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Masters Theses 1911 - February 2014
1996
Mosquito vectors of eastern equine encephalomyelitis virus in Massachusetts.
Rajeev Vaidyanathan University of Massachusetts Amherst
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MOSQUITO VECTORS OF EASTERN EQUINE
EN®P^®miYELITIS VIRlIi IN MASSACHUSETTS
A Thesis Presented
by
RAJEEV VAIDYANATHAN
Submitted to the Graduate School of the
University of Massachusetts Amherst in partial fulfillment
of the requirements for the degree of
MASTER OF SCIENCE
May 1996
Entomology MOSQUITO VECTORS OF EASTERN EQUINE
TOCEPHALOMYELITIS VIRUS IN MASSACHUSETTS
A Thesis Presented
by
RAJEEV VAIDYANATHAN
Approved as to style and content by:
D, Edman, Chair
T. Michael Peters, Member
John P. Burand, Member
Thomas W. Scott, Member
T. Michael Peters, Department Head
Entomology Dedicated to the memory of
George C. Eickwort
He taught us how to fly. ACKNOWLEDGMENTS
University of Massachusetts at Amherst Dr. John Edman Dr. T. Michael Peters Dr. John P. Burand Dr. John Buonaccorsi Geoff Attardo
University of Maryland at College Park Dr. Thomas W. Scott Lynn A. Cooper Les Lorenz
University of Massachusetts Cranberry Experimental Station Dr. Anne Averill Marty Averill Jay O’Donnell
Massachusetts Department of Health Dr. Ralph Timperi Dr. Barbara Werner Donald Buckley
Massachusetts Mosquito Control Projects John Smith (Norfolk County) Dr. Ken Ludlam (Plymouth County) Ken Ek (Plymouth County) Alan DeCastro (Bristol County)
Family and friends Madurai Meenakshisundaram and Radha Vaidyanathan Dr. Myong Won Kahng Joseph and Julie Lakshmanan Nikhil and Varuni Mallampalli Nicholas Komar The Apiary Crew All the Beans
IV ABSTRACT
MOSQUITO VECTORS OF EASTERN EQUINE ENCEPHALOMYELITIS VIRUS IN MASSACHUSETTS
MAY 1996
RAJEEV VAIDYANATHAN
B.S., CORNELL UNIVERSITY
M.S., UNIVERSITY OF MASSACHUSETTS AMHERST
Directed by: Professor John D. Edman
The current study was conducted in 1994 and 1995 to determine vector competence of six local mosquitoes for a strain of eastern equine encephalomyelitis
(EEE) virus isolated from Culiseta melanura in Easton, Massachusetts in 1992.
Aedes canadensis, Ae. vexans, Coquillettidia perturbans, Anopheles quadrimaculatus, An. punctipennis, and Culex salinarius are potential vectors to humans and horses because of their geographic and seasonal overlap with EEE cases in mammals, isolations of EEE virus, and host preference. Nulliparous females
(collected as late larvae, pupae, or teneral adults) were fed on an EEE-viremic chick.
Mosquito saliva and bodies were collected 7 and 14 days post blood feeding and tested for cytopathic effect on a monolayer of baby hamster kidney cells. A laboratory colony of Cs. melanura, the enzootic vector of EEE virus, was used as a control.
Aedes canadensis, Cx. salinarius, Cq. perturbans, and An. quadrimaculatus maintained infections and transmitted EEE virus via saliva. Aedes canadensis (13%) and Cx. salinarius (10%) demonstrated higher transmission rates and appear to feed more readily on avian and mammal hosts than Cq. perturbans (4%) and An. quadrimaculatus (10%). Coquillettidia perturbans and An. quadrimaculatus may play a role in equine or early human cases when nestling birds manifest a high viremia .
Neither Ae. vexans nor An. punctipennis transmitted virus, although both became
v infected (27 and 100%, respectively). Culiseta melanura, the enzootic vector of EEE virus, had the highest infection (100%) and transmission rates (94%).
In addition, eight trapping methods for epidemic EEE vectors were compared with one another and to human biting rates. Supplemented with carbon dioxide, a light trap designed by the American Biophysics Company ® (ABC) best reflected human biting risk for most species. An ABC light trap supplemented with carbon dioxide and octenol was second best. A New Jersey light trap, flickering light ABC trap, steady light ABC trap, and resting boxes had nearly equal mean trap yields.
Addition of octenol to an ABC light trap did not significantly improve trap yield over the CDC light trap.
An ABC light trap with carbon dioxide best reflected biting risk from Ae. canadensis, Cq. perturbans, and other nuisance mosquitoes, such as Ae. triseriatus,
Ae. cinereus, Ae. aurifer, and Ae. abserratus. An ABC light trap with carbon dioxide and octenol best reflected biting risk from Cx. salinarius. Resting boxes collected the most numbers of An. quadrimaculatus and An. punctipennis, but females often were engorged. An ABC light trap with either CO2 alone or with CO2 plus octenol was a better measure of human biting risk by anophelines. Natural populations of Ae. vexans were low, and no useful prediction could be made regarding trap efficiency.
