index(#
mosquitoes captured in sock+ CO2 baited trap/
mosquitoes captured in the CO2 only baited traps)
remained relatively consistent ( 2. 13, 2. 16, 2. 28, 1. 73,
2. 21, 1. 68, 2. 84, and 1. 75 for days 1 through 8,
respectively).
Another encouraging factor is that different kinds
socks and the same kind of socks worn
by two
different people showed the
and subsequently slowly released. The relative collection
response patterns.
The data reported herein are also in basic agreement
with recent results obtained in Europe. Mboera et al.
1998 reported that in dualchoice olfactometer studies
to JDKWS appeared
female Cx. quinquefasciatus were significantly more
than to DLKWS. This is consistent
attracted to foot skin emanations collected on nylon
with observations made over several years on relative
stockings( by wearing them for four to five days between
individuals
0600 hr and 1800 hr) than clean stockings. In contrast to
to
be slightly
greater
mosquito attractive responses to these two
unpublished
data).
these
data,
no significant
interaction between
CO2
and
192
Journal of Vector
TABLE 5.
December, 1998
Ecology
CO2 and/ or Limburger cheese near Gainesville, Fl( May 30- June 02, 1997).
Bait
Species
None
CO22
Cheese
Cheese+
Index'
CO2
7. 50 ( 3. 28) B
8. 75 ( 4. 21)
313. 00 ( 22. 89) A
305. 75 ( 86. 58) A
0.98
4. 50 ( 2. 72) B
5. 25 ( 2. 87) B
115. 50 ( 30. 01)
A
109. 25 ( 36. 83) A
0. 89
0. 00 ( 0.00) B
0. 75 ( 0. 48) B
37. 25 ( 6. 18)
A
34. 25 ( 10. 83) A
0. 73
Ae. dupreei
0.00 ( 0. 00) B
0. 50 ( 0. 50) B
14. 50
A
Ae. fulvus- pallens
0. 25 ( 0. 25) B
0. 00 ( 0. 00) B
Ae. infirmatus
0. 00 ( 0. 00) B
Ae. triseriatus
Ae.
All
species
Aedes
Ae.
atlanticus
canad'' nsis
B
( 7. 35)
9. 25 (
1. 75) A
0. 98
1. 25) A
10. 75 ( 5. 34) A
2. 73
0. 00 ( 0. 00) B
24. 75 ( 7. 39) A
35. 50 ( 13. 15) A
1. 05
0. 00 ( 0.00) A
0. 00 ( 0. 00) A
0. 50 ( 0. 29) A
0. 00 ( 0.00) A
0. 00 ( 0. 00) A
0. 00 ( 0. 00) A
A
0. 00 ( 0.00) A
0. 50 ( 0.29) B
0. 75 ( 0. 48) B
61. 50 ( 32. 12) A
55. 75 ( 15. 02) A
0. 63
0. 00 ( 0.00) B
0. 00 ( 0. 00) B
0. 25 ( 0. 25) A
1. 00 ( 0.00) A
4. 00
0. 25 ( 0. 25) B
0. 25 ( 0. 25) B
8. 50 ( 4. 50) A
12. 00 ( 3. 14) A
0. 85
Cx. nigripalpus
0. 25 ( 0.25) B
0. 25 ( 0.25) B
2. 25 ( 0. 48) A
5. 00 ( 1. 78)
A
1. 22
1. 00 ( 1. 00) B
0. 50 ( 0. 50) B
8. 50
( 3. 97) A
6. 50 ( 4.27) A
0. 91
Cx.
quinquefasciatus
0. 25 ( 0.25) A
0. 00 ( 0.00) A
0. 00
( 0. 00) A
0.00 ( 0.00) A
Cx.
salinarius
0. 50 ( 0. 50) B
0. 00 ( 0.00) B
6. 50
( 1. 19) A
8. 75 ( 3. 09) A
0. 00 ( 0.00) B
0. 50 ( 0.50) B
5. 50
( 1. 32) A
6. 25 ( 0.95) A
Psorophora ferox
0. 00 ( 0.00) C
0. 00 ( 0.00) C
23. 50
( 7. 10) B
Ps. howardii
0.00 ( 0. 00) A
0. 00 ( 0. 00) A
0. 25
( 0. 25) A
vexans
Anopheles
An.
crucians
quadrimaculatus
Coquillettidia
Culiseta
perturbans
melanura
2. 75 (
1. 00
( 0. 41)
4 nights; means in the same row followed by the same letter are not significantly
n=
2CO2
mean#
emanations
experiments.
to
attract
Mboera,
was
observed
Cx.
results of
encouraging.
Limburger cheese in the
Similar
responses were
cheese
as was noted with
In fact, the
the
addition of
Limburger
combination of
Limburger
Culex spp.,
numbers),
and Ae. infirmatus
CO2.
