SOCIETY FOR VECTOR ECOLOGY - SOVE Home Page folder/journal/sovejournal74-2000/SOVE 1992, VO… · BULLETIN OF THE SOCIETY FOR VECTOR ECOLOGY ... S. K. Wikel, R. N. Ramachandra, and

BSOVE 17( 1): 1- 84 ( 1992)

ISSN 0146-6429

Bulletin of the

SOCIETY FOR

VECTOR ECOLOGY

1 11 1 I

4: Nr

K::: JVolume 17, Number 1 June, 1992

BULLETIN OF THE

SOCIETY FOR VECTOR ECOLOGYVolume 17- Number 1 June 1992

Marc J. Klowden, Editor

Division of EntomologyUniversity of IdahoMoscow, ID 83843

EDITORIAL BOARD

Mir S. Mulla, Chairman,

University of CaliforniaRiverside, CA 92521

R. C. Axtell W. J. Crans R. S. NasciNorth Carolina State University Rutgers University CDC- DVBIDRaleigh, NC New Brunswick, NJ Fort Collins, CO

N. Becker C. I. Dahl M. W. Service

University of Heidelberg University of Uppsala Liverpool School of Tropical Med.Germany Uppsala, Sweden Liverpool, England

D. R. Barnard F. R. Holbrook E. D. WalkerMed. Vet. Entomol. Lab USDA-ARS Michigan State UniversityUSDA, Gainesville, FL Laramie, WY East Lansing, MI

H. Briegel R. S. Lane S. K. Wikel

University of Zurich University of California Oklahoma State UniversityCH- 8057, Zurich, Switzerland Berkeley, CA Stillwater, OK

E. P. Catts G. G. Marten S. C. WilliamsWashington State University New Orleans MCD San Francisco State UniversityPullman, WA New Orleans, LA San Francisco, CA

L. S. Mian

County Vector ControlSan Bernardino, CA

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ii

BULLETIN

OF THE

SOCIETY FOR VECTOR ECOLOGY

Volume 17 June, 1992 Number 1

CONTENTS

In Memoriam- Edgar Alvin Smith( 1916- 1992) v

In Memoriam- Allan Marion Barnes( 1924- 1992) vii

BOOK REVIEW

Biology of Ticks: Volume I Daniel E. Sonenshine Reviewed by C. F. Hopla 1

PROCEEDINGS

5th SOVE European Region Meeting, Uppsala, SWEDEN2- 6 September 1990

Intermediate Hosts of Schistosomes: Ecology and Control H . Madsen and N . 0. Christensen 2

23rd SOVE Annual Conference, Sparks ( Reno), Nevada

17-20 November 1991

Immunological Strategies for Suppression of Vector Arthropods: Novel Approaches in Vector Control..S. K. Wikel, R. N. Ramachandra, and D. K. Bergman 10

SUBMITTED PAPERS

Lack of Correlation Between Peripheral Receptor Sensitivity and the Daily Pattern of Host-SeekingBehavior in Female Culex pipiens Mosquitoes M. F. Bowen 20

The Feeding and Resting Behavior of Anopheles gambiae on the Islands of Sao Tome and Principe, WestAfrica H. Ribeiro, H. C. Ramos, and J. G. Janz 25Analysis of Temperature-Dependent Stage- Frequency Data of Aedes vexans ( Meigen) PopulationsOriginated From the Magadino Plain( Southern Switzerland)

F. Fouque, J. Baumgartner, and V. Delucchi 28

A New Species of Eusaperium ( Acari: Trombiculidae) From an Incan " Rat" Opossum, Lestoros incaMarsupialia: Caenolestidae) in Peru M. L. Goff 38

Laboratory and Field Efficacy of Bacillus sphaericus 2362 Against Culex pipiens in the River LlobregatDelta( Barcelona, Spain) C. Aranda and R. Eritja 41

A Field Study of Bacillus sphaericus for the Control of Culicine and Anopheline Mosquito Larvae inTanzania R. N. Ragoonanansingh, K. J. Njunwa, C. F. Curtis, and N. Becker 45

Efficacy and Selectivity of Two Pyrethroid Insecticides Against the Predator Triops longicaudatusNotostraca: Triopsidae) and Culex tarsalis Larvae

M. S. Mulla, M. Zgomba, H. A. Darwazeh, and J. D. Chaney 51

The Distribution and Seasonal Abundance of Deer Keds( Diptera: Hippoboscidae) on Columbian Black-tailed Deer( Odocoileus hemionus columbianus) in Northern California

D. R. Westrom and J. R. Anderson 57

Absence of Ixodes dammini( DeerTicks) on Peromyscus leucopus( White-Footed Mice) in Brown and DoorCounties, Wisconsin T. D. Cleven, E. C. Burgess, R. W. Howe, and A. I. Goldsby 70

Borrelia burgdorferi in Ticks on Angel Island, California: Low Risk of Human Infection R. S. Lane 75

SCIENTIFIC NOTE

Effectiveness of a Soap Repellent Against Mansonia Mosquitoes in a Fresh Water Swamp Forest inNorthwestern Peninsular Malaysia A. Abu Hassan and V. Narayanan 83

VOLUME 17 JUNE, 1992 NUMBER 1

IN MEMORIAM

EDGAR ALVIN SMITH

1916- 1992

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On February 2, 1992, Edgar A. Smith, who devoted medical entomologist, and malaria control officer.his entire professional life to the control of mosquito- After the end of World War II, Ed returned to theborne diseases on a worldwide basis, died of cancer in San Joaquin Valley tojoin the Merced County MosquitoFairfax, Virginia. Ed will be remembered for this Abatement District. He later became manager, and inoutstanding organizational skills and for his unflagging 1953 moved to the Santa Clara County Healthzeal in solving problems associated with mosquito- Department to organize their vector control division.borne diseases in developing nations of the world. Ed used his organizational skills to great advantage in

Ed was born December 4, 1916, in Los Banos, this assignment, as well as his experience as aCalifornia. He graduated from high school in Los Banos photographer, to create a model program of publicin 1935 and attended San Jose State College, receiving education and awareness. This program was one of thea BA in biological sciences in 1939. He then did first to deal with vectors other than mosquitoes.graduate work at the University ofCalifornia at Berkeley In 1956, Ed accepted the first of many overseasand at Stanford University, receiving a secondary assignments with the Federal Government Foreignteaching credential from the latter institution in 1940. Assistance Program when he became malaria advisor inEd taught high school biology for two years, then Sumatra. Over the next 15 years he served in a similarentered the U. S. Army in 1942, serving as a photographer, role in Indonesia, Thailand, and India. During this

v

NUMBER 1 JUNE, 1992 VOLUME 17

period, Ed was involved in all aspects of these programs, began while in high school where he organized the Los

including budgeting, training, operations, and Banos Hobby Club and extended to San Jose Stateestablishment of technical specifications. In 1971, Ed where he organized the Comstock Entomology Club,moved to Washington, D. C. to become ChiefofMalaria and even to Thailand where he organized the Thailand

and Vector-borne Diseases for the U. S. Agency for Entomology Club. Ed was an active member of manyInternational Development. In this capacity, Ed was professional organizations, including the Californiaresponsible for a wide- ranging and vital program which Mosquito and Vector Control Association and the Societyincluded vector control operations and research, as well for Vector Ecology.as a malaria vaccine development program. Ed is survived by his wife, Joyce, and sons, Brandon,

Ed returned in 1982, but continued to be active Garret, and Kirk. He also leaves his twin brother, Arthur

professionally, first in organizing a training program at C. Smith, also an entomologist. Vector control has lost

the International Center for Public Health Research at an effective spokesman, as well as an interesting and

the University of South Carolina, and later as a freelance colorful friend.

consultant in vector control.

Ed Smith was adynamic and tireless spokesman for

vector control. His interests extended to all subjects in Bruce F. Eldridge

natural history. He was a highly skilled photographer Department of Entomologyand his intimate knowledge of world geography was University of California

legendary. He was also a consummate organizer. This Davis, CA 95616

vi

VOLUME 17 JUNE, 1992 NUMBER 1

IN MEMORIAM

ALLAN MARION BARNES

1924- 1992

immis

Dr. Allan M. Barnes, Chief of the Centers for Allan coauthored over 70 scientific papers rangingDisease Control Plague Section, died April 30, 1992. in topic from control of house flies and lice to fleaHe had been a member of the Society for Vector Ecology taxonomy and an overview of plague surveillance andsince 1985 and provided annual updates on the control throughout the United States. He served as a

worldwide status of plague at SOVE meetings for many consultant to the United States Agency for Internationalyears. Development and The World Health Organization' s

Allan was born in Stirling City, California, Expert Panel on Vector Biology and Control.December 8, 1924. He earned an associates degree from Allan was active in Rotary International for manyChaffee Junior College in 1951, a bachelors degree in years to promote youth exchange around the world.

Life Sciences from San Diego State College in 1953, Many students, both American and foreign exchange,and a doctorate from the University of California at have called the Barnes' residence their home for several

Berkeley in 1963. He worked for the California months at a time.

Department of Public Health, B ureau of Vector Control Besides his accomplishments as a scientist and

as a vector control specialist from 1955 to 1967. During scholar, Allan was a friend to many of us. We willthat time he was instrumental in organizing theCalifornia remember him as a man with whom we spoke about

State Zoonoses Surveillance Network. Since that time, topics as diverse as orchid culture and salmon or trout

until his death, Allan served as Assistant Chief and then fishing. Many of us will remember him as aChiefof the Plague Branch at the CDC, National Center compassionate person concerned with the welfare of his

for Infectious Diseases, Fort Collins, Colorado. His fellowman, as well as a talented teacher of science. He

laboratory was given Plague Reference Center will be missed by his friends, his scientific collegues,designation by the World Health Organization due to and indirectly by those who will be spared plaguethe worldwide scope of his knowledge, and that of his infection thanks to Allan' s many contributions to controlstaff,on the epidemiology and epizootiology of plague. of this disease.

His expertise was in demand in such countries as China,Saudi Arabia, Burma, Indonesia, India, Vietnam, Rex E. Thomas

Tanzania, and many others. During the Vietnam conflict Paravax, Inc.

Allan was stationed in- country to advise on mitigation 2301 Research Blvd., Suite 110

of exposure of troops to endemic plague. Fort Collins, CO 80526

vii

BULL. SOC. VECTOR ECOL., 17( 1): 1 JUNE, 1992

BOOK REVIEW

Title: BIOLOGY OF TICKS: Volume I1

Embryogeny; Endocrine/Neuroendocrine Regulation ofAuthor: Daniel E. Sonenshine Development and Reproduction; Water Balance in Non-Publication Date: 01/ 02/ 92 feedings Ticks; Literature Cited and Index.ISBN: 0- 19- 505910-7 Volume II will also consist of three parts: Part IV:Publisher: Oxford University Press, Inc., 200 Madison Ecology, Behavior, and Host Parasite Interactions; PartAvenue, New York, New York 10016 V: Tick-Borne and Tick Caused Diseases; and Part VI:447 pp., Cloth Price: $ 95.00 Control of Ticks and Tick-Borne Diseases. Publication

of Volume II is planned forearly 1993, probably January.The present text is the first part of a work that will Previous major works dealing with ticks contain

ultimately comprise two volumes. Volume I consists of much of value but of necessity omitted needed infor-447 pages divided into three parts. They are Part I: mation on many aspects of tick systematics, biology,Introduction, consisting of three chapters, two of which ecology, and disease relationships. Therefore, there isare separately developed to discuss Evolution and a clear need for an up- to-date general text on theseSystematics Relationship of Ticks and the Life Cycle of subjects. Volume I fills this need admirably and theTicks. Part II: Outline of Tick Body Structure, Dealing forthcoming Volume II will be of equal caliber.with External Anatomy; The Integument and Internal To the author' s credit, the most recent studies and

Anatomy: General Features. Part III is devoted to interpretations have been stressed; but where appropriate,

Structure and Function ofTick Body Organs andTissues older studies have been included, not ignored as so often

and consists of Chapter 7 through 22. They are: The happens in present day publications.Mouth Parts and Foregut, Salivary Glands; Midgut, The text is amply illustrated by the careful use ofHindgut, Malpighian Tubules, and Coxal Glands; high quality illustrations of various types. The volumeCirculatory System and Hemolymph; Respiratory is highly recommended, not only to serious students ofSystem; Fat Body; Nervous andNeuroendocrineSystem; acarology, but to medical personnel, vector controlSense Organs and Sensory Physiology; Female officers, and all libraries. The author has worked

Reproductive System; Male Reproductive System; diligently for several years to produce this tome. HisCytogenetics and Genetics of Ticks; Tick Pheromones; careful consideration shows throughout the text.

Reviewed by C. E. Hopla, Department of Zoology, University of Oklahoma, Norman, OK 73019- 0235, U.S.A.

BULL. SOC. VECTOR ECOL., 17( 1): 2- 9 JUNE, 1992

INTERMEDIATE HOSTS OF SCHISTOSOMES:

ECOLOGY AND CONTROL'

H . Madsen2 and N. 0. Christensen2

ABSTRACT: The biology, ecology, and habitats of the snail intermediate hosts of human schistosome species arebriefly reviewed and the role of snail control in schistosomiasis control is reviewed and discussed. The presentstrategy of schistosomiasis control comprise morbidity control within the framework of the primary health careapproach. Maintenance of a low level of morbidity requires repeated chemotherapy at a level which is neitherlogistically nor economically feasible and this underlines the necessity of parallel preventive measures, such as snailcontrol, to reduce the level of exposure to cercariae- infested water. Snail control through focal molluscicide

application is most likely to contribute to reduced morbidity in highly endemic areas where transmission is focal andthe transmission pattern is easy to identify. Furthermore, in order to secure community participation in snail controloperations, schistosomiasis should be perceived as a major health problem. Environmental control measures have

proven particularly effective against amphibious snails, but less promising against aquatic species. Biological

control of snails is still at an experimental stage.

INTRODUCTION formosana ( Pilsbry and Hirase), chiui ( Habe and

Miyazaki), nosophora( Robson), and lindoensis Davis

Schistosomiasis is endemic in many countries in and Carney) are intermediate hosts for S. japonicum.Africa, South America, and Asia with an estimated Tricula aperta( Temcharoen) acts as host ofS. mekongi,

number of200 million infectedpeople( Doumenge et al. while Robertsiella kaporensis Davis and Greer is

1987). There are three important species that cause intermediate host for the S. japonicum-like species in

schistosomiasis in humans, namely, Schistosoma Malaysia and Thailand( Jordan and Webbe 1982).

mansoni Sambon in Africa, the Middle East, and South

America, Schistosoma haematobium( Bilharz) in Africa The Intermediate Hosts

and in the Middle East, and Schistosoma japonicum Oncomelania hupensis ssp. are small, amphibious,Katsurada in South- East Asia( Jordan and Webbe 1982). and dioecious snails inhabiting flood plains andIn addition, there are some species of more limited especially man- made habitats resulting from agriculturaldistribution, i.e., Schistosoma intercalatum Fisher in development( Mao 1948, McMullen et al. 1951, Pesig anZaire, Cameroon, and Gabon, Schistosoma mekongi et al. 1958, Cheng 1971, Blas 1976, Shou- Pai and Bao-Voge, Bruckner, and Bruce in Southern Laos and Ruo 1982). Irrigation and drainage canals, roadside

Kampuchea and a S. japonicum- like species in Malaysia ditches, and rice fields are thus important transmission

and Thailand( Jordan and Webbe 1982). sites. Snails are found primarily outside water on theEach of these species has its characteristic inter- banks but some are also found in very shallow water,

mediate snail host spectrum. The intermediate hosts of i.e., at a depth of less than approximately 20 cm( Shou-S. mansoni, S. haematobium, and S. intercalatum belong Pai and Bao- Ruo 1982). Snails may also be found in theto the Pulmonata, family Planorbidae ( Brown 1980), mud, mostly in the upper 2 cm. Shaded habitats with awhile those of S. japonicum and S. mekongi belong to moderate and relatively constant temperature arethe Prosobranchia, family Pomatiopsidae ( Kitikoon preferred by O. hupensis subspecies. A water velocity1984). Various species of Biomphalaria serve as above 0. 14 m/ s is generally unfavourable for O. hupensisintermediate hosts for S. mansoni, and various Bulinus ( Cheng 1971).species are intermediate hosts for S. haematobium and Depending on the O. hupensis subspecies, eggs areS. intercalatum. Certain subspecies of Oncomelania laid singly either on solid objects in the water or on thehupensis( i.e., hupensis Gredler, quadrasi( MSllendorff), mud or even on objects outside the water. Hatching

Presented at the Fifth European Regional SOVE Meeting, Uppsala, SWEDEN, September 2- 6, 1990.

2Danish Bilharziasis Laboratory, WHO Collaborating Centre for Applied Medical Malacology and SchistosomiasisControl, Jaegersborg All6 1 D, 2920 Charlottenlund, DENMARK.

JUNE, 1992 BULL. SOC. VECTOR ECOL. 3

occurs after 7- 61 days, depending on the temperature, commonly being of a focal and seasonal nature. Theand the newly hatched snails pass through an aquatic focality and seasonality in the transmission ofstage of some weeks before entering the amphibious schistosomes are key issues for snail control.stage. The snails reach sexual maturity in 9- 16 weeksand may live for 24- 35 weeks on the average, but snailshave been reported to live for more than 2- 3 years. Their Schistosomiasis Controlreproductive potential is low compared to that of the Snail control was earlier considered to be a keypulmonates. Populations of O. hupensis spp. undergo factor in the control of schistosomiasis and was attemptedrelatively small fluctuations in density. mainly through area-wide application ofmolluscicides.

Tricula aperta is found in parts of the Mekong and However, this approach has changed during recentMun rivers where it clings to rocks, sticks, and other years due to the development of safe and effective anti-solid objects in running, well-aerated, clear water schistosomal drugs, the development of simple andUpatham et al. 1980). Population densities appear to be rapid diagnostic techniques, and the increased emphasis

controlled by the annual rise and fall in the water level on the primary health care approach to schistosomiasisof the river. During the rainy season the flow is control. In addition, the high cost of molluscicides andtorrential and the species seems to persist as eggs the relatively unsatisfactory results of large- scale area-attached to the underside of stones. Female snails live wide mollusciciding campaigns have further contributedless than ayear and apparently lay large numbers ofeggs to this development.prior to the onset of the rains ( Upatham et al. 1980). The present strategy of schistosomiasis control isRobertsiella kaporensis may be found in small streams based on morbidity control within the framework of theattached to rocks, leaves, and twigs, but are most abundant primary health care system( Mott 1984). Operationalin overgrown areas where they attach to roots( Greer et experience with this strategy is rather limited and theal. 1980). few control programmes that have been conducted have

Biomphalaria and Bulinus species are aquatic and largely been based on chemotherapy. The experiencehermaphroditic snails which can be found in almost all from these programmes reveals that in areas highlytypes of water bodies, ranging from small temporary endemic for schistosomiasis, maintenance ofa low levelponds or streams to large lakes and rivers( Brown 1980). of morbidity requires repeated chemotherapy at aMan- made water bodies like irrigation canals and darns frequency which is neither logistically nor economicallyare, in particular, excellent habitats; and because of the feasible( Butterworth et al. 1987, Wilkins 1989). Thisintense human water contact, these may play a very had led to the conclusion that morbidity reductionimportant role in the transmission of schistosomiasis. achieved through chemotherapy can best be maintainedEggs ofBiomphalaria and Bulinus species are deposited through parallel preventive measures reducing the levelin masses on plants or solid objects in the water. Under of exposure to cercariae- infested water( Wilkins 1989).optimal temperature conditions hatching takes place Such reduction in risky contacts with cercariae-after 7- 10 days. Snails may reach sexual maturity after infested water may principally be achieved through4- 6 weeks. The average longevity varies among species water supply and sanitation programmes backed up byand with local conditions. Their reproductive capacity intensive health education. If the necessary communityis high and populations of these species may undergo collaboration is established, such programmes maymarked seasonal fluctuations in density. contribute to reduced water contacts related to domestic

Snails may be widely distributed in an area, but and personal activities( Jordan 1977, 1988). However,there is a tendency for Schistosoma infected snails to be such programmes will not significantly contribute tofocally distributed due to the focality of human water reducing water contacts of a recreational nature amongcontacts. Focal infection rates with schistosomes may the younger segment of the human population, as wellbe quite high and this is the case both for the prosobranchs as occupational water contacts will not be affectedOemijati 1976, Sudomo 1984) and the pulmonates ( Jordan 1977). Water and sanitation programmes shouldMadsen et al. 1987). In large irrigation schemes, most therefore be supplemented by provision of snail- free

infected Biomphalaria and Bulinus snails are found water bodies suitable for sustaining recreational andclose to human settlements( Babiker et al. 1985, Madsen domestic activities. It would appear realistic that healthet al. 1987). education directed towards reduc ing risky watercontac ts

Snail populations undergo great seasonal variations might be successful once suitable and infection- freeindensity and infection rates, particularly the pulmonates. water bodies are provided. Snail control thereforeRainfall and/ or temperature are the main causative becomes central to schistosomiasis morbidity controlfactors. This results in the pattern of transmission programmes( Fenwick 1987, Klumpp and Chu 1987).

4 BULL. SOC. VECTOR ECOL. JUNE, 1992

Snail Control care approach rather than the prospect of finding aSnail control may be achieved principally through compound which is superior to synthetic molluscicides

chemical, environmental, or biological means. At in efficiency or less toxic to the environment. Attemptspresent, the most reliable method of achieving drastic to involve the community in the preparation andreductions in snail population density in the short term applicationofaS. madagascariensisbasedmolluscicide

is the use of molluscicides( McCullough 1986). Many in Tanzania, however, was not successful since this

different molluscicides have been used during the past, activity was considered as an overload which did notbut at present only one product is readily available, produce perceivable results ( Mutakyawa et al. 1990).

namely, niclosamide ( McCullough 1986). The high This emphasizes the fact that community participationcost of niclosamide( McCullough and Mott 1983) and in snail control can only be anticipated ifschistosomiasisits environmental impact( i. e., primarily toxicity to fish) is recognized as a major health problem. Validation of

Andrews et al. 1983) make it desirable to search for the plant molluscicide approach to snail control awaits

new approaches to snail control. Such include modified further chemical, ecotoxicological, and especially

techniques and strategies for molluscicide application, operational research. However, recent safety testing ofenvironmental control, and biological control methods P. dodecandra( Endod) following OECD guidelines forso as to either reduce amounts of niclosamide used or to pre- market chemicals was satisfactory and it wasprovide more long- lasting effects. This need is concluded that schistosomiasis intervention trials usingfurthermore enforced by the possibility of the Endod are now justifiable( Lambert et al. 1990).

intermediate hosts developing resistance to niclosamide New procedures of molluscicide delivery are beingJelnes 1977, 1987). Presently, however, this pheno- investigated. Interest has thus been paid to the develop-

menon has not yet hampered snail control operations ment ofimproved controlled release formulations, such

using niclosamide ( McCullough and Mott 1983). A as soluble glasses releasing copper compounds

number of environmental control measures exists that ( Chandiwana et al. 1987). Third generation controlled

could be used to reduce snail population densities release formulation, as described by Thomas et al.

especially in irrigation schemes. These methods have ( 1980), is another interesting approach which meritsproved to be particularly effective against the amphibious further investigation. Such formulations release specific

snails, but less promising against aquatic species. attractants, arrestants, and phagostimulants attracting

Biological control of snails is still at an experimental the snails and inducing them to ingest the matrix withstage. Presently, the most promising candidates are subsequent release of a molluscicide into the snails' gut.

other non-susceptible snails which may act as competitors Thomas et al. ( 1983, 1986, 1989) have identified

of the intermediate hosts. propanoic and butanoic acids as attractants and maltose

as a phagostimulant for Biomphalaria glabrata( Say).Transmission of schistosomiasis is generally

Molluscicides focalized rather than widespread and focal- seasonal

At present, only one molluscicide is commercially application, i.e., application of molluscicide at the most

available, namely, niclosamide; and new molluscicides important transmission sites during the most importantare being evaluated only on a limited scale as there is no transmission period, is one way to reduce the quantity ofsecure market for these products( McCullough 1986). molluscicide used. Obviously, focal molluscicideHowever, B- 2 ( B- 2- sodium,2, 5 dichloro-4- bromo- application in lotic habitats poses special problems

phenol) ( Wong et al. 1988), chloroacetamide and ( Madsen et al. 1986). In the Gezira Agricultural Scheme

bromoacetamide ( Zhu and Yin 1986) have shown in Sudan, a standard procedure of applying molluscicide

promising effects on 0. hupensis. Some encouraging to all major human water contact sites, has been

effects against aquatic snails have also been obtained developed( Gaddal 1985), and another example of focal

with organotin compounds incorporated in slow- release application with niclosamide in Mali was given bymatrices( McCullough et al. 1981). There has also been Madsen et al.( 1986). Admittedly, however, operationalan increasing interest in the development of plant experience with this approach has been less than

molluscicides prepared locally and based on local plants, satisfactory( Werler 1986) pointing out that support andin particular, Phytolacca dodecandra( L' Herit), Swartzia supervision are crucial to achievement of effectiveness

madagascariensis( Desv.), and Tetrapleura tetraptera in this approach.

Taub.) ( Suter et al. 1986, Mott 1987, Maillard et al. Much criticism has recently been paid to snail1989). The main stimuli for this interest are the possibility control in schistosomiasis control. Admittedly, area-

of spending less foreign currency and of incorporating wide control is neithereconomic nor logistically feasiblesnail control into the community based primary health and thus presently non- existent, while criticism directed

NNE, 1992 BULL. SOC. VECTOR ECOL. 5

at focal snail control has taken the starting point in the phase of new irrigation projects, for example bybelief that comprehensive knowledge concerning the constructing weirs or drop structures that will completelytransmission is required for implementing this approach. drain out when canals are not used ( Bolton 1988).Application of molluscicide by the community in Structural changes in already existing schemes may notschistosomiasis morbidity control programmes would be possible for practical and/or economical reasons.admittedly require the development of a simple standard Water storage reservoirs may constitute importantmethodology for molluscicide application and for transmission sites in irrigation schemes. Regulartransmission site identification. However, identification fluctuations in water level in such reservoirs mayof major water contact sites used for recreational and significantly affect snail populations ( Oomen et al.domestic activities is usually possible, and simple 1990). If access to reservoirs is prevented, for exampleecological criteria could probably be developed for by fencing, the possibility should, however, beidentifying the main period of transmission. Such considered that habitats outside the scheme may becomecriteria would obviously differ from area- to-area, but important sites for recreational water contact.parameters to be considered comprise presence of host In large irrigation schemes, snails are oftensnails, water velocity, presence ofsubmergedor floating associated with aquatic macrophytes( Hilali et al. 1985,aquatic macrophytes, permanence of habitats, etc.( see Madsen et al. 1987, 1988), and control of these mightfor example Ali and Byskov 1986). contribute significantly to reducing snail populations.

The backbone in schistosomiasis control should be However, experience from the Gezira Agriculturalchemotherapy combined with health education, water, Scheme shows that mechanical weed removal alone didand sanitation programmes. Snail control should never not substantially reduce snail populations and the weedsstandalone. Snail control, however, couldbe an effective soon repopulated the canals ( Hilali et al. 1985).community based supplement to these measures in Mechanical weed control, both by machinery and byareas where transmission is highly focal and possibly man power, is extremely expensive and difficult, as italso seasonal. In areas where the transmission pattern is has to be repeated at short intervals( Oomen et al. 1990).less clear-cut and transmission widespread, snail control Mechanical removal of macrophytes is normally donewould be extremely difficult to incorporate into the to secure optimal operation of the canal system.

primary health care system. Such snail control operations The search for alternative measures, such asshould be combined with health education and it should biological methods for weed control, should bebe realized that community participation can only be encouraged. For example, Schayck ( 1986) found thatexpected if the community perceives schistosomiasis as biological control of macrophytes by means of the grassa major public health problem. carp, Ctenopharyngodon idella( Val.), resulted in lower

total plant cover than both chemical and mechanicalEnvironmental Control methods and greater reductions in the densities of the

A number of environmental control measures exist intermediate hosts.

that could be used to reduce snail population densities,

especially in irrigation schemes. Amphibious snails are Biological Control

particularly sensitive to such measures owing to the Biological control of the intermediate host snails ofnature of their habitats( Cheng 1971, Blas 1976, Shou- schistosomes is at present mainly in the experimentalPai and Bao-Ruo 1982). Such measures include stream phase. The most promising approach is the introductionchannelization, seepage control, canal lining, canal of non- susceptible snail species which may act asrelocation with deep burial of snails, proper drainage in competitors of the intermediate hosts ( World Healthirrigation schemes, removal of vegetation, earth filling, Organization 1984, Madsen 1990). The most extensivelyponding, and improved agricultural practices. These studied competitors are Marisa cornuarietis ( L.),measures are expensive to implement but they are Helisoma duryi( Wetherby), Thiara granifera( Lamarck)effective in the control of Oncomelania hupensis ssp. and Melanoides tuberculata( Muller), and among these

Some of these methods may be of value also in the the most encouraging results for the time being are thosecontrol of aquatic snails but to a much lesser extent than reported for the thiarid snail species ( T. granifera andin the control of amphibious snails. Often the design of M. tuberculata) in the Caribbean area ( Pointier andsluice gates and various weirs, especially inverted weirs, McCullough 1989). There is evidence that theseissuchthattheycreatefavourablebreedingsites( refugia) competitors may be effective in certain habitats, butfor snails, even in irrigation schemes where most canals there seems to be the important limitation in their useare concrete- lined. Improved designs of such structures that their habitat preferences only partially overlap withshould be considered, particularly during thepreparation those of the intermediate hosts. There is evidence that


Biomphalaria straminea, which is relatively poorly approach, has been shown to be effective in the short

susceptible or insusceptible to S. mansoni, is replacing term, but sustainability is problematic ( Tanner 1989).Biomphalaria glabrata in some areas ( Barbosa et al. However, it is still felt that there is a great potential for

1981, Guyard et al. 1982) and its use in biological the integration of a substantial part of control activities

control ofschistosomiasis has been suggested. However, in a primary health care system, provided that the aimsthis approach was not recommended by a WHO and sequences of control interventions are well matched

consultation ( World Health Organization 1984). The with the needs felt by the communities concerned( Tannerspecies is an important intermediate host in parts of 1989).