VI TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... iv
ABSTRACT...... v
LIST OF TABLES...... ix
LIST OF FIGURES...... xi
Chapter 1. LITERATURE REVIEW....1
Introduction...... 1
Epidemiology...... 1 Epizootiology.2 Virus Isolation...... 3 Vectorial Capacity and Vector Competence...... 5
Rationale: Why these six species?...... 6
Aedes canadensis (Theobald)...... 7 Aedes vexans (Meigen)...... 8 Coquillettidia perturbans (Walker).....9 Anopheles punctipennis (Say) and An. quadrimaculatus Say.....10 Culex salinarius Coquillett.....11
Monitoring Methods...... 12
Carbon dioxide and Octenol...... 12 Landing Rates on Humans...... 14
2. VECTOR COMPETENCE OF MASSACHUSETTS MOSQUITOES FOR A LOCAL STRAIN OF EEE VIRUS...... 16
Introduction...... 16 Materials and Methods...... 17
Collection of Test Mosquitoes...... 17 Blood Feeding and Saliva Collection...... 17 Virus Assay...... 18
VII Results... 19
Host Feeding and Survival.19 Infection and Transmission.22
Discussion.25 Conclusions. 30 Summary...30
3. SURVEILLANCE FOR EPIDEMIC VECTORS OF EEE VIRUS.31
Introduction.32 Materials and Methods.32
Site Selection.32 Experimental Design.32 Data Analysis. 33
Results.34
Trap Comparisons of All Species Combined.34 Trap Specificity.38
Discussion.42 Conclusions.45
4. REFERENCES. 47
VIII LIST OF TABLES
Table Page
1. Vims titers for blood from chicks injected with EEE virus (92-983 strain). Titers are given in PFUs, the reciprocals of logio plaque forming units per 1.0 ml of blood. Aedes canadensis, Cq. perturbans, and Anopheles were fed twice in 1995.....20
2. Feeding and survival data for females fed on an EEE-viremic chick, 1994.... 21
3. Feeding and survival data for females fed on an EEE-viremic chick, 1995. 21
4. Mosquitoes and saliva producing CPE on a BHK monolayer 7 and 14 days post blood feeding on an EEE-viremic chick, 1994...... 24
5. Mosquitoes and saliva producing CPE on a BHK monolayer 7 and 14 days post blood feeding on an EEE-viremic chick, 1995.24
6. Mosquitoes and saliva producing CPE on a BHK monolayer 7 and 14 days post blood feeding on an EEE-viremic chick, 1994 and 1995 combined...... 25
7. Nine monitoring methods for mosquitoes: trap total, nights in use, mean, and percent total yields...... 35
8. Pairwise comparisons using the Wilcoxon Signed Rank Test between monitoring methods for all species. If p > 0.05, then difference is not significant; the probability that the two methods are the same is > 5%. If p < 0.05, then difference is significant.. 37
9. Nightly means for nine monitoring methods for female Aedes. Species included Aedes canadensis, Ae. vexans, Ae. stimulans s.l., Ae. triseriatus, Ae. cinereus, Ae. aurifer, Ae. abserratus, and Ae. punctor...... 41
10. Nightly means for eight monitoring methods for female Coquillettidia perturbans...... 41
11. Nightly means for nine monitoring methods for female Culex salinarius...... 41
IX 12. Nightly means for eight monitoring methods for female Anopheles quadrimaculatus. An. punctipennis, and An. walkeri.41
13. Nightly means for eight monitoring methods for female Culiseta melanura.41
x LIST OF FIGURES
Figure Page
1. Mean nightly catches for nine monitoring methods for mosquitoes.36
xi CHAPTER 1
LITERATURE REVIEW
Introduction
Epidemiology
Eastern equine encephalomyelitis (EEE) virus is enzootic in passerine birds
(Dalrymple et al. 1972, Hayes et al. 1962b, Kissling et al. 1957, Srihongse et al. 1978,
Stamm 1968), may cause high viremias in snakes, turtles, and frogs (Dalrymple et al.
1972, Hayes et al. 1964), and causes debilitating or fatal encephalitis in humans, horses, whooping cranes, and exotic game birds such as ring-necked pheasants and
Pekin ducks (Ayres & Feemster 1949, Beaudette & Black 1948, Beaudette & Hudson
1945, Clark 1987, Dein 1986, Dougherty & Price 1960, Eklund & Blumstein 1938,
Tyzzer et al. 1938, Wesselhoeft et al. 1938). EEE virus is maintained in a sylvatic avian cycle by the ornithophagic mosquito Culiseta melanura (Coquillett) and is transmitted from birds to mammals by other mosquitoes (Morris 1988). EEE virus is a member of the family Togaviridae, genus Alphavirus, possessing a lipid bilayer and a single-stranded RNA genome. It is related to but antigenically distinct from Western equine encephalomyelitis (WEE) and Highlands J (HJ) virus (Levy et al. 1994).
Human EEE infection is manifested by initial fever, nausea, vomiting, and convulsions, and can lead to meningitis, coma, and death (Manson-Bahr & Bell 1987,
Scott & Weaver 1989). Inflammation of the brain and spinal cord, leukocytosis, and increased lymphocytes and protein in the cerebrospinal fluid can result in neurological sequelae and mental retardation in non-fatal cases (Manson-Bahr & Bell 1987).
Human cases in Massachusetts during an epidemic year appear in September and early October. Epidemics the subsequent year appear in late July or early August
(Edman et al. 1993). Twenty-five persons out of 34 confirmed cases and at least 269 horses died of EEE in 1938 in Massachusetts (Getting 1941). Four cases in 1955 and twelve cases in 1956 resulted in ten human deaths (Feemster 1957, Webster 1956).
1 Seven human cases from 1970-75 resulted in four deaths (Edman et al 1993). Thirteen human cases from 1980-89 resulted in four deaths (Villari et al 1995). To date, 72 human cases with 43 deaths and 630 horse cases have been reported in Massachusetts
(Edman et al. 1993).
Hanson (1958) retrospectively documents the first horse epizootics of EEE in
Massachusetts in 1831, when 75 horses died out of 100 putative cases in Plymouth
County. Cases peaked in August and ended by late September. Horse deaths in New
Jersey in 1933 yielded the first isolates of EEE virus (Giltner & Shahan 1933, Ten
Broeck & Merrill 1933). Horse cases in the San Joaquin Valley of California yielded what was suspected to be EEE virus in 1930 (Meyer et al. 1931), but existing serology may have been unable to distinguish between EEE and WEE (Ten Broeck & Merrill
1933). Studies of the New Jersey epizootic confirmed that EEE virus was the etiologic agent of equine encephalitis (Feemster 1957, Getting 1941, Hayes 1981, Webster
1956).
Epizootiology
Culiseta melanura is the principal enzootic vector of EEE virus, capable of transmitting virus after 3 days of extrinsic incubation (El) (Scott & Burrage 1984).
Culiseta melanura maintains a summer reservoir of virus in wild birds in wooded, permanent swamps (Burbutis & Jobbins 1957, Dalrymple et al. 1972, Hess & Holden
1958, Morris 1988). Larval habitats along the Atlantic and Gulf coasts are characterized by red maple/white cedar stands and include shaded, cold pools in depressions of sphagnum bogs, the bases of trees, or tree blowout, and spring-fed acidic pools surrounded by hemlock and cypress (Joseph & Bickley 1969). This mosquito mainly rests and feeds in wooded swamps and adjacent hardwood forest
(Joseph & Bickley 1969). Culiseta melanura feeds predominantly (96-99%) on passerine and wading birds in Florida (Edman et al. 1972), North Carolina (Irby & Apperson 1988), Virginia (Tempelis 1974), Maryland (Joseph & Bickley 1969), New
2 Jersey (Crans 1964), Connecticut (Magnarelli 1977), Massachusetts (Nasci 1980,
Nasci & Edman 1981), and New York (Means 1968, Schober & Collins 1966).