Why our Limburger cheese failed to yield a good
aegypti to
No
significant
unbaited versus
There
CO2
was
cheese resulted
cheese+
CO2
no
cheese+
baited traps.
resulted
0. 05) in Ps. ferox
olfactometer or field studies is interesting since Knols
in
a
(
1996) reported such good responses for An. gambiae
s. s. Knols( personal communication) suggested that too
much cheese was used in these studies, which may have
resulted in repelling some mosquitoes. He suggested
that a study needs to be conducted on the dose- response
behavior of Ae. aegypti to Limburger cheese. Further
discussion with Knols concerning Limburger cheese
revealed that there are many different kinds of Limburger
cheese.
Knols ( 1996) did not indicate the brand of
The
Limburger cheese used for tests in The Netherlands.
in
Indeed, other Dutch colleagues have since provided
a
collections
several brands of Limburger cheese from The
only baited traps. In contrast
perturbans, An. quadrimaculatus
Netherlands for evaluation. Preliminary studies in the
to
Cq.
Ae.
overall mosquito collection size.
decrease( p<
in very low
occurred
of cheese+
studies
relatively
noticed for most
studies.
worn sock+
in
when compared
0. 05);
collections were slightly increased with the combination
stockings
between Limburger
slight reduction
significant
of
for any species in
only baited traps.
similar synergistic effect
CO2
(
in Tanzania to
worn
olfactometer was
was noted
Limburger
different( P>
attractive response from mosquitoes either in the
Response
in the field
mosquito species
with
the Limburger cheese
were not as
difference
laboratory
data).
unpublished
low ( 6. 4%).
in these
quinquefasciatus
CDC traps baited
The initial
0. 00 ( 0. 00) A
in Limburger Cheese+
Also, these investigators have so far failed
wild
unlighted
0. 57
CO2 baited trap
mosquitoes
mean#
foot
1. 22
A
500 ml/ min.
release rate=
Index=
11. 50 ( 2. 63)
0. 96
CO2
olfactometer with one of
these brands against A e.
aegypti
December, 1998
TABLE 6.
Journal of Vector
193
Ecology
CO2 and/ or White human worn socks( DLKWS) near Gainesville, Fl( June 8- 12, 1997).
Bait
Species
All
None
species
Aedes
Ae.
atlanticus
DLKWS
CO2
2
Index3
DLKWS+ CO
2
1. 50 ( 1. 22) B
4. 00 ( 1. 22) B
178. 50 ( 36. 97) A
237. 00 ( 37. 68) A
1. 33
0. 00 ( 0. 00) C
1. 25 ( 0. 48) C
15. 75 ( 3. 86) B
34. 25 ( 3. 50) A
2. 17
0.25 ( 0. 25)
0.50 ( 0. 29)
23. 19 ( 11. 59) A
B
B
18. 75 ( 5. 45)
A
Ae. dupreei
0.00 ( 0. 00) B
0.00 ( 0. 00) B
0. 75 ( 0. 48)
B
3. 00 ( 0. 71)
A
4. 00
Ae. fulvus-pallens
0.25 ( 0. 25)
0. 00 ( 0. 00) B
0. 50 ( 0. 29)
B
4. 25 (
1. 55)
A
8. 50
Ae. infirmatus
0.00 ( 0. 00) B
0. 00 ( 0. 00)
B
4. 50 ( 1. 19)
A
5. 75 (
1. 93)
A
1. 28
Ae. triseriatus
0.00 ( 0. 00) A
0.00 ( 0. 00) A
0. 25 ( 0. 25)
A
0. 00 ( 0. 00) A
Ae.
0.00 ( 0. 00) B
0.00 ( 0. 00)
B
1. 50 ( 0. 65)
A
0.00 ( 0. 00) B
1. 00 ( 0. 41)
B
67. 00 ( 20. 24) A
0.00 ( 0. 00) A
0. 00 ( 0. 00) A
0. 00 ( 0. 00) A
21. 00 ( 5. 70) A
17. 50 ( 9. 54) A
0. 83
3. 25 ( 1. 03) A
7. 00 ( 1. 22) A
2. 15
canadensis
vexans
Anopheles
An.
crucians
quadrimaculatus
B
A
1. 17
74. 00 ( 19. 40) A
1. 18)
1. 10
0. 25 ( 0. 25)
A
0. 25 ( 0. 25) B
0. 50 ( 0. 50) B
Cx. nigripalpus
0.00 ( 0. 00) B
0. 50 ( 0. 29)
0.50 ( 0. 50) B
0. 00 ( 0. 00) B
Cx.