Brazil (Paraense 1983, Malek and Rouquayrol 1986), Hopefully, future developments in molluscicideand although insusceptible strains can be found, there is delivery methods, environmental orbio-control methodsno guarantee that they stay resistant and that they are for example, will provide more simple and operationally

resistant to newS.mansoni strains that might be imported. feasible tools for snail control within the framework of

Introductions of new snail species to an area should a community based primary health care system.obviously only be done after a careful evaluation of theirpotential status as carriers of parasites of medical or

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IMMUNOLOGICAL STRATEGIES FOR SUPPRESSION OF VECTOR

ARTHROPODS: NOVEL APPROACHES IN VECTOR CONTROL'

S. K. Wike12, R. N. Ramachandra2, and D. K. Bergman2

ABSTRACT: Significant medical and veterinary public health benefits are gained from the effective control ofvector-borne diseases. Reduction of vector arthropod populations is a central approach for disease prevention. Use

of insecticides and acaricides has been the long- standing means of achieving that objective. Significant problemsand concerns have arisen from the use of these chemicals. These include:( a) resistance of arthropods to the control

compounds,( b) their impact upon nontarget species and the environment,( c) meat/milk residues,( d) cost, and( e)

human health concerns. Alternative methods of vector control need to be developed which could be used alone or

as a component in an integrated vector suppression strategy. A particularly promising area of investigation is theimmunological control of blood feeding arthropods. Studies described focus upon host- tick associations. Thecomplex array of host immune responses stimulated by tick infestation include antibody, cytokines, complement,and cell mediated effector mechanisms. The feeding tick modulates the host immune system by altering cytokineelaboration by macrophages and T- lymphocytes. Tick induced immunosuppression is expressed by reducedresponses of host lymphocytes to T-cell mitogens and reduced antibody responses to thymic dependent antigen.Reduced host immune competence could facilitate tick feeding and pathogen transmission. Thorough understandingof the immune mechanisms involved in acquired resistance is essential for the development of immunological

control strategies for ticks. Immunogens used to induce anti- tick immunity include extracts of whole ixodids,salivary glands, reproductive tract, and gut. Recent studies have focused upon identification, isolation, and

characterization of novel immunogens from tick digestive tract and integument. Specific protection inducingmolecules have been identified and a number of vaccine trials are in progress. Immunization based control can likelybe applied to a number of arthropod species of medical and veterinary public health importance.

INTRODUCTION Ticks are the most important vectors of infectious

agents to domestic and wild animal species, rankingControl ofblood feeding arthropodsandthediseases behind mosquitoes as transmitters of pathogens to

they transmit is a significant medical and veterinary humans ( Balashov 1972). An increasing variety andpublic health concern. Vector suppression remains the complexity of tick-borne diseases have been recognizedkey element for controlling this large complex of diseases ( Hoogstraal 1981). Worldwide incidence of LymeLaird 1985). Insecticides and acaricides are the central borreliosis continues to increase for humans, companion

elements of effective arthropod control programs. animals, and domestic animals( Steere 1989, Szczepanski

However, resistance of vectors to these compounds is a and Benach 1991). Extensive research efforts have

serious threat to animal production ( Brown 1983, focused upon control of the tick vectors, vaccine

Solomon 1983). Resistance to a vector control chemical development, and discovery of more effectivecan occur within a few years after the introduction of the pharmacological agents. Ehrlichiosis is emerging as acompound. A variety ofcharacteristics ofboth the com- tick-borne disease of increasing medical and veterinarypound and vector influence the rate of resistance develop- importance ( Magnarelli 1990). Effective control of

ment. Elements of resistance development related to anaplasmosis remains a significant veterinary publicarthropods include genetic variability of the population, health concern( McCallon 1973). Significant gaps exist

reproductive capacity, and the rate at which mut:itions in our knowledge of these and other tick-borne diseases

occur. Factors related to the control compound include in regard to transmission dynamics, development of the

frequency of application, persistence, and exposure to a pathogen within the vector, host responses to infection,

compound with a related structure or mode of action. and pathogenesis.

1Presented at the Annual SOVE Conference, Reno, Nevada, 20 November 1991.

2Department of Entomology, 127 Noble Research Center, Oklahoma State University, Stillwater,Oklahoma 74078,U.S. A.


The Food and Agriculture Organization ( 1984) TICK SALIVA

estimated the economic impact due to ticks and the

diseases they transmit at seven billion dollars per annum. Salivary gland expressed proteins change duringInfestation and tick-borne diseases are a major constraint the course of tick feeding ( Oaks et al. 1991). Ixodid

on agricultural development in tropical and sub- tropical salivary secretions contain a variety of immunogens andregions. Quality of life in these regions could be greatly pharmacologically active molecules ( Wikel 1988;enhanced by effective tick control. Significant concerns Ribeiro 1987a, 1987b; Ribeiro et al. 1992). Anti-

include vector resistance to acaricides, impact of these hemostatic, anti- inflammatory, and immunosuppressivecompounds upon the environment, meat/milk residues, factors have been identified in the saliva of Ixodes

and human health implications. The need clearly exists dammini( Ribeiro et al. 1987b). Platelet aggregation is

for development of alternative control strategies. One inhibited by salivary apyrase. Prostaglandin E2 from 1.potentially very useful approach is the induction of dammini saliva inhibited IL-2 production by a T-immunologically mediated host resistance to the vector lymphocyte hybridoma, thus, identifying one potentialby vaccination. immunosuppressive substance. Salivary kinase would

antagonize pain and edema promoting properties ofHOST IMMUNITY TO TICKS prostaglandin E2 associated with the inflammatory

response.

Cattle and laboratory animals acquire resistance to Salivary glands of feeding D. andersoni containtick infestation as a consequence of exposure to ixodid active agents that act against coagulation factors V and

feeding, and the immunological basis of the anti- tick VII( Gordon and Allen 1991a). Ixodes dammini saliva

response has been investigated( Willadsen 1980; Wikel contains anaphylatoxin inactivating activity, which1982a, 1984, 1988). Expression of acquired resistance inhibited smooth muscle contraction and edema

results in reduced numbers ofengorged ticks, decreased formation; however, neutrophil aggregation was not

volumes of blood meals, reduced numbers and viability altered by tick saliva ( Ribeiro and Spielman 1986).of ova, increased feeding times, impaired pathogen Alternative pathway of complement activation, whichtransmission, and tick death. can result in production of the anaphylatoxins C3a and

Immunological effector elements involved in the C5a, was shown to be involved in expression of anti- tick

expression of host acquired anti- tick resistance include immunity ( Wikel 1979). Gordon and Allen ( 1991b)

circulating and homocytotropic antibodies, cell mediated reported that tick salivary antigens activated complement

immunity expressed as cutaneous basophil hyper- component C5 to produce chemotactic activity.

sensitivity, complement, and other soluble mediators Inhibition of the alternative complement activation

Willadsen 1980; Wikel 1982a, 1984, 1988; Brossard et pathway was subsequently shown to be associated withal. 1991). a 49 kilodalton ( kdal) molecule of I. dammini saliva

Host immune competence is modulated by the ( Ribeiro 1987a). Ixodes dammini saliva contains

feeding tick, thus, facilitating engorgement and pathogen prostacyclin, which could increase blood flow at the bite

transmission. Tick induced host immunosuppression site and prevents leukocyte degranulation( Ribeiro et al.

results in impaired in vitro proliferative responses to T- 1988). Impact of this molecule upon neutrophil function

lymphocyte mitogens ( Wikel, 1982b) and reduced includes inhibition of aggregation, granule enzyme

primary antibody responses to a thymic dependent secretion, and phagocytosis ( Ribeiro et al. 1990).

antigen( Wikel, 1985a). FemaleDermacentorandersoni Immunological and pharmacological interactions at the

salivary gland extracts, prepared daily during the course host-vector interface are extremely dynamic. It is likelyofengorgement, suppressed in vitro responses ofnormal that adaptations to host defenses and activities of

lymphocytes to the T-cell mitogen concanavalin A, substances introducedby the vector are important factorselaboration of the macrophage cytokines interleukin- 1, in determining the range of effective host- vector-IL- 1, and tumor necrosis factor alpha, TNF, and the pathogen relationships.

lymphocyte cytokines interleukin-2, IL-2, and gamma Production ofhost antibodies is induced by a varietyinterferon, IFN-G ( Ramachandra and Wikel 1992).

of molecules contained in tick saliva. Specificity ofDisruption of the normal host cytokine immuno-

many of these immunoglobulins have been identified byregulatory network appears to impact the macrophageand T-helper- 1 lymphocyte subpopulation. Most intense immunoblotting. Rabbits hypersensitized by infestation

suppression of host cytokine responses correlateswith Hyalomma anatolicum anatolicum developed

positively with the time interval during which most tick- antibodies that reacted with nine saliva proteins and 17

borne pathogens are transmitted ( Ramachandra and bands in an electrophoretogramof salivary gland extract

Wikel 1992). prepared from female H.anatolicum anatolicum allowed


to feed for 96 hours ( Gill et al. 1986). Guinea pigs Ultrastructural studies performed in our laboratoryresistant to Rhipicephalus appendiculatus infestation revealed that cellular elements attracted to the tick

developed antibodies reactive with salivary gland attachment site by the immune response of resistantcomponents over a molecular weight range of 16 to 120 animals damaged the digestive tract of the feedingkdal. ( Shapiro et al. 1986). Salivary gland components ixodid( Voss- McCowan 1991). Intact cells and granules

of female D. andersoni reactive with host antibodies of basophils and eosinophils were phagocytized by gutwere observed to vary in concentration during the epithelial cells, and death of ticks in situ was correlated

course of engorgement ( Gordon and Allen 1987). with observed ultrastructural changes.

Salivary glands obtained from female Amblyomma Knowledge about the immune responses stimulated

americanum at selected times during the course of by tick feeding and the impact of these responses uponengorgement contained several polypeptides that tick biology are increasing. TABLES 1 and2provideanconsistently reacted with host antibodies ( Jaworski et overview of the immune responses induced and elicited

al. 1990). Tick infested cattle, guinea pigs, and rabbits by initial and repeated host exposures to tick feeding. Adeveloped cross- reactive antibodies to A. americanum thorough knowledge of the immunology of acquiredand D. andersoni as a consequence of infestation with resistance to ixodid infestation is necessary in order toeither species( Wikel and Whelen 1986). Exposure to develop rational strategies for the design of anti- ticktick feeding induces a broad range of tick salivary gland vaccines.

reactive immunoglobulins. Antibodies are clearlyimplicated as effector elements in the expression of VACCINATION

acquired resistance to infestation ( Willadsen 1980;

Wikel, 1982a, 1984). Initial efforts to vaccinate cattle and/or laboratoryLaboratory animal species and bovines develop animals against tick infestation involved use of crude

antigen specific cell mediated immune responses to tick extracts of whole ixodids or isolated tissues ( Wikel

infestation, as measured by delayed type hypersensitivity 1982a, 1984, 1988). Recent studies have focused uponskin reactivity and in vitro lymphocyte proliferation the use of defined immunogenic molecules andWikel et al. 1978, George et al. 1985). Salivary gland innovative approaches to resistance induction ( Wikel

extracts were used as the antigen sources in these 1984, 1988; Rand et al. 1989; Rutti and Brossard 1989).studies. Histopathology at tick attachment sites on Trager ( 1939) immunized guinea pigs with ananimals expressing acquired resistance is characterized extract of whole larvae of Dermacentor variabilis.

by a cutaneous basophil hypersensitivity response( Wikel Induced host- immune response protected against a1982a), a reaction regulated in part by cell mediated challenge infestation with D. variabilis larvae. Animmune effector mechanisms( Askenase et al. 1979). extract of Ixodes holocyclus larvae was used to induce

TABLE 1. Acquisition of immune responsiveness by host during first exposure to tick infestation.

Tick attachment and introduction of changing array of immunogens and pharmacologically active molecules.

Antigen processing by Langerhans cells and macrophages and presentation of immunogen to B and T-lymphocytes.

Primary antibody response and stimulation of initial regulatory and effector T-lymphocyte response.

Activation of classical and alternative complement pathways.

Antigen clearance.

Generation of memory cells.

PRIMARY EXPOSURE CHARACTERIZED BY HIGH LEVEL OF ENGORGEMENT AND LIMITEDRESPONSE TO INFESTATION. FEEDING COMPLETED BEFORE PRIMARY IMMUNE RESPONSESTRONGLY EXPRESSED.


TABLE 2. Expression of acquired resistance to tick infestation.

Tick attachment and introduction of saliva.

Antigen processing and presentation to antigen reactive B and T-lymphocytes. Memory cells activated.

Circulating antibody and complement with direct impact upon ticks.

Cutaneous basophil hypersensitivity response.

Anaphylatoxins and homocytotropic antibodies result in release of mediators from basophils and mast cells.

Attraction of cells to the reaction site.

Direct damage to feeding tick and antigen clearance.

ACQUIRED RESISTANCE CHARACTERIZED BY CUTANEOUS HYPERSENSITIVITY AT TICK

ATTACHMENT SITES, IMPAIRED ENGORGEMENT, REDUCTION IN OVA PRODUCTION, AND

REDUCED VIABILITY OF CHALLENGE TICKS.

resistance to a challenge with larvae of the same species animals. Cutaneous reactions at tick feeding sites onBagnal11975). Vaccination inducedresistanceresulted immunized animals can serve as foci for secondary

in rejection of 38 to 68 percent of infesting larvae. An infections and infestations. Proper selection of

important finding was the presence ofcutaneous basophil immunogens, adjuvants, and delivery systems must

hypersensitivity reactions at tick attachment sites on focus on effective induction of resistance and

vaccinated animals. An extract of whole H. anatolicum development of responses that do not result in" lumps,

anatolicum was used to induce a marked degree of bumps, and lesions" upon challenge infestation.

resistance in rabbits to subsequent infestation with the Significant progress is being made in the identification,same species( Manohar and Banerjee 1992). isolation, and characterization of potential protection

A logical extension of research focusing upon inducing immunogens. An important commercial

characterization of immunity to tick bite was the consideration is to obtain sufficient quantities ofmaterial

development of vaccination regimens that used tick for vaccine production. Biotechnology can provide asalivary glandderived molecules as immunogens. Calves variety of effective solutions to that problem.immunized with salivary glands of partially engorged Cattle have been protected against infestation with

Boophilus microplus developed significant resistance H. anatolicum anatolicum by immunization with wholeto infestation( Brossard 1976). Salivary glands obtained salivary gland extract or a 10,000 x g supernatant offrom partially engorged, unfertilized, female D. homogenized salivary glands obtained from partiallyandersoni were disrupted and a 10,000 x g supernatant fed female ixodids( Banerjee et al. 1990). The 10,000 x

was used to vaccinate guinea pigs ( Wikel 1981). g pellet did not induce a protective response. It is

Immunization induced resistance did not differ important to note that the 10, 000 x g supernatant wouldsignificantly from that of animals expressing acquired contain membrane fragments. Protection inducinganti- tick immunity. Hypersensitivity reactions elements in these extracts induced both antibody anddeveloped at tick attachment sites on immunized animals. cell mediated immune responses. Importance of

Rabbits were protected against Rhipicephalus assessing both immunoglobulin and cell mediatedappendiculatus infestation by immunization with immune reactions at each step ofimmunogenpurificationextracts obtained from adult females and males of that cannot be overemphasized. Cell mediated reactivity isspecies, which had engorged for four to five days essential for regulation of the host immune response and

Nyindo et al. 1989). for anti- tick effector functions.

A significant concern relative to a commercially Broadly recognized T-lymphocyte epitopes mustpractical anti- tick vaccine is development of cutaneous be a component of any vaccine in order to assure

hypersensitivity at tick attachment sites on vaccinated responsiveness of individuals within a randomly bred


population( Sher 1988). Recombinant produced subunit tick feeding. An adult female ixodid increases from anvaccines might be effectivelyprocessedbyonlyalimited unfed weight of ten milligrams to a final engorgement

range of major histocompatibility antigens. Therefore, weight of 1, 000 milligrams and proces ses approximatelythe responsiveness of the general population could be 4,000 milligrams of blood( Kaufman 1989). Therefore,

highly variable. Some individuals might respond very it is clear that tick digestive tract epithelial cells are

well, while others might be unresponsive. Screening for potentially exposed to a considerable amount of hostT- lymphocyte reactivity is essential at each immunogen immunoglobulin.

purification step. It would be tragic to develop a vaccine Willadsen et al. ( 1988) reported successful

only to find that many animals in the general population vaccination of cattle with material isolated from semi-

were incapable of responding to the immunogen. The engorged female Boophilus microplus. Protection

key to responsiveness is effective processing and inducing immunogens were isolated from crudepresentation of immunogen by class II major membrane preparations by detergent extraction, gelhistocompatibility antigens( Berzofsky et al. 1988). permeation chromatography, and preparative isoelectric

The most effective assay for vaccine potency is the focusing. Further purification resulted in identificationability of individual fractions to induce resistance to of the protection inducing molecule as a membraneinfestation. These studies are time consuming and bound glycoprotein with a molecular weight of 89 kdal,

costly due to the number of animals required. an isoelectric point 5. 1 to 5. 6, and an affinity for wheatDevelopment of an in vitro assay that correlates with germ lectin( Willadsen et al. 1989). Immunogen was invaccine potency would be a tremendous asset. very low abundance in partially engorged female B.

Molecules not associated with saliva and induction microplus. Vaccination with microgram quantities ofof acquired resistance have shown significant potential immunogen induced host immunity, which reduced tickas immunogens for vaccines to induce anti- tick engorgement, egg production, and survival( Willadsenimmunity. Guinea pigs were immunized with either et al. 1989). Antibodies obtained from immunizationmidgut/ reproductive tract or all internal organs of protected cattle inhibited endocytotic activity of tick gutpartially engorged female D. andersoni ( Allen and cells( Willadsen et al. 1989). Since ixodid ticks utilizeHumphreys 1979). Administration ofcombined midgut intracellular digestion, the impact of the anti- tickand reproductive tract immunogens induced a host response could be significant. Protection inducingresponse that resulted in reduced engorgement and glycoprotein was subsequently cloned and expressed byinhibition of the hatching of ova. It is likely that damage Escherichia coli as a fusion protein, consisting of 599to the gut allowed immune effector elements increased amino acids of tick origin and 651 amino acids of beta-access to reproductive tissue epitopes. Rats immunized galactosidase. Fusion protein containing E.coli inclusionwith midgut tissue of female and male D. variabilis bodies was used to induce a bovine anti- tick responseinduced a significant level of resistance to challenge ( Rand et al. 1989).infestation ( Ackerman et al. 1980). These studies Opdebeeck et al.( 1988) immunized Hereford steersrepresent the first use of" novel" immunogens for the with soluble 100,000 x g supernatant, and membraneinduction of anti- tick immunity. enriched extracts of B. microplus midgut. Membrane

Several research groups have focused their fraction immunized cattle expressed 91 percent resistanceinvestigations on obtaining molecules of ixodid gut to three challenges, each consisting of 20,000 larvae.origin, which can be administered to induce anti- tick Detergent solubilized B. microplus membranes were

immunity. Use of gut derived immunogen has distinct found to contain three antigens ( MB5, MB 14, andtheoretical advantages, which have been largely proven MB20) that had a significant impact upon levels ofin practice( Willadsen and Kemp 1988, Opdebeeck et al. protective immunity ( Wong and Opdebeeck 1989).1988, Wikel 1988). Host immune effector elements are Cattle vaccinated with midgut membrane antigen incontained within the blood meal obtained from the association with the adjuvant Quil A developedelevatedimmunized host. Host antibodies, complement, and IgGI and IgG2 levels, and protection was correlatedpossibly effector cells damage the gut of the feeding with presence of complement fixing antibodies( Jacksontick. Impact of host mediated damage can result in and Opdebeeck 1990). Antibodies induced byreduced engorgement, reduction or prevention of egg immunization with tick midgut membrane immunogensproduction, and death of the tick. Immunization with reacted with larval membranes, soluble larval extracts,tick digestive tract epitopes stimulates a host response, gut membranes, soluble gut extracts, salivary glandwhich induces little or no reactivity to ixodid salivary membranes, and soluble salivary gland extractsgland antigens. Therefore, challenge infestation is ( Opdebeeck and Daly 1990). Positive cellular responsescharacterized by minimal or no reactivity to subsequent developed to gut and salivary gland antigens( Opdebeeck


and Daly 1990). dimensional sodium dodecyl sulfate polyacrylamideAn extremely interesting study by Rutti and Brossard gel electrophoresis and reactive on immunoblotting had

1989) found that greater than 50 percent of sera derived molecular weights of 64, 66, 68, 113, 125, 133, 137,

from mice infested with Ixodes ricinus nymphs and 141, 144, 153, and 157 kdal. Unimmunized and

rabbits infested with I. ricinus adults reacted with a 25 uninfested control animals had immunoglobulins that

kdal antigen detected in integument and in an extract of reacted with a 61 kdal polypeptide.

whole female I. ricinus. Antibodies stimulated by Similar nitrocellulose immobilized electro-infestation with I. ricinus cross- reacted with a 20 kdal phoretograms were reacted with biotinylated lectins in

molecule of R. appendiculatus females, which was an effort to identify glycoconjugates within thefound at a high level in integument. Brossard and Rais immunogen fractions. Concanavalin A reacted with D-

1984) established that intact immunoglobulin crossed mannose containing residues over a molecular weightthe midgut epithelium of I.ricinus. A damaged midgut range of 27 to 154 kdal. Wheat germ agglutinin

would facilitate access of host antibodies to the complexedtoN-acetyl-D- glucosamineresiduesonbands

hemolymph and cuticle epitopes. A vaccine might with a molecular weight range of 37 to 140 kdal. Peanut

contain combined immunogens from tick gut, agglutinin reacted with D-galactose containing moietiesreproductive tract, and cuticle. Anti- tick immunity, over a molecular weight range of 30.5 to 144 kdal. Ulex

based upon vaccination induced disruption of cuticle, europaeus agglutinin I bound L-fucose on extract

should be thoroughly investigated. components with a molecular weight range of 37 to 127

Tick tissue culture cells represent another source of kdal.

immunogens for stimulation of anti- tick immunity Immunoblot and lectin blot reactivities were

Wikel 1985b). Primary in vitro cultured cells of compared. Wheat germ agglutinin bound 66, 68, 141,

developing A. americanum larvae were administered and 144 kdal moieties, while peanut agglutinin

subcutaneously at a concentration of one million cells complexed with 64, 68, 141, and 144 kdal

on clays zero, seven, and 21. A significant degree of glycoconjugates. Initial lectin affinity purification isresistance to challenge with adult female and male A. performed with columns containing immobilized wheatamericanum was induced. Mean engorgement weight germ agglutinin and peanut agglutinin. Protection

of female A. americanum feeding upon vaccinated inducing glycoconjugates have been obtained by thisanimals was reduced 74.8 percent and 54.6 percent of isolation strategy.female ticks died. All ticks obtaining a blood meal from Fractions obtained at each purification step arefirst exposure animals were viable. screened for specificity of antibody reactions by

Animals immunized with A. americanum primary immunoblotting and for their ability to stimulate celltissue culture cells were not only resistant to challenge mediated reactivity, as measured by in vitro lymphocytewith A. americanum adults, but they also inhibited responsiveness. Fractions can be further purified for

engorgement ofD. andersoni females by a mean of 71. 3 lymphocyte proliferation assays by preparative sodiumpercent ( Wikel 1985b). Cross resistance between A. dodecyl sulfate polyacrylamide gel electrophoresis,

americanum and D. andersoni could be interpreted as followed by detergent removal or solubilization ofindicating the presence of broadly shared protection nitrocellulose immobilized bands ( Young and Lambinducing epitope( s). The protection inducing cells and 1986).

epitopes in primary tick tissue cultures remain to be Immunogen purification can be achieved through

identified. the following steps: ( a) differential centrifugation to

Research in our laboratory( Wikel 1988) continues enrich for tick gut absorptive surface membranes, ( b)

to focus upon the use of tick digestive tract brush border non- ionic detergent solubilization of integral membrane

molecules as vaccine immunogens. Resistance induced proteins, ( c) lectin affinity chromatography, ( d) gelwith absorptive surface fragments reduced mean filtration high performance liquid chromatography,( e)engorgement of female challenge ticks up to 69.8 percent. chromatofocusing, and ( f) immunoaffinity chroma-Fewer ova were produced and 37.5 to 71. 5 percent of tography performed with monoclonal immunoglobulinschallenge ticks died. Cutaneous hypersensitivity to molecules of interest. Biotechnological methods

reactions did not occur at tick attachment sites on exist for the commercial scale production of immuno-

vaccinated animals. gens. Vaccine immunogen delivery systems can beSera of animals immunized with absorptive surface formulated, which will require minimal handling of

fragment enriched immunogen preparations were used animals and the production of a sustained immune

in immunoblotting studies to identify reactive molecules. response.

Absorptive surface molecules separated by one Vaccination induced anti- arthropod immunity can


be an effective control strategy for long and short- term REFERENCES CITED

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BULL. SOC. VECTOR ECOL., 17( 1): 20-24 NNE, 1992

LACK OF CORRELATION BETWEEN PERIPHERAL RECEPTOR

SENSITIVITY AND THE DAILY PATTERN OF HOST-SEEKING

BEHAVIOR IN FEMALE CULEX PIPIENS MOSQUITOES

M. F. Bowen'

ABSTRACT: A daily pattern of host- seeking behavior exists in both field and laboratory populations of Culexpipiens mosquitoes. Females are non- host-responsive during the daylight hours but actively host- seek during theevening and night. Actively host- seeking females possess antennal olfactory receptors that are highly sensitive tothe host attractant lactic acid. This study demonstrates that in mature non-diapausing Culexpipiens females olfactorysensitivity is high during both the dark and light phases of the light-dark cycle in both host-seeking and non- host-seeking females, respectively. Thus, the circadian expression of host- seeking behavior in this species is notdependent upon peripheral sensory input and control of the daily manifestation of host responsiveness must resideelsewhere.

INTRODUCTION Bowen et al. 1988), do not possess such receptors. We

have postulated that the presence ofhigh sensitivity hostMosquitoes display daily periods of activity and attractant receptors is a requirement for the expression

inactivity that are the external manifestations of of host- seeking behavior and that the responsiveness ofendogenous circadian oscillators ( Jones 1976, 1981; these receptors is modulated throughout the life cycle inJones et al. 1972). These circadian rhythms are apparent such a way as to determine the behavioral mode of theboth in the field and in the laboratory and the pattern of female( Bowen 1991).

activity, whether diurnal, nocturnal, or crepuscular, is In aprevious publication( Bowen et al. 1988) Culexspecies- specific( Clements 1963, Gillett 1971). Culex pipiens females were examined electrophysiologicallypipiens is active nocturnally in the field and females only during the dark phase of the environmental lightexhibit a circadian pattern of host- responsiveness in the cycle as that was the only time of day at which host-laboratory that approximates this natural pattern( Bowen seeking behavior could be observed. These non-

et al. 1988). Host- seeking behavior is absent during the diapausing host- responsive females consistentlyday but can be readily elicited shortly after" lights-off." displayed high lactic acid receptor sensitivity at thatThe behavior reaches a peak during the dark phase and time ofday. Since non-diapausing Culexpipiens femalesdecreases prior to lights- on, being completely absent do not host- seek during the light phase of the light-darkupon the onset of,and during, the light phase. This host- cycle ( Bowen et al. 1988), the question arose as to

seeking strategy is just one among many within the whether this cyclic lack of behavioral responsiveness to

Culicidae( Edman and Spielman 1988) and its advantages a host was due to a daily variation in the sensitivity of theare clear. Feeding on diurnal hosts during their periods lactic acid receptors.

of inactivity will minimize host-defensive behavior and The experiments described here detail anthus maximize blood- feeding success( Edman and Scott electrophysiologic al examination of lactic acid receptors

1987, Edman and Spielman 1988, Edman et al. 1985). in Culex pipiens females during both the light and theHost- responsiveness in mosquitoes is closely dark phase of the environmental light-dark cycle.

correlated with the state of the peripheral sensory systemDavis 1984a, 1984b; Bowen et al. 1988). Host- MATERIALS AND METHODS

responsive females always have antennal olfactoryreceptors that are highly sensitive to the host- attractant Animals, Rearing Procedures, and Environmentallactic acid. Females that do not exhibit host- seeking Conditions

behavior, such as those with developing eggs ( Davis The mosquitoes used in this study were reared from1984a) or individuals in a state of ovarian diapause material obtained from Dr. Carl Mitchell ( CDC, Fort

1SRI International, Life Sciences Division, Insect Neurobiology Group, 333 Ravenswood Ave., Menlo Park, CA94025, U.S. A.


Collins, CO) that had been originally collected from isdeliveredintoacarrierairstreamofwarmed, humidified

sites in the Fort Collins area. The parental generation synthetic air that is constantly flowing over theconsisted of females collected from the field in the experimental preparation. Stimulus intensity is variedsummer of 1989 that were blood- fed in the laboratory. by adjusting the rate of flow of the stimulus airstream.The mosquitoes used in this study were F2 generationfemales that had been reared from the first larval instar RESULTS

under short-day( LD 10: 14) photoperiodic conditions at22° C. These rearing conditions give rise to diapausing The results presented here were obtained from 19

adults( Wilton and Smith 1985, Bowen et al. 1988). At sensillae on 14 animals. None of the females examined

approximately six weeks ofage, these diapausing females during the light phase were host- responsive. All of thewere transferred to long- day photoperiodic conditions females examined during the dark phase were host-LD 14: 10) and 27° C. After one to two weeks of this responsive.

treatment they had terminated diapause and initiated As shown in Figure 1, lactic acid-excited cells

post-diapause development as indicated by an overall displayed high sensitivity to stimulus during the lightaverage ovarian follicle length of 55. 0 g( range= 43. 3- phase of the photoperiodic cycle despite the fact that the

69. 2µ, n=( 1) 4 individuals). Each female was tested mosquitoes are unresponsive to a host at this time of dayfor host- responsiveness prior to electrophysiological ( Bowen et al. 1988). The averaged dose response curves

analysis. Electrophysiological recordings were carried from receptor cells of non- diapausing host-responsiveout between 5 and 11 hours after" lights-on" or between mosquitoes that were examined during the dark phase1 and 6 hours after lights-off. Ovarian follicle length are also shown in Figure 1. These, as expected( Bowen

was determined for each female after each recording et al. 1988), were of high sensitivity. The dynamicsession. range ( range of stimulus intensities to which a given

class of receptor cell responds) was the same( 1. 4 to 32.0

Electrophysiological Recording x 1011 moles/ sec) in both groups and the average

Single unit extracellular recordings were made maximum evoked response was also very similar( AF=using standard e lectrophysiologic al recording techniques approximately 40 impulses/ sec).similar to those described previously ( Davis andSokolove 1976, Bowen et al. 1988). This study reports DISCUSSION

the results of recordings made from cells associated

with the grooved- peg type A3 sensillae( McIver 1974), Host- seeking behavior in non- diapausing femalewhich are sensitive to the host attractant lactic acid Culex pipiens is expressed with a pronounced dailyBowen et al. 1988). Individual female mosquitoes rhythmicity under defined photoperiodic conditions

were glued to a brass mount and the antennae secured on ( Bowen et al. 1988). Non- diapausing females are host-double- stick scotch tape. An uninsulated tungsten responsive during the dark phase but not during the lightmicroelectrode was inserted through the cuticle into the phase of the photoperiodic cycle.

base of the sensillum. The signal was amplified, filtered, The sensitivity of the antennal receptors for the hostdisplayed on an oscilloscope, and routed through an attractant lactic acid have been shown to be positivelyamplitude analyzer and counter, from which correlated with host- seeking behavior in both CulexinstantaneOus spike frequencies from individual cells pipiens and Aedes aegypti ( Bowen et al. 1988; Davis

were plotted on a chart recorder. The peak phasic 1984a, 1984b). Concurrent daily variations in sensitivityresponses to stimuli of various intensities were measured of the lactic- acid receptors could account for the absence

and plotted against stimulus intensity to yield dose- of host- seeking during the day and the presence of host-response functions for individual neurons. These dose- responsiveness during the night. Similar variations inresponse functions are graphic representations ofreceptor receptor function have been described for certain

cell sensitivity( Davis and Sokolove 1976, Davis 1988). chemoreceptor cells( Omand and Zabara 1981, Blaneyand Simmonds 1983, Blaney et al. 1987).

Stimulus Delivery In this study it is shown that lactic acid-excitedAirborne stimuli were delivered over the antennae neurons of Culexpipiens females do not show variations

using the technique ofOugh and Stone( 1961). Synthetic in sensitivity with time ofday. Non-diapausing femalesair is bubbled through a solution of the stimulus chemical are not host- responsive during the light phase yet receptormaintained at a constant temperature. Theconcentration sensitivity remains high and unchanged from thatof the substance in the saturated gaseous phase is a observed after the onset ofdarkness. Thus, there appears

function of temperature and vapor pressure. The stimulus to be no temporal variation in receptor sensitivity

22 BULL. SOC. VECTOR ECOL. NNE, 1992

50

40 -

s

30 -

A

20 - -

10 -

0 - =

1 10 100

Lactic Acid Concentration

Figure 1. Averaged dose response functions recorded from 10 individual lactic acid- excited neurons of non-host

responsive Culex pipiens females during the light phase( open symbols) and from 9 neurons of host-responsive females recorded during the dark phase ( closed symbols) of a long-day ( LD 14: 10)photoperiodic cycle. The difference in nerve spike frequency between spontaneous activity and the peakphasic response to stimulus presentation( SF) is plotted against stimulus intensity( x 10- 11 moles/ sec).

concurrent with the temporal pattern of host-seeking seeking behavior is inhibited by signals mediated bybehavior, and variations at the peripheral level cannot stretch receptors that apparently reside in the anterioraccount for the behavioral rhythmicity. The rhythm of abdomen and which are activated by midgut distentionhost responsiveness must be controlled at a more central ( Klowden and Lea 1979a). This" distention inhibition"

level, being either driven directly by the light-dark cycle of host- seeking is followed by a humorally inducedor gated by a circadian oscillator. inhibition that is maintained until after oviposition

A similar confluence of high receptor sensitivity ( Klowden and Lea 1979b), during which host-seekingand lack of a behavioral response occurs in Aedes behavior is tightly coupled with lactic acid sensitivityaegypti immediately after ablood meal above a threshold ( Davis 1984a).

volume( Klowden and Lea 1979a). In this case host- Although the presence of high sensitivity lactic


acid receptors is a requirement for the expression ofhost grooved-peg sensillaof the mosquitoAedes aegypti.

seeking behavior ( Davis 1984a, 1984b; Bowen et al. J. Insect Physiol. 34: 443- 449.

1988), such behavior is not the inevitable result of high

lactic- acid receptor sensitivity. Peripheral sensitivity Davis, E. E. and P. G. Sokolove. 1976. Lactic acid-

changes can predict host responsiveness in the maturing sensitive receptors on the antennae of the mosquito,

female ( Davis 1984b), after the blood meal ( Davis Aedes aegypti J. Comp. Physiol. 105: 43- 54.1984a), and during diapause( Bowen et al. 1988), but thedaily control of host seeking behavior must reside Edman, J., J. Day, and E. Walker. 1985. Vector-hostelsewhere. interplay- Factors affecting disease transmission.

Acknowledgments Pp. 273- 285 in Ecology of Mosquitoes: Proceedingsof a Workshop.( L. P. Lounibos, J. R. Rey, and J. H.

The author is indebted to E. E. Davis and Carl J. Frank, eds.). Florida Medical EntomologyMitchell for critically reviewing the manuscript and to Laboratory, Vero Beach, Florida.Adeline Will for her editorial assistance. These studies

were supported by NIAID grant AI23336 to the author. Edman, J. D. and T. W. Scott. 1987. Host defensive

behavior and the feeding success of mosquitoes.REFERENCES CITED Insect Sci. Appl. 8( 4- 6): 617- 622.

Blaney, W. M. and M. S. J. Simmonds. 1983. Edman, J. D. and A. Spielman. 1988. Blood feeding byElectrophysiological activity in insects in response vectors: physiology, ecology, behavior, andto antifeedants. P. 219 in TDRI Misc. Pub. Lond. vertebrate defense. Pp. 153- 189 in Epidemiology

of Arthropod-Borne Viral Diseases, Vol. 1. ( T.

Blaney, W. M., M. S. J. Simmonds, and L. M. Monath, ed.). CRC Press, Boca Raton, Florida.

Schoonhoven. 1987. Inconstancies of

chemoreceptor sensitivities. Pp. 141- 145 in Insects- Gillett, J. D. 1971. Mosquitoes. Weidenfeld and

Plants. ( V. Labeyrie, G. Fabres, and D. Lachaise, Nicolson, London. 274 pp.eds.). Dr. W. Junk, Netherlands.

Jones, M. D. R. 1976. Persistence in continuous light

Bowen, M. F. 1991. The sensory physiology of host- of a circadian rhythm in the mosquito Culexpipiens

seeking behavior in mosquitoes. Ann. Rev. fatigans Wied. Nature 261: 491- 492.

Entomol. 36: 139- 158.

Jones, M. D. R. 1981. The programming of circadianBowen, M. F., E. E. Davis and D. A. Haggart. 1988. A flight activity in relation to mating and the

behavioural and sensory analysis of host- seeking gonotrophic cycle in the mosquito, Aedes aegypti.

behaviour in the diapausing mosquito Culexpipiens. Physiol. Entomol. 6: 307.

J. Insect Physiol. 34: 805- 813.

Jones, M. D. R, C. M. Cubbin, and D. Marsh. 1972. The

Clements, A. N. 1963. The Physiology of Mosquitoes. circadian rhythm of flight activity of the mosquito,Vol. 17 in Monographs on Pure and Applied Biology Anopheles gambiae: the light response rhythm. J.

G. A. Kerkut, ed.). Pergamon, New York. Exp. Biol. 57: 337- 346.

Davis, E. E. 1984a. Regulation of sensitivity in the Klowden, M. J. and A. 0. Lea. 1979a. Abdominal

peripheral chemoreceptor systems for host- seeking distention terminates subsequent host- seeking

behaviour by a haemolymph- borne factor in Aedes behavior ofAedes aegypti following a blood meal.aegypti. J. Insect Physiol. 30: 179- 183. J. Insect Physiol. 25: 583- 585.

Davis, E. E. 1984b. Development of lactic acid- Klowden, M. J. and Lea, A. 0. 1979b. Humoral

receptor sensitivity and host- seeking behaviour in inhibition of host-seeking in Aedes aegypti during

newly emerged female Aedes aegypti mosquitoes. oocyte maturation. J. Insect Physiol. 25: 231- 235.

J. Insect Physiol. 30: 211- 215.McIver, S. B. 1974. Fine structure of antennal grooved-

Davis, E. E. 1988. Structure- response relationship of pegs of the mosquito, Aedes aegypti. Cell Tissue

the lactic acid- excited neurons in the antennal Res. 153: 327- 337.


Omand, E. and J. Zabara. 1981. Response reduction in rapid and critical odor measurement. J. Food Sci.

dipteran chemoreceptors after sustained feeding or 26: 452- 456.

darkness. Comp. Biochem. Physiol.( A) 70: 469-478. Wilton, D. P. and G. C. Smith. 1985. Ovarian diapause

in three geographic strains of Culexpipiens( Diptera:

Ough, C. S. and H. Stone. 1961. An olfactometer for Culicidae). J. Med. Entomol. 22: 524- 528.


THE FEEDING AND RESTING BEHAVIOR OF ANOPHELES GAMBIAE ON

THE ISLANDS OF SAO TOME AND PRfNCIPE, WEST AFRICA

H. Ribeirol, H. C. Ramos1' 2 and J. G. Janzl

ABSTRACT. The proportions of Anopheles gambiae populations that were endophagic and endophilic were

estimated using data on the human-biting rate, human blood index, indoor abundance of gravid females in relationto humans, daily survival rate, and length of the gonotrophic cycle, as proposed by Ribeiro and Janz( 1990). Usingthis new approach, high exophagy and exophily by the local malaria vector were suggested.

INTRODUCTION The ovarian condition of all females was classified

according to Christophers' stages and was recorded.In a previous paper, Ribeiro and Janz ( 1990) Following Carnevale et al.( 1982), a child was counted

proposed four indices for the measurement of the as only one- third of an adult for the calculation of theendophagy and endophily of malaria vectors according number of persons sleeping in each house.to the mathematical expressions in TABLE 1. As The length of the gonotrophic cycle and the dailydefined, all the indices will theoretically vary from zero survival rate were determined through laboratoryno endophagy and no endophily) to one ( complete breeding in loco and from the observed parous rates,

endophagy and complete endophily). The whole indices respectively. Lastly, the human blood index waswill be less than the corresponding simple indices if estimated through counterimunoelectrophoresis on a

anthropophily, endophagy, and endophily are not sample of 27 freshly fed An. gambiae females caught incomplete. The smaller the whole indices, the greater several kinds of resting places, both indoors and outdoors.will be the portion of the local vector population that

does not exhibit the behavioral characteristic described RESULTS

by the index.In the present article we present the values obtained TABLE 2 shows the values of the six basic ento-

for these indices for Anopheles gambiae populations on mological variables in Sao Tome and in Principe, as

the islands of Sao Tome and Principe, which were reported by Ribeiro et al. ( 1990). The indices of

already thought to be markedly exophagic and exophilic endophagy and endophily are presented in TABLE 3.Ribeiro et al. 1990). The field work was carried out On the island of Sao Tome, only 14 percent of the

during April and May, at the end of the rainy season. anthropophilic An. gambiae females bite humans indoors

ENGI = 0. 144), and these females represent only aMATERIALS AND METHODS slightly smaller proportion of all the biting An. gambiae

females( WENGI= 0. 138). On the other hand, only twoThe methods used were described previously by out of every thousand anthropophilic, endophagic An.

Ribeiro et al. ( 1990) and followed standard techniques gambiae females rest indoors after feeding until at leastWHO 1975, Molineaux and Gramiccia 1980, Werns- the ovariolar stage III( ENLI= 0. 002), and the proportion

dorfer and McGregor 1988). of these anthropophilic, endophagic, and endophilic

Indoor and outdoor human-biting rates( IMBR and females represents only one- eighth of all the active An.OMBR) were estimated by human bait catches from gambiae females, i. e., one in four thousand( WENGI=

18. 00 to 06.00 hours( 40 man- nights in Sao Tome and 0.00025).

and 20 man- nights in Principe). The indoor-resting On the island of Principe, about 28 per cent of the

density of the vector in relation to humans was estimated anthropophilic An. gambiae females ( about twice that

by pyrethrum spray collections carried out during the on Sao Tome) bite humans indoors ( ENGI=0. 279),

day in 14 houses in Sao Tome and 4 houses in Principe. representing a slightly smaller proportion of all the

1Centro de Doer-was Infecciosas e Parasitairias( INIC), Institute de I ligiene e Medicina Tropical, Universidade Novade Lisboa, Junqueira, 1300 Lisboa, Portugal.

2Centro de Zoologia, Instituto de Investigacao Cientffica Tropical, Junqueira, 1300 Lisboa, Portugal.


TABLE 1. Formulae used in this study to evaluate mosquito feeding and resting habits.

Endophagic index( ENGI)= IMBR/(OMBR+IMBR).

Whole endophagic index( WENGI)=( IMBR* HBI)/(OMBR+IMBR).

Endophilic index( ENLI)= IRDG/[ IMBR(i- 1) j].

Whole endophilic index( WENLI)=( IRDG)(HBI)/[(OMBR+IMBR)( i- 1) j].

IMBR= Indoor human-biting rate, the number of females biting indoors per night.

OMBR= Outdoor human-biting rate.

HBI= Human blood index( Garret-Jones, 1964).

IRDG= Diurnal indoor-resting abundance in relation to humans of all late fed, half gravid, andgravid females( Christopher' s ovary stages III, IV, and V).

i= Length of gonotrophic cycle, in days.

i- 1

j= I pk

is a function of the daily survival rate( p) so thatk= 1

IRDG=IMBR( i- 1) j when all the indoor human- biting females are endophilicand k takes successive values of 1, 2, 3... i- 1.

TABLE 2. Estimates of the basic entomological parameters for the An. gambiae populationsof the islands of Sao Tome and Principe.

Sao Tome Principe

Indoor human-biting rate( IMBR) 8. 1 9. 9

Outdoor human-biting rate( OMBR) 48.2 25. 6

Indoor-resting density of gravids( IRDG) 0.05 0

Length of gonotrophic cycle( i) 3 days 3 days

Daily survival rate( p) 0.92 0.88

Human blood index( HBI) 0.96 0.96

TABLE 3. Indices of endophagy of Anopheles gambiae in the islands of Sao Tome andPrincipe.

Sao Tome Principe

Endophagic index( ENGI) 0. 144 0. 279

Whole endophagic index( WENGI) 0. 138 0. 268Endophilic index( ENLI) 0.002 0

Whole endophilic index( WENLI) 0.00025 0


activeAn. gambiae females each day( WENGI= 0.268). REFERENCES CITED

On the other hand, as no gravid females were collected

resting indoors in the four houses treated with pyrethrum Bushrod, F. M. 1981. The Anopheles gambiae Giles

sprays in Principe, the value of the endophilic indices complex and Bancroftian filariasis transmission in

will be zero, or in any case, nearly so. a Tanzanian coastal village. Ann. Trop. Med.It may be useful to adopt the five basic categories of Parasitol. 75: 93- 100.

Bushrod( 1981) that qualify the local vector populationas to its feeding and resting behavior and to then quantify Carnevale, P., M. Molinier, J. Mouchet, and J. Coz.

them using the computed values of the endophagic and 1982. L' 6valuation des seuils critiques de

endophilic indices, as follows: transmission du paludisme en zone d' endemie

stable. Rev. Epidr m. et Santry Publ. 30: 49- 70.

Indices Designations

Garrett- Jones, C. 1964. The human blood index of

ENGI or ENLI= 1 Absolute endophagic or malaria vectors in relation to epidemiological

endophilic. assessment. Bull. Wld. Hlth. Org. 30: 241- 261.

0.50< ENGI or ENLI< 1 Preferential endophagic or Molineaux, L. and G. Gramiccia. 1980. The Garki

endophilic. Project. Research on the Epidemiology and controlof Malaria in the Sudan Savanna of West Africa.

ENGI or ENLI= 0.50 Indifferent. World Health Organization, Geneva.

0< ENGI or ENLI< 0.50 Preferential exophagic or Ribeiro, H. and J. G. Janz. 1990. Exophagy andexophilic. exophily in malaria vectors. Bull. Soc. Vector

Ecol. 15: 185- 188.

ENGI or ENLI= 0 Absolute exophagic or

exophilic. Ribeiro, H., H. C. Ramos, and C. A. Pires. 1990. Sobre

os vectores da malaria em Sao Tom6 e Principe.

If we adopt these designations, the An. gambiae Garcia de Orta, S6r. Zool. 15: 135- 152.

populations of Sao Tom6 and Principe should be

classified as preferentially exophagic and near-absolute Wemsdorfer, W. H. and I. McGregor. 1988. Malaria.

exophilic. These marked behavioral characteristics of Principles and Practice of Malariology. 2 vols.the local vector populations, together with their proven Churchill Livingstone, Edinburgh.

physiological resistance to organochiorine insecticides,

may be among the reasons for the failure of past malaria World Health Organization. 1975. Manual on Practical

control campaigns in the Democratic Republic of Sao Entomology in Malaria. Parts I and II. World

Tome and Principe. Health Organization, Geneva.


ANALYSIS OF TEMPERATURE- DEPENDENT STAGE-FREQUENCY

DATA OF AEDES VEXANS( MEIGEN) POPULATIONS ORIGINATED

FROM THE MAGADINO PLAIN (SOUTHERN SWITZERLAND)'

F. Fouque2, J. Baumggrtner2, and V. Delucchi2

ABSTRACT: This work is part of a research project design to develop a mosquito control program against Aedesvexans( Meigen) in the Magadino plain( southern Switzerland). Single and multi-cohort data of the aquatic life-

stages were studied both under controlled conditions and in the field. The data were compiled into stage- frequencytables and analyzed by means ofManly' s( 1987) multiple-regression model. The stage- specific developmental timesand survival rates vary with temperatures in a stage- specific manner. These relationships improve our understandingof the dynamics of the aquatic life- stages of Ae. vexans under field conditions.

INTRODUCTION were Ae. vexans, research on factors affecting itspopulation dynamics were considered the highest

In recent years mosquitoes have become a nuisance priority.

in the Magadino plain of southern Switzerland( canton Although Ae. vexans is found in diverse habitats

of Ticino), and for the first time in the history of the within the Magadino plain ( e. g., temporary streams,region it became necessary to apply control measures. ephemeral puddles in corn fields), this work focused on

At the beginning of the century, the area was a marshland populations developing in temporary pools in the forestedcreated by the delta of the river Ticino. Only a small areas of the natural reserve and its surroundings. This

portion of the area bordering the lake remains, the rest habitat was chosen because it produces the greatest

being eliminated by economic development. Although numbersofAe. vexans( Fouque 1991). An understandingthe area is a suitable breeding place for mosquitoes, no of the population dynamicsofAe. vexansis aprerequisite

mosquito outbreaks were reported until the last decade. to the planning of rational control measures, such asIn 1974, the swampy area was declared a natural reserve application of B. thuringiensis var. israelensis. Eggto protect rare species of plants and migrating birds. hatching and subsequent development depend largelyConsequently, the environment slowly changed, creating on water availability and favourable environmentalnew breeding sites for Aedes species. In addition, new conditions. The temperature was the first environmental

recreational areas expanded near the reserve. The factor considered in the research program, and its

concomitant increases in mosquito populations and in influence on the development and survival of the aquatic

recreation around the area created intolerable conflicts life-stages ofAe. vexans are the focusof this investigation.

requiring governmental intervention. At this stage, the Therefore, developmental and survival rates of cohorts

canton government funds control mosquito larvae using of the aquatic- stages ofAe. vexans reared in the laboratoryBacillus thuringiensis var. israelensis serotype H14 were estimated using Manly' s( 1987) method of cohort

Liithy, 1987, 1988, 1989) and a three year research analysis. These cohort statistics were expressed as

program to collect the basic ecological data to develop functions of the temperature. The predicted results were

an IPM program for mosquitoes( Fouque 1991). then compared to those derived from field cohorts.

As few studies had been carried out on Culicidae in

the Ticino( Borrani 1937, Briegel 1973), it was necessaryMATERIALS AND METHODS

to first make a faunistic survey to characterize themosquito species responsible for the nuisance. Nineteen Observations Under Natural Conditions

species were identified (Fouque et al. 1991), but only The fluctuations of Ae. vexans larval populations

Aedes cinereus, Aedes intrudens, Aedes punctor, Aedes were followed in several temporary pools from 12 Aprilsticticus and Aedes vexans were found to bite humans. 1988 to 18 July 1991. In eight temporary pools, a totalBecause 50 percent of the captured biting mosquitoes of 109 samples was collected from 1989 to 1991.

1Presented at the Vth European Regional Meeting of the Society for Vector Ecology, Uppsala, Sweden, 3 September1990.

2lnstitute of Plant Sciences, ETH, CH- 8092 Zurich.


Among them, 49 samples were collected during the Ae. samples. The macroorganisms were eliminated byvexans infestation periods, with 609 of the units filtration and this filtered water is referred to as filtratedcontaining some larvae or pupae. In this paper, however, pool water. Fouque( 1991) found that this filtrated poolonly the data collected in 1989 are used for illustrative water allowed in some cases 100 percent of Ae. vexanspurposes. The data collected in a single pool( number 34 eggs to develop to adults in experiments with 10 orin Fouque 1991) during an infestation period, starting fewer individuals/ 1 and at mean temperatures betweenon 5 June 1990 and ending on 11 July 1990, were used 20° C and 30°C. The containers were then placed at oneto illustrate the statistical analysis. Additional data from of six constant temperature treatments( 12. 3° C, 20°C,the same and other temporary pools were also subjected 25° C, 30° C, 35° C, and 40°C). The larvae and pupaeto statistical analysis. were counted daily and assigned to instars until the

This infestation period started with eggs hatching emergence of the adults. Two replicates, i. e. two con-and ended when no more mosquito larvae and pupae tainersoflarvae, wereusedpertreatmentintheanalysis.

were found. Samples of water containing larvae and/ or Other experiments were carried out in the laboratorypupae were taken every two or three days and brought with inundated soil samples maintained at time-varyingto the laboratory, where the larvae were counted by temperatures during the aquatic development. The

instar and species. The pupae were also counted but had results of the tests run at the constant temperatures 20° Cto be kept in the laboratory until adult emergence to and 25° C are not included in the analysis because thedetermine the species. The adult emergence in the field temperature cabinet failed. Instead of them, the analysiswas not monitored. The sample unit was a 0.25 I dip of uses the results of one time-varying temperaturewater taken randomly with a standardized mosquito pint experiment, having a mean temperature value ofdipper( Service 1976). The number of units sampled 22.3° C.

was between 10 and 20 if the larval and pupal densities

were less then 30 individuals per dip, and between Construction of the Stage-Frequency Matrices5 and 10 at higher densities. The precision of the The data originating from field and laboratoryestimated mean density( m) per dip could be evaluated studies were compiled in stage- frequency matriceswith a coefficient of variability ( C), Karandinos ( Manly 1987, 1989, 1990; Severini et al. 1990). The

1976): columns represent the different stages as defined below,

the lines correspond to the days, and the elements of the

C=( s/ fin)/ m, 1] matrix are the number ofAe. vexans in the same stage at

the same date.

where s is the standard deviation and n is the number of The developmental phenology of Ae. vexans isunits ( dips). If C is below 0.2, the precision was normally structured into four larval instars, the pupalconsidered sufficient. However, if C was greater than stage, and the adult stage( TABLE 1). In our analysis,

0. 2, more units were taken and incorporated into the however, larval development was arbitrarily dividedsample for reevaluation of precision. This procedure into two stages, i. e., young larvae ( first and second-was repeated until C was below 0. 2. instars) and old larvae( third and fourth- instars). This

The circumference and the water level of the procedure was selected because these two stages could

temporary pools were measured at each sampling date be easily identified with small error while differentiatingand used to, estimate the volume of the water in the fourinstarswouldhaverequiredexcessivehandlingand

pools. concomitant increased larval mortality. In our analysisstage 3 and stage 4 are pupae and adults, respectively.

Experiments and Observations under LaboratoryConditions Description of the Model for Analyzing the Stage-

Aedes vexans eggs are found in the soil at the edge frequency Matricesof pool water and at higher density in areas where the Manly' s ( 1987) regression model was fit to allwater had recently receded( 10- 30 cm from the bottom matrices. The general equation for this model for qof the pool in Fouque 1991). Some soil samples were stages and I sampling dates is as follows:taken from these areas around the pools and were

transported to the laboratory where the eggs were Fit = B li_1 + 01 fi_i i + 02 4_12+ + 0q fi_l q, [2]

inundated for hatching. The first instar larvae were

removed within 24 hours and groups of 10 to 30 of them where i= 1, 2,... I refers to sampling dates and j= 1, 2,... qwere placed in containers with 41 of water collected on refers to stages. Fi 1 is the number of individuals in stagethe day of hatching from the same pools as the soil 1 and higher at time i. In other words, Fi1 is the number


TABLE 1. Stage- frequency data obtained from the sampling of an Aedes vexans populationin a temporary pool( number 34) during an infestation period starting 5 June 1990and ending 13 July 1990. The density of larvae and pupae are in number ofindividuals per liter( L1, L2, L3, and L4 are respectively the 1st, 2nd, 3rd, and 4thlarval instars).

Date of Sampling Ll L2 L3 L4 Pupae

5 June

9 June 2 76

12 June 4 122

15 June 14 126

18 June 562 40

20 June 2 42 2

22 June 28

25 June 44

28 June 2 76

3 July 1. 25 10

5 July 7

7 July 1 2

9 July 4

11 July13 July16 July

entering the stage 1 between the times i- 1 and i plus the other parameters explaining the stage- frequency matricessurvivors from different stages higher than stage 1 ( Manly 1987). Among the most important parameterswhich were in the population at the time i- 1. are the mean duration dj( days) of stage j, and the stage-B 1i- 1 is the number of entries to stage 1 between times specific survival rate wj. In the field, appreciable

i- 1 and i that are still alive at the time i; 0j is the mortality may occur, hence the parameters were correctedprobability of surviving from one sample time to the according to Manly' s ( 1987) method. The correctednext for an individual that is in stage j at the time i, and estimates are written as dj* and wj* in Manly' s( 1987)fi_1j is the number in stage j at time i- 1. This analysis publication, but we simplify this notation and substituteassumes that the observations are taken at equal intervals Dj for dj* and Wj for wj*.of time. For single-cohort data, the unit- time-survival- Functions were fit to laboratory data onrates 0j are the unknown parameters which can be developmental rates and survival rates with respect to

estimated via least square regression. For multi-cohort constant temperatures. These functions were validated

data, equation [ 2] is modified and the unknown with field data.

parameters are both the numbers of entries ( i. e., the

daily cohorts), and the unit-time-survival- rates 0i( Manly RESULTS AND DISCUSSION

1987).

The data from the laboratory experiments were Figure 1 shows that several periods of infestation

obtained daily and can be compiled directly in stage- may occur during one year. Each infestation period isfrequency matrices. On the other hand, data from field caused by an increase of the water level in the breedingconditions were not collected daily, hence the stage- sites. Ae. vexans egg hatching responds to rainfall, tofrequency matrices had to be corrected on a daily inundations as water level rises in the lake, or to an

basis and thus contain counts as well as interpolated increase of the underground waters. The level of the

estimates. water in the Ae. vexans breeding sites can increase by 70The estimates of daily survival rates for each stage cm within 24 hours due only torainfall( Fig. 2). However,

0j) in both laboratory and field data depend on the egg hatch occurs only when the water level increase isenvironmental conditions. This enabled us to calculate 8 cm or higher.


20 —

a)

a

g 10 -a)

0I1 ii

1 365

Days

Figure 1. The occurrences( density per 1) of the Aedes vexans infestation periods in a temporary pool( number 31)under study in 1989.

80 —

i0D

M

80 . . ., ,,,, • • , ., •••, • • • • •••• I • • • 11 ••• 1

1 10 100 1000 10000

Log( Daily Mean Rainfall)

Figure 2. Water levels[ cm] in the Aedes vexans breeding sites according to the log of daily mean rainfall[ mmper day], in the Magadino plain, from 6 April 1989 to 1 September 1990. ( The daily mean rainfalls arecalculated between 2 measures of the water level).

Using a 1990 infestation period as an example, individuals inhabit the uppermost layer of the water

patterns of aquatic life- stages fluctuate and are ( Clements 1963). However, it is recognized that the

characterized by a time- varying age- structure( Fig. 3). movements of feeding larvae may introduce a systematicThe absolute numbers were estimated on each sampling error in the estimates. This error is considered as

date for the upper 10 cm of the pool volume( TABLE 2) negligible because of the small size of the pool under

by multiplying the surface of the pool. This is justified study. In one sample the depth of the pool was less thanby the fact that, for respiration purposes, most of the 10 cm( TABLE 2) and the calculation of the volume was


2.0e+ 6 —

Pupae

3rd and 4th- instar Larvae

z 1. 0e+ 6 - lrst and 2nd-instar Larvae

0.0e+ 0

0 10 20 30 40

Days

Figure 3. Estimated total numbers ofAedes vexans aquatic life-stages during an infestation period starting 5 June1990(= day 1) and ending 13 July 1990(= day 35), in a temporary pool in the Magadino plain.

modified accordingly. The total number of individuals in TABLE 4 show that the daily survival rates Ojentering the first larval stage within the same time increase with age ( stage), the developmental time Djinterval ( i.e., one day in our investigation) is called a and survival rates Wj differ between the life stages.cohort. When all the newborn larvae hatch on the same However, these values are specific for this infestation

day, a single cohort is created, but under field conditions period, and may differ radically for others.the infestation is due to multiple cohorts. The fluctuations

of the population can be due to sampling errors, to the Mean Duration Time Temperature and Survival

mortality factors, or even to the dynamics of multiple- Rate Temperature Relationships

cohort infestation. The laboratory experiments under constantThe following properties characterize the stage- temperatures yielded the stage- specific parameters Dj

frequency data obtained under both laboratory and field and Wj which vary, as expected, according to theconditions: 1) under field conditions, multiple- cohort temperatures( Figs. 4 and 5). The shortest time from eggstage- frequency data are obtained while in the laboratory hatch to pupae in this study was about five days for thesingle-cohort are produced; 2) during development, temperature range 30° C <_ T <_ 35° C, whereas, Brust

losses occur because of intrinsic mortality, predation in ( 1968) found a value of five days at 26° C. Furthermore,the field, and other causes; 3) the probability of an for T equal to 40°C, the pupae do not survive. Theindividual surviving from one stage to the next may be longest time required from egg to pupae was about 30different for each stage and consequently the survival days at 12° C, but this temperature was also found to berates per unit time vary from stage to stage; and 4) lethal for the pupae which survived only a few days.empirical data show that each stage has a stage- specific The stage- specific threshold temperatures to completemean duration. The method of Manly ( 1987) is the development of aquatic life-stage were estimated

particularly appropriate to estimate the confounding from the linear functions of the developmental rates oneffect of mean duration time and survival in such data the temperatures ( Fig. 4). A linear function satisfac-set. torily describes the relationships, the high coefficients

The estimated mean densities in the temporary pool of determination indicate that the major portion of theunder study( TABLE 1) were transformed in TABLE 3 regression is explained. The developmental threshold

and analyzed as explained above. Theresulting estimates is about 3° C for young larvae ( Fig. 4a), 5° C for old


TABLE 2. Fluctuations of the water level( H in cm) and the volume of the water

V in 1) in the upper 10 cm of the temporary pool( number 34) understudy during the same infestation period as in TABLE 1.

Date of Sampling H [ cm] V[ 1]

5 June 0.00 0.00

9 June 65. 00 21, 529.00

12 June 25. 00 6, 467.00

15 June 14. 00 3, 346.00

18 June 8. 00 1, 734.00

20 June 14. 00 3, 346.00

22 June 42.00 12, 110.00

25 June 29.00 7, 651. 00

28 June 27. 00 7, 166.00

3 July 41. 00 11, 196.00

5 July 36.00 9, 204.00

7 July 27.00 7, 166.00

9 July 14.00 3, 346.00

11 July 16.00 4,031. 00

13 July 7. 00 1, 339.00

16 July 0.00 0.00

larvae( Fig. 4b), and was set at 12° C for pupae because Comparison of DJ and Wj Estimated fromthey fail to develop at this temperature ( Fig. 4c). The Laboratory and Field Datatemperature range in Fig. 4 is limited to T <_ 35° C The functions describing the developmental rate-because it was the highest temperature under study temperature relationships( Fig. 4) and the survival ratewhere any individual develops to adult. temperature relationships( Fig. 5) enable us to compare

Figure 5 shows the stage- specific survival rates W1. the same parameters obtained from the field data usingThe survival rates of the young larvae remained very the Manly' s model ( 1989) and estimated from thehigh across temperatures in the range 12° C< T<_35° C. function. To do this, we use the mean temperature of the

W1 varies from 0. 8 to 1. 0( Fig. 5a), and this indicates water ( 16. 3° C) during the infestation period andthat a high proportion of first and second- instar larvae substitute it in the function depicted in Fig. 4 and 5. Thesurvive and enter the third- instar. However, the mean durations predicted by the functions for young andsurvivorship should be zero at the threshold of 3° C old larvae were five and eight days, respectively. Fieldsuggesting a curvilinear relationship as found for old pupae werekept at 20° C andsubstituting this temperaturelarvae ( Fig. 5b) and pupae ( Fig. 5c). The maximum in the function, a mean duration of three days is predicted.

value of the old larvae and pupae survival rate is rapidly Those values are reasonably close to the Manly' sreached when the constant temperatures are higher than estimates reported in TABLE 4.

20° C. Unfortunately, the experiments at 20° C and 25° C The stage- specific survival rates at 16. 3° C were 91

failed and hence we cannot characterize the function percent for the young larvae, 50 percent for the oldaccurately for the old larvae. For this reason, we larvae, and 99 percent for the pupae held at 20° C. The

considered a laboratory experiment with fluctuating young larvae had a lower survival rate under fieldtemperature and roughly fit the same shape function to conditions ( TABLE 4) indicating that additionaleach data set. Thus, the filtrated pool water and the mortality factors affect the young larvae. The predictedconstant temperature regimes between 12° C and 35° C survival rate of old larvae is lower than the observed

permit the survival of all life- stages. The food contained one, and this is probably due to the roughly fitted curve.in the filtrated pool water is not a limiting factor, but The predicted survival rate of pupae is closed to the

rather the limiting factors are low and high tempera- estimated one.

tures which affect each stage differently. Additional comparisons with data from other


TABLE 3. Matrix of transformed stage- frequency data from TABLE 1( Stage 1= Ll-L2; Stage 2= L3-

IA, Stage 3= pupae). The data under this form can be subjected to Manly' s( 1987) multiple-regression analysis( fi- 11, fi- 12, fi- 13, fi-14, Fil are variables defined in the text).

days 4- 11 fi- l2 fi- 13 fi- 14 Fi 1

1 410,500 0 0 0 842,000

2 842,000 0 0 0 1, 282,500

3 1, 282,500 0 0 0 1, 679, 262

4 1, 679,262 0 0 0 1, 389,800

5 1, 389,800 0 0 0 1, 111, 000

6 1, 111, 000 0 0 0 814,842

7 814,842 0 0 0 712,000

8 547,000 165, 000 0 0 594,000

9 288,500 305, 500 0 0 468,440

10 0 468,440 0 0 667,500

11 0 639,000 23, 000 5, 500 889,000

12 0 831, 500 47,000 10,500 1, 082, 870

13 0 998,010 69,360 15, 500 601, 308

14 0 540,500 40,000 20,808 165, 912

15 0 147, 220 6, 692 12,000 239, 845

16 0 233, 500 3, 000 3, 345 340,780

17 0 339,080 0 1, 700 340,000

18 0 340,000 0 0 340,000

19 0 340,000 0 0 336, 640

20 0 336,640 0 0 575,000

21 0 225,000 280,000 70,000 605,000

22 0 120,000 350,000 135,000 766,948

23 0 14,332 544,616 208,000 746,492

24 0 14, 200 460,000 272,292 626, 100

25 0 14, 100 375,000 237,000 496,050

26 0 14,050 280,000 202,000 377,000

27 0 14,000 200,000 163,000 250,955

28 0 13, 995 111, 960 125,000 192, 820

29 0 7, 250 96,000 89,570 136, 428

30 0 0 64,428 72,000 97, 222

31 0 0 42,000 55, 222 47,332

32 0 0 14,332 33,000 28,331

33 0 0 14,000 14, 331 23,384

34 0 0 13, 384 10,000 12, 691

35 0 0 6,000 6,691 0

temporary pools under study with approximated mean diapausing eggs. Early in the spring, the eggs hatch andtemperatures and from experiments with fluctuating the breeding sites often contain many first-instar larvaetemperatures give similar results( TABLE 5), including but very few fourth-instar larvae and hardly any pupae.the lack of correspondence for the survival rate of the The only plausible reason for the disappearance of theyoung larvae in the field. The high survival of old larvae late preimaginal stages, in the apparent absence of

under both field and laboratory conditions suggests that predators and parasites, seems to be low temperatures.

they suffer little from other than intrinsic mortality However, this fact needs to be confirmed by more fieldfactors in the field conditions. investigations. The demographic parameters could be

In the study area, Ae. vexans pass the winter as satisfactorily deduced from the study of the fieldcohorts


TABLE 4. Estimated life table parameters for a population of Aedes vexans, after the data from TABLE 1.

Stage( j)Life- table Parameters 1 2 3

Daily Survival rates( 0j) 0.80 0.95 1. 00

Number entering(Mj) 2, 191, 800 1, 092,000 746, 620

Mean time of entry in days(µ j) 8. 41 18. 55

Stage- durations in days( Dj) 6. 94 10. 14 3. 40

Stage- Specific Survival rates( Wj) 0.50 1. 00 0.93

0. 6— A

0.6— B1g

ii32 •

it

25 • •

G40. 3 '

110. 3' 11

3111

2326

0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35

Temperature Temperature

1. 0 C 9

il

cv

0. 577 •

9C4

3 70. 0

0 5 10 15 20 25 30 35

Temperature

Figure 4. Stage- specific developmental rates Aedes vexans young larvae ( A, rate = - 0.047 + 0.O16T, r2 =

coefficient of determination= 0.827), old larvae( B, rate=- 0.06+ O. 012T, r2= 0. 638), and pupae( C,

rate = 0.04[ T - 12. 3], r2 = 0.83), reared in laboratory, in filtrated pool water and under constanttemperatures. The numbers in the figures represent the numbers of individuals used in each replicate.

The numbers in italics represent the time-varying temperature experiment.

with a rather simple experimental method. In particular, Acknowledgments

the influence of constant temperatures on mosquito

development and survival in laboratory has been assessed We are especially grateful to the Dipartimento dell'and compared with field estimates based on cohort Ambiente, Bellinzona, for financial support, and to Dr.

dynamics. A simulation model of theAe.vexans population A. Barbieri, Dr. F. Fi l ippini and Ing. G. Righetti of thedynamics, with time-varying temperatures, may be more Dipartimento dell' Ambiente, Bellinzona for thegeneral and more useful not only for an understanding permission to use the laboratory facilities and forof the dynamics of Ae. vexans population but also for providing technical assistance. Special thanks are alsodecision-making concerning control practices. addressed to Dr. A. P. Gutierrez of the Department of


31 11

1. 0- 11 1. 0- B 11 •

11

A_

32 1325

1

0. 5-

26

A

7/11'73

23

0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35

Temperature9

Temperature

1. 0 - f- lame 1117 9

0. 5 -0

30.0 .... ,

0 5 10 15 20 25 30 35

Temperature

Figure 5. Stage- specific survival rates of Aedes vexans young larvae( A, survival= 0. 91( 1- exp[- 2.77{ T-2. 9}])),old larvae( B, survival= 1( l- exp[- 0.06{ T- 5}])), and pupae( C, survival= O. 99( 1- exp[- 2.65{ T- 12. 3)])),reared in laboratory, in filtrated pool water, and under constant temperatures. The numbers in the figuresrepresent the number of individuals used in each replicate. The numbers in italics represent the time-

varying temperature experiment

Biological Control, University of Berkeley, California, Clements, A. N. 1963. The physiology of mosquitoes.for the careful revision of the manuscript. Finally, we Pergamon Press, Oxford, 393 pp.also thank Dr. H. Briegel of the Department ofZoology,University of Zurich, for the identification of some Fouque, F. 1991. La d6moustication de la plaine de

species. Magadino. These EPFZ N° 9603, Zurich, 172 pp.

REFERENCES CITED Fouque, F., V. Delucchi, and J. Baumgartner. 1991.

La demoustication de la Plaine de Magadino.

Borrani, E. 1937. Osservazioni e ricerche sulla I. Inventaire faunistique et identification des especes

distribuzionedeiCulicidiesopratuttodelleanofelini nuisibles. Bull. Soc. Ent. Suisse 64: 231- 242.

del Cantone Ticino in relazione con gli antichi

focolaimalarici. Istituto d' Igiene Sperimentale e di Karandinos, M. G. 1976. Optimum sample size and

parassitologia dell' University di Losanna, Tesi, comments on some published formulae. Bull. Ent.

Facolta di Scienze dell' Universita di Losanna. Soc. Am. 22: 417- 421.

Tipografia Commerciale G. Pedrazzini, Locarno,

44 pp. Lathy, P. 1987. Das Stechmuckenmuckenproblem imEinzugsbereich der Bolle di Magadino.

Briegel, H. 1973. Zur Verbreitung der Culicidae Unpublished Report, Mikrobiologisches Institut

Diptera: Nematocera) in der Schweiz. Revue ETH, Zurich, 21 pp.Suisse de Zool. 80: 447- 472.

Lathy, P. 1988. La lotta alle zanzare nel corso dell' annoBrust, R. A. 1968. Effect of starvation on molting and 1988 nel basso piano di Magadino. Unpublished

growth in Aedes aegypti and Aedes vexans. J. Econ. Report, Mikrobiologisches Institut ETH, Zurich,

Ent. 61: 1570- 1572. 18 pp.


TABLE 5. Comparison of observed mean duration times and survival rates with the mean duration times andsurvival rates as predicted by the functions given in Figures 4 and 5.

Mean Temperatures

Stage Field Laboratory

Life- table Parameters 15. 4 16. 2 20.8 22.6

Young Larvae

Observed duration( Dl) 7.07 1. 50 6. 69 3. 86Predicted duration( D' 1) 5.02 4.71 3. 50 3. 18

Observed survival rates( W1) 0. 15 0.01 1. 00 0.50Predicted survival rates( W' 1) 0.91 0.91 0.91 0.91

Old Larvae

Observed duration( D2) 8. 15 5. 03 6. 84 3. 53Predicted duration( D' 2) 8. 01 7. 44 5. 27 4. 73

Observed survival rates( W2) 0. 94 0.62 0. 85 0. 61Predicted survival rates( W' 2) 0.45 0.47 0.59 0.63

Pupae

Observed duration( D3) 1. 61 1. 39

Predicted duration( D' 3) 8. 06 6. 41 2.94 2. 43

Observed survival rates( W3) 0.76 1. 00

Predicted survival rates( W' 3) 0. 99 0. 99 0.99 0.99

Lathy, P. 1989. Rapporto sulla lotta alle zanzare nel Manly, B. F. J. 1990. Stage- Structured PopulationsCorso dell' anno 1989 nel basso piano di Magadino. Sampling, Analysis and Simulation. Chapman

Unpublished Report, Mikrobiologisches Institut Hall, London, 187 pp.ETH, Zurich, 8 pp.

Service, M. W. 1976. Mosquito Ecology: Field SamplingManly, B. F. J. 1987. A multiple regression method for Methods. Applied Science Publishers, London, 450

analyzing stage- frequency data. Res. Popul. Ecol. pp.29: 119- 127.

Severini, M., J. Baumgartner, and M. Ricci. 1990.

Manly, B. F. J. 1989. A review of methods for the Dalle osservazioni fenologiche alla costruzione dianalysis of stage- frequency data. Pp. 3- 69 in un modello demografico. Society Italiana diEstimation and Analysis of Insect Populations( L. Fitoiatria, Convegno nazionale modelli euristici eMc-Donald, B. F. J. Manly, J. Lockwood, and J. operativi per la difesa integrata in agricoltura,Logan. eds.). Lecture Notes in Statistics, Springer, Caserta, 27/ 29 settembre, 1990, Notiziario sulleBerlin. Malattie delle Piante 111: 41- 56.


A NEW SPECIES OF EUSAPERIUM( ACARI: TROMBICULIDAE)

FROM AN INCAN " RAT" OPOSSUM, LESTOROS INCA

MARSUPIALIA: CAENOLESTIDAE) IN PERU

M. L. Goffl

ABSTRACT: Eusaperium spinicoxa is described as new from specimens collected from two mean rat opossums,

Lestoros inca( Marsupialia: Caenolestidae) taken in Matchu Picchu, Peru. A key to the known species ofEusaperiumis given.

Examination of a small collection of chiggers biconvex; AM base posterior to level of AL bases; SB

removed from museum specimens in the U. S. National slightly anterior to level of PL bases; AL2 PL>AM;MuseumofNaturalHistorybythelateDr. F.S. Lukoschus sensillae globose, head with setules; PW/SD = 1. 55-

has revealed a new species in the genus Eusaperium 1. 81. Scutal measurements of holotype followed byBrennan 1970). The host for this new species is the means and ranges of holotype and four paratypes in

Incan Rat Opossum, Lestoros inca, collected in Peru. parentheses: AW 64( 67, 64-73); PW 85( 90, 85- 94); SB

The genus Lestoros ( Marsupialia: Caenolestidae) is 19( 20, 19- 21); ASB 33( 36, 32- 39); PSB 17( 18, 17- 19);

monotypic and appears to be restricted in distribution to AP 33( 35, 31- 39); AM 41( 46, 41- 51); AL 50( 55, 50-

the Andean zone of southern Peru at elevations above 60); PL 42( 49, 42-55); sens. 32 X 18( present only on2,900 m. While this opossum appears to be common in holotype). Legs. All seven- segmented, terminating in acool forests throughout its range, little is known of its pair of claws and a clawlike empodium. Onychotriches

life history( Walker 1968). absent. IP= 809- 894. Leg I. 279- 289; coxa with oneThe holotype of the new species is in the collection branched seta( 1B); trochanterlB; basifemurlB; telofemur

of the U. S. National Museum of Natural History, 5B; genu 4B, two genualae, microgenuala; tibia 8B, two

Smithsonian Institution ( chigger collection currently tibialae, microtibiala; tarsus( 64 X 23) 21B, tarsala( 13-

housed in the Acarology Laboratory, University of 15), microtarsala, subterminala, parasubterminala,

Hawaii at Manoa) and paratypes there and in the pretarsala. Leg II. 236- 280; coxa 1B; trochanter 1B;collection of the B. P. Bishop Museum, Honolulu. All basifemur 2B; telofemur 4B; genu 3B, genuala; tibia

measurements are given in micrometers. Terminology 6B, 2 tibialae; tarsus ( 55 X 21) 16B, tarsala ( 16- 18),

follows Goff et al.( 1982). microtarsala, pretarsala. Leg III. 294- 325; coxa 1B;trochanter 1B; basifemur 2B; telofemur 3B; genu

Eusaperium spinicoxa Goff, new species Fig. 1 3B, genuala; tibia 6B, tibiala; tarsus ( 75 X 19) 15B.

Coxa III with prominent ventrodistal spine directed

Description of species. Larvae. Idiosoma. Measuring posteriorly( Fig. IE).275 X 240 in unengorged specimen. Eyes 2/2, anterior Type Data. Holotype and seven paratypes, PERU,

slightly larger, on ocular plate. Humeral setae not Torentoy, Matchu Picchu, 4.XI.1915, ex two Lestorosdistinguishable, ca. 150 dorsal idiosomal setae with 6- inca( Smiths.= USNM 194935 and 194936), E. Heller,

10 thick lateral branches and a series of short dorsal collector.

branches ( Fig. 1D), arranged in irregular rows, setae Remarks. In the original proposal of the genus

inserted on platelets, measuring 29-48, anterior setae Eusaperium, Brennan( 1970) included three species: E.

longer; one pair of sternal setae at level of coxae I, vangelderi ( Brennan 1970), from an area near the

measuring 36- 38; ca. 170 ventral idiosomal setae, similar Bolivian-Brazilian border; Euschoengastia pamelae

in form to dorsal idiosomal setae, arranged irregularly Brennan, 1963, and Euschoengastia colombiana, both

posterior to level of coxae I, measuring 29- 39; total from Colombia. All of these species have, to date, been

idiosomal setae ca. 322. Gnathosoma Palpal setal collected only from rodents( Brennan and Goff 1977).formula BB/BBB/ 6B; palpal claw three-pronged, In this respect, E. spinicoxa represents an extension of

slender; galealaN; cheliceral blade( 32- 36) with tricuspid both geographic and host group range for the genuscap; gnathobase moderately punctate, bearing a pair of Eusaperium. The presence of the spine on coxa RI

branched setae. Scutum. Moderately punctate with serves to separate this species from others in the genus

biconcave anterior margin; posterior margin shallowly as well as providing the basis for the species name.

Department of Entomology, 3050 Maile Way, University of Hawaii at Manoa, Honolulu, Hawaii 96822, U.S.A.


D01 ::::' :

1,

9. ®

CtP,

3/1/1""/ A t,\\‘\k• i

i i ti ilk D

c, ...Vf

B E23

418

14 16

o' ;•• 14

25

F 1.1: 1 r . i v. Ie

t• : Q 1 L q; i

moo`,f ' F.,) I

219 14 12

N. 16

3o

16

1514

Q'. -.a 0 (

tea1

H J:: •: 0...2:--)-:0 .

Figure. 1. Larva of Eusaperium spinicoxa Goff, n. sp. A, scutum; 13, dorsal aspect ofgnathosoma; C, ventral aspectof palpal tibia and tarsus; D, dorsal idiosomal seta; E, coxa III ventral aspect; F, leg I distal 3 segmentsshowing specialized setae ( measurements in micrometers) and bases of branched setae; G, leg II asabove; H, leg III as above.


Key to Larvae of Species of Eusaperium

1. Dorsal idiosomal setae with 6- 10 thick basal branches and series of short branches 2Dorsal idiosomal setae with only short branches 3

2. Coxa III with ventrodistal spine E. spinicoxaCoxa III lacking such a spine E.pamelae

3. Dorsal idiosomal setae more than 55 micrometers in length E. colombianaDorsal idiosomal setae less than 35 micrometers in length E. vangelderi

Acknowledgments Brennan, J. M. and M. L. Goff. 1977. Keys to the generaof chiggers of the Western Hemisphere( Acarina:

This is Journal Series No. 3661 of the Hawaii Trombiculidae). J. Parasitol. 63: 554-566.Institute ofTropical Agriculture and Human Resources.

Goff, M. L., R. B. Loomis, W. C. Welbourn, and W. J.

Wrenn. 1982. A glossary of chigger terminologyREFERENCES CITED Acari: Trombiculidae). J. Med. Entomol. 19: 221-

38.

Brennan, J. M. 1970. A collection ofchiggers( Acarina:Trombiculidae) from rodents in southwestern Walker, E. P. 1968. Mammals of the World. 2nd ed.,Colombia. J. Parasitol. 54: 679- 685. vol. 1. The John Hopkins Press, Baltimore, 644 pp.


LABORATORY AND FIELD EFFICACY OF BACILLUS

SPHAERICUS 2362 AGAINST CULEX PIPIENS IN THE

RIVER LLOBREGAT DELTA (BARCELONA, SPAIN)

C. Arandal and R. Eritjal

ABSTRACT: Laboratory and field tests were undertaken during 1988 and 1989 to determine lethal concentrations,the effective field dose, and the period of efficacy of Bacillus sphaericus strain 2362 for Culex pipiens. The LC90at 48 hours was 0.016 mg/ I. Subsequent tests carried out in water highly polluted by urban sewage in small ditches350 m3) showed that in doses of 0.5, 1. 0, and 1. 51/ Ha, only the latter dose allowed effective control( 83%) after seven

days. In subsequent tests it was found that this dose prevented the appearance of pupae for up to nine days aftertreatment, and that a higher dose of 3. 0 1/ Ha increased this period to a total of 12 days.

INTRODUCTION MATERIAL AND METHODS

The development of bacterial insecticides, such as Laboratory TestsBacillus thuringiensis var. israelensis and Bacillus Laboratory tests were performed to determine thesphaericus has provided new possibilities for the different lethal doses. These were repeated twice; and

management of mosquitoes that are resistant to four concentrations were evaluated each time, together

conventional insecticides. In Spain, B. sphaericus was with a control in four replicates, using the methods

selected because of its suitability for use in polluted widely described and standardized by the World Healthwater where Culex pipiens breed and because of the Organization. Twenty- five third and early fourth- instarneed to provide authorities with performance data prior larvae were placed in 400 ml plastic cups containing

to the registration and marketing of this material. different concentrations of B. sphaericus in 200 ml of

Although B. sphaericus does appear to be affected distilled water. The larvae were obtained from rural

by the high degree ofpollution( Mulla et al. 1984, 1988), breeding sites that had not been treated for a sufficientits use, especially strain 2362( Mulla 1986, Mulla et al. period, transferred to tap water, and after 24 hours of1986), is more advisable than other bacterial insecticides, observation, were selected and washed in distilled water.

such as B. thuringiensis var. israelensis under these Small quantities of finely shredded fish food wereconditions( Berry et al. 1987) and against mosquitoes of provided and temperature was maintained at between

the genus Culex( Lacey et al. 1988). 24° and 26° C. Larvae were examined at 24 and 48 hours

A number of studies have been conducted to assess and mortality corrected according to the Abbott formulathe possible recycling potential and persistence in the ( Abbott 1925). The results were analyzed statistically

environment of B. sphaericus ( Mulla et al. 1984, by the probit method( Finney 1971).Davidson 1985, Karch et al. 1986). In some cases more

importance was given to persistence based on spores Field Tests

present in the sediment ( Matanmi et al. 1990), but To assess the most suitable dose, three tests were

factors such as recycling in larval cadavers ( Hougard carried out; two of them preliminary ones. The selectedand Nicolas 1986, Liu and Chen 1988), the influence of field area was within the delta plain formed by the

other filtering organisms, and the physical environment Llobregat River. At that point the land is divided into

Karch et al. 1990) should also be taken into con- cultivated fields often separated by narrow drainagesideration. Although mechanisms of this type occur, as ditches, which are parallel to each other and connected

in the case of larval cadavers, long- terme fficacy depends to other larger ones. The quality of the water is extremelymore on initial concentration and spore persistence than poor as it takes in sewage water from urban areas, and

on recycling. This study was undertaken during 1988 for most of the year there is no appreciable flow. The

and 1989 to determine the activity of B. sphaericus in limnological population of arthropods mainly consisted

the laboratory and the most suitable dose and period of of Cx.pipiens larvae and other diptera, such as Eristalis

efficacy in field tests. spp. Due to the high degree of eutrophy there are large

1Mosquito Control Department, B aix Llobregat District Council, Crtra. N. 340- Parc Torreblanca, 08980 Sant FeliuDe Llobregat, SPAIN


masses of Cyanophyceae and Chlorophyceae in part of treatment, we wished to observe how frequently thethe sediment and on the surface. The ditches chosen for treatment had to be repeated under normal workingtreatment measured 125 m in length by 2. 8 m wide and conditions. Thus, rather than noting the percent ofhad an average depth of 1 m. One of these was selected reduction in the population in this case, we observed thefor each concentration and another for control; the time elapsing before further treatment had tobe applied,sampling points being selected at random. that is, the moment when the first pupae made their

A semirigid net was used at five points for each appearance. Each ditch was treated individuallyditch and the part closest to the connection with the main whenever a pupa was observed at any point in theditch was eliminated to avoid interference. The samples sample. The number of days in which the treatment waswere taken by sweeping along 1 mprior to treatment and considered effective was calculated as the number ofthen at 24° and 48 hours, 5 days, and 7 days, except in days from obtaining effective treatment to the appearancethe preliminary test which was only at 48 hours, and in of the pupa, minus one. The sampling system was thethe following at 24 and 48 hours. Larval cadavers were same as in the previous case, the temperature varyingdiscarded in all counts. between 22° and 27° C, and the quality of the water

Analyses of the water in the same area revealed that being comparable to that of 1988.conductivity oscillated between 4,000 and 6, 000µ S/ cm Statistical methods were applied by means of a one-and was always above 3, 500µ S/ cm. The pH value was way ANOVA after normalizing transformation by the7.5 and the phosphate levels were over 25 µ g- at/ l, aresineof the square root ofrelativefrequency. Checkingeventually raising to over 1, 000 µ g- at/ 1. The nitrite was also made by using non-parametrical methodsconcentration was 3. 8µ g- at/ l, locally exceeding 10µ g- ( Kruskal-Wallis test). The differences were analyzed byat/ 1. Nitrates were found in values of 4 itg- at/ 1 and the Tukey test.dissolved oxygen was under 2. 7 ppm, generally withvalues equal to zero. Water temperature varied between RESULTS24 and 26° C. Experiments were carried out in the Laboratory Testsmonths of July and August, 1988. The results obtained for sensitivity of Cx. pipiens

In the preliminary tests, B. sphaericus concentrations third and early fourth-ins tar larvae to B. sphaericus areof 1. 2, 1. 6, 3. 0, 3. 2, 5. 0, and 7. 01/ Ha were used. In the summarized in TABLE 1. In the case of this product,final test the doses were 0.5, 1. 0, and 1. 5 1/ Ha. Twelve the data should be compared at 48 hours as it acts muchtreatments repeated in the summer of 1989 were carried more slowly than other compounds( Mulla et al. 1986)out to assess the period of efficacy, eight of these being but produces the greatest toxicity after 36 hours( Sinegreat doses of 1. 51/ Ha, and the rest at 3. 01/ Ha. As we were 1990).

mainly interested in obtaining data with a view to future The data from the two tests ( A and B) were

TABLE 1. Sensitivity of Culex pipiens to Bacillus sphaericus 2362 at river Llobregat delta as determined in thelaboratory.

Lethal Concentration( mg/ 1)Confidence Confidence

Test LC50 Interval LC90 Interval

A 24h. 0.0195 0.0167- 0.0222 0.0446 0.0388- 0.0528

B 24h. 0.0143 0.0117- 0.0172 0.055 0.0430-0.0770

Mean 0.0169 0.0498

A 48h. 0.0111 0.0095- 0.0130 0.0183 0.0155- 0.0236

B 48h. 0.006 0.0050-0.0070 0.014 0.0117-0.0178

Mean 0.0085 0.0161

Significance level P<0.05


comparable except for the LC50 at 48 hours in which the DISCUSSION

margins do not overlap. The average LC90 at 48 hourswas 0.016 mg/ l. We were able to determine the efficacy of B.

sphaericus against Cx. pipiens in the laboratory andField Tests under the particular field conditions in our area. The

Under field conditions and after having carried out LC90 of 0.016 14/ 1 at 48 hours that we obtained wastwo preliminary studies we were able to determine the similar to that reported in other studies( Mulla 1986).

most suitable dose. In these two tests all the However, few of the studies were done on Cx. pipiens;

concentrations chosen( 1. 17, 1. 58, 3.0, 3. 17, 5. 0, and 7.0 most laboratory data existing in the literature came from1/ Ha) allowed an effective control of between 95 and 99 experiments with Cx. quinquefasciatus. Our results are

percent reduction both at 24 or 48 hours. therefore within the range ofothers; and, from a practical

On finding optimum results at 24 hours, bearing in viewpoint, enabled us to decide whether to use them as

mind that it is after 36 hours that the highest mortality is an alternative to substances used hitherto without this

to be produced, and taking into account that we were initially implying a considerable increase in the doses totrying to find the minimum dose in order to produce be applied.

effective control, it was decided to repeat the test, this The comparison of field results is where greater

time with doses of 0.5, 1. 0, and 1. 5 1/ Ha( TABLE 2). discord might be expected due to the variation of

The dose of 1. 5 1/ Ha maintained a mortality close to 97 conditions. Sinegre et al.( 1990) found that with a dose

percent until the fifth day and one of 83 percent on the of 1. 51/ Ha in highly polluted water, the colonization byseventh day. Doses of 0. 5 and 1. 01/ Ha allowed a much Cx. pipiens would be renewed on the eighth day ofquicker recovery of the population with a mortality that treatment. Also, in a contaminated but shaded area 1. 0

did not reach 50 percent in five days. It should be 1/ Ha provided 76 percent control on the twelfth day. Apointed out that in all concentrations small quantities of dose of 2. 21/ Ha of formula BSP2 produced a control of

pupae were found on the seventh day, especially at the seven days while 4. 41/ Ha achieved this in a four week

lower concentrations. period against Culex stigmatosoma in polluted water

Matanmi et al. 1990). However, Arredondo-Jimenez

Period of Efficacy et al. ( 1990) found that the larval population of Culex

Tests to assess the period of efficacy were carried spp. could be controlled to 87 percent at a dose of 1. 25out during three months using doses of 1. 5 and 3.01/ Ha. 1/ Fla in a 12 week period in water exposed to the sun.

For the second dose only four repetitions were made due Our results show that under conditions of highlyto the drought affecting the area. The lowest dose gave polluted water, extreme turbidity, and maximuma mean of9. 3 days during which no pupa appeared, with exposure to the sun, 1. 51/ Ha gave a control of80 percent

a 7 to 12 day variation. The dose of 3. 0 1/ Ha in four by the seventh day for the whole of the larval population,repetitions exceeded that figure, attaining a mean of producing the appearance of pupae on the ninth day of12.5 days, with variations of from 10 to 17 days. treatment. The dose of 1. 01/ Ha, however, was unable to

TABLE 2. Percent of Culexpipiens larval mortality in highly polluted ditches at varioustimes after treatment.

Dose( 1/ Ha) 24 h 48 h 120 h 168 h

0.5 71. 68a 67. 1a 31. 78a 32.9a

1 80.22a 89.68b 14.0b

1. 5 94.26a 99.52b 96.8c 82.6b

Numbers followed by the same letters in each column are not significantly differentP< 0.05).

Population recovered.

No significant reduction found in control pool.


produce an effective control even on the fifth day. Karch, S., J. Coz, J. L. Jullien, G. Vigo, and G. Sinegre.Repeated treatment at 3. 0 1/ Ha was successful in 1986. Viabilite et persistance des spores de Bacillus

preventing the appearance of pupae until the twelfth sphaericus dans divers milieux aquatiques. 4eme

day. Dispersion in the results at the time of comparison congres sur la protection de la sante humaine et desis explained by the wide variation of conditions at the cultures en milieu tropical, Marseille 1986: 374-time of carrying out the field tests. The quantity of 379.

organic matter in suspension, the degree of sunlight, the

presence of abundant accompanying fauna, or even the Karch, S., N. Monteny, J. L. Jullien, G. SinBgre, and J.larvae density were influential at the time of obtaining Coz. 1990. Control of Culex pipiens by Bacillusthe data. The data presented in this paper have enabled sphaericus and role of nontarget arthropods in itsus, together with the extensive literature available, to recycling. J. Am. Mosq. Contr. Assoc. 6: 47- 54.select B. sphaericus as a promising candidate in thecontrol of Cx.pipiens populations in the Llobregat delta. Lacey, L., C. Lacey, B. Peacock, and I. Thiery. 1988.

Mosquito host range and field activity of BacillusAcknowledgments sphaericus isolate 2297 ( serotype 25). J. Am.

Mosq. Contr. Assoc. 4: 51- 56.We wish to thank Imma Busquet for the work she

did during the period of sample taking and analysis ofLiu, W. and H. Chen. 1988. Directly received. Shangai

some data. We would also like to thank Dr. Antoni Institute of Entomology. Academia Sinica, 19 pgs.Arcas for his help in performing the probit analysis.

Matanmi, B. A., B. A. Federici, and M. S. Mulla. 1990.REFERENCES CITED Fate and persistence of Bacillus sphaericus used as

a mosquito larvicide in dairy wastewater lagoons.Abbott, W. 1925. A method of computing the J. Am. Mosq. Contr. Assoc. 6: 384- 389.

effectiveness of an insecticide. J. Econ. Entomol.

18: 265- 267. Mulla, M. S. 1986. Efficacy of the microbial agentBacillus sphaericus Neide against mosquitoes

Arredondo-Jimenez, J., T. Lopez, M. Rodriguez, and D. Diptera: Culicidae) in southern California. Bull.Brown. 1990. Small scale field trials of Bacillus Soc. Vector Ecol. 11: 247- 245.sphaericus ( Strain 2362) against anopheline andculicine mosquito larvae in southern Mexico. J.

Mulla, M. S., H. Axelrod, H. Darwazeh, and B. A.Am. Mosq. Contr. Assoc. 6: 300- 305.

Matanmi. 1988. Efficacy and longevity ofBacillussphaericus 2362 formulations for control of

Berry, W., M. Novak, S. Khounlo, W. Rowley, and G. mosquito larvae in dairy wastewater lagoons. J.Melcior. 1987. Efficacy of Bacillus sphaericusAm. Mosq. Contr. Assoc. 4: 448- 452.

and Bacillus thuringiensis var. israelensis for controlof Culex pipiens and floodwater Aedes larvae in

Mull a, M. S., H. Darwazeh, and C. Aly. 1986. LaboratoryIowa. J. Am. Mosq. Contr. Assoc. 3: 579- 582. and field studies on new formulations of two

Davidson, E. 1985. Bacillus sphaericus as a Microbialmicrobial control agents against mosquitoes. Bull.

Control Agent for Mosquito Larvae.Soc. Vector Ecol. 11: 255 263.

g q Pp. 213- 226in Integrated Mosquito Control Methodologies,Vol II( M. Laird and J. Miles, eds.). Mulla, M. S., H. Darwazeh, E. Davidson, and H.

Dulmage. 1984. Efficacy and persistence of theFinney, D. J. 1971. Probit Analysis. 3d. ed. Cambridge microbial agent Bacillus sphaericus against

University Press, London. mosquito larvae in organically enriched habitats.J. Am. Mosq. Control. Assoc. 4: 166- 173.

Hougard, J. M. and L. Nicolas. 1986. Evaluation deBacillus sphaericus dans les gites larvaires a Culex Sinegre, G., B. Gaven, J. L. Jullien, and 0. Moussiegt.quinquefasciatus en Afrique Tropicale. 4eme 1990. Utilisation de Bacillus sphaericus dans lacongres sur la protection de la sante humaine et des lutte contre les moustiques: presentation,

cultures en milieu tropical, Marseille 1986: 369- performances et limites d' emploi. Document

373. EIDLM Num 57, 13 pgs.


A FIELD STUDY OF BACILLUS SPHAERICUS FOR THE CONTROL OF

CULICINE AND ANOPHELINE MOSQUITO LARVAE IN TANZANIA

R. N. Ragoonanansinghl, K. J. Njunwa2, C. F. Curtis3, and N. Becker4

ABSTRACT: Liquid and briquet formulations of Bacillus sphaericus were tested against larvae of Culex

quinquefasciatus and Anopheles funestus. Laboratory tests showed that the LC95 was 240 times higher foranopheline larvae compared with the culicines. Two or more weeks of control were obtained in natural breedingsites with 2. 5 ppm of a liquid formulation against Cx. quinquefasciatus but 60 ppm were needed for similar prolonged

control of An. funestus. The briquets were ineffective against both species in natural breeding sites.

INTRODUCTION Summit Chemical Co., Baltimore, MD, U.S.A.). The

susceptibility of larvae of Culex quinquefasciatus ( aThe microbial control agent Bacillus sphaericus filariasis vector) and Anopheles funestus ( a malaria

Neide has shown considerable activity against several vector) to each preparation was evaluated under

genera of mosquitoes. This activity has been laboratory and field conditions in Tanzania.demonstrated both in the laboratory and under fieldconditions ( Davidson et al. 1981; Lacey and Singer1982; Mulla et al. 1982, 1984, and 1986). MATERIALS AND METHODS

The larvicidal activity is based on the endotoxincrystals produced by B.sphaericus during the sporulation Laboratory Assaysprocess. Following their ingestion by mosquito larvae, Spherimos FC was tested in triplicate laboratorythe crystals are broken down within the larval gut into bioassays to assess the lethal concentrations( LC50 and

the active toxic compounds, which rapidly disrupt the LC95). A stock solution was prepared by adding 50µ lmidgut cells. This leads to paralysis and the death of the of homogenized Spherimos FC to 10 ml of distilled

larvae. The toxic effect appears to be restricted to larval water, and 0. 1 ml of this mixture was added to 9. 9 ml of

mosquitoes. It does not affect nontarget organisms distilled water and agitated to give a 50 µ 1/ liter stock

sharing the aquatic habitat or other invertebrates or solution. Samples of 20 An. funestus or Cx. quinque-

vertebrates( Mulla et al. 1984). fasciatus, collected in the field, were placed in plastic

Factors, such as the quality and temperature of the bowls with 100 ml of water. Using an Eppendorf Digitalwater, the sensitivity of different larval instars of the Pipette 4710, 1, 2, 4, 8, 10, and 12 ml volumes of the

same species or of different species, the larval density, stock solution were added to the plastic bowls containingand the occurrence of filter feeding nontarget organisms, An. funestus larvae to obtain final concentrations of 0.5,

all influence the effect of the microbial agents( Mulla 1, 2, 4, 5, and 6 ppm.

1990). It has been shown that first instar larvae of Culex In the bioassay with Cx. quinquefasciatus, finalpipiens are two to five times more susceptible to B. concentrations of 0.0005, 0.001, 0.0025, 0. 005, and

sphaericus products than are fourth- instar larvae 0.05 ppm were tested by adding 1, 2, 5, 10, and 100 glWraight et al. 1981). Finally there are differences in the of stock solution to the corresponding bowls. In each

potency of available formulations of B. sphaericus. test series, three bowls were left untreated as controls.

The present study was conducted in order to evaluate Larval mortality was recorded after 24 and 48 hours andthe larvicidal activity of two B. sphaericus formulations: the LC50 and LC95 values were estimated after plottingSpherimos FC( a fluid concentrate from Duphar B. V., probit mortality values against dosage on a logarithmicWeesp, Netherlands) and solid Spherimos briquets scale.

1Caribbean Epidemiology Centre, Box 164, Port of Spain, Trinidad.

2Amani Medical Research Centre, Box 4, Amani, Tanga, Tanzania.

3London School of Hygiene and Tropical Medicine, London WC1E 7HT, U.K

4German Mosquito Control Association( KABS), Postfach 210780, Ludwigshafen, Germany.


Field Tests LC95 in the laboratory for Cx. quinquefasciatus and 2xSmall scale field tests were conducted within a five and 10x the LC95 in the laboratory for An.funestus. The

kilometre radius of the Ubwari Field Station near the Spherimos briquets were placed on the water surface attown of Muheza in northeastern Tanzania during the a rate of one briquet per square meter.

short rainy season from mid-October to mid-December, The toxicity ofB. sphaericus and temephos( Abate)1990. Well-defined typical breeding sites of Cx. to notonectid predators of mosquito larvae was studied.quinquefasciatus and An. funestus, such as pools in A pond was treated with Spherimos FC( dosage: 1 ppm),blocked streams, were chosen as test sites. They were and two weeks later the same pond was treated withmore or less polluted and the sizes varied from 0.5 to 25 temephos at a dosage of 1. 0 ppm.m2. The depths were not accurately measured but were The density of the notonectids was determined byassumed to be 10 cm when estimating the dosage to be careful observation of the water, one square meter at aapplied. time. This was done one day before, immediately before

Larval density was monitored at four day intervals the treatments, and then daily up to one week afterwards.before treatment, immediately before treatment, andone day, two days, and one week after treatment, and RESULTS

later on a weekly basis up to five weeks. The larvalpopulation was determined by taking 10 dips( 250 ml Laboratory tests of Spherimos FC showed that aftereach) from different positions and the center of each 48 hours ofexposure, concentrations of 0.002 and 0.025site. Specimens obtained from each site were classified ppm caused approximately 50 percent and 95 percentaccording to instar ( first-fourth). Samples of fourth- mortality, respectively, of early fourth- instar larvae ofinstar larvae were identified by species. Two sites had Cx. quinquefasciatus. Anopheles funestus larvae wereboth Cx. quinquefasciatus and An. funestus; the others found to tolerate concentrations 240 to 500 times higherhad only one of these species. At each breeding site an than larvae of Culex; the LC50 and LC95 values for earlyindex of the level of organic pollution, pH, conductivity, fourth- instar larvae of An. funestus were estimated aschloride concentration, and intensity of sunlight were 1. 0 and 6. 0 ppm, respectively.recorded. In field tests, Spherimos FC caused a reduction of

The diluted liquid formulation was applied with a the Culex larval populations even at a dosage of 0.05hand-held flit gun ( calibrated for volume emitted per ppm( 2 x LC95). At a dosage of 2.5 ppm( 100 x LC95),pump stroke) so as to completely cover the water surface. complete control was observed in one site over a periodThe final concentrations were 2x, 100x, and 2400x the of five weeks( see TABLE 1; site no 4). In the untreated

TABLE 1. Field evaluation of Bacillus sphaericus ( Spherimos FC and Spherimos Briquets) against Culexquinquefasciatus larvae.

Mean Number of Larvae/DipSite Before After Treatment( Days)

No. Dose 16 12 8 4 0 1 2 7 14 21 28 35

1) 0.05 ppm FC 2 6 5 2 2 1 < 1 0 0 0 4 2

2) 0.05 ppm FC 1 3 1 3 2 1 0 0 0 < 1 0 < 1

3) 2.5 ppm FC 50 25 23 - 0 0 1 0 8 2 7

4) 2. 5 ppm FC 5 3 5 3 4 1 0 0 0 0 0 0

5) 60 ppm FC 3 6 1 1 6 1 0 0 0 0 < 1 0

6) 60 ppm FC 1 1 1 2 1 0 0 0 0 0 0 0

7) 1 briquet/m2 17 23 18 18 22 13 6 < 1 6 7 13 16

8) 1 briquet/m2 7 15 5 6 4 4 4 2 3 - - -

9) Control 4 4 4 5 4 7 6 4 4 4 5 3

10) Control 16 6 12 3 8 22 16 7 5 6 18 18


check sites the larval densities were consistently higher preparations. These include the potential for toxin

than those in treated sites. recycling and extended res idual effectiveness, the limitedCulex egg rafts were frequently noted during the potential for the development of resistance, and the lack

five weeks of posttreatment examination of breeding of toxicity to the environment andtonontargetorganisms,sites, long before larvae were observed. Extended particularly predators ofmosquito larvae. This durabilitycontrol was therefore due to the larvicidal efficacy and and specificity are coupled with rapid effectiveness,

persistence of B. sphaericus and not to the absence of even in polluted habitats, and safety during handling byoviposition by the mosquitoes. mosquito control operators. They are also safe for

In contrast with Spherimos FC, the briquet human consumption when applied to drinking waterformulation was ineffective in the natural breeding sites and are stable in storage.

of Cx. quinquefasciatus( TABLE 1, sites 7, and 8). This study was conducted in order to determine theTo fully control An. funestus larvae, 10 x LC95( 60 relative susceptibility of Culex and Anopheles larvae to

ppm) of Spherimos FC was required. Application at this two different formulations of B. sphaericus larvicide.

dosage completely controlled anopheline larvae within Spherimos FC was also compared to an

24 hours and continued to do so up to 28 days, after organophosphorus insecticide, temephos, in regard to

which first instars reappeared( TABLE 2, site 15). toxicity to notonectids, predators of mosquito larvae.TABLE 3 shows the environmental profiles ofeach The fact that in these studies An.funestus exhibited

site averaged from data collected 12 days prior to a lower susceptibility to Spherimos FC than Cx.treatment and 14 days after it. quinquefasciatus agrees with the results ofother workers

Notonectid ( Anisops and/ or Enithares spp.) and may have several explanations. Anopheles larvaepopulations were not affected by the application of feed at the air-water interface, rarely lower than 1- 2 mmSpherimos FC at 1 ppm. By comparison, the presence beneath the surface( Aly et al. 1987). Bacillus sphaericusof temephos reduced the notonectid density by 80- 90 spores are denser than water and sink to the bottom after

percent with many dead notonectids being observed application. In comparison, Culex larvae feed at all

TABLE 4). depths and are, therefore, more likely to encounter thetoxin. Also, while larvae of both genera are filter-

DISCUSSION feeders, the filtration rates ofAnopheles larvae are 10 to

20 times less than those of Culex larvae( Aly 1988).Microbial larvicides, such as B. sphaericus, offer The degree to which these behavioral differences

several advantages over traditional synthetic chemical may protect Anopheles from receiving a fatal dose of

TABLE 2. Field evaluation of Bacillus sphaericus( Spherimos FC and Spherimos Briquets) against Anopheles

funestus larvae. Note: Sites 14 and 15 in this TABLE were the same as sites 6 and 5, respectively, inTABLE 1.

Mean Number of Larvae/Dip

Site Before After Treatment( Days)

No. Dose 16 12 8 4 0 1 2 7 14 21 28 35

11) 12 ppm FC 3 1 1 2 2 1 2 0 0 < 1 < 1 1

12) 12 ppm FC 2 1 3 1 3 0 0 < 1 2 2 1 1

13) 12 ppm FC 7 1 2 4 2 1 < 1 0 0 0 2 2

14) 60 ppm FC 3 3 3 3 4 0 0 0 0 0 0 < 1

15) 60 ppm FC 1 2 1 3 3 1 0 0 0 0 < 1 0

16) 1 briquet/ nu 11 10 - - - 4 1 3 1 < 1 < 1 < 1

17) 1 briquet/ m2 8 3 2 8 - 4 < 1 < 1 < 1 < 1 3 -

18) Control 2 5 1 9 6 6 5 4 10 6 4 5

19) Control 2 7 2 2 2 2 2 2 1 2 5 2


TABLE 3. Environmental profile of each site ( average of readings taken 12 days before and 14 days aftertreatment).

a) Culex Sites:-

Site * Index of Organic Sunlight Conductivity ChlorideNo. Pollution Lux x 100) pH TDS) mg/ I)

1) 203 7.4 42 37

2) 849 7. 1 21 54

3) 407 5. 1 44 61

4) 385 9.0 39 103

5) 83 7. 4 45 76

6) 363 8. 4 76 126

7) 1028 7. 4 41 36

8) 0.67 10. 1 75 176

b) Anopheles Sites:-

11) 240 8.0 37 32

12) 774 7.0 30 61

13) 275 9. 2 73 181

14) 363 8. 4 76 126

15) 83 7. 4 45 76

16) 422 8. 1 23 35

17) 132 7. 7 14 23

On a scale from++++= highly polluted to+= clean.

TABLE 4: Comparison of the effects of Bacillus sphaericus and temephos( both at dosages of 1 ppm) onnotonectid( Anisops and/ or Enithares spp.) density.

Number of Notonectids per Square Meter

Before After Treatment( Days)

Larvicide 1 0 1 2 3 4 5 6

Spherimos FC 13 10 9 7 8 9 7 9

Temephos 9 2 1 2 1 1 1


toxin is not known. However, one would expect that the the laboratory the LC95 values were found to be 0.025Spherimos briquets, which float and release the active ppm for Cx. quinquefasciatus and 6 ppm forAn.funestus.

material at the water surface, would exhibit corn- 2) The liquid formulation yielded mediocre control in

paratively higher efficacy against Anopheles larvae the field at 2x these LC95 values for both Culex andthan the rapidly sinking liquid formulation. The findings Anopheles larvae. 3) Control of Culex was obtained at

of this study do not support this expectation for reasons 2.5 ppm and it persisted for two weeks or more. 4) At

that are not clear. Distribution of the toxin maybe more the highest concentration of 60 ppm, excellent control

restricted when the briquet formulation was used. At of sites containing populations of both larvae waseach of the four sites treated with a single Spherimos sustained for four weeks or longer. 5) The briquet

briquet ( sites between 2. 5 and 10 square meters), no formulation ( 10% active ingredient) compared

larvae were detectable in the vicinity of the briquet but unfavorably with the liquid formulation. This may bethey were readily found elsewhere within the site. In because of insufficient distribution of larvicide from

breeding areas where the free drift of the briquet would briquets. 6) Notonectid species were observed as a

be limited by vegetation or debris, the liquid formulation representative nontarget species and a known predator

should offer greater control. Alternatively, one could of mosquito larvae and were found not to be adverselyenvisage a role for a formulation in which the toxin is affected by the larvicide Spherimos FC.bound to small buoyant particles, thereby incorporatingthe advantages of wide distribution and concentration at

the water surface and thus offering more effective Acknowledgments

control of Anopheles.

Of the two, the briquet formulation may provide We thank the Tanzanian Science and Technologygreater efficacy when used in small breeding sites, Commission, Professor W. Kilama and Dr. S. bare of

which are subject to repeated cycles of flooding and the Tanzanian National Institute for Medical Research

drying. The floating briquet would continue to release for authorizing this study.toxin in the treated site with each successive flooding At the Ubwari Field Station, our sincere thanks are

while the liquid formulation might become bound to extended to Jasper Ijumba, acting Head of the Station,bottom sediments. This possibility was not tested in this and to Tony Wilkes, Maggi Phillip Kivuyo, Selemanistudy. Twahili, and Charles Chambika.

The efficacy and longevity of the larvicide may be Funding for this project was generously suppliedinfluenced by the degree of water pollution. Higher by the Sir Halley Stewart Trust.levels of control in clean, as opposed to polluted, waters

were demonstrated by Mian et al. ( 1983). However, REFERENCES

longer residual effects were obtained at higher levels of

pollution in a study by Nicolas et al.( 1987). The degree Aly, C., M. S. Mulla, W. Schnetter, and Bo-Zhao Xu.of pollution was noted for our sites( TABLE 3) and the 1987. Floating bait formulations increaserange of this variable for either genus alone was limited. effectiveness of Bacillus thuringiensis var.

As expected, Culex were found to tolerate higher levels israelensis against Anopheles larvae. J. Am. Mosq.of pollution than Anopheles larvae. Control Assoc. 13: 583- 588.

An important advantage of B. sphaericus is that

nontargetspecies, andparticularlypredatorsofmosquito Aly, C. 1988. Filtration rates of mosquito larvae in

larvae, remain unharmed by the toxin. Notonectids suspensions of latex microspheres and yeast cells.

represent one such group of predators and B. sphaericus Entomol. Exp. Appl. 46: 55- 61.was compared to temephos with respect to adverse

effects on notonectid numbers ( TABLE 4). Bacillus Davidson, E. W., A.W. Sweeney, and R. Cooper. 1981.sphaericus did not reduce notonectid populations Comparative field trials of Bacillus sphaericus

significantly. Temephos application, however, resulted strain 1593 and Bacillus thuringiensis var.

in a notable reduction of their numbers for up to one israelensis commercial powders. J. Econ. Entomol.

week with numerous dead notonectids observed. Just 74: 350- 354.

how important notonectid predation is in the control of

mosquito larvae has not been established, but it seems Lacey, L. A. and S. Singer. 1982. Larvicidal activity oflikely that their feeding activity would supplement new isolates of Bacillus sphaericus and Bacillus

larval control by a larvicide. thuringiensis against mosquito larvae. Mosq. NewsThis study led to the following conclusions: 1) In 42: 537- 543.


Mian, L. S. and M. S. Mulla. 1983. Factors influencing microbial agent Bacillus sphaericus against

activity of the microbial agent Bacillus sphaericus mosquito larvae in organically enriched habitats.against mosquito larvae. Bull. Soc. Vector Ecol. 8: Mosq. News 44: 166- 173.128- 134.

Mulla, M. S., H. A. Darwazeh, and C. Aly. 1986.Mulla, M. S. 1990. Activity, field efficacy, and Laboratory and field studies on new formulations

use of Bacillus thuringiensis israelensis against of two microbial control agents against mosquitoes.

mosquitoes. Pp. 134- 160 in Bacterial Control Bull. Soc. Vector Ecol. 11: 255- 263.of Mosquitoes and Blackflies ( H. de Barjac

and D. J. Sutherland, eds.), Unwin Hyman, Nicolas, L., J. Doussou- Yovo, and J. M. Hougard.London. 1987. Persistence and recycling of Bacillus

sphaericus 2362 spores in Culex quinquefasciatusMulla, M. S., B. A. Federici, and H. A. Darwazeh. 1982. breeding sites in West Africa. Appl. Microbiol.

Larvicidal efficacy of Bacillus thuringiensis Biotech. 25: 341- 345.

serotype H- 14 against stagnant water mosquitoesand its effect on nontarget organisms. Environ. Wraight, S. P., D. Molloy, and H. Jamnback. 1981.Entomol. 11: 788- 795. Effect of Bacillus sphaericus strain 1593 against

the four instars of laboratory reared and fieldMulla, M. S., H. A. Darwazeh, E. W. Davidson, and H. collected Culexpipienspipiens and laboratory Culex

T. Dulmage. 1984. Efficacy and persistence of the salinarius. Can. Entomol. 113: 379- 386.

BULL. SOC. VECTOR ECOL., 17( 1): 51- 56JUNE, 1992

EFFICACY AND SELECTIVITY OF TWO PYRETHROID INSECTICIDESAGAINST THE PREDATOR TRIOPS LONGICAUDATUS

NOTOSTRACA: TRIOPSIDAE) AND CULEX TARSALIS LARVAE

M. S. Mullal, M. Zgomba2, H. A. Darwazehl, and J. D. Chaney1

ABSTRACT: The tadpole shrimp, Triops longicaudatus, is an effective predator of Culex tarsalis larvae inexperimental field mesocosms. This predator interferes with the production of mosquito larvae for experimentalpurposes and it has become necessary to control hatched populations of tadpole shrimp with an effective pesticidethat will have a higher level of activity against the predator than the prey, mosquito larvae. Two new syntheticpyrethroids ( bifenthrin and lambdacyhalothrin) were evaluated against tadpole shrimp and Cx. tarsalis larvae inmesocosms. Tadpole shrimp were completely controlled at the very low dosages of 0.25 to 0.50 g AI/hectare. Thesetwo materials were also highly active against mosquito larvae; somewhat higher rates of 1. 0 to 1. 5 g AI/ha producedcomplete control of larvae. A three to five- fold difference in activity( at the 100% mortality level) against the twogroups was noted, which may allow for selective control of tadpole shrimp where this predator and pest must bemanaged. Since it takes three to five times more insecticide to eliminate mosquito larvae than the rates needed fortadpole shrimp control, the predator can be eliminated without affecting mosquito larvae. In repeated experimentsthese two pyrethroids at the rates of 0. 25-0.50 g AI/ha have controlled tadpole shrimp without adversely affectingmosquito larvae.

INTRODUCTION 1989, 1990, 1991). Tadpole shrimp also have beenfound to be pests in rice paddies in some special situations

Tadpole shrimp Triops longicaudatus( Le Conte) where rice culture involves seeding and flooding,are floodwater organisms. They breed or are found in especially where water covers the field during seedephemeral aquatic situations where the total habitat or germination ( Grigarick et al. 1985, Rosenberg 1947).portions of it are subjected to intermittent flooding and Due to their burrowing and swimming activity, thedrying. When eggs are deposited, they sink to the shrimp dislodge and feed on rice seedlings and in thesebottom or are attached to substrata in the breeding situations their control is an essential part of ricehabitats. In the moist habitat, eggs undergo production( Grigarick et al. 1961, 1985).embryogenesis and conditioning. After conditioning, During the past two decades we have beenthe eggs remain viable in completely dry habitats for conducting fundamental and applied research on themonths or years, and hatch within 24-72 hours after biology, ecology, and control of stagnant- waterreflooding. Mosquitoes also breed profusely in mosquitoes, using experimental field ponds orintermittently flooded and dried habitats. Soon after mesocosms at two sites. The primary purpose of theseflooding of suitable breeding sources, a brood of flood- ponds is to raise natural populations of mosquitoes andwater mosquitoes( Aedes and Psorophora) will develop other biota for experimental purposes. However, duringwithin five to seven days after flooding. If the water the past five years or so, heavy populations of tadpoleremains for longer periods, a succession of Culex and shrimp have decimated mosquito larvae and to someother stagnant water mosquitoes ensue within 7- 15 extent othermacroinvertebratesaswell. To utilize thesedays. In this situation, where tadpole shrimp and established mesocosms in the intended research, itmosquito larvae are found together, this predator plays became essential to control tadpole shrimp after eachan important role in regulating mosquito larval flooding.populations. The current studies were initiated to evaluate

Tadpole shrimp have been reported to be effective chemical control agents that would eliminate hatchedbiological control agents of Culex mosquitoes in some tadpole shrimp without impairing the subsequentsemipermanently flooded habitats ( Tietze and Mulla recruitment or survival of mosquito larvae. Some of the

1Department of Entomology, University of California, Riverside, CA 92521, U.S.A.

2Current address: College of Agriculture, University of Novi Sad, Novi Sad, Yugoslavia.


synthetic pyrethroid insecticides that are highly active various experiments.

against mosquito larvae ( Mulla and Darwazeh 1985, Mosquito larvae were sampled before and after

Mulla et al. 1978, 1980, 1981, 1982b) have been found treatment by the following dipping technique. In eachto be also highly active against the shrimp( Walton et al. mesocosm four dips( 400 ml each) were taken in or near

1990). each corner( where larvae occurred at higher density)

In the present studies we assessed the efficacy of and a fifth dip sample was taken at random along one oftwo new pyrethroid insecticides against both young the margins. The samples were composited,

tadpole shrimp and mosquito larvae with the hope that concentrated, and preserved in 70 percent ethanol. All

these insecticides would have a higher level of activity larval instars were counted under a stereomicroscope

against the shrimp and thus spare mosquito larvae which and grouped as first and second- instars and third and

appear a few days later. fourth- instars. The reduction was calculated on the

basis of the mean counts of third and fourth- instar

MATERIALS AND METHODS larvae, which receive the maximum exposure. The firstand second- instar larvae were not employed since the

Natural populations of the tadpole shrimp Triops first- instar larvae could have hatched out recently but

longicaudatus, a predator, and mosquito larvae, the would not have had adequate exposure.

prey, were experimentally produced in previously Bifenthrin ( formulation Capture® EC 2, FMC

described field mesocosms ( Mulla et al. 1982a) in Corporation, Princeton, NJ) and lambdacyhalothrin

which the water level was maintained by float valves. ( formulation Karate® EC 1, ICI Americas Inc.,

Mesocosms located at the Aquatic and Vector Control Richmond, CA) were applied as aqueous sprays three to

Research Facility at the University of California, four days after flooding when most shrimp wttehatched

Riverside, were 27 m2 in surface area and 35 to 40 cm out. In preliminary studies these two compounds were

deep. Rooted plant growth in these mesocosms was found to be extremely active against both tadpole shrimpcontrolled by physical removal and herbicidal and mosquito larvae, so very low dosages had to beapplication. At Oasis in theCoachella Valley ofsouthern applied. To apply these very low dosages to the smallCalifornia the mesocosms were 30 m2( water depth 35- mesocosms, the EC formulations were diluted in tap

40 cm) and fully vegetated with Bermuda and crab grass water to obtain 1 percent A.I. stock suspensions which

5- 10 cm high). Water temperature at both sites was were serially diluted with water to obtain the 0. 1 andmonitored by placing maximum-minimum thermo- 0.01 percent suspensions that were employed in the

meters in the mesocosms. treatments. To treat a mesocosm, the required aliquot of

Before flooding, 1 kg of chicken laying mash was the dilute suspension was added to 120 ml water in a

added to each mesocosm to organically enrich the plastic squeeze bottle. The total volume was then

habitat for Culex tarsalis Coquillett production. sprayed onto the water surface of a mesocosm. Several

Mosquito oviposition usually occurs for the first time in rates of application( 0. 25 to 1. 5 g/ha) were tested against

four to seven days after flooding, and third and fourth- both shrimp and mosquito larvae. Each treatment andinstar larvae generally become abundant for testing 8 to control in each test were replicated three times with the

12 days after flooding. In contrast, shrimp eggs usually treatments randomly assigned. The concentrations ofhatch within 24 to 72 hours after flooding and are the various dosages were calculated on the basis of

noticeable within three to four days. The shrimp have a 10,000 liters the total volume of water per pond. The

synchronous mode of development, whereas, Cx. tarsalis magnitude of percent reduction of third and fourth-

is asynchronous. instar mosquito larvae and tadpole shrimp was calculated

Pretreatment and posttreatment density of tadpole by the method of Mulla et al.( 1971).shrimp was monitored by dragging a D- frame coarse netbase width 33 cm, mesh 9 X 11 opening/ cm, opening RESULTS AND DISCUSSION

size 60 X 80 micrometers), 150 cm handle through thewater along the perimeter of each mesocosm. Materials Bifenthrin EC 2 was tested against the tadpole

collected in two drag samples, each covering two sides shrimp in three experiments in the vegetated mesocosmsof the mesocosm( Walton et al. 1990) were transferred at Coachella Valley while lambdacyhalothrin was testedto an enamel pan filled with water. The sampled in one experiment in vegetation devoid mesocosms at

material was spread in the water and live shrimp were Riverside. In the first two experiments in the Coachella

counted and their size estimated. After counting the Valley ( A and B) ( TABLE 1) each treatment level

shrimp, each sample was returned intact to its respective produced 100 percent mortality. In the third experimentmesocosm. Their size ranged from 4 to 8 mm in the at this location ( C), the administered dosages were


TABLE 1. Efficacy of two pyrethroids bifenthrin( Capture® EC 2) and lambdacyhalothrin( Karate EC 1)against natural populations of the tadpole shrimp Triops longicaudatus in field experimentalmesocosma.

Mean no. of tadpole shrimp/sampleRate Concentration 2 days

g/ha ppb Pretreatment posttreatment reduction

Test A( May 1990) bifenthrinb

0.25 0.0725 17 0 1000.50 0. 145 14 0 1001. 00 0.290 8 0 100

Check 0 17 10 41

Test B ( June 1990) bifenthrinc

0.25 0.0725 11 0 1000. 50 0. 145 12 0 100

Check 0 17 17 0

Test C( June 1990) bifenthrind

0.05 0.0145 18 13 410. 10 0.0290 17 3 860.25 0.0725 28 0 100

Check 0 18 22 0

Test D( July 1990) lambdacyhalothrine

0.05 0.0145 34.5 33. 3 4

0. 10 0,0290 77.0 102. 5 00. 25 0.0725 116.3 1. 5 99

0.50 0. 1450 75. 8 0.0 100Check 0 70.4 70. 1 1

aTests A, B, C in mesocosms in the Coachella Valley, test D in mesocosms at Riverside, California.bWater temperature mean min.- max. 19- 30° C, TPS size 5- 7 mm.eWater temperature mean min.-max. 24. 5- 29° C, TPS size 4- 6 mm.dWater temperature mean min.- max. 20- 30° C, TPS size 5- 7 mm.eWater temperature mean min.- max. 25- 32°C, TPS size 5- 7 mm.

lowered and the activity range for bifenthrin was found. respectively( TABLE 1).At the dosage of 0. 1 g/ ha, bifenthrin yielded 86 percent From these studies it is evident that bifenthrin andreduction, while at 0. 25 g/ ha, as in experiments A and B, lambdacyhalothrin have similar activity against tadpoleit yielded 100 percent control of shrimp populations. shrimp. Essentially similar results were obtained in

Lambdacyhalothrin EC 1 evaluated in the tests conducted at the two sites with the two materials.unvegetated mesocosms ( test D) at Riverside yielded A minimum dosage of 0. 25 g/ ha of either materiallittle or no reduction at the very low rates of application produced 100 percent control. To compensate forof 0. 05 and 0. 10 g/ ha( TABLE 1). However, at 0.25 and uncontrollable variables in rice paddies or other natural0.5 g/ ha, it yielded 99 and 100 percent reduction, field situations where shrimp control is desired, a


maximum dosage of 1 g/ ha( which is still very low) may From the data generated in these studies, it is quite

be required for their effective control. The efficacy of apparent that both bifenthrin and lambdacyhalothrin

bifenthrin and lambdacyhalothrin( both with an effective have an extremely high level of activity against tadpoledosage of 0. 25 g/ ha) under comparable conditions is shrimp and mosquitolarvae. However, both compoundshigher than that of another closely related pyrethroid, have higher activity ( three to five-fold at the 100%cypermethrin ( effective dosage of 0.6 to 1. 1 g/ ha as mortality level) against shrimp than against mosquitoes.reported by Walton et al. 1990). Compared to the This differential activity and selectivity will allow theorganophosphate insecticides, both bifenthrin and use of these two pyrethroids at the very low rates of 0.25lambdacyhalothrin have 400 to 500- fold higher activity to 1. 0 g/ ha to control the shrimp without appreciablythan fenthion and about 100- fold higher level of activity impacting the larvae. However, if the situation calls foras compared to chlorpyrifos against tadpole shrimp the control of mosquitoes as well, then the dosage

populations. The activity range of these two pyrethroids should be increased to 1 to 2 g/ ha rates which is still quiteagainst the shrimp is even much greater than the activity low.

of DDT, Diazinon, and malathion which were found to Timing of application will also allow for effectivebe effective against shrimp in rice fields in the range of control of the shrimp predator without diminishing the2 lb/a as reported by Grigarick et al. ( 1961). The potential for mosquito production. Since the shrimppyrethroids, therefore, are over 2200- fold more active hatch peaks at three to four days and mosquito larvae

than these conventional insecticides. With such an appear seven to ten days after flooding, an early treatment

extremely high level of activity of bifenthrin and with the effective pyrethroid three to four days

lambdacyhalothrin, these materials can be used postflooding will avoid impact on mosquito larvae. Theselectively to control tadpole shrimp and not impact very low dosages applied for shrimp control have nomosquito larvae( see below) and other biota. persistence and will likely lose effectiveness within a

Both bifenthrin and lambdacyhalothrin, when day or two after treatment, a time when no Culex larvaeevaluated against larvae of Cx. tarsalis, showed a very are present. To further document this feature of these

high level of effectiveness. At 0.25 and 0.50 g/ ha, two pyrethroids, we have used the dosage of 0.25 and

bifenthrin yielded 21 and 80 percent control two days 0.50 g/ ha that eliminated shrimp but did not impactposttreatment, and the level of control improved with mosquito larvae that appeared as young instars in thean increase in dosage( TABLE 2). At 1. 0 and 1. 5 g/ha mesocosms three to seven days after treatment with the

control of third and fourth- instar larvae, equalled or minimal effective dosages for tadpole shrimp control.exceeded 92 and 97 percent, respectively, for 14 days. Cypermethrin, a closely related pyrethroid, isBifenthrin as well as lambdacyhalothrin showed similar another compound that exhibited higher activity againstlevels of activity against larvae of Cx. quinquefasciatus tadpole shrimp ( with an effective dosage of 0. 6 g/ ha,Say in the laboratory( see footnotes C in TABLE 2 and Walton et al. 1990) than against Cx. tarsalis larvae with

TABLE 3). an effective dosage of 2. 8 to 5.5 g/ ha as evaluated in theThe efficacy of lambdacyhalothrin was also very same mesocosms( Mulla and Darwazeh 1985, Mulla et

high against Cx. tarsalis larvae but somewhat lower al. 1982b). On the basis of these findings, these three

than the efficacy ofbifenthrin. At the rates of 1. 0 and 1. 5 pyrethroids could be employed for shrimp control in

g/ ha, lambdacyhalothrin produced 62 and 100 percent rice culture as well as in experimental mesocosms

reduction two days posttreatment; 92 to 100 percent where production of mosquitoes and other biota is

reduction, respectively, was observed after seven days desired.

with 0.5, 1. 0, and 1. 5 g/ha( TABLE 3). The highest rate The synthetic pyrethroids have been known to

1. 5 g/ ha) provided 94 percent reduction 14 days show toxicity to fish( at higher dosages than the targetposttreatment. One possible reason for the somewhat dosage) and some groups ofnontarget organisms( Mulla

lower activity of lambdacyhalothrin may be the et al. 1978, 1980). However, it is anticipated that the

temperature differential during the testing period of this impact of the low dosages employed here would be

compound and bifenthrin. During the bifenthrin test, minimal as two other related pyrethroids, permethrin

water temperature was lower ( mean min.-max. 15. 1° and decamethrin, were found to be innocuous in

and 25. 7° C), while during the lambdacyhalothrin test, experiments on the survival andreproduction ofmosquito

the water temperature was higher ( mean min.-max. fish, Gambusia affinis Baird and Girard, using multiple21. 4° and 31. 8° C). Since mostpyrethroids have negative applications at five-fold mosquito larvicidal rates( Mulla

temperature- activity relationship( more active at lower et al. 1981). Impact studies on macroinvertebrates and

temperatures), it is likely that the lower temperature fish using bifenthrin and lambdacyhalothrin areinfluenced the level of mortality slightly. warranted using the extremely low rates of application.


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REFERENCES CITED Mulla, M. S., H. A. Darwazeh, and L. Ede. 1982b.

Evaluation of new pyrethroids against immature

Grigarick, A. A., W. H. Lange, and D. C. Finfrock. mosquitoes andtheireffectsonnontargetorganisms.

1961. Control of tadpole shrimp, Triops Mosq. News 42: 583- 590.longicaudatus, in California rice fields. J. Econ.Entomol. 54: 36- 40. Mulla, M. S. and H. A. Darwazeh. 1985. Larvicidal

activity of two new pyrethroids against mosquitoGrigarick, A. A., J. H. Lynch, and M. 0. Way. 1985. larvae. Bull. Soc. Vector. Ecol. 10: 1- 6.

Controlling tadpole shrimp. Calif. Agr. March-April 1985: 12- 13. Rosenberg, L. E. 1947. Apus as a pest in California rice

fields. Calif. Dept. Agric. Bull. 36: 42- 48.Mulla, M. S., R. L. Norland, D. M. Fanara, H. A.

Darwazeh, and D. W. McKean. 1971. Control of

Tietze, N. S. and M. S. Mulla. 1989. Prey- size selectionchironomid midges in recreational lakes. J. Econ.

by Triops longicaudatus( Notostraca: Triopsidae)Entomol. 64: 300-307.

feeding on immature stages of Culex

quinquefasciatus. J. Am. Mosq. Control Assoc. 5:Mulla,M. S., H.A.Navaab-Gojrati, and H. A.Darwazeh.

392- 396.1978. Biological activity and longevity of newsynthetic pyrethroids against mosquitoes and some

nontarget insects. Mosq. News 38: 90- 96. Tietze, N. S. and M. S. Mulla. 1990. Influence of

tadpole shrimp, Triops longicaudatus( Notostraca:

Mulla, M. S., H. A. Darwazeh, and M. S. Dhillon. 1980. Triopsidae), stocking rate on Culex tarsalis

New pyrethroids as mosquito larvicides and theirdevelopment in experimental field microcosms. J.

effects on nontarget organisms. Mosq. News 40: 6- Am. Mosq. Control Assoc. 6: 265- 269.

12.

Tietze, N. S. and M. S. Mulla. 1991. Biological control

Mulla, M. S., H. A. Darwazeh, and M. S. Dhillon. 1981. of Culex mosquitoes ( Diptera: Culicidae) by theImpact and joint action of pyrethroids and freshwater tadpole shrimp Triops longicaudatus( Notostraca:fishes on mosquitoes. Bull. Environ. Contamin. Triopsidae). J. Med. Entomol. 28: 24- 31.

Toxicol. 26: 689- 695.

Walton, W. E., H. A. Darwazeh, M. S. Mulla, and E. T.

Mulla, M. S., B. A. Federici, and H. A. Darwazeh. Schreiber. 1990. Impact of selected synthetic

1982a. Larvicidal efficacy ofBacillus thuringiensis pyrethroids and organophosphorous pesticides on

serotype H- 14 against stagnant- water mosquitoes tadpole shrimp, Triops longicaudatus Le Conteand its effect on nontarget organisms. Environ. Notostraca; Triopsidae). Bull. Environ. Contamin.

Entomol. 11: 788- 795. Toxicol. 45: 62- 68.

BULL. SOC. VECTOR ECOL., 17( 1): 57-69 JUNE, 1992

THE DISTRIBUTION AND SEASONAL ABUNDANCE OF DEER KEDS (DIPTERA:

HIPPOBOSCIDAE) ON COLUMBIAN BLACK-TAILED DEER ( ODOCOILEUS

HEMIONUS COLUMBIANUS) IN NORTHERN CALIFORNIA

D. R. Westroml and J. R. Anderson2

ABSTRACT: The seasonal abundance and distribution of deer keds( Lipoptena depressa Say and Neolipoptenaferrisi Bequaert) on Columbian black- tailed deer in northern California were studied over a three year period. Adigestion technique enabled quantitative collection of ectoparasites from the hide of the entire host. Absolutepopulation densities of deer keds were compared on the basis of host anatomy, age, and sex and on the season andhabitat of collection. Lipoptena depressa was present on all 71 deer examined; a total of 30, 091 specimens wascollected with a mean infestation of 424 keds per infested deer( range 21 to 1, 563). Seasonal abundance data showed,

for the first time, that this was a bivoltine species with populations on deer peaking in midsummer and early winter.Neolipoptena ferrisi was found on 60 of 71 deer with a significantly lower prevalence on younger deer and deercollected in the spring. This univoltine species reached its greatest density on deer during the summer. The 60infested animals yielded 3, 660 N.ferrisi with a mean infestation of 61 keds per deer( range 1 to 481). Alate( winged)forms ofboth species were far less abundant on deer than were the apterous forms. Most L depressa( 64%) occurred

on the posterior regions of the deer with only 1. 5 percent on the head. Conversely, most N.ferrisi were found onthe head, with only 5 percent collected from the posterior regions. Factors potentially influencing the disparatedistributions of the two species are analyzed and discussed.

INTRODUCTION and on Lipoptena mazamae( Samuel 1969, Samuel and

Trainer 1972, Davis 1973). Unfortunately, data on theHippoboscid flies ( deer keds, louse flies) are seasonal abundance and distribution of L depressa and

common hematophagous ectoparasites of deer N.ferrisi are fragmentary and inconsistent. Previousworldwide. In northern California two species, Lipoptena studies have emphasized qualitative aspects and usuallydepressa Say and Neolipoptena ferrisi Bequaert, have been hampered by a paucity of hosts examined,parasitize the Columbianblack- tailed deer( Odocoileous limited seasonal data, or inadequate collectionhemionus columbianus Richardson). The literature on techniques.

the population dynamics of these insects consists The objectives of this study were to: 1) determineprimarily of records of specimens collected from hosts, the spatial distribution of deer keds on individual hosts;which for the most part, have been sampled using 2) determine the prevalence and seasonal abundance of

handpicking or combing of selected anatomical areas of deer keds occurring on a representative number of hosts;the deer( Dixon 1934, Dixon and Herman 1945, Shaw and 3) evaluate selected factors( intrinsic and extrinsic)1947, Leopold et al. 1951, Holdenreid et al. 1951, that could contribute to the observed seasonal abundance

Longhurst et al. 1952). Only Linsdale and Tomich and distribution of the deer keds.

1953) have published quantitative data on hippoboscidinfestations of black- tailed deer. Senger and Capelle STUDY AREA

1959) used a semiquantitative combing techniquewhen sampling mule deer and white- tailed deer in All deer were collected at the University ofMontana. California Hopland Field Station( HFS) in Mendocino

Studies on other species ofLipoptena that parasitize County, California. This station is located 160 km northvarious species of cervids in North America, Europe, of San Francisco and 64 km inland from the Pacificand Asia have contributed significantly to our knowledge Ocean. It extends from the floor of the Russian River

ofdeerkedpopulations, especially the work on Lipoptena Valley to the crest of the Mayacamas Mountains, whichcervi( Haarlov 1964, 1965, Popov 1965, Ivanov 1974) separate the Russian and Sacramento river drainages.

1# 11 Doctors Park Drive, Santa Rosa, CA 95405, U.S. A.

2Department of Entomological Sciences, University of California, Berkeley, CA 94720, U.S. A.


The terrain is hilly and interlaced with small creeks and anatomical area, and released them again onto previouslyravines. The elevation ranges from 150 to 900 m. combed areas. After release of 435 marked keds, the

Eighty percent of the station is mixed oak woodland- deer carcasses were treated as all others in the studygrassland habitat and 15 percent is chaparral. More except that they were combed again for keds after aboutdetailed descriptions of the station have been presented an hour instead of being frozen and soaked in 5 percentby Heady 1961, Hoy and Anderson 1978, and Anderson, KOH. All hippoboscids recovered in the second combingOlkowski, and Hoy 1974. were examined with a dissecting microscope under

During the three years of this study the black- tailed ultraviolet light.

deer population on the HFS numbered from 550 to 900, Specimens were identified using the key,as estimated by the methods of Anderson et al.( 1974). descriptions, and figures of Peterson and Maa( 1970)

Deer were available for study from two sources: animals and Bequaert( 1957). Both L depressa and N.ferrisi

killed during the hunting season in August and September have two easily recognized morphological forms. Newlyand deer collected at other times on a scientific permit. emerged flies have fully developed, functional wings,The latter deer also were used for several other studies but after these alate(= volant) forms have located a host

e. g., Connolly and Longhurst 1975, Longhurst et al. deer, they soon lose their wings, and the apterous forms1979). begin to increase in size.

Deer ked numbers were compared on the basis of

MATERIALS AND METHODS host anatomy, age, sex, the season of collection, andwhether the deer was from chaparral or oak woodland-

A total of 24 male and 47 female black- tailed deer, grassland habitat. An analysis of variance with split-

ranging in age from 5 months to 12 years, was collected plot design was used for interpretation of the abundance

and examined during the period October 1970 to October data. Duncan' s multiple range test was used to test for

1973. The deer were shot in the neck or head. The significantly different means. Prevalence data were

number examined by season was fall( 18), winter( 12), analyzed by chi square tests using 2X2 contingencyspring ( 23), and summer( 18). Hosts that were not of tables. A log( n+ 1) transformation was used to normalizeknown age were placed into year classes on the basis of the data prior to analysis. Goodman' s log linear modelmolar wear and tooth eruption on the lower jaw( Larson was used to analyze the data on distribution( Goodman

and Taber 1980). The carcasses were bagged separately 1978).

in large plastic bags within 5- 15 minutes after death andtransported by truck to station headquarters. After RESULTS

animals were skinned in a postmortem laboratory, thehides were placed into separate plastic bags and kept Seasonal Abundance

frozen until examined. The time elapsed between death Lipoptena depressa was the most common

and skinning almost always was less than one hour and ectoparasite encountered, being found on all 71 deernever more than two hours. examined. These 71 deer yielded 30,091 L depressa

Each hide was thawed prior to processing and 42 of with a mean infestation of 424 keds per deer and a range

the 71 deer were divided into six sections: head, anterior of 21 to 1, 563. The frequency distribution was skeweddorsum, anterior venter, posterior dorsum, posterior with most deer being infested with fewer than 600 fliesventer, and tail including the perianal area( Fig. 1). The ( Fig. 2). There was no significant difference in the

separate sections, and the whole hides of other deer, prevalence or the mean number of L depressa on hosts

were soaked for 36 to 48 hours in separate containers of of different ages or sexes nor from the two different

5 percent KOH to promote sloughing of the hair from habitats. However, the seasonal occurrence of L

the skin. The loose hair, along with the ectoparasites depressa on deer ( Fig. 3) was bimodal; peaks inand detritus that it contained, was then heated to boiling midsummer and early winter indicate that this is ain fresh 5 percent KOH. After being filtered, rinsed in bivoltine species.

tap water, and suspended in 70 percent ethanol, the keds The winged forms were far less abundant on deer

were identified, sexed, and counted while viewed with than the apterous flies, with only 45 of 71 deer( 63.4%)a stereoscope. being infested. Alates also were absent from deer for

To study possible movement of keds after host nearly four months( December to late March) in winterdeath, we captured keds on six dead deer by combing and again for about two to four months in summer,

within a few minutes of host death, marked them with depending on the year( Fig. 3). The largest number of

different colors of Helecon® fluorescent dusts( United alates collected from one deer was 15, and the mean

States Radium Corporation, Morristown, NJ) for each density was 4. 7 per deer.


hi.'j

v. it f f.:j •f_..

1v

041- pv

pd

C•:

j

t

Figure 1. Sections of deer hide processed separately for a

3to.partasites. The percent surface area for each section

was: h= 14.9; ad= 20.0; av= 22.4; pd=145; pv=2 . 9; t= 4.3.


30—

25—

20—

8 Lipoptena depressa

0

15—

100 300 500 700 900 1100 1300 1500

Number of Hippoboscids

Figure 2. Frequency distribution of Lipoptena depressa collected from 71 black- tailed deer at the Hopland FieldStation( 1970- 1973).

Neolipoptena ferrisi was found on 60 of 71 deer A total of 3, 660 N.ferrisi was collected from 6084. 5%); the mean number and prevalence( TABLE 1) infested deer with a mean of 61. 0 flies per deer and a

were significantly lower on fawns( 1- 12 months) than range of 1 to 481. Like L depressa, the frequencyon middle-aged ( 13- 36 months) or old ( 37+ months) distribution of N.ferrisi was also skewed( Fig. 5) withdeer. There was no significant difference in the mean very few heavily infested deer.number and prevalence ofN.ferrisi on deer of different

sexes or from the two different habitats. The data show Sex ratio

that this is a univoltine species( Fig. 4) that was far more The overall sex ratio of L depressa did not differprevalent in the summer( JJA) than in the spring( MAM). significantly from 1: 1. However, deer examined inThe alates of N. ferrisi were less prevalent than the summer had a significantly smaller percentage of malesapterous forms, infesting only 24 of 71 deer ( 33. 8%). ( p<0.05) than deer from fall or winter. The sex ratio ofThe mean density of alates was 2. 8 per deer, and the L depressa was also related to location on the host.maximum number collected from one deer was 10. No There was a smaller proportion of males( p< 0.05) on thealates were collected from December through March, tail( 46.2%) and anterior ventral regions( 46. 8%) than

and in April and November only 21 percent and 16 on other parts of the deer( 49.4- 51. 9%). Neolipoptena

percent, respectively, of the deer examined had alates. ferrisi showed no significant difference in sex ratio,From May through September, 50 to 62 percent of the either among seasons or among different areas on thedeer examined had alates. host.


1400 —

01- 1

Q1200 — apterates4.

aco

1000 -

p, 800 — dQ Qo

elatesw

h 800 — O

n

Z 400 — 4 z

a n 3

200 — 2

rur,:.`v Y' 3`' 34. hFk

A S O N D J F M A M J J AS

Months

Figure 3. Seasonal density of alate and apterous forms of Lipoptena depressa on black- tailed deer at the HoplandField Station( 1970- 1973).

TABLE 1. Prevalence of Neolipoptena ferrisi on black- tailed deer of different ages.

No. of Deer No. of Deer Percent of Chi SquareHost Age Examined Infested Deer Infested Probability

1- 12 months 21 13 62

0513- 36 months 22 20 91

1- 12 months 21 13 62

005

37+ months 28 27 96

113- 36 months 22 20 91

05

37+ months 28 27 96


1400 —

1200 —

I

tu

apterates1000 —

a

ct 800 —y

as

elates

600 — 6 w

0 0

5v:n

E 400 — rah q

z 3 Zz

t :t£s

200 — 2 2 a)

J F M A M J J A S O N DJ

Months

Figure 4. Seasonal density of alate and apterous forms of Neolipoptenaferrisi on black- tailed deer at the HoplandField Station( 1970- 1973).

Distribution ranked on the basis of keds per unit of surface area

The distribution of L depressa on the various ( TABLE 2) the highest population density ofN.ferrisiregions of the deer was not equal ( p< 0.05). Most occurred on the head. In fact, there were nearly as manyindividuals( 64%) were found on the posterior regions N.ferrisi per unit area sampled on the head( 1. 70) as on

tail, post. dors., post. vent.), and only 1. 5 percent were all other host surface areas combined( 1. 77).

collected from the head ( TABLE 2). Except for the Limited data on alate hippoboscids ofboth species

smaller proportion of males on the tail, male and female indicated that they were distributed much like theL depressa had similar posterior orientations on the apterous flies; the anterior regions harbored 91. 7 percent

hosts. When ranked on the basis of keds per unit of of the winged N.ferrisi and only 15. 6 percent of thesurface area( TABLE 2), the highest population density winged L depressa.

occurred on the tail, where 9. 5 percent of all L depressa Examination of plastic bags after deer carcasses

obtained were collected fromjust 4.3 percent of the total were removed in the postmortem laboratory revealedsurface area sampled. Although the distribution shifted that both species of keds had remained on the dead

slightly on a seasonal basis, the same general pattern animals. Visual inspection of the carcasses prior to

persisted throughout the year. skinning revealed that keds had remained at the sameIn contrast to L depressa, only 5. 0 percent of the N. anatomical areas where they had been observed

ferrisi population was collected from the posterior immediately after death. Keds also remained in specificregions ( TABLE 2) compared to 95 percent on the anatomical areas during skinning, and they were foundanterior areas ( head, ant. dors., ant. vent.). When in these same areas when sections of thawed hides were


60—

50-

40—

a>a)

0 30—

Neolipoptena ferrisi

5

Z 20"

10—

1 1 1

12 37 62 87 112 137 162 187 212

Number of Hippoboscids

Figure 5. Frequency distribution of Neolipoptenaferrisi collected from 71 black- tailed deer at the Hopland FieldStation( 1970- 1973).

TABLE 2. Number of keds per area sampled and density per unit of host area sampled.

Host Area Percent of TotalSampledl Surface Area L depressa2 N.ferrisi3

h 14. 9 6.4/ 0.43 25. 3/ 1. 70

ad 20.0 70.0/ 3. 50 15. 6/ 0.78

av 22.4 76.3/ 3. 41 17. 1/ 0.76

pv 23. 9 133. 6/ 5. 59 0.9/ 0.04

pd 14.5 97.5/ 6. 72 1. 8/ 0. 12

4.3 40.3/ 9. 37 0. 3/ 0.07

1Areas of host sampled are illustrated in Fig. 1.2No. L depressa per area sampled/density per unit of host area sampled, based on a mean of424 keds/ host.

3No. L ferrisi per area sampled/density per unit of host area sampled, based on a mean of 61keds/host.


processed and examined. the digestion technique used here to combing the hidesIn the mark- release study, the 435 marked hippo- with a fine- toothed comb revealed that the efficacy of

boscid flies were recombed from three body regions of combing varied widely between individual deer( 37 tothe six dead deer. The number of flies on the head, 63 percent positive) and among different areas of theanterior body, and posterior body did not change same deer( 0 to 83 percent positive). Because of the

significantly ( p> 0.05) from the mark- release time mobility and small size of alates, we believe that theimmediately after host death) until deer hides were combing and hand-picking collection methods used in

recombed about an hour after release of the flies. The other studies resulted in underestimates of hippoboscidnumber of marked flies from each area and their change prevalence. Also, combing or hand searches ofrestrictedin location between release and recapture is shown in areas on hosts would give a distorted estimate ofparasite

TABLES. The greatestmovement was fromtheposterior prevalence since, as our data show, the two species of

to the anterior body area; 32 flies representing 12. 3 flies have distinctly different distributions.percent of the original posterior population changed The lower prevalence of N. ferrisi on fawns asposition. No movement was detected between the head compared to older deer apparently is a sampling artifact;and posterior regions. The initial distribution of all many fawns were examined in the early spring when themarked flies was: head, 9.6 percent; anterior body, 30.6 majority of alates had not yet emerged. If the digestionpercent; posterior body, 59. 8 percent. The final process used to obtain specimens somehow damaged

distribution of marked flies was: head, 9.4 percent; the delicate wing joints of some older alates, it isanteriorbody, 35.4percent; posteriorbody, 55. 2percent. possible that our prevalence figures for winged forms

may be slightly low. However, we wish to point out thatDISCUSSION this process had not removed the wings from the large

numbers of alates recovered in our samples, and thatPrevalence of Hippoboscids on Deer isolated wings never were found in any samples. Alates

The prevalence of L depressa and N.ferrisi on deer reportedly lose their wings within 48 hours of landingwas higher in this study than recorded in previous on a deer( Hare 1953). A noteworthy difference in thestudies. Senger and Capelle( 1959) found 60 percent of prevalence of the winged forms of L depressa and N.their deer infested with L depressa, whereas, Cowan ferrisi was that L depressa, being bivoltine, had two1946) reported a 95 percent infestation rate with the seasonal flightperiods resulting in its alates being active

same species. Prevalence figures for N.ferrisi on deer longer than those of N.ferrisi.

range from 32 percent( Senger and Capelle 1959) to 44

percent( Spencer 1939). There are no previous reports Seasonal Abundance

on the prevalence of either species of hippoboscid in The seasonal fluctuations of hippoboscids on deerrelation to host age, sex, habitat, or season. revealed by this study were, to a large extent, a reflection

The higher prevalence of hippoboscids and the of the activity of alate flies. The winged forms of N.higher prevalence of mixed infestations found on deer in ferrisi were collected from deer in those months whenthis study probably resulted from the efficient collection the wingless keds also were most abundant ( Fig. 4);technique used. A study ( Westrom 1975) comparing alates were absent from early November until about

TABLE 3. Movement of marked hippoboscid flies on dead black- tailed deer.

Initial Position of Marked Fies Final Position of Marked Flies

Number Anterior Posterior

of Flies Head Body Body

Head 42 39( 92.9) 1 3( 7. 1) 0( 0)

Anterior Body Area 133 2( 1. 5) 119( 89. 5) 12( 9.0)

Posterior Body Area 260 0( 0) 32( 12. 3) 228( 87. 7)

Number( percent).


mid-April, the period of lowest N.ferrisi abundance on mid-March/early-April. Summer generation pupae dropdeer. Winged forms of L depressa were most common to the ground from about July through September and

shortly before the wingless forms were numerous. Peak the resulting winged adults emerge and seek hosts fromnumbers occurred in October and April( Fig. 3). about August through November.

Populations of both species on deer declined in All data forN.ferrisi indicate that this is a univolti ne

October, possibly as a result of the fall molt which species( Fig. 4). Except for the Montana data of Sengeroccurs in September( Taber and Dasmann 1958). The and Capelle( 1959), who reported N.ferrisi to be most

population of L depressa, however, was able to rebound abundant during January and February and absent inbecause a new population of alates was still emerging as July, records of N.ferrisi indicate peak populations onlate as November, whereas, the winged forms of N. deer from May to July( Longhurst and Douglas 1953) orferrisi disappeared in early November. In the absence August and September ( Linsdale and Tomich 1953).

ofyoung, winged, host- seeking flies in the winter months Our results for N.ferrisi are most similar to the results

both species declined to their lowest levels on deer by of Linsdale and Tomich( 1953), although the period of

spring. activity in our study was longer than that reported byThe absence of the winged forms ofboth species on these workers. The preponderance of N.ferrisi on deer

deer in the winter months was consistent with results of in the summer was significant, but deer harbored large

other studies ( Cowan 1943, Hare 1945, Linsdale and numbers of flies in May and September as well.Tomich 1953, Bequaert 1953). The few available Because bivoltinism had not been reported

records of the seasonal abundance of L depressa on previously for hippoboscids and because our sampledeer suggest that keds are scarce in late winter and early sizes of winged keds from deer were relatively small, to

spring and most numerous in the summer and fall further examine the univoltine-bivoltine hypothesis we

Cowan 1943, Longhurst and Douglas 1953, Linsdale increased our sample size of winged forms byand Tomich 1953, Senger and Capelle 1959, Taber and subsequently checking the collection data associatedDasmann 1958). Our data confirm the late winter- early with all pinned specimens in the museum collections of

spring population decline, but they do not support a the California Insect Survey, University of California,population peak in the summer- fall period. Berkeley, and the California Academy of Science, San

The month- to-month population fluctuation in the Francisco, California. Because winged forms of both

mean number of L depressa on deer at our study site species are attracted to and easily collected fromhumansreflected the midsummer and early winter generations, ( Hare 1945, 1953, and personal observations), they arewith the population density of the winter generation relatively common in museum collections. Of the 351being the greatest. The largest numbers of L depressa winged L. depressa examined from the museum

1, 433; 1, 299; 1, 209) were collected from deer in collections, the 160 specimens shown in TABLE 4 were

December and January. At our HFS study site, peak collected from the adjacent north coastal counties

abundance in December and January is predictable surrounding our study area. Like our samples from deerbecause alates are being added to the population as late hides, the seasonal collection records for these L.

as December when older wingless flies, which arrived depressa alates also reflect a bimodal peak of abundance,

on deer in August and September, may still be alive. and, thus, support our conclusion that this species is

Hare( 1953) found that adult L depressa could survive bivoltine in northern California. Most of these specimens

for at least four months. had been collected during the past 25 years; the threeAn analysis of the seasonal population data for both oldest specimens were collected in 1919, 1924, and

the winged and wingless forms of L depressa led us to 1932. The 22 winged museum specimens of N.ferrisi

conclude that this species is bivoltine at the study area examined all had been collected between May and

Fig. 3). This is the first time this phenomenon has been November and, hence, support the concept of

recognized for this group of parasites. There are about univoltinism for this species.

five months between the summer and winter population Few workers have reported the mean number of L

peaks and about seven months between the winter and depressa or N.ferrisi on deer, and their estimates are

summer peaks. This could result from prolonged considerably lower than our figures of 424 and 61,development of pupae at lower temperatures. The respectively. Spencer( 1939) found 35 L depressa perlarger population peak in winter suggests abetter survival deer, Linsdale and Tomich ( 1953) 225 per deer, and

of pupae in warmer( and drier) months. Pupae produced Taber and Dasmann( 1958) estimated 200 per deer in the

by reproductive flies of the winter generation drop to the summer. The mean number of N. ferrisi has been

ground from about January- March, and the resulting reported as 10. 3 per deer( Spencer 1939) and 38. 5 per

winged adults begin to emerge and seek hosts about deer( Linsdale and Tomich 1953). There are records of


TABLE 4. Number of volant Lipoptena depressa examined from museum collections.

Month Number of Volants1 Seasonal Totals

March 8

April 17

May 57 Spring- 94

June 12

July 12

August 9 Summer- 21

September 18

October 35 Fall- 61

November 8

December 2

January 1 Winter- 5

February 2

All specimens included in this table were collected in adjacent north coastal counties

surrounding our Hopland Field Station study site in Mendocino County.

individual deer infested with as many as 1, 350 L depressa occasions. The maximum numberofN.ferrisi collectedDixon and Herman 1945) and 3,493 keds of both from a single deer( 481) was only slightly higher than

species( Linsdale and Tomich 1953). Considering that the mean number of L depressa( 424) on all 71 hosts.these collections were made by handpicking or combing, The 1: 1 sex ratio of L depressa found in this studythe actual number of flies was probably much higher agrees with the results of Senger and Capelle( 1959), butthan reported. Our maximum number was 1, 563 for L differs from the 70 male/ 175 female ratio of Spencerdepressa and 481 for N.ferrisi. 1939) and the 1: 28 ratio of Bequaert( 1953). Because

Dual infestation of black- tailed deer with both our results were based on a much larger sample( 30,091)species of keds in this study was substantially higher than those ofother workers and were taken over a longerthan previously recorded. Spencer ( 1939) observed period of time, we consider the 1: 1 sex ratio to be moremixed infestations on 2 of 9 deer in British Columbia, accurate. Sex ratios of related deer keds, L mazamaewith L depressa outnumbering N. ferrisi 245 to 4. ( Samuel and Trainer 1972, Davis 1973) and L cerviSenger and Capelle( 1959) found 15 of 51 mule deer and ( Haarlov 1964), also were found to be 1: 1.3 of 9 white-tailed deer infested with both ked species. The high proportion of female L depressa found inNeolipoptena ferrisi was the predominant species in 6 summer was previously reported by Senger and Capelleof 15 mixed infestations on mule deer. Twenty-six ( 1959). The reason for this phenomenon is unknown,percent( 105/ 404) of the randomly collected keds from but a later emergence of females in the spring or athese mule deer were N.ferrisi, representing 33 percent differential mortality of the sexes would account for theof the deer keds collected from hosts infected with both predominance of females in summer.

species. Linsdale and Tomich( 1953) did not provideprevalence figures but observed that L depressa was far Distribution

more abundant than N.ferrisi; of6,582 keds taken from Virtually nothing has been reported about the25 deer, 85. 4 percent were L depressa. distribution of N. ferrisi or L depressa on deer other

In the 60 instances of dual infestation in our study, than the observation by Cowan ( 1943) that female LN. ferrisi outnumbered L depressa on only two depressa tended to congregate on the ventral regions of

JUNE, 1992 BULL. SOC. VECFOR ECOL. 67

the host. Haarlov( 1964) described the distribution ofL Haarlov' s ( 1964) analysis of skin temperatures,cervi on Danish deer and made a detailed analysis of hair composition, and degree of subcutaneoushost factors that might account for the observed vascularization of red deer( Cervus elaphus), if similardistribution of flies. However, his technique ofcombing for black- tailed deer, would not account for the observedfor keds may have disrupted their original distribution distribution of either L depressa or N. ferrisi. Evenand probably contributed to the small number of flies though Haarlov was able to detect temperaturerecovered( 181 from 34 deer). Haarlov found the keds differences as great as 4° C between some skin surfaces,distributed as follows: neck 25 percent, groin 23 percent, he found no correlation between the distribution offlank 20 percent, anal region 16 percent, axilla 12 temperatures and the distribution of keds. Furthermore,percent, head 3 percent, belly 2 percent, and back 2 a study by Freel ( 1972) of thermoregulation by black-percent.

tailed deer revealed that the surface temperature of aSamuel ( 1969) determined the numbers and single area on a deer can vary by as much as 25° C over

locations of L mazamae on two white-tailed deer from a 24 hour period, suggesting that small variations( 4° CTexas; one was infested with 5 flies, the other with 256. or less) from one part of the body to the next would haveHe concluded that the keds preferred the lateral surface a minimum influence on hippoboscid distribution.of the chest, the ventral abdominal area, and the lateral Without further knowledge of the biology of N.and medial surface of the hind legs. No flies were found ferrisi or L depressa, it is difficult to ascertain withon the head or neck. certainty what determines their disparate distributions.

Results of our mark-release study were similar to Competition between the two species is probably not athat reported by Samuel and Trainer( 1972), who found majordeterminant since the mean numberofL depressano significant change in the number of L mazamae on on deer with mixed infestations was higher than on deerwhite- tailed deer within an index area on the posterior withoutN.ferrisi. The difference in distribution was notregion of a deer during the first two hours after host the result of ked movements after the death of the deerdeath. Although we found a small amount of ked because this did not occur in a separate study usingmovement from one body region to another ( mostly marked hippoboscids ( unpublished data). Speciesbetween the anterior and posterior regions), the net distribution patterns were related to where the alateschange in the distribution of marked hippoboscids initially landed, with winged flies on hosts beingactually was smaller than the total amount of movement distributed like the older wingless flies. In this regard,TABLE 3) because flies moving from the anterior to the landing behavior of hippoboscid alates is like that of

the posterior body were replaced by flies moving from other host- seeking hematophagous flies whose variousthe posterior to the anterior body. The mark- release species feed on specific anatomical regions of deerresults thus show that the interval between host death ( Anderson et al. 1974, Hoy and Anderson 1978,and the collection ofkeds from deer had very little affect Smith et al. 1970) and other mammals ( Magnarelliin altering the original distribution of keds on their and Anderson 1980, Mullens and Gerhardt 1979,hosts. Schmidtmann et al. 1981). However, since morpho-

Previous studies of the distribution ofhippoboscids logical adaptations account for the distribution of manyare difficult to interpret or compare with our databecause wingless ectoparasite species( Nelson et al. 1975), suchof different collection techniques, different divisions of adaptations also might, in part, influence the disparatehost surface area, and different host- parasite systems. distributions of wingless deer keds adults.

Nevertheless, it is apparent that the density of infestationsfor the deer-ked species studied so far varies considerably Acknowledgments

among body areas of the same host. The reasons forthese differences in density are obscure, although We thank A. H. Murphy, former Superintendent,variability in skin temperature, structure, and hair for the use of facilities at the Hopland Field Station, W.composition have been suggested as influencing the M. Longhurst, former resident Zoologist,( University ofprevalence and abundance of keds ( Haarlov 1964, California, Davis), for allowing us to examine deerMarshall 1981). Freedom from host defensive collected on scientific collecting permits, and G. F.grooming) behavior or reduced interspecific com- Connolly, HFS, for help in collecting and aging deer.

petition with other ectoparasites might also determine We also thank B. C. Nelson( deceased), California Stateinhabitation of certain regions on deer. Nelson et al. Department of Health Services, and R. S. Lane,1975) also have suggested that localized acquired Department of Entomological Sciences, University of

resistance may be important in the distribution of California at Berkeley, for reviewing various drafts ofectoparasites on a given host. the manuscript, W. W. Jederberg and V. L. Gildengorin,


Letterman Army Institute of Research, who wrote the Dixon, J. S. and C. M. Herman. 1945. Studies on the

computer program and helped with the statistical analysis condition of California mule deer at Sequoia

of the data on distribution, and Mariko Yasuda for National Park, Calif. Fish and Game. 31: 3- 11.

typing the manuscript. This research was supported, inpart, by the University of California. Agricultural Freel, M. C. 1972. Thermal ecology of the mule deerExperiment Station and by a training grant from the U. Odocoileus hemionus). M.S. Thesis. Calif. State

S. Public Health Service, National Institute of Health, Univ., Hayward, CA, 60 pp.No. A.I. 00218.

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Diptera: Hippoboscidae). Can. Entomol. 102: distribution of ecoparasites on black- tailed deer

1117- 1122. Odocoilus hemionus columbianus) in Mendocino

County, California. Ph.D. Thesis. Univ. California,Popov, A. V. 1965. The life cycle of the louse flies Berkeley, CA, 184 pp.


ABSENCE OF IXODES DAMMINI( DEER TICKS) ON

PEROMYSCUS LEUCOPUS( WHITE-FOOTED MICE) IN

BROWN AND DOOR COUNTIES, WISCONSIN

T. D. Clevenl, E. C. Burgess', R. W. Howe2, and A. I. Goldsby2

ABSTRACT: Peromyscus leucopus ( white- footed mice) were trapped from June through August, 1989 at theUniversity of Wisconsin- Green Bay and in several sites in Door County, Wisconsin to determine the incidence ofIxodes dammini ( deer ticks) near a large population center ( Green Bay) and a popular recreational area ( DoorCounty). No I.dammini were found on the 96 P. leucopus trapped. One Ixodes sculptus and one Ixodes cookei werefound on P. leucopus from Door County. The apparent lack of an established population of I.dammini is consistentwith the low incidence of Lyme disease reported in these areas.

INTRODUCTION 1988). In the northeastern part of the state, limited

surveys of hunter-killed deer had not found viableLyme disease is a multisystemic inflammatory breeding populations of I.dammini( Davis et al. 1984).disease caused by the bacterium Borrelia burgdorferi

Northeast Wisconsin is an important area for severalBurgdorfer et al. 1982) which is primarily transmitted

reasons. First, it supports a large and growing population.by Ixodes dammini( deer tick)( Steere et al. 1977, Steere

Second, the northern portions( Door County andNicoletand Malawista 1979, Burgdorfer et al. 1982). The

National Forest) are heavily used by tourists. Third, itdisease often is characterized by a bull' s- eye rash calledis an area believed not to be endemic for I dammini. The

erythema chronicummigrans( ECM)( Steere et al. 1977). objective of this study was to determine the incidence ofIf left untreated, the disease progresses toward the1. dammini on P. leucopus in the vicinity of Green Baydevelopment of cardiac, neurologic, and arthritic (Brown County) and in Door County.

complications( Steere et al. 1977).While other small mammals can also serve as hosts

In 1968, the first reported case of ECM in thefor I.dammini, P. leucopus was found to be the primaryUnited States occurred in a grouse hunter in Taylorhost in Wisconsin( Godsey et al. 1987) and also the most

County, Wisconsin( Scrimenti 1970). In retrospect, thisabundant mammal caught in this survey. For these

is believed to be the first case of Lyme disease in thereasons we chose to focus only on P. leucopus.United States. In 1975, Steere and coworkers ( 1977)

We attempted to determine the presence of I.recognized an epidemic of arthritis in eastern Connecticut

dammini by trapping P. leucopus, an abundant rodentas beinganewcl inicaldiseasebecauseof thegeographic

that is known to be the primary host for larval andclustering of patients in Old Lyme and because of the

nymphal stages of I. dammini ( Godsey et al. 1987).characteristic preceding ECM in some patients. The

Monitoring rodents for the presence of ticks probably isdisease was later named after one of the communities ina more sensitive indicator of tick presence than usingwhich it was recognized.

CO2-baited traps or dragging vegetation ( Wilson andIn 1977, three Wisconsin residents developedSpielman 1985).

symptoms ( Dryer et al. 1979) consistent with thosedescribed by Steere et al.( 1977). This prompted state-

MATERIALS AND METHODSwide acaralogic surveys in 1981 and 1982 in Wisconsin

that revealed that I.dammini were in the western half of Peromyscus leucopus were trapped during 1989the state ( Davis et al. 1984). In 1986 and 1987, B. from the University of Wisconsin-Green Bay( UWGB)burgdorferi infection was found in Peromyscus leucopus Cofrin Arboretum and from four sites in Door Countywhite- footed mice) and I. dammini in the LaCrosse, ( Fig. 1). These sites were chosen for various reasons.

Wisconsin area( Anderson et al. 1987, Callister et al. The UWGB site was chosen because it is a large natural

1Department ofMedical Sciences, University of Wisconsin-Madison School of Veterinary Medicine, Madison, WI53706, U.S. A.

2Department of Natural and Applied Sciences, University of Wisconsin Green Bay, Green Bay, WI 54311- 7001,U.S.A.


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Figure 1. Map of 1989 study sites in Brown and Door Counties are the University of Wisconsin-Green Bay( SiteA), Peninsula Sanctuary( Site B), Toft Point Natural Area and Lighthouse Point( Site C), and private dairyfarms( Site D). The dairy farms are not individually designated on the map, but both are in the generalarea shown.


area supporting white- tailed deer ( Odocoileus ( quaking aspen).virginianus) and P. leucopus populations close to the Traps at one of the farms in Door County werecity of Green Bay. Peninsula Sanctuary, Toft Point, and placed in moderately shaded forest dominated by AcerLighthouse Point were selected because they are large saccharum ( sugar maple), Betula papyrtfera ( whitenatural areas in close proximity to major tourist areas of birch), and Popular tremuloides( quaking aspen). Trapsnorthern Door County. Two farms in Door County were at the other farm were in lightly wooded swamplandselected with the assistance of Cliff G. Ehlers, Door dominated by Populus spp. and Betulapapyrifera( whiteCounty UW-Extension agriculture agent, because cattle birch).on the farms had previously tested positive for B. Mammal species were identified as described byburgdorferi antibodies. Burt ( 1972) and Stromberg ( 1979). Each captive P.

Sherman live traps( 7. 5 x 9.0 x 23. 0 cm), baited with leucopus was inspected for ticks around the eyes, ears,peanut butter and oatmeal, were placed 10 to 30 meters face, anus, and toes. Inspection was carried out in theapart in grids ranging from 300 to 40,000 square meters. field for a minimum of five minutes per animal. An earBecause our intent was to maximize P. leucopus trapping punch was used to punch a hole in the center of the rightsuccess, traps were placed in the most likely habitat for ear of each P. leucopus captured. If P. leucopus wereP. leucopus at each site( i. e., forest or forest edge) and recaptured, they were immediately released and notwere not restricted to a standardized grid pattern. The rechecked for ticks. Trapped species other than P.number of traps at a single station ranged from 20 to 100, leucopus were likewise immediately released and wereaveraging 30 per night. Traps were set in the evening not checked for ticks. Ticks were identified by Phillipand checked the following morning. They remained set J. Pellitteri, Senior Outreach Specialist, U.W. Insectat each site no longer than two nights to minimize Diagnostic Lab, Madison, Wisconsin 53706.recaptures. If traps were used for a second night oftrapping at a site, they were closed during the day, RESULTS

rebaited, and reset in the evening.Trapping at UWGB was carried out between June One nymphal Ixodes sculptus was found on a P.

22 and July 16, and again on August 12 and 13( Site A, leucopus from Lighthouse Point and one larval IxodesFig. 1). Traps were set in Door County at Peninsula cookei was found on a P. leucopus from Toft Point. NoSanctuary on July 21 and 22 ( Site B), at Toft Point I. dammini were found at any site.Natural Area on July 22 and 23, at Lighthouse Point on Peromyscus leucopus captured at the four sites isJuly 24( Site C), and on two private dairy farms south of summarized in TABLE 1. One P. leucopus from UWGBSturgeon Bay on August 6 and 7( Site D). and one from Toft Point appeared unhealthy. The

Sampling consisted of a total of 995 trap-nights for mouse from UWGB was a male exhibiting emaciation,all sites, where one trap night is a single trap monitored unthriftiness, and a roughened hair coat. It was heavilyover a single night. The trapping sites at UWGB were parasitized by fleas, and it exhibited paresis in its hindlocated in or near lightly to heavily shaded deciduous legs. The male from Toft Point only exhibited paresis inforest. Tree species included Fraxinus pennsylvanica its hind legs.

green ash), Populus deltoides ( eastern cottonwood),Populus tremuloides ( quaking aspen), Acer negundo DISCUSSIONboxelder), Acer spp. ( maples), Ulmus americanaAmerican elm), Quercus spp.( oaks), and at one site, The absence of I. dammini in this survey supports

the coniferous Thuja occidentalis( northern white cedar). the findings ofothers( Knipping et al. 1950, Davis et al.Sites at Peninsula Sanctuary were located in heavily 1984, Wis. Dept. of Health 1990, and Phillip J. Pellitteri,

shaded forest of Thuja occidentalis ( northern white- pers. commun.), which indicate that deer ticks have notcedar), Tsuga canadensis ( eastern hemlock), Picea established viable breeding populations in Brown andglauca ( white spruce), and Acer saccharum ( sugar Door Counties. Since I.dammini has been incriminatedmaple). At Toft Point, traps were placed in the lightly as the primary vector of B. burgdorferi ( Burgdorfer etshaded boreal forests which were dominated by Thuja al. 1982), our findings suggest that there is not a highoccidentalis( northern white-cedar), Picea glauca( white risk to humans of acquiring Lyme disease in the studyspruce), Abies balsamea( balsam fir), Tsuga canadensis areas. Current data of human Lyme disease caseseastern hemlock), and Acerspicatum( mountain maple). support this assertion. Most counties of eastern

Traps at Lighthouse Point were placed in lightly shaded Wisconsin have fewer confirmed Lyme cases acquiredmixed forest. Alnus rugosa ( speckled alder), Betula within the county than in thel.dammini-endemic countiespapyrifera ( white birch), and Populus tremuloides of northwestern and western Wisconsin( Wis. Dept. of


TABLE 1. Results of Peromyscus leucopus trapping in Brown and Door Counties, Wisconsinfrom June through August, 1989.

Trap TotalSitea County Nightsb Species Captured Numbers

A Brown 495 Peromyscus leucopus 57

B Door 130 Peromyscus leucopus 11

C Door 230 Peromyscus leucopus 7

D Door 140 Peromyscus leucopus 21

aLetters preceding each site correspond to the locations in Figure 1.A" is the University of Wisconsin- Green Bay.B" is Peninsula Sanctuary.C" is Toft Point and Lighthouse Point.

D" is two private dairy farms.

bOne trap night is a single trap monitored over a single night.

cTotal P. leucopus numbers exclude recaptures.

Health 1991).

More research is needed on Lyme disease in REFERENCES CITED

northeastern Wisconsin and other areas near the margin

of!. dammini' s geographic range. Our study sites provide Anderson, J. F., P. H. Duray, and L. A. Magnarelli.areas from which comparisons can be made with I. 1987. Prevalence of Borrelia burgdorferi in

dammini-endemic regions of the state. Future research white- footed mice and Ixodes dammini at Fort

should focus on habitat, hosts, meteorological and McCoy, Wisconsin. J. Clin. Microbiol. 25: 1495-environmental influences, and the interactions of these 1497.

in determining the distribution and abundance of I.dammini. Our study provides a baseline for such work. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L.

Benach, E. Grunwaldt, and J. P. Davis. 1982.

Acknowledgments Lyme disease— A tick-borne spirochetosis? Science

216: 1317- 1319.

Dr. Keith White is appreciated for giving theinspiration for this project. We would also like to Burt, W. H. 1972. Mammals of the Great Lakes Region.

acknowledge the entomologic expertise of Phillip J. University of Michigan Press, Ann Arbor, MI, 240Pellitteri and Dr. Charles F. Koval, the trapping assistance pp.

of Donald P. Cleven, the help and kindness of severalDoor County farmers, the botanical expertise of Gary Callister, S. M., W. A. Agger, R. F. Schell, and J. L. E.

Fewless and Jim Moore, the guidance and hospitality in Ellingson. 1988. Borrelia burgdorferi infection

Door County provided by Dr. Cliff G. Ehlers and Dick surrounding LaCrosse, Wisconsin. J. Clin.

Weidman, and the technical assistance of Fred Baliweg, Microbiol. 26: 2632- 2636.

John B. French, and Paula Fleurant. We also thank Dr.

James J. Kazmierczak, Dr. John M. Gustafson, Phillip J. Davis, J. P., W. L. Schell, T. E. Amundson, M. S.

Pellitteri, Leonore Neumann, and Dr. Stanley A. Temple Godsey Jr., A. Spielman, W. Burgdorfer, A. G.for reviewing various drafts of this manuscript. This Barbour, M. LaVenture, and R. A. Kaslow. 1984.

research was funded by an undergraduate research grant Lyme disease in Wisconsin: Epidemiologic, clinical,

from the University of Wisconsin-Green Bay Cofrin serologic, and entomologic findings. Yale J. Biol.

Arboretum Foundation. Med. 57: 685- 696.


Dryer, R. F., P. G. Goellner, and A. S. Carney. 1979. Steere, A. C., S. E. Malawista, J. A. Hardin, S. Ruddy,Lyme arthritis in Wisconsin. J. Am. Med. Assoc. P. W. Askenase, and W. A. Andiman. 1977.

241: 498- 499. Erythema chronicum migrans and Lyme arthritis:

The enlarging clinical spectrum. Ann. Intern. Med.Godsey, M. S., Jr., T. E. Amundson, E. C. Burgess, W. 86: 685- 698.

Schell, J. P. Davis, R. Kaslow, and R. Edelman.

1987. Lyme disease ecology in Wisconsin: Stromberg, M. R. 1979. Field identification ofDistribution and host preferences oflxodesdammini, Peromyscus leucopus and P. maniculatus withand prevalence of antibody to Borrelia burgdorferi discriminant analysis. Wis. Acad. Sci. Arts Lett.in small mammals. Am. J. Trop. Med. Hyg. 37: 67: 159- 164.180- 187.

Wilson, M. L. and A. Spielman. 1985. Seasonal activityKnipping, P. A., B. B. Morgan, and R. J. Dicke. 1950.

of immature Ixodes dammini( Acari: Ixodidae). J.Notes on the distribution of Wisconsin ticks. Wis.

Med. Entomol. 22: 408- 414.Acad. Sci. Arts Lett. 40: 185- 197.

Scrimenti, R. J. 1970. Erythema chronicum migrans. Wisconsin Department of Health and Social Services,

Arch. Dermatol. 102: 104- 105. Division of Health. 1990. Lyme disease updates-

epinotes. Wis. Epidemiol. Bull. 12: 2- 6.

Steere, A. C. and S. E. Malawista. 1979. Cases of Lyme

disease in the United States: Locations correlated Wisconsin Department of Health and Social Services,with distribution of Ixodes dammini. Ann. Intern. Division of Health. 1991. Lyme disease update.Med. 91: 730- 733. Wis. Epidemiol. Bull. 13: 1- 4.

BULL. SOC. VECTOR ECOL., 17( 1): 75- 82 NNE, 1992

BORRELIA BURGDORFERI IN TICKS ON ANGEL ISLAND,

CALIFORNIA: LOW RISK OF HUMAN INFECTION

R. S. Lanes

ABSTRACT: The human risk of exposure to ticks and Lyme disease was assessed at Angel Island State Park, a

heavily used recreational area in San Francisco Bay, and at two comparison areas on the California mainland. Toevaluate risk, a questionnaire was distributed to outdoor workers in all three localities to determine their exposurehistories to ticks and tick-borne diseases. Also, a tick- spirochete survey was conducted on Angel Island to determinethe relative abundance of human-biting ticks and the prevalence of spirochetal infection in such ticks. None of fiveoutdoor workers on Angel Island reported a history of tick-bite, whereas, 15 workers in the comparison areasaveraged between 0. 1 and 0.2 tick-bites/ year, and one individual experienced a relapsing fever- like illness. Twohuman-biting ticks, Ixodes pacificus and Dennacentor occidentalis, were collected on Angel Island. Four( 0.27%)of 1, 464 I.pacificus adults versus zero of 25 I.pacificus subadults and 183 D. occidentalis adults were found tocontain spirochetes; spirochetal infection rates determined previously in I.pacificus adults from both comparisonareas were somewhat higher( 0.86- 1. 35%). One of the infected ticks fromAngel Island yielded an isolate ofBorreliaburgdorferi, the first from an island in western North America. This isolate was determined to be similar

antigenically to tick-derived isolates from the adjacent mainland. Collectively, these findings demonstrate that therisk of being bitten by ticks and acquiring Lyme disease is low in all three localities, but it is lowest on Angel Island.

INTRODUCTION vectors and vertebrate hosts/ reservoirs ( birds, lizards,mammals) in three north coastal counties, i.e., Marin,

Lyme disease is endemic throughout much ofCalifornia, Sonoma, and particularly Mendocino, as well as inbut most cases reportedly occur in the north coastal Contra Costa County in the San Francisco Bay area, andregion from Marin County in the San Francisco Bay Yuba County in north central California. Nothing hasarea northward to Humboldt County ( R. A. Murray, been published previously, however, about the occur-pers. commun.). Within this region, hyperendemic foci rence of Lyme disease on islands in western North

have been identified including a small rural community America.

population, - 150) where about 20 percent of the Angel Island, or Angel Island State Park( AISP), is

residents are seropositive for Lyme disease( Lane et al. located in northern San Francisco Bay. Approximately

1990). Human cases have been reported from 48 of the 175, 000 persons visit the island each year for various

58 counties statewide and spirochete- infected ticks, recreational activities including birdwatching, bicycling,

principally the western black- legged tick, Ixodes hiking, and picnicking. During the early 1980' s, apacificus, have been collected from vegetation, preliminary tick/ spirochete survey conducted on AISPvertebrates, or both in- 32 counties through April 1992 ( i. e., as part of a broader survey in northern CaliforniaBurgdorfer et al. 1985, B issett and Hill 1987, Lane and and southern Oregon) revealed that I. pacificus was

Pascocello 1989, Monsen et al. 1990, Lane et al. 1991, well-established there. Also, spirochetes resembling B.

Lane and Brown 1991, Boyce et al. 1992, R. A. Murray, burgdoiferi were detected in one adult tick that was

pers. commun.). Ixodes pacificus has been implicated collected from vegetation, but unfortunately they could

as the primary tick vector of Borrelia burgdorferi to not be cultured and, therefore, their specific identity

humans in northern California( Naversen and Gardner remained uncertain ( B urgdorfer et al. 1985).

1978, Burgdorfer et al. 1985, Lane et al. 1991). Concurrently, a questionnaire was distributed to parkAll published studies of the enzootiology of B. personnel at AISP to determine their personal exposure

burgdoiferi, the etiologic agent of Lyme disease, in histories to ticks and tick-borne diseases. A similar

California have been conducted on the mainland( Lane questionnaire was administered to personnel ofSugarloaf

et al. 1991). Investigations performed by the writer and Ridge State Park( SRSP) in adjacent Sonoma County,

coworkers have focused largely on the complex and to employees of the University ofCaliforniaHoplandinterrelations of B. burgdorferi with its potential tick Field Station( HFS) in Mendocino County, as comparison

1Department of Entomological Sciences, University of California, Berkeley, CA 94720, U.S.A.


areas. Both SRSP and the HFS are located on the land, woodland grass, and dense woodland.mainland of north coastal California in areas that areendemic for Lyme disease. Survey of Outdoor Workers

Between 1988 and 1991, AISP was revisited a few A questionnaire was distributed to employees oftimes each year during the activity period of adult I. AISP, SRSP, and the HFS. The questionnaire elicitedpacificus to quantify the relative abundance of host- information concerning length of employment, theseeking individuals of that tick species as well as adults number of ticks found crawling on clothing/ skin orof another human-biting tick found there, Dermacentor attached to skin, the seasonality of tick exposure, andoccidentalis. Individuals of both ticks were examined illnesses that could be attributed either directly to tick-immunologically for the presence ofspirochetes, and an bite or to potential tick-exposure.attempt was made to isolate spirochetes from the tissuesof infected ticks to determine their taxonomic identity Tick Collectionsand antigenic properties. Moreover, subadult I.pacificus Adult D. occidentalis and I.pacificus were collectedcollected by flagging leaf litter or removed from two from vegetation on nine dates ( eight in winter, one inspecies of lizards were also examined for spirochetes. early spring) by flagging with tick-drags composed ofHere I present the results of the recent tick/spirochete white flannel having surface areas of - 0. 9- m2. Thesurvey as well as findings arising from the earlier tick- number of ticks collected hourly was recorded to obtainexposure survey of outdoor workers. a rough estimate of relative abundance between years.

In 1988 and 1989, flagging was carried out in themorning(- 0900 to 1200 hr), whereas, in 1990 and 1991

MATERIALS AND METHODS it was performed in the afternoon(- 1330 to 1630 hr).

The sampling schedule was switched from morning toThe Study Areas afternoon in 1990 because in prior years the vegetation

AISP is located in southern Marin County, where it was often still damp with dew until mid-morning, whichis separated by less than 1. 6 km from the Tiburon seemed to hamper the efficiency of flagging. All ticksPeninsula by Raccoon Strait. The island comprises 299 collected were identified as to species and sex, and theha ranging in elevation from sea level to 238 m. Several tissues of most individuals were examined for thedifferent plant communities occur on the island including presence of spirochetes by direct immunofluorescencechaparral, coastal scrub, grassland, and oak woodland. ( DI) as described previously( Burgdorfer et al. 1985).

Two sites, each of which bordered< 0. 25 km of the Subadult I.pacificus were collected on three dates.former Northridge Trail at elevations ranging from One alligator lizard collected in the principal study area

152 to 183 m, were selected for studying populations in March, and three western fence lizards( Sceloporusof adult ticks. Grasses, other herbaceous vegetation, occidentalis), captured elsewhere on the island in earlyand some chaparral ( e.g., coyote bush, Baccharis July, were examined for the presence of ticks. Also,pilularis) were flagged ( see below) on flats and nymphal I.pacificus were collected by flagging leafmoderately sloping hillsides bordering each side of the litter for- four hours on a north- facing, heavily-woodedridge. Trees growing there include oaks( Quercus spp.) hillside in early August. Most subadult ticks removedand pines ( Pinus sp.). Deer sign ( Columbian black- from lizards and all nymphs flagged from leaf litter weretailed deer, Odocoileus hemionus columbianus), ground- tested for spirochetes by DI following preparation offrequenting birds, and alligator lizards ( Gerrhonotus whole- body smears.sp.) were observed in the study area.

SRSP is located mainly in east central Sonoma Isolation of Spirochetes

County( a small portion of it lies within west central All tissues remaining from adult ticks that wereNapa County). Elevations at the 1, 012-ha park range found to contain spirochetes by DI were trituratedbetween 183 and 832 m. Plant communities present individually in 1- ml of Barbour-Stoenner-Kelly( BSK)include chaparral, grassland, meadows, oak-woodland, II medium ( Barbour 1984), and the resultant tickand dense woodland. suspension was injected into 9- ml of BSK H. Dilutions

The HFS is located largely in southeastern of 10- 2 and 10- 3 were prepared similarly. To reduceMendocino County on the western slopes of the bacterial contamination, 50 ug/ml of the antibioticMayacmas Mountains in the Russian River Valley. It rifampin were added to all culture tubes. Cultures wereencompasses 2, 168 ha of rolling hills interspersed with incubated at 34.5° C; wet mounts prepared from eachravines; elevations vary between- 152 and 914 m. The culture were examined for spirochetes by dark- fieldpredominant vegetational types are chaparral, grass- microscopy weekly for one month.


Antigenic Characterization of Spirochetes 5 to 15 years per employee group. None of the fiveThe singleisolateofB. burgdorferiobtainedduring respondents from AISP reported a history of

this study was characterized with six monoclonal occupationally- related tick-bite, but one individual,antibodies by indirect immunofluorescence. The who had worked there for five years, found a tick on hismonoclonal antibodies were the Borrelia ( genus)- clothing twice in spring. At SRSP and the HFS, thespecific 119724, four others that are specific for two mean numbers of tick- bites reported annually perouter surface proteins( OspA, OspB) of B. burgdorferi individual were only about 0.2 and 0. 1, respectivelyi.e., 115332 and H3TS for OspA, H6831, and H5TS for ( TABLE 1). Spring and especially summer were

OspB), and 114825 that is specific for the outer membrane mentioned as the seasons of peak tick exposure at the

protein of the relapsing fever spirochete Borrelia hermsii. HFS; comparable data were unavailable for SRSP. OneFurthermore, the protein profile of Coomassie blue- of 11 employees ofthe HFS reported an illness followingstained, whole cell lysates of the AISP isolate were tick attachment that was similar to classical tick-borne

characterizedbysodiumdodecylsulfate-polyacrylamide relapsing fever( i.e., characterized by several bouts ofgel electrophoresis ( SDS- PAGE) and compared with fever alternating with afebrile periods). Another

three previously characterized strains ofB. burgdorferi individual who worked briefly at the HFS before theB31, CA3, CA9), strain HS 1 of Borrelia hermsii, and employee survey was undertaken also had experienced

strain C053 ofBorrelia coriaceae. The B31 type strain a relapsing fever- like illness that was apparentlywas derived from an Ixodes dammini tick collected on contracted at the station.

Shelter Island, New York, and strains CA3 and CA9

were isolated from I. pacificus ticks obtained at the Tick Collections

Point Reyes National Seashore in Marin County, Adults of two human-biting ixodid ticks, D.California( Lane and Pascocello 1989). Details of the occidentalis and I. pacificus, were flagged from

indirect immunofluorescence test and of the SDS- PAGE vegetation each year( TABLE 2). In total, 256( 136 d' ,

were presented earlier( Lane and Pascocello 1989). 120 4) D. occidentalis and 1, 552 ( 748 d' , 804 g) 1.pacificus were collected. The number ofD. occidentalis

RESULTS collected hourly ranged from 3. 1 to 14.4 in differentyears, whereas, that of I.pacificus varied from 15.4 to

Survey of Outdoor Workers 106.5 ticks per hour. The sex ratio( d' :4) was 1. 1: 1. 0

Twenty employees of AISP, SRSP, and the HFS for D. occidentalis and 1. 0: 1. 1 for I.pacificus.

responded to the questionnaire ( TABLE 1). The One adult alligator lizard collected in March was

average duration of employment ranged from about infested with one larva and two nymphs of I.pacificus,

TABLE 1. History of tick exposure and tick-borne diseases among employees of two California state parks andthe University of California Hopland Field Station.

Length of Employment( yrs.) No. Reported Tick Bites No. Illnesses

No. X Per Individual Following

Locality Respondents Total X ± SD( range) Total Per Year Tick Bite

Angel Island

State Park 5 24. 5 4.9± 2.4( 2- 8. 5) 0

Hopland Field

Station 11 160.5 14.6± 10.7( 1. 5- 32) 10* 0. 08 I**

Sugarloaf Ridge

State Park 4 19. 3 4. 8± 4.3( 0.8- 9. 0) 4 0. 21 0

Data available for only 10 respondents who were employed a total of 128. 5 years.

Relapsing fever-like.


and three western fence lizards ( two juveniles, one characterized, tick-derived isolates of B. burgdorferi

adult) captured in early July were parasitized by a total ( Fig. 1). In particular, the abundance and relative

of three larval andsix nymphal I.pacificus. Additionally, mobilities of the OspA, OspB, and flagellin proteins

16 nymphal I.pacificus were collected from leaf litter ( i.e.,- 31- 32, 33- 34, and 40-41 kilodaltons, respectively)during- four hours of flagging in early August. were comparable. All four isolates of B. burgdorferi

Direct immunofluorescence examination revealed differed from B. coriaceae and B. hermsii in several

spirochetes in smears of midgut tissues prepared from major protein bands( Fig. 1).four ( 0.27%) of 1, 464 adult I.pacificus ( TABLE 3).

Spirochetes were not detected in midgut smears from DISCUSSION

183 adult D. occidentalis( TABLE 3) or in whole bodysmears prepared from 16 nymphal I.pacificus collected Risk of Borrelia burgdorferi Infection

by flagging and nine sub adult I.pacificus( two larvae, The absence of reported tick-bites among the fiveseven nymphs) that were removed from lizards. Tissue outdoor workers at AISP, whose collective employment

suspensions from all four spirochete- infected Lpacificus totaled 24.5 years( TABLE 1), and the low prevalence

were injected into BSK II medium, but only one of them ( 0 to 0.3%) of detectable spirochete- infection in host-

yielded an isolate. This isolate, designated CA 129, was seeking D. occidentalis and I.pacificus adults( TABLEidentified as B. burgdorferi by its reactivity with all four 3), demonstrate that the risk of people acquiring B.B. burgdorferi-specific monoclonal antibodies. It also burgdorferi on the island is lower than it is in the two

bound with the Borrelia ( genus)- specific monoclonal comparison areas( TABLES 1 and 3). Tick-exposure

antibody H9724, but it did not bind to the B. hermsii- histories ( TABLE 1) and spirochete- infection rates

specific monoclonal antibody H4825. reported earlier for adult I.pacificus( Burgdorfer et al.

The identity of isolate CA 129 was further 1985) were both somewhat higher at SRSP( 0.86%, n=

established by the overall similarity of its protein profile 348) and the HFS ( 1. 35%, n = 518) than at AISP.

with the protein components of three formerly Infection rates in I. pacificus from SRSP versus the

1 2 3 4 5 6

66K- h _

fir400000.

4iii

31K - 411 # r.,

21K -

Figure 1. SDS- PAGE Coomassie blue- stained proteins of whole-cell lysates of B. burgdorferi from Ixodes spp.ticks( lanes 1- 4), B. hermsii( lane 5) and B. coriaceae( lane 6). Lanes: 1, B31 type strain froml.dammini;

2, CA 129 from!. pacificus; 3, CA 3 from/. pacificus; 4, CA 9 froml.pacificus; 5, B. hermsii strain HS 1;

6, B. coriaceae strain C053. All spirochetes were grown in BSK II medium. The approximate positions

of the low-molecular-weight protein standards(103)

are shown to the left of the first lane.


HFS, and from SRSP versus AISP, did not differ for ixodid ticks by drag sampling to determine the risksignificantly( Fisher' s exact tests, 2- tailed, p> 0. 05). In of human exposure to ticks( Falco and Fish 1989). Risk

contrast, the prevalence of spirochetes in ticks was was evaluated by means of an Ixodes dainmini" encountersignificantly higher at the HFS than it was at AISP( p= distance," which was defined as the average number of

0.01). meters dragged before a nymphal or adult I. dammini

Too few adult D. occidentalis( n= 183) were tested was encountered. The encounter distance ranged from

for spirochetes to permit meaningful comparison with 36 to 118 min high- risk parks and from 208 m to infinityresults reported here for I.pacificus. In earlier tick/ ( no ticks) in low-risk parks, and it was generally higherspirochete surveys, however, spirochetes were detected in high- use areas, presumably because of habitatrarely in D. occidentalis( Lane and Lavoie 1988, Lane alteration. In Illinois, an overall encounter distance of

and Loye 1991); and, more recently, this tick was found 78 m was reported for I.dammini during peak nymphalto be an inefficient vector of B. burgdorferi( Brown and density at a state park where five km of hiking trails wereLane 1992). dragged for the presence of ticks( Siegel et al. 1991),

Angel Island park employees restrict most of their which is comparable to that of high- risk parks in New

activities to modified habitats or established trails when York State.

working outdoors in natural areas and their regulation More recently, 14 recreational areas and eight schoolgarb ( e.g., boots, full- length pants, shirt tucked into properties in a Lyme disease endemic area of New

pants) affords some protection against ticks. Also, Jersey were evaluated for risk of transmission with anmuch of the island is steep and heavily wooded, and the ecological index( Schulze et al. 1991). To predict risk

distribution of/. pacificus seems tobe restrictedprimarily in different sites, an index was developed that was based

to grassy flats and moderately sloped hillsides. These on information about various plant communities and the

factors may account for the low tick-exposure reported abundance ofspirochete- infected adult!. dammini. These

by park personnel even though the overall collection workers present field data which demonstrate that the

rate for adult I.pacificus at AISP( mean= 46.4 ticks per proposed index is a cost- effective way to identify areashour, n= 33 hours) was comparable to or higher than it where people would be at risk of contracting Lymewas in each of eight Lyme disease case areas and two disease in the northeastern United States.

comparison areas( including the HFS) on the mainland I assessed year- to-year variation in tick numbers

of northern California( Lane and Lavoie 1988). Rates with a temporal index ( i.e., mean number of ticks

such as these cannot be compared statistically, however, flagged hourly), and risk of exposure to ticks/Lyme

unless flag sampling is standardized diurnally, disease by surveying outdoor workers and by examiningseasonally, and by habitat type and operator. numerous ticks for the presence of spirochetes. Flagging

In southern New York State where Lyme disease is was not confined to the margins of hiking trails becausehighly endemic, eight recreational parks were surveyed insufficient numbers of ticks would have been obtained

TABLE 2. Mean number of adult ixodid ticks flagged hourly from vegetation at AngelIsland State Park, California, 1988- 1991.

No. Hours X No. Ticks Per Hour± SD( Range)

Year Flagging D. occidentalis L pacificus

1988 4 7. 8± 2. 6 22.0± 12. 7

4- 10) 10- 37)

1989 9 3. 1 ± 3.4 15. 4± 12. 8

0- 8) 2- 40)

1990 12 6. 8± 5. 5 39.4± 18. 9

1- 21) 19- 82)

1991 8 14.4± 11. 8 106. 5± 41. 4

1- 33) 39- 178)


to accurately determine spirochete- infection rates in on other vertebrates including humans ( Furman andthem, i.e., due to the typically low spirochete- infection Loomis 1984). The infectivity and pathogenicity of thisrates recorded for diverse populations ofadult I.pacificus spirochete for humans, currently unknown, are underin northern California which usually average only—1- 6 investigation.

percent( Lane et al. 1991). Nevertheless, the encounter

distance for adult I.paccficus along the trail connecting Potential Vertebrate Reservoirs

my two principal study areas at AISP would have been Wildlife that have been implicated as probableequivalent to that of high-risk parks in New York( R. S. reservoirs of B. burgdorferi in nearby mainland areas,Lane, unpub. observations). The actual risk of acquiring such as the deer mouse ( Peromyscus maniculatus),Lyme disease in some recreational areas of the dusky- footed woodrat ( Neotoma fuscipes), and

northeastern United States, however, may be 100 or Californiakangaroo rat( Dipodomys californicus)( Lanemore times greater than it is on Angel Island since in 1990a, Lane and Brown 1991, Brown and Lane 1992),general —25% of host-seeking nymphal and —50% of are absent on AISP. In fact, the island has a depauperatehost- seeking adult I.dammini in enzootic areas harbor mammalian fauna comprised of only six species ( G.spirochetes( Lane et al. 1991). In highly enzootic areas, Maier, pers. commun.). The Angel Island mole

up to 100 percent of adult I.dammini have been found ( Scapanus latimanus insularis), Townsend' s big-earedto be infected with spirochetes( Burgdorfer 1984). bat ( Plecotus townsendii), California vole ( Microtus

The occurrence of relapsing fever- like illnesses californicus), Norway rat( Rattus norvegicus), raccoonamong two former workers at the HFS is of special ( Procyon lotor), and Columbian black- tailed deer ( 0.interest. The clinical spectrum of Lyme disease hemionus columbianus) inhabit the island. Procyonsometimes includes a recurrent type of febrile illness lotorhasbeenimpl icatedasareservoirofB. burgdorferisimilar to that of relapsing fever ( P.E. Lavoie, pers. in the northeastern United States ( Fish and Danielscommun.), and both individuals contracted their 1990), and R. norvegicus has been found infectedinfections before Lyme disease was even known to be naturally with B. burgdorferi in China( Fangzhen andendemic in California( i. e., one person reported onset of Wannian 1990). Further studies are needed to determineillness in 1960, the other about 1976). On the other the reservoir competence of raccoons and Norway ratshand, another spirochete, Borrelia coriaceae, occurs in for B. burgdorferi on Angel Island.all stages of its soft tick vector, Ornithodoros coriaceus, Also, six reptilian species( two lizards, four snakes)at the HFS( Lane et al. 1985, Johnson et al. 1987). This and 125 resident or migratory avian species have beentick feeds principally on cattle and deer, and occasionally observed at AISP. Various lizards and birds serve as

TABLE 3. Prevalence of spirochete- infected adult ixodid ticks from Angel Island StatePark, California, 1988- 1991.

No. Ticks Examined( sex) No. Ticks Infected(%)

Year D. occidentalis I.pacificus D. occidentalis 1. pacificus

1988 40 113 0 0

23d , 174) 56d' , 574)

1989 15 91 0 0

50' , 104) 39d , 529)

1990 75 450 0 1 d' , 19 ( 0. 44)480' , 274) 195M, 2554)

1991 53 810 0 29 ( 0. 25)150' , 384) 392d' , 4189)

Total 183 1, 464 0 1 d ,39 ( 0. 27)91d' , 929) 682d' , 7829)


hosts of subadult I.pacificus( Arthur and Snow 1968, Cooley and Kohls, 1943: its life-history andFurman and Loomis 1984, Manweiler et al. 1990), and occurrence. Parasitology 58: 893- 906.both species of lizards present at AISP( i. e., an alligator

lizard, Gerrhonotus sp., and the western fence lizard, S. Barbour, A. G. 1984. Isolation and cultivation of Lyme

occidentalis) may be locally important as hosts of I. disease spirochetes. Yale J. Biol. Med. 57: 521-

pacificus ( Furman and Loomis 1984, Lane and Loye 525.

1989, Manweiler et al. 1990). All four lizards examined

during this study ( one Gerrhonotus sp., three S. Bissett, M. L. and W. Hill. 1987. Characterization of

occidentali.$)wereparasitized by one to five I.paqficus. Borrelia burgdorferi strains isolated from Ixodes

Nonetheless, the reservoir competence ofS. occidentalis pacificus ticks in California. J. Clin. Microbiol. 25:

for B. burgdorferi seems to be low( Lane 1990b). The 2296- 2301.

potential involvement of birds in the enzootiology of B.burgdorferi in northern California is poorly understood, Boyce, W.M., R. N. Brown, B. C. Zingg, R. B. LeFebvre,though in one study birds appeared to be relatively and R. S. Lane. 1992. First isolation of Borrelia

unimportant as hosts of subadult I.pacificus in an oak- burgdorferi in southern California. J. Med.

woodland community located in the Sierra Nevada Entomol. 29: 496- 500.

foothills( Manweiler et al. 1990).

Brown, R. N. and R. S. Lane. 1992. Lyme disease in

Antigenic Characteristics of Isolate California: a novel enzootic transmission cycle of

The isolate ofB. burgdorferi obtained from AISP is Borrelia burgdorferi. Science 256: 1439- 1442.

the first to be characterized from an island in western

North America. Given the close proximity of the island Burgdorfer, W. 1984. The New Zealand white rabbit:

to the Tiburon Peninsula and the likely transport of I. an experimental host for infecting ticks with Lymepacificus subadults to and from the island via birds, it is disease spirochetes. Yale J. Biol. Med. 57: 609-

not surprising that the isolate from Angel Island is 612.

similar antigenically to two tick-derived isolates fromthe mainland of Marin County ( Fig. 1). The slight Burgdorfer, W., R. S. Lane, A. G. Barbour, R. A.

amount of antigenic variation observed between these Gresbrink, and J. R. Anderson. 1985. The western

strains is well within the normal range of variation black- legged tick, Ixodes pacificus: a vector of

observed for other tick-derived strains ofB. burgdorferi Borrelia burgdorferi. Am. J. Trop. Med. Hyg. 34:from California ( Bissett and Hill 1987, Lane and 925- 930.

Pascocello 1989).Falco, R. C. and D. Fish. 1989. Potential for exposure

Acknowledgments to tick bites in recreational parks in a Lyme disease

endemic area. Am. J. Pub. Hlth 79: 12- 15.

I thank the California State Department of Parks

and Recreation for permission to conduct this study atFangzhen, Z. and L. Wannian. 1990. A strain of

Angel Island State Park, employees of this park andBorrelia burgdorferi isolated from R. norvegicus.

those of Sugarloaf Ridge State Park and the UniversityChinese J. Epidemiol. 11: 16- 18.

of California Hopland Field Station who voluntarily Fish, D. and T. J. Daniels. 1990. The role of medium-completed the questionnaire, and Ranger G. Maier for

sized mammals as reservoirs ofBorrelia burgdorferiproviding the checklist of vertebrates on Angel Island. in southern New York. J. Wildl. Dis. 26: 339-L. E. Casher, J. Kleinjan, J. A. Pascocello, and G. B.

345.Schoeler are gratefully acknowledged for their technicalassistance. R. N. Brown provided helpful comments on Furman, D. P. and E. C. Loomis. 1984. The ticks ofthe manuscript. J. Piesman and G. Maupin are thanked California ( Acari: Ixodida). Bull. Calif. Insect

for help in flagging nymphal ticks. This research was Surv. 25: 1- 239.

funded in part by a grant( AI22501) from the NationalInstitutes of Health. Johnson, R. C., W. Burgdorfer, R. S. Lane, A. G.

Barbour, S. F. Hayes, and F. W. Hyde. 1987.

REFERENCES CITED Borrelia coriaceae sp. nov.: putative agent of

epizootic bovine abortion. Int. J. Syst. Bacteriol.

Arthur, D. R. and K. R. Snow. 1968. Ixodes pacificus 37: 72- 74.


Lane, R. S. 1990a. Infection of deer mice and pifion California: interrelationship of ixodid ticks( Acari),mice( Peromyscus spp.) with spirochetes at a focus rodents, and Borrelia burgdorferi. J. Med. Entomol.

of Lyme borreliosis in northern California, U.S. A. 28: 719- 725.

Bull. Soc. Vector Ecol. 15: 25- 32.

Lane, R. S. and J. A. Pascocello. 1989. Antigenic

Lane, R. S. 1990b. Susceptibility of the western fence characteristics ofBorrelia burgdorferi isolates fromlizard ( Sceloporus occidentalis) to the Lyme ixodid ticks in California. J. Clin. Microbiol. 27:

borreliosis spirochete( Borrelia burgdorferi). Am. 2344-2349.

J. Trop. Med. Hyg. 42: 75- 82.Lane, R. S., J. Piesman, and W. Burgdorfer. 1991.

Lane, R. S. and R. N. Brown. 1991. Wood rats and Lyme borreliosis: relation of its causative agent tokangaroo rats: potential reservoirs of the Lyme its vectors and hosts in North America and Europe.disease spirochete in California. J. Med. Entomol. Ann. Rev. Entomol. 36: 587- 609.28: 299- 302.

Manweiler, S. A., R. S. Lane, W. M. Block, and M. L.Lane, R. S., W. Burgdorfer, S. F. Hayes, and A. G. Morrison. 1990. Survey of birds and lizards for

Barbour. 1985. Isolation of a spirochete from the ixodid ticks ( Acari) and spirochetal infection in

soft tick, Ornithodoros coriaceus: a possible agent northern California. J. Med. Entomol. 27: 1011-

of epizootic bovine abortion. Science 230: 85- 87. 1015.

Lane, R. S. and P. E. Lavoie. 1988. Lyme borreliosis in Monsen, S. E., W. E. Hazeltine, and T. L. Henderson.California: acarological, clinical, and 1990. Lyme borreliosis surveillance in Butte

epidemiological studies. Ann. N.Y. Acad. Sci. County, California. Bull. Soc. Vector. Ecol. 15:539: 192- 203. 63- 72.

Lane R. S., E. T. Lennette, and J. E. Madigan. 1990. Naversen, D. N. and L. W. Gardner. 1978. Erythema

Interlaboratory and intralaboratory comparisons of chronicum migrans in America. Arch. Dermatol.

indirect immunofluorescence assays for 114: 253- 254.

serodiagnosis of Lyme disease. J. Clin. Microbiol.

28: 17741779. Schulze, T. L., R. C. Taylor, G. C. Taylor, and E. M.

Bosler. 1991. Lyme disease: a proposed ecologicalLane, R. S. and J. E. Loye. 1989. Lyme disease in index to assess areas of risk in the northeastern

California: interrelationship of Ixodes pacificus United States. Am. J. Pub. Hlth. 81: 714- 718.

Acari: Ixodidae), the western fence lizard

Sceloporusoccidentalis), andBorreliaburgdorferi. Siegel, J. P., U. Kitron, and J. K. Bouseman. 1991.

J. Med. Entomol. 26: 272- 278. Spatial and temporal distribution of Ixodes dammini

Acari: Ixodidae) in a northwestern Illinois stateLane, R. S. and J. E. Loye. 1991. Lyme disease in park. J. Med. Entomol. 28: 101- 104.


Scientific Note

EFFECTIVENESS OF A SOAP REPELLENT AGAINST MANSONIA

MOSQUITOES IN A FRESH WATER SWAMP FOREST INNORTHWESTERN PENINSULAR MALAYSIA

A. Abu Hassan 1 and V. Narayananl

ABSTRACT: Soap repellent containing 20 percent deet and 0.5 percent permethrin applied to exposed limbs ofhuman bait was effective against Mansonia mosquitoes in a Malaysian fresh water swamp forest.

Mosquitoes are common insect vectors of disease did not apply the repellent soap. The soap was washedin many parts of Malaysia. One way to reduce contact off after each night of testing and a fresh application wasbetween mosquitoes andhumans is to deter the mosquito applied the next night. After five nights the teamsfrom biting. Tests have shown that skin application of switched roles so that the untreated pair were nowthe repellent deet ( N,N-diethyl-3- methyl benzamide) treated and the treated pair were now controls. The hutwas effective against mosquitoes in the nipah palm test sites were also changed. The results after ten nightsmangrove swamps of Peninsular Malaysia( Traub and of testing are shown in TABLE 1. Ninety- five percentElisberg 1962a, 1962b). Deet is also an important of the mosquitoes caught in the test were Mansoniamosquito repellent against Aedes and Anopheles in the indiana, three percent Mansonia uniformis, one percentUnited States ( Rutledge et al. 1989a, 1989b; Schreck Mansonia annulifera, and one percent Culex spp.1985). A chi- square analysis showed the treatments were

Permethrin- treated bed nets are employed in significantly different( X2= 17. 3, df= 9, P< 0.05)( Zarmalarious areas to protect against Anopheles( Hossain 1974). The experiment demonstrated that the soapand Curtis 1989, Snow et al. 1988). In the present study, repellent reduced the number ofMansonia biting humans.a soap repellent containing 20 percent deet and 0.5 Yap ( 1986) also showed that a soap formulationpercent permethrin( cis: trans ratio: 25: 75) was evaluated. containing deet and permethrin applied to exposed armsThe soap was supplied by WHO/ WPRO, Manila, and legs of volunteers was effective in reducing thePhilippines. The study area was an open swamp in landing/ biting rates of mosquitoes in Penang, Malaysia.northwestern Peninsular Malaysia. The area is divided The soap repellent should be very useful for hunters,by the Kerian River and its tributaries. During the night fishermen, and forest product gatherers because it canthe swamps are frequented by people engaged in their be carried easily and is safe. The two active ingredientslivelihoods of fishing and hunting. Several species of of the soap formulation possess low mammalian toxicityMansonia mosquitoes are abundant in the swamps and ( Kenaga and Morgan 1978). In Malaysia, commercialcases of Brugian filariasis were reported from the mosquito repellent products containing deet in the formsurrounding villages ( Ministry of Health, Malaysia, of a solid bar or liquid sell for around US$ 2. 70 per item.unpub. data). Mansonia are nocturnal biters and are The soap formulation containing deet and permethrinknown vectors of filariasis. Thatched huts constructed costs around US$ 0.35 ( WHO/WPRO, per. commun.)

along the river are usedby fishermen to anchor equipment and, therefore, is much more cost effective. The use offor catching fish. Hunters also use these huts for resting. the soap formulation provides personal protection againstTwo huts of approximately equal size( 3 m X 3 m), 500 the Mansonia vectors of Brugian filariasis and maymeters apart were chosen as a test site. A team of two provide an additional weapon in the overall strategy ofmen wearing T-shirts and long trousers was stationed in vector and disease control.

each hut from 1800 hours to 0100 hours and collected

landing/ biting mosquitoes. The mosquitoes were col- Acknowledgments

lected by a battery operated aspirator. One of the teamslathered their exposed limbs and faces with the repellent This study was financed by Universiti Sainssoap five minutes before sunset, while the other team Malaysia R& D grant No. 123/ 3305/ 2403.

1School of Biological Sciences, Universiti Sains Malaysia, 11800 Pulau Penang, Malaysia.

84 BULL. SOC. VECFOR ECOL. JUNE, 1992

TABLE 1. Numbers of Mansonia mosquitoes collected landing/ biting in two huts with and withoutmosquito repellent.

Hut

1 2

With Without Percent

Day Repellent Repellent Protection

1 0 31 100

2 1 90 99

3 5 172 97

4 19 227 92

5 10 92 90

Without With

Repellent Repellent

6 30 0 100

7 27 1 96

8 59 5 92

9 45 2 96

10 87 4 96

REFERENCES CITED manus. J. Am. Mosq. Contr. Assoc. 1( 1): 98- 100.

Snow, R. W., S. W. Lindsay, R. J. Hayes, and B. M.Hossain, M. I. and C. F. Curtis. 1989. Assay of Greenwood. 1988. Permethrin treated bed nets

permethrin impregnated fabrics and bioassays mosquito nets) prevent malaria in Gambian

with mosquitoes( Dip: Culicidae). Bull. Ent. Res. children. Trans. Roy. Soc. Trop. Med. and Hyg. 82:79: 299- 308. 838- 842.

Kenaga, E. E. and R. W. Morgan. 1978. Commercial Traub, R. and B. L. Elisberg. 1962a. Field test on

and experimental organic insecticides ( 1978 diethyltoluamide ( DEET), a highly effectiverevision). Spec. Pub. 78- 1. Entomol. Soc. Am., 79 repellent against mosquitoes in the nipah palm

pp. mangrove swamps in Malaya. Pacific insects. 4( 2):

303- 313.

Rutledge, L. C., R. L. Hooper, R. A. Wirtz, and R. K.

Gupta. 1989a. Efficacyofdiethylmethylbenzamide Traub, R. and B. L. Elisberg. 1962b. Comparative

DEET) against Aedes dorsalis and a comparison efficacy of diethyltoluamide skin applicationof two end points for protection time. J. Am. Mosq. DEET) and M- 1960 clothing impregnant againstContr. Assoc. 5( 3): 363- 368. mosquitoes in nipah palm mangrove in Malaya.

Pacific Insects. 4( 2): 314-318.

Rutledge, L. C., R. L. Hooper, R. A. Wirtz, and R. K.

Gupta. 1989b. A field trial of ethyl hexanediol Yap, H. H. 1986. Effectiveness of soap formulationsagainst Aedes dorsalis in Sonoma County, containing deet and permethrin as personalCalifornia. J. Am. Mosq. Contr. Assoc. 5( 3): 374- protection against outdoor mosquitoes in Malaysia.

376. J. Am. Mosq. Contr. Assoc. 2( 1): 63- 67.

Schreck, C. E. 1985. The status of DEET( N,N,Diethyl- Zar, J. H. 1974. Biostatistical Analysis. Prentice Hall

M-Toluamide) as a repellent for Anopheles albi- Inc., New Jersey, pp. 59- 69.

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