Frequent virus isolations from high densities of Cs. melanura correlate with epizootics in wild passerine birds (Sudia et al. 1968), ring-necked pheasants
(.Phasianus colchicus), emus (Dromaius novaehollandiae) and horses (Anon. 1995,
Wallis et al. 1974, Williams et al. 1974). Epizootics in birds usually occur during late summer and fall when avian population density is highest and birds are most active
(Dalrymple et al. 1972). EEE virus has been recovered from almost every state of the
U.S. east of the Mississippi River, with epizootics and epidemics from Massachusetts to the Gulf states, and isolated foci in southern Michigan, northern Indiana, and upstate New York (Boromisa et al. 1987, McLean 1991). The bridge vectors, or vectors that transfer virus from the epornitic (epizootic in birds) cycle to horses and humans during late summer epidemics, have not been determined in Massachusetts.
Recovery of EEE virus from a rock dove, Columba livia (Fothergill & Dingle
1938), ring-necked pheasants, Phasianus colchicus (Tyzzer et al. 1938), and an apparently healthy grackle, Quiscalus quiscula (Kissling et al. 1951), implicated birds as both reservoir and amplification host. Squirrels (Sciurus spp.), woodchucks
(.Marmota monax), raccoons (Procyon lotor), opossums (Didelphis virginiana), skunks
(Mephitis mephitis), swine (Sus scrofa dome Stic us), some turtles (Chrysemys picta,
Terrapene Carolina, Chelydra serpentina, and Clemmys guttata), and some lizards
(.Basiliscus sp., Ameiva sp., and Cnemidophorus sp.), presumably naturally infected, possess antibodies against EEE virus, although these animals are not proven reservoirs for EEE virus (Craighead et al. 1962, Dalrymple et al. 1972, Karstad et al. 1960).
Virus Isolation The seasonal nature of EEE suggested an arthropod vector, but the role of
mosquitoes as vectors of EEE was enigmatic until the 1950s. The chicken mite
Dermanyssus gallinae (De Geer) and the chicken louse Eomenacanthus stramineus
(Nitzsch) yielded the first isolations of EEE virus from field-caught arthropods
3 (Howitt et al. 1948). Wild-caught Coquillettidia perturbans were the first mosquitoes from which EEE virus was detected (Howitt et al. 1949).
The role of Cs. melanura in sylvatic viral maintenance was considered when this mosquito first yielded EEE virus in Louisiana (Chamberlain et al. 1951).
Subsequent isolations of EEE virus from Cs. melanura in Georgia (Chamberlain et al.
1969), Maryland (LeDuc et al. 1972), Connecticut (Wallis 1959), Massachusetts
(Feemster et al. 1958, Hayes 1961) and New Jersey (Burbutis & Jobbins 1957,
Chamberlain 1960, Chamberlain et al. 1958, Holden et al. 1954) reinforced its role in maintaining avian infection. Chamberlain (1958b) established Cs. melanura as the enzootic vector of EEE virus by avian blood meal identification, frequent virus isolation from fed females, and proximity of roosting birds to feeding mosquitoes.
Culiseta melanura is not considered the epidemic vector because of its strong avian feeding preference (Hayes 1961b).
Sudia et al. (1956) first suspected that the saltmarsh mosquito Aedes sollicitans
(Walker) transmits EEE virus to horses. Hayes et al. (1962a) incriminated Ae. sollicitans as the vector of EEE in coastal New Jersey. Crans (1977) discussed virus isolation during human outbreaks, host preference, and geographic distribution overlapping human cases to emphasize Ae. sollicitans as an epidemic vector.
Epidemic foci in southeastern Massachusetts are not coastal or estuarine; therefore, Ae. sollicitans is considered negligible in epidemic transmission in the Commonwealth
(Edman et al. 1993). The current study examines six other mosquito species which could play a role in EEE virus transmission in Massachusetts.
Karstad et al. (1957) isolated EEE virus from Aedes mitchellae (Dyar),
Anopheles crucians, and an unidentified Culicoides midge in Georgia. Aedes sticticus and engorged, ornithophilic black flies, Eusimulium johannseni (Hart) and Simulium meridionale Riley, yielded EEE isolates in Wisconsin (Anderson et al. 1961).
During the 1963 outbreak in Georgia, Chamberlain et al. (1969) isolated EEE virus from Aedes atlanticus and Cs. melanura. Srihongse & Galindo (1967) first isolated EEE virus from Culex (Melanoconion) taeniopus Dyar and Knab in Panama.
4 Wellings et al. (1972) isolated EEE virus from Ae. triseriatus, Culex (.Melanoconion) spp., Cx, quinquefasciatus, Cx. salinarius and Cs. melanura from the Tampa Bay area of Florida from 1963-70. During equine epizootics in New Jersey, Crans & Schulze
(1986) and Clark et al. (1985) isolated EEE virus from Cs. melanura and Cq. perturbans. Fourteen isolations of EEE virus were made from Aedes albopictus collected at a tire depot in Polk County, Florida (Anon. 1992, Mitchell et al. 1992).
Scott & Weaver (1989) listed EEE virus isolations from naturally infected arthropods of North and South America. Isolations of virus prove that the mosquito fed on an infected host or was transovarially infected. Virus isolations do not prove the mosquito’s ability to transmit virus or its vector competence.
Vectorial Capacity and Vector Competence
Vectorial capacity is affected by extrinsic and intrinsic factors (Hardy et al.
1983). Extrinsic factors include geographic, seasonal, and diel overlap between potential vectors and their hosts; mosquito and host population density; and climate.
Intrinsic factors governing vectorial capacity include age; host preference; and vector competence. Vector competence is an arthropod’s susceptibility to infection of midgut, hemocoel, and salivary glands and ability to transmit virus by bite or by egg.
Temperature influences vector competence by affecting mosquito metabolism, viral replication, and possibly genetic expression of vector competence.
Chamberlain et al. (1954) established certain criteria for incriminating a vector of an arthropod-borne virus. Transmission rate is the percentage of female mosquitoes transmitting infection by bite to a susceptible host after imbibing a meal of high viral titer after a suitable extrinsic incubation period (El). Infection rate is the percentage of females which, regardless of transmission rate, harbor virus. Several investigators have described techniques for experimentally determining vector competence of mosquitoes for EEE virus (Chamberlain et al. 1954, Davis 1940, Kelser 1933, Merrill
et al. 1934, Scott et al. 1990, Ten Broeck & Merrill 1935).
5 Kelser (1933) tested Aedes aegypti and first proved that mosquitoes could transmit EEE virus. Merrill et al. (1934) tested mosquito transmission of EEE virus in the laboratory, incriminating Aedes sollicitans and Ae. cantator as potential vectors.