0.00 ( 0. 00) B
0. 25 ( 0. 25) B
5. 25 ( 1. 55) A
5. 25 ( 2. 46) A
1. 00
0. 25 ( 0. 25)
B
0. 00 ( 0. 00) B
20. 75 ( 9. 08) A
15. 50 ( 6. 17) A
0. 75
Coquillettidia
perturbans
salinarius
Culiseta
melanura
B
12. 25 ( 5. 71)
A
19. 75 ( 9. 68)
A
1. 61
0.00 ( 0. 00) A
0. 00 ( 0. 00) A
0. 75 ( 0. 75) A
0. 25 ( 0. 25) A
0. 33
Ps. ferox
0.00 ( 0. 00) C
0. 00 ( 0. 00) C
4. 75 ( 1. 80) B
11. 00 ( 2. 27) A
2. 32
Ps. howardii
0.00 ( 0. 00) B
0. 00 ( 0. 00) B
1. 50 ( 0. 65)
2. 00 ( 1. 68) A
1. 50
Psorophora
ciliata
A
by the same letter are not significantly
4
n=
2
1. 75 (
1. 89
CO2
nights;
means
release rate=
3Index=
mean#
in the
followed
same row
500 ml/ min.
in DLKWS+
CO2 baited trap
mosquitoes
mean#
using the same amount of cheese as before has resulted
in a mean attractive response of 24. 3% compared to
6. 4% for our American brand. Thus,
the
brand of cheese
behavior
may also be
of the test mosquitoes. Further studies are planned to see
acontributing factor to the
if these trends
different( P> 0. 05);
response
studies.
I want to thank H. T. McKeithen for his
technical assistance and for performing the statistical
analyses of these data. I also want to thank K. Posey for
his assistance in the conduct of the olfactometer tests.
REFERENCES CITED
remain consistent.
Cooperative studies are in progress with analytical
chemists
far 24
worn
with
determine
to
two types
of
in
have
elicited>
in
combinations are
in the
worn socks.
So
have been identified from the
testing in the dual
these compounds has begun.
socks and
aegypti alone and
these
cheese and
volatile compounds
compounds
Ae.
what volatiles are present
Limburger
50%
port olfactometer
Several
of
these
attractive response
combination.
in the planning
Field tests
by
with
stage.
De Jong, R. and B. G. J. Knols. 1995. Selection of biting
sites on man by two malaria mosquito species on
man. Experientia 51: 80- 84.
Hall, D. R., P. S. Beevor, A. Cork, B. F. Nesbitt, and G.
A. Vale. 1984. 1- Octen- 3- ol: A potent olfactory
stimulant and attractant for tsetse isolated from
cattle odours. Insect Sci. Appl. 5: 335- 339.
Kline, D. L. 1994. Olfactory attractants for mosquito
surveillance
and
Kline, D. L. 1998.
I
want
to thank American Biophysics
control:
1-
octen-
3-
ol.
J. Am.
Mosq. Contr. Assoc. 10: 280- 287.
Acknowledgments
Corporation,
East Greenwich, RI, for supplying the traps for these
Comparison of two American
Biophysics mosquito traps: the professional and a
new
counterflow
geometry trap.
J. Am.
Mosq.
194
Journal of Vector
Contr. Assoc. ( In Press).
Knols, B. G. J.
behaviour
Odour- mediated host- seeking
the Afro- tropical malaria vector
Anopheles gambiae Giles. Ph. D. Thesis,
of
December, 1998
Culex quinquefasciatus Say( Diptera: Culicidae) in
1996.
of
Ecology
a dualchoice olfactometer. J. Vect. Ecol. 23( 2):
107- 113
University
Posey, K. H., D. R. Barnard, and C. E. Schreck. 1998.
Wageningen, The Netherlands, 213 pp.
and R. De
Jong. 1996. Limburger cheese
A triple cage olfactometer for evaluating mosquito
Knols, B. G. J.
as an attractant
gambiae s. s.
Knols, B. G. J.
mosquito
for the malaria mosquito Anopheles
Parasitol.
and
Today
12: 159- 161.
behavior. Sci. Med. 4: 56- 63.
van
W. Takken. 1997. Behavioural
responses of the
Anopheles
Limburger
gambiae (
female
Jong, and
and electrophysio-
malaria mosquito
Diptera: Culicidae)
cheese volatiles.
to
Bull. Entomol. Res.
87: 151- 159.
Mosquito attraction to substances from the skin of
different humans. J. Am. Mosq. Contr. Assoc. 6:
406- 410.
Schreck, C. E., N. Smith, and D. A. Carlson. 1981. A
material isolated from human hands that attracts
female mosquitoes. J. Chem. Ecol. 8: 429- 438.
Mboera, L. E. G., B. G. J. Knols, W. Takken,
T. Huisman. 1998.
ed. SAS Institute, Cary, NC
Schreck, C. E., D. L. Kline, and D. A. Carlson. 1990.
Loon, A. Cork, R. D.
Robinson, W. Adam, J. Meijerink, R. De
logical
SAS Institute. 1985. SAS Procedure Guide, version 6
J. Meijerink. 1997. Odors influence
Knols, B. G. J., J. J. A.
Diptera: Culicidae) attraction responses. J. Med.
Entomol. 35: 330- 334.
Olfactory
and
responses of
P. W.
female
1- octen- 3- ol as mosquito attractants. J. Am. Mosq.
Contr. Assoc. 5: 311- 316.
Journal of Vector
23( 2): 195- 201
Ecology
Egg Maturation in Neotropical Malaria Vectors:
One Blood Meal is Usually Enough
L. P. Lounibos',
D. Couto Lima2, R. Lourenco- de- Oliveira2,
R. L. Escher', and N. Nishimura'
University of Florida, Florida Medical Entomology Laboratory,
200 9th St. SE, Vero Beach FL 32962, USA
2Departamento de Entomologia, Instituto Oswaldo Cruz,
Av. Brasil 4365, 21045- 900 Rio de Janeiro, RJ-Brazil
Received 30 June 1998; Accepted 28 September 1998
ABSTRACT: The requirement of more than one blood meal to complete a gonotrophic cycle is reported
here to be infrequent among field-collected Anopheles( Nyssorhynchus), including Anopheles darlingi,
South America' s most important malaria vector. The overall frequencies of host-seeking females that did
not
develop
5. 0%),
and
eggs after one
Anopheles
blood
feeding
were
low for Anopheles darlingi ( 6. 6%), Anopheles rondoni
triannulatus( 2. 2%);
the majority of wild-caught females that did not develop eggs
after one blood meal were nulliparous. Laboratory- reared Anopheles albimanus and Anopheles albitarsis,
other species of the same subgenus, were grown on enriched and impoverished larval diets to yield a range
of adult sizes and to examine relationships between egg maturation, body size, and blood meal intake.
10. 7%
of nulliparous
An.
albimanus and
22. 9%
of An. albitarsis failed to mature eggs after sugar and one
blood meal, and shorter wings and smaller blood meals predisposed females to require multiple blood feeds
for egg maturation. Unmated An. albitarsis were significantly more likely than mated females not to
develop eggs. Multiple blood meals within a single gonotrophic cycle appear to be less important in the
life histories of neotropical Nyssorhynchus species compared to afrotropical malaria vectors.
Keyword Index:
Anopheles( Nyssorhynchus), egg development, blood feeding, female mass, wing
lengths.
INTRODUCTION
1993). In West African localities, 42% of An. gambiae
and 63% of An. funestus required more than one
s. s.
The
occurrence of multiple
single gonotrophic cycle
from vector ecologists studying
For Africa'
s
Anopheles( Cellia)
funestus,
this
gambiae s.
habit is
l.
disease vectors.
(
Molineaux
and
Grammicia
1980).
Field studies
transmitters
elsewhere in the Old World tropics have established that
Anopheles( Cellia)
other important malaria vectors commonly imbibe blood
more than once during the first gonotrophic cycle( e. g.,
malaria
and
blood meal to develop eggs( Brengues and Coz 1973),
and pre- gravids were more common in the dry season
within a
renewed attention
mosquito
important
most
blood feeds
has received
associated with
endophily
and
the
easy accessibility of human blood as a nutrient source
Beier 1996, Lyimo and Takken 1993). Gillies( 1954,
1955) demonstrated that the majority of field- collected
females of these two species that required blood more
Reisen and Aslamkhan 1976, Mogi et al. 1995).
Laboratory investigations have demonstrated that
the pre- gravid condition of African anophelines may
result from inadequate larval nutrition ( e. g. El-Akad
than once to mature eggs were
and Humphreys 1990, Takken et al. 1998). The use of
for
the first blood meal to satisfy energy requirements in-
which
these
he
coined
recently emerged nullipars,
the term ' pre- gravids' to describe
new emergences
meals
to
Approximately
20%
that needed two or more
the
complete
of
first
gonotrophic
host- seeking An.
the
incidence
of
this
Aedes
mosquitoes (
Briegel 1990). Observations
that several species of sugar- deprived Anopheles took
in Tanzania,
repeated blood meals within a single gonotrophic cycle
phenomenon
seasonally ( Gillies 1954, 1955; Lyimo
stead ofvitellogenesisismorecharacteristicofAnopheles
than
l.
gambiae s.
and An.funestus were pre- gravid at two sites
where
blood
cycle.
and
varied
Takken
have led to the conclusion that this behavior" prevails in
many
anopheline( s)"(
Briegel
and
Hurler 1993).
Journal of Vector
196
Blood
requirements
relatively poorly
although multiple
cycle
for egg
in
studied
blood feeding
has been documented in
Anopheles( Nyssorhynchus)
Briegel
and
Anopheles)
from field-
meals might predispose females to take blood more than
butnot several species
once to mature eggs. Finally, our discussion compares
and
An.
important
meals
the multiple blood feeding habit in neotropical and
to
field- collected in
western
meal
Only
gonotrophic discordance( Rao 1947), and gonotrophic
disharmony( Adam et al. 1964) to describe the absence
Venezuela, where this species
or incompletion of egg development after a single blood
humans,
develop
to
The older literature on this topic proposed terms
such as gonotrophic dissociation( Swellengrebel 1929),
nuneztovari,
transmits Plasmodium vivax to
blood
afrotropical malaria vectors.
neotropical
Deane 1988, Zimmerman 1992).
Anopheles ( Nyssorhynchus)
of
than one
blood
subgenus
albimanus, regarded as
malaria vectors(
4- 10%
reported
required multiple
The latter
1992).
in Argentina that Anopheles
and
includes the majority
such as Anopheles( Nyssorhynchus) darlingi
mature eggs.
of species,
most important malaria vector ( Zimmerman
Briegel 1994),
Secondly, we describe results of experiments on
laboratory colonies of two other Nyssorhynchus species
to examine whether small body size or small blood
pseudopunctipennis,
Nyssorhynchus,
of
colony of
Briegel 1990,
in Central America
vector
malaria
specimens
that multiple blood meals are not required for egg
maturation by most wild- caught, host- seeking
Nyssorhynchus, including An. darlingi, South America' s
laboratory
a
albimanus(
Frederickson 1993). Davis( 1928)
collected
The purpose of the present paper is to demonstrate
within one gonotrophic
Wirier 1993, Klowden
important
an
have been
anophelines,
maturation
neotropical
December, 1998
Ecology
eggs (
required more
meal. We consider these terms, which predate a better
Lounibos
understanding of the physiological processes involved
e. g., Washino 1977, Briegel 1990), to be unnecessary
and
Conn 1991).
By contrast, most unmated, sugar- deprived females
in the context of the present paper.
of An. nuneztovari would not mature eggs after one
blood meal in the
laboratory(
Lounibos
and
Conn 1991).
Subsequently, circumstantial evidence indicated that
Anopheline mosquitoes were collected 1800- 2100
mating may be necessary for egg development in some
Nyssorhynchus species, such as An. nuneztovari; during
females
copulation
needed
sory
to
for ovarian maturation from enlarged male acces-
glands(
complete
earlier
of such species receive substances
(
hours from human bait, using protocol approved by the
University of Florida' s Institutional Review Board
Projects No. 94. 299 and 96. 011), in the vicinity of
Lounibos 1994a). A requirement for mating
Porto Velho( site CR), State of Rondonia and Peixoto de
egg development had been reported decades
Azevedo( site PX), State of Mato Grosso, in Amazonian
for Anopheles( Cellia)
An.
1940).
subpictus
from India(
the
TABLE 1.
Rates
of insemination,
parity,
Brazil( TABLE 1).
Roy
probability
Among
maturing some eggs was significantly increased by
large blood meal( Lounibos and Conn 1991).
nuneztovari,
Mosquitoes were transported live in
of
humidified containers to laboratories and, on the morning
a
after collection, lightly anesthetized for species
identifications under a dissecting microscope. Females
and need
for another blood meal to
develop eggs(= To with stationary
ovaries) among field-collected Anopheles( Nys.) females.
No. with
Collections
Date( s)
Site
Species
No.
No.
stat.
dissect.
insemin.
ovaries
Blood
sources
Parous
%
rateb( n)
An. darlingi
CR
21- 28/ VI1195
M, Q
48
11
22.9
An. darlingi
CR
11/ X- 9/ XI/ 95
140
140
2
1. 4
—
An. darlingi
CR
4/ IV- 9/ V/ 96
Q
Q
120
119
8
6. 7
—
An. darlingi
PX
20NII/96
G
83
5
6. 0
30. 5( 59)
An.
rondoni
PX
14- 18/ VII/96
G
40
2
5. 0
49. 0( 37)
An.
triannulatus
CR
26/ V11/ 95
26
0
0. 0
triannulatus
CR
Q, H
Q
26
4/ IV- 9/ V/ 96
20
20
1
5. 0
all= human, G=
guinea
An.
bBased
on
dissections
pig, M=
of non-
mouse,
fed females
Q=
quail
collected
from
the same site on the same
date.
39. 4( 94)
7. 7( 13)
—
December, 1998
Journal of Vector
blood were confined
with no signs of recent
in 9- dram
vials
fitted
and
were
for
restrained animal or,
blood
three
excrete
23- 40%
fluid( Perez de Valderrama
and
by weight of ingested
Scorza 1976). Ovarian
development was allowed to proceed for three days at
27± 0.5°C for An. albimanus or 28± 1° C for An. albitarsis,
for
whereafter stages of development were assessed, as
investigators. After
indicated previously, by dissection of females that did
low
on
risk
for two to
not oviposit in vials. Individual An. albitarsis, but not
days without access to sugar in ambient- temperature
An. albimanus, were examined for insemination. In
meal,
insectaries
the
Nyssorhynchus
a
repletion
species considered
at
saline(
period prior
was
2
stage
3
stage
from
ovaries
which were classified
19111)
Christophers). For the
of
the
of specimens,
dissection.
Christophers[
of
during
oviposited
to
assessed
Hayes 1953),
stationary(<
developing >
majority
holding
development
Egg
dissected in
were maintained
26± 2°C. Females rarely
post- blood
as either
females
paper
of
blood- fed to
malaria transmission, on one of the
the
filter
lids. Within two days
screened
with
females
collection,
individually
moist cotton and
containing
197
Ecology
classification
or
vast
decision
was
developing follicles being in stages
straightforward, most
comparable conditions of a previous study ( Lounibos
1994a), all females of colony An. albimanus held in
cages with males were inseminated. Wing lengths were
measured
as
described in Lounibos ( 1994b). The
relationships between wing lengths, mass after blood
feeding, and the proportions of females developing eggs
were tested by logistic regression with the procedure
CATMOD of SAS( 1985) for fit to the sigmoid function:
4 or 5 of Christophers at the time of dissection. The
detection of stationary
ovaries
blood
be
meal
would
indicated that a subsequent
to
required
maturation.
Ovaries
in distilled
water and allowed
classified as
complete
to
dry
egg
were rinsed
stationary
on microscope
for parity determinations by the tracheolar skeining
method ( Detinova 1962). Among certain collections,
ln[ f/( 1- f)]= A+ B( wing length)+ C( blood meal mass)
where f is the proportion maturing eggs and A, B, and
C are parameters to be estimated ( Trexler and Travis
1993).
Previous studies support the use of similar
slides
spermathecae of
dissected females
in saline
were placed
and examined for the presence of sperm under high
logistic regression models to describe the relationship
between measures of mosquito body size and such
binary variables as egg development or infection with
parasites ( e.
power
of
compound
a
Non- blood fed
microscope.
from four collections were dissected to
specimens
parity incidence
estimates
of
the
g., Lounibos 19946, Lyimo and Koella
1992).
obtain
sampled
field
RESULTS
populations.
used
Laboratory colonies of two An.( Nys.) species were
to obtain females of known developmental history
Estimates From Field Collections
Four collections of An. darlingi from two sites at
from 1. 4 to 22. 9%
General colony maintenance was as described
previously for An. albimanus originating from El
three times
Salvador( Ford and Green 1972)
and
from Brazil ( Horosko
1997). Larvae
Only 6. 6% of all An. darlingi dissected( n= 391) failed
to develop eggs after one blood meal. The frequency of
stationary ovaries after blood feeding was also low for
and age.
fed
albimanus were
powder and
a suspension of
Brewer'
a pollen- fish
s yeast and
food- dog
sizes (
females
day
albitarsis( s. s.)
finely
those of
Lounibos
1994b).
to 10%
of
to
An.
liver
ground
An.
albitarsis
Lounibos
were adjusted
were allowed access
yield adults
Newly
emerged
sucrose on
the
first
presence of an
for the first two days. On
3, females
day
An.
triannulatus(
of
2. 2%) and An. rondoni( 5. 0%).
Among An. darlingi with stationary ovaries( n= 26)
et al.
in the
after eclosion and maintained
excess of males
An.
chow mixture (
1998). Diet concentrations
of varied
al.
et
of year yielded a range
females that did not mature eggs after blood( TABLE 1).
after
blood, three ( 11. 5%)
were parous. Insemination
rates of field-collected females were high forAn. darlingi
( 99. 6%)
and
estimates at
An. triannulatus( 100%)(
times of
TABLE 1). Parity
collection ranged
from 7. 7% for
An. triannulatus to 49% for An. rondoni.
were allowed to consume blood from a restrained chicken
An. albimanus)
two
hours
complete,
after
or a
human
blood
masses
of
arm(
An. albitarsis). Within
Size Variables and Egg Maturation among Nyssor-
but
hynchus Colonies
after diuresis was
feeding
anesthetized specimens
lightly
determined gravimetrically on an electrobalance
Lounibos and Conn 1991), after which females
were
recovered
relative
rather
in individual vials. The purpose was to estimate
blood mealintakeofalargenumberofspecimens,
than precisely measure
Briegel
1986).
During
blood
diuresis,
meal volume( e.
related
species
g.,
of
Among laboratory- reared, nulliparous females,
10.7%
of
An.
albimanus and
22. 9% of An. albitarsis
failed to develop eggs after one blood meal( TABLE 2).
The distribution of wing lengths was significantly skewed
to the right among An. albimanus only, indicating an
overabundance of larger females of this species. Wing
lengths and mass after blood feeding were significantly
198
Journal of Vector
correlated
in both
Of the An.
inseminated,
and
species.
11. 0% (
albitarsis,
65. 8%(
n=
25)
these
of
38)
n=
did
were
not mature
A G- test for independence using Williams'
correction( Sokal and Rohlf 1995) indicated that failure
develop
eggs
lack
and
associated (
significantly
insemination
observations suggest a different sequence ofreproductive
were
events for the neotropical anophelines after emergence
35. 2, P< 0. 001) for An.
compared to afrotropical malaria vectors. Whereas
of
Gadi=
albitarsis.
Both
teneral An. gambiae usually seek blood before mating,
mass
blood
after
meal
logistic
for An.
nearly all An. darlingi and An. triannulatus were
inseminated before their first blood meal. This trend
proportion of An. albimanus maturing
blood meal( TABLE 3). ForAn. albitarsis,
was also observed for Nyssorhynchus field-captured in
mass after
Likelihood
the
in the logistic
for the
eggs after one
only
wing length
and
regression
explained significant variation
model
December, 1998
was relatively uncommon among these Nyssorhynchus
species. Most collected females were nulliparous and,
in contrast to pre- gravid afrotropical malaria vectors
caught athosts( Gillies 1954, 1955), inseminated. These
not
eggs.
to
Ecology
blood meal
Trinidad,
ratios showed a
statistically significant.
significant deviation from
regression model
for An.
albitarsis
but
where
more
than
90%
of host- seeking
Anopheles aquasalis and more than 97% ofAn. albitarsis
was
( s. l.) had been inseminated( Senior White 1955). Since
the principal afrotropical vectors are more endophagic
not
albimanus.
and endophilic than their neotropical counterparts( e. g.
Beier 1996 cf. Zimmerman 1992), the availability of
human blood lends itself to more multiple blood feeding
DISCUSSION
in the African situation. Less frequent contact with
Our preliminary data from field collections of hostseeking females indicate that the pre- gravid condition
TABLE 2.
human hosts by Nyssorhynchus species may be one
cause of low malaria infection rates among neotropical
Mean wing lengths and masses after blood feeding, plus associated statistics, for laboratory- reared
Anopheles( Nyssorhynchus).
Mean
with
n
wing
length
blood
stat.
Species
Mean
mass
after
ovaries
(
mg)
S. D.
(
mm)
S. D.
C. V.
ra
skewness
An.
albimanus
564
10. 7
2. 84
0. 67
3. 26
0. 17
5. 22
0. 567**
An.
albitarsis
347
22. 9
2. 58
0. 46
3. 56
0. 20
5. 62
0. 434**
-
0. 37
3. 60***
0. 04
0. 30ns
a product-moment correlation between mass after blood and wing length
bt- test for significance of skewness( Sokal and Rohlf 1995)
P< 0. 01, ***=
P< 0. 001,
TABLE 3.
ns=
non- significant
Maximum likelihood analyses of variance for logistic regressions of egg
development( dependent, binary variable) versus wing length and mass after
blood feeding( independent, numeric variables).
Species
An.
albimanus
n
564
source
mass after
blood
wing length
likelihood
An.
albitarsis
347
mass after
ratio
blood
wing length
likelihood
ratio
d. f.
P
xz
1
52. 02
<
0.001
1
18. 65
<
0.001
494
124. 33
1. 000
1
6. 64
0.010
1
3. 17
141
206. 01
0.075
<
tb
0.001
December, 1998
Journal of Vector
Arruda
vectors( e. g.,
1986, Oliveira- Ferreira
et al.
distribution
insemination of An. albitarsis confirms other evidence
An.
albimanus
derived from
for a role ofmating in egg maturation( Lounibos 1994a)
impoverished larval diets
mimicked the
but does not, by itself, explain the relatively high
wing lengths and their coefficients of
in natural populations of related
pre- gravid rate in the experimental cohort of this
size
enriched and
range
of
of
variation and skewness
Lounibos 1994b). The
species(
females in
sized
biting
large-
species. The contrasting results between An. albimanus
and An. albitarsis may be attributable to differentexperi-
Lounibos 1994b)
mental protocols, species- specific genetic properties,
preponderance of
collections (
suggests that Nyssorhynchus species
in
larval
nature to the
stresses
blood
malaria vectors to seek
1998).
et al.
An.
Approximately
low incidence
a
frequently(
more
90%
Lounibos 1994a). The
after post- blood
for An.
blood
Valderrama
based
wing length
Lounibos et al. 1995).
The probability
positively
wing length
square value
likelihood
and Briegel 1994), there is reason to doubt that this
species of similar size
populations (
Lounibos 1994b,
developing
both
to
some
An.
observed
are
higher for
RL-O, unpublished), and Senior White( 1955) reported
only 2% of wild- caught An. aquasalis in stages 3- 5.
by
Although malaria transmission by An. albimanus and
An. darlingi may occur indoors in some circumstances
chi-
maximum
post- blood mass than
for An.
100%)
likely
more
gambiae(
higher percentage
much
examined
be
to
Lyimo
and
of the small
Takken
by
Ovaries of host-
feeding
Although the
albimanus.
Trexler and Travis 1993). Although
albimanus
1993).
seeking An. darlingi captured in Brazil are rarely
(
wing length, multicollinearity of these two variables
impedes estimates of their independent impact on egg
maturation(
Frederickson
was
eggs
blood
mass after
regression estimates
was much
during one egg cycle( Briegel and Hurler 1993, Klowden
albimanus
advanced to stage 3 of Christophers ( DCL, LPL, and
An.
of
from
An.
behavior is common in natural, largely exophilic
comparisons(
of
correlated
a
An. darlingi ( Perez de
Scorza 1976),
and
in
similar to values calculated
and
upon
normal behavior for An. albimanus, An. darlingi, and
related species in the field. Even though sugar- deprived
females of a strain of the former species were attracted
repeatedly to hosts and took multiple blood meals
feeding,
of the same species
average mass of
nuneztovari
The present study calls into question whether
repeated host- seeking within a gonotrophic cycle is
Takken
of pre- gravids observed
diuresis was
or both.
the experimental
of
females
smaller sample of unmated
and
may be less subject
that induce afrotropical
albimanus matured eggs after one
confirming
199
relationship between the pre- gravid state and lack of
et al.
1990, Frederickson 1993).
The
Ecology
feeding ( Loyola et al. 1993, Lourenco- de- Oliveira
1994). Thus, replete females would need to reinitiate
the host- location process to refeed which, based upon
smaller
pre- gravid,
as
Takken 1993),
the data reported here, is usually unnecessary for egg
a
An. gambiae( 69. 21998)
et al. (
Zimmerman 1992), females of these species typically
leave dwellings during the night or morning of blood
maturation. The exophilic behavior of these neotropical
required at
species suggests that they might more readily consume
least two experimentally delivered blood meals to mature
plant sugar for energy, a behavior nearly discarded by
eggs.
the endophilic afrotropical vectors ( Beier 1996). The
The An.
females
generally larger
scanty field data available indicate that nectar feeding
may be attributable to rearing
richer larval diet, or the temperate
is common in female Nyssorhynchus ( Senior-White
albitarsis
than An. albimanus,
were
which
conditions, such as a
1952).
source of this species from southern Brazil( Horosko et
al.
1997). The
laboratory
An.
rearings of
albitarsis on
Acknowledgments
high and low diets did not yield a skewed size distribution,
in field
which
samples
of
related
species
is
commonly negatively skewed ( Lounibos 1994b).
Moreover, An. albitarsis females took a smaller blood
meal, on average,
their
body
Perhaps
source
1972),
related
than
as
higher
a
this
explaining
albimanus
nutritious
percent(
compared to
regression
data for
An.
estimated
to the smaller
from the less
pre- gravid
logistic
size,
22. 9%)
An.
from
blood
significant
with
S. Horosko and J. Bento graciously provided An.
lengths.
albitarsis s. s. from their colony. We are grateful to S.
its
Juliano for statistical advice and to R. Darsie, C. Lord,
W. Takken, R. Zimmerman, and an anonymous reviewer
for critical comments on an earlierdraftofthemanuscript.
meal
of
An.
and/ or
albitarsis were
albimanus (
only
variation.
a
10. 7%).
poorer
mass
The
FNS) for hospitality and assistance in Porto Velho and
of USAMRU- Rio for the same in Peixoto de Azevedo.
proportion to
wing
human host ( Shelton
model provided
species,
in
We thank personnel of Fundacao Nacional da Saude
(
The
fit to the
after
blood
significant
Research was supported in part by grants from NIH( AI31034) of the USA and CNPq, PAPES/ FIOCRUZ and
FNS of Brazil. This is Florida Agricultural Experiment
Station, Journal Series No. R- 06536.
Journal of Vector
200
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Volume 23, Issue 2, December 1998

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