Journal

of
Vector Ecology
Journal of the Society for Vector Ecology

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Volume 21, No. 2 December 1996

JVE 21( 2): 105- 201 ISSN 1081- 1710
 Journal of Vector Ecology
Volume 21 - Number 2 December 1996

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 @[Link]

EDITORIAL BOARD

M. S. Mulla, Chair W. J. Crans R. S. Nasci

University of California Rutgers University CDC-DVBID
Riverside, CA, USA New Brunswick, NJ, USA Fort Collins, CO, USA

R. C. Axtell C. I. Dahl M. W. Service

North Carolina State University University of Uppsala Liverpool School of Tropical
Raleigh, NC, USA Uppsala, Sweden Medicine, Liverpool, England

N. Becker F. R. Holbrook E. D. Walker

KABS, Waldsee, Germany USDA- ARS Michigan State University
Laramie, WY, USA East Lansing, MI, USA
D. R. Barnard
Medical & Veterinary R. S. Lane S. K. Wikel
Entomolology Laboratory University of California Oklahoma State University
Gainesville, FL, USA Berkeley, CA, USA Stillwater, OK, USA

H. Briegel G. G. Marten S. C. Williams

University of Zurich New Orleans MCD San Francisco State University
CH- 8057, Zurich, Switzerland New Orleans, LA, USA San Francisco, CA, USA

L. S. Mian
County Vector Control
San Bernardino, CA, 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, P. O. Box 87, Santa Ana, California 92702 USA.

Subscription Rates: Membership, including the Journal of Vector Ecology,$ 50.00, Student membership$ 25. 00,
Institutional subscription $ 50.00,
Sustaining membership$ 100.00
 SECOND INTERNATIONAL CONGRESS
OF VECTOR ECOLOGY

Theme: Achieving Sound Vector Control Through Ecological Understanding

Sponsored by the
SOCIETY FOR VECTOR ECOLOGY

OCTOBER 19- 24, 1997

HOLIDAY INN INTERNATIONAL DRIVE RESORT
ORLANDO, FLORIDA, U. S. A.

The Second International Congress of Vector Ecology will be held in Orlando, Florida,
U.S. A., on October 19- 24, 1997. It is being sponsored by the Society for Vector Ecology.
Facilities for the Congress will be in the Holiday Inn International Drive Resort, which is 15
minutes from the airport and downtown Orlando.
The City of Orlando is world renowned for its Disney World Resort, Universal Studios,
and Sea World. A short distance away is Cape Canaveral and the Kennedy Space Center. It
offers a subtropical climate. Florida is a state that offers leading edge technology, great
agricultural production, and a wide variety of vector problems.

Scientific Program
The Scientific Program will include plenary sessions, symposia, and poster sessions.
Only invited papers will be presented at plenary sessions and symposia. Submitted papers
will be accepted for poster sessions. Among the topic areas to be presented are:

Changing Patterns of Vector-Borne Molecular Biology

Disease Genetics of Vectors

Parasitic Disease Trends Vector- Pathogen Interactions

Viral and Bacterial Disease Trends Surveillance and Reporting Systems
Vector Control Programs Worldwide Immunology and Vaccine Development
Vector- Host Interactions Educational Programs

Biogeography New Approaches to Control

Systematics of Vectors Future Predictions and Needs

Human Ecology and Sociology

For information contact: Gilbert L. Challet, Secretary, 2nd International Congress of

Vector Ecology, P. O. Box 87, Santa Ana, CA 92702, Phone ( 714) 971- 2421, ext. 148, Fax
714) 971- 3940.
 JOURNAL OF VECTOR ECOLOGY

Volume 21 DECEMBER, 1996 Number 2

CONTENTS

International Congress of Vector Ecology Frontispiece

Board of Directors
ii
Russell E. Fontaine Memoriam
iii
E. Paul Catts Memoriam
Guidelines for Contributors
vii

Submitted Papers

A Concise Review of Chironomid Midges( Diptera: Chironomidae) as Pests and Their Management
Arshad Ali 105
An Ecological Survey of Anopheles albimanus Larval Habitats in Colombia
Gerald G. Marten, Marco F. Suarez, and Rodrigo Astaeza 122
Reanalysis of the C. G. Macnay Mosquito Repellent Data L. C. Rutledge R. K. Gupta
and 132
Life-Table Characteristics of Toxorhynchites splendens( Diptera: Culicidae) Cohorts Reared Under
Controlled Food Regimens D. D. Amalraj and P. K Das 136
Laboratory and Field Trials of Fenthion and Cyfluthrin Against Mansonia Uniformis Larvae
Han- Heng Yap, E. S. Annie, Chow- Yang 146
Lee Foo, Chong Ngo-Long, Abdul Hamid Awang, Baba Rohaizat, and Abdul Malik Yahaya Natural
Variation in Blood- Feeding Kinetics of Four Mosquito Vectors Dave D. Chadee and John C. Beier 150
Effects of Age and Mating on the Host- Seeking Behavior of Aedes aegypti Mosquitoes
M. J. Klowden and N. M. Fernandez 156
Vertical Distribution of Adult Mosquitoes ( Diptera: Culicidae) in Southern and Central Sweden
Jan O. Lundstrom, Jan Chirico, Anders Folke, Christine Dahl 159
Use of the Fly Grill for Assessment of House Fly Populations: An Example of Sampling Techniques
That Create Rough Fuzzy Sets I. Scudder
Harvey 167
Bacterial Abundance in Larval Habitats of Aedes albopictus( Diptera: Culicidae) in a Florida Cemetery
Edward D. Walker, George F. O' Meara, and William T. Morgan 173
Interrelationships between Different Borrelia Genospecies and Their Principal Vectors
E. I. Korenberg and G. G. Moskvitina 178
Identification of Larval Instars of Aedes albopictus ( Skuse) Aedes triseriatus( Diptera:
and
Say) (
Culicidae) Based on Head Capsule Size Hwa- Jen Charles. S. Apperson
Teng and 186

Proceedings
27th SOVE Annual Meeting, Fort Collins, Colorado, 10 October 1995

From Mouse to Sequence and Back to Mouse: Peregrinations of an Arbovirologist

Charles H. Calisher 192

Scientific Note

Breeding of Anopheles plumbeus in Tires in France S. Karch 201

 SOCIETY FOR VECTOR ECOLOGY

BOARD OF DIRECTORS

OFFICERS

President Michael W. Service

President- Elect Rex E. Thomas

Vice- President David A. Dame

Past- President B. Fred Beams

Gilbert L. Challet
Secretary- Treasurer

REGIONAL DIRECTORS

Southwestern Minoo B. Madon

Northwestern Steven V. Romney
North Central Thomas R. Wilmot
South Central Cluff E. Hopla
Northeastern Durland Fish
Southeastern Jonathan F. Day
European Isabelle Thiery

ii
 NUMBER 2 DECEMBER, 1996 VOLUME 21

IN MEMORIAM

Russell E. Fontaine
1914- 1996

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The Society of Vector Ecology lost a leader and a Russ was born on October 9, 1914 in Worcester,
central figure when Russell E. Fontaine passed
away on
Massachusetts. He is survived by his wife Vera, sons
August 6, 1996 after an extended illness. He was most Bob ( M. D.) and Ted, daughter Lola Craft, four
active as the President of the Society in 1982 and grandchildren, and brother Leo.
thereafter editor for the all- important Newsletter. Using Russ did his entire undergraduate work at the
his global network from his he
previous experiences,
University of Toronto and graduated in 1939 with a
played a major role
enabling the Society to reach the B. Sc. in agricultural sciences. Through his contact with
level of national and international prominence that it Richard Peters, he became involved in medical
currently enjoys. Most recently he had served very
entomology in the Armed Forces during World War II
effectively as Coordinator of the University of in Australia. He continued to serve as a medical
California Mosquito Research Program from 1976 until
entomologist in the U. S. Army, first in Seoul, Korea,
his retirement in 1986.
from 1947 through 1948 and subsequently with the

iii
VOLUME 21 DECEMBER, 1996 NUMBER 2

Caribbean Command from 1948 through 1950. In 1950 Chief, Aedes aegypti Eradication Program from 1964
he was enticed to join Richard Peters again, this time through 1966 and later appointed Chief, Operations
with the Bureau of Vector Control, California Section Malaria Program International ( 1966- 1969).
Department of Public Health. It was during this tenure He continued his involvement in global malaria control
that he was dubbed " the Senator" because of his programs with the World Health Organization from
statesman- like, professional approach to
conducting 1969 through 1976. In 1976 he left to accept a statewide
business at hand. In 1958 he left to serve as Senior responsibility for coordinating the mosquito research
Malaria Advisor in Ethiopia where he directed amalaria program in the University of Californian system where
pilot project and assisted the host government in he remained until his retirement in 1986.
planning, developing, and implementing a countrywide
malaria eradication program. From 1960 to 1964, he
served as Regional Malaria Advisor, Latin America for Dr. Robert K. Washino, University of California,
U. S. A.I.D. Subsequently, he joined CDC as Assistant Davis.

iv
NUMBER 2 DECEMBER, 1996 VOLUME 21

IN MEMORIAM

E. Paul Catts
1930- 1996

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The scientific
community lost one of its brightest was during this labor that Paul developed the work ethic
and most talented members when E. Paul Catts, 66, that he practiced throughout the rest of his professional
pr
entomology at Washington State University and personal life.
WSU), died 5 April 1996 after suffering a heart attack
He married Margaret Seavy in 1952. Themarriage,
while
playing lacrosse with the university team.
m. Paul which ended in 1978, produced two sons, Glen and
one of the finest, most creative people I have
was
Wade. During the Korean conflict, Paul was a corn
known. He life" his 66 years than
squeezed more" out of
missioned officer in the United States Army, serving as
most of us could only dream of. Just being around him a Battery Commander of an antiaircraft artillery unit.
almost always made a person feel happier. Catts earned his B. S. and M. S. degrees in
Paul was born 3 April 1930 to Helen Gleason and
entomology from the University of Delaware( UD) and
E. Paul Catts, Sr., in Elizabeth, New Jersey. He was
a Ph. D. in parasitology from Berkeley. In 1964 he was
graduated from Cranford High School, spending his hired by UD as an assistant professor and, within 10
summers raising tomatoes for the Heinz Company. It years, was promoted to professor. Paul taught and lived

v
VOLUME 21 DECEMBER, 1996 NUMBER 2

the importance of ecology. While at UD, he led field several hours of sawing, hammering, bending, cutting,
trips to the Delaware marshes, the Okefenokee Swamp, etc., it was done. And a beauty it was although I would
and the Hawaiian Islands while part of his research suggest that it was no threat to the Trojan Horse! I regret
examined how wildlife adapted to infringements of to inform the readers that five minutes after placing our
human activity in woods, swamps, and marshes. fake horse in the field, it was unceremoniously smashed
He married Dana Ketner in April 1979 and, in to pieces by the true equines. Paul' s comment at the
keeping with " Cattsian tradition," the wedding was in time was" nothing beats a trial but a failure."
an old church ( built in 1773!) that had no heat and the He was a giver. He was truly concerned about the
reception was in the local fire house hall! In 1980 they way we treated Mother Earth. He spent much time with
moved to Pullman, WA, where Catts served as chair of youngsters and adults at a church camp called Camp N-
the entomology department at WSU. For the next 16 Sid- Sen in Washington. His story- telling skills were
years, Paul taught various courses in entomology while legendary, especially around a campfire. He was an
he and Dana enjoyed the great outdoors with their active scout leader and a member of the Community
daughter, Summer and son, Ketner. Congregational United Church of Christ. Other little-
The dictionary defines ` Renaissance Man' as fol- known talents included building turtle boats, archery,
lows: a present- day many broad interests
person with sand sculpting, skier, birder, reciting poetry, and playing
who has the opportunity to indulge in them so as to the ukulele.

acquire a knowledge of each that is more than superficial. He was humble. Although outspoken at times, he
Paul Catts was such a person. He was a scientist. He was was well- liked. His popularity did not breed arrogance,
internationally recognized in medical and forensic however; it was not his nature. One of his descriptive

entomology, having published over 40 papers in both words was " shirty," a term he used to describe people
areas. Paul worked on over 50 homicide investigations, whom he thought took themselves much too seriously.
attempting to use insects to clarify murder investigations. He practiced simple yet active living, reusing what most
He was an author. He coauthored two editions would discard, and making needed items from scratch.
1970 1982) Manual of Medical He would undoubtedly be amused and perhaps slightly
and of the Entomology);
Entomology and Death: A Procedural Guide; and Insects embarrassed by all the nice things that have been written
Did it First, all of which he illustrated. He authored two about him.
Annual Review of Entomology articles.
Many of his He was a friend; a friend to the earth, a friend to the
writings have been published in the Delaware arts, a friend to his students, and a friend to all those who
Conservationist. were lucky enough to know him.
He was a teacher. He taught by and through Paul' s efforts did not go unrecognized. In 1977 he
inspiration, thereby instilling in his students an unending received Order of the Arrow, Order of Merit, and the
desire to learn the subject. He made a point of photo- Silver Beaver Award from the Boy Scouts of America
graphing students in his class so he could call them all for his distinguished service to youth. In 1972 he was
One his Insects as Outstanding Educator in America. In
by name. of most popular courses," and recognized

People," was highlighted by a bug luau featuring such 1975 and 1989 the Entomological Society of America
culinary delights as fried grubs and bee pupae muffins! presented him with its Excellence in Teaching Award
He was an artist. In addition to illustrating his and Award for Outstanding Service respectively.
scientific publications, he was an accomplished wildlife Michael Costello, a research technician at WSU,
and historical artist. A number of his works remain on wrote the following about Paul Catts: " It' s difficult to

display at the Delaware Nature Education Society, the convey the magnitude of the void Paul Catts' passing
National Headquarters for Sigma Nu, and the WSU leaves behind. He was the smartest, most intellectually
James Entomology Museum. He also created several honest, generous, and sincerely humble person I have
duck and trout stamps in Delaware and Washington. ever known.
I always had difficulty calling him my
Even his class handouts carried one or more of his friend because to do so seemed like immodest, self-
drawings. congratulatory name dropping. If he knew that, it would
He was a creator/ inventor. One of his maxims was have embarrassed him. He was the sort of person I want
why buy it when you can make it." During our bot fly to be when I grow up. And I miss him terribly."
research together, Paul decided we should build a fake That sums up the feelings for a lot of us about Paul
horse to try and figure out what exactly about a horse Catts.
attracted the adult flies. To this end, I met him at his
house early one Saturday. Having never constructed a By Stanton E. Cope, Ph.D., Lieutenant Commander,
horse myself, I was curious, to say the least. After United States Navy.

vi
 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.

Page charges, which

defray the cost of publication, are $ 35 per printed page.
partially
SOVE members who are unable to pay page charges may apply for a limited number of waivers.
Reprint charges are shown in the table below.

Pages 1- 4 5- 8 9- 12 13- 16 17- 20

50 copies 60.00 105. 00 150. 00 195. 00 240.00

or less

Each add' l 20.00 38. 00 56. 00 74.00 92. 00

50 copies
Same order

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

P. O. Box 87
Santa Ana, CA 92702

vii
VOLUME 21 DECEMBER, 1996 NUMBER 2

ADVERTISING

Commercial advertising space is available in the Journal; full

page ( black and white) at $ 150. 00 per issue, half page ( black
and white) at $ 90. 00 per issue. Inquiries may be addressed to
H. B. Munns, Advertising Manager, SOVE, P. O. Box 338,
Pioneer, California 95666, telephone ( 209) 295- 3540 or Fax
209) 295- 3563.

The publisher reserves the right to approve or refuse any

advertisement.
The publisher is not responsible for any
claims, litigations, or expenses resulting from the advertiser' s
unauthorized use of any name, photograph, sketch, or words
protected by registered trademark or copyright.

viii
Journal of Vector Ecology 21( 2): 105- 121

A Concise Review of Chironomid Midges (Diptera:

Chironomidae) as Pests and Their Management

Arshad Ali

University of Florida, IFAS, Central Florida Research & Education Center,

2700 East Celery Avenue, Sanford, FL 32771- 9608.

Received 18 May 1995; Accepted 25 October 1995

ABSTRACT. Increasing eutrophication of inland urban and suburban natural and man- made aquatic
ecosystems resulting from natural and anthropogenic factors in recent years has been conducive to
population increases of aquatic chironomid midges in many parts of the world. Adult midges from these
habitats frequently emerge in large numbers, causing nuisance problems, human allergies, and also have
severe adverse economic impact. Globally, nearly 100 of the 4, 000 known chironomid species are
documented as pestiferous. Numerous laboratory and field studies to reduce midges by physical and
cultural, biological, and chemical means have been conducted. Organochlorines, organophosphates( OPs),
pyrethroids, and insect growth regulators( IGRs) have been evaluated against midge larvae in the laboratory
and/ or used in the field. The best results for chemical control have come from OP insecticides( chlorpyrifos
and temephos), and IGRs ( diflubenzuron, methoprene, and pyriproxyfen). The OP insecticides have

generally provided larval field control for two to five weeks at rates< 0.56 kg AI/ha resulting in insecticidal
concentrations of< 1 to 5 ppm, but increased tolerance by midge larvae to some materials has been reported.
Insect growth regulators( especially pyriproxyfen) have provided> 90% suppression of midge emergence

for several weeks at< 0.25 kg Al/ha. Pyriproxyfen, methoprene, and diflubenzuron warrant registration for
chironomid control in the USA. Attempts at physical and cultural control of chironomids have given mixed
results. Biological control agents, such as Bacillus thuringiensis serovar. israelensis, the flatworm Dugesia
dorotocephala, as well as several fish species are useful but only in smaller habitats. The midge habitats,
which cover hundreds or thousands of ha, require appropriate investigations on the biology, ecology, and
behavior of larval and adult pestiferous midge species to formulate suitable control strategies.

Keyword Index: Chironomidae, midges, pests, nuisance problems, control, management.

INTRODUCTION numbers of species, biomass, or both. However, in

recent years, chironomid densities amounting to

Chironomids ( non- biting midges) are one of the thousands of larvae/ m2 of habitat have been reported
most ubiquitous, diverse, ecologically important
and
from many countries of the world( Ali 1991a). Prevalence
macroinvertebrates ( Coffman and
groups of aquatic of such high midge densities in many habitats are
Ferrington 1984), the most widespread insects in generally due to increasing eutrophication of the habitat
freshwater ( Thienemann 1954), and occur on all from anthropogenic sources, which causes a decline of
continents including Antarctica. The larvae occupy a macrofaunal species diversity. As a result, populations
broad spectrum of aquatic and semi- aquatic habitats of some pollution- tolerant organisms, including some
including brackish and marine waters, and even inhabit species of Chironomidae, increase greatly in such
some semi- terrestrial and terrestrial biotopes( Oliver et habitats. Rapid human population growth and related
al. 1990). increases in human activities around the globe in recent
In inland natural or man- made aquatic ecosystems, decades necessitate construction or creation of a variety
chironomid larvae are usually abundant in terms of of new temporary or permanent aquatic habitats amid
106 Journal of Vector Ecology December, 1996

urban and suburban population centers. Some of these

Accumulations of dead adult midges and the unsightly
habitats, such as reservoirs and channels used for some
webs spun by the predaceous spiders around resting
industrial discharges, ponds and effluent discharge midges on buildings, stucco, and fascia deface structures
channels at
processing facilities, residential-
sewage
and require frequent cleaning and washing of properties
recreational lakes, irrigated agriculture, and others remain for proper maintenance. Adult midges clog air-
heavily infested with chironomid larvae. Consequently, conditioning wall units and automobile radiators. Some
frequent emergence of adult midges in large numbers
species stick to car paint causing damage. Dead midges
from these habitats cause a varietyofnuisance, economic,
can have the odor of rotting fish, which persists for
and in some situations medical problems for the human several days after removal of the dead adults. At times,
populations within the dispersal range of these insects. the dead adults accumulate on roads in such quantities
that they make the roads slippery and dangerous for
NATURE OF PROBLEMS traffic. Such conditions often prevail in the multistoried
car- parking lots along waterfronts in Venice, Italy,
Chironomidmalestypicallyproduceaerialswarms. where also at the Marco Polo Airport, there is great
Females usually do not swarm, but rest on marginal
concern regarding the possibility of airplanes skidding
shore vegetation and structures until
they fly into a over massive accumulations of dead midges on
swarm of males, select a male, and copulate. Size of the
waterfront runways. Entry of adult chironomids into
swarms
may vary from as few as 10 to many millions delicate equipment mounted on airplanes poses danger
Sublette and Sublette 1988). Dense midge swarms
and additional economic loss to the aviation industry
often limit human activity outdoors because the adults ( Barbato et al. 1990).
can be inhaled or fly into the mouth, eyes, or ears; adult Chironomid midges are not known to be vectors of
swarms
may cause asphyxia in cattle( Grodhaus 1963). any disease organism. However, adult midges are
The adults deposit meconium or release
egg masses on associated with human allergic reactions, such as asthma,
resting surfaces which frequent washing or
require rhinitis, and conjunctivitis( Cranston 1988). The adults
repainting. Midges soil automobiles, cover headlights
emerging from some polluted habitats may transport
and windshields, and the swarms may create
risky bacteria ( Steinhaus and Brinley 1957) and organic
driving conditions. They are hazardous to passengers insecticides( Larsson 1984) to terrestrial environments.
and crews on trains, buses, cargo vessels, and boats in Globally, there are estimated 20, 000 species of
Venice, Italy ( Ali et al. 1992). Attraction of adult
chironomids, with 4, 000 species presently known
midges to light causes great discomfort in residential ( Cranston 1995). Of these, nearly 100 species in the
areas because the adults swarm around lighted outdoors subfamilies, Orthocl adii nae, Tanypodin ae, and
electrical fixtures and other objects which serve as
Chironominae have been reported to emerge in large
swarm markers. Small- sized adult midges enter homes numbers and pose pest problems. A list of the nuisance
and cause a of nuisance and economic problems
variety midge species was provided by Ali( 1995).
indoors ( Ali 1995). They cause
may a considerable
Chironomid larvae also can produce certain
economic loss to the hotel and tourism industry in many undesirable effects, such as contamination of drinking
situations worldwide. An economic impact study in water Langton 1988).
supply systems ( et al. Midge
Florida, USA( Anonymous 1977), revealed that at least
larvae transported in potable water find their way into
10 counties in Florida were economically affected by bottled beverages; they have also been seen in toilet
chironomids; and specifically swarms
emanating from bowls, causing concern for homeowners ( Mulla, pers.
Lake Monroe, Sanford, central Florida, inflicted a commun.). Larvae of some midge species are agricultural
business loss of$ 3 to 4 million annually on the City of pests, damaging rice seeds and plants ( Stevens and
Sanford, with just one lakefront motel spending nearly Warren 1992) and several horticultural and aquatic
50, 000 each year on maintenance and control
property plants as reviewed in Ali( 1991a).
attempts of the pest. Similar, but unquantified, economic
loss caused by adult midges has been in the
reported MANAGEMENT A'I' I EMPTS
literature from many parts of the USA, Japan, Italy, and
Australia. Midges also can be a problem for paint, Numerous chemical, biological, and physical and
pharmaceutical, and food processing industries where
cultural techniques to control midges in a variety of
hordes of adults
may contaminate the final products. habitats have been employed in the past 50 years. Of
There can be a considerable disruption and loss of
these, chemical control has been the most practiced.
working time because of midge swarms, impairing Midge control in some natural or man-made habitats by
work, and/ or complete stoppage of operation.
causing mechanical means, such as application of surface oils to
December, 1996 Journal of Vector Ecology 107

trap emerging adults ( Lewis 1957), removal of midge

proliferation could be discouraged by manipulating
egg masses from habitats, and dredging and
mixing of physical, chemical, and/ or biological conditions
substrate materials( Shimizu 1978) proved ineffective, conducive to chironomid breeding and rapid propagation.
while removal of substrate materials
containing midge
Attempts to control midges biologically are
larvae( Ali et al. 1976) and continuous water
spraying to relatively limited. The reported pathogens of chirono-
prevent midge oviposition ( Flynn and Bolas 1985) mids include viruses( Harkrider and Hall 1979, Majori
caused considerable midge reductions. Electrocutor et al. 1986), rickettsiae( Federici et al. 1976), and fungi
traps of various sizes and designs are perhaps the most ( Weiser and
McCauley 1971). Protozoan parasites of
commonly used means of adult midge control worldwide, chironomids are the microsporidia( Hunter 1968), and
but their effectiveness in reducing midge nuisance has the ciliophores( Corliss 1960). Several mermithid nema-
not been documented in any quantitative manner. Also, todes, Gastromermis, Hydromermis, Octomyomermis,
these traps in
often malfunction situations where midge
Orthomeris, and Paramermis have a variety of chiro-
swarms are dense and recurring( Ali 1991a). nomid hosts( Johnson 1963, 1965; Parenti 1966; Poinar
Behavioral manipulations of some midges through 1964, 1968). However, none of the above- mentioned
attraction of the adults to various light intensities( Ali et pathogens and parasites has thus far been studied
al. 1984, 1994a) or wavelengths( Kokkinn and Williams objectively enough to develop for midge control
1989, Hirabayashi et al. 1993) could reduce midge purposes.

nuisance in some situations. The adults could be drawn The bacteria, Bacillus thuringiensis serovar.
from heavily populated residential and business centers israelensis ( B. t. i.) and Bacillus sphaericus have been
to relatively less- inhabited areas by using brighter lights evaluated as midge larvicides in the
laboratory. The
at the latter sites where suitable adult control may be LC90 values for Glyptotendipes paripes, Chironomus
implemented. Keeping the lights off in some situations, crassicaudatus, Chironomus decorus, and Tanytarsus
where possible, may provide some relief from midge spp. ranged from 4. 6 to 47 ppm for three wettable
nuisance ( Bay, pers. commun.). For light spectrum- powder ( WP) and one flowable formulation of B.t. i.
specific attraction, commercially available lamps which
containing 1, 000 to 3, 500 international toxicity units
emit light with peaks in these parts of the spectrum may ( ITU)/ mg ( Ali et al. 1981). The former two midge
be employed for adult midge diversion, trapping, or species were insensitive to several toxic strains ( to
decoy purposes. In case of adulticiding, applications of
mosquitoes) of B. sphaericus, with LC5))values exceeding
insecticides could be synchronized with emergence 50 ppm in the laboratory ( Ali and Nayar 1986). In

periodicity of the adults ( Ali 1980), their range of outdoor ponds in Florida, a WP of B.t. i., containing
dispersal ( Ali and Fowler 1983), and short- term and 1, 000 ITU/ mg, gave 18 to 88% larval reductions of

long- term patterns of abundance( Ali et al. 1983, 1985a). Chironominae ( Chironomini and Tanytarsini) for two
This practice should not only reduce the land area to be weeks at rates ranging from 1 to 10 kg/ ha. The same
treated, but also the amount of insecticide needed.
formulation applied at 3 kg/ ha to a pond on a golf course
Reduction of midge larvae in some habitats is in central Florida, yielded up to 67% reductions of larval
possible through habitat management, such as source Chironomini for four weeks posttreatment( Ali 1981a).
reduction by alternate operation of sludge lagoons In a man- made lake in southern California, a technical
Anonymous 1963), and rotational flooding and drying powder( Vectobac®, 5, 000 ITU/mg) of B. t.i. produced
of partial areas ofspreading systems used for aquifer > 90% larval reductions of C. decorus for two to four
recharge ( Anderson et al. 1964). Proper designing of weeks at rates of 4. 4 to 6. 7 kg/ ha; a flowable formulation
new reservoirs and man- made lakes would be conducive ( Vectobac® 12 AS, 1, 200 ITU/mg) applied to the same
to low midge production( Magy 1968). In some habitats, lake also resulted in excellent control of the midge for
midge populations may be manipulated by understanding four to five weeks when applied at 9. 4 and 18. 7 liters/ha
their interaction with prevailing macrophytes( Johnson ( Mulla et al. 1990). At a lower rate of 5. 2 liters/ ha, the
and Mulla 1983), by reducing
or some larval food flowable formulation provided little or no control of the
components( Ali 1990). Environmental management to midge. In other studies in California, Rodcharoen et al.
reduce midge populations is highly desirable in large ( 1991) used technical powders( 5, 000 and 12, 430 ITU/
natural midge habitats, each spread over thousands of mg), corngrit granules ( 200 ITU/mg), and an aqueous
hectares. In such habitats, physical and chemical
suspension( Vectobac® 6AS, 600ITU/mg) formulations
composition of substrate materials and
chemistry of
of B.t. i. in 30 m2 and 0.3 m deep ponds and/ or in man-
overlying water in relation to spatial and seasonal made lakes ranging from 8. 4 to 21. 4 ha and 1. 8 to 2. 1 m
abundance of larvae of pestiferous midges may provide in depth. In these studies, > 80 to 100% control of

a clue to the ecological basis of midge production. Their chironomine midges was achieved for two to three
108 Journal of Vector Ecology December, 1996

weeks at the rather high rates of treatment employed, but Anderson 1965) and whitefish ( Rasmussen 1990)
Tanypodinae in general had remained unaffected. produced short- term reduction of midge larvae.
Specifically, a technical powder( 5, 000 ITU/ mg) at 6. 7 Mosquito fish did not produce any significant midge
kg/ ha gave complete control of Chironomus midges in reduction even when stocked at 276 kg/ ha ( Bay and
a lake( Rodcharoen et al. 1991). Thus, B. t. i. is effective Anderson 1966). In one situation, the introduction of
mostly against Chironominae midges, but relatively catfish remained ineffective in reducing midge nuisance
high rates of treatment( at least 10X or higher than the because of the immediaterecolonizationbymidgelarvae,
rates established for mosquito larvicidal activity) are replacing the supply of fish food as it was consumed
required to achieve satisfactory chironomid control in ( Hayne and Ball 1956). The use of exotic fish, such as
some situations. The use of such elevated rates of B. t. i. Tilapia spp. for chironomid control( Legner and Medved
for midge control in California, Florida, and elsewhere 1973) involves the risk of causing temporary or
may be possible in habitats ranging up to 100 ha, but permanent environmental and faunal alterations in the
would not be economical or practical in natural large aquatic ecosystem ( Hurlbert et al. 1972). In general,
lakes spread over thousands of hectares. Field studies short- term and partially effective biological control of
on B. sphaericus in California( Rodcharoen et al. 1991) chironomids through predatory fish alone may be
confirmed the ineffectiveness of this bacterium against possible only in small(< 20 ha or so) and closed habitats.
chironomids when applied at a rate as high as 22. 4 kg/ In open ( connected to river systems) and large lakes,
ha. Therefore, the presently available strains of B. which extend over thousands of hectares, the use of
sphaericus do not appear to offer
any potential for predatory fish would have to be considered as one
midge control. There is a need to discover more toxic component in the overall integrated approach to
and effective strains of B. t. i. and B. sphaericus against chironomid management.

chironomid larvae. There are a few reports of chironomid larvae and

The invertebrate predators of chironomid larvae pupae forming significant proportions of food consumed
and pupae include a wide range of predators, both small
by amphibian newts ( Avery 1968, Strohmeier et al.
Hydra ( Grzybkowska 1988)] and large [ Odonata 1989). Frogs and toads also were reported to consume
Johnson 1985)]. Armitage( 1995) lists 11 invertebrate adult midges( Wilson 1969).
groups that consume aquatic stages of chironomids in a Among birds, the groups that consume chironomids
wide range of aquatic habitats, and also specifies include flycatchers, gulls, martins, plovers, quail, rails,
terrestrial invertebrates predaceous and scavengers on swallows, swifts, terns, waders, wagtails, waterfowl,
adult midges. Although numerous invertebrates have and others. Armitage( 1995) provided Latin equivalents
been reported in the literature as predators of chironomid of English common names of some birds which consume
larvae and pupae, the planarian, Dugesia dorotocephala, chironomids as food. In northern latitudes, chironomids
is the only invertebrate studied in a quantitative manner formed a large part of the diet of mallard ducklings
for the biological control of chironomids under field ( Chura 1961), so much so that in one artificial lake high
conditions ( Ali and Mulla 1983). Since mass rearing mortality of mallards in their first week of life was
and maintenance of D. dorotocephala seems feasible attributed to the low availability of chironomids( Street
Tsai and Legner 1977), this flatworm, and perhaps 1977).
other Dugesia species, such as tigrinamay have potential The mammals which consume chironomids include
for chironomid control in some habitats. bats and humans. Large numbers of Chironomidae in
Chironomid predation
by fish has been reported in stomach and fecal samples of bats have been reported,
the literature by large biologists.
a number of aquatic
and it was suggested that the bats cue in on the humming
Armitage( 1995) has provided selected references
citing sound emanating from large aggregation of swarming
chironomids as food of at least 19 fish groups( families), Griffith Gates 1985).
midges( and
Swarming dipterans
including bream, bullhead, char,
grayling, gudgeon, (
including chironomids) are used as food by humans
minnow, perch, pope, tench, Tilapia, brown and rainbow
living around some large African lakes( Armitage 1995).
trout, The flies that are attracted to lights, fall to the ground and
walleye, whitefish, and mormyrids.
Additionally,
young bass, carp, catfish, mosquito fish, desert pupfish, are collected, boiled, and made into small cakes( Kungu
gilthead seabream, and sunfish were reported to feed on cake) which are said to taste similar to caviar or salted
chironomids( Ali 1991a). Despite the numerous accounts locust.
on chironomid predation
by fish in the literature, field The above given account on amphibians, birds, and
assessments revealing any successful biological control
mammals are merely reports of natural predation on
of Chironomidae through the use of fish are limited. In Chironomidae by these animal groups. None of these
quantitative field studies, only the carp ( Bay and animals has been assessed or developed in a quantitative
December, 1996 Journal of Vector Ecology 109

manner to reduce or control midges

biologically. Japan were susceptible to chlorpyrifos(
LC90= 0.0139
The chemical control of midges primarily by ppm)( Tabaru 1985a), but tolerated fenitrothion( LC90=
larviciding has been attempted mostly in the USA and >
4 to 8. 87 ppm)( Sato and Yasuno 1979, Tabaru 1985a).
Japan( Ali 1991a, Tabaru et al. 1987). A few scattered Species of Chironomus and Tanytarsus in the Santa Ana
and limited chironomid chemical control studies in the
River water percolation system, California, were highly
laboratory and/ or field have also been conducted in
susceptible to chlorpyrifos and temephos( LC90< 0.005
Europe, United Kingdom( Edwards et al. 1964), France ppm),
but Cricotopus spp. in the same habitat were
Sinegre et al. 1990), Germany( Buchmann 1932), Italy tolerant to the two insecticides as well as to the other
Ali and Majori 1984, Ali 1985b, 1992), Africa OPs tested (
et al.
LC90 = 0. 12 to 2. 1 ppm) ( Ali and Mulla
Abul- Nasr et al. 1970, Brown et al. 1961), and Australia 1976). Chironomid fauna of concrete sewage channels
Stevens 1992, Trayler et al. 1994).
and storm drains in California were generally tolerant to
The use of pyrethrum powder against midge larvae chlorpyrifos, fenthion, malathion, and temephos ( Ali
was attempted in Germany more than six decades ago and Mulla 1980). In natural lakes of Florida, G. paripes
Buchmann 1932). In the USA, pyrethrins, rotenone, were susceptible to most OPs while C. crassicaudatus
orthodichlorobenzene, and trichlorobenzene were the in the same lakes tolerated chlorpyrifos, fenthion,
first chemicals used as midge larvicides ( Fellton 1940,
malathion, and temephos, with LC9os ranging from 0. 14
1941). Later, organochlorines, such as DDD, DDT, to 0. 48 Ali 1981b).
ppm(
In the lagoon of Venice, Italy,
dieldrin, BHC, and others were used against midge
Chironomus salinarius were generally susceptible to
larvae( Brown et al. 1961, Patterson 1964, Anderson et the OPs tested( TABLE 1). The test pyrethroids were
al. 1964). However, the problems of bio- magnification
highly toxic to many chironomid species occurring
of organochlorines in the aquatic food- chain, occasional in a of habitats ( TABLE 1), but use of pyre-
variety
fish mortality, and development of resistance to throids as midge larvicides in natural or man- made
organochlorines in midge larvae( Lieux Mulrennan
and
lakes would involve a great risk because they likely
1956) necessitated the use of organophosphates( OPs), would have simultaneous adverse effects on nontarget
such as Dipterex, DDVP, EPN, and malathion which invertebrates and fish ( Mulla et al. 1978a, 1978b).
proved highly toxic to Chironomus plumosus at 0. 11 kg However, pyrethroids may be useful for midge control
AI/ ha in laboratory and field trials ( Hilsenhoff 1959, in sewage ponds, wastewater channels, and perhaps
1962). The OPs, fenthion, and temephos also were rice fields( Stevens 1993) where nontarget biota would
successfully used nearly 30 years ago as larvicides of G. be of minimal concern. Pyrethrins have been success-
paripes in some small- sized natural lakes in central fully used to control chironomid larvae infesting
Florida( Patterson and Wilson 1966). public water supply systems ( Burfield and Williams
In the past 25 years, the
majority of midge chemical 1975).
control studies have been conducted in the USA In field studies, many OP insecticides including
particularly California and Florida), Japan, Italy, and chlorfenvisfos, chlorpyrifos, chlorpyrifos methyl,
Australia. These studies include OP insecticides and diazinon, fenitrothion, fenthion, malathion, methyl
insect growth regulators( IGRs), such as chitin synthesis parathion, phenthoate, temephos, and trichlorfon have
inhibitors( CSIs) and juvenile hormone analogs( JHAs) been used since the early 1970s to reduce midge larvae
Ali 1995). Severalpyrethroidsalsowerehighlyeffective in a variety of habitats worldwide ( Mulla et al. 1971,
against midge larvae in the laboratory ( Ali 1981b). 1975; Tabaru et al. 1987; Stevens and Warren 1992).
Larval susceptibility of selected chironomid species to Among these insecticides, chlorpyrifos and temephos
various OPs and pyrethroids in the laboratory in different
have been the most extensively used because of their
geographical regions of the world are summarized in higher levels of effectiveness against a variety of midge
TABLE 1. species. In California, chlorpyrifos applications at rates
Data in TABLE I reveal that susceptibility of a
of 0. 11 to 0.28 kg AUha to shallow( 1 to 5 m deep) man-
chironomid species to the OPs or the pyrethroids made lakes covering< 10 to 100 ha resulted in excellent
including numbered experimental materials) varies midge control for over one month( Mulla et al. 1973, Ali
considerably, and different chironomid species or genera and Mulla 1977b).
The same insecticide at 0. 25 kg AU
occurring in the same habitat may respond
differently to ha also was successfully used for control of Chironomus
a test compound in terms of susceptibility. For example, riparius in a sewage effluent channel in Chicago, Illinois
Chironomus tepperi populations in rice fields in Australia ( Polls et al. 1975). In Japan, chlorpyrifos methyl
were highly susceptible to chlorpyrifos( LC90= 0. 0019 concentrations up to 2 ppm maintained for 20 to 30
ppm), but were tolerant to fenthion( LC90= 0. 447 ppm)
minutes provided satisfactory control of C. yoshimatsui
Stevens 1992). Similarly, Chironomus yoshimatsui in in wastewater gutters, disinfectant tanks, and discharge
110 Journal o Vector E 20! December, 19 6

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December, 1996 Journal of Vector Ecology 113

channels at sewage treatment facilities( Tabaru 1985b). has indicated that these OP larvicides were effective and
More recently, an emulsifiable concentrate ( EC) and suppressed larval populations of midges for at least
a pellet formulation of chlorpyrifos at rates of 0. 07 two weeks and longer, at rates below 0.56 kg AUha,
ppm( EC) and 0. 2 to 0. 6 kg AUha( pellets) yielded
up to resulting in insecticidal concentrations of< 1 to 5 ppm.
100% control of chironomid larvae for several days in Granular and pellet formulations of these OP compounds
rice fields in Australia ( Stevens and Warren 1992, have produced better chironomid control in terms of
1995). magnitude and duration in various habitats than the EC
Temephos has also been used in California as a formulations.
midge larvicide ranging from 0. 17 to 0. 84 kg All
at rates There are no specific data in the literature on
ha in water percolation basins and man- made lakes. chironomid adult control although in Florida in the
These rates yielded a wide range of midge control 1950s and 1960s, malathion and malathion- lethane or
depending upon the nature of habitat and its midge naled applied as thermal aerosol fogs from trucks, boats,
composition. For example, in water percolation basins, and airplanes at 0. 14 to 0. 27 kg AUha( malathion) were
temephos at 0. 27 to 0.38 kg AUha gave a maximum of effective, giving up to four days control of G. paripes
78% control of Tanytarsus, Chironomus, and Procladius ( Patterson et al. 1966). In Japan, there are no significant
midges for one week posttreatment( Johnson and Mulla reports on the use of insecticides as midge adulticides;
1980), while rates of 0. 56 to 0. 84 kg AI/ha in man- made but in Italy, deltamethrin and malathion have been
recreational lakes controlled midges for four to five employed to control adult C. salinarius in Venice( Ali
weeks ( Mulla et al. 1971, 1975). By contrast, much et al. 1992). Presently, no specific insecticide is registered
lower rates of 0. 17 to 0. 28 kg AUha were needed to in the USA for the sole purpose of adulticiding midges.
achieve satisfactory control of midges in water- However, some OP compounds and synthetic pyre-
percolation basins( Ali and Mulla 1976) and recreational throids, such as Scourge®( resmethrin) labeled for adult
lakes in California( Ali and Mulla 1977b) and Florida mosquito control ( Rathburn 1988), can be used for
Xue et al. 1993). However, repeated use of temephos midge adulticiding as included on the label.
in a lake in California resulted in poor or lack of control In the past two decades, IGRs have provided
of C. decorus and Procladius spp. even at application additional options for midge In laboratory
control.

rates of 0. 33 to 0. 56 kg AI/ ha( Johnson and Mulla 1981). evaluations, several JHAs ( including methoprene and
In Japan, temephos at concentrations of
up to 2 ppm pyriproxyfen) and CSIs( including diflubenzuron) were
maintained for 60 minutes gave
satisfactory control of effective at ppb levels against a variety of midge species
C. yoshimatsui in polluted rivers( Tabaru 1975, Tabaru in the USA( California and Florida), Japan, and Australia
et al. 1978, Ohno and Shimizu 1982). In eel
culturing ( TABLE 2). Among the JHAs, pyriproxyfen and
ponds, chironomid populations were
significantly methoprene were the most active. In Australia, pyri-
0. 05 1 of temephos ( Ohkura and proxyfen caused 90% inhibition of adult emergence of
reduced by to ppm

Tabaru 1975, Yasuno et al. 1982). In wastewater P. nubifer at 10 ppb( Trayler et al. 1994); similar results
habitats, 2 ppm of temephos maintained for 20 to 30 were obtained with methoprene against Chironomus sp.
minutes produced excellent control of C. yoshimatsui 51 and Tanypus grodhausi midges in California( Mulla
Inoue and Mihara 1975, Tabaru 19856). In Italy, et al. 1974). In Japan, 0. 65 ppb of methoprene resulted
temephos at rates of 0. 2 to 0. 4 kg AUha reduced C. in 50% inhibition of adult emergence of C. yoshimatsui
salinarius populations by 82 to 92% in the saltwater ( Kamei et al. 1982). The benzoylphenylurea CSIs
lagoon of Venice( Ali et al. 1992). In shallow wetlands including diflubenzuron, Bay SIR- 8514, and several
in Australia, granular temephos at 0.26 kg AI/ ha gave 72 other experimental( numbered) compounds were highly
to 92% control of Polypedilum nubifer after seven days active with LC90 values in the range of 2.0 to 22 ppb
Pinderet al. 1993), and in 80 m2
experimental rice bays, ( TABLE 2). A comparative laboratory study on the
a pellet formulation of temephos applied at rates ranging activity of methoprene and diflubenzuron against C.
from 0. 042 to 0. 73 kg AI/ ha significantly suppressed C. yoshimatsui revealed diflubenzuron to be more toxic
tepperi- type larvae for at least 13 days at all treat- than methoprene( Tabaru 1985c).
ment rates utilized, with the higher rates yielding con- Field assessments of IGRs against chironomids
trol for longer periods ( Stevens and Warren 1994). have previously been reported from the USA( California
Temephos, in general, yields control of midges for and Florida), Japan, and more recently from Australia.
shorter durations than chlorpyrifos even when applied In California, Pelsue et al. ( 1974) reported 60 to 86%
at higher rates than the latter insecticide and in most midge control for one to three weeks with methoprene
situations is innocuous to Tanypodinae at field- use ( Altosid®, SR- 10) and diflubenzuron ( 25% WP)

rates. Overall, field use of chlorpyrifos and temephos concentrations of 0. 1 ppm in water spreading basins.
114 Journal of Vector Ecology December, 1996

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December, 1996 Journal of Vector Ecology 115

Several formulations of these IGRs were employed for methoprene concentrations of 0. 13 and 4 ppm controlled
chironomid control in man- made lakes, while
C. yoshimatsui only at the higher concentration
diflubenzuron also was utilized in flood control channels ( Tsumuraya et al. 1982).
and
spreading basins ( used to replenish subsurface
Laboratory and field studies of IGRs on chironomids
water) in California. In the lakes, 0. 11 to
methoprene at have shown that most of these compounds were effective
0.34 kg AUha inhibited adult emergence ofChironominae
at very low concentrations( ppb range) against a variety
and Tanypodinae midges
by 47 to 100% for 8 to 19 days of chironomid species. Some of these compounds
Mulla et al. 1974, 1976), while diflubenzuron at 0. 11 to induced midge control in the field for several weeks at
0. 28 kg AUha induced 48 to 100% for five 0. 25 AUha.
midge control rates <
kg Due to their unique mode of
to eight weeks( Mulla et al. 1976), and> 80% for two to action, the IGRs would be desirable for midge control
five weeks ( Ali Mulla 1977b, Ali 1978). In
and et al.
because they may not decimate midge larval biomass( a
flood control channels
spreading basins,
and vital component of the aquatic food chain) to the degree
diflubenzuron at 0. 11 kg AUha gave 88 to 100%( four that OP and other larvicides do.
weeks) and 59 to 64% ( three weeks) control of midges The use of OP insecticides and IGRs for chironomid
in a channel and
spreading basins, respectively( Ali and control in aquatic environments has been shown to have
Mulla 1977a). Evaluation of 25% wettable powder
temporary or chronic effectsonnontargetbiotacoexisting
WP) and 0. 5% granular( G) formulations of the IGR, with midge larvae( Ali and Mulla 19786, 1978c; Ali and
SIR- 8514 at 0. 11 and 0.28 kg AUha in ponds on a golf
Stanley 1981). Therefore, the use of these compounds
course in California resulted in 50 to 100% and 70 to in aquatic environments needs cautious evaluation of
100% control of Chironomus spp. and Procladius sp. their environmental implications and cost benefits.
midges for five weeks with the WP and the G formulation, However, adverse effects of chemical control agents on
respectively( Johnson and Mulla 1982). The level and nontarget biota would be of minimal concern in sewage
duration of control given by the lower rate of each ponds and polluted rivers.
In some situations, only
formulation were almost the same as produced by the partial areas of a habitat that support large populations
higher rate. In Florida, evaluations of methoprene, of midge larvae could be treated( Ali and Mulla 1977b,
diflubenzuron, and several new experimental IGRs in Ali et al. 1992). This practice would reduce some midge
various formulations against natural populations of
nuisance and simultaneously would be conducive to
chironomids in experimental ponds showed that among quicker restoration of the lost nontarget organisms from
SIR- 8514, MV- 678, diflubenzuron, UC- 62644, UC- areas of a habitat left untreated. Toxic effects of the
84572, methoprene, and pyriproxyfen, UC- 62644 was chemical used in such a practice would also diminish
the most active, inhibiting 94 to 99% adult emergence of sooner due to dilution.
midges for four 28 g Al/ ha.
weeks at a rate as low as
At present, temephos( G formulations) is the only
Other IGRs, SIR- 8514, diflubenzuron, and UC- 84572, chemical registered by the United States Environmental
also caused significant reductions of adult midge
Protection Agency for use against chironomid larvae in
emergence for several weeks posttreatment at rates standing waters in the USA, and diflubenzuron has a
ranging from 24 to 200 g AUha ( Ali et al. 1994b, Ali special local need registration for midge control in the
1995). Sustained release methoprene, Altosid® XR State of California. Repeated and prolonged use of only
briquet and Altosid® pellet formulations, reduced midge one insecticide, such as temephos as a midge larvicide
emergence from the ponds by 38 to 98% and 64 to 98% could result in buildup of resistance in midge larvae as
for seven weeks at rates of 0. 82 kg AI/ha( briquet) and already evidenced( Pelsue and McFarland 1971, Ohno
0. 22 kg AUha( respectively( Ali 1991 b). A 3%
pellets), and Okamoto 1980, Johnson and Mulla 1981). Therefore,
sand granule formulation of pyriproxyfen ( Nylar®) chemical control of midges in a habitat requires a
applied to the ponds at 50 g AI/ ha gave long- term( nine specific strategy of avoiding frequent and indiscriminate
weeks) midge control ranging from 81 to 100%( Ali et use of a chemical and promoting rotational use of
al. 1993). In a wetland in western Australia, 0. 5% G alternate effective materials where possible. To facilitate
pyriproxyfen at 50 g AUha inhibited> 80% emergence this, registration of methoprene, pyriproxyfen, and a
of chironomids ( predominantly P. nubifer) for three broader registration of diflubenzuron for midge control
weeks( Trayler et al. 1994). In Japan, diflubenzuron and in the USA is warranted.
methoprene applied at 1 ppm maintained for 60 minutes
for midge control in two rivers resulted in satisfactory Acknowledgments

control of C. yoshimatsui, with the former IGR being

more effective ( Tabaru 1985c). In the gutters and This is Florida Agricultural Experiment Station
discharge channels of sewage treatment plants, Journal Series No. R- 04545.
116 Journal of Vector Ecology December, 1996

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chironomid larvae ( Diptera: Chironomidae) in Evaluation of the juvenile hormone mimic
establishing rice crops using starch- based pyriproxyfen ( S- 31183) against nuisance chiro-
chlorpyrifos pellets. J. Am. Mosq. Contr. Assoc. nomids ( Diptera: Chironomidae), with particular
11: 206- 210. emphasis on Polypedilum nubifer( Skuse). J. Aust.
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December, 1996 Journal of Vector Ecology 121

37: 474- 478.

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Journal of Vector Ecology 21( 2): 122- 131

An Ecological Survey of Anopheles albimanus

Larval Habitats in Colombia

Gerald G. Marten', Marco F. Suarez2, and Rodrigo Astaeza3

Servicio Nacional de Erradicacion de Malaria, Bogota, Colombia

School of Policy Studies, Kwansei Gakuin University, Sanda, Hyogo 669- 13, Japan
2Instituto de Inmunologia del Valle, Universidad del Valle,
Apartado aereo 25360, Cali, Colombia
3Floramerica, Calle 93 No. 19- 25, Santafe de Bogota, Colombia

Received 13 December 1994; Accepted 20 March 1996

ABSTRACT: The flora and fauna of 69 aquatic sites in Colombia were surveyed to identify ecological
conditions that favor production of Anopheles albimanus. Anopheles albimanus larvae were most
numerous at sun- exposed sites with abundant Culex larvae and grass at the edge of the water. Only 29%
of the sites with An. albimanus larvae contained pupae, suggesting that poor larval survival prevented the
production of adult mosquitoes at many sites. In the Atlantic region, An. albimanus production was highest
from large ponds with an abundant and varied aquatic insect fauna, including many kinds of predators of
An. albimanus larvae. Although productive sites were often covered with water hyacinth, aquatic
vegetation was generally not a reliable indicator of An. albimanus production. In the Pacific region, An.
albimanus production was highest from small water bodies with few aquatic macrophytes and an
abundance of cladocera, reflecting an abundance of microalgal food for mosquito larvae. In both regions,
An. albimanus production was negatively associated with a complete cover of Lemna, fish, hydrometrid
nymphs, large species of cyclopoid copepods, and dragonfly or mayfly nymphs. Anopheles albimanus
production was also negatively associated with dytiscid beetle larvae in the Pacific region.

Keyword Index: Mosquito, ecology, habitat, larvae, biological community.

INTRODUCTION Anopheles albimanus larvae are generally found at

sites that are well exposed to sunlight( Breeland 1972).
Anopheles albimanus Wiedemann is a common One way to expand and refine this characterization is to
and widely- distributed neotropical mosquito species look for discernable communities of aquatic flora and
that breeds in a variety of aquatic habitats ( Breeland fauna. Plants and animals that share aquatic ecosystems
1972): ditches, temporary pools, ponds of all sizes, with An. albimanuslarvaeshouldhaveprofoundimpacts
lakes, streams, and estuaries. Source reduction for such on the larvae as food, shelter, predators, or competitors.
extensive larval habitat would appear to be an over- Savage et al. ( 1990), Rejmankova et al. ( 1991,

task. 1992), and Rodriguez et al. ( 1993) surveyed aquatic

whelming
The prospects for source reduction might be habitats on the Pacific coastal plain of southern Mexico,
improved if ecological common denominators could be where An. albimanus larvae were associated with
identified that cut across the apparent diversity of habitats. emergent aquatic plants, planktonic algae, pasture

Such information might provide ecological indicators grasses, or water hyacinth. In Belize, Rejmankova et al.
for recognizing sites that are most important for An. ( 1993) observed An. albimanus larvae to be associated
albimanus production, so that larviciding or other forms with cyanobacterial mats and submerged plants covered

of source reduction could be focused on those sites. with periphyton.

Ecological information might also help to identify key Anopheles albimanus is common throughout the
characteristics of An. albimanus larval habitats that coastal zone of Colombia( Quinones et al. 1987) ( Fig.
could be modified to render the habitats unsuitable for 1). We conducted a field survey of aquatic habitats in
An. albimanus production. Colombia to identify ecological conditions that support
 December, 1996 Journal of Vector Ecology 123

the production ofAn. albimanus. We particularly wanted

Aquatic fauna, including mosquito larvae and pupae,
to know if the habitat associations were the same in were collected with a plankton
net( 120 pml mesh). The
Colombia' s Pacific and Atlantic regions. The survey
mouth of the net was attached to a square frame, 20 cm
emphasized aquatic flora and fauna, their organization
on a side, which was dragged with a pole through water
into biotic communities, and their associations with An.
albimanus larvae and pupae.
up to 50 cm in depth. The total distance sampled by the
net at each site varied from 10 m to 50 m, depending on
the size of the body of water. Wherever possible, the net
MATERIALS AND METHODS
was dragged along a transect from one side of the water
The survey
to the other, but it was necessary to drag the net only near
was designed to cover the full range of
the shore if the water was too deep in the middle.
aquatic habitats that might produce An. albimanus on
the Pacific
Sampling at the largest sites was at intervals along the
and Atlantic coasts of Colombia. Twenty- shore.
seven sites were sampled on the Pacific coast from
Animals collected in the plankton net were preserved
October 1986 to April 1987 in the vicinity of Tumaco
1)
in formalin for identification and counting in the
Fig. and
extending 50 km inland along the highway laboratory. Counts were by taxonomic groups( TABLE
east of Tumaco. Mangroves dominate the coastal part 2); subsamples were counted when animal numbers
of the area surveyed, while farther inland the landscape
is dominated
were large. Mosquito larvae were counted by instar.
by small- scale agriculture ( coconut and
The large numbers of animals collected by the plankton
palm plantations, sugar cane, and subsistence crops).
net provided quantitatively reliable samples of most
The rainy season on the Pacific coast extends from
kinds ofanimals present at each site, including substantial
September to June with peaks in January, February, and numbers of mosquito pupae ( if present), which we
May. Small bodies of water are numerous because considered to reflect the production of adult mosquitoes.
much of the land is only slightly above sea level, rainfall
All animal counts, including mosquito larvae and pupae,
is plentiful, and the water table is high. Sample sites on
were expressed as numbers per meter dragged by the
the Pacific coast included borrow pits, stream
plankton net. A log( X+ 1) transformation was applied
impoundments, roadside ditches, and
temporary pools.
to animal counts to bring them closer to a normal distri-
Forty- two sites were sampled on the Atlantic coast bution before including them in the statistical analysis.
from May 1987 to October 1987) in the vicinity of Las Gut contents ofAn. albimanus larvae were examined
Flores, Santa Catalina, and Carmen de Bolivar(
Fig. 1). under the microscope to assess the quantity ofmicroalgae,
Cattle ranches dominate the Atlantic landscape along bacteria, detritus, and mineral particles. Gut content
with small- scale agriculture. There are two rainy seasons data were not included in the statistical analyses.
on the Atlantic coast— September to November and
Two kinds of correlations were calculated between
May to June— with distinct dry seasons between them. the 56 quantitative variables( physical, chemical, floral,
Small bodies of water are unusual on the Atlantic coast. and faunal): conventional linear correlation coefficients
Large cattle ponds and impoundments to store water for and nonparametric Spearman rank- order correlation
household use are common.
coefficients. A positive correlation between two kinds
A total of seven physical/ chemical factors, ten
of plants or animals reflected a tendency for both to be
categories of terrestrial or aquatic plants, and 39 abundant at the same site, as well as scarce or absent at
categories of aquatic animals ( five stages of An. the same sites. A negative correlation reflected a
albimanus plus 34 other kinds of animals) were assessed
tendency to be present or abundant at different sites.
at each site( TABLE 1, TABLE 2). Physical and chem-
The rank correlations were helpful for identifying
ical properties of the water at each site were measured
associations that were not apparent from conventional
between 10 AM and 2 PM. The pH was measured with
correlations because the associations were not linear.
colorimetric paper. Temperature, salinity, and Factor analysis with varimax rotation was applied
conductivity were measured with a YSI meter( Yellow to the 1, 540 conventional correlation coefficients
Springs Instrument Co., Yellow Springs, Ohio). Oxygen
between all physical/ chemical, floral, and faunal
was measured
by the Winkler method( Ruttner 1963).
variables to identify intercorrelated groups of variables.
Terrestrial vegetation was assessed visually as
Factors with eigenvalues> 2 were considered significant.
percent ground cover of trees, bushes, flowering plants, A variable was considered to be part ofthe intercorrelated
or grasses within one meter of the water' s edge. Aquatic group represented by a particular factor if its factor
vegetation was assessed
visually as percent cover of
loading exceeded 0.3.
submersed, emergent, or
floating macrophytes over the The factor analysis was repeated with a single
entire body of water. juvenile stage( larval instar or pupae) of An. albimanus
 Journal of Vector Ecology December, 1996
124

Atlantic Ocean

Panama Q Venezuela

Colombia
Pacific Ocean

CO

I
eo
foe

Brazil

Ecuador

Distribution of Anopheles albimanus

1. Las Flores
2. Santa Catalina
3. Carmen de Bolivar
7. La Ensenada Fl
8. El Charco
9. Salahonda
10. Bucheli

Figure 1. Geographic distribution of Anopheles albimanus in Colombia. Sampled sites were at locations 1- 3 in the
Atlantic region and locations 7- 10 in the Pacific region.
 December, 1996 Journal of Vector Ecology 125

TABLE 1. Physical/ chemical characteristics evaluated at each sample site.

Atlantic Pacific

Mean± SE Range Mean± SE Range

Area(
m2)'

1500± 272 144 - 4500 27± 6 1- 150

Depth( m)' 1. 54± 0. 10 0. 4 - 4. 0 0. 34± 0.04 0. 15 - 1. 0
Temperature(° C) 2 29. 3± 0. 3 27. 5 - 32 27. 7± 0. 4 24- 33
2
Conductivity( mhos) 566± 106 80- 4000 558± 252 110- 7000
2
Salinity( ppt) 0. 10± 0.04 0- 1. 5 0. 25± 0. 15 0- 4. 0
2
Oxygen( ppm) 4. 29± 0.30 0. 5 - 8. 0 3. 98± 0. 50 1. 0- 8. 5
pH2

6. 28± 0. 04 6. 0 - 7. 0 6. 10± 0. 04 5. 8 - 6. 4

Area and depth of the sampled water body.

2Measured between 10 AM and 2 PM.

TABLE 2. Flora and fauna evaluated at each sample site.

FLORA

Terrestrial vegetation at edge of water. Trees, bushes, flowering plants( Compositae, Verbinaceae), grasses
Gramineae).

Aquatic plants. Submersed plants( e.g., Chara, Elodea), emergent plants( e.g., reeds), floating-leaved plants
FAUNA
e.g., Nuphar, Nymphea, Brasenia), water hyacinth( Eichornia), duckweed( Lemna), water lettuce( Pistia).
Crustaceans. Cladocera, shrimp( malacostraca), ostracods, large cyclopoid copepods, small cyclopoid
copepods, calanoid copepods.
Aquatic bugs( nymphs and adults).
Hebrids, naucorids, hydrometrids, notonectids, nepids, belostomatids,
gerrids, veliids, mesoveliids, corixids, plaeids.
Aquatic beetles( larvae and adults).
Dytiscids, hydrophilids, hydraenids, miscellaneous Coleoptera.
Aquatic diptera larvae. An. albimanus( each instar and pupae), other Anopheles, chironomids,
stratiomyids, Culex( 1111 instars, III/IV instars), miscellaneous Diptera( e. g., ceratopogonids, tipulids)
Odonata nymphs. Mayfly, damselfly, dragonfly.
Other aquatic insects. Collembola.
Aquatic mites. Hydracarina.
Aquatic vertebrates. Fish, tadpoles.

in each run. The for

reason not including all stages of
RESULTS
An. albimanus in the same run was to avoid creating an
artificial " An. albimanus" factor due to the high Atlantic Region
correlation between different stages of the same species.
Anopheles albimanus larvae were present at 78%
The floral/ faunal associations identified by the factor
of the sites that were sampled in the Atlantic region. The
analysis were the same regardless of the stage of
An.
albimanus that was included in
number offirst instars per meter dragged by the plankton
a particular run.
net ranged from 0. 3 to 55; second instars ranged from
Stepwise multiple regression analysis was applied

to each of the
0. 1 to 40, third instars ranged from 0. 03 to 25, and fourth
instars ( and pupae) of An. albimanus as instars ranged from 0. 05 to 12. Only 31% of the sampled
dependent variables, using all floral, faunal and physical/
chemical variables as independent variables. Variables
sites had An. albimanus pupae, ranging from 0. 1 to 3. 5
pupae per net meter.
were considered significant if the F- value exceeded 4. 0
P< 0. 05).
The factor analysis did not reveal discrete floral/
faunal communities, but it did five
identify significant
 Journal of Vector December, 1996
126 Ecology

groups of associated flora and fauna( TABLE 3). One plants and animals associated with sites that had a
or sometimes two) of the groups was prominently minimum of bushes or trees at the edge of the water,
represented at every site we sampled. Anopheles so the water was fully exposed to the sun. Duckweed
albimanus larvae or pupae had a significant positive ( Lemna sp.) was often the dominant plant. If the pond
association with every group except one. was not heavily covered with duckweed, first instar
The most distinct floral/ faunal group in the Atlantic An. albimanus larvae were more abundant in asso-
region ( Atlantic Group# 1, TABLE 3)
was associated ciation with this floral/ faunal group than any other
with water hyacinth ( Eichornia sp.). Mid- day water group in the Atlantic region. Other larval instars of
27. 5°- 30° C); An. albimanus, as well as pupae, were common as well.
temperatures were relatively low( range:

water temperatures at sites with a heavy cover of hya- However, there were virtually no An. albimanus larvae
cinth averaged 2° C less than sites with few or no floating or pupae if the duckweed cover was greater than
diverse and abundant 85%.
plants. Group # 1 contained a

An. Anopheles pseudo- Anopheles albimanus larvae and pupae were a

fauna, including albimanus,

punctipennis Theobald, Anopheles triannulatus( Neiva significant part of Atlantic Group# 3, which included
Pinto), and Culex Anopheles albimanus submersed vegetation and large numbers of aquatic
and spp.
and higher instar larvae were particularly abundant [Link] instar An. albimanus larvae were
pupae

at sites where other animals in Group # 1 were also positively associated with Atlantic Group # 4, which
included grass at the water' s edge and crustaceans such
abundant.

Atlantic Group # 2 ( TABLE 3) consisted of as shrimp and small species of cyclopoid copepods.

TABLE 3. Groups of associated flora and fauna in the Atlantic region, based on factor analysis of all variables in
TABLE 1 and TABLE 2.'

Group 1 ( 15. 8%).

Dytiscids (. 87), plaeids (. 82), Anopheles triannulatus and Anopheles pseudopunctipennis (. 80), damselflies
78), stratiomyids(. 74), hydrophilids(. 73), Culex( all instars)(. 70), naucorids(. 58), chironomids(. 56), water
52), An. albimanus pupae (. 51), large
hyacinth (. 55), grass at edge of water (-. 53), dragonfly nymphs (.

cyclopoids (. 50), temperature (-. 50), cladocera (. 49), ostracods (. 47), fourth instar An. albimanus (. 46),

belostomatids(. 44), notonectids(. 37), third instar An. albimanus (. 36), nepids(. 35), mesovelids(. 34).

Group 2( 7. 9%).
Veliids (.70), tadpoles (. 70), duckweed(. 67), first instar An. albimanus (. 63), mesoveliids (. 63), calanoids
53), second instarAn. albimanus(. 52), fourth instarAn. albimanus(. 49), third instarAn. albimanus(. 47),
41), hydrophilids (. 39), belostomatids (. 38), large cyclopoids (-. 36), shrimp (-. 33),
An. albimanus pupae (.

collembola(. 33), corixids(. 32), trees and bushes(-. 30), fish(-. 30).

Group 3 ( 6.9%).
Hebrids(. 84), nepids(. 72), third instarAn. albimanus (. 54), An. albimanus pupae(. 53), dragonfly nymphs
53), second instarAn. albimanus (. 51), belostomatids(. 50), fourth instarAn. albimanus(. 43), naucorids
38), mayfly 34), first instar An. albimanus (. 33),
38), submersed plants (. 38), chironomids (. nymphs (.

damselfly nymphs(. 30).

Group 4( 6. 1%).
Shrimp(. 83), small cyclopoids(. 71), mayfly nymphs(. 67), notonectids(. 64), pH(. 41), grass at edge of water
36), flowering plants(. 34), plaeids(. 32), first instar An. albimanus (. 30).

Group 5 ( 5. 9%).
diptera (. 67), fish (. 56), 56), misc. coleoptera (. 52), ostracods (-. 51),
Small cyclopoids (. 72), misc. mites (.

cladocera(-. 45), grass at edge of water(. 36).

Factor loadings
group is in the group
parentheses after number.
Percentage of total variation explained by each

A factor indicates negative association with the group.

are in parentheses after each variable. negative loading
 December, 1996 Journal of Vector
Ecology 127

Atlantic Group# 5 was characterized

by the presence of TABLE 4.
fish, the presence of small species ofcyclopoid copepods
Results of stepwise multiple regression
analysis for the Atlantic region.
instead of large species), and the absence of ostracods
and cladocera( TABLE 3). Anopheles albimanus larvae Stage' Significant independent variables2
and pupae were not a significant part of Atlantic Group 1st instar
5. Positive: Culex4(. 50±. 10)
Results from stepwise multiple regressions( TABLE R2= . 3
( 74) veliids(. 34±. 09),
4) reflected of the associations
many identified by the water depth(. 32±. 09)
factor analysis. Each of the positive regression tadpoles(. 26±. 10)
coefficients in TABLE 4 represents a of
group small cyclopoids(. 20±. 09)
intercorrelated variables in TABLE 3. ( i. e., most of the Negative: large cyclopoids(-. 39±. 11)
variables listed in TABLE 4 are also in TABLE 3, where hydrometrids(-. 25±. 09)
they have the same relation with An. albimanus. The 2nd instar
variables selected Positive: veliids(. 35±. 11)
by multiple regression for TABLE 4
R2=' 3
are the best predictors of juvenile An. 75) nepids(. 35±. 09)
albimanus (
abundance.)
Culex4(. 32±. 09)
Faunal variables predominate in TABLE 4; no grass at edge of water(. 28±. 11)
physical/ chemical or floral variables had significant small cyclopoids(. 26±. 09)
regression coefficients, except for grass at the water' s Negative: hydrometrids(-. 21±. 09)
edge. Third/ fourth instar Culex larvae, veliid or nepid
3rd instar Positive: veliids(. 45±. 07)
bugs, and grass at the edge of the water were the best R2= . 3
predictors of sites with large 92) nepids(. 43±. 06)
numbers of An. albimanus (
larvae or pupae. Hydrometrid bugs were a Culex4(. 41±. 06)
strong
negative predictor for all juvenile stages of An. area of water body (. 35±. 07)
albimanus.
small cyclopoids(. 23±. 06)
All variables with strong negative regression grass at edge of water(. 19±. 07)
coefficients also had strong negative rank correlations, Negative: salinity(-. 27±. 07)
typically in the range of- 0. 3 to- 0. 5. Although fish did hydrometrids(-. 26±. 05)
not have regression coefficients strong enough to appear
4th instar Positive: veliids(. 44±. 05)
inTABLE4, therewasastrongnegativerankcorrelation
2
between fish An. R =. 95) nepids(. 42±. 05)
and albimanus larvae( r=-. 45, P<. 01) (
51, P<.
and pupae( r=-. 001). Culex4 (. 41±. 06)
dytiscids(. 32±. 07)
Pacific Region grass at edge of water(. 30±. 06)
There wereAn. albimanus larvae at 81% of the sites Negative: salinity(-. 47±. 06)
sampled in the Pacific region. Where larvae were hydrometrids(-. 34±. 07)
present, the number of first instars per meter dragged beetles(-. 27±. 05)
by misc.

the plankton net ranged from 0. 1 to 127, second instars

Pupae Positive: nepids(. 46±. 09)
ranged from 0. 1 to 31, third instars ranged from 0. 03 to
R2= '

8, fourth instars 77)' Culex4(. 43±. 09)

and ranged from 0. 05 to 52. Only 26% (
of the sampled sites had An. albimanus pupae, small cyclopoids(. 41±. 09)
ranging
from 0. 1 to 5 pupae per net meter. grass at edge of water(. 27±. 09)
The factor analysis revealed five Negative: hydrometrids(-. 28±. 09)
groups of fauna
and flora in the Pacific region ( TABLE 5). Anopheles mayfly nymphs(-. 27±. 10)
albimanus larvae or pupae were positively or
negatively '
associated with each of the groups except one. Dependent variables are each juvenile stage of An.
albimanus.
The faunal composition Pacific
of
Group # 1
5) to Atlantic
2Significant
Significant independent variables( P< 0.05). Normalized
was similar
Group# 1 ( TABLE
3), Pacific Partial regression coefficients and their standard errors
except
Group # 1 had lower diversity of
animals than did Atlantic Group # 1. First instar An. are shown in parentheses.
albimanus larvae were negatively associated with Pacific 3R2= percent of total variation in the dependent variable
Group # 1, in part because Pacific Group # 1 included explained by the listed independent variables.
duckweed, and An. albimanus larvae were absent if the 4II1/ IV instars.
 Journal of Vector December, 1996
128 Ecology

cover of duckweed was complete. Mid- day oxygen was larvae were a major part of this group.
low( 5. 8- 5. 9). Pacific Group # 5 ( TABLE 5) was associated
relatively low( 0. 5- 1. 7
ppm), and pH was

Water hyacinth was not part of Pacific Group# 1. No with small water bodies that lacked aquatic plants
hyacinth was found at the sites sampled in the Pacific such as reeds and floating- leafed plants. Group# 5 had
region.
an abundance of zooplankton ( clacoderans and small
of cyclopoid copepods), which were not a
Pacific Group# 2( TABLE 5) was associated with species

small, shallow bodies of water fully exposed to the sun. prominent part of the other floral/ faunal group
Mid- day water temperatures( 27°- 30° C) and dissolved associated with small water bodies ( Pacific Group
oxygen ( 3. 5- 8. 5 ppm) were higher than at other sites. # 2). Anopheles albimanus pupae and higher instar

There were seldom fish, and there were large numbers larvae were more abundant in association with Group
of Culex larvae. Early instar An. albimanus larvae were # 5 than any other floral/faunal group in the Pacific
more associated with Pacific Group# 2 than region.
positively
other
In stepwise multiple regressions for the Pacific
any group.
Pacific Group# 3( TABLE 5) was associated with region( TABLE 6), third/ fourth instar Culex larvae and
flowering plants cladocera were the best predictors of the abundance of
floating- leafed plants, as well as along
the shore. Tadpoles were usually abundant. Anopheles late- instar An. albimanus larvae and pupae. Dytiscid
albimanus larvae and pupae were neither positively nor beetle larvae were the best negative predictors of An.
Pacific 3. albimanus larvae and pupae.
negatively associated with Group#
Pacific Group# 4( TABLE 5) was associated with As in the Atlantic region, rank correlations for the
bushes or trees at the edge of the water. The water was Pacific region were in agreement with negativeregression
shaded, so mid- day water temperatures( 24°- 26° C) and coefficients. In addition, malacostracan shrimp had
relatively low. First instar
oxygen( 0. 8- 2. 5 ppm) were significant negative rank correlations withAn. albimanus

larvae( r=-. 42, P<. 01) and pupae( r=-. 32, P<. 01) in the
An. albimanus larvae were conspicuously absent from
but Anopheles Dyar and Knab Pacific region.
Group# 4, punctimacula

TABLE 5. Groups of associated flora and fauna in the Pacific region, based on factor analysis of all variables
in TABLE 1 and TABLE 2. 1

Group 1 ( 13. 0%).

Misc. coleoptera(. 93), plaeids(. 87), misc. diptera(. 86), duckweed(. 79), hydrometrids(. 55), mayfly nymphs
52), shrimp(. 51), pH(-. 45), notonectids(. 45), chironomids(. 45), dytiscids(. 40), veliids(. 36), first instarAn.
albimanus(-. 30).

Group 2( 9. 9%).
Culex( all instars)(. 85), dragonfly nymphs(. 77), first instarAn. albimanus(. 76), second instarAn. albimanus
67), water temperature(. 66), chironomids(. 66), oxygen(. 52), water depth(-. 49), fish(-. 33), area(-. 30).

Group 3( 9. 4%).
Belostomatids (. 81), tadpoles (. 80), mesoveliids (. 76), damselfly nymphs (. 62), floating- leaved plants (. 59),
flowering plants(. 42), pH(. 37).

Group 4( 9. 3%).
Anopheles punctimacula (. 91), hydraenids(. 81), grass at edge of water(-. 74), dytiscids(. 68), oxygen(-. 50),

trees and bushes(. 50), first instarAn. albimanus (-. 30), water temperature(-. 30).

Group 5 ( 6. 7%).
Emergent plants(-. 74), cladocera(. 67), third instarAn. albimanus(. 66), fourth instar An. albimanus(. 63),
An. albimanus pupae (. 56), area of water body (-. 63), small cyclopoids (. 56), mites (. 50), pH(. 34),
oxygen(-. 33), floating- leaved plants(-. 32), collembola(. 30).

Factor loadings
Percentage of total variation explained by each group is in parentheses after the group number.

A factor indicates negative association with the

are in parentheses after each variable. negative loading group.
 December, 1996 Journal of Vector Ecology 129

TABLE 6. Results of stepwise multiple regression

Although we did not find biological communities
analysis for the Pacific region.
that were completely distinct from one another, we did
Stage' Significant independent variables
z find consistent associations among many of the aquatic
plants and animals. Anopheles albimanus larvae and
1st instar Positive: temperature(. 51±. 15) pupae had a discernable relation ( positive or negative)
2
R = . 45)
3
collembola(. 50±. 15) with most of these floral/ faunal groups, the connection
Negative: ( no significant variables)
generally being stronger with the fauna than with the
flora. Among the physical/ chemical factors that we
Culex4(.
2nd instar Positive: 67±. 17) measured, only salinity appeared to be of consequence
4
R2= .
44) Negative: dytiscids(-. 51±. 17) to the distribution of An. albimanus larvae.
Culex4(.
Although there were similarities between the floral/
3rd instar Positive: 94±. 11)
faunal groups in the Pacific and Atlantic regions
R2= . 3
88) cladocera(. 50±. 09) particularly Group# 1 in each region), the floral/ faunal
grass at edge of water(. 24±. 09) groups in the two regions were far from identical. This
small cyclopoids(. 20±. 09) is not surprising, considering the physical differences
Negative: dytiscids(-. 41±. 11) between aquatic habitats of the two regions. We found
dragonfly nymphs(-. 50±. 15) a greater diversity of flora and fauna in the Atlantic
hydraenids (-. 31±. 10) region, apparently because many of the water bodies
Culex4(
that served as larval habitat for An. albimanus in the
4th instar Positive: 1. 15±. 11)
Atlantic region were larger than those in the Pacific
R2= . 3
89) cladocera(. 72±. 10) region.

belostomatids((. 24±. 09) One of the most important findings of the survey
Negative: dytiscids(-. 69±. 11) was that some sites had large numbers of all larval
dragonfly nymphs(-. 37±. 10) instars of An. albimanus as well as pupae, while other
Pupae
sites had large numbers of early instar larvae but no
Positive: cladocera(. 87±. 10)
R2= . 3 Culex4 (.
pupae. Most sites without pupae also lacked fourth
89) 72±. 10)
instar larvae. Sites that have large numbers of larvae
duckweed(. 72±. 10)
because they are attractive to oviposition are not
stratiomyids(. 31±. 10) necessarily the best sites for larval survival and the
belostomatids(. 27±. 09) production of adult mosquitoes.
Negative: dytiscids(-. 75±. 12)
Despite differences between the floral/ faunal groups
large cyclopoids(-. 45±. 11) of the Pacific and Atlantic regions, the relation of An.
hydrometrids(-. 35±. 09) albimanus larvae and pupae to floral/ faunal groups was
similar in both regions. In both regions first instar An.
Dependent variables are each juvenile stage of An. albimanus larvae ( which we consider to reflect
albimanus. oviposition) were associated with sun- exposed sites,
2Significant independent variables( P< 0. 05). Normalized Particularly sites with grass at the edge of the water.
partial regression coefficients and their standard errors Sites that were shaded by trees or bushes at the edge of
are shown in parentheses. the water, or completely covered with floating plants
3
R
2=_
percent of total variation in the dependent variable
such as duckweed, were least favored for oviposition.
explained by the listed independent variables.
Anopheles albimanus pupae ( and presumably the
production of adult mosquitoes) were associated with
4III/ IV instars.
two ecological factors. First was food supply, as indicated
by the abundance of An. albimanus pupae at sites with
an abundance of animals( e. g., Culex larvae or cladocera)
DISCUSSION
that feed on algae. Sites with large numbers of these
animals had abundant phytoplankton ( or submersed
Ideally, it be desirable to discrete
would
identify vegetation covered with periphyton), and the guts ofAn.
floral/ faunal communities, some of which include An. albimanus larvae at these sites contained large quantities
albimanus and others of which do not. Dominant
of microalgae. The hypothesis that microalgae are a key
vegetation or other flora/faunal indicators in each
resource for An. albimanus production is compatible
community could facilitate prediction of the magnitude with the observation of Savage et al. ( 1990) and
of Anopheles production.
Rejmankova et al. ( 1993) that An. albimanus larvae
130 Journal of Vector Ecology December, 1996

were associated with planktonic algae and periphyton in regard to the kind of algae, because some algae are
Mexico and cyanobacterial mats in Belize. nutritious for An. albimanus larvae and others are not
Predation was the second factor of importance to ( Marten 1986). It is possible that An. albimanus
the abundance of An. albimanus pupae in our survey. production could be reduced if microalgae were

Curiously, most predators of mosquito larvae were suppressed by chemical or biological means or if
positively associated with An. albimanus larvae and nutritious algae were replaced by algae that are not
pupae, apparently reflecting a positive response of all nutritious.

fauna, whether predator or prey, to sites with a high level Results from the survey point to specific predators
of biological productivity. Only two kinds of predators— of possible use for biological control: fish, hydrometrid
fish and hydrometrid nymphs— had a consistent negative nymphs, large cyclopoid copepods, and dytiscid larvae.
association with An. albimanus larvae and pupae in both While fish are in common use, copepods have been used
Atlantic and Pacific regions. The negative association for Anopheles control only in field trials( Marten et al.
with hydrometrids was most striking. No An. albimanus 1994). Dytiscids and hydrometrids are known to prey
pupae were observed at any sites in the Pacific or on mosquito larvae ( Mijares and Broche 1985; G. G.
Atlantic regions where hydrometrids were present, Marten, personal observation), but they have not been
though An. albimanus pupae were found at 44% of the used for operational mosquito control.

sites without hydrometrids ( contingency table chi-

square= 7. 07, df= 1, P<. 01). Dytiscid larvae and dragonfly Acknowledgments

nymphs were negatively associated with An. albimanus

in the Pacific region. Guillermo Berruecos, Aquileo Bornacelli, Flavio
Marten et al. ( 1989) reported a strong negative Castillo, Parmenides Churta, Moises Cortes, Jose Vicente
association between large cyclopoid copepods and Manotas, Cesar Monje, and Jose V. Santander assisted
juvenile An. albimanus from the first 42 sites sampled in with field laboratory work. Financial support
and

this study. After all 69 sites were sampled, large ( Grant MVR-CO- 2- 85- 46) was provided by the National
cyclopoids had negative rank correlations with An. Research Council, U.S. National Academy of Sciences,
albimanus larvae and pupae ranging from-. 24 to-. 28. It through a grant from the U. S. Agency for International
appears the full magnitude of negative association Development. The New Orleans Mosquito Control
between the most effective cyclopoid predators and An. Board and Tulane University provided computer
albimanus was obscured
by grouping all larger cyclopoid facilities for statistical analyses. We thank Eliska
species for the statistical analyses reported here; large Rejmankova for comments on the manuscript.
cyclopoids included Mesocyclops longisetus ( a more
effective predator) and Mesocyclops venezolanus ( a REFERENCES CITED
less effective predator).
What are the implications of this study' s findings Breeland, S. G. 1972.
Studies on the ecology of
for control of An. albimanus? While water hyacinth was Anopheles albimanus. Amer. J. Trop. Med. Hyg.
identified as an indicator of An. albimanus production, 21: 751- 754.
the study did not identify other macrophytes to signal Hobbs, J. and P. Molina. 1983. The influence of aquatic
production at sites without water hyacinth. However, fern Salvinia auricultata on the breeding of
some plants appear to be reliable as indicators of sites Anopheles albimanus in coastal Guatemala. Mosq.
that do not produce An. albimanus. Production was low News 43: 456- 459.
from sites that were completely shaded by trees, and a Marten, G. G. 1986. Mosquito control by plankton
complete cover of small floating plants such as duckweed management: the potential of indigestible green
excluded An. albimanus larvae from a site. It might be algae. J. Trop. Med. Hyg. 89: 213- 222.
practical to plant shade trees around small water bodies Marten, G. G., R. Astaeza, M. F. Suarez, C. Monje, and
that would otherwise produce An. albimanus. Small J. W. Reid. 1989. Natural control of larval

floating plants ( e. g., duckweed or Salvinia) might be Anopheles albimanus ( Diptera: Culicidae) by the
used to render breeding sites unsuitable ( Hobbs and predator Mesocyclops( Copepoda: Cyclopoida). J.
Molina 1983; Margaret Dix, personal communication). Med. Entomol. 26: 624- 627.
Planktonic, epiphytic, and benthic microalgae Marten, G. G., E. S. Bordes, and M. Nguyen. 1994. Use
appear to be the most reliable indicators of a site' s of cyclopoid copepods for mosquito control.

capacity to produce An. albimanus. The practical Hydrobiologia 292/ 293: 491- 496.
significance of microalgae for An. albimanus control Mijares, A. S. and T. R. Broche. 1985. Control
requires further study, which should be specific with biologico de larval de mosquito mediante
 December, 1996
Journal of Vector Ecology 131

coleopteros acuaticos en condiciones

de laboratorio. L. Legters. 1993.
Rev. Cub. Med.
Trop. 37: 354- 358.
Environmental and regional
Quinones, M. L., M. F. Suarez, and G. A. determinants of Anopheles ( Diptera: Culicidae)
Fleming. larval distribution in Belize, Central America.
1987. Distribucion y bionomfa de los anofelinos
de
la costa pacffica de Colombia. Colombia
Environ. Entomol. 22: 978- 992.
Modica
18: 19- 24. Rodriguez, A. D., M. H. Rodriguez, R. A. Meza, J. E.
Rejmankova, E., H. M. Savage, J. I. Arredondo- Jimenez, Hernandez, E. Rejmankova, H. M. Savage, D. R.
Roberts, K. 0. Pope, and L. Legters.
and D. R. Roberts. 1991. Multivariate 1993.
analysis of
relationships between habitats, environmental Dynamics of population densities and vegetation
factors and occurrence of anopheline
associations of Anopheles albimanus larvae in a
mosquito
larvae Anopheles albimanus and An.
coastal area of southern Chiapas, Mexico. J. Am.
pseudo-
Mosq. Contr. Assoc. 9: 46- 58.
punctipennis in Southern Chiapas, Mexico. J. Ruttner, F. 1963.
Applied Ecol. 28: 827- 841. Fundamentals of Limnology.
Rejmankova, E., H. M. Savage, M. H. Rodriguez, D. R. University of Toronto Press, 295 pp.
Roberts, and M. Rejmanek. 1992.
Savage, H. M., E. Rejmankova, J. I. Arredondo- Jimenez,
Aquatic D. R. Roberts, and M. H. Rodriguez.
vegetation as a basis for classification 1990.
of Anopheles
albimanus Weidemann( Diptera: Culicicae) larval Limnological and botanical characterization of
habitats. Environ. Entomol. 21: 598- 603. larval habitats for two primary malarial vectors,
Rejmankova, E., D. R. Roberts, R. E. Harbach, J. Pecor, Anopheles albimanus and Anopheles pseudo-
E. L. Peyton, S. Manguin, R. punctipennis, in coastal areas of Chiapas state,
Krieg, J. Polanco, and Mexico. J. Am. Mosq. Contr. Assoc. 6: 612- 620.
Journal of Vector Ecology 21( 2): 132- 135

Reanalysis of the C. G. Macnay Mosquito Repellent Data

Gupta2

L. C. Rutledge' and R. K.

111 Circle Way, Mill Valley, CA 94941- 3420

2Department of Entomology, Walter Reed Army Institute of
Research, Washington, D. C. 20307- 5100

Received 17 July 1995; Accepted 19 October 1995

ABSTRACT: Data reported by C. G. MacNay on the protection periods of 38 repellents tested in the field
against Aedes sticticus, Aedes stimulans, Aedes vexans, and Aedes trichuris in 1937 were reanalyzed by
current methods. Protection periods decreased by 7. 6 min. for each 1° C increase in air temperature over
the range 17- 27° C. Treatment means were adjusted to the 22.2° C level to permit comparison of the
treatments on an equal basis. Pyrethrum extract, pine tar oil distillate, thyme oil, thymol, and geraniol
provided 3 or more hr. protection. Geranium oil, citronella oil, cedar oil, American pennyroyal oil, and
camphor provided relatively short protection. Confidence limits and multiple comparison data are
provided for the adjusted treatment means.

Keyword index: Mosquitoes, repellents, mosquito repellents.

INTRODUCTION be costly today, because of modern requirements for use

of human subjects in research. The cost of toxicology
In the 1992 keynote address to the annual conference testing, alone, could easily exceed$ 100,000 for a single
for Vector Ecology in San Francisco, A. test material. The present report was prepared to restore
of the Society
R. Barr stated that" one of our highest priorities should the MacNay data to currency by republication of the
be the systematizing of the older literature in such a original data with addition of confidence limits and
has been multiple comparison data based on current methods of
fashion that we can more easily determine what
done"( Barr 1993). The work of Focks et al. ( 1993) is statistical analysis.

a good example of the effective use of data dating back

Focks 1994) have shown that MATERIALS AND METHODS
as far as 1901. et al. (

reanalysis of previously published data may be justified,

if it is very recent.
Test Materials
even

The mosquito repellent data of MacNay( 1939) are Active ingredients included pine tar oil and pine tar
unique in that they represent tests of mostly botanical oil distillates, essential oils, organic compounds, and an
unspecified material, Pamph. 55 ( 8). Materials
natural association of northern
repellents against a

mosquitoes. Interest in botanical repellents has increased containing more than l active ingredient were pyrethrum-
in recent years due to public concern for safety of thyme( thyme oil and pyrethrum extract, 1: 2), citronella-
synthetic chemicals. Although the northern mosquitoes spike( citronella oil and spike lavender oil, 1: 1), Pamph.
55 ( 5) ( salol, 1: 1), and Pamph. 55 ( 7)
they have not camphor and
include several important pest species,

had a high priority in repellent testing programs. cedar oil, citronella oil, and spirits of camphor 1: 2: 2).
However, since the data bases for most computer Most materials were diluted 1: 1 in olive oil for
searches of the literature do not go back to i939, the testing. Thymol was diluted 1: 2 in olive oil. Pyrethrum
MacNay data are little known and little used
today. In extract( 1 test only), camphor, and paradichlorobenzene
become the were diluted 1: 3 in olive oil. Pyrethrum- thyme was
addition, statistical analysis had not yet

norm for scientific papers in 1939, and statistical data diluted 1: 1 in castor oil. Citronella- spike was diluted
were not included in the report when it was published. 2: 1: 1 in beeswax and lanolin. Pamph. 55( 5) was diluted
3: 2 in petrolatum. Phenyl salicylate was tested as the
Duplication of tests conducted by MacNay would
 December, 1996 Journal of Vector Ecology 133

saturated solution in olive oil. Pamph. 55( 7) was tested

in the undiluted form.
ted air temperatures were converted to° C and rounded
to the nearest whole number for analysis. Pyrethrum
Test Species extract in olive oil( 1: 1) and pyrethrum extract in olive
oil ( 1: 3) were treated as different test materials.
Repellents were tested against a natural association
of Aedes sticticus( Meigen), Aedes stimulans( Walker), RESULTS
Aedes vexans( Meigen), and A edes trichuris( Dyar) in a
wooded area in the vicinity of Ottawa, Ontario.
Biting Results of the analysis of covariance are shown in
rates determined prior to the tests ranged from 2. 3 to
17. 3 bites/ min. Tests were conducted TABLE 1. Effects of the treatments( test materials) and
the period during the covariable ( air temperature) on protection period
25 May- 1 July 1937. were significant at the
5% level. The coefficient of

Test Method
regression of protection period on air temperature was
All tests were
7. 6 min./° C, indicating that for each increase of 1° C in
conducted by MacNay himself,
working alone. Two ml of the test material
air temperature there was a corresponding decrease of
were applied
7. 6 min. of protection.
to each forearm, and the time until 4 or 5 bites were
received was
To permit comparison of the treatments on an equal
subsequently recorded. Air temperature,
basis, mean protection periods were adjusted for air
relative humidity, wind speed, cloud cover,
biting rate,
and time
temperature( TABLE 2). The adjusted treatment means
of
day were recorded as concomitant variables.
Data
shown are the means expected at 22. 2° C, the average air
were reported for 62 tests of 41 materials, but
5 tests were terminated
temperature recorded in the study.
prior to completion because of
Pyrethrum extract( 1: 1) provided 265. 1 min. ( 4. 4
rain or other reasons. The reduced data set includes 57 hr) protection at 22. 2° C ( TABLE 2). This period was
tests of 38 materials. Within this set, 21 materials were
tested one time
significantly longer than those of all other test materials
only and 17 materials were tested 2 or

more times.
except tar oil distillate II, pyrethrum extract( 1: 3), thymol,
thyme oil, and geraniol.
The term " tar oil ( rectified)" (
Data Analysis
TABLE 2) is a
synonym of pine tar oil, a dark, reddish- brown liquid
MacNay ( 1939) compared the test materials
by
rank, based on the means of the observed protection
derived from pine tar. MacNay tested pine tar oil and 3
periods. The present analysis
distillates prepared by F. A. Herman. The intermediate-
used the Statistical Analysis
System for Microcomputers,
boiling fraction( distillate II) provided 261. 3 min. ( 4.4
Version II. 0, 1984 hr) protection at 22. 2° C.
Statistical Consultants, Inc., Lexington,
Kentucky) for
Thyme oil ( Labiatae: Thymus vulgaris) provided
a more precise analysis.
199. 4 min. (3. 3 hr) protection( TABLE 2), and thymol
Preliminary analysis indicated that the effects of (
2- hydroxy- 1- isopropyl- 4- methylbenzene), a principal
relative humidity, wind speed, cloud cover, mosquito
constituent of thyme oil, provided 212. 3 min. ( 3. 5 hr)
biting rate, and time of day on protection period were not protection. Geranium oil (" geranium bourbon" of
statistically significant. The model fitted in the final
analysis included the response variable,
TABLE 2) provided 140.5 min.( 2. 3 hr) protection, and
protection period geraniol ( 3, 7- dimethyl- trans- 2, 6- octadien- 1- ol), a
quantitative), and 2 explanatory variables, test material
principal constituent of geranium oil, provided 181 min.
qualitative), and air temperature( quantitative). Repor- ( 3. 0 hr) protection.

TABLE 1. Analysis of covariance.

Source of
Sum of Mean
Variation df Squares Square F P

Test material 37 329, 451 8, 904 7. 20 0. 00

Air temperature 1 6, 043 6, 043 4. 89 0. 04
Error 18 22, 257 1, 236
Total 56 357, 751
 Journal of Vector December, 1996
134 Ecology

N), mean protection periods( min), 95% confidence intervals,

TABLE 2. Test materials, numbers of replicates(

and significance of differences between means at the 5% level. a

Protection Confidence

Period Interval Significance'

Test N

1: 1 4 265. 1 225. 6- 304. 6 a

Pyrethrum extract

distil. II 1 261. 3 179. 8 - 342. 8 ab

Tar oil

Pyrethrum extract 1: 3 1 260. 8 186. 4- 335. 2 ab

2 212. 3 157. 5- 267. 1 abc

Thymol
Thyme 2 199. 4 140. 7- 258. 1 abed

1 181. 0 106. 9- 255. 1 abcde

Geraniol
2 162. 0 106. 2- 217. 8 bcde
Pyrethrum- thyme
2 153. 6 99. 8- 207. 4 bcdef
Phenyl salicylate

laurel 1 148. 6 73. 6- 223. 6 bcdefg

Bay bcdefghi
Geranium bourbon 1 140. 5 57. 6- 223. 4
128. 4 54. 5 - 202. 3 bcdefghi
Tar oil distil. III 1
125. 8 51. 4- 200. 2 bcdefghi
Cresol 1
123. 6 48. 6- 198. 6 bcdefghi
Tar oil distil. I 1
2 123. 5 71. 0- 176. 0 cdefgh
Pine oil

2 117. 4 60. 0- 174. 8 cdefghi

Tar oil( rectified)

2 117. 3 64. 2- 170. 4 cdefghi

Citronella
113. 3 37. 7- 188. 9 bcdefghi
Pamph. 55 ( 5) 1
2 103. 6 47. 6- 159. 6 cdefghi
Geranyl acetate

2 102. 3 47. 5 - 157. 1 defghi

Citral
1 96. 3 14. 8 - 177. 8 cdefghi
Sweet almond

Citronella- spike 1 93. 3 17. 7- 168. 9 cdefghi

1 91. 0 16. 9- 165. 1 cdefghi

Bitter orange

1 91. 0 16. 9- 165. 1 cdefghi

Cedar
2 78. 6 24. 8- 132. 4 efghi
Benzyl alcohol
75. 8 1. 4- 150. 2 defghi
Pamph. 55( 7) 1
2 63. 7 4. 8 - 122. 6 efghi
Hemlock
Hedeoma 2 55. 8 1. 2- 110. 4 efghi

2 52. 1 0. 0- 104. 6 fghi

Anethole
1 48. 7 0. 0- 127. 5 efghi
Caprylic alcohol

2 42. 0 0. 0- 95. 7 ghi

Camphor
1 38. 4 0.0- 112. 3 efghi
Spike
1 33. 4 0. 0- 107. 3 efghi
Terebinth
Eucalyptus 1 31. 1 0.0- 107. 7 efghi

23. 1 0. 0- 103. 0 fghi

Pamph. 55( 8) 1
2 18. 3 0. 0- 72. 9 hi
Nutmeg i
2 14. 5 0. 0- 70. 3
Amyl salicylate

1 5. 8 0. 0- 80.2 ghi
Paradichlorobenzene
1 0. 0 0. 0- 68. 1 i
Olive oil( check)

Mean protection periods and confidence intervals adjusted for comparison at 22.2° C.
bTerminology is that of MacNay( 1939). The spelling of" anethole" has been corrected. The terms" tar oil"and" tar
oil ( rectified)" refer to pine tar oil. The term " bay laurel" usually refers to the sweet bay or Grecian laurel
Lauraceae: Laurus nobilis). The material designated as" geranium bourbon" is now known as" oil of geranium
Reunion", Bourbon being the former name of Reunion Island. The identity of the plant source is not known to the
latifolia( Labiatae). The bitter orange
The term" spike" refers to the spike lavender, Lavandula
authors. probably
is Citrus aurantium( Rutaceae). " Hedeoma" refers to the American pennyroyal Hedeoma pulegioides( Labiatae).
distinguishes edible varieties of the almond, Prunus amygdalus ( Rosaceae), from
The term " sweet almond"

inedible varieties, known as " bitter almond". The term" eucalyptus" is generic; eucalyptus oil is obtained from
several species, notably the blue gum, Eucalyptus globulus( Myrtaceae). The term" hemlock" usually refers to the
poison hemlock, Conium maculatum( Umbelliferae).
Means followed letter do not differ significantly at the 5% level.
by the same
 December, 1996 Journal of Vector Ecology 135

Several well- known botanical repellents provided

significantly longer than that of citronella oil ( 1: 1 in
shorter periodsofprotection: Citronellaoil( Gramineae: olive oil)( TABLE 2).
Cymbopogon nardus) provided 117. 3 min. ( 2. 0 hr)
Pine tar oil is also a traditional repellent that has
protection. Cedar oil( Cupressaceae: Thujaoccidentalis) persisted in use to recent times( Allen 1986).
MacNay
provided 91. 0 min. ( 1. 5 hr) protection. American (
1939) reported that the intermediate boiling fraction
pennyroyal oil ( Labiatae: Hedeoma pulegioides)
provided longer protection than the oil itself, without its
Hedeoma" of TABLE 2) provided 55. 8 min. ( 0. 9 hr)
objectionable color and staining properties. The present
of protection. Camphor ( Lauraceae: Cinnamomum
study shows that the observed difference was statistically
camphora) provided 42. 0 min. ( 0. 7 hr) protection. significant ( TABLE 2). The protection period of the
intermediate boiling fraction was also significantly longer
DISCUSSION than that of citronella oil.
Although thymol and geraniol have had
The present paper quantifies the relation of
comparatively little use as repellents, their protection
protection period and air temperature in the field for the
periods did not differ significantly from those of
first time. This relationship was
previously evaluated in pyrethrum, pine tar oil, and citronella oil in the present
the laboratory by Khan et al. ( 1973),
using deet( N, N- study. In addition, the protection periods of thymol and
diethyl- 1, 3- methylbenzamide) againstAe. aegypti( L.).
geraniol did not differ significantly from those of the
As calculated from TABLE 1 of Khan et al.( 1973), the
source materials, thyme oil, and geranium oil(" geranium
coefficient of regression of protection period on air bourbon" TABLE 2).
of
Osmani et al.( 1972) reported
temperature was - 2. 4 min./° C
in that study, compared
that geranium oil provided 230 min. ( 3. 8 h) protection
with- 7. 6 min./° C in the present study. The magnitude against Culex Linn. ( as
pipiens Culex fatigans
of the difference in values obtained reflects
difficulty
the Wiedemann) in India.
of extrapolating field results from limited laboratory Results of this study therefore suggest that pine tar
data.
oil, thyme oil, and/ or geranium oil, or their derivatives,
Variation of air temperature over the range recorded
could be commercially developed to be competitive
in the study( 17- 27° C) could account for as much as 76
with pyrethrum and citronella oil. This conclusion was
min. ( 1. 2 hr) of the observed variation in protection
not apparent prior to the present analysis.
periods. Accordingly, treatment means adjusted for
comparison at 22. 2° C( TABLE 2) differed substantially REFERENCES CITED
from those of the original report(
MacNay 1939). Only
2 treatments retained the same rank in the reanalysis:
Allen, M. 1986. Irving' s Fly Dope. Yankee 50( 5): 15-
Pyrethrum extract( 1: 1) at no. 1 and pine oil at no. 14. On 16.
the other hand, 7 treatments shifted
up or down in rank Barr, A. R. 1993. Critical issues in vector-borne
by 5 or more places: Citronella- spike, geranyl acetate,
disease. Bull. Soc. Vector Ecol. 18: 33- 37.
sweet almond, Pamph. 55 ( 8), hemlock, caprylic acid,
Focks, D. A., D. G. Haile, E. Daniels, and G. A. Mount.
and eucalyptus.
1993. Dynamic life table model for Aedes aegypti
However, confidence limits obtained in the analysis Diptera: Culicidae): Analysis of the literature and
of covariance ( TABLE 2) provide more precise
model development. J. Med. Entomol. 30: 1003-
comparisons of the adjusted treatment means than rank 1017.
alone. TABLE 2 shows that 134 differences among
Focks, D. A., S. B. Linda, G. B. Craig, W. A. Hawley,
adjusted treatment means were significant at the 5% and C. B. Pumpuni. 1994. Aedes albopictus
level. Adjusted treatment means and their associated
Diptera: Culicidae): A statistical model of the role
confidence limits may be preferable to unadjusted
of temperature, photoperiod, and geography in the
treatment means alone as product data
performance
induction of egg diapause. J. Med. Entomol. 31:
submitted to the Environmental Protection Agency for 278- 286.
registration of products and approval of product labels. Khan, A. A., H. I. Maibach, and D. L. Skidmore. 1973.
Prior to the advent of synthetic repellents, pyrethrum
A study ofinsect repellents. 2. Effect of temperature
and citronella oil were
widely used in repellent lotions, on protection time. J. Econ. Entomol. 66: 437- 438.
sprays, smokes, and candles. Pyrethrum is still used in
to
MacNay, C. G. 1939. Studies on repellents for biting
mosquito coils" produce repellent smoke, and flies. Can. Entomol. 71: 38- 44.
citronella oil is still available in repellent lotions and Osmani, Z., I. Anees, M. B. Naidu.
and 1972. Insect
candles. In the present study, the protection period of repellent from
creams essential oils. Pesticides
pyrethrum extract ( 1: 1 and 1: 3 in olive oil) was 6( 3): 19- 21.
Journal of Vector Ecology 21( 2): 136- 145

Life-Table Characteristics of Toxorhynchites splendens

Diptera: Culicidae) Cohorts Reared Under Controlled Food Regimens

D. D. Amalraj and P. K Dast

Vector Control Research Centre, Indian Council of Medical Research,

Medical Complex, Indira Nagar, Pondicherry- 605 006, India

Received 24 August 1995; Accepted 27 December 1995

ABSTRACT: Larval and adult life table characteristics of Toxorhynchites splendens were studied under
different food regimens. The survivorship from first instar to adult ranged from zero to 0. 27 and was
greatest in food regimens of> 6 mg( dry) prey/ predator/day. Median developmental time from first instar
to adult( E50) ranged from 15. 83 to 64. 63 days but was only 15. 83 to 19. 8 days in food regimens of >7 mg
dry) prey/predator/ day. Except at the lowest feeding rate, the proportion of the offspring that were female
did not differ significantly among treatments. Life expectancy( e!) ranged from 1. 93 to 50. 23 days for males
and from 11. 64 to 47. 84 days for females. Daily survivorship of adults reared under food regimens of> 2. 8
mg( dry) prey/predator/ day was> 0.94. The net reproductive rate( R0) ranged from zero to 518. 33 living
female offspring/female/ generation and was related linearly to the amount of food offered. The generation
times( G) ranged from 40. 18 to 81. 99 days and the instantaneous rate of increase in living female/ female
rm) ranged from zero to 0. 14. Female survivorship patterns approximated Slobodkin' s Type-II curve and
males exhibited either Type- II or Type- III curve. The number of eggs laid was greatest in the second week
of adult life and oviposition continued for six to seven weeks. The suitability of this species for the
biological control of container breeding mosquitoes is discussed in relation to its relatively high Ro, rm, and
long adult life span.

Keyword Index: Life- table, Toxorhynchites splendens, food regimens.

INTRODUCTION survival, development rate, and adult fitness( Fish and

Carpenter 1982, Mogi Hawley 1985). When
1984,
The usefulness of life tables as tools for studying determined under insectary conditions with different
insect population dynamics has been recognized as rates of feeding but keeping the other variables constant,
early as 1954 ( Morris and Miller 1954). Age- specific the life table of a predator will express a species- specific
horizontal life tables present a succinct tabular summary genetic potential and may be used to study the effect of
of mortality and reproductive schedules. It measures the factors on the survivorship and reproductive strategies.
fate of a real cohort, such as a number of individuals of Recently, the life table approach has been applied to
a single population( Reisen and Mahmood 1980). The studies of the survivorship and reproductive strategies
advantage of the laboratory analysis of life tables of of colonized culicines ( Crovello and Hacker 1972,
mosquito predators of that life table characteristics can Walter and Hacker 1974, Reisen et al. 1979) and
be by manipulating biological factors which are
studied anophelines ( Reisen and Mahmood 1980). However,
known to influence the life structure of an organism. complete survivorship and fecundity- fertility sche-
Several studies indicated that larval stress, primarily dules have not been compiled for the genus
caused by food limitation within habitats, not only Toxorhynchites.
produced small adults but adversely affected larval A review of the control of vector mosquitoes using
predators indicated that species of the genus
Address for Correspondence Toxorhynchites have the greatest potential to effectively
 December, 1996 Journal of Vector Ecology 137

control container breeding mosquitoes ( Hoick 1988) dried larvae were allowed to cool in a desiccator and
including Aedes aegypti, the major urban vector of
weighed with 0. 1 mg of precision. The dry weight of an
yellow fever, dengue, and dengue hemorrhagic fever
individual prey was then calculated. The number of
DHF) ( Rudnick 1967). Horsfall ( 1955) first reviewed
prey offered per predator per day was multiplied by the
the biology of Toxorhynchites and it was followed dry weight of prey to calculate the amount of food
by the review of systematics,
biology, and biological offered ( Amalraj and Das 1994). Early instar Tx.
control potential of the members
belonging to this genus splendens had great difficulty killing and consuming
Steffan 1975, Steffan and Evenhuis 1981). Several Ae. aegypti; and because their development time was
workers studied the biology and life table characteristics
of Toxorhynchites splendens in the
found to be very short, more growth and prey
laboratory ( Paine consumption occurred in the late instars( Chowanadisai
1934, Newkirk 1947, Muspratt 1951, Chan 1968, et al. 1984). Minimum food required for completion of
Furumizo and Rudnick 1978, Chowanadisai 1984).
et al.
the fourth instar larval development was found to be
These studies showed that adults of Tx. splendens between 60- 70%
of the total consumption during the
survived longer when compared to prey species. Other entire larval stage ( Vongtangswad and Trpis 1980).
desirable attributes of these biological control agents
Therefore offering early fourth instar Ae. aegypti to
are high daily consumption, ability of the larvae to young Tx. splendens would not have affected the overall
survive long periods of starvation when prey are not consumption rate. Each cohort consisted of 200 eggs in
available ( Trpis 1972, 1981), the phenomena of
enamel trays measuring 0. 45 x 0.4 x 0. 05 m and filled
compulsive killing ( Corbet 1963, Trpis 1972, Crans
with 3 1 tap- water. Each food regimen was replicated
and Slaff 1977) and a high in
success rate
locating three times ( n = 3). The proportion of egg hatch was
artificial oviposition sites ( Focks et al. 1979, 1983a,
calculated from the number of first instars emerging
1983b). However, earlier attempts to keep the vector
from these eggs. Old prey were replaced daily with fresh
population below the threshold level by
releasing ones.

Toxorhynchites were ineffective mainly due to

Numbers oflarvae pupating each day were counted
difficulties in mass-
rearing and releasing a sufficient and the pupae were kept separately in 500 ml beakers
number( Steffan 1975). This could be attributed to the
containing fresh tap water. Water in the trays was
poor
understanding of the factors affecting the sur-
replaced daily. Cannibalism and compulsive killing
vivorship and reproductive strategies of the predator.
took place mostly before pupation. The generally
On the Indian subcontinent, only Tx. splendens has
accepted theory to explain the compulsive killing
been reported to breed in coastal and forest areas
behavior in Toxorhynchites is protection ofthe relatively
Barraud 1934, Nagpal and Sharma 1987). Therefore, vulnerable pupae from cannibalism( Corbet and Griffiths
we carried out this study to assess the effect of food 1963). Therefore cannibalism is not considered the
availability on survival of immatures and adults and
major source of pupal mortality. Hence separating the
the reproductive schedule of Tx. splendens. The data pupae for adult emergence would not affect the life table
collected would help in mass-
producing this predator characteristics of the predator.
at the optimum food level for field releases and
Although there are many reports of cannibalism
should be an important background for evaluating among larvae of Toxorhynchites( Newkirk 1947, Trpis
future field predator- prey interactions during control 1973, Trimble and Carbet 1975, Furumizo and Rudnick
attempts.
1978, Focks and Boston 1979, Lounibos 1979, Steffan
et al. 1980, Steffan and Evenhuis 1981, Annis and
MATERIALS AND METHODS
Rusmiarto 1988), a recent study showed the relationship
between the rate of cannibalism among different instars
A colony of Tx. splendens was initiated from fourth
and prey density( Amalraj and Das 1992). In their study
instar larvae collected from tire dumping yards in 1977
it was found that first instar Tx. splendens consumed
and has been continuously maintained at the Vector larvae of its own species higher than the other instars in
Control Research Centre. Aedes aegypti mosquitoes
the absence of prey. However, when prey were provided,
were used as the prey. Cohorts of Tx. splendens were there was a significant fall in the rate of cannibalism.
reared from egg to adult by offering 0.35, 0. 85, 1. 4, 2. 8,
Because cannibalism is the function of prey density and
3. 4, 4. 95, 6. 8, 7. 15, 19. 8, 28. 6, and 39. 11 mg.( dry wt.)
the objective of the present study was to examine the life
early fourth instarAe. aegypti/ predator/ day. The amount characteristics of the predator in relation to food
of food consumed/ predator/ day was not estimated. To availability, predator larvae were not protected from
determine their dry weight, 200 early fourth instar Ae.
cannibalism by their density in the rearing containers.
aegypti were placed in an oven at 50° C for 24 h. The Dates on which adults of each sex emerged were noted.
138 Journal of Vector Ecology December, 1996

The time taken for 50% of the first instar larvae to RESULTS

become adults( E50) was calculated by fitting regressions

of the form Probit( P)= a+ b lnx, where P= proportion Larval Life Table Characteristics
on each day ( x) transformed to probits. The Mean proportions of eggs hatched for different
emerging
E50 values were then calculated by solving the equation cohorts ranged from 0. 81 to 0. 96. One way analysis of
for P= probit 50% ( Reisen and Mahmood, 1980). variance showed no significant difference in the egg
Females and males emerged from each set of hatch among cohorts of different batches( P> 0.05).

into 0. 3 The food regimens clearly had a significant effect

m3

experiments were released mosquito cages

and offered honey and glucose pads and oviposition on the survival and development of immatures.
trays with water. The honey pads were changed biweekly, Survivorship from first instar to adult was significantly
whereas the glucose pads were changed on alternate ( P< 0.001) less in cohorts that were reared under the
days. The oviposition containers were changed daily food regimens of< 6 mg( dry) prey/ predator/ day than
and the number of eggs counted. Each morning all dead those reared on larger quantities of food ( TABLE 1).
were recorded according to sex and then discarded.
adults
Median developmental time( E50) was influenced also
All experiments were conducted in controlled significantly( P< 0. 001) by the food regimens. When the
temperature( 25°- 27° C) and relative humidity( 60- 70). cohorts were offered> 7 mg ( dry) prey/ predator/ day,
Adultlife- table characteristicswerecalculatedfollowing the E50 was only 15. 83- 19. 8 days, whereas, predators
the methods of Reisen et al. ( 1979) and Reisen and reared under food regimes of< 3 mg( dry) prey/ predator/
Mahmood( 1980). day required as long as 39. 73- 64. 63 days for 50% adult
Analysis of variance( ANOVA) was performed on emergence( TABLE 1). There was no adult emergence
the life table parameters to determine significant when Tx. 0. 35 mg( dry) prey/
splendens was offered<

differences among food regimens. Comparison among predator/ day. Except at the lowest feeding rate, the
life table statistics was made by correlation analysis proportion of the offspring that were female did not
from mean values for each food regimen. The functional differ significantly ( P > 0.05) among food regimens
TABLE 1).
relationship between food regimens and the net (

reproductive rate per cohort ( R°) was described by

regression analysis following the method of Sokal and Adult Life Table Characteristics
Rohlf( 1981). The different food regimen treatments also had a

TABLE 1 Immature developmental attributes of Toxorhynchites splendens as functions of food regimens.

Immature developmental attributes of Tx. splendens#( S. E.)

Food regimens Survivorship Median emergence time( days)

mg. dry wt./ 1st instar adult) 1st instar adult) Sex ratio*
predator/ day) Male*** Female*** Male*** Female*** ( female/ total)

0. 35
0. 85 0.007( 0. 003) 0. 003( 0. 002) 64. 63 ( 8. 18) 56. 33( 12. 33) 0.22 ( 0. 11)
1. 40 0. 02 ( 0.01) 0.02 ( 0.00) 50. 64 ( 10. 36) 55. 00 ( 7. 33) 0. 56 ( 0. 12)
2. 80 0. 06 ( 0.05) 0. 05 ( 0.01) 39. 73 ( 2. 75) 50. 11 ( 4. 86) 0. 57 ( 0.07)
3. 40 0. 08 ( 0. 03) 0. 07 ( 0.003) 23. 69 ( 0. 12) 27. 48 ( 0. 64) 0. 55 ( 0.02)
4. 95 0.08 ( 0. 01) 0. 09 ( 0. 01) 25. 12 ( 1. 37) 29. 78 ( 0.51) 0. 55 ( 0.01)
6. 80 0. 19 ( 0. 02) 0. 16 ( 0. 01) 22. 36 ( 3. 09) 23. 48 ( 3. 81) 0. 52 ( 0.02)
7. 15 0. 10 ( 0. 01) 0. 09 ( 0.01) 18. 54 ( 1. 03) 19. 80 ( 0.76) 0. 50 ( 0.01)
19. 80 0. 27 ( 0. 01) 0. 20 ( 0.00) 17. 79 ( 0.61) 17. 95 ( 0. 63) 0. 44 ( 0.003)
28. 60 0.20 ( 0. 08) 0. 17 ( 0. 06) 16. 22 ( 0.06) 15. 83 ( 0.02) 0. 46 ( 0. 003)
39. 11 0.24 ( 0. 00) 0. 25 ( 0. 04) 18. 32 ( 1. 42) 18. 43 ( 1. 47) 0.55 ( 0. 03)

S. E)= Mean( Standard error); n= 3; attributes marked with asterisks denote significant difference among
the treatments using 1- way ANOVA. df= 10, 22. *= P 0. 01 < 0. 05; ***= P< 0.001.
December, 1996 Journal of Vector Ecology 139

significant ( P< 0. 001) effect on the adult life table Rowas obtained in the 39. 11 mg( dry) prey/ predator/day
Tx. TABLE 2). Among the
parameters of splendens (
food regimen. Ro was not significantly correlated with
eleven larval food regimens, the range of adult life female life expectancy at emergence( r= 0. 16, P> 0.05).
expectancy ( was 1. 93- 50. 23 days for males and
Age in days at mean cohort reproduction( To) was
el)
11. 64- 47. 84 days for females. Adult daily
survivorship significantly( P< 0. 001) lower( occurred earliest in life)
s) was lower among adults that emerged from larvae in females from cohorts fed< 2 mg( dry) prey/ predator/
food 2. 83 mg ( dry) prey/
reared under regimens of<
day food regimen. Conversely, To was high for the
predator/ day. females from > 2 mg ( dry) prey/ predator/day food
There was a linear relationship between the
regimens. To was not significantly correlated with Ro( r
food 0. 17, P > 0. 05).
amount of offered and the net reproductive rate =
The capacity for increase, re was
b= 12. 34, t= 17. 61, P< 0.001)( Fig. 1). Highest significantly greater at high food regimens. It should be
Rd(

TABLE 2. Adult life- table attributes of Toxorhynchites splendens as functions of food regimens.

Food regimens Adult- life- table characteristics of Tx. splendens#( S. E.)

mg. dry wt./

day) female)*** S( S( female)***
predator/
el ( male)*** e1 ( male)***
To***

0.35
0. 85 29. 73( 15. 08) 42. 07( 21. 04) 0. 57 ( 0. 003) 0.001 ( - ) 16. 84 ( 8. 53)
1. 40 1. 93 ( 0.96) 11. 64 ( 5. 83) 0. 00 ( 0.00) 0. 66 ( 0. 33) 00. 00 ( 0.00)
2. 80 35. 59 ( 5. 85) 47. 84( 12. 11) 0. 97 ( 0.01) 0.97 ( 0. 02) 17. 92 ( 2. 75)
3. 40 27. 29 ( 7. 41) 28. 06 ( 2. 63) 0. 97 ( 0. 003) 0. 97 ( 0. 003) 18. 69 ( 1. 85)
4.95 50. 23 ( 1. 56) 32. 98 ( 0. 52) 0. 98 ( 0.00) 0.95 ( 0. 01) 14. 32 ( 0.77)
6. 80 36. 01 ( 6. 79) 27. 13 ( 3. 18) 0. 97 ( 0.003) 0.95 ( 0. 006) 14. 87 ( 1. 59)
7. 15 48. 48 ( 2. 41) 36. 57 ( 2. 09) 0. 98 ( 0.006) 0.98 ( 0. 01) 14. 96 ( 0. 38)
19. 80 28. 02 ( 0. 15) 34. 68 ( 3. 87) 0. 96 ( 0.003) 0.95 ( 0. 01) 18. 78 ( 2. 99)
28. 60 45. 49 ( 5. 57) 36.01 ( 1. 36) 0. 97 ( 0.006) 0.96 ( 0. 003) 14. 87 ( 0. 55)
39. 11 24. 75 ( 0. 83) 32. 53 ( 2. 1) 0. 94 ( 0.005) 0. 94 ( 0.00) 13. 86 ( 0. 13)

r *** r *** G*** b*** d***

m c

0. 35
0. 85 0. 008( 0. 004) 0.03 ( 0. 02) 64. 70( 33. 64) 0. 68 ( 0. 34) 0. 67 ( 0. 34)
1. 40 0. 00 ( 0.00) 0.00 ( 0. 00) 00. 00 (-) 0. 00 (- ) 0.00 (- )
2. 80 0. 03 ( 0. 001) 0. 16 ( 0. 04) 78. 15( 10. 34) 1. 05 ( 0.01) 1. 02 ( 0. 01)
3. 40 0. 03 ( 0. 01) 0. 14 ( 0. 04) 81. 99( 14. 53) 1. 07 ( 0.006) 1. 04 ( 0. 006)
4. 95 0. 07 ( 0.004) 0.29 ( 0. 02) 61. 78 ( 2. 54) 1. 09 ( 0.006) 1. 02 ( 0. 003)
6. 80 0. 08 ( 0.01) 0.32 ( 0. 02) 58. 83 ( 4. 23) 1. 13 ( 0.006) 1. 04 ( 0. 006)
7. 15 0. 10 ( 0.003) 0. 30 ( 0. 02) 44. 25 ( 1. 42) 1. 12 ( 0.01) 1. 02 ( 0.01)
19. 80 0. 10 ( 0.01) 0. 30 ( 0. 06) 54. 24 ( 3. 31) 1. 13 ( 0.02) 1. 03 ( 0.003)
28. 60 0. 12 ( 0.01) 0. 34 ( 0.06) 40. 18 ( 4. 56) 1. 16 ( 0. 02) 1. 04 ( 0.01)
39. 11 0. 14 ( 0.001 0.44 ( 0. 02) 45. 39 ( 2. 21) 1. 18 ( 0. 006) 1. 04 ( 0. 006)

mean life expectancy from emergence in days; s= constant daily survivorship assuming a type II
el=
survivorship curve; To= age in days at mean cohort reproduction; rm= instantaneous rate of increase in living female/
female; capacity for increase; G= mean generation time in days; b= instantaneous birth and d= death rate,
rc =
assuming a stable age distribution.

S. E.)= Mean( Standard error); n=

3; attributes marked with asterisks denote significant difference among the
treatments using ANOVA. *** = P< 0.001.
140 Journal of Vector Ecology December, 1996

600 -

t= 17. 61; P< 0. 001

Observed
450 - — Regression line

0a
ri 300 -

b0
a

150 -

0
0 10 20 30 40

Amount of Food Offered/ Predator/Day

mg. dry weight)

Figure 1.
Functional relationship between net reproductive rate in living female progeny/ female/ generation( R0)
and food regimens.

pointed out that

rc utilized
To in its calculation, and thus either Type II or III curve( Fig. 2).
was based only on the adult age. The number of eggs laid was high in the second
The instantaneous rate of increase( found to
rm) was
week of adult life and egg laying activity was observed
increase significantly ( P< 0. 001) with increases in the for six to seven weeks from adult emergence. Females
amount of food. was
significantly correlated with r reared from higher food regimens laid more eggs than
rm
r= 0.94, P< 0. 001). R( 0. 76, P< 0. 001). The
and r=
the female reared from low food regimens( Fig. 3).
mean generation time ( G) of Tx. splendens was
significantly( P< 0. 001) lower in> 2. 83 mg( dry) prey/ DISCUSSION

predator/day. G was significantly correlated with To( r

0.41, P< 0.05). As in the
rm/ b increased
seen r , ratio
The present study made it easy to compute the per
significantly( P< 0. 001) with increase in amount of food capita rate of increase of Tx. splendens population under
offered. The instantaneous birth( b) and death( d) rates different food regimens so as to produce the best possible
and the b/ d ratio were
significantly( P< 0. 001) higher in quantification of population performance. High egg
2 mg ( dry) prey/ predator/ day. The birth rate( b) and hatching rate( range 81 to 96%) reported in the present
the death rate ( d) estimated from the stable age
study suggests an effective fertilization and is in
distribution werefound to be closely correlated with agreement with Furumizo and Rudnick ( 1978) who
rm
r= 0. 74, 0. 68 respectively, P< 0. 001). Lower birth and reported
hatching rate of 77 to 91% in Tx. splendens.
egg
death rates accompanied
by a lower
rm would suggest a
However, egg viability was reported to range from 25 to
trend toward greater population
stability ( Reisen et al. 100% in other species of the genus Toxorhynchites
1979).
Trimble 1979, Steffan et al. 1980). Greater immature
Age specific survivorship curves for males and survival was obtained when the predator was reared
females and
fecundity curves for females have been under food regimens of>
19. 8 mg( dry) prey/ predator/
depicted graphically in Figures 2 and 3. In general,
day. This clearly shows that both food availability and
female survivorship patterns approximated Slobodkins'
cannibalism are interrelated and are the contributing
1962) Type II curve with little increase in mortality
factors for the survival of the immatures( Amalraj and
during early age intervals, whereas male curves exhibited Das, 1992). The median developmental time(
E50) from
December, 1996 Journal of Vector Ecology 141

w1 w1 of x
kr)
t wl wT 0f
1
N 4 N
0:a

N N

1.)

q:, ;)

M F M

i
00 D
s• ' I' N O 00 ,. 0 ' 1' N O
O O O O O O O O

alvIAI) XI alnuiad) XI

fr<
1 iPPiUf to
Trf = #
U1
in

Q E

N N

I- I

Airy

7,04-.•

1,
0•P'

M cn

O O

00 vO t N O 00 Z) N O
O O O O O O O O
alcIAD XI alnuiad) XI

Figure 2.
Age- specific survivorship in individuals/ individual/ day( 1X) plotted as a function of age in weeks under
different food regimens. Each point represents the mean of three replicates.( A 1) 0. 85,( A2) 2. 80,( B) 3. 40,
C) 4. 95,( D) 6. 80,( E) 7. 15,( F) 19. 80, ( G) 28. 60,( H) 39. 60 food/ predator/ day.
mg. ( dry wt.)
142 Journal of Vector Ecology December, 1996

1200
a—

B
900
AE-

m-

e" 600 - D

kanui
300 -

0
0 3 6 9 12 15
Week

1200

F
900

N--

03--

E" 600
H

300

0 3 6 9 12 15
Week

Figure 3.
The number of female offspring produced/ living female/ week( mx), plotted as a function of age in weeks
under different food regimens. Each point represents the mean of three replicates.( A) 2. 80,( B) 3. 40,( C)
4. 95,( D) 6. 80,( E) 7. 15,( F) 19. 80,( G) 28. 60, ( H) 39. 60 mg. ( dry wt.) food/ predator/ day.
December, 1996 Journal of Vector Ecology 143

first instar to adult ranged from 15. 83 to 64. 63 when the

theoretical colonizing ability ( MacArthur and Wilson
highest and lowest food regimens were provided. Paine 1967), were relatively low. Thus, this species would be
1934) similarly reported a mean development time of
considered" r" strategist, but a relatively poor colonizer.
23 days for well fed larvae and 110 days for starved Another adaptive character found in this species is
larvae. Chan ( 1968) reported an immature duration of
elongation of G when food is scarce. High R and rm
39 days in Tx. splendens. Furumizo and Rudnick( 1978) values at high food level as well as longer adult lifetime
reared Tx. high prey density and observed
splendens at
than those of the prey species give hope for exploitation
the immature developmental duration to be 21. 5 days.
of the species against container breeding mosquitoes
By delaying pupation when food is scarce, Tx. splendens such as Ae. aegypti.
maintains its food ingested above the basal metabolism
rate to accrue sufficient food reserves in the form of Acknowledgments
lipids and glycogen to initiate pupation. Further the total
energy stored in adults provides a measure of the energy We are grateful to Dr. Vijai Dhanda, Director,
reserves built up during the larval The
stage. massive
Vector Control Research Centre, Pondicherry, for his
larval abdominal muscles, which are histolysed during critical comments on the manuscript that facilitated
the first days of adult life must be a valuable source of
great improvement in the quality of the manuscript. The
energy for young adults( Briegel 1990a, 1990b). technical assistance of Messrs K. Balarajan, G.
Males survived slightly longer than did females.
Meganathan, and G. Sathianathan is gratefully
This is presumed to be a behavioral requirement for acknowledged.

minimal swarm size. Focks et al. ( 1977) reported that

females of Tx. rutilus rutilus survived an average of 49 REFERENCES CITED
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Rudnick ( 1978) estimated an adult life span of 28 to 35 Amalraj, D. D. and P. K. Das. 1992. Cannibalism and
days for Tx. splendens. However, a maximum survival
Carnivory in Toxorhynchites splendens ( Diptera:
of 120 days by Steffan 1980). In the Culicidae).
was reported et al.(
Southeast Asian J. Trop. Med. Pub.
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survivorship was 0. 98. The laboratory estimates of
Amalraj, D. D. and P. K. Das. 1994. Time to death from
survivorship and longevity were presumed to depict the
starvation and compulsive killing by the larvae of
genetic potentiality of this species. The contribution of Toxorhynchites splendens ( Diptera: Culicidae).
individual males and females to the population increase Acta Trop. 58: 151- 158.
would be directly related to life expectancy at emergence Annis, B. and S. Rusmiarto. 1988. Trial of a quantified
and the number of fertile eggs produced. The highest R. mass rearing method for Toxorhynchites splendens.
value of 518. 33 was obtained by offering Tx. splendens J. Am. Mosq. Cont. Assoc. 4: 368- 369.
39. 11 mg ( dry) prey/ day and was found to be
many Barraud, P. J. 1934. The fauna of British India, including
times higher than 16. 3 reported by Chowanadisai et al.
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The presentstudy indicated that the oviposition period Briegel, H. 1990a. Metabolic relationship between
was extended
up to 50 days for females reared on high female body size, reserves and fecundity of Aedes
food regimens. Steffan et al. ( 1980) reported an aegypti. J. Insect Physiol. 36: 165- 172.
85 to 95 days in Tx.
oviposition period of amboinensis.
Briegel, H. 1990b. Fecundity, metabolism and body
This is much longer than the 27 days reported for Tx. size in Anophelines ( Diptera: Culicidae), vectors
splendens ( Furumizo and Rudnick 1978). A of malaria. J. Med. Entomol. 27: 839- 850.
comparatively high rm of 0. 14 was estimated when the Chan, K. L. 1968. Observation on Toxorhynchites
predator was reared under the food regimen of 39. 11 mg splendens ( Wiedemann) ( Diptera: Culicidae) in
dry) prey/ predator/ day. Higher values of generally Singapore. Mosq. News. 28: 91- 95.
rm
to be for Chowanadisai, L., N.
were considered an
evolutionary adaptation
Benjaphong, and B.
existing in or colonizing variable environments( Hairston Phanthumachinda. 1984. Laboratory observations
et al. 1970, Pianka 1972). Hence, with
relatively high on Toxorhynchites splendens Wiedemann in
potential value of Tx. be to
rm, splendens would able
Thailand. Southeast Asian J. Trop. Med. Pub. Hlth.
exploit favorable conditions such as the availability of 15: 337- 341.
prey in large numbers and rapidly increase its population Corbet, P. S. 1963. Observations on Toxorhynchites
size. However, the b and b/ d ratios, an indication of brevipalpis Conradti Grunb in Uganda. Bull.
rm/
144 Journal of Vector Ecology December, 1996

Entomol. Res. 54: 9- 17. Hu, S. M. K. 1955. Progress report on biological control
Corbet, P. S. and A. Griffiths. 1963. Observation on the of Aedes albopictus skuse in Hawaii. Proc. Calif.
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Journal of Vector Ecology 21( 2): 146- 149

Laboratory and Field Trials of Fenthion and

Cyfluthrin Against Mansonia uniformis Larvae

Han- Heng Yap, Annie E. S. Foo, Chow- Yang Lee, Ngo-Long Chong,
Abdul Hamid Awang, Rohaizat Baba, and Abdul Malik Yahaya

Vector Control Research Unit, School of Biological Sciences,

Universiti Sains Malaysia, 11800 Penang, Malaysia.

Received 19 November 1995; Accepted 12 January 1996

ABSTRACT: A laboratory bioassay of insecticides against laboratory- reared late third/early fourth- instar
larvae of Mansonia uniformis indicated that the synthetic pyrethroids tested( cyfluthrin and permethrin)
were more active than the organophosphate compounds tested( chlorpyrifos, fenthion, and temephos). The
LC50 values ranged from 0. 09( cyfluthrin) to 7. 68 pg/ I( temephos). Field trials of fenthion( Baytex GR2)

and cyfluthrin ( Baythroid H10WP) against laboratory- cultured late third/early fourth- instar larvae of
Mansonia uniformis in floating screened cages were conducted in small plots on Penang Island, Malaysia.
At respective doses of 0. 50 and 0.30 g/ m2( 5. 0 and 3. 0 kg/ ha), both fenthion and cyfluthrin caused more
than 90% mortality of the Mansonia larvae exposed at 0 hour post- treatment. The use of these larvicides

for the control of Mansonia larvae in open field environments is discussed.

Keyword Index: Mansonia uniformis, larvicide, fenthion, cyfluthrin.

INTRODUCTION laboratory( Yap et al. 19956).

We report here the efficacy of two synthetic
Mansonia uniformis is one of the major vectors of insecticides, namely fenthion and cyfluthrin, as tested in
Brugian filariasis in Southeast Asia ( Mak 1981, Yap the laboratory ( compared with chlorpyrifos, temephos
1985). Approximately one billion people lived in and permethrin) and field against Ma. uniformis larvae.
endemic areas with approximately 5. 8 million cases of
infection in this region ( World Health Organization MATERIALS AND METHODS
1994). Currently, Brugian filariasis is controlled through
mass drug treatment using diethylcarbamazine citrate,
The Ma. uniformis larvae used in both laboratory
and new drugs such as ivermectin are still under and field assessments were from well- established
experimentation( Mak 1981, 1990, Ramachandran 1993, laboratory colonies at the Vector Control Research
World Health Organization 1994). Unit, Universiti Sains Malaysia. The colony was initiated
In comparison with other mosquito genera, there
from collections in Permatang Damar Laut, Penang
have been few larvicidal tests against Mansonia ( Yap Island in 1990. The mosquitoes used in this study were
1985, Yap et al. 1995a). Chapman ( 1955) conducted late third and early fourth- instar larvae.
laboratory and field tests with 15 insecticides against Technical grade fenthion ( o, o- dimethyl- o-[ 4-
Mansonia dubitans and concluded that parathion and ( methylthio)- m- tolyl] phosphorothioate, temephos( o, o-
EPN were most effective. Further laboratory studies on ( thiodi- 4- 1- phenyl ene) o, o, o, o- tetramethyl phosphor-
Mansoniaperturbans( Yap et al. 1968) and Ma. uniformis othioate), chlorpyrifos( o, o- diethyl o- 3, 5, 6- trichloro- 2-
Yap and Sulaiman 1976) indicated good efficacy of pyridyl phosphorothioate), cyfluthrin [ alpha- cyano( 4-
temephos and chlorpyrifos. Field studies further fluoro- 3- phenoxyphenyl) methyl 3-( 2, 2- dichloro-
confirmed the efficacy of temephos against Mansonia ethenyl)- 2, 2- dimethyl- cyclopropanecarboxylate] and
larvae( Gass 1985). More recently, etofenprox, a
et al. permethrin[ 3- phenoxybenzyl- 3- 2, 2- dichlorovinyl- 2, 2-
relatively new insecticide with low mammalian
toxicity, dimethylcyclopropane carboxylate] were used in the
was also found to provide good larvicidal
activity in the laboratory bioassay. The organophosphates chosen for
December, 1996 Journal of Vector Ecology 147

thisstudy are currently being used as mosquito larvicides

small plot trials followed essentially those of Yap et al.
in Malaysia. Temephos is used for clear water breeders, ( 1991). Efficacy and residual effects of the insecticides
such as the Aedes species, whereas fenthion and
againstMansonia larvae were determined by introducing
chlorpyrifos are used for the polluted water breeding batches of larvae into additional cages at intervals of 0,
Culex species. The choice of the two residual pyrethroids
24, 48, 72, and 168 hours post- treatment. The mortality
permethrin and cyfluthrin) takes into consideration the readings were conducted 24 hours after the introduction
greater use of pyrethroids for household and public
of each batch of larvae at the above designated intervals.
health insect control in recent based their
years on
For both formulations tested, application rates
selective toxicity. ranged from 0.01 to 2. 00 g/ m2. The fenthion granule
The bioassay method was essentially that established formulation( Baytex GR2) was mixed thoroughly with
by the World Health Organization for larvicidal fine sand and dispersed by hand. The cyfluthrin wettable
susceptibility ( World Health Organization 1981) with powder formulation ( Baythroid H10WP) was mixed
the following modifications:( 1) technical grades of test
with seasoned tap water( local tap water kept for more
insecticides were diluted to test concentrations using than 72 hours) and applied using a Geizhal ES 10
analytical grade acetone as solvent;( 2) twenty late third/ pressurized knapsack sprayer( Dr Stahl and Sohm Gmh
early fourth- instar larvae in three replicates were used
and Co, Uberlinger, Germany).
for each concentration of insecticides per The
test. Rainfall, temperature, pH, dissolved oxygen, and
experiment was then repeated three times using different water conductivity at the field sites were recorded daily.
batches of larvae and insecticide preparations;( 3) total
Measurements were made using portable meters
volume of test solution was 100 ml instead 250 ml
of per
including a membrane pH meter( Hanna HI 8314, Italy),
beaker and;( 4) a small piece of styrofoam was placed in
dissolved oxygen meter( Yellow Spring Instrument, YS
each beaker for attachment of Mansonia larvae after the
IM67, USA) and conductivity meter( WIW LF 91 with
addition of insecticide. probe KLEI/ T, USA). Data were subjected to probit
For field efficacy tests, only fenthion granule
analysis ( Finney 1971) using a computer program by
Baytex GR2) and cyfluthrin wettable powder( Baythroid Daum ( 1970).
H10WP) formulations were used. The field trials were
conducted by exposing mosquito larvae to insecticide RESULTS AND DISCUSSION
applications in floating screened cages in small plots( 15
18 Test had
m2).

plots a vegetative cover of water

Based on thelaboratory bioassays, the Ma. uniformis
hyacinth ( Eichhornia crassipes Solm), which is the larvae appeared to be more susceptible to the two
natural host plant of Mansonia. Six treated and three pyrethroids ( cyfluthrin and permethrin) than to the
untreated control plots were used. The nine plots totaled organophosphates ( chlorpyrifos, temephos, and
145 The field
m2.

plots were located in

swampy ditch a fenthion) ( TABLE 1). The LC„ values for these
abandoned coconut plantation in
of an
Permatang Damar insecticides ranged from 0. 09 to 7. 68 tg/ 1. Cyfluthrin
Laut on the southern coastal alluvial plain of
Penang seemed to be the most effective of all the insecticides
Island, Malaysia. The detailed protocols for tests in tested. Among the organophosphates, chlorpyrifos and

TABLE 1.
Activity of five insecticides against late third/ early fourth- instar Mansonia
uniformis larvae in the laboratory.'

LC( 95% fiducial limit) [in [Link]/ 1J

Insecticide 50 90 Slope± SE

fenthion 4. 96( 4. 81- 5. 10) 7. 54( 7. 21- 7. 95) 7. 04± 0.25

temephos 7. 68 ( 7. 32- 8. 05) 18. 54( 17. 17- 20.22) 3. 35± 0.09
chlorpyrifos 3. 85 ( 3. 36- 4. 27) 6. 70( 5. 87- 8. 31) 5. 33± 0. 59
cyfluthrin 0. 09( 0. 08- 0. 10) 0. 31 ( 0. 26- 0. 37) 2. 34± 0. 10
permethrin 1. 74( 1. 63- 1. 84) 3. 93 ( 3. 61- 4. 36) 3. 61 ± 0. 15

Based on pooled data of three experiments.

148 Journal of Vector Ecology December, 1996

fenthion were more effective than temephos. Dose- same environmental conditions.

response values of some of the insecticides tested here The meteorological and water quality conditions
agree with those from earlier publications (
Yap et al.
for the field site in Permatang Damar Laut, Penang
1968, Yap and Sulaiman 1976). Some minor Island were similar to those recorded in the same plots
discrepancies occured due to species differences( Yap in earlier publication( Yapetal. 1991). The mean values
et al. 1968) and the use of laboratory- cultured versus (± S. E.M) for temperature, pH, dissolved oxygen, and
field collected mosquito larvae ( Yap and Sulaiman conductivity of the field water were 27. 4± 0. 3° C, 6. 7±
1976). 0. 1, 1. 8± 0. 1 mg/ l, and 10. 1± 2. 1 mmho/ cm, respectively.
Both fenthion and cyfluthrin proved to be effective Field trials indicated that at respective application
in the simulated field control of Ma. uniformis. The rates of0. 50 and 0. 30 g/ m2( equivalent to 5. 0 and 3. 0 kg/
larvicidal efficacy of fenthion and cyfluthrin against ha), fenthion and cyfluthrin caused more than 90%
sentinel Ma. uniformis in small plots in swampy ditches mortality of the Mansonia larvae at 0 hour post- treatment
showed that the latter had a higher efficacy than fenthion in natural larval habitats with high organic and ion
at the initial introduction of Mansonia larvae contents. At subsequent intervals( 24, 48, 72, and 168
immediately after insecticidal application) ( TABLE hours post- treatment), there was a decrease in efficacy
2). When new Mansonia larvae were introduced at 24 of insecticides against the introduced larvae ( TABLE
and 48 hours post- treatment, some significantmortalities 2). Thus, both fenthion and cyfluthrin showed only
of larvae occured at the higher dosages used. For the slight residual effects against Mansonia larvae in the
cyfluthrin formulation, introduction of new batches of natural polluted habitats except at considerably higher
larvae at 24, 48, 72, and 168 hours post- treatment dosages. However, such lack of persistence at lower
indicated significant residual effects of up to seven days dosages should still provide reasonable effective control
highest dosages 1. 00 2. 00 g/ if the application routine was conducted monthly. This
m2,

at the two used ( and

TABLE 2). The effective dosages for both insecticides is because Mansonia mosquitoes in general need a much
for Mansonia control in such habitats appeared to be longer time ( 20- 25 days) to complete their larval
lower than those of microbial insecticidessuchasBacillus development ( Wharton 1962) as compared with other
thuringiensis H- 14 ( Foo and Yap 1983) and Bacillus mosquito species.

sphaericus(
Yap 1990, Yap et al. 1991) tested under the Results from the present field studies indicated that

TABLE 2. Small- plot field trials on fenthion and cyfluthrin against Mansonia uniformis larvae placed in floating
cages.

Dosage No. live larvae over 50 introduced at following intervals after 24 hours exposure'
Insecticide ( g/
m2)

0 hr 24 hr 48 hr 72 hr 168 hr

fenthion control 44. 0± 2. 6 44. 3± 2. 1 41. 6± 1. 5

0. 01 43. 6± 1. 2 41. 0± 2. 0 38. 3± 7. 0
0. 10 26. 0± 10. 5 43. 7± 4. 6 37. 3± 1. 5
0. 30 16. 3± 18. 0 28. 3± 16. 1 42. 0± 2. 6
0. 50 2. 3± 3. 2 18. 7± 12. 7 42. 3± 6. 4
1. 00 0. 7± 0. 6 7. 3± 0. 6 7. 3± 3. 8
2. 00 0 5. 0± 4. 6 30. 6± 16. 3

cyfluthrin control 46. 0± 1. 0 41. 3± 1. 2 43. 7± 0.6 44. 7± 1. 5 46.3± 2. 5

0.01 37. 3± 1. 2 38. 0± 1. 7 37. 7± 0. 6 42. 7± 4. 0 41. 7± 2. 9
0. 10 8. 3± 4. 6 32. 6± 2. 1 35. 7± 2. 9 42. 0± 1. 0 45. 3± 1. 5
0. 30 3. 3± 4. 0 19. 3± 0. 6 25. 7± 6.0 36. 0± 5. 0 42. 3± 3. 1
0. 50 1. 3± 1. 2 7. 0± 5. 6 18. 7± 8. 1 31. 7± 4. 2 40.0± 2. 0
1. 00 1. 0± 1. 7 6. 7± 3. 1 13. 0± 6. 9 26. 3± 1. 2 31. 0± 3. 5
2. 00 0. 3± 0. 6 0 0 2. 0± 1. 7 6. 7± 3. 1

All values are mean± S. E of three experiments of live larvae recovered after 24 hours exposure.
 December, 1996 Journal of Vector Ecology 149

both fenthion and cyfluthrin have the potential to be Ramachandran, C. P. 1993. Control of lymphatic
effective larvicides for Mansonia control in natural
filariasis and onchocerciasis, World Health
breeding habitats. However, the choice of control
any Organization mimeograph document WHO/ CTD/
agents, whether chemical or biological, should take into
MIP/ WP.93. 5, 17 pp.
consideration the environmental impacts of such agents Wharton, R. H. 1962.
under operational usage.
The biology of Mansonia
mosquitoes in relations to the transmission of
filariasis in Malaya. Bull. Inst. Med. Res. 11: 1- 113.
Acknowledgments World Health Organization. 1981. Instruction for

The authors wish to thank the IRPA R& D

determining the susceptibility or resistance of
program, larvae
mosquito to insecticides. WHO/ VBC/
Ministry of Science, Technology and Environment,
81. 807, 6 pp.
Malaysia and UNDP/ World Bank/ WHO Special
World Health Organization. 1994. Lymphatic filariasis
Program for Research and
Training in Tropical Diseases, infection and disease: control strategies. Report of
Geneva( Grant No. 800040) for their research support;
a consultative meeting held at the Universiti Sains
Bayer AG, Monheim, Germany for the supply of Malaysia, Penang, Malaysia. World Health
formulated insecticides; Professor M. S. Mulla
Organization: Division of Control of Tropical
University of California, Riverside) for comments and

suggestions on draft manuscript; the Dean, School

Disease. TDR/CTD/FIL/PENANG/94. 1, 30 pp.
of
Yap, H. H. 1985. Review on control of Brugian
Biological Sciences Coordinator, Vector Control
and

Research Unit( VCRU), Universiti Sains Malaysia for filariasis vectors, especially Mansonia species. Pp.
131- 135 in Proceedings of the World Health
the use of
laboratory facilities; H. T. Tan, H. Ali Zaini
Organization Regional Seminar on Control of
and other staff of VCRU for technical assistance; and C.
Brugian Filariasis, Kuala Lumpur, Malaysia. 1- 5
L. Tang for clerical assistance.
July. World Health Organization, Geneva, 174 pp.
Yap, H. H. 1990. Field trials of Bacillus sphaericus for
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use of Bacillus thuringiensis H- 14
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Laboratory larvicidal efficacy of etofenprox
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Journal of Vector Ecology 21( 2): 150- 155

Natural Variation in Blood- Feeding

Kinetics of Four Mosquito Vectors

Dave D. Chadee' and John C. Beier2

Insect Vector Control Division, 3 Queen Street, St. Joseph, Trinidad, West Indies

2Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University
Medical Center, 1501 Canal Street, New Orleans, LA 70112- 2824, USA.

Received 21 November 1995; Accepted 15 March 1996

ABSTRACT. The blood- engorgement kinetics of Anopheles aquasalis, Aedes aegypti, Haemagogus
janthinomys, and Culex quinquefasciatus were determined under laboratory conditions using females
collected from three field sites in Trinidad. Most An. aquasalis, Ae. aegypti, and Hg. janthinomys
completed probing within 70 s but Cx. quinquefasciatus averaged> 180 s. Anopheles aquasalis ( 67. 0 s)

had the shortest gut filling time while Cx. quinquefasciatus had the longest gut filling time( 222. 8 s). Aedes
aegypti and Hg. janthinomys had similar gut filling durations, 87. 7 s and 90.4 s, respectively. More than
70% of An. aquasalis, Ae. aegypti, Cx. quinquefasciatus, and Hg. janthinomys showed prediuresis.
Anopheles aquasalis exhibited the shortest mean duration of prediuresis( 77. 3 s) and Cx. quinquefasciatus
the longest ( 526. 9 s). Most individual mosquitoes spent less time filling their guts than in prediuresis.
Overall, An. aquasalis exhibited the shortest total feeding time ( 135. 7 s) and Cx. quinquefasciatus the
longest( 661. 9 s). Individual An. aquasalis mosquitoes showed a high degree of variability in their ability
to agglutinate human erythrocytes immediately upon blood engorgement, with individuals agglutinating
25% of the ingested erythrocytes and others>_90%. Among Ae. aegypti and Cx. quinquefasciatus, the
proportion showing agglutination varied from 0%( 67% and 69% respectively) to< 50%. The parity of the
field collected mosquito vectors had no significant effect on probing, duration of blood- feeding, duration
of prediuresis, or erythrocyte agglutination. The inter- and intra- specific variation in blood- feeding and
erythrocyte processing observed for the four vector species collected from the field in Trinidad are
described in terms of vector competence and evolution of these behaviors.

Keyword Index: Mosquito, blood- feeding, excretion, prediuresis.

INTRODUCTION time, gut- filling time, and duration of prediuresis. Indeed,

information on this aspect of the biology of each species
In the Americas, four significant vector mosquitoes
is long overdue and vital to the understanding of the
include Anopheles aquasalis Curry, the coastal vector transmission dynamics ofyellow-fever, dengue, malaria,
of malaria from Venezuela to Brazil and including and filariasis.
Trinidad and Tobago( Faran 1980); Aedes aegypti( L.), Although Trinidad and Tobago are currently free
the cosmopolitan vector of dengue and urban yellow of malaria, yellow- fever, and- bancroftian filariasis,
fever ( Christophers 1960); Haemagogus janthinomys the presence of large numbers of potential vectors
Dyar, the sylvan vector of yellow fever in the Latin means that the risk of disease outbreaks remains high.
American and Caribbean region( Arnell 1973, Chadee Recently, Chadee and Beier ( 1995) developed
et al. 1992); and Culex quinquefasciatus Say, the vector methodology for evaluating the blood- feeding beha-
of bancroftian filariasis worldwide ( Nathan 1981, vior of four anopheline mosquitoes from Trinidad and
Macdonald 1991). Tobago. Here we present the results of the duration
Much information is available on the host- seeking and kinetics of blood- feeding of field-collected, An.
activity of these four vectors but little is known about the aquasalis, Ae. aegypti, Hg. janthinomys, and Cx.
duration of blood engorgement which includes probing quinquefasciatus.
December, 1996 Journal of Vector Ecology 151

MATERIALS AND METHODS

of probes, duration of probing, duration of the gut-
filling phase, duration of prediuresis, proportion of
Field collections of Ae. aegypti and Cx. quinque-
females exhibiting prediuresis, and total feeding time
fasciatus were made at St. Joseph( 10° 38' N; 61° 25' W),
could be calculated( Vaughan et al. 1991, Chadee and
a small town, 16 km east of Port of Spain, Trinidad. The
Beier 1995). After blood- feeding, the mosquitoes were
study area,meteorology, topography, vegetation, and immediately killed and dissected. Both ovaries of each
two mosquito populations were described
by Chadee female mosquito were examined and scored as
and Corbet ( 1987) and Chadee ( 1994). Anopheles
nulliparous and parous using Detinova' s method,
aquasalis collections were carried out in the rice- fields according to the presence of tracheolar skeins( Detinova
of Frederick Settlement( 10° 36' N; 61° 25' W), a village 1962).
5 km west of Piarco International Airport. The anopheline
populations, meteorology, topography, and vegetation Patterns of Prediuresis
of this site previously described by Chadee( 1992)
were
To determine whether these mosquitoes excreted
and Chadee and Beier( 1995). Field collections of Hg.
tubular fluid ( prediuresis) during feeding, 20 to 60
janthinomys were conducted at the Pt. Gourde Forest mosquitoes were placed into an ice-cream cylinder cage
10° 40' N; 61° 40' W), a site
approximately 16 km west
containing filter paper on which the insects could
of Port of Spain, the capital of Trinidad. The mosquito prediurese and were offered a blood meal on a human
populations, meteorology, vegetation, and topography forearm. Once the mosquitoes had fed to repletion, the
of this area were described by Chadee and Tikasingh filter paper was removed from the cage, examined, and
1989).
the pattern of prediuresis( as indicated by spots of blood
Host- seeking mosquitoes were captured excreted) recorded( Vaughan et al. 1991).
using
human bait and standard procedures ( Haddow 1954)
during the hours of peak landing for each species; 16: 00- Haemagglutination of Ingested Blood
18: 00 hours forAe. aegypti( Chadee 1988), 22: 00- 02: 00
The degree of erythrocyte aggregation was
hrs. for Cx. Nathan 1981), 18: 30-
quinquefasciatus ( determined at 250 or 400 x in wet mount preparations of
19: 00 hrs. for An. aquasalis( Senior- White 1953), and
blood meals dissected from freshly fed mosquitoes into
10: 00- 14: 00 hrs. for Hg. janthinomys ( Chadee et al. physiological saline. This was scored 0, 1, 2, 3, 4, or 5,
1992). Mosquito collectors were stationed at ground for 0, 1%- 25%, 26%- 50%, 51%- 70%, 71%- 90%, and
level with flashlights, collecting nets, and aspirator. The > 90% of red cells agglutinated, respectively.
mosquito collection methodology, handling, storage,
and transportation from the field to the
laboratory were Statistical analysis
described by Chadee and Beier ( 1995). At the Insect Data were subjected to a Duncan multiple range
Vector Control Division laboratory, the mosquitoes
test( P= 0. 05) to compare means within each category,
were anaesthetized
lightly with chloroform, examined that is, within probing times, gut filling times, prediuresis
under a microscope at 40 x magnification, identified, times, and total blood- feeding times ( Sokal and Rohlf
and counted. Mosquitoes with
any trace of blood were 1980). In addition, the data were transformed into
rejected. Mosquitoes were segregated by species and
contingency tables and subjected to a G- test to determine
placed into 450 ml, net- screened, cylindrical ice- cream the inter and intra specific differences in erythrocytic
containers ( 18 cm in height x 16. 5 cm in diameter).
agglutination levels and differences in patterns among
Mosquitoes held for 24 hours
were and tested during nulliparous and parous mosquitoes ( Sokal and Rohlf
their peak biting times. 1980).

Duration of Engorgement RESULTS

To study blood- feeding, each mosquito was placed
in a 5 ml glass tube covered with mosquito
netting and TABLE 1 summarizes the probing behavior of the
allowed to engorge on a human forearm. Mosquitoes
field- collected An. aquasalis, Ae. aegypti, Hg.
were allowed to probe into the skin ( number of times
janthinomys, and Cx. quinquefasciatus under laboratory
recorded) and the duration of probing was timed with a conditions. No significant ( P= 0. 09) differences in the
stopwatch. Another stopwatch was activated at the first
mean number of probes was found among the four
signs of engorgement( red color and abdominal
swelling). mosquito species belonging to four genera. The duration
A third stopwatch was activated at the first sign of
of probing varied significantly ( P< 0.0003) between
prediuresis( anal excretion). This process was observed species ( TABLE 1),
with An. aquasalis( 19. 6 s) being
under a
dissecting microscope( 60 x) so that the number much faster than Cx. quinquefasciatus( 148. 3 s).
152 Journal of Vector Ecology December, 1996

TABLE 1. Probing, gut filling, prediuresis, and total feeding times of four field- collected mosquito vector
species.*

No. of Time( s)

Species probes Probing Filling gut Prediuresis Feeding

An. aquasalis 1. 6± 0. 9 19. 6± 17. 0 67. 0± 39. 0 77. 3± 33. 6 135. 7± 48. 8
Ae. aegypti 1. 3± 0.5 48. 0± 40. 0 87. 7± 66. 0 205. 8± 77. 7 266. 0± 110. 7

Hg. janthinomys 1. 0± 0. 2 61. 9± 39. 8 90.4± 51. 4 177. 1 ± 74. 8 214. 4± 120. 4
Cx. quinquefasciatus 1. 1 ± 0. 4 148. 3± 73. 4 222. 8± 127. 9 526. 9± 195. 0 661. 9± 268. 5

P= 0. 1 P= 0.003 P= 0. 001 P= 0. 006 P= 0. 05

are means± S. D.

The times it took Ae. aegypti ( 87. 7 s) and Hg. in the incidence and duration of prediuresis between
janthinomys ( 90. 4 s) to fill their guts were not parous and nulliparous females of any of the four
significantly different ( P< 0. 001), but Cx. quinque- mosquito species. In addition, within each mosquito
fasciatus ( 222. 8 s) was slower ( P< 0.01). In fact, An. species, blood- feeding times were similar for parous
aquasalis ( P< 0. 001), Ae. aegypti ( P< 0. 001), and Hg. and nulliparous females.
janthinomys ( P< 0.001) weresignificantly faster than The duration of feeding among individual
Cx. quinquefasciatus( TABLE 1). mosquitoes varied significantly( P< 0.001) according to
TABLE 1 shows the mean prediuresis times for all species and genera, with the range of feeding times
four mosquito species. Aedes aegypti( 205. 8 s) and Hg. being 133. 0 s to 650.0 s, 135. 0 s to 650.0 s, 66. 0 s to
janthinomys ( 177. 1 s) were similar ( P< 0.01). Culex 505. 0 s, and 263. 0 s to 1287. 0 s for An. aquasalis, Ae.
quinquefasciatus, however, took longer over pre- aegypti, Hg. janthinomys, and Cx. quinquefasciatus,
diuresing ( 526. 9 s) and was significantly ( P< 0.006) respectively.
slower than An. aquasalis( 77. 3 s). There were both inter( P< 0.001) and intra- specific
The total feeding time for the four mosquito differences among the four species of different genera in
species, of which prediures times were a large com- the level of erythrocyte agglutination in the blood meal.
ponent, are summarized in TABLE 1. All species The inter- specific variation in An. aquasalis, with 72%
exhibited prediuresis but the proportion of individuals ( G= 15. 5; df = 6, P< 0. 01) being scored 2 and 3. In
that did so varied with the mosquito species; An. aquasalis contrast, Ae. aegypti( 67%) ( G= 14. 9; df= 6; P< 0. 01),
86. 1%), Ae. aegypti ( 86. 7%), Cx. quinquefasciatus Hg. janthinomys( 67%)( G= 14. 8 df= 6 P< 0.01), and Cx.
86. 1%), and Hg. janthinomys ( 70. 0%) ( TABLE 2). quinquefasciatus ( 60%; G= 12. 0 df= 6; P< 0.05) were
The lowest level of prediuresis was observed among scored 0( see TABLE 3). Within each species, there
Hg. janthinomys ( 70. 0%) but this difference was not
were no significant differences in agglutination by
significant( P= 0. 9). There were no significantdifferences nulliparous and parous mosquitoes.

TABLE 2. Patterns of prediuresis exhibited by the four species of

mosquitoes

No. No. and(%)

Species feeding prediuresing

An. aquasalis 30 26 ( 86. 7%)

Ae. aegypti 36 31 ( 86. 1%)
Hg. janthinomys 30 21 ( 70. 0%)
Cx. quinquefasciatus 30 25 ( 83. 3%)
December, 1996 Journal of Vector Ecology 153

DISCUSSION the filariasis vector Cx. quinquefasciatus averaged 11

min. per feeding time. These results are probably
Partitioning the blood- feeding process for representative of natural
feeding events, as> 80% of the
mosquitoes into the number
components, such as of
field-collected females fed readily on volunteers when
probes, duration of probing, duration of the gut-
filling tested under laboratory conditions.
phase, duration of prediuresis, proportion of females Without exception, the four vector species showed
exhibiting prediuresis, and total feeding time provides a tremendous intra- specific differences for each of the
functional basis for interspecific comparisons of
parameters of the blood- feeding process. That is,
Vaughan 1991, Chadee
mosquito species ( et al. and
individual mosquitoes varied significantly in their timing
Beier 1995). We employed this strategy for evaluating with respect to probing, gut-filling, prediuresis, and
four vector species of mosquitoes from Trinidad: An. total feeding time. For example, female Cx.
aquasalis, Ae. aegypti, Hg. janthinomys, and Cx. quinquefasciatus varied eight- fold in their duration of
quinquefasciatus. In contrast to laboratory- based studies probing, from 40 to 320 s. For each species, the degree
using colonized mosquito species( Vaughan et al. 1991), of intra- specific variation is expressed in TABLE 1 as
we used field collected females captured
coming to feed the standard deviation for each mean value per blood-
on humans within their ecological niche in Trinidad.
feeding parameter.
The use of field- collected females for studies of blood- In groups of species tested in our studies, the
feeding represents an ideal way to obtain relevant duration of prediuresis usually accounted for most of the
information on natural behaviors in the absence of
interspecific differences in total feeding times; duration
biases laboratory colonization.
selection associated with
of gut- filling tended to be less variable among species
Notably, if blood- feeding behavior has a genetic basis, ( Vaughan et al. 1991, Chadee and Beier 1995). This was
then slow- feeding individuals would be selected against not true in our current evaluation, as Cx. quinquefasciatus
and lost very quickly in colonization efforts.
spent an average of 3. 7 min. in the gut- filling stage,
Significantly different patterns of blood- feeding more than twice as long as the other three species. This
were observed for the four vector species from Trinidad. species spent an even longer time undergoing prediuresis.
Except for the number of probes per
feeding event, the Unlike some mosquito species that fail to exhibit
four species showed significant differences with respect prediuresis ( Vaughan et al. 1991, Chadee and Beier
to duration of probing, duration of gut-
filling, duration 1995), from 70- 86% of all the vector species tested in
of prediuresis, and total feeding time. Overall, mean
the current study exhibited prediuresis. Notably, the
total feeding time varied almost five- fold among the timing of prediuresis as a proportion of the total feeding
four species. The malaria vector An. aquasalis was the time differed among the species, with An. aquasalis
quickest feeder, with total feeding time averaging just having the shortest period of prediuresis and Cx.
over 2 min. per
feeding event. The two arbovirus vectors, quinquefasciatus the most pronounced.
Ae. Hg. janthinomys, intermediate in
aegypti and were
Erythrocyte agglutination is a trait whereby
duration, totaling3- 4min. perfeeding. Mostsurprisingly, substances in the saliva promote red blood cell

TABLE 3. Patterns of erythrocyte agglutination displayed by the four mosquito vectors.

No. of Mosquitoes
Agglutination An. Ae. Hg. Cx.
score aquasalis aegypti janthinomys quinquefasciatus

0 0 20 20 18
1 3 9 8 10
2 12 1 1 2
3 14 0 1 0
4 7 0 0 0
5 0 0 0 0

Total 36 30 30 30
154 Journal of Vector Ecology December, 1996

in the midgut. Interestingly, a tremendous

agglutination
I. Bandoo, and A. Charran for field and laboratory
degree of inter- specific variation in agglutination patterns assistance. The research was supported by the National
was seen for the vector species from Trinidad. Institutes of Health Grant RO1 AI29000 and the Fogarty
Agglutination was observed for all 30 oftheAn. aquasalis International Research Collaborative Award R03
tested. In contrast, for the other three mosquito species, TW00240.

only 33- 40% exhibited agglutination. Inter-specifically,

An. aquasalis had the most pronounced variation in REFERENCES CITED
agglutination patterns, with 10% of the females
agglutinating 1- 25% of the red cells and 29% Arnett, H. J. 1973. ArevisionofthegenusHaemagogus.
agglutinating 71- 90% of the erythrocytes ingested. Less Contr. Am. Entomol. Inst. 10: 1- 174.
intra- specific variation is normally found in laboratory Chadee, D. D. 1988. Landing periodicity of the mosquito
Vaughan et al. 1991).
colonized species(
Aedes aegypti in Trinidad in relation to the timing
The process of blood- feeding behavior does not of insecticide space- spraying. Med. Vet. Entomol.
appear to change significantly as mosquitoes become 2: 189- 192.

physiologically chronologically older. None of the

or
Chadee, D. D. 1992. Indoor and outdoor host- seeking
species tested showed any signs of behavioral differences rhythms of Anopheles albitarsis ( Diptera:
between nulliparous and parous females. As the Culicidae). J. Med. Entomol. 29: 567- 569.
chronological ages of the parous mosquitoes were not Chadee, D. D. 1994. Life table characteristics of a
known in this study because all were field- collected, laboratory population of Culex quinquefasciatus
there was no possibility for testing directly for age- Say, the vector of Bancroftian filariasis. Bull. Soc.
related changes in the various parameters of blood- Vector Ecol. 19: 121- 124.
feeding behavior. Chadee, D. D. and J. C. Beier. 1995. Blood- engorgement
In conclusion, the parameters of blood- feeding kinetics of four anopheline mosquitoes from
measured in this study are extremely relevant to Trinidad, West Indies. Ann. Trop. Med. Parasitol.
understanding the dynamics of pathogen transmission 89: 55- 62.
by mosquitoes and other insects. Under the standard Chadee, D. D. and P. S. Corbet. 1987. Seasonal incidence
conditions of our test system, the field- collected and diel patterns of oviposition in the field of the
mosquitoes evaluated showed tremendous inter- and mosquito, Aedes aegypti( L.)( Diptera: Culicidae)
intra- specific differences in the timing of various in Trinidad, West Indies: a preliminary study. Ann.
parameters of the blood- feeding process.
Presumably, a Trop. Med. Parasitol. 81: 151- 161.
pronounced genetic component governs the degree of Chadee, D. D. and E. S. Tikasingh. 1989. Observations
variation seen in the study. Functionally, pathogens, on the seasonal incidence and diel oviposition
such as arboviruses, protozoa, and nematodes have periodicity of Haemagogus mosquitoes ( Diptera:
unique requirements for transmission that depend closely Culicidae) in Trinidad, W.I. Part I. Haemagogus
on the characteristic parameters of the blood- feeding janthinomys Dyar. Ann. Trop. Med. Parasitol. 83:
process. There are a number of consequences of having 465- 475.
a long feeding time and contact with the host( Molyneux Chadee, D. D., E. S. Tikasingh, and R. Ganesh. 1992.
and Jefferies 1986). For example, it is not hard to see
Seasonality, biting cycle and parity of the yellow
why Cx. quinquefasciatus is a successful vector of fever vector mosquito Haemagogus janthinomys in
filariasis, as females average 11 min. feeding contact Trinidad. Med. Vet. Entomol. 6: 143- 148.
with humans( e. g., ample time for infective L3 stages of Christophers, S. R. 1960. Aedes aegypti ( L.). The
the parasite to escape the thorax and navigate to host Yellow Fever Mosquito. Cambridge University
tissues through the mouth parts of the mosquito). Further Press, 739 pp.
evaluations of mosquito blood- feeding behavior are
Detinova, T. S. 1962. Age- grouping methods in Diptera
needed, especially with respect to establishing a better of medical importance with special reference to
understanding of pathogen transmission under field some vectors of malaria. Monograph Series No. 47.
conditions.
Geneva: World Health Organization, 213 pp.
Faran, M. E. 1980. Mosquito studies( Diptera: Culicidae).
Acknowledgments XXXIV. A revision of the Albimanus section of the
subgenus Nyssorhynchus of Anopheles. Contr. Am.
We thank Dr. R. Doon, Specialist Medical Ent. Inst. ( Ann Arbor) 15: 1- 215.
Officer, for valued help, R. Mohammed, W. Ramdath, Haddow, A. J. 1954. Studies on the biting habits of
R. Ganesh, S. Seesahai, A Hamid, F. Pierre, L. Punnette, African mosquitoes. An appraisal of methods
December, 1996 Journal of Vector Ecology 155

employed, with special reference to twenty- four- Soc . Trop. Med. Hyg. 75: 721- 730
hour catch. Bull. Ent. Res. 45: 199- 242.
Senior-White, R. A. 1953. On the evening biting activity
Macdonald, W. W. 1991. Control of Culex quinque- of three neotropical Anopheles in Trinidad, B. W.I.
fasciatus in Myanmar ( Burma) and India: 1960- Bull. Ent. Res. 44: 451- 460.
1990. Ann. Trop. Med. Parasitol. 85: 165- 172. Sokal, R. R. and F. J. Rohlf. 1980. Biometry. The
Molyneux, D. H. and D. Jefferies. 1986. Feeding principles and practice of statistics in biological
behaviour of pathogen- infected vectors. Parasitol. research. 2nd. Freeman, New York, 859 pp.
92: 721- 736. Vaughan, J. A., Noden, B. H., and J. C. Beier. 1991.
Nathan, M. B. 1981. Bancroftian filariasis in coastal
Concentration ofhuman erythrocytes by anopheline
North Trinidad, West Indies: intensity of
mosquitoes( Diptera: Culicidae) during feeding. J.
transmission by Culex quinquefasciatus. Trans. R. Med. Entomol. 28: 780- 286.
Journal of Vector Ecology 21( 2): 156- 158

Effects of Age and Mating on the Host-Seeking

Behavior of Aedes aegypti Mosquitoes

Marc J. Klowden and Noemi M. Fernandez

Division of Entomology, University of Idaho, Moscow, ID 83844- 2339, USA

Received 27 December 1995; Accepted 29 March 1996

ABSTRACT: Chronologically older female Aedes aegypti mosquitoes that had previously undergone a
gonotrophic cycle were more likely to respond to host stimuli when gravid than those that had not blood-
fed previously. Mating or injection with male accessory gland ( MAG) homogenates decreased the
percentage responding to host stimuli in both groups of old females and there was a greater reduction by
those that had previously ingested blood.
Older females that were mated late in life were less likely to
respond to host stimuli than were old females mated early. Injection of MAG homogenates into older
females that mated early in life caused a significant decline in host- seeking behavior. This difference in
the age- related change in host- seeking behavior during oogenesis appeared to be due to the decline in the
activity of MAG substances within the females.

Keyword Index: Mosquito, male accessory gland, host- seeking behavior

INTRODUCTION
in the host- seeking behavior of older mosquitoes when
they are gravid.
The host- seeking behavior of Aedes aegypti
mosquitoes is often inhibited during egg development MATERIALS AND METHODS
until after oviposition occurs, when new attempts to
locate a host define the beginning of the next The UGAL strain of Ae. aegypti ( L.) wasusedinall
gonotrophic cycle. Compared to females in their first experiments. Larvae were reared at 27° C on a standard
gonotrophic cycle, older females of this species show diet of rat chow, brewer' s yeast, and lactalbumin hydro-
less of an inhibition of host- seeking behavior lysate ( 1: 1: 1
and are
by weight). Adults were maintained at
more likely to approach a host when gravid. This effect 27° C and 80% RH under a 14: 10( L: D) photoperiod and
is accentuated if they have undergone several had access to 10% sucrose available from cotton wicks,
gonotrophic cycles and are
gonotrophically as well as
except for two days prior to olfactometer testing. Males
old ( Klowden and Lea 1984). Other
chronologically and females were separated by sex in the pupal stage
differences in mosquito behavior have also been
using the criterion of the presence of the projecting plate
associated with aging ( Gillies 1957, Hitchcock 1968, of the ninth sternite in the female and its absence in the
Crans et al. 1976). Christophers 1960).
male (
Blood meals were usually
We recently demonstrated that male accessory obtained from human hosts, but in some experiments
gland ( MAG) substances that are transferred
during measured volumes of rat blood were also administered
mating modulate the host- seeking behavior of gravid
by enema( Briegel and Lea 1975).
Ae. aegypti, making them less likely to express host-
Newly emerged females were initially maintained
seeking behaviors compared to unmated gravid females
without males. In the early mating group, males were
that were
developing Fernandez Klowden,
eggs ( and
introduced into their cage three days after female
1995). Because females usually mate early in their lives
emergence. In the late mating group, the males were not
and
generally do not remate as a result of the inhibition introduced until day 22. The females were blood- fed on
of subsequent mating by MAG substances ( Craig day 22 and their host- seeking behavior was evaluated in
1967), we tested the hypothesis that the inactivation of an olfactometer ( Klowden and Lea 1978) three days
MAG substances with age is responsible for the increase later. In another experiment,
mating occurred on day 3
December, 1996 Journal of Vector Ecology 157

and
additionally, 0. 2 p1 of a 4 MAG/ pd homogenate( 0. 8 Controls were injected with the same volume of saline
MAG equivalents) was injected
shortly before I p1 of alone.
Three days later when their host- seeking
blood was introduced as an enema on day 22. The host-
behavior was evaluated, controls that had mated on day
seeking behavior of these females was tested day 25.
on

Other mosquitoes were treated

3 and that received blood for the first time on day 22
identically, but were less than half as likely to respond than were
additionally blood- fed on days 5 and 10 post- females of the same age that had blood- fed twice
emergence. Control groups were injected with saline or
previously( TABLE 2), demonstrating that gonotrophic
heat- inactivated MAGs. To compare responses
aging reduced the inhibition of host- seeking behavior
between groups, we used the test based
z- on the during oogenesis. Within each group, those injected
percentages responding ( SigmaStat Statistical with the MAG homogenate were less likely to respond
Software, ver. 1. 0. Jandel Scientific Software, San
to host stimuli, and the one group additionally injected
Rafael, CA).
with heat- inactivated MAG substances responded no
differently than did controls.
RESULTS

DISCUSSION
Effects of Age and Natural Mating on Host-Seeking
Behavior
Aging and mating both modulate the host-seeking
Females were mated either at three days post-
behavior of gravid Ae. aegypti mosquitoes. Our data
emergence or on day 22, ingested blood on
day 22; and
suggest that the increased host- seeking behavior that is
their host- seeking behavior 25
was measured on
day characteristic of the unmated and older gravid females
before oviposition occurred. As shown in TABLE 1, appears to result from the absence or inactivation of
the time that the mating occurred determined the MAG substances after they are introduced into the
degree to which host- seeking behavior was affected.
female during mating. When females mated early, the
About half of the unmated females responded, and those
inhibition of host- seeking behavior after gonotrophic
that were mated on day 3 showed a significantly reduced aging occurred was less than if they mated later in life
response. Those females that did not mate until day 22, ( TABLE 1).
Injections ofMAG substances into already
however, showed an even greater inhibition that was mated females increased the degree of behavioral
typical of that displayed by younger mosquitoes.
inhibition that was expressed when the mosquitoes were
older( TABLE 2). The absence of any effects from the
Effects of MAG Homogenates and Repeated Blood-
injection of heat- treated homogenates suggests that this
Feeding on the Host- Seeking Behavior of Older
response was not a result of non- specific trauma from
Females

Females mated on day 3 were either given two

blood meals on days 5 and 10 after emergence or were TABLE 2 Effects of physiological and gonotrophic
not allowed to feed, and on
day 22, were all given I ill
age and MAG injection on the host- seeking
of blood by enema. Other groups were additionally behavior of 25- day- old females mated on
injected with a MAG homogenate on
day 22 or a day 3.
homogenate first heated at 100° C for 15 minutes.
Treatment of MAG Percent Responding
Females Injected to Host Stimuli± SE
TABLE 1. Host- seeking behavior of 25- day- old
mosquitoes that were mated either or
early 1 pi of blood on - 12. 1± 4. 2
late in life.
day 22 only 3. 3± 2. 3*
heat 12. 2± 4. 2
Percent Responding inactivated)
Treatment to Host Stimuli (± SE)

Blood meals on
Unmated 56. 0± 6.
4a

days 5 and 10; - 28. 1± 5. 8

6b
Mated on day 3 25. 6± 5. I IA blood on 16. 5± 4. 8*
Mated on day 22 1. 4± 1. 4° day 22

Values followed by the same letter do not differ * signifies significant differences from non- injected
significantly( P< 0.05). controls( P< 0. 05).
158 Journal of Vector Ecology December, 1996

the presence of large amounts of protein in the behavior of gravid Aedes aegypti mosquitoes. J.

hemolymph. Insect Physiol. 41: 965- 970.

Previous studies identified age- related behavioral Gillies, M. T. 1957. Age- groups and the biting cycle in
and physiological changes in Ae. aegypti( Klowden and Anopheles gambiae. A preliminary investigation.
Lea 1980, 1984). females that Bull. Entomol. Res. 56: 237- 262.
Chronologically older

were mated soon after emergence were more likely to Gomulski, L. 1990. Polyandry in nulliparousAnopheles
host than gambiae mosquitoes ( Diptera: Culicidae). Bull.
seek a while gravid when they were younger;

Entomol. Res. 80: 393- 396.

but when
they had undergone several gonotrophic

cycles, there was an probability of

even greater Hitchcock, J. R., Jr. 1968. Egg retention in Anopheles
engaging in host- seeking ( Klowden and Lea 1984). quadrimaculatus Say in relation to the physiological
Male accessory gland substances also prevent the age of the mosquito. J. Med. Entomol. 5: 8.

female from engaging in subsequent remating ( Craig Klowden, M. J. and A. 0. Lea. 1978. Blood meal size as

1967), but these substances no longer provide as a factor affecting continued host- seeking by Aedes
effective a barrier to mating after several gonotrophic aegypti( L.). Am. J. Trop. Med. and Hyg. 27: 827-

Williams Berger 1980, Downe 831.

cycles( and Young and

1982). However, polyandry, when reported from field Klowden, M. J. and A. 0. Lea. 1980." Physiologically
is infrequent( Mahmood and Reisen 1980,
populations, old" mosquitoes are not necessarily old
Reisen et al. 1984, Gomulski 1990, Villarreal et al. physiologically. Am. J. Trop. Med. Hyg. 29: 1460-
1994, Yuval and Fritz 1994); and there is little evidence 1464.

that multiple mating would be likely to enhance the Klowden, M. J. and A. 0. Lea. 1984. Blood feeding
early receipt of MAG substances in natural
effects of an affects age- related changes in the host- seeking
populations. The increased tendency to seek a host by behavior of Aedes aegypti ( Diptera: Culicidae)

chronologically older, more epidemiologically during oocyte maturation. J. Med. Entomol. 21:
important mosquitoes, appears to result from an age- 274- 277.

related inactivation of MAG substances. Mahmood, F. and W. K. Reisen. 1980. Anopheles

culicifacies: the occurrence ofmultiple insemination
Acknowledgments under laboratory conditions. Entomol. Exp. Appl.
27: 69- 76.
We thank Gail Chambers for her technical assis- Reisen, W. K., B. G. Evans, and M. E. Bock. 1984.
tance. Supported by NSF grant IBN- 9224357 to MJK. Reinsemination of parous Culex tarsalis females.
Mosq. News 44: 580- 582.
REFERENCES Villarreal, C., G. Fuentes- Maldonado, M. H. Rodriguez,
and B. Yuval. 1994. Low rates of multiple

Briegel H. and A. 0. Lea. 1975. Relationship between fertilization in parous Anopheles albimanus. J. Am.
protein and proteolytic activity in the midgut of Mosq. Contr. Assoc. 10: 67- 69.
mosquitoes. J. Insect Physiol. 21: 1597- 1604. Williams, R. W. and A. Berger. 1980. The relation of
Christophers, S. R. 1960. Aedes aegypti( L.) the Yellow female polygamy to gonotrophic activity in the
Fever Mosquito. Cambridge University Press. 739 Rock strain of Aedes aegypti. Mosq. News 40: 597-
pp.
607.

Craig, G. B., Jr. 1967. Mosquitoes: female monogamy Young, A. D. M. and A. E. R. Downe. 1982. Renewal of
induced by male accessory gland substance. Science sexual receptivity in mated females, Aedes aegypti.
156: 1499- 1501. Physiol. Entomol. 7: 467- 471.
Crans, W. J., J. D. Downing, and M. E. Slaff. 1976. Yuval, B. and G. N. Fritz. 1994. Multiple mating in
Behavioral changes in the salt marsh mosquito female mosquitoes - evidence from a field
Aedes sollicitans as a result of increased population of Anopheles freeborni ( Diptera:
physiological age. Mosq. News 36: 437- 445. Culicidae). Bull. Entomol. Res. 84: 137- 140.
Fernandez, N. M. and M. J. Klowden. 1995. Male
accessory gland substances modify the host- seeking
Journal of Vector Ecology 21( 2): 159- 166

Vertical Distribution of Adult Mosquitoes

Diptera: Culicidae) in Southern and Central Sweden

Jan 0. Lundstrom'' 2, Jan Chirico3, Anders Folke3, and Christine Dahl2

Virology Unit, Swedish Institute for Infectious Disease

Control, S- 10521 Stockholm, Sweden

Department of Zoology, Uppsala University, Villavagen 9,

S- 75236 Uppsala, Sweden

Department of Parasitology, National Veterinary Institute,

Box 7073, S- 75007 Uppsala, Sweden

Received 23 January 1996; Accepted 20 July 1996

ABSTRACT: The vertical distribution of adult mosquitoes was studied in southern ( Norra Asum) and
central Sweden ( Tarnsjo) during August 1995, with an emphasis on the ornithophilic species Culiseta
morsitans( Theobald), Culex pipiens L., and Culex torrentium Martini. A modified slingshot method was
used to suspend Centers for Disease Control ( CDC) miniature light traps, in combination with CO2 as
additional attractant, into the canopy of deciduous trees. Altogether 15, 186 mosquitoes, including 93 Cs.
morsitans and 182 Cx. pipiens/ torrentium, were collected at various heights above ground during 43 trap-
nights. The numbers of Cs. morsitans and Cx. pipiens/ torrentium per trap- night were not significantly
influenced by height, but the number of total mosquitoes per trap- night decreased with increasing height.
Significantly larger proportions( a<0.001, chi- square test) of both Cs. morsitans and Cx. pipiens/ torrentium,
to the total of other mosquitoes, were collected in the canopy than at chest height. The proportions of Cs.
morsitans were 18% at 12 to 15. 5 m and 2. 2% at 1. 5 m in Norra Asum, and 49% at 14 to 18 m and 0. 6% at

1. 5 m in Tarnsjo. The proportions of Cx. pipiensltorrentium were 36% at 12 to 15. 5 m and 6. 9% at 1. 5 m in

Norra Asum, and 5. 1% at 6 to 9 m and 0.2% at 1. 5 m in Tarnsjo. Our method of suspending traps in the canopy
and use of a simplified protocol to quickly distinguish the relevant species was efficient for placing the traps
and for sampling adult females of the ornithophilic species Cx. pipiens/ torrentium and Cs. morsitans in
Sweden. We suggest that this procedure might also be of use to investigate these and other ornithophilic
mosquitoes for bird-associated arboviruses in woodlands in the European region.

Keyword Index: Ornithophilic mosquitoes, Culex, Culiseta, vertical distribution, arbovirus.

INTRODUCTION second enzootic vector, Culiseta morsitans( Theobald)

Lundstrom 1993). Transmission to humans is probably
Several human pathogenic arboviruses of the genera accomplished by Aedes cinereus Meigen( Francy et al.
Alphavirus( Sindbis, eastern equine encephalomyelitis, 1989, Turell et al. 1990) that feed predominantly on
western equine encephalomyelitis) and Flavivirus( Saint mammals but also on birds( Service 1971 a, Jaenson and
West Niklasson 1986). Francy et al.( 1989) found that Culex
Louis encephalitis, Murray Valley encephalitis,

Nile) exploit birds as reservoir hosts and specialized pipiens L./ torrentium had a 28- fold higher Ockelbo
ornithophilic mosquitoes of the genera Culex and virus field infection rate, and that Cs. morsitans had a
Culiseta as enzootic vectors in their natural transmission 10- fold higher rate, than had Ae. cinereus. None of the
cycles( Niklasson 1988, Reisen andMonath 1988, Morris other 17 species investigated hosted Ockelbo virus.
1988, Tsai and Mitchell 1988, Marshall 1988, Hayes, Thus, specific sampling of Cx. pipiens/ torrentium and
1988). Ockelbo virus, a north European subtype of Cs. morsitans, species specialized in feeding on birds in
Sindbis virus( Lundstrom et al. 1993), is maintained in northern Europe ( Service 1969, 1971a, Jaenson and
the passerine bird population by CulextorrentiumMartini Niklasson 1986), would greatly improve our ability to
which is the main enzootic vector, and by a potential detect Ockelbo virus activity. These ornithophilic
 160 Journal of Vector Ecology December, 1996

mosquitoes are, however, difficult to collect as adults.

Collection and Identification of Mosquitoes
Most of the mosquito that have been
species
In order to count the absolute numbers of ornitho-
examined are active at lower levels, usually because philic mosquitoes in relation to other species, we used
little sampling is done
or no at higher levels. In the USA,
the Centers for Disease Control( CDC) miniature light
the ornithophilic Culiseta melanura ( Coquillett) was
trap with carbon dioxide as additional attractant( Sudia
found be evenly distributed up to 7. 6
to metres in and Chamberlain 1962), placed at different heights.
Massachusetts ( Nasci and Edman 1981), and Culex
Each CDC trap was baited with 1- 2 kg of dry ice, kept
salinarius Coquillett
evenly distributed up to 25 m
was
in an insulated envelope to allow a slow release of
in Tennessee ( Snow 1955). The British Cs. morsitans
carbon dioxide. In Norra Asum, two CDC traps were
and Cx. pipiens/ torrentium were more active at 5. 5 m suspended in alder trees at 12 and 15 m, and one was
than at ground level ( Service 1971b). The sampling of suspended in a willow tree at 15. 5 m. Ten traps were
mosquitoes well above ground level in the
canopy may placed at 1. 5 m, six of these traps were positioned above
therefore provide more specific samples of ornithophilic
the water surface in the marshy biotope, while the
mosquito species.
remaining four traps were set in a similar biotope but
The aims of the study were to investigate the without a visible water surface. All traps were operated
vertical distribution of adult mosquitoes in wooded
on three consecutive nights between July 31 and August
biotopes in southern and central Sweden during the 3, 1995.
latter part of summer, and to establish for
a protocol
In Tarnsjo, two CDC traps were suspended in beech
efficient sampling of ornithophilic mosquitoes with
trees( Betula pubescens) at 8 m and at 9 m, and one trap
emphasis on the enzootic vectors of Ockelbo virus.
was suspended in a dead Norwegian spruce at 6 m. Two
traps were placed at 1. 5 m. In Tarnsjo the traps were
MATERIALS AND METHODS operated on two consecutive nights, between August 9
and 11, 1995. A second trial was conducted in Tarnsjo
Study Sites
with six traps suspended at 14, 15. 5, 16. 5, 17, 17. 5, and
Two study sites were chosen. The first was situated 18 m in aspen, and two traps at 1. 5 m. These traps were
in Norra Asum ( 55° 59' N, 14° 10' E) at the western
operated the night between August 22 and 23, 1995. All
shore of the Lake Hammarsjon, Province of Sickle,
mosquito collections were performed between 6 p. m.
in southern Sweden. The shore of this shallow lake and 7 a. m.
was covered
by extensive reed( Phragmites communis) To place the traps in tree canopies at the required
belts and of
periodically flooded open areas mainly height, the modified slingshot method ( Novak et al.
covered by Carex spp. Outside the Carex belt the
1981) was used. A 90 g pellet of lead or tin was fitted to
area is dominated by extensive farmland and patches
the nylon monofilament line of a spinning reel mounted
of deciduous forest. The sampling of mosquitoes was
on a spinning rod attached to the side of a 230 cm
conducted in one of these deciduous forest patches
wooden plank that also served as the holder of a slingshot
approx. 30 hectares), at two
study sites approxi- (
Fig. 1). By placing the lower end of the plank against
mately 50 m apart. Alder trees ( Alnus glutinosa), 15 the ground, both hands could be used to stretch the
to 20 m tall, dominate the forest. The ground was
elastic band of the slingshot, and the pellet with attached
covered with dense herb vegetation and willow bushes line could thereby be catapulted with great precision
Salix spp.) up to 2 m high. One site was
marshy, over a selected robust branch at considerable height( at
with extensive areas of water of less than 10 cm least 25 m), and the mass of the pellet was sufficient to
deep. pull the line up over the branch and back to the ground.
The second study site, in Sweden,
central was near
Once the line had been placed over the selected branch,
the southern shore of the Lake Nordmyrasjon, Tarnsjo it was used to draw a 50- meter 1. 0 mm diameter
60° 10' N, 16° 54' E), Province of Vastmanl and, in central interwoven multifilament nylon rope( rope A), over the
Sweden. This lake has been almost
completely over- branch. A metal loop was attached to one end of rope A,
grown with reed, Carex spp., willow, etc., and is now a
and a slightly shorter rope( B) was inserted through the
wetland periodically flooded
by the river Dalalven. loop, which was then suspended from the canopy so that
Mosquitoes were collected in the borderland between
both ends of rope B reached the ground. The trap was
the wetland and a mixed deciduous/ coniferous forest.
then attached to one end of rope B so that it could easily
Aspen( Populus tremula) and Norwegian spruce( Picea
be raised and lowered for collection( Fig. 2). For each
abies), 25 to 30 m tall, are the most common trees on the
trap, the total length of rope B was measured and divided
study site; and the ground is covered by dense vegetation by two, to determine the height of the trap.
up to 1 m tall. Collected mosquitoes were anaesthetized with
December, 1996 Journal of Vector Ecology 161

ill!
a
i

14;;/'
f!rl

b.

i 11,/

R l•
r

r
v

L
1

r l

fr

Figure 1. The design of the slingshot used for catapulting the 90 g lead pellet with attached line over a branch in
dense canopies of deciduous trees.
162 Journal of Vector Ecology December, 1996

rc, 6_,., cr
c\
o
nr
Eic-'.\v ,,-
‘ --, ..,'---
r) t,. 0 It - / -
Ljc -
1'
1.°P .'------''' -- rill l, /
V

J
i'lkt1' i/ 1 - rat

V
4116
i

cC.. w I
t
C G _,
A/ % rr '

illi
Rope A

I, f/ j
I
II'
II
Rope B

ii
1

I + 111
Ii li

ik 1,
I

Will
1!'.
I'
6 1
I
rfi Ii

r„
r„,0
iir ir.,,
s._
r.,. e"" I, \,,
v,,,,,,,

Figure 2
The method for suspending and rapid lowering of CDC miniature light traps and the envelope with dry
ice in the canopy of deciduous trees.
December, 1996 Journal of Vector Ecology 163

carbon dioxide, dispensed in 8 ml plastic vials of the eventual difference between the proportions of
approximately 350 mosquitoes/ vial), and transported certain mosquito species collected in the canopy versus
on dry ice to the laboratory at the Swedish Institute for at chest height.
Infectious Disease Control in Stockholm for
identification. Mosquitoes were identified according to RESULTS

the species descriptions in Mohrig ( 1969) under a

stereomicroscope. Specimens of Cs. morsitans, Culiseta Altogether 15, 186 mosquitoes, including 93 Cs.
annulata ( Schrank), Ae. cinereus, Coquillettidia morsitans and 182 [Link]/ torrentium, were collected
richiardii ( Ficalbi), and Anopheles claviger( Meigen) at various heights above ground on 43 trap- nights in the
were identified to species. Females of Cx. pipiens and
two study areas during three study periods( TABLE 1).
Cx. torrentium could not be reliably distinguished on The proportions of these species to the total number of
morphological criteria and were therefore grouped
mosquitoes collected were significantly higher in the
together as Cx. pipiens/ torrentium. Similarly, Anopheles canopy than at chest height. However, the numbers of
spp., other than An. claviger, were identified as An. Cs. morsitans and Cx. pipiens/ torrentium per trap- night
maculipennis sensu latu. To increase the speed of were similar at all heights because the average number
processing specimens for identification, and because of total mosquitoes per trap- night decreased with
Aedes spp. other than Ae. cinereus have not been increasing height.
considered vectors of Ockelbo virus in Sweden, other
Culiseta morsitans constituted significantly higher
species in this genus were grouped together as Aedes proportions of the total number of mosquitoes collected

spp. at canopy level than at chest height in both study areas

The chi- square test, corrected for continuity( Siegel during all three study periods. Traps at 12 to 15. 5 m in
and Castellan 1989), was used for statistical evaluation Norra Asum collected 18% Cs. morsitans, while traps at

TABLE 1. Vertical distribution of mosquitoes collected during July and August 1995 in southern
Sweden( Norra Asum) and in central Sweden( Tarnsjo) using CDC miniature light traps
with carbon dioxide as attractant.

Norra Asum Tarnsjo Tarnsjo

July 31 to August 3 August 9 to 11 August 22 to 23

51
Height( meters) 12- 15. 5 1. 5 1. 6- 9 1. 5 14- 18 1. 5

Trap- nights( no.) 9 12 18 4 4 6 2

Species

torrentium2

Culex pipiens/ 40 54 85 0 0 2 1

Culiseta morsitans 20 17 27 3 3 19 4

Culiseta annulata 0 2 1 0 0 0 0

Anopheles claviger 4 46 18 0 0 0 0

Anopheles maculipennis sl. 0 1 0 0 0 0 0

Coquillettidia richiardii 15 216 188 55 48 6 9

Aedes cinereus 0 11 3 192 1, 976 2 93

3
Aedes spp. 32 435 204 509 10,321 10 514

Total 111 782 526 759 12, 348 39 621

Total/ trap- night 12. 3 65. 2 29. 2 190 3, 087 6. 5 310

These traps were operated over shallow water pools in the forest.

2Female Cx. pipiens and Cx. torrentium both occur in the study areas but could not be distinguished
reliably to species.
3Numbers pooled of all Aedes other than Ae. cinereus.
164 Journal of Vector Ecology December, 1996

1. 5 m collected 2. 2% in the drier biotope( different from

morsitans at 5. 5 m than at ground in Britain. The activity
57. 5, df=1, a< 0.001) and
x2=
5. 1% in the marshy
canopy of ornithophilic mosquitoes in the canopy is probably a
biotope( different from canopy, x2=

24. 2, df= 1, a<0. 001). reflection of the spatial distribution of their bird hosts.
The traps in Tarnsjo collected 0. 4% Cs. morsitans at 6 The energy demands of sustained search flight at various
to 9 m versus 0.02% at 1. 5 m during August 9 to 11
heights and the problems associated with locating small
30.4, df= 1,
x2=

a< 0. 001), and 49% 14 to 18

at m versus
birds in the canopy should decrease the proportion of
0. 6% at 1. 5 m
during August 22 to 23 ( x2=
267, df= 1, non- ornithophilic species in the canopy, which is in line
a<0. 001). However, the numbers of Cs. morsitans
with our results. Thus, mosquito collection at canopy
collected per
trap- night were not different in the canopy level was shown to be efficient for sampling ornithophilic
than at chest- height. mosquitoes.

Similarly to Cs. morsitans, the numbers of Cx. The mosquito fauna of both south and central
pipiens/ torrentium per trap- night were not influenced Sweden includes Cx. pipiens as well as Cx. torrentium
by height, but the proportions of these species to the total ( Dahl 1977, Franey et al. 1989). The males could be
number of mosquitoes collected were
significantly larger identified to species based on the morphology of the
in the canopy than at chest height at all comparisons. genitalia, while reliable morphological methods to
Traps at 12 to 15. 5 m in Norra Asum collected 36% Cx.
distinguish between the females of these species are not
pipiens/torrentium, while traps at 1. 5 m in the marshy
available( Dahl 1988). Thus, we are presently unable to
biotope collected 16%( different from canopy, 24. 2,
x2=

report these species separately and have decided to

df= 1, a< 0. 001), and traps at 1. 5 m in the drier biotope report them as Cx. pipiens/ torrentium, although we are
collected 6. 9% of these species( different from canopy, aware of the differences in their experimental vector
x2=
84. 5, df= 1, a<0. 001). The mosquito collection during competence for Ockelbo virus( Lundstrom 1994). Future
August 22 to 23 in Tarnsjo gave 5. 1% Cx. pipiens/ studies may also show differences in the behaviour of
torrentium at 14 to 18 m, and 0. 2% of these species at 1. 5 these two species.
32. 5, df= 1, a< 0.001).
x2=
m (
The greater number of Cx. pipiensltorrentium
Coquillettidia richiardii was collected at all heights collected in Norra Asum than in Tarnsjo during August
in both Norra Asum and Tarnsjo( TABLE 1). In Norra ( late summer) was probably not caused by a major
Asum this species made up 14% of mosquitoes collected
difference in the overall population size because very
at 14 to 18 m and 28% of mosquitoes collected at 1. 5 m large numbers of Cx. pipiens/ torrentium larvae were
different, 0. 02, df= 1, a= 0. 90). However,
x2=

not
Cq. observed in Tarnsjo one month prior to the study
richiardii was more prevalent at 14 to 18 m ( Lundstrom, information).
significantly unpublished
More likely,
15%) than at 1. 5 m ( 1. 4%) during August 22 to 23 the paucity of Cx. pipiensltorrentium in the Tarnsjo
38. 7, df= 1, a< 0. 001),
x2=

6 to 9 7. 2%)
and at m( versus
samples was caused by replacement of the host- seeking
1. 5 m( 0. 4%) during August 9 to 11 in Tarnsjo( x2=

440, summer generation by the strictly nectar- feeding pre-

df= 1, a< 0. 001).
hibernating generation that are not attracted by CO2.
Aedes mainly collected at 1. 5 m in
cinereus was
The shift between summer and pre- hibernating
both study sites( TABLE 1). However, in Tarnsjo during generations occur during the first half of August in
August 9 to 11 it constituted 25% of the catch at 6 to 9 central Sweden( Jaenson et al. 1986).
m versus 16% at 1. 5 m, and two Ae. cinereus were col- Similar numbers of Cs. morsitans were collected in
lected 14 to 18 in Tarnsjo during August 22 to 23.
at m
both study areas, which is in line with the phenology of
this species in Sweden, with a single summer generation
DISCUSSION
that takes multiple blood meals during June to October
Jaenson et al. 1986).
We investigated the vertical distribution of adult
The frequent host- seeking of Cq. richiardii at
mosquitoes in woodlots of south and central Sweden, canopy level, both in Norra Asum and in Tarnsjo,
and found a significantly larger proportion of the indicates that it searches for bird hosts because birds
ornithophilic species Cs. morsitans and Cx. pipiens/ are the most common vertebrates in the canopy. Also,
torrentium in the canopy than at chest height during all
search flight to find mammals in the canopy as corn-
three study periods in both study sites. Studies in North
pared to at ground level is a waste of energy because
America have also shown increased proportions of ground dwelling mammals of several species are
ornithophilic Culex and Culiseta species in the mosquito
common while only one species of squirrel occur in the
fauna canopy level ( Snow 1955, Nasci and Edman
at
canopy in the study areas. Observations in Africa and in
1981, Novak et al. 1981). Service ( 1971b) reported North America have shown that mosquitoes of the
larger numbers of Cx. pipiens/ torrentium and Cs. genus Coquillettidia feed readily in the and that
canopy
December, 1996 Journal of Vector Ecology 165

birds generally are the preferred hosts ( Edman 1971, investigatedforarbovirusesalthoughtwobird- associated
McIntosh et al. 1972, Rickenbach et al. 1974, Chandler and mosquito- borne viruses that cause disease in humans
et al. 1976). Further studies on the host preference of have been identified in Europe( Lundstrom 1994).
Cq. richiardii and assay of the species for bird-associated
arboviruses could therefore be
interesting. Acknowledgments
We found that Ae. cinereus was active both at
ground level and in the canopy, and our collection at 6 We thank Ms. Ingallordgrenforprovidinghousing
to 9 m and at 14 to 18 m in Tarnsjo is the first evidence
in Tarnsjo during the study, Professor Bo Niklasson for
that the species is active well above ground level. lending us CDC traps, and two anonymous reviewers
Mohrig ( 1969) and Jaenson and Niklasson ( 1986) for valuable suggestions to improve the manuscript.
reported that Ae. cinereus appears only close to the This research was funded by a grant from the Swedish
ground and that the species feeds mainly on small Society for Medicine to J. O. Lundstrom.
rodents. When taking blood from larger mammals,
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Mohrig, W. 1969. Die Culiciden Deutschlands. edition, McGraw- Hill Book Company, New York,
Untersuchungen zur Taxonomie, Biologie and
399 pp.
OkologiedereinheimischenStechmucken. Parasit. Snow, W. F. 1955. Feeding activities of some
Schr. Reihe 18: 260 pp. bloodsucking diptera with reference to vertical
Morris, C. D. 1988. Eastern equine encephalomyelitis. distribution in bottomland forest. Ann. Entomol.
Pp. 1- 20 in The Arboviruses: Epidemiology and Soc. Am. 48: 512- 51.
Ecology ( T. P. Monath, Volume III, CRC
ed.),
Sudia, W. D. and R. W. Chamberlain. 1962. Battery-
Press, Boca Raton, Florida, USA, 234 pp.
operated light trap, an improved model. Mosq.
Nasci, R. S. and J. D. Edman. 1981. Vertical and News 22: 126- 129.
temporalflight activity of the mosquito Culiseta Tsai, T. F. and C. J. Mitchell. 1988. St. Louis encephalitis.
melanura ( Diptera: Culicidae) in southeastern
Pp. 113- 143 in The Arboviruses: Epidemiology
Massachusetts. J. Med. Entomol. 18: 501- 504. and Ecology( T. P. Monath, ed.), Volume III, CRC
Niklasson, B. 1988. Sindbis, and Sindbis- like viruses.
Press, Boca Raton, Florida, USA, 234 pp.
Pp. 168- 176 in The Arboviruses: Epidemiology Turell, M. J., J. 0. Lundstrom, and B. Niklasson. 1990.
and Ecology( T. P. Monath, ed.), Volume III, CRC Transmission of Ockelbo virus by Aedes cinereus,
Press, Boca Raton, Florida, USA, 234 pp. Ae. communis, and Ae. excrucians ( Diptera:
Novak, R. J., J. Pelequin, and W. Rohrer. 1981. Vertical Culicidae) collected in an enzootic area in central
distribution of adult mosquitoes( Diptera: Culicidae) Sweden. J. Med. Entomol. 27: 266- 268.
Journal of Vector Ecology 21( 2): 167- 172

Use of the Fly Grill for Assessment of House Fly Populations:

An Example of Sampling Techniques That Create Rough Fuzzy Sets

Harvey I. Scudder

7409 Hansen Drive, Dublin, CA 94563- 2724 USA

Received 21 February 1996; Accepted 30 April 1996

ABSTRACT: Widely used since its initial development in 1944, the " Scudder Fly Grill" has been a
successfully employed sampling system without any theoretical statistical validation. Yet, this
unconventional method has been shown to be empirically sensitive for guiding fly control programs, and
has shown a good correlation of fly population levels with fly- borne disease. This report provides the first
workable explanation that grill sampling is really the use of" rough fuzzy sets" of data, part of a new
computational system now becoming widely used in industry. Parallels of fly population sampling are
made to other biological sampling problems where the use of fuzzy logic might also be useful.

Keyword Index: Fly, grill index, contagious distribution, sampling method, fuzzy logic.

HISTORY OF THE SAMPLING METHOD way of quantitatively evaluating competing baits.

Extended field observations of fly behavior, such
When the U. S. Public Health Service reassigned as their mobility, socializing, and in particular, their
the author to the Henry Rose Carter Laboratory in selection of edges for resting, finally led the author to
Savannah, Georgia in October 1944, his task was to design a replacement uniform resting surface, the fly
determine the efficacy of that very new insecticide, grill( Fig. 1), on which one could very rapidly count the
DDT, for the control of adult flies. At the outset, in the resting flies with minimal disturbance of the general
author' s analysis, the first and most difficult step in the population( Scudder 1947, McGuire and Lindsay 1950,
research was to be the requirement for the formulation West 1951, Schoof 1955, Ehlers and Steel 1965).
and design of a dependable and sensitive method for The grill proved to be an easy, quick, and
assessing fly populations in order that all treatments reproducible way of measuring fly populations, and was
undertaken could be evaluated by quantitative adopted as the standard for studies of DDT- fly control
measurements. in the Savannah area in 1945; and in the following year
The study required that the census method be in the control of flies in nine Lower Rio Grande towns
feasible for determining all naturally occurring fly in Texas in a large scale experiment to determine the
population levels prior to and after treatments, and role of flies in the transmission of endemic( bacillary)
therefore be able to validly reflect any difference in dysentery ( Shigella) ( Watt and Lindsay 1948). This
dosage levels of the insecticide, duration and degradation was followed next year by a large scale study in
of effect, difference in treatment techniques, and physical Thomasville, GA( Lindsay et al. 1953).
differences in situations treated. The data must be The Savannah study was successful, the use of the
reproducible for validity of comparison. grill method was validated in many studies, and became
Study of house fly populations revealed that they the standard for measurement of fly populations in
were very mobile, very gregarious, sensitive to light,
many control programs throughout the world( Holway
wind, and temperature levels, and very responsive to a et al. 1951, Ricciardi and Paulini 1955, Dhillon and

variety of attractants. No sampling method in a fixed Challet 1985).

position could be valid. No attractant ( bait) could be Extensive experience in using the grill successfully
used because there would be no way of evaluating it to guide and evaluate fly control programs led in 1978
against those attractants already present in the to incorporation of the method into the California Code
environment being sampled, nor was there any practical of Regulations( Title 14-§ 17682 California Integrated
 168 Journal of Vector Ecology December, 1996

4 ta
e
a

g
e

r . r

II

11511WININIIIIMI

1:
41. 1111111111. 11111111
Y
r

44 4

It

f
Y •... R
w f ;.._
a; .. r art,

ti
y, r gip.

Figure 1.
The fly grill( 3' square) in use, placed over a fly population point, and showing about 469 houseflies
resting on it.
December, 1996 Journal of Vector Ecology 169

Waste Management Board) as the required procedure

major feeding and resting places available in the local
for determining the effectiveness of landfill cover for situation under study. Brief note is taken of those
the prevention of fly infestations( Barclay' s 1992). locations, the attractants present, their exposure to wind
Submission of the original paper on the fly grill
and sun, as well as time of day. By this procedure one
method for publication ( Scudder 1947) went through becomes familiar with the local extent and situations of
the official channels of the U. S. Public Health Service, greatest activity of the population about to be evaluated.
where at NIH, an authority on conventional statistical
Next, the grill counts are made only of the most
techniques stripped the author' s text of all interpretative dense aggregations of flies, by momentarily placing the
statements. This" correction" by the then" conventional grill, with as little disturbance as possible, in the
wisdom" reduced the paper to only a " cook book" approximate center of each group of flies within the
technique, which is where it has stood until this present principal area in which the mobile population is
paper, which proposes that its appropriate status should
circulating at the time of sampling. Only the highest
be as a sampling concept within the new computational three counts are used, but for the first inspection more
method, " fuzzy logic." should be taken and the lower numbers discarded, with
In all the years since the original formulation of
retention of the highest three. This would appear arbitrary,
grill sampling, no analytical explanation has ever but experience shows that it is sufficient to produce a
appeared to explain why the method has proved to be valid index average.
empirically sound, has been accepted, and is widely Perhaps for greater clarity in explaining the
used.
procedure, one might more easily compare it with
The chance now to explain the grill sampling system sampling a human population in a given community.
in light of the newer concepts of fuzzy logic has been The procedure would be to count the number of people
most compelling. The mystery can now be clarified, at each of their principal points of aggregation in town

fifty years after the development of its methodology. within a short period of time, with careful consideration

The author has come to this conclusion with all the of weather, and at a time when the population is free of
confidence of a" Eureka" event, born of his experience constraints, such the workday imposes. Selected for
in cognition studies during the last twelve years, and counting would be the number of persons at the most
more
directly to the point, in coming to a recent clearer popular athletic game in town, or the number seated in
understanding of " fuzzy logic" that provides a very the largest theater for a leading performance, or the
good fit for the sampling enigma of the ever- changing dense crowd listening to an important speaker in the
fly population. Pawlak( 1992) states: public park, or the number in a train or bus station at train
The basic idea of rough set theory consists in or bus time. The purpose would be to find at least the

replacing vague concepts with a pair of precise highest three concurrent population aggregations of a
concepts ( so that classical set theory can be limited or definable population, such as one town. The
applied. This is called lower and upper physical size of the locale counted should not be the
approximation. . . With each vague concept a limiting factor; in other words the numbers should not
boundary region is associated, which consists be constrained by the site, but rather by the choice of the
of all objects that cannot be placed clearly persons attending.
within the concept... The" size" of the boundary Only the highest three numbers are used to produce
region can be used as a measure of vagueness the index. Whatever averaging is chosen should be
of the vague concept.( The greater the boundary consistently used for the same class of situation. The
region, the more vague is the concept; precise central idea is to determine the average largest number
concepts do not have
boundary regions at all.) of individuals the population delivers to its most choice
Obviously the boundary region is the difference places of aggregation at peak times, and only at those
between theupperandlowerapproximationof peaks. This is what the index number determined by
the concept." grill sampling is, an approximate number, the upper
fuzzy set boundary, derived as a function of the size of
THE SAMPLING PROCEDURE the uncounted base population, sampled at optimal
points in its most gregarious or contagious behavior.
For each premise or localized situation to be
evaluated, the resident fly population, consisting of CHARACTER OF THE GRILL INDEX
several groups of differing sizes and numbers of flies,
is first carefully located and its several aggregations are
To speak figuratively, one is trying to determine
noted throughout its range, which centers around all the the elevation of a mountain
top by taking several
170 Journal of Vector Ecology December, 1996

measurements of what are conceived as minor variations

A very broad range of significance has been
in configuration at its Exactness the figures is
peak. of
determined for grill indices relative to very diverse
not the critical point, but rather the consistency in the situations, depending upon a variety of public acceptance
manner of securing the average or index datum. A and public health expectations. For each class of situation
parallel assumption might be one that the mountain has the grill readings found satisfactory for control are the
a given slope of repose, and therefore its volume is following upper approximations, above which is the
derivable solely from the rounded- off peak figure.
boundary region considered as exemplifying inadequate
If one again uses the example of human populations control. The lower approximation becomes the level
illustration, then consider that
as an a
city with maximum below which control activity is either discontinued or
local residential densities of 25 houses( say 60 persons) put on a standby status. Each of these paired levels is
per block, must be a small village, while one with 40 or now proposed as the upper and lower levels of a rough
more story condominia, like Victoria in Hong Kong, fuzzy set as defined by Pawlak( 1992). Some sample
must have very many thousands. Though this is not an upper level indices which have proved useful for
activity index, it is still reflective of gregariousness, and
maintenance of fly control are:
shows the lack of any immediate relationship of the
linearity of physical space to density of occurrence of Restaurant kitchen 2 flies
the life form. Residential back yard 2- 3
One arrives at a reasonably valid assumption that City block 5
the maxima in the aggregation patterns of a gregarious
Milking parlor 15
population are
collectively a function of the total General farm 20
population which produces them, up to discernible
physical limitations of the immediate environment. When the index rises above the standard given
above for a specified area, field inspections and control
INDEX INTERPRETATION measures should respond. Field use has verified that
this is far more sensitive than ordinary visual inspection,
The next step is interpreting the significance
one of
so that by using effective control measures guided by
and the fly index for
utility of grill each situation, or
grill indices, one may manage a control program very
community being sampled, for the purpose of a sensitive
accurately to the particular standard selected, avoiding
assessment and management of a fly control program.
any perceptible outbreaks, if the sampling covers a large
In this step
very broad range of significance is
a enough control area, provided some unusual event does
determined for fly grill indices in diverse situations.
not intervene, such as wind blowing an outside population
One establishes an acceptable upper level for the presence into the control area. This event is uncommon, as the
of flies in each human is
contact situation, and able with
house fly is unusually very domiciliary, and tends to
grill indices to fine- tune control measures to meet the stay within its small universe unless its population
desired standards, whether one is the level of nuisance
overflows, or winds become unusual; in strong contrast
within a food or disposal industry, a public social to the green and blue- bottle flies that are scavengers
gathering indoors or outdoors, or a disease transmission ranging over large territories and are not gregarious.
situation, actual or potential. This is accordingly the
upper fuzzy set
boundary," above which active control THE ROUGH FUZZY SET
measures are immediately invoked.
The diversity of acceptance of flies in the
The fly grill indices are now being proposed in this
environment would run from none for a microbiologist
paper as the parameters of" rough fuzzy sets," interpreted
in a laboratory or a couple
dining in an good restaurant, as good control even if the upper approximate level of
to very few for a family at home depending on their control connotes rather different but very practical
social level, to
progressively more in each situation for concepts of tolerance for a fly population. Used for the
a family on a picnic, a shopper in an open public market,
past fifty years as a derivative of a very unorthodox
a farmer on his ranch, an employee in a food- processing
algorithm, it has functioned effectively in evaluating the
plant, or an employee in a garbage dump. Grill indices significance of maximum gregariousness of given house
have proved very constant over a few days time when
fly populations at points in time, in each localized area
taken under comparable conditions of weather.
They of the ecology in which that population moves freely
have proved dependable, and are accepted as essential
about, whether it is a home, dairy, chicken ranch,
for evaluation and maintenance of fly control programs restaurant, or city block. This datum, a simple average
at whatever level. of population maxima, represents at one point in time
December, 1996 Journal of Vector Ecology 171

the highest level of discernible gregarious activity which cow just mentioned, since one plant might be very
the locally resident fly population produces. This figure heavily infested, while an adjacent one might be nearly
has been found so reliable as a measure of population free of mites. I suggest that for contagious distributions,
level and so sensitive to change that it has been used the preoccupation with linear equivalence of the substrate

successfully to guide fly control programs. for the event or for the life form creates a parameter of
In 1946 in the lower Rio Grande Valley, the little or no value in analysis. Consider a computation of
transmission epidemiology of endemic bacillary the average number of persons per square mile, as a

dysentery was found to show a good correlation with the randomized nonvalue. For example, using California,
community fly grill indices ( Watt and Lindsay 1948, how can one rationally contemplate a per square mile
Lindsay et al. 1953). This verification, that the level of calculation which would combine 20,000 or more persons

fly- vector populations was in fact being accurately in a square mile of downtown Los Angeles with vast
measured, supported the author' s search for basic factors areas of zero in a California desert, yielding a very low
which would explain the empirical. A general discussion and meaningless figure for the entire state?
of flies and disease is given in Lindsay and Scudder Another window of understanding for this problem
1956). can be illustrated by the following: a measure of a
contagious distribution is nothing new to hunter-
AN ALIEN PARADIGM gatherers and the hunted, whether man or other animals,
whose survival has long been tied to searching carefully
In general the gregarious behavior of populations and successfully for the prime locations of food/prey/
of many kinds was qualified as contagious by Neyman enemies, and calculating the search/ avoidance effort
1939) and more extensively by Neyman and Scott needed on the basis of a conservative comparison of the
1959). Flies, human beings, and cosmic matter all searched area to past experience. Is this not in each such
exhibit " contagious distributions," which commonly case, a rough fuzzy set?
disregard linear factors of the substrate upon, or medium I offer fly grill data collection and interpretation as
in which,
they are distributed, and cannot therefore be a very real candidate for inclusion in rough fuzzy logic
sampled
by conventional systems. Very commonly, sets, essentially the same as survival calculations.
such populations are counted in full, instead of counted Variations in even the same perceived information

by sampling some aliquot. means a variety of valid conclusions to different

With respect to a plague research project, in which perceivers, interpretable in each instance by the
the investigator wished to make counts of the number of experience/ need of the hunter/ hunted. The innate hunter-
fleas on domestic rats, the difference in flea infestation gatherer/ hunted instincts have long been validated by
levels between individual rats in the same colony was successful survival levels because they are successful
found to be most extreme, to the point of very major for species after species. Is any other basis ofexplanation
differences between counts on cohabiting animals. The needed as a workable theory of what actually operates?
investigator quite properly felt that derivation of an Some years after the beginning of extensive fly
average number of fleas per rat was meaningless and grill sampling, those doing fly control studies at the
could not be a usable parameter. A contagious Savannah Technical Development Laboratories of
distribution analysis on the fly model was offered, but CDC were able to show that an experienced field team
turned down by statisticians. was capable of visually estimating fly population data
Quite comparable to the flea/ rat picture is one without using the grill. They had learned their
easily observed on individual cattle in a pasture. One observational discipline so well that their field estimates
animal may have dozens of horn flies while an adjacent could be verified by another team using the grill( Welch
animal may have them by the thousands. One may easily and Schoof 1953). This successful experiment in data
conclude that the hosts have comparatively little gathering was conducted after the team had two years of
ecological equivalence, one to another, so that any experience in grill sampling. Certainly this experience
sampling validity must be based upon factors more supports the analogy of the " hunter-gatherer/hunted"
consistent with representative ecological values than concept, and would justify the rough fuzzy logic nature
the simple assumption that all animals are so equal that of the data.
random
sampling of them is valid. In conclusion, this discussion proposes that the
Another project was the study of red spider mites on history and acceptance of the fly grill for determining fly
roses in a greenhouse, where the investigator wished to population indices may be a valid interpretation of
evaluate populations and control measures. The finding contagious distributions by their analyses as rough
was also one of major host difference, like the rat or the fuzzy logic sets. A corollary would be that classical
172 Journal of Vector Ecology December, 1996

statistical methods are not applicable to such data. A

moderate morbidity. Pub. Hlth. Rpt. 68: 361- 367.
prospect would seem to exist that
many additional data McGuire, J. U. and D. R. Lindsay. 1950. Considerations
complexes in natural history would be better analyzed
in sampling fly populations. CDC, PHS, DHEW,
by methods comparable to grill
sampling, which Atlanta, GA Bull. 5: 31- 35.
deliberately samples the most populated points in a Neyman, J. 1939. On a new class of " contagious
distribution.
distributions, applicable in entomology and
The author would invite comment this
on
bacteriology. Ann. Math. Stat. 10: 35- 57.
interpretation, inclusive of a wide range of questions
Neyman, J. and E. L. Scott. 1959. Stochastic models of
and ideas. So far this may be the first basic explanation population dynamics. Science 130: 303- 308.
to appear in all these years of successful use of the grill Pawlak, Z. 1992. Rough sets: a new approach to
method.
vagueness. Pp. 105- 118 in Fuzzy Logic for the
Management of Uncertainty ( Zadeh, Loffia,

REFERENCES CITED Kaeprzyk, and Janusz, John Sons,

eds.).
Wiley&
New York, 675 pp.
Barclay' s California Code of Regulations. 1992. Title Ricciardi, J. and E. Paulini. 1955. Normas gerais para
14: Natural Resources. California Integrated Waste
determina ao da densidade de Musca domestica em
Management Board, Section 17682- Cover, and uma localidade. Rev. Brasileira de Malariologia e
Section 17683- Performance Standards, pp. 734-
Doenas Trop. 7: 93- 101.
735.
Schoof, H. F. 1955. Survey and appraisal methods for
Dhillon, M. S. and G. L. Challet. 1985. The evaluation community fly control programs. Public Health
of three
sampling techniques for the determination Monogram No. 33. Public Health Service
of
fly( Diptera) densities at four sanitary landfills in Publication No. 443. USDHEW, Public Health
southern California. Bull. Soc. Vect. Ecol. 10: 36- Service, Washington, DC. IV+ 20 pp.
40.
Scudder, H. I. 1947. A new technique for sampling the
Ehlers, V. M. and E. W. Steel. 1965. Municipal and
density of housefly populations. Pub. Hlth. Rpts.
Rural Sanitation, 6th Pp. 326- 328 and figs. 9-
ed, 62: 681- 686.
17. McGraw- Hill Book Company, New York, 663 Watt, J. D. R.
and
Lindsay. 1948. Diarrhea] disease
pp. control studies. I. Effect of fly control in a high
Holway, R. T., W. A. Mitchell, and A. A. Sal- ah. 1951. morbidity area. Pub. Hlth. Rpts. 63: 1319- 1334.
Studies on the seasonal prevalence and dispersal
Welch, S. F. and H. F. Schoof. 1953. The reliability of
of the Egyptian house fly ( Musca domestica). I.
surveys in evaluating fly densities for
visual"

The adult flies. Ann. Entomol. Soc. Am. 44: 381-

community control programs. Am. J. Trop. Med.
398.
Hyg. 2: 1131- 1136.
Lindsay, D. R. and H. I. Scudder. 1956. Nonbiting flies West, L. S. 1951. The Housefly, its Natural History,
and disease. Ann. Rev. Entomol. 1: 323- 346. Medical Importance, Control.
and
Pp. 420-422
Lindsay, D. R., W. H. Stewart, and J. Watt. 1953. Effect and figs. 140- 141. Comstock Publishing Company,
of fly control on diarrheal disease in an area of Ithaca, New York, 584 pp.
Journal of Vector Ecology 21( 2): 173- 177

Bacterial Abundance in Larval Habitats of Aedes albopictus

Diptera: Culicidae) in a Florida Cemetery

Edward D. Walker', George F. O' Meara2, and William T. Morgan'

Department of Entomology, Michigan State University, East Lansing, MI 48824

2Florida Medical Entomology Laboratory, Institute of Food and Agricultural
Sciences, University of Florida, 200 9th Street SE, Vero Beach, FL 32962

Received 23 February 1996; Accepted 20 May 1996

ABSTRACT: Bacterial abundance in bronze and non- bronze containers was studied in relation to
distribution of immature Aedes albopictus( Skuse) mosquitoes in a cemetery in northern Florida. In non-
bronze containers, immature Ae. albopictus occurred at higher density and greater frequency than in bronze
containers, yet bacteria were more abundant in water from bronze containers( range, 2. 59 x 106/ ml to 2. 25
10A/ 105/
x ml) than in water from non- bronze containers ( range, 8. 01 x ml to 5. 57 x 10'/ ml). Thus, the

hypothesis that chemicals such as copper would leach from the bronze, kill bacteria, and thereby eliminate
the larval food supply was not supported. Other factors affecting skewed distribution of larvae away from
bronze containers need to be studied.

Keyword Index: Aedes albopictus, larvae, bacteria, container habitats

INTRODUCTION aegypti and Ae. triseriatus larvae in larval habitats

Livdahl and Willey 1991, Juliano 1994). However, it
Intra- and interspecific competition for food among is not known how food resource abundance and
mosquito larvae in container habitats is an important distribution affects these interactions, and the resulting
developmental patterns of distribution of Ae. albopictus. Recently,
process affecting larval growth, success,

and population regulation ( see, for example, relevant O' Meara et al. ( 1992a, 1992b) observed that Ae.
papers in Frank and Lounibos 1983, and Lounibos et al. albopictus and Ae. aegypti distributions in water- filled
1985). Consequently, studies that incorporate analysis containers in cemeteries in Florida were skewed away
of the nature, distribution, and abundance of larval food from bronze containers. These authors hypothesized
can offer insights into the patterns of larval mosquito that the copper in the bronze vases may be directly
abundance observed in natural settings. For example, toxic to larvae, or to microorganisms that serve as
supplementation of larval habitats with plant detritus larval food ( Merritt et al. 1992). Therefore, in this
improved growth of Aedes triseriatus( Say) mosquitoes study we investigated the abundance of bacteria, one
in container habitats in south Florida( Lounibos et al. component of larval food ( Walker et al. 1988, Merritt
1993), and total mass of organic material in tree holes et al. 1992), in relation to distribution and abundance
was correlated with total numbers of Aedes sierrensis of Ae. albopictus and other mosquitoes in bronze and
Ludlow emerging from tree holes in northern California other types of containers in a cemetery in Florida, to
Colwell et al. 1995). determine if bacterial densities were lower in the bronze
The invasion and establishment of Aedes albopictus containers.

Skuse) in Florida has attracted considerable attention

because it apparently has displaced Aedes aegypti( L.) MATERIALS AND METHODS
in certain landscapes with various types of artificial
containers, particularly in northern Florida( O' Meara et The study site was a cemetery in Jacksonville,
al. 1995). The mechanisms by which Ae. albopictus Florida, that has been described elsewhere( O' Meara et
invades and displaces Ae. aegypti are not clear, but they al. 1992a). Many of the grave sites have water-holding
may involve superior competitive interactions with Ae. containers including those made of plastic, stone, and
 174 Journal of Vector Ecology December, 1996

bronze. Thirty bronze containers and thirty containers examined, number of bacteria counted, filter area, and
of one of the other materials(` non- bronze') were sampled field area.
as follows, on August 5, 1992. A water sample was
The number of Aedes larvae per container, number
drawn with a sterile syringe and preserved as4% formalin of larvae in
per ml of water containers ( i. e., larval
to a final volume of 5 ml. Then, the entire contents of the
density), container volume, and bacterial density( number
container were emptied with a siphon, the volume of
of bacteria per ml) were compared among bronze and
water from each container measured, andAedes mosquito
non- bronze containers using either the unpaired t-test or
larvae and pupae retained for counting. On four previous the nonparametric Mann- Whitney U- test. Bacterial
occasions in 1990 and in 1991, containers in this cemetery
for immature Aedes.
density data were transformed with log10 prior to
were sampled At those times,
statistical analysis. The frequency of containers with or
among the Aedes- positive containers, nearly all ( 209/ without larvae, between the two container types, was
211) had immature Ae. albopictus, while Ae. aegypti
compared with a 2 x 2 contingency table for test of
was found in less than 5% of the samples( 6/ 211). In the independence with the G statistic. Correlation of larval
current study, a smaller portion of the total collection
density and log1o( bacterial density) was examined with
21 immatures from 6 bronze containers and 98 the correlation coefficient( Sokal and Rohlf 1969).
immatures from 11 non- bronze containers) were
identified to species. RESULTS
Bacteria in water samples were enumerated using a
direct count method after
staining of bacterial DNA Nine of 30( 30%) of bronze containers held Aedes
with 4' 6 diamidino- 2- phenylindole( DAPI)( Porter and larvae, while 30 of 30( 100%) of non- bronze containers
Feig 1980, Walker et al. 1988). Subsamples of the held Aedes larvae. All specimens identified to species
original water sample were exposed to DAPI in solution
were Ae. albopictus. There were significantly fewer
final concentration of 2- 4 pg/ ml) and held for 15- 30 bronze containers with larvae compared to non- bronze
min. on ice in the dark. The sample was then transferred
containers( G- test on 2 x 2 contingency table, G= 41. 0,
to the glass chimney ( 17 mm diam.) of a filtering P< 0. 001).
apparatus, and the sample drawn by low vacuum pressure
TABLE 1 shows summarized data by container
30 cm Hg) onto a black, polycarbonate NucleporeTM type, of water volume, numbers of larvae recovered per
filter ( 0. 22 pm pore size, 25 mm diam.), thus leaving container, larval density, bacterial density in con-
particulate material of bacterial size and larger on the
tainers, and the results of t-tests or Mann- Whitney U-
filter surface. The Nuclepore filter was backed with a tests. Bronze containers held more water on average
0.45 pm pore size, HA- type Millipore filter to provide than did non- bronze containers, but harbored fewer
even pressure to eliminate
clumping of material. After larvae per container and had a much lower larval density
filtration, Nuclepore filters were removed from the
than did non- bronze containers. Bacterial density was
holder and placed on slides
previously prepared with a significantly higher ( t-test on log10- transformed data;
thin film of type B Cargille immersion oil. A drop of oil see TABLE 1) in water of bronze containers( range, 2. 59
was added to the top of the filter and a cover slip x 106/ ml to 2. 25 x 10"/ ml) than in water of non- bronze
applied. Slides were stored in the dark at 4° C until
containers( range, 8. 01 x 105/ ml to 5. 57 x 10'/ m1). There
examined.

Bacteria
was no correlation between larval density and login
were enumerated
by exciting the DAPI- ( bacterial density) for bronze( r= 0.08, P> 0.05) or non-
bacterial DNA complex with 365 nm wavelength of bronze( r= 0. 149, P> 0. 05) containers.
ultraviolet light using a JenaLumar A/ D fluorescence
microscope in the epifluorescent mode. Individual DISCUSSION
bacteria appear bright blue against a black background
in such preparations. Counts of bacteria were performed In this study, Aedes larvae were more common in
at 1, 000 times magnification using an ocular grid, until non- bronze containers. These observations confirm the
a minimum of 200 bacteria least
were counted
among at earlier finding
by O' Meara et al. ( 1992a, 1992b) that
15 different randomly- chosen fields on the filter mosquito larvae were less common and less abundant in
preparation, to satisfy statistical assumptions of
bronze vessels compared to non- bronze vessels.
estimation of bacterial numbers( Kirchman et al. 1982). Although the reason for this difference is not known,
Bacterial density ( i. e., numbers of bacteria per ml of one
possibility ( discussedby O' Meara et al. [ 1992a,
original water sample) were calculated from the counts 1992b]) is that copper ions from the bronze metal leach
using a standard formula that accounts for sample into the water and accumulate to a sufficient
volume, dilution owing to formalin, number of fields concentration that is toxic to mosquito larvae.
December, 1996 Journal of Vector Ecology 175

TABLE 1. Water volume, number of Aedes larvae, larval density per ml, and bacterial density per ml in
bronze and non-bronze containers at the Evergreen Cemetery, Jacksonville, Florida, August
5, 1992. N= 30 containers for each group. **, P< 0.01, ***, P< 0. 001.

Container Type

Variable Bronze Non- bronze Statistical test

Water volume( ml)

Mean 633. 3 382. 8 t= 4. 77***
Range 350- 900 10- 1000
SEM 5. 9 7. 6

No. of larvae
Mean 8. 8 95. 5 U= 57. 5***
Range 0- 88 5 - 462
SEM 3. 8 17. 7

Larval density ( per ml)

Mean 0. 01 0. 35 U= 33. 5***
Range 0.00- 0. 18 0.02- 1. 55
SEM 0. 007 0.070

Bacterial density( per ml)

Mean 2. 66 x 10' 1. 13 x 10' t= 2. 87**
106- 108
Range 2. 59 x 2. 25 x 8. 01 x 105- 5. 57 x 10'
106
SEM 7. 70 x 2. 51 x 106

An alternative hypothesis is that copper ions are studies, we have demonstrated that larval feeding by Ae.
toxic to the microorganisms, such as bacteria, that form triseriatus reduces microbial densities in field and
the larval food( Walker et al. 1988, Merritt et al. 1992, laboratory microcosms ( Walker et al. 1991), whereas

Sota and Kato 1994). Copper compounds, such as selective removal of Ae. triseriatus larvae from tree
copper sulfate, are often used to control unwanted holes and tires is followed rapidly by increases in
aquatic, vascular plants and algae in recreational lakes microbial densities( Morgan and Merritt 1992; R. Merritt,
and other bodies of water( Ware 1989). However, the W. Morgan, and E. Walker, unpublished data). Copper
effect of copper ions on bacteria in containerized habitats may also be toxic to bacteria adhering to the inner
of mosquito larvae is not known. Here, we found that surface of the bronze containers, or to larvae that ingest
bacteria were more abundant in bronze than in non- copper as they feed. Aedes larvae browse underwater
bronze containers, thus we must reject our hypothesis surfaces as part of their normal feeding behavior
that the skewed distribution of Aedes larvae in bronze ( Khawaledetal. 1988, Walker and Merritt 1991, Juliano
and non- bronze containers is mediated through a toxic and Remminger 1992).
effect of copper on the microorganisms forming the O' Meara et al.( 1992a) noted that organic material,
larval food. Although the ranges of bacterial densities such as leaves and flower parts, accumulate in containers
in larval habitats of the two container types overlapped, in cemeteries, particularly after holidays, and that such
yet the higher densities of bacteria observed in water of material may form a food supply for larval growth( cf.
bronze containers could be related to the absence, or Barrera- Rodriguez et al. 1979, Lounibos et al. 1993).
much lower densities, of mosquito larvae in those Such detritus aggregates cannot, in general, be used
containers compared to non- bronze ones. Although directly by mosquito larvae, but must be mineralized
there was no correlation between larval density and and transformed through decomposition processes by
bacterial density in non- bronze containers here, yet we microorganisms( Cummins and Klug 1979, Bengtsson
did not take into account variation in larval stage in the 1992) which then become food items directly( Walker
samples which could account for this result. In controlled et al. 1988). Whether detritus decomposition is inhibited
176 Journal of Vector Ecology December, 1996

or altered in bronze containers compared to other ones Bull. W.H. O. 44: 643- 649.
is not known, however, larval mosquito growth was Colwell, A. E., D. L. Woodward, and N. L. Anderson.
inhibited in bronze containers even when they were
1995. Environmental factors affecting the western
supplemented with a lab chow( O' Meara et al. 1992b). treehole mosquito ( Aedes sierrensis). Northwest
Our study provides information on the variation in Science 69: 151- 162.
abundance of larval mosquito food in the
setting where Cummins, K. W. and M. J. Klug. 1979. Feeding ecology
displacement of Ae. aegypti by Ae. albopictus has of stream invertebrates. Annu. Rev. Ecol. Syst. 10:
apparently occurred. One hypothesis regarding this 147- 172.
species displacement phenomenon would suggest that Frank, J. H. and L. P. Lounibos, eds. 1983. Phytotelmata:
Ae. albopictus larvae outcompeteAe. aegypti larvae for terrestrial plants as hosts for aquatic insect com-
food resources, or are more efficient in utilizing available
munities. Plexus, Medford, New Jersey, USA, 293
food resources( Juliano 1994). Investigations of larval pp.
food of Ae. aegypti and Ae. albopictus are scant. We Ho, B. C., A. Ewert, and L. Chew. 1989. Interspecific
note that most of the published experimental studies
competition among Aedes aegypti, Ae. albopictus,
e.g., Black et al. 1989, Chan et al. 1971, Ho et al. 1989) and Ae. triseriatus ( Diptera: Culicidae): larval
that have examined competitive outcomes have utilized development in mixed cultures. J. Med. Entomol.
laboratory mosquito chow as food, despite recent 26: 615- 623.
evidence that Ae. albopictus and Ae. aegypti larvae Juliano, S. A. 1994. Interspecific competition, apparent
grow when provided cultures of bacteria ( Sota and competition, and the replacement of Aedes aegypti
Kato 1994). Thus, further studies on the nature of
by Aedes albopictus: a field experiment with
larval mosquito food are needed to the role of
clarify container- dwelling mosquitoes. Bull. Ecological
larval nutrition and
efficiency of utilization of food by Soc. Am. 75: 125- 126( Suppl). ( Abstract).
larvae of different species in these competitive
Juliano, S. A. andL. Remminger. 1992. The relationship
interactions.
between vulnerability to predation and behavior of
larval treehole mosquitoes: geographic and
Acknowledgments ontogenetic differences. Oikos 63: 465- 476.
Khawaled, K., Z. Barak, and A. Zaritsky. 1988. Feeding
We thank Len Evans, of the Florida Medical
behavior of Aedes aegypti larvae and toxicity of
Entomology Laboratory, for assistance with field dispersed and of naturally encapsulated Bacillus
sampling at the Evergreen Cemetery. This study was thuringiensis var. israelensis. J. Invertebr. Pathol.
supported in part by contracts from the Florida 52: 419- 426.
Department of Health and Rehabilitative Services, and Kirchman, D., J. Sigda, R. Kapuscinski, and R. Mitchell.
by NIH grant AI21884. 1982. Statistical analysis of the direct count method
for enumerating bacteria. Appl. Environ. Microbiol.
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Barrera- Rodriguez, R., C. E. Machado- Allison, and L. invasion: competition between Aedes albopictus
A. Bulla. 1979. Criaderos, densidad larval y and native Aedes triseriatus. Science 253: 189-
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Culex fatigans Wied., C. Theo. y Aedes
corniger
Lounibos, L. P., J. R. Rey, and J. H. Frank, eds. 1985.
aegypti L.) en el cemeterio de Caracas. Acta Ecology of mosquitoes: proceedings of a workshop.
Cientifica Venezolana 30: 418- 424.
Florida Medical Entomology Laboratory, Vero
Bengtsson, G. 1992. Interactions between fungi, bacteria,
Beach, Florida, USA, 579 pp.
and beech leaves in a stream microcosm. Oecologia Lounibos, L. P., N. Nishimura, and R. L. Escher. 1993.
89: 542- 549. Fitness of a treehole mosquito: influences of food
Black, W. C., IV, K. S. Rai, B. J. Turco, and D. C. type and predation. Oikos 66: 114- 118.
Arroyo. 1989. Laboratory
study of competition Merritt, R. W., R. H. Dadd, and E. D. Walker. 1992.
between United States strains of Aedes albopictus Feeding behavior, natural food, and nutritional
and Aedes aegypti ( Diptera: Culicidae). J. Med. relationships of larval mosquitoes. Annu. Rev.
Entomol. 26: 260- 271. Entomol. 37: 349- 376.
Chan, K. L., Y. C. Chan, and B. C. Ho. 1971. Aedes
Morgan, W. T. and R. W. Merritt. 1992. Conspicuous by
aegypti ( L.) and Aedes albopictus ( Skuse) in absence? Protozoa in the diet of Aedes triseriatus.
Singapore City. 4. Competition between species. Vector Control Bull. North Central States 1: 80.
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O' Meara, G. F., A. D. Gettman, L. F. Evans, Jr., and F. mosquito larvae Aedes ( Stegomyia) ( Diptera:
D. Scheel. 1992a. Invasion of cemeteries in Culicidae): preliminary experiments with
Florida by Aedes albopictus. J. Am. Mosq. Control Pseudomonas fluorescens. Appl. Entomol. Zool.
Assoc. 8: 1- 10. 29: 598- 600.
O' Meara, G. F., A. D. Gettman, L. F. Evans, Jr., and F. Walker, E. D., E. J. Olds, and R. W. Merritt. 1988. Gut
D. Scheel. 1992b. Reduced mosquito production content analysis of mosquito larvae ( Diptera:
in cemetery vases with copper liners. J. Am. Mosq. Culicidae) using DAPI stain and epifluoresence
Control Assoc. 8: 419- 420. microscopy. J. Med. Entomol. 25: 551- 554.
O' Meara, G. F., L. F. Evans, Jr., A. D. Gettman, and J. Walker, E. D., D. L. Lawson, R. W. Merritt, W. T.
P. Cuda. 1995. Spread of Aedes albopictus and
Morgan, and M. J. Klug. 1991. Nutrient dynamics,
decline ofAe. aegypti( Diptera: Culicidae) in Florida. bacterial populations, and mosquito productivity in
J. Med. Entomol. 32: 554- 562. tree hole ecosystems and microcosms. Ecology 72:
Porter, K. G. and Y. S. Fieg. 1980. The use of DAPI for 1529- 1546.

identifying and
counting aquatic microflora. Walker, E. D. and R. W. Merritt. 1991. Behavior of
Limnol. Oceanogr. 25: 943- 948. larval Aedes triseriatus ( Diptera: Culicidae). J.
Sokal, R. R. and F. J. Rohlf. 1969. Biometry. W. H. Med. Entomol. 28: 581- 589.
Freeman, San Francisco, USA, 776 pp. Ware, G. W. 1989. The pesticide book, 3rd edition.
Sota, [Link] K. Kato. 1994. Bacteria as diet for the Thomson Publications. Fresno, CA, 340 pp.
Journal of Vector Ecology 21( 2): 178- 185

Interrelationships between Different Borrelia

Genospecies and Their Principal Vectors

E. I. Korenberg' and G. G. Moskvitina'

Laboratory of Vectors of Infections and Center on Borrelioses for Russia,

Gamaleya Institute for Epidemiology and Microbiology, Russian Academy
of Medical Sciences, 18 Gamaleya Street, Moscow, 123098 RUSSIA

Received 23 February 1996; Accepted 11 July 1996

ABSTRACT: Under conditions of similar prevalence of Borrelia infection in tick populations, a

generalized infection with the presence of spirochetes in the salivary glands develops much more
frequently in the unfed adult Ixodes persulcatus ticks, infected by Borrelia afzelii and Borrelia garinii in
Russian natural foci, than in [Link] ticks from the northeastern United States, infected with Borrelia
burgdorferi sensu stricto. During the first two to three days after infected [Link] begin to engorge,
the proportion of ticks with spirochetes in the salivary glands did not increase. In [Link], therefore,
Borrelia migration from the gut into the salivary glands at the beginning of the blood- sucking phase is not
a necessary or even important condition for Borrelia transmission with saliva. These data provide evidence
for different interrelationships between particular Borrelia genospecies and their principal tick vectors.

Keyword Index: Borrelia burgdorferi sensu lato, vectors, ticks, generalized infection.

INTRODUCTION garinii and Borrelia afzelii spirochetes are transmitted

by adult Ixodes persulcatus ticks, the disease is
Borrelia causing Lyme borreliosis are transmitted manifested in almost 90%
clinically of cases even
to vertebrates, including humans, in the saliva of tick though the period of tick attachment to the patient' s
vectors( Ribeiro et al. 1987, Zung et al. 1989). However,
body is usually less than one day ( Korenberg et al.
the Ixodes
salivary glands of unfed adult scapularis 1994). The facts described above allowed us to suggest
ticks, the main vectors of Borrelia burgdorferi sensu that interrelations between particular Borrelia
stricto,
rarely contain spirochetes; the latter are usually
genospecies and their main vectors are characterized by
found in the gut of infected ticks ( Burgdorfer 1984,
a certain individuality. The latter may account for
1989a, 1992; Burgdorfer 1988). Hence,
et al. some
differences in the frequency of generalized infection
researchers questioned the very possibility of (
accompanied by the presence of Borrelia in the
inoculation of the Lyme borreliosis pathogen with salivary glands of unfed ticks), in the fate of Borrelia in
saliva of Ixodid ticks( Benach et al. 1987). Although this
the tick gut after the initiation of blood-feeding, and in
mode of Borrelia transmission is now beyond question,
the probability of their penetration into the salivary
experiments have demonstrated that the tick must be glands
during this period ( Korenberg 1994).
attached to the host for at least 48 h for successful Consequently, different pathogens of Ixodid tick-borne
inoculation to occur. During this period, spirochetes borrelioses ( ITBBs), which we regard as a group of
migrate from the into the salivary glands where they
gut
etiologically individual infectious diseases( Korenberg
accumulate ( Piesman et al. 1987, 1991; Ribeiro et al. 1993a, b, 1994), should have certain peculiarities of
1987; Burgdorfer 1989b; Piesman 1993, 1994). their horizontal and vertical transmission. In this
Therefore, the transmission of B. burgdorferi Gem ( 1994), it is important to
sensu context, as noted
by
stricto during the first two days after the I. scapularis demonstrate whether the development of different
tick attachment to the host body is unlikely, and the Borrelia genospecies in I. persulcatus and I. ricinus
prompt removal of attached ticks reduces risk of Lyme ticks is similar to that of B. burgdorferi sensu stricto in
borreliosis( Piesman et al. 1987, 1991). I. scapularis ticks.
On the other hand, in Russia, where Borrelia In this paper, we describe the results of our studies
December, 1996 Journal of Vector Ecology 179

aimed at revealing generalized infection in unfed adult ( 1967). Briefly, ticks were half-embedded into a
1. persulcatus ticks from Russian natural foci. These paraffin wax- rosin mixture, washed with ethanol and

results are compared with corresponding data on I. saline, and dissected under a binocular microscope by
scapularis from the USA. In addition, we analyzed cutting the cuticle at the sides of the body. Isolated gut,
parameters of infection in [Link] ticks during the salivary glands, ganglia, and gonads were used for
first days after the initiation of blood- feeding. preparing smears on glass slides. We made two
preparations of each organ and used the second

MATERIALS AND METHODS reserve) preparation for better resolving any ambi-
guous cases. Preparations were air-dried and fixed in
Unfed adult ticks were collected from vegetation flame, stained with the Romanovsky- Giemsa mixture
in two ITBB foci located in the European crystal violet ( Kovalevskii et al. 1988), and
by flagging and

part of Russia and in one focus located in the analyzed under a light microscope with oil immersion at
northeastern United States. magnification of 1125x. After detecting spirochetes in
In May and June of 1992, I. persulcatus ticks were
the salivary glands, we always analyzed preparations of
collected in the natural focus located in the Kirovskii ganglia and reproductive organs of the same ticks to

district( Leningrad region of Russia). In this focus, des- confirm generalized infection.
cribed in detail previously ( Korenberg et al. 1991 a, b), In another group of partially fed ticks, screening for
the average prevalence of infection in I. persulcatus is Borrelia was performed using only the gut contents.
about 34% ( Kovalevskii et al. 1993). Spirochetes Standard live preparations were made as described by
isolated from ticks were identified as B. afzelii and B. Kovalevskii et al. ( 1991) and analyzed under a dark-
garinii( Baranton et al. 1992, Canica et al. 1993). field microscope with water immersion at a mag-
Ticks of the same species were also collected in nification of 600x ( 40x1. 5x10). Both methods for

May and June of 1992- 1993 in the suburbs of Perm' revealing Borrelia produce similar results( Kovalevskii
near the Ural Mountains), where B. garinii and B. et al. 1988).
afzelii circulate enzootically ( Korenberg et al. 1994), In smears and live preparations, all Borrelia in 250
and the prevalence of infection in ticks is about 38%. microscopic fields were counted. The concentration of
Ixodes scapularis ticks were collected in October spirochetes was expressed as counts per 100 micro-
of 1993, in the Lyme borreliosis focus located near scopic fields. These values were recalculated with
Ipswich ( eastern Massachusetts), which has been respect to differences in magnification and assessed
documented in previous publications ( Spielman et al. using the following grades of Borrelia abundance
1985, Lastavica et al. 1989). Borrelia burgdorferi sensu ( concentration in a particular organ), which were

stricto circulates there, as it does throughout the proposed previously for fixed preparations analyzed at
northeastern states ( Baranton et al. 1992), and the a magnification of 700x: low, 0. 1- 5. 0; medium, 5. 1-
prevalence of infection in adult ticks reaches 38% 25. 0; high, 25. 1- 125. 0; and very high, more than 125
Piesman et al. 1986). spirochetes per 100 microscopic fields ( Levin et al.,
Some I. persulcatus ticks from the Leningrad 1993). In live preparations, corresponding values were
region were pooled into groups with
approximately 0. 1- 10, 10. 1- 50.0, 50. 1- 250. 0, and more than 250
equal proportions of males and females ( to stimulate spirochetes per 100 microscopic fields, respectively
blood- feeding) and fed on white mice placed under ( Kovalevskii et al. 1991).
individual " hoods," according to the approved Calculated mean values and percentages were
procedure. Partially fed ticks were removed one to three analyzed statistically using a confidence interval based
days after they had attached to mice, and subsequent on a double error of mean or sampling error. Signi-
analysis was performed using only females. ficance of differences between mean values( atp< 0.05)
In 1992- 1994, we collected I. persulcatus ticks was determined using Student' s t- test. Coefficient of
removed from patients at Perm' medical institutions. As correlation, designated r, was calculated by the usual
these patients entered into contact with ticks largely on formula.
weekends while working on their garden plots or A total of 1962 [Link] and 1561. scapularis

visiting suburban forests, the time of tick attachment ticks was analyzed individually using some of the
and removal was easy to determine. In each case, the methods described above
period of tick attachment was documented.
Live unfed ticks and some partially fed ticks were RESULTS

individually analyzed for the presence of Borrelia in

internal organs using the technique of Sidorov et al. According to the preliminary data ( see above),
 180 Journal of Vector Ecology December, 1996

zones of the Leningrad and Perm' regions of Russia

where ticks were sampled are characterized
parameters of infection in unfed and partially engorged
by similar
[Link] more accurately. Out of 359 unfed ticks
prevalence of infection in I. persulcatus ticks. This
collected from the vegetation 110 were infected; 92 of
allowed us to pool data on unfed ticks of this species and
them with Borrelia in the gut and 18 with Borrelia both
analyze them as a single, more representative sample.
in the gut and salivary glands. A total of 1, 481 ticks was
Thus, 740 I. persulcatus were analyzed, and Borrelia removed from human bodies after different periods of
was detected in 194 of them. In 169 ticks, spirochetes
attachment. Borrelia was found in 364 of these ticks
were detected in the gut only, whereas the
remaining 25 ( infection 24. 6%),
prevalence but individual organs
ticks also carried them in the salivary glands and other
were analyzed in only 301 of them. In the latter sample,
organs. In the case of I. scapularis, 41 out of 156 ticks 62 ticks( 20. 6%) were infected: 39 with spirochetes in
were infected, but only one tick contained spirochetes
the gut only and 23 with spirochetes both in the gut and
both in the gut and salivary glands. These results were
salivary glands. Parameters of tick infection calculated
used for calculating indices ofBorrelia infection in ticks from these data are shown in TABLE 2. General indices
TABLE 1). On the whole, the proportions of infected of infection and proportions of ticks with spirochetes in
ticks and of ticks
containing Borrelia only in the gut the salivary glands among unfed and partially engorged
were similar in both species. However, the proportion of
ticks removed from people were similar. The
ticks with generalized infection among all infected ticks
prevalence of infection in unfed ticks was slightly
in I. persulcatus greatly exceeded that in I. scapularis, higher than in partially engorged specimens ( t= 2. 2),
and this difference was highly significant( t= 3. 1). and the difference in the proportion of ticks with
Ticks with low and medium Borrelia populations
spirochetes in the salivary glands was not statistically
in their guts prevailed
among infected ticks of both significant( t= 1. 4). However, incidence of Borrelia in
species: 81% in [Link] and 83% in I. scapularis.
salivary gland preparations of partially engorged
In I. persulcatus, we recorded a similar proportion
infected ticks was significantly higher than in
86%) of ticks with low and medium concentration of preparations of unfed infected ticks( t= 3. 3).
spirochetes in the salivary glands. A single Borrelia-
TABLE 3 shows the results of screening for
positive preparation of I. scapularis salivary glands
Borrelia in the gut and salivary glands of all I.
contained spirochetes at low concentration of 0. 4 per
persulcatus ticks, both fed on mice and removed from
100 microscopic fields.
humans, after tick organs were analyzed at different
In I. persulcatus, the presence of higher Borrelia
periods of blood- feeding. A total of 423 such ticks were
populations in the gut did not correlate with an increased studied; 76 of them were infected and 27 had Borrelia
percentage of ticks with spirochetes in their salivary
both in the gut and salivary glands.
glands. The difference between the minimum and
It appeared that 24, 48, 72, and more hours after
maximum percentages registered in this study was not the initiation of blood-
feeding, the proportion of I.
statistically significant( t= 1. 3). We also compared the
persulcatus infected with Borrelia in the salivary glands
series of Borrelia counts( per 100 microscopic fields) in
remained virtually unchanged. Although this should be
the gut andsalivary glands of 251. persulcatus ticks with verified by other methods of spirochete detection, our
generalized infection and found no correlation between
data indicate that Borrelia concentration in the salivary
them ( r=- 0. 23).
glands of unfed and partially engorged 1. persulcatus
We used data on unfed ticks from the Perm' region remained at the same level ( 10- 14 spirochetes per 100
only ( i. e., from the same region where
partially microscopic fields). On the other hand, the number of
engorged ticks were collected) to the
compare Borrelia in preparations from the gut of ticks fed for 24

TABLE 1. Indices of Borrelia infection in unfed tick vectors.

Proportion of ticks with Borrelia, % Prevalence of

generalized
in the gut in the gut and infection in
Species total only infected ticks,%
salivary glands

I. persulcatus 26. 2± 3. 2 22. 8± 3. 1 3. 4± 1. 3 12. 9± 4. 8

1. scapularis 26. 3± 7. 0 25. 6± 7. 0 0. 6± 1. 2 2. 4± 4. 8
December, 1996 Journal of Vector Ecology 181

hours was approximately the same as in preparations of DISCUSSION

unfed ticks ( on average, 22- 25 per 100 microscopic
fields). After 25- 48 hours of blood- feeding, this number The results of this study show that, at a similar
decreased to 9- 16; and after 49- 72 hours, to 2- 9 prevalence of B. garinii or B. afzelii infection in 1.
spirochetes per 100 microscopic fields. persulcatus and B. burgdorferi sensu stricto infection in
TABLE 4 shows more detailed data on the I. scapularis and even at similar proportions of ticks
proportions of ticks with different concentrations of with different concentrations of spirochetes in the gut in
Borrelia in the gut and
salivary glands among unfed and corresponding natural foci, generalized infection in I.
engorged I. persulcatus. These proportions, as
partially persulcatus ticks develops much more frequently
well as the average number of Borrelia in the gut, ( TABLE 1). Our results with I. scapularis agree with
remained
virtually unchanged during the first days after
data obtained previously. Thus, 77 infected adult ticks
the beginning of blood- feeding. It is particularly of this species, collected in New York state, contained
noteworthy that we did not find any tendency toward an spirochetes
only in the gut ( Burgdorfer et al., 1982).
increase in the incidence and concentration of Borrelia
According to our estimates, the proportion of ticks with
in the salivary glands of ticks during the first days of
generalized infection among 179 infected [Link]
blood- feeding. analyzed in subsequent studies( Burgdorfer et al. 1988,

TABLE 2. Indices of Borrelia infection in unfed and partially engorged Ixodes persulcatus
ticks from the Perm' region of Russia.

Proportion of ticks with Borrelia, % Prevalence of

generalized

Analyzed in the gut in the gut and infection in

Material Total only salivary glands infected ticks, %

Unfed ticks
collected from 30.6± 4. 9 25. 6± 4. 6 5. 0± 2. 3 16. 4± 10. 2
vegetation

Partially engorged
ticks removed from
people:

total 24. 6± 2. 2 no data available

analyzed for
Borrelia in 20. 6± 5. 2 13. 0± 3. 9 7. 6± 3. 0 37. 0± 10. 2
individual
organs

TABLE 3. Proportions of Ixodes persulcatus ticks with Borrelia in the salivary

glands at different time after initiation of blood- feeding.

Prevalence of
generalized

Period of Number of ticks Proportion of infection in

blood- feeding studied infected ticks, % infected ticks, %

up to 24 hours 245 24. 5± 5. 5 35. 0± 12.4

25- 48 hours 80 8. 7± 6. 3 42. 9± 40. 1
49- 72 hours and more 98 9. 2± 5. 9 33. 3± 33. 3
182 Journal of Vector Ecology December, 1996

Burgdorfer 1989a) was only 2. 2± 2. 2%. microscopic analysis of fixed and live preparations is a
This figure contrasts sharply with the data on 1. reliable method for detecting Borrelia in the internal
pacificus ticks from California, which can transmit not
organs of partially engorged ticks as well as of unfed
only B. burgdorferi sensu stricto but also Borrelia of the ticks. However, the Borrelia concentration in the tick
original DN 127 group ( Postic and Baranton 1994). gut decreased with an increase in the amount of fluid
Only 25 of 1687 adult I. pacificus were infected, but a
consumed during blood- feeding. Consequently, the
generalized infection was recorded in eight of them number of spirochetes in preparations of this material
32± 19%)( Burgdorfer 1985).
et al.
also decreased, which reduced the probability of
In Central Europe, the proportion of 1. ricinus ticks identifying infected ticks. We believe that these
with systemic infection can be estimated at 5. 5± 5. 4% " technical" inconsistencies account for lower indices of
see: Burgdorfer et al. 1989) or at 5. 3± 4. 2% ( see:
infection in partially engorged I. persulcatus as
Burgdorfer et al. 1983). Gem ( 1994) suggested that compared with unfed ticks from the same focus. In
systemic infection ( including the presence of
blood- feeding ticks of this species, Borrelia is found in
spirochetes in the salivary glands) in 1. ricinus is the salivary glands with the same frequency, and in the
actually more frequent than indicated by the previous gut, with a lesser frequency than in unfed ticks;
studies. In
any case, the values given above exceed those consequently, the proportion of ticks with spirochetes in
determined for 1. scapularis but are far lower than those the salivary glands among all infected ticks is also
for I. persulcatus. If confirmed in subsequent studies, greater. This is a purely" arithmetic" effect, and a more
this fact, in combination with other factors, would detailed analysis ( see TABLES 3 and 4) shows that it
explain the lesser vector
efficiency of I. ricinus, should not be regarded as evidence for an actual increase
compared with I. persulcatus, in ITBB foci( Korenberg in the number of ticks containing spirochetes in the
et al. 1991a, b, Korenberg 1993b). salivary glands or for higher concentrations of
The results described here suggest that direct spirochetes in them. A similar situation was described

TABLE 4.
Proportions of ticks with different concentrations of Borrelia in the gut and salivary glands
among infected Ixodes persulcatus.

Concentration of Borrelia Mean number of

Borrelia per 100
Analyzed Number high and microscopic fields
Material of ticks low medium very high approximated)

Unfed ticks
collected from
vegetation:

gut 359 40 41 19 22

salivary glands 359 50 33 17 10

Partially
engorged ticks

removed from
people:

gut 1477 44 39 17 24
salivary glands 326 33 50 17 12

Partially
engorged ticks

fed on laboratory
animals:

gut 122 36 50 14 17
salivary glands 122 50 50 4
December, 1996 Journal of Vector Ecology 183

for adult I. ricinus collected in a natural focus: the owing to low probability of pathogen transmission atthe
prevalence of infection in partially engorged females beginning of blood- feeding, as in the case of I.
decreased from 36. 3± 5. 6% ( characteristic of unfed scapularis ( Piesman et al. 1987; Ribeiro et al. 1987,
ticks) to 21. 6± 6. 1%, whereas the prevalence of Burgdorfer 1989b). In [Link], Borrelia are often
generalized infection in infected ticks remained found in salivary glands of the unfed ticks, i.e., the
unchanged( Burgdorfer et al., 1983). essential condition for pathogen transmission during
We regard the peculiarities of the relationship of B. the first hours of blood- feeding is satisfied. Thus, we
garinii and B. afzelii spirochetes with I. persulcatus took into account indices of spontaneous infection and
ticks to be important. These spirochetes are
frequently the frequency of Borrelia presence in the salivary
detected in the salivary glands as well as in the gut of glands of I. persulcatus prior to blood- feeding and, on
unfed ticks. As infected ticks engorge, the proportion of this basis, estimated that B. garinii and B. afzelii
ticks with spirochetes in the salivary glands remains transmission by recently attached ticks occurs in four to
five cases per 100 tick bites ( data from the Perm' and
virtually unchanged for at least two to three days
TABLES 2 and 3), as concentration of spirochetes in Leningrad regions of Russia). Our experience shows
this organ does ( TABLE 4). In other words, Borrelia that people do become infected in such a situation,
migration from the gut into the salivary glands in I. although the duration of tick attachment is usually less
persulcatus ticks during blood- feeding is not a than one day ( Korenberg et al. 1994).
or even important condition for the The results of our studies confirm that each of the
necessary
transmission of ITBB pathogens with their saliva. The ITBBs is characterized by a certain frequency of
frequency of this transmission, which occurs within the generalized infection in unfed ticks, its principal
first 24 hours after the tick attachment to the human vectors. This phenomenon is based on the obscure

body ( Korenberg et al. 1994), depends largely on the specificity of relationships established between each of
initial proportion of unfed ticks containing Borrelia in Borrelia species and its vectors. It agrees well with a
their salivary glands. more general principle formulated by Balashov( 1995):
Based on our data, we cannot exclude the the extent of generalization of infection is apparently a
possibility of Borrelia migration from the gut into the species- specific feature of the microorganism and its

salivary glands in adult [Link] on the second or vector.

third day of blood- feeding. The role of such a

phenomenon ( if it exists) in the epizootic process Acknowledgments

remains unknown. However, it is apparently

insignificant from an epidemiological standpoint, We are grateful to Professor Andrew Spielman of
because adult ticks are usually physically removed at the Harvard School of Public Health( Boston, MA) and
the onset of blood- feeding, and it is mainly adult I. personnel of his laboratory for their help in collecting!.
persulcatus ticks that attack humans. scapularis ticks. This comparative study would be
Data obtained in the laboratory and in the field impossible without their kind cooperation.
showed that I. scapularis nymphs and adult ticks

usually transmit B. burgdorferi sensu stricto only after REFERENCES CITED

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Piesman, J. 1994. Dispersal of Borrelia burgdorferi to Ribeiro, J. M. C., T. N. Mather, J. Piesman, and A.
the salivary glands of nymphal ticks during the act Spielman. 1987. Dissemination and salivary
of feeding. VI International Conference on Lyme delivery of Lyme disease spirochetes in vector tick
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Microbiol. 25: 557- 558. Spielman, and S. R. Telford. 1989. Fine structural
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burgdorferi with description of a needle aspiration Zool. 67: 1737- 1748.
 Journal of Vector Ecology 21( 2): 186- 191

Identification of Larval Instars of Aedes albopictus (Skuse) and Aedes

triseriatus ( Say) ( Diptera: Culicidae) Based on Head Capsule Size

Hwa-Jen Tengt•2 and Charles. S. Apperson"'3

IDepartment of Entomology, Box 7613, North Carolina State

University, Raleigh, NC 27695- 7613, USA
2Current address: National Institute of Preventive Medicine,
Entomology Division, 161, Kun- Yang St. Nan-Kang,
3 Taipei, Taiwan 11513, Republic of China
T whom requests for reprints should be addressed.

Received 4 March 1996; Accepted 3 July 1996

ABSTRACT: Effects of constant temperature ( 15, 23, 31° C) on the head capsule widths of the larval
instars ofAedes
species, head
albopictus( Skuse) and Aedes triseriatus( Say) were determined in the laboratory. For both
capsule widths were
significantly different between instars ( P< 0. 001). Temperature signi-
ficantly affected the size of head capsules of larvae of both species. For Ae. albopictus, except for the 1st
instar, mean head capsule widths of 2nd- 4th instars tended to be larger at 23°C than at 15 or 31° C; while for
Ae. triseriatus, mean head capsule size was largest only at 23°C for 3rd and 4th instars. However, for both
species, mean head capsules widths were significantly ( P<0.05) larger at 23°C than at the other two
temperatures only for 4th instars. The head capsule widths of all instars of Ae. triseriatus were larger than
for the corresponding instars of Ae. albopictus. Using classification criteria developed through discriminant
analysis,> 98%
of larvae of both species were identified to instar regardless of the temperature in which the
larvae were reared.

Keyword Index: Aedes albopictus, Aedes triseriatus, head capsule width, discriminant analysis.

INTRODUCTION Accordingly, we determined effects of temperature

on head capsule width of Aedes albopictus( Skuse) and
Investigations of the population dynamics of
Aedes triseriatus ( Say) in the laboratory, and used
mosquitoes often require estimates of age- specific
discriminant analysis to develop classification criteria
mortality in larvalpopulations ( Hawley 1985). For for identification of the instars of both species.
mosquito larvae, instar provides an estimate of the
relative age of mosquitoes. Head capsule width and MATERIALS AND METHODS
body length have both been used to differentiate the
instars of mosquitoes, such as Culex quinquefasciatus
Mosquito Strains and Rearing Conditions
Say, Aedes aegypti( L.)( Rueda et al. 1990), Aedes
and
A colony of Ae. albopictus ( ALBO- ROC) was
vigilax( Skuse)( Shinkarenko et al. 1986). Shinkarenko established from eggs that were obtained from the late
et al.( 1986) found that head capsule width was a more
Dr. George Craig( Department of Biological Sciences,
reliable measure of instar than was body length for Ae.
Notre Dame University, Notre Dame, IN). This mosquito
vigilax. Rueda et al. ( 1990) and Shinkarenko et al.
1986)
strain was originally collected in Rockingham County,
reported temperature significantly affected the NC in June 1987. Eggs of a Walton strain of Ae.
head capsule width of mosquito instars. Thus, cohort triseriatus ( TRIS- WAL) were also obtained from Dr.
structure could
potentially be altered

as a result of the
seasonally or
Craig. This colony has been maintained in his laboratory
geographically temperature of
breeding for an unspecified number of years.
sites.
Mosquito colonies were maintained in an insectary
December, 1996 Journal of Vector Ecology 187

at 28° C, 85% RH, and a 16: 8 h light:dark cycle. Females calibrated ocular micrometer contained in a Wild M5
were fed citriated pig blood ( Benzon and Apperson microscope.

1987) and provided with a 10% sucrose solution.

Occasionally, mosquitoes were allowed to feed on the Statistical Analyses

forearm of a human. Eggs were collected on white filter Effects of instar and temperature on head capsule
paper placed inside a black cup that was partially filled widths for each species were analyzed by two- way
with water. Eggs were stored between moistened analysis of variance ( ANOVA) ( Neter et al. 1985),
paper towels in plastic bags in an incubator at 21° C using a general linear model procedure( SAS 1985). To
and a 16: 8 h light:dark cycle until needed for experi- determine if differences in head capsule size for each
ments. instar were statistically significant between temperatures
P< 0. 05), probability of difference values were
Size of Instars calculated in least significant difference tests for least
To obtain first instars, eggs of both species( ALBO- square mean ( LSM) head capsule widths ( SAS 1985)
ROC and TRIS- WAL) were
separately immersed in under the hypothesis H°: LSM(.)=
LSM6). Discriminant
0. 1% ( wt:vol) Bacto Nutrient
a
hatching medium of analysis was used to estimate probabilities of correct

Broth ( Difco Laboratories, Detroit, MI) in distilled classification and to set up a classification criterion
water( Novak and Shroyer 1978). After six hours, first ( Johnson and Wichern 1988, SAS 1985) to identify
instars were placed individually in shell glass vials larvae of each species to instar.
1. 5 cm dia. x 6 cm ht.) and 2 ml of a 0. 1%( wt./

vol.) food suspension in distilled water was added per RESULTS

vial. The food consisted of a 1: 1: 1 mixture( v: v: v) of
brewer' s yeast, lactalbumin, and ground Purina rabbit Head Capsule Widths
chow( Chambers 1985). Groups of vials were placed in Frequency distributions of head capsule widths for
each of three reach- in environmental chambers
larval instars of each species were constructed( Fig. 1).
maintained at 15, 23, and 31° C) in the S. E. Plant Generally, head capsule sizes for each instar of each
Environmental Laboratory at N. C. State University. species were discretely distributed. However, there was
Larvae were observed daily at 10x to determine if they some overlap in the size of head capsules for 3rd and 4th
had molted. Groups of 15 to 40 larvae for each instar at instars of Ae. albopictus, and the 1st and 2nd, and 2nd
each temperature were preserved in 70% ethanol after and 3rd instars of Ae. triseriatus( Fig. 1).
larval head capsules had darkened. When head capsule Head capsule width varied significantly( P<0. 005)
measurements were taken, larvae were placed on a between instars and temperatures ( TABLE 1) for each
glass microscope slide, and excess moisture was re- species. A significant temperature* instar interaction
moved with a paper towel. Care was taken to assure was found for both species ( TABLE 1). Probability
that each larva was positioned such that its head capsuie of difference values calculated ( SAS 1985) for least
was perpendicular to the plane of view. The head square means of each instar within each temperature
capsule of each larva was measured at the widest point revealed that only 4th instars reared at 23°C had
across the developing compound eyes at 30- 60x using a significantly larger head capsule sizes( TABLE 2). The

TABLE 1. ANOVA of effects of temperature and instar on head capsule widths of

Aedes albopictus and Aedes triseriatus larvae.

Aedes albopictus Aedes triseriatus

Sources DF Type III SS DF Type III SS

Temperatures

2 0. 020* 2 0.031***
Instar 3 26.011*** 3 22. 885***
Temperature* Instar 6 0.016 6 0. 044**
Error 408 0. 538 359 0. 506

Significant at P= 0.05; ** Significant at P= 0.0005; *** Significant at P= 0.0001.

aTemperature was not replicated at the chamber level.
 188 Journal of Vector Ecology December, 1996

15
Aedes albopictus

0 15° C

0 23° C
10:
ir 31° C
r
a

0 I II III IV
MIIM

IttAil
Z 5-

4 fiajlk 4\

4;
II •

0 v / iii••
14 . 22 . 30 . 38 . 46 . 54 . 62 . 70 . 78 . 86 . 94 1. 02 1. 10 1. 18
18 . 26 . 34 . 42 . 50 . 58 . 66 . 74 . 82 . 90 . 98 1. 06 1. 14

Head Capsule Width( mm)

15 -
Aedes triseriatus

g 10 '

0 I II . III IV
i.u .
E
Z 5'
I•
iiis.
IA
Ir AT•••• twit• ,
0 AAA*
AAA• A 0
1. T. J, t • J

14 . 22 . 30 . 38 . 46 . 54 . 62 . 70 . 78 . 86 . 94 1. 02 1. 10 1. 18
18 . 26 . 34 . 42 . 50 . 58 . 66 . 74 . 82 . 90 . 98 1. 06 1. 14

Head Capsule Width( mm)

Figure 1.
Head capsule widths( mm) of Aedes albopictus and Ae. triseriatus larvae that were reared at three
different temperatures.

head capsule widths of all instars of Ae. triseriatus

Mean head capsule widths ranged between 0. 271-
larger than for the
corresponding instars of Ae.
were
0. 934 mm for 1st- 4th instars of Ae. albopictus, and
albopictus; however, differences between species for

larval head capsule sizes could not be analysed

between 0.342- 1. 046 mm for corresponding instars of
Ae. triseriatus. Coefficient of variations( CV' s)±
SEcv
statistically because effects of temperature on instars (± standard errors of the CV) of 1st instars were larger
were not determined concurrently for both species. than for other instars,
indicating that variations in
December, 1996 Journal of Vector Ecology 189

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 190 Journal of Vector Ecology December, 1996

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head capsule widths for this larval growth stage Identification of Instars
were
significantly larger than for other stadia( TABLE
In the ANOVA, type III SS for the main effect of
2). The same trend in head capsule size variations
temperature were relatively small compared to instar
held true for Ae. triseriatus except that the CV' s± ( TABLE 1), and the graphical plot of head capsule
SEcv' s, generally, were not larger than for other
width for each temperature( Fig. 1) indicated that instar
instars, indicating that variation of head capsule size
within an instar did
head capsule sizes were not strongly affected by
not
vary significantly between temperature. Consequently, discriminant analyses were
instars. conducted for each species, without regard to
temp-
December, 1996 Journal of Vector Ecology 191

erature, to estimate the probabilities of correct classi- REFERENCES

fication of larvae for each instar. For Ae. albopictus, no

misclassification would occur for the 1st instar and 3rd Benzon, G. L. and C. S. Apperson. 1987. An electrically
instar ( TABLE 3). However, for the 2nd or the 4th heated membrane blood- feeding device for
instar, one out of 1001arvae might be misclassified. For mosquito colony maintenance. J. Am. Mosq.
Ae. triseriatus, there was a 1 in 100 chance that a 2nd, Control Assoc. 3: 322- 324.

3rd, or 4th instar might be misclassified. Chambers, R. C. 1985. Competition and predation

among larvae of three species of treehole- breeding

DISCUSSION mosquitoes. Pp. 25- 53 in Ecology of mosquitoes:
Proceedings of a workshop( P. Lounibos, J. R. Rey,
As in our investigation, Shinkarenko et al. ( 1986) and J. H. Frank, eds.). Florida Medical Entomology
and Rueda et al. ( 1990) found that temperature Laboratory, Vero Beach, 579 pp.
significantly affected the head capsule width of instars Hard, J. J., W. E. Bradshaw, and D. J. Malarkey. 1989.
of some Aedes and Culex mosquitoes. In contrast to our Resource- and density- dependent developement in
results, Rueda et al.( 1990) found that head capsules for tree- hole mosquitoes. Oikos 54: 137- 144.

Cx. quinquefasciatus and Ae. aegypti were wider for all Hawley, W. A. 1985. Population dynamics of Aedes
instars at 15°C relative to 25 and 30°C. sierrensis. Pp. 167- 184 in Ecology of mosquitoes:
Our results for Ae. albopictus and Ae. triseriatus Proceedings of a workshop( P. Lounibos, J. R. Rey,
indicate that head capsule width is a relatively fixed and J. H. Frank, eds.). Florida Medical Entomology
morphometric character regardless of the temperature Laboratory, Vero Beach, 579 pp.
at which larvae are reared. A high level of precision in Johnson, R. A. and D. W. Wichern. 1988. Applied

instar identification was achieved despite the significant multivariate statistical analysis. Prentice- Hall, Inc.
effects of temperature on head capsule widths of both Englewood Cliffs, NJ, 607 pp.
species. Furthermore, our results indicate that the Moore, C. G. and B. R. Fisher. 1969. Competition in
classification criterion developed can be used to age mosquitoes: density and species ratio effects on
grade field- collected larvae of Ae. albopictus and Ae. growth, mortality, fecundity, and production of
triseriatus. In their study of Ae. vigilax, Shinkarenko et growth retardant. Ann. Entomol. Soc. Am. 62:
al. ( 1986) found that head capsule width was a more 1325- 1331.
Neter, J., W. Wasserman, M. H. Kutner. 1985.
reliable indicator of instar than was body length. Using and

discriminant analysis, 1986) correctly

Shinkarenko et al.( Applied linear statistical models: regression,

identified> 99% of larvae to instar based on head capsule analysis of variance, and experimental designs.
width despite the effects of temperature and salinity on Richard D. Erwin, Inc. Homewood, IL, 1, 127 pp.
head capsule size. However, it should be noted that Novak, R. J. and D. A. Shroyer. 1978. Eggs of Aedes
other environmental factors such as larval density and triseriatus and Aedes hendersoni: a method to
food ration also affect larval growth rates ( Hard et al. stimulate optimal hatch. Mosq. News 38: 515- 521.
1989, Moore and Fisher 1969). Therefore, under field Rueda, L. M., K. J. Patel, R. C. Axtell, and R. E. Stinner.
conditions, the size of larvae could potentially be affected 1990. Temperature- dependent development and
the interactions of a number of environmental factors. survival rates of Culex quinquefasciatus and Aedes
by
Larval instar is a suitable measure of relative age, aegypti( Diptera: Culicidae). J. Med. Entomol. 27:
and an appropriate parameter to include in life table 892- 898.

models of stage- specific mortality. However, since the SAS. 1985. SAS user' s guide: statistics, version 5
rate of larval development is temperature dependent edition. SAS Institute Inc., Cary, NC, 1, 290 pp.
Rueda et al. 1990), larval instar conveys little Shinkarenko, L., K. Hulsman, P. Mottram, P. Dale, and
information about the physiological age of mosquitoes. B. H. Kay. 1986. Reliability of using head capsule
Consequently, instar would not be an appropriate width and body length to identify instars of Aedes
parameter to include in mathematical models of vigilax( Diptera: Culicidae). J. Aust. Soc. 25: 37-

temperature- dependent development. 40.

 Journal of Vector Ecology 21( 2): 192- 200

From Mouse to Sequence and Back to Mouse:

Peregrinations of an Arbovirologistt

Charles H. Calisher

Arthropod-Borne and Infectious Diseases Laboratory, Foothills

Campus, Colorado State University, Fort Collins, CO 80523

Received 15 January 1996; Accepted 31 January 1996

ABSTRACT: When the albino( laboratory) mouse was found to be useful for the production ofhigh titering
improvements in diagnostic
reagents, methods soon followed. Dalldorf, Bugher, Casals, Lennette,
Koprowski, Theiler, and Webster each made contributions that were central to the generation of assays
allowing much more precise measurements of virus than had been possible.
With the discovery of
hemagglutination by Hirst and its application to both hemagglutination-inhibition tests for antibody to
viruses and the study of virus-cell attachment mechanisms, a relatively simple and inexpensive tool became
available. Subsequent efforts by Sabin and Buescher and by Clarke and Casals applied this method as well
as complement- fixation and neutralization to epidemiological and laboratory studies of arboviruses and
arboviral diseases. Worldwide studies of arboviruses, supported by the Rockefeller Foundation, led to the
discovery of newly recognized viruses and their geographic distributions. Later, electron microscopic
studies by Holmes and Murphy corroborated the antigenic studies of Casals, Shope, and others and the
cascade of information regarding the molecular characteristics and genomic sequences of viruses
subsequently provided powerful other analytical tools. In 1993 a previously unrecognized hantavirus, Sin
Nombre virus, was shown to be the etiologic agent of Hantavirus Pulmonary Syndrome in the U.S., and
various techniques were used to determine the epidemiology and natural history of this virus. Longitudinal
studies of hantaviruses in the southwestern U.S. are yielding information useful for understanding the
fundamentals of transseasonal transmission, epizoology, epidemiology, evolution, epidemic potential,
prevention, and control of hantaviruses, here and elsewhere. Mice of various species, sizes, shapes, and
colors have been central, if involuntary, participants in these hantavirus studies and advances. We have,
in a way, come full circle.

Keyword index: Bunyaviridae, hantavirus, Sin Nombre, mouse, history, arboviruses.

The laboratory mouse had not been used as a test

in virology
to develop neutralization tests, particularly those to
animal until 1930, when Max Thei ler reported
detect antibody or to identify western equine encephalitis,
that newly born inoculated
mice
intraperitoneally with
eastern equine encephalitis, and Venezuelan equine
yellow fever virus died of encephalitis( Theiler 1930). encephalitis viruses( Lennette and Koprowski 1944).
In 1935 it that the
was reported
laboratory mouse was
When Jordi Casals was beginning his scientific
suitable for use as a test animal for the diagnosis of
career in 1936, he went to work with Leslie Webster of
rabies( Webster and Dawson 1935). Then, in 1941, John
the Rockefeller Institute in New York. Two years later
Bugher reported that when mice were inoculated when
an outbreak of encephalitis occurred in Massachusetts
they were younger than 10 days of age,
they sickened and Casals and Webster became interested in the etiologic
and died after
being subcutaneously inoculated with agent, eastern equine encephalitis virus, and soon were
yellow fever virus( Bugher 1941) and, in 1944, Edwin
involved in studies of antigenic comparisons of the
Lennette and
Hilary Koprowski reported taking ad-
viruses that were known at that time to cause encephalitis:
vantage of the high susceptibility of three- day old mice
rabies, eastern equine encephalitis( EEE), western equine

Presented at the 27th Annual Meeting of the Society for Vector Ecology, Fort Collins, Colorado, October 10, 1995.
 Journal of Vector Ecology 193
December, 1996

encephalitis ( WEE), Russian spring- summer reagents, marvelous collegiality, and appropriate
financial and administrative support. Thus armed, they
encephalitis, louping ill, and poliomyelitis( Casals 1944).
the meticulous methods needed to properly began the classic investigations that were so integral to
Applying
perform the complement- fixation test taught him by our knowledge of the interrelationships among
Jules Freund working on tuberculosis, Casals found arboviruses( Theiler and Downs 1973).

relationships between the viruses of Russian spring- In 1943, during studies of yellow fever in Uganda,
summer encephalitis and louping ill and no relationship Kenneth Smithburn and his colleagues of the Rockefeller
between rabies, poliomyelitis, and other viruses( Casals Institute had made the initial isolation of Bunyamwera
and Webster 1944). Casals also found that St. Louis, virus from Aedes species mosquitoes( Smithburn et al.
Japanese, and Murray Valley encephalitis viruses were 1946); the isolation was made using mice. A few years
related to Russian spring- summer later, Richard M. Taylor, Telford H. Work ( Fig. 1) a
antigenically
encephalitis and louping ill viruses but that antigenic former member of SOVE who died early this year, and
varied; that is, whereas all were related, some others, isolated Sindbis virus from mosquitoes, birds,
proximity
were more distantly and some more closely related one and humans in Egypt( Taylor et al. 1955); the isolations
to another( Casals 1957). were made using mice. Smithburn scarcely could have
The hemagglutination technique, developed at the imagined that Bunyamwera virus would become the
Rockefeller laboratories by George Hirst working with type species of both the genus Bunyavirus and of the
influenza A virus in embryonated hens' eggs ( Hirst family Bunyaviridae, nor could Taylor and Work have
1941), useful in early studies of virus- cell attachment imagined that Sindbis virus would become the prototype
mechanisms, was shown by Casals and Brown to be virus of the genus Alphavirus and of the family
Casals and Brown Togaviridae.
suitable for work with arboviruses (

1954). Then, using antigens produced in the brains of The classical grouping fluids produced by Shope
mice, Albert Sabin found that yellow fever and dengue and others; the application of complement- fixation
related and, with Edward ( Casals 1949), HI, and neutralization tests ( Casals
viruses are antigenically
Buescher, demonstrated that the hemagglutination- 1963a); the use of suckling mice, of acetone extraction
inhibition ( HI) technique could be applied to other of antigens and sucrose in antigen preparations( Clarke
Buescher 1950). When Max and Casals 1958)— all were used to define antigenic
arboviruses ( Sabin and

test for evidence groups and to determine antigenic differences.

Theiler suggested using HI as a serologic

of infection with arboviruses, a formidable, yet Casals formulated three dicta, which can be
tool became available to show paraphrased as follows:( 1)" No virus can belong to two
simple,
exquisitely
differences. antigenic groups,"( 2)" If two viruses cross- react, they
antigenic similarities rather than

viruses, antigens, and antibodies are are related," and ( 3) " If two viruses of two different
High titering
essential for detecting distant antigenic relationships groups cross- react, they do not belong to two different
between viruses, so that the finding by Gilbert Dalldorf groups." These simple but effective premises have
that coxsackieviruses replicate to high titer in newborn never been disproved. Perhaps their significance can

mice( Dalldorf and Melnick 1965) led Casals and others best be understood if one thinks of them in terms of
to replace adult mice with newborn mice for reagent phenotypic expression of genotypes and in terms of
production and for other purposes. By 1951, Casals and phylogeny.

coworkers had adapted human poliomyelitis type 2 By 1960, it was known that EEE and WEE and
virus to replicate in newborn mice and developed a certain other viruses were related. These were placed in
what was termed the " Group A arboviruses" (
now
complement- fixation test to detect it( Casals et al. 1951a;

Casals et al. 1951b). they began using suckling

When known as the genus Alphavirus in the family
borne viruses, the results Togaviridae). Japanese encephalitis, Murray Valley
mice for the study of arthropod-

were shorter incubation periods, higher titers, and better encephalitis, and St. Louis encephalitis viruses, West
polyclonal antibodies.
Nile, yellow fever, Russian spring- summer encephalitis,
At that time the Rockefeller Institute was supporting louping ill, and others were known to be related to each
laboratories studying yellow fever and other diseases in other and were placed in what was termed the" Group B
Africa, South America, and elsewhere, and the New arboviruses"( now known as the genus Flavivirus in the
York laboratory was receiving scores of virus isolates family Flaviviridae). The laboratory mouse was central
from arthropods and vertebrates ( Theiler and Downs to all this work and to these findings.
1973). Casals, Robert Shope, Loring Whitman, and In the following decades many more newly
their coworkers were in the unique position of having recognized arboviruses were isolated and workers at the
a profusion of viruses, but excellent Rockefeller Foundation laboratories and elsewhere used
available not only
 194
Journal of Vector Ecology December, 1996

meticulous testing to detect relationships between and

among many of them and to establish serogroups in

these viruses all were interrelated, albeit distantly and
which to place them( Theiler
enigmatically. These cross- reactions between one or
and Downs 1973). Within
about a ten- year period, not
another member of the various serogroups often were
only had the Bunyamwera
weak and some appeared tenuous but all were repeatable.
serogroup been recognized( Casals and Whitman 1960),
but serogroups C( Casals and Whitman 1961), Guama, "
Therefore, Casals suggested placing them in a
Bunyamwera Supergroup" ( Casals l963b).
California, Capim, Anopheles A, Simbu, Bwamba,
Patois, Koongol, Tete, and others had been distin-
In the late 1960s, sophisticated electron microscopic
guished ( Hammon and Reeves 1952;
studies by Ian Holmes in Australia( Holmes 1971) and
Whitman and
Casals 1961; Theiler and Downs 1973; Whitman and
Fred Murphy at the Centers for Disease Control( CDC)
Shope 1962). Because at least one virus in in Atlanta( Murphy et al. 1973) recognized the morpho-
each of these
groups reacted
serologically with at least one virus( or
logical similarities of Bunyamwera Supergroup viruses
antibody to it) and supported and extended the Supergroup concept in
of another
group, Casals suggested that
that they were able to show that these viruses were not

Ar'' ';.."'': .

4000:::'!%0? 0:'''''' ''.' *

Figure 1.
Telford H. Work, photo taken about 1964 while he was Chief, Virology Section and Arbovirus Unit,
National
Georgia.
Communicable Disease Center( now Centers for Disease Control and Prevention), Atlanta,
 195
December, 1996 Journal of Vector Ecology

distinguishable by size, morphology, or ( Bunyaviridae in general) provide a milieu in which

clearly
morphogenesis in infected cells. Some of these viruses reassortment of RNA segments can occur.

were later belong to separate genera within the

shown to Recent advances in molecular genetics, molecular

family Bunyaviridae( Murphy et al. 1995). epidemiology, and molecular evolution have been the
Subsequent studies of the phenotypic characteristics result of a combination of developments, including
of these viruses and of viral structure- function access to computer-assisted analyses, availability of
relationships, replicative mechanisms, biologies, and techniques for rapid and accurate sequencing of nucleic
ecologies has led us first to classification, then to acids, and most importantly the appearance of highly
taxonomy, nowphylogeny. Whether or not one
to skilled molecular biologists who can apply these
considers classification to be simply the obsessive
techniques with imagination and who can articulate

collecting of things, it
follows that taxonomy and their findings. It has been only since these occurrences
are the twin goals of such a compulsion. coincided that we have been able to scrutinize viral
phylogeny
In the 1960s the Virology Section at the CDC was genomes. Viral classification, previously based solely
headed by Telford Work. That was a time when virus on phenotypic characters, became gene- based and has
research was not prioritized as a Disease- Of-The- Week evolved, and is evolving, into a taxonomy parallel to but
Still,
Club. The methodical characterization of viruses, newly very different from that of cellular organisms.

recognized or not, associated with disease or not, along when attempting to identify a virus, it is useful to
with the emphasis placed on field studies, and the inoculate it into suckling mice and see what happens. If
epidemiologic brilliance of Work, ran together to actually one knows the terrain and the species of arthropod, one

discover things rather than to solve only acute pro- can obtain a rough, but fairly reliable, idea as to which
blems. On the staff at that time were Roy Chamberlain, virus an isolate might be simply by determining how
Philip Coleman, Bernard Fields, Martin Hirsch, Brian long it takes to kill the mouse.
Henderson, Blaine Hollinger, Rexford Lord, Fred I find it extraordinary that the serologic and antigenic
Murphy, Daniel Sudia, and others, many if not all of studies done many years ago, using relatively crude
whom are familiar to the readers of this journal. I was techniques that never were more than an indication of
fortunate enough to have been added by Tel Work to that phenotype, have been so predictive of genotype. Clearly,
select In relatively short order, Murphy' s viruses with similar gene sequences produce similar
group.
laboratory showed that the alphaviruses and flavi- proteins; it usually follows that the more dissimilar the
viruses differed from what were being called rhabdo- gene sequence, the more dissimilar the antigens
viruses, reoviruses, arenaviruses, and filoviruses. Nearly produced. Therefore the early tests and conclusions of
as as viruses could be amplified in mice, they Casals, Shope, and others provided a reliable set of
quickly
were passaged in cell cultures, and had their photos indicators of genetic relationships although they did not
taken. Still, while results of such biological and think of them in this way at that time. The original
morphological analyses were instructive, they also wee intent, of course, was not to improve taxonomy but to
incomplete. Fortunately, there has been a cascade of improve laboratory diagnosis of arboviral and other
information about viral nucleic acid sequences. viral infections; this they did, mostly with the help of
Arboviruses have the advantage of passing through untold numbers of laboratory mice.
atleast two taxonomically distinct hosts during their life In mid-May 1993, an outbreak of fatalities in adult
cycles. One host, the vertebrate, lives in a relatively humans with acute respiratory failure was recognized in
constrained ecosystem, a defined ecological niche, New Mexico and, shortly thereafter, in Colorado and
and has othermechanisms that protect Arizona. The first evidence that these infections were
produces antibody,
it from the potentially deleterious effects of the virus, caused by a hantavirus ( genus Hantavirus, family
while at the same time providing selection pressures that Bunyaviridae) was that case- patients and rodents trapped

lead to diversity of virus genotypes. The mouse is a near the homes of these patients had antibody to one or
useful model for much of this. The arthropod seems to more hantavirus not known to occur in this country. It
be mostly unaffected by infection with a virus and does was serologic evidence that was first used to classify the
not have immune mechanisms, in effect serving only as hantaviruses. Soon thereafter, a newly recognized virus,
an incubator for amplification, maintenance, and Sin Nombre virus( SNV), was shown to be associated
transport of a virus population. Viruses, such as hanta- with this disease, now called hantavirus pulmonary
from to rodent, syndrome ( HPS) ( Elliott et al. 1994). Elegant and
viruses, which are transmitted rodent

by innovative molecular epidemiologic studies of specimens

have no such protection, being constantly assaulted

antibodies that may provide a selective mechanism. from HPS patients and from deer mice, the putative
hantaviruses natural vertebrate hosts of this virus, captured in case
Further, dual infections with closely related
 196 Journal of Vector Ecology December, 1996

residences, showed that SNV is distinct from other (

known hantaviruses.
also called Epidemic hemorrhagic fever and Korean
Analyses and comparisons of hemorrhagic fever).
nucleic acid sequences of this and other
hantaviruses
Thus, patients with pulmonary
indicated that SNV is widely distributed in the U. S. and
disorders were not what one would expect if one were
that this virus
predicting an American disease caused by a hantavirus.
naturally infects rodents, principally
Peromyscus maniculatus, throughout that rodent' s range. That inability to predict is one ofthe many reasons many
Since 1993, hantaviruses have been implicated in
ofus studied biology in the first place. Other hantaviruses
illnesses in 124 people in the U. S., most of whom have
in other parts of the world can cause renal failure and
hemorrhagic illnesses( TABLE 1). The hantaviruses in
been residents the area
of
adjoining New Mexico, the U.S., when they are associated with human illness,
Colorado, Arizona, and Utah; six of these cases have
been in Colorado and five of these six died. The
are associated with pulmonary disease.
One fascinating aspect of the hantaviruses is their
mortality rate has been slightly less than 50%.
apparent specific association with specific rodent species
Fortunately, not many people get this disease.
Most of the first cases in 1993 were detected in
Lee et al. 1991); Hantaan virus with Apodemus agrarius
(
Native Americans because the outbreak first was
striped field mouse); Seoul virus( Kariwa et al. 1994)
in an area with predominant populations of
recognized
with Rattus norvegicus and Rattus rattus; Puumala
virus ( Brummer- Korvenkontio et al. 1980) with
Native Americans. In more recent studies, however, the Clethrionomys glareolus ( bank vole); Prospect Dill
proportions of caseshave been: Hispanic ( 8%), non-
virus ( Lee et al. 1985) with Microtus pennsylvanicus
Hispanic Caucasian ( 56%), Native American ( 34%), (
and African- American ( 2%).
meadow vole); SNV and Convict Creek( Schmaljohn
Ages of patients have
et al. 1995) virus with Peromyscus maniculatus( deer
ranged from 12 to 69 years, with a median of 32 years;
55% have been
mouse); El Moro Canyon virus( Hjelle et al. 1994) with
males.
Reithrodontomys megalotis ( western harvest mouse);
Because hantaviruses in other parts of the world
had been
Thottapalayam virus ( Zeller et al. 1989) with Suncus
associated only with rodents and not with
murinus( shrew); Black Creek Canal virus( Rollin et al.
arthropods, rodents
immediately became suspect
1995) from Sigmodon hispidus( hispid cotton rat); Tula
reservoirs. Indeed, epidemiologic evidence suggests
that the natural
virus( Plyusnin et al. 1994) from Microtus arvalis and
history
of SNV involves
cycling in Microtus rossiaemeridionalis( voles); and Bayou virus
rodents,
principally P. maniculatus, the deer mouse (
Childs et al. 1994). The virus appears to be transmitted
Morzunov et al. 1995) from Oryzomys palustris( rice
rat). A hantavirus associated with a human infection in
between rodents, in subclinical infections, and from
the northeastern U.S. has been isolated from Peromyscus
rodents to humans, in whom it disease.
may cause severe
Hantavirus pulmonary
leucopus ( white-footed mouse; Song et al. 1994) and
syndrome now is known to
other, apparently heretofore unrecognized, hantaviruses
occur throughout most of the U. S., in Canada, and in
Brazil
have been isolated from Neotoma mexicana in the
and Argentina. Retrospective evidence indicates
southwest and west( B. Hjelle, personal communication,
the occurrence of
previously undiagnosed cases of HPS
in the U. S.
1995) and from harvest mice in Costa Rica( Hjelle et al.
well prior to the 1993 outbreak,
making this 1995). It is likely that, as more tests are done with
an
emerging but not a new disease. Patients with HPS
have acute pulmonary
different rodent species in different locations, and even
edema and shock. Pathogenesis
appears to be
in the same species in different ecological and
related to the presence of viral antigens in
environmental circumstances ( altitude, latitude, mean
pulmonary capillaries( Zaki et al. 1995). temperature, rain fall, etc.), additional unique
The story of the determination of the etiologic hantaviruses will be discovered. Whether the gene
agentis a classic in
epidemiology and a tribute to the
knowledge, intellect, and organizational abilities sequences of these viruses are sufficiently divergent to
of the
CDC, the U. S.
warrant considering them distinct from SNV ( i. e., Do
Army, and many others. Because much
of this work has been
the viruses cross- protect and therefore have
published, there is little reason to
epidemiologic significance?) remains to be determined,
repeat the details of this remarkable story. However, I
but it appears they do.
will stay with the general theme of this paper
by focusing Two bonuses from all this have been the focus
on the vertebrate host of SNV.
When the first required by the scientific community on the definition
serologic tests indicated that the
New Mexican patients of" virus" and whether a virus that has not been iso-
might have had infections with a
hantavirus, the scientific scurrying began. The prototype lated, but whose nucleotide sequence is entirely known
hantavirus, Hantaan virus, causes a disease known
can legitimately be called " a virus" and registered as
hemorrhagic fever
such. Keep in mind that the work of Stuart Nichol and
principally as with renal syndrome coworkers allowed not
only the sequencing of SNV and
December, 1996 Journal of Vector Ecology 197

TABLE 1. Viruses of the family Bunyaviridae, genus Hantavirus( Hantaan group; hantaviruses) by
geographic distribution and whether they cause human illness.

Complex Virus Geographic distribution Human illness

KHFa, HFRSb

Hantaan Hantaan Asia, Europe

Seoul Asia, Europe, mild HFRS
N. America, S. America
Dobrava( Belgrade) Europe HFRS
Thailand Asia
NEc

Puumala Puumala Europe, Asia

Prospect Hill N. America none known
HPSd

Sin Nombre Western U. S.

Convict Creek California
unplaced) Thottapalayam India none known
Bayou Louisiana HPS
El Moro Canyon N. America, S. America none known
Black Creek Canal Florida HPS

Tula Europe none known

Rio Mamore Bolivia HPS

aKHF= Korean hemorrhagic fever

bHFRS= hemorrhagic fever with renal syndrome
cNE= Nephropathia Epidemica
dHPS= hantavirus pulmonary syndrome

the production of antigens and antibodies against it but those patients showed that the viruses from the patients
had the potential for producing a vaccine against a virus were essentially identical to the viruses from the rodents.
that had not been isolated. This situation is becoming Sequences of a hantavirus from a patient who died in
quite common in virology. Arizona were the same as those of hantaviruses from
Hantaviruses are notoriously difficult to coax into people and rodents in the southwest corner of Colorado,

replicating in laboratory systems. Blind passages, long which was puzzling. Additional, epidemiologic
incubation periods, indirect detection systems, and investigations determined that the patient had come
significant biohazards are some of these difficulties. At from the southwest corner ofColorado; thus his infection
the CDC in Atlanta, ateam led by C. J. Peters approached likely had been acquired there and not in Arizona. This
this diagnostic problem on a variety of fronts. When was a slick bit of molecular epidemiology.
serologic tests provided evidence that a hantavirus was Availability of sophisticated molecular tools
the etiologic agent of HPS, virus isolation became a notwithstanding," the mouse" continues to play a major
priority for diagnosis and for research; however, it was role in diagnostic studies. Although laboratory diagnosis
not until six months after the first case was detected that of HPS has been made more rapid by inserting SNV
the etiologic agent was isolated ( Elliott et al. 1994). gene sequences into Escherichia coli and allowing these
Deoxyoligonucleotide primers ( from Puumala and bacteria to express nucleocapsid proteins useful as
Prospect Hill hantaviruses) were constructed and used antigens in serologic tests, including IgM antibody
in PCR assay to detect and define the gene sequences
a capture and IgG ELISAs; E. coli- expressed nucleo-
of the responsible hantavirus. Utilizing PCR practically capsid proteins of SNV have been used to immunize
as an art form, the molecular biology team, directed by laboratory mice for production of reference
Nichol, was able to retrieve hantavirus sequences from hyperimmune mouse ascitic tluids. However, detection
the lungs of HPS patients and, later, from rodent tissues. of hantaviral RNA continues to be the assay of choice
Comparisons of nucleotide sequences from case- patients for both detection and definition of hantaviruses from
and from infected rodents collected near the homes of clinical and field- collected specimens.
 198 Journal of Vector Ecology December, 1996

The emergence of SNV caused enormous concern

Personally, this has been a great opportunity for
in local Native American populations, in other residents
me. I get out of the house a bit and have a chance to meet
of the Four Corners area and of the respective states, and
mice other than white ones. The work has helped me
in potential tourists and visitors to the area. Fora while,
creep up the learning curve; until I began working with
lack of information concerning geographic distribution
Barry Beaty and doing this field work I thought
of the virus, its
transseasonality in rodent hosts, and the polyestrous was a fabric. We put four kids through
risk factors for its transmission aggravated the situation
college with the help of lab mice; I hope now to work
and led to baseless associations of the virus with Native
with a different set of mice and buy a rocking chair. But
Americans.
before I sit down one last time, I would like to know all
Because there is little information about the ability there is to know about these fascinating viruses. This
of SNV to be maintained in
transseasonally endemic may take some time.
areas, there is no reliable means of determining risks of
infection in human populations in ensuing years. For Acknowledgments
example, little is known about the origin of the 1993
epidemic. Among many possible causes are: emergence
I thank Jordi Casals, Telford Work, Robert E.
of a new virus, evolution of established viruses, genetic
Shope, Thomas P. Monath, Frederick Murphy, and
changes within vertebrate populations or genetic
many, many others for having taken the time, over many
selection of vertebrate hosts, and other epidemiologic
years, to discuss with me how people, viruses,
factors as yet unrecognized. SNV appears to be a newly institutions, and epidemics fit together.
recognized but not a new virus,
fitting the definition of
This work was supported in part by U.S. P.H. S.
emerging." Grant AI-34454 from the National Institutes of Health
Beginning in June 1994, we established
study sites
and in part by a contract" Hantaviruslongitudinalstudies
in southeastern, southwestern and, west central Colorado in Colorado,"
awarded to Colorado State University,
with
Barry Beaty, Director of the Arthropod- Borne and
Fort Collins, Colorado, by the U.S. Centers for Disease
Infectious Diseases Laboratory, Colorado State Control and Prevention, Atlanta, Georgia, through the
University. Currently, performing longitudinal
we are
Colorado Department of Health and Environment,
studies of the hantaviruses we find in those places, Denver, Colorado.
trapping rodents at each site
every six weeks. The
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Journal of Vector Ecology 21( 2): 201

Scientific Note

Breeding of Anopheles plumbeus in Tires in France

S. Karch

SIAAP- Syndicat Interdepartemental pour l' Assainissement

de 1' Agglomeration Parisienne, 14 Allee Francois ler
93600, Aulnay Sous Bois, FRANCE

Received 10 September 1995; Accepted 13 October 1995

Keyword Index: Anopheles plumbeus, mosquito, ecology, FRANCE.

Anopheles plumbeus Stephens, 1828 is widespread Sardinia, Logan ( 1953) observed that 20% of An.

in Europe, Western and Central Asia, and North Africa plumbeus breeding did not occur in tree holes, including
Knight and Stone 1977). It is one of the few species of springs, river pools, and containers.

anophelines that can breed in tree holes. It overwinters This anopheline is not considered as an efficient

in the egg stage and has two generations per year( Snow vector of malaria but in certain circumstances it can
1986). We found this species breeding in used and old transmit Plasmodium vivax. Due to its low biting
tires in a garbage dump near Paris in the summers of density it was never considered as a dangerous mosquito
1994 and 1995. More than 50% of the tires filled with in France. However, its adaptations to tires can greatly
water harbored An. plumbeus larvae. The garbage dump increase its populations in areas where the number of
was surrounded by small trees. immigrants is large. This phenomenon requires further
Anopheles plumbeus commonly feeds on humans surveillance.

at night along the edges of the Seine river, but it is

Most the colonization of tires REFERENCES CITED
relatively scare. likely,
is a secondary biotope for a mosquito population that
breeds mainly in tree holes. However, these new Knight, K. L. and A. Stone. 1977. A catalog of the
established and around mosquitoes of the world ( Diptera: Culicidae).
breeding sites are now well

them adult anophelines were biting humans in large Thomas Say Foundation, 611 pp.
numbers until the tires were treated with temephos Logan. 1953. The Sardainian project, John Hopkins,
Abate®). Baltimore.

Abnormal breeding sites of An. plumbeus have Senevet, G., L. Andavelli, and R. Adda. 1955. Presence

already been reported in flower vases

in cemeteries in d' Anopheles plumbeus surle littoral algerien. Arch.
Algeria( Senevet et al. 1955). It was thought that leaves Inst. Pasteur d' Algerie 23: 138- 139.
in the increased the content of the organic Snow, K. R. 1986, Mosquito nuisance and control in
falling vases

matter, conditions similar to tree holes. In U. K. Environmental Health 94: 265- 268.
creating