Culex pipiens and Anopheles quadrimaculatus failed to transmit EEE virus. Ten
Broeck & Merrill (1935) demonstrated that Aedes taeniorhynchus and Ae. vexans could transmit EEE virus, but, as before, An. quadrimaculatus and Cx. pipiens could not. Davis (1940) demonstrated transmission of EEE virus by Aedes vexans, Ae. sollicitans, Ae. cantator, Ae. atropalpus, Ae. triseriatus, and Ae. aegypti, but not by
Anopheles punctipennis, Culex salinarius, Cx. pipiens, or Coquillettidia perturbans.
Chamberlain et al. (1954) compared the infection and transmission rates of twenty species of mosquitoes for EEE virus. Aedes sollicitans, Ae. aegypti, and Ae. triseriatus all had a 100% infection rate and 75, 56, and 56% transmission rates respectively. Culex salinarius, Anopheles quadrimaculatus, An. crucians, and Cx. quinquefasciatus did not transmit, although An. quadrimaculatus had a 79% infection rate. The infection threshold, or titer of virus necessary to cause infection, varied among species. To cause a 1-5% infection rate, a 102'7 virus titer was necessary for
Ae. aegypti, whereas a virus titer of 108 0 was necessary to achieve the same infection rate for Cx. salinarius, Cx. quinquefasciatus, and An. crucians.
More recently, Scott et al. (1990) demonstrated laboratory transmission of EEE virus by the Asian tiger mosquito Aedes albopictus (Skuse).
Rationale: Why these six species?
A dual vector (enzootic and epidemic) of an avian-reservoired zoonosis must have a broad host preference (Edman & Taylor 1968). Culiseta melanura feeds almost exclusively on birds, and hence cannot function as a dual vector. During the 1973
Massachusetts epidemic, about 1.7 million acres were treated with malathion, at a cost of $600,000 (Edman et al. 1993). Determination of the true epidemic vectors could focus larvicidal control and aerial spraying, thereby lowering insecticide volume and cost and reducing human risk for EEE.
6 One or more of six Massachusetts mosquito species are possible bridge vectors of EEE virus from the epornitic cycle to horses and humans based on their field prevalence prior to horse and human cases, flight ranges overlapping horse and human habitats, host feeding patterns, EEE virus isolation, and vector competence for EEE virus. The current study tests the susceptibility to infection and potential for transmission of these six local species for a Massachusetts strain of EEE virus.
Aedes canadensis (Theobald)
This spring or vernal pool mosquito breeds in temporary woodland pools containing decaying leaves. It may re-emerge if heavy rains during mid- to late summer reflood these sites, which are often in close association with the permanent swamp habitats of Cs. melanura and enzootic EEE transmission (Carpenter et al.
1946, Main et al. 1968). Females rest in woodlands and begin seeking hosts around sunset. They remain close to the forest margin and mainly bite at dusk and in early evening (Hayes 1962, Trueman & Mclver 1986).
Aedes canadensis is collected in a variety of mosquito traps (Hayes et al. 1962,
Howard et al. 1988). Females are eclectic but feed mainly on mammals. Hayes
(1961b) found equal engorgement rates on reptiles, birds, and mammals and some engorgement on amphibians. LeDuc et al. (1972) detected feeding on ruminants, raccoons, rabbits, and passerine and columbiform birds. Magnarelli (1977) identified blood meals from canines, squirrels, sheep, humans, passerines, and frogs. Means
(1968) recorded blood meals from rabbits, rodents, bats, blue jays, bobwhite quail, sparrows, starlings, snakes, turtles, and frogs. Nasci & Edman (1981) identified horse, rabbit, pig, bovine, and human blood meals. Aedes canadensis is the most common mosquito feeding on turtles from spring to late summer (Crans & Rockel 1968, Irby &
Apperson 1988). It readily bites humans but is usually only a serious pest in early summer.
Although vector competence for EEE virus has not been proven for Ae. canadensis, California encephalitis virus has been shown to multiply in this species
7 (McLean & Wilson 1972). Isolations have been made of EEE virus from Ae. canadensis in Massachusetts (Grady et al. 1978) and upstate New York (Howard et al, 1988).
Aedes vexans (Meigenl
This common inland floodwater mosquito develops in rain-filled open grassy depressions, floodplains of streams and rivers, poorly drained alluvium with accumulated rain or irrigation water, and margins of impoundments (Carpenter et al.
1946, Horsfall et al. 1973). Although periods of maximum emergence may vary locally and annually, adults emerge from mid-June to mid-September, with highest densities in July (Horsfall et al. 1973). Aedes vexans is a crepuscular biter throughout the summer and early fall. It rests in woods during the daytime, but is more likely to invade suburban areas to feed and range farther than Ae. canadensis (Edman et al.
1993). Aedes vexans was abundant during the 1959 New Jersey EEE epizootic (Hayes et al. 1962a) and overlapped horse and human cases during the 1938 Massachusetts outbreak (Getting 1941).
Aedes vexans is abundant in CCVbaited CDC light trap collections (Buckley et al. 1994). It is the most common pest mosquito in many parts of the U.S. Females are persistent biters of horses and cattle (Nasci 1984, Nasci & Edman 1981) and often take multiple blood meals (Edman & Downe 1964). Burkot & Defoliart (1982) identified
362 mammal blood meals, the majority deer, out of 365 bloodfed females tested. The other three blood meals belonged to two birds and one reptile. Cupp & Stokes (1973) identified 115 mammal blood meals (largely human, horse, and dog) out of 125 bloodfed females tested. The other 10 blood meals were avian. Edman (1971) identified 1249 mammal blood meals out of 1252 bloodfed females tested. The other three blood meals belonged to two birds and one snake. Irby & Apperson (1988)
identified 15 mammal blood meals out of 18 bloodfed females tested. The other three blood meals were reptile or amphibian. Magnarelli (1977) identified 60 mammal
blood meals out of 62 bloodfed females tested. The other two blood meals belonged
8 to passerine birds. Ritchie & Rowley (1981) identified 104 mammal blood meals out of 108 bloodfed females tested. The other four blood meals were avian. Suyemoto et al. (1973) detected only mammal blood meals out of 112 blood fed females. Murphey et al. (1967) recorded a greater proportion of mammals fed upon than birds, but Ae. vexans did feed on ciconiine and passerine birds.
Ten Broeck & Merrill (1935) and Davis (1940) incriminated Ae. vexans as a vector of EEE virus. Chamberlain et al. (1954) reported a 13% transmission rate for
Ae. vexans, the lowest rate for ten competent mosquito species tested. Researchers have isolated EEE virus from Ae. vexans in Alabama (Sudia et al. 1968), Connecticut
(Wallis et al. 1960), Florida (Wellings et al. 1972), and New Jersey (Kandle 1960).
Coquillettidia perturbans (Walker)
Larvae of this cattail mosquito pierce the submerged roots of emergent aquatic vegetation in permanent freshwater marshes, usually created by human disruption of natural drainage systems (Crans et al. 1986, Morris et al. 1990). Larvae are associated with cattail (Typha latifolia), water lettuce (Pistia stratiotes), water willow or swamp loosestrife {Decodon verticillata), arrow arum {Peltandra virginica), and maidencane
{Panicum hemitomon) (Lounibos & Escher 1985, Slaff & Haefner 1985). Females are most active in mid-summer but persist into late summer in southeastern Massachusetts
(Main et al. 1968). Females are strong fliers and bite in unforested areas in the early evening in New England (Capotosto & Boyes 1984, Hayes 1962) or later in the night in more northern latitudes (Trueman & Mclver 1986).
Coquillettidia perturbans is abundant in human biting collections and light traps baited with CO2 (Buckley et al. 1994, Kline et al. 1991a, Schreck et al. 1972).
Females feed predominantly on large mammals and humans but may feed on amphibians, reptiles, ciconiines, passerines, and fowl (Edman 1971, Hayes 1961b,
Magnarelli 1977, Means 1968, Murphey et al. 1967). Coquillettidia perturbans collected in Alabama (Chamberlain et al. 1954), Connecticut (Jaynes et al. 1962), and
Indiana (Boromisa et al. 1987) have transmitted EEE virus experimentally. EEE virus
9 has been isolated from females in Alabama (Sudia et al. 1968), Georgia (Howitt et al.
1949), Ohio (Nasci et al. 1993), Massachusetts (Edman et al. 1993), New Jersey
(Clark et al. 1985, Crans & Schulze 1986), and New York (Ninivaggi & Guirgis 1994,
Srihongse et al. 1978).
Anopheles punctipennis (Say) and An. quadrimaculatus Sav
Both species breed throughout the eastern and central United States in sluggish freshwater canals, ponds, and lake borders with surface emergent vegetation or floating debris (Carpenter et al. 1946). They are pests of humans and domestic mammals from mid- to late summer, reaching a plateau by mid-August, and declining as water levels recede (Weathersbee & Meisch 1991). Females are nocturnal, their activity usually peaking just after dark. Anopheles quadrimaculatus can disperse about 2 km from its forest edge resting sites (Weathersbee & Meisch 1990).
A North Carolina study revealed 81% mammal and 19% avian blood meals from An. punctipennis and 77% mammal and 23% avian blood meals from An. quadrimaculatus (Irby & Apperson 1988). Murphey et al. (1967) detected An. quadrimaculatus feeding on waterfowl, passerines, small mammals, and to a lesser extent, on turtles. Cupp & Stokes (1973) identified 12% avian blood meals and
Schaefer & Steelman (1969) identified only 1% avian blood meals from An. quadrimaculatus in Louisiana. Apperson & Lanzaro (1991), Edman (1971), and
Suyemoto et al. (1973) identified exclusively mammal (ruminant, deer, pig, and rabbit) blood meals from An. quadrimaculatus. Nasci & Edman (1981) identified
horse, rabbit, pig, dog, and bovine blood meals from An. quadrimaculatus, and only
horse and bovine blood meals from An. punctipennis. Murphey et al. (1967) detected
An. punctipennis feeding predominantly on mammals, but feeding on birds might be
influenced by host availability. Tempelis (1974) identified primarily cattle and horse
blood meals from An. punctipennis in the Midwest.
Anopheles punctipennis and An. quadrimaculatus actively feed on humans, but
rarely are collected and tested for EEE virus (Edman et al. 1993). Anopheles
10 quadrimaculatus has yielded EEE virus isolates in Suffolk County, New York
(Ninivaggi & Guirgis 1994), and both species have yielded EEE virus isolates in
Delaware (C. Stachecki, Delaware Division of Fish and Wildlife). Two out of 20 An. quadrimaculatus from Georgia that fed from a 107 6 titer of an EEE virus strain from
New Jersey transmitted virus after ten days (Collins et al. 1965). Females that fed on lower titers did not transmit. Experimental vector competence for EEE has not been demonstrated for An. punctipennis (Chamberlain et al. 1954, Davis 1940, Merrill et al. 1934, Ten Broeck & Merrill 1935, Wallis & Main 1974).
Culex salinarius Coquillett
The larval habitat, seasonal abundance, and adult feeding periodicity of this species are similar to that of the anophelines. Host-seeking females can disperse 1-2 km from their forest resting sites (LaSalle & Dakin 1982). Culex salinarius usually feeds on domestic mammals adjacent to swamps, but its host choice is influenced by host availability, and it may feed equally on birds and mammals (Edman 1974, LeDuc et al. 1972, Murphey et al. 1967). Edman (1974) identified 53% avian blood meals from Cx. salinarius collected by aspirators in a swamp-hammock site, but only 5% of females collected near a cattle ranch had fed on birds. Females in Texas fed entirely on mammals, whereas females in Minnesota fed principally on passerine birds
(Tempelis 1974). About 3% of blood meals from Cx. salinarius were from birds in
Kansas (Edman & Downe 1964) and in Louisiana (Schaefer & Steelman 1969). Hayes
(1961b) detected nearly equal engorgement rates (40-50%) on birds, mammals, and reptiles and some feeding on amphibians in Raynham, Massachusetts. Murphey et al.
(1967) detected equal engorgement rates on birds and mammals but not on reptiles.
Suyemoto et al. (1973) identified 96% mammal, 2% bird, and 2% reptile blood meals.
Ritchie & Rowley (1981) detected culicines (not all Cx. salinarius) feeding on passerines in May and on mammals in July. Cupp & Stokes (1973) identified 45% avian, 17% equine, and 15% canine blood meals from 328 bloodfed females. Irby &
11 Apperson (1988) identified 38 mammal blood meals out of 44 bloodfed females tested; the remaining six bloodmeals were avian.
Culex salinarius is abundant in avian biting collections (Kissling et al. 1957) and light traps with C02 (Carestia & Savage 1967, Schreck et al. 1972). EEE virus has been recovered from females in New Jersey (Burbutis & Jobbins 1957,
Chamberlain et al. 1958), Maryland (Muul et al. 1975), Alabama (Sudia et al. 1968), and Florida (Wellings et al. 1972). Vector competence for EEE virus has not been demonstrated for this species (Chamberlain et al. 1954, Davis 1940). Kandle (1960) suggests: “previous laboratory data that Culex salinarius might be involved...” in EEE transmission.
Monitoring Methods
In Massachusetts, veterinarians, physicians, and hospitals in an enzootic area are alerted when EEE virus is isolated from Cs. melanura by public health officials in early summer. When birds have manifested a viremia high enough to infect Cs. melanura, potential epidemic vectors may have emerged as adults. Larvicidal control often is precluded, and mosquito abatement districts resort to adulticiding.
Widespread application of broad spectrum adulticides like malathion encounters public opposition because of environmental and health concerns (Edman et al. 1993).
Upon determination of the true epidemic vectors, surveillance for these mosquitoes should be specific. Surveillance should correlate well with human biting risk and be convenient and cost effective for mosquito abatement projects to focus potential fogging areas. Historically, mosquito control personnel have used New Jersey
(Headlee 1932) or CDC light traps (Sudia & Chamberlain 1962), sometimes supplementing them with chemical attractants to increase the trap catch.
Carbon dioxide and Octenol Among the chemicals used with light traps to enhance biting fly attraction, especially for species with minimal attraction to light, carbon dioxide (C02) and 1-
12 octen-3-ol (octenol) have shown the most promise. Carbon dioxide activates resting females to flight, prolongs host seeking, and orients females to the host (Gillies 1980).
Visual components of the host, warm and moist air convection, and odor cues also influence female host-seeking response (Allan et al. 1987, Bowen 1991). Rudolfs
(1922) and Reeves (1951) first reported C02 as a female mosquito attractant.
Mosquito control projects have used either dry ice (Newhouse et al. 1966) or compressed gas cylinders (Gamble 1994) to deliver C02 to a light trap. The addition of C02 to light traps increased numbers of Ae. canadensis and Ae. vexans (Buckley et al. 1994), Cq. perturbans (Magnarelli 1975, Schreck et al. 1912), An. quadrimaculatus
(Newhouse et al. 1966), and Cx. salinarius (Carestia & Savage 1967).
Hall et al. (1984) first isolated the volatile compound octenol from ox breath.
Kline (1994) reviews octenol as a biting fly attractant. Takken & Kline (1989) found that octenol alone attracted mosquitoes in numbers similar to C02 released at 200 cc/min in the Everglades. A 3.0 mg/h release rate of octenol alone increased catches relative to no bait for Ae. taeniorhynchus, Cx. nigripalpus Theobald, and Wyeomyia, but not for Culex {Melanoconion) spp. (Kline et al. 1991b). Kline et al. (1990a) showed that octenol alone attracted more Cq. perturbans than C02 alone. Octenol also acted synergistically with C02 to increase catches of anophelines.
In a study in Arkansas, octenol plus C02 increased collections of Cq. perturbans and Cx. salinarius but yielded ambiguous results with An. quadrimaculatus (Kline et al. 1991a). Kline et al. (1990b) noticed a significant attraction to octenol relative to no attractant for Cq. perturbans and Cx. salinarius but a negative octenol response for An. quadrimaculatus in Polk County, Florida. Octenol plus 500 cc C02/min attracted fewer Anopheles spp. and Culex (.Melanoconion) spp. than C02 alone. Significantly greater numbers of Ae. vexans, Cq. perturbans, Cx. salinarius, and An. quadrimaculatus were collected in Louisiana and Colorado when octenol was added to C02 (Kline 1994).
13 Traps baited with light or octenol caught few mosquitoes in the Upper Rhine
Valley of Germany (Becker et al. 1995), whereas CO2 alone or in combination with
light or octenol caught the most Ae. vexans and Ae. cinereus.
Kemme et al. (1993) and Van Essen et al. (1994) in Queensland, Australia,
noticed increased Aedes funereus and Ae. vigilax numbers with CO2 supplemented
with octenol. Culex annulirostris and Cx. sitiens were not enhanced by addition of octenol at any emission rate. Collections of Culex generally are not improved by the
addition of octenol to CO2 (Takken & Kline 1989, Kemme et al. 1993). However, collections of the more mammalophagic Cx. salinarius have increased with the
addition of octenol (Kline et al. 1991a, Kline et al. 1990b) or C02 (Carestia & Savage
1967, Schreck et al. 1972).
Landing Rates on Humans
Service (1993) discussed sampling host seeking females with animal or human bait. Human biting collections directly measure the biting rate of anthropophagic mosquitoes, thereby representing the vector population responsible for disease transmission. Light trap collections frequently are compared to landing rate collections to evaluate how efficiently light traps reflect actual human biting risk.
Slaff et al. (1983) compared the NJ light trap and CDC light trap supplemented
with dry ice (CO2) to human biting risk in northwestern New Jersey. The CDC trap
with CO2 best reflected human biting risk by Ae. canadensis, Ae. vexans, Cq. perturbans, An. quadrimaculatus, and An. punctipennis, but the NJ trap
underestimated species diversity and biting risk. Acuff (1976) collected more Ae.
canadensis, Ae. vexans, and An. punctipennis with NJ light traps and C02-baited CDC
traps than with human biting collections. Odetoyinbo (1969) first studied unbaited CDC light traps for sampling malaria
vectors in Africa. Working in Tanzania, Davis et al. (1995) collected 1.23 times the
number of anophelines with CDC traps versus human bait collections, and Lines et al.
(1991) found three light traps collected as many Anopheles and Culex as two human
14 volunteers. Light traps have underestimated actual biting risk when anopheline abundance varied (Mbogo et al. 1993), but regression correlations based on low numbers may emphasize local population density over distribution (Smith 1995).
Geography, microhabitat, time of year, time of evening, and mosquito abundance influence how mosquito traps reflect actual human biting risk.
Surveillance for the epidemic vectors of EEE should be specific, convenient, cost effective for mosquito control projects, and accurately reflect human biting risk and disease transmission.
15 CHAPTER 2
VECTOR COMPETENCE OF MASSACHUSETTS
MOSQUITOES FOR A LOCAL STRAIN OF EEE VIRUS
Introduction
In Massachusetts one or more of six different mosquito species are possible vectors of EEE virus from the epornitic cycle to horses and humans. Incrimination is based on seasonal prevalence prior to horse and human cases, flight ranges overlapping horse and human habitats, host preference, history of EEE virus isolation, and vector competence for EEE virus. Determination of the true epidemic vectors could focus larvicidal control or aerial spraying, thereby lowering insecticide volume and cost and reducing human risk for EEE.
Aedes vexans (Chamberlain et al. 1954, Davis 1940, Ten Broeck & Merrill
1935), Coquillettidia perturbans (Boromisa et al. 1987, Chamberlain et al. 1954,
Jaynes et al. 1962), and Anopheles quadrimaculatus (Collins et al. 1965) were found to experimentally transmit EEE virus; Aedes canadensis, Culex salinarius, and
Anopheles punctipennis were unable to transmit EEE virus (Chamberlain et al. 1954,
Davis 1940, Merrill et al. 1934, Ten Broeck & Merrill 1935, Wallis & Main 1974).
Previous data on the vector competence of these six species are equivocal, because mosquitoes from one geographic region were tested with EEE virus from another region. Vector competence was proven if mosquitoes were found to transmit
EEE virus from another region; however, colonized or geographically isolated populations may be refractory to infection for an exotic EEE viral strain (Hardy 1988).
Recent isolates of EEE virus from Massachusetts provided local geographic and genetic strains to re-evaluate the vector competence of field-caught species from the same region.
16 Materials and Methods Collection of Test Mosquitoes
Mosquitoes were collected from natural breeding sites as fourth instars, pupae, young adults, or a combination thereof. Test females collected as late larvae or pupae were nulliparous. Females collected as young adults were probably nulliparous, because they were collected soon after emergence, either during initial host seeking or at resting boxes near larval sites.
Aedes canadensis larvae and pupae were dipped from vernal pools throughout
May in Hampshire County, Massachusetts. Coquillettidia perturbans females were collected with an aerial net the last week of June and first week of July, as they began seeking hosts at dusk, near a cattle farm in East Bridgewater, Massachusetts. Aedes vexans larvae were dipped from a grassy depression flooded with rain in July, August, and September in Amherst, Massachusetts. Culex salinarius larvae and pupae were dipped from a reflooded grassy pool in August in Amherst, Massachusetts. Anopheles punctipennis and An. quadrimaculatus adults were aspirated from resting boxes facing a sluggish stream throughout August in Hadley, Massachusetts. A laboratory colony of Cs. melanura, the enzootic vector of EEE virus, was the control (Wallis & Whitman
1969). Pupae were allowed to eclose in a 20 x 23.5 x 30 cm cage. Wild-caught adults were transferred to similar cages. One hundred males of each species were collected and held at -70° C to test for evidence of transovarial transmission. Adults were held for 7-14 d post eclosion at 22° C and 90% humidity, on a 14:10 lightidark schedule.
Females were given a 5% sucrose solution ad lib, which was removed 24 h before feeding on an EEE-infected chick.
Blood Feeding and Saliva Collection
A stock solution of the 92-983 strain of EEE virus, isolated from Cs. melanura in Easton, Massachusetts, on 16 August 1992, contained 108 plaque-forming units
17 (PFUs)/1.0 ml (B. Werner, Massachusetts Department of Public Health). This stock solution was diluted 1:10, resulting in a dosage of 107 PFU/ml.
One day-old chicks were inoculated intramuscularly with 0.1 ml of this viral dilution (106 PFU/ml). One day after inoculation, chicks were restrained in a nylon stocking and mosquitoes were allowed to feed for 2 hr. Immediately after feeding, 0.2 ml of blood was drawn from the chick to assess circulating viremia. Blood was mixed with 0.9 ml of diluent and stored at -70° C until virus assay. Diluent was minimum essential medium (MEM) for growth in baby hamster kidney (BHK) cell cultures plus
20% fetal calf serum (FCS). Engorged females were separated from unfed, and held at
25° C and 90% humidity, on an 18:6 light:dark schedule until saliva collection. Water and 5% sucrose solution were provided ad lib.
Seven days post blood feeding, saliva was collected from one-third of the surviving females, as described by Weaver et al. (1990). Double-sided tape was applied lengthwise to a clean 25 x 75 mm microscope slide. Microtiter tubes (50 pi) were cut into 2 cm sections and dipped into mineral oil. Six of these sections were laid perpendicular to the tape, the section with mineral oil on the slide. Wings and legs were removed from each female, prior to inserting the proboscis into the mineral oil. Mosquitoes were allowed to salivate into the oil for 20-30 mins.
Following the salivation period, the proboscis was removed from the capillary tube, and each female was placed in a centrifuge tube. Capillary tubes were removed from the slide, placed in centrifuge tubes with 200 pi of 25% FCS, and centrifuged twice for 10 s each at 104 rpm to recover and resuspend virus. Fourteen days post blood feeding, the remaining females were processed identically to those on day 7.
Mosquitoes and salivas were numbered accordingly and held at -70° C until virus assay.
Virus Assay BHK cell monolayers were grown on 24-well microtiter plates and overlaid with BHK 21 maintenance media (MEM, 10% FCS, L-Glutamine, Penicillin and
18 Streptomycin, 7.5 % NaHCCb). Stored mosquitoes and tubes of saliva in FCS were thawed in a water bath warmed to 37° C. Mosquitoes then were triturated with 500 |il of cold mosquito diluent (MEM, 22% FCS, 1% Fungizone®), centrifuged for 2 mins at 104 rpm, and kept on wet ice. Thawed tubes of saliva also were kept on ice. The
BHK 21 maintenance media overlay was discarded, and 100 pi of mosquito homogenate supernatant and mosquito saliva in FCS were overlaid in alternate wells.
Plates were incubated at 37°C for 1 hr and gently agitated every 10 mins. Each well was overlaid with MEM plus 5 or 10% FCS. Three, four, and seven days after inoculation, plates were read for cytopathic effect (CPE). Mosquitoes and saliva were scored either positive (virus present) or negative (virus absent); viral titers were not calculated.
Results
Host Feeding and Survival
In 1994, 242 females fed on EEE-viremic chicks; 360 fed in 1995. Table 1 shows the viral titers for blood from injected chicks. Tables 2 and 3 show feeding and survival data for these two years. Survival data were recorded 7 and 14 days post blood feeding. One-third of the surviving females were sampled on day 7; therefore, the data for day 14 represent percent survival from day 7, not from day 0.
Usually less than one-third of females fed on the chick, even though mosquitoes were starved for 24 hours. Few patterns emerge regarding engorgement, except for the 13 and 14% engorgement rates for Ae. canadensis in 1994 and 1995.
Percent engorgement varied from 1994 to 1995 for Ae. vexans (29 and 14%), Cq. perturbans (39 and 29%), An. punctipennis (1 and 17%), and An. quadrimaculatus (40 and 10%). Culex salinarius and Cs. melanura, which were tested in 1995 only, exhibited 53 and 82% engorgement rates respectively.
Culiseta melanura, a documented ornithophagic species, had the highest engorgement rate (82%), whereas An. punctipennis had the lowest (1 and 17%, average 9%). Although field evidence suggests Ae. canadensis is an eclectic feeder,
19 only 13-14% of females fed on a restrained domestic chick. An average of 20-30% of
the principally mammalophagic Ae. vexans, Cq.. perturbans, and An. quadrimaculatus
engorged on the chick. Fifty-three percent of Cx. salinarius engorged, but females
would feed only 1-2 weeks post eclosion; attempts to feed them within the first week
after emergence were unsuccessful.
Most species exhibited less than 25% mortality one week after feeding, except
Cq. perturbans, which exhibited 39 and 76% mortality in 1994 and 1995. Except for
Ae. vexans and Cq. perturbans, on average more than half of engorged females
survived for two weeks after feeding.
Table 1. Virus titers for blood from chicks injected with EEE virus (92-983 strain). Titers are given in PFUs, the reciprocals of logio plaque forming units per 1.0 ml of blood. Aedes canadensis, Cq. perturbans, and Anopheles were fed twice in 1995.
Species Chick titer
1 1994 Ae. canadensis 7.26 Cq. perturbans 7.60 Ae. vexans 6.30 Anopheles 6.15
1995 Ae. canadensis 8.78, 9.00 Ae. vexans 6.00 Cq. perturbans 4.30, 9.00 Anopheles 8.00, 8.00 Cx. salinarius 7.38 Cs. melanura 8.15
20 Table 2. Feeding and survival data for females fed on an EEE-viremic chick, 1994. Species Number fed/total % alive at day 7 % alive at day 14
Ae. canadensis 26/200 80.8 78.6
Ae. vexans 72/250 83.3 12.5
Cq. perturbans 78/200 61.5 25.0 % 1 An. punctipennis 1/90 100 NA
An. quadrimaculatus 65/160 92.3 53.8
Table 3. Feeding and survival data for females fed on an EEE-viremic chick, 1995.
Species Number fed/total % alive at day 7 % alive at day 14
Ae. canadensis 28/200 75.0 41.7
Ae. vexans 11/80 72.7 NA*
Cq. perturbans 115/400 24.4 17.6
An. punctipennis 10/60 60.0 30.0
An. quadrimaculatus 8/80 100.0 66.7
Cx. salinarius 106/200 77.4 61.0
Cs. melanura 82/100 89.0 73.2
* NA = not applicable. All females tested on day 7; none remained until day 14
21 Infection and Transmission
Three hundred and five females (out of 602 that fed) were tested for
susceptibility to infection and ability to transmit EEE virus. Almost all viremic chicks
had virus titers between 105-109 PFUs (Table 1). One chick on which Cq. perturbans
A 1 fed in 1995 had a titer of 10 . Infection and saliva transmission rates for 1994 and
1995 are presented in Tables 4 and 5; averaged results for both years are presented in
Table 6. Average infection rates remained the same from 7 to 14 days for Ae.
canadensis (100 to 94%), Ae. vexans (29 to 20%), An. punctipennis (100 to 100%),
and Cs. melanura (100 to 100%). Infection rates increased for Cq. perturbans (38 to
64%) and Cx. salinarius (6 to 22%) and decreased for An. quadrimaculatus (54 to
24%).
Average transmission rates remained the same from 7 to 14 days for Ae. vexans
(0 to 0%), An. punctipennis (0 to 0%), and Cs. melanura (94 to 93%). Transmission rates increased for Ae. canadensis (0 to 27%) and Cx. salinarius (0 to 13%) and
decreased for Cq. perturbans (6 to 0%) and An. quadrimaculatus (13 to 0%).
A stable, increasing, or decreasing infection rate corresponded with a stable,
increasing, or decreasing transmission rate for all species except Ae. canadensis and
Cq. perturbans. Average infection rate from 7 to 14 days for Ae. canadensis remained the same, but the transmission rate increased. Average infection rate from 7 to 14 days
for Cq. perturbans increased, but the transmission rate decreased.
Colony Cs. melanura, used as a control, had a 94% transmission rate, highest
for the seven species tested. The highest average transmission rate for a potential
epidemic vector was 13% for Ae. canadensis. Ten percent of Culex salinarius and An.
quadrimaculatus transmitted virus through saliva. The three lowest transmission rates
were those of Cq. perturbans (4%), Ae. vexans (0%), and An. punctipennis (0%).
Infection is an unreliable estimate of potential for transmission. The average
infection rates for Ae. canadensis (97%) and An. punctipennis (100%) were
comparable to that of Cs. melanura (100%). Whereas infected Cs. melanura had a
94% transmission rate, only 13% of infected Ae. canadensis and none of the infected
22 An. punctipennis transmitted virus. Culex salinarius had the lowest infection rate
(15%), but its 10% transmission rate was comparable to those of Ae. canadensis (13%) and An. quadrimaculatus (10%). Forty-three percent of Cq. perturbans and 41% of
An. quadrimaculatus were infected, but only 4% of infected Cq. perturbans transmitted as opposed to 10% of An. quadrimaculatus. Aedes vexans had almost twice the infection rate (27%) of Cx. salinarius (15%), but Cx. salinarius had a 10% transmission rate compared to 0% for Ae. vexans.
One hundred triturated males tested for five species did not produce CPE on a
BHK monolayer at 3 or 4 days incubation. Male Ae. vexans did produce cell sloughing and clumping.
23 Table 4. Mosquitoes and saliva producing CPE on a BHK monolayer 7 and 14 days post blood feeding on an EEE-viremic chick, 1994. day 7 day 14 total
mosq saliva mosq saliva mosq saliva species positive/tot positive/tot positive/tot positive/tot positive/tot positive/tot
Ae. canad 7/7 (1.0) 0/7 (0.0) 11/11 (1.0) 3/11 (.27) 18/18(1.0) 3/18 (.17) Ae. vexans 5/20 (.25) 0/5 (0.0) 1/5 (.20) 0/1 (0.0) 6/25 (.24) 0/6 (0.0) Cq. perturb 10/16 (.63) 1/10 (.10) 6/8 (.75) 0/6 (0.0) 16/24 (.67) 1/16 (.06) An. quadr 9/20 (.45) 1/9 (.11) 5/21 (.24) 0/5 (0.0) 14/41 (.34) 1/14 (.07)
An. punct 1/1 (1.0) 0/1 (0.0) - - 1/1 (1.0) 0/1 (0.0)
Table 5. Mosquitoes and saliva producing CPE on a BHK monolayer 7 and 14 days post blood feeding on an EEE-viremic chick, 1995. day 7 day 14 total
mosq saliva mosq saliva mosq saliva species positive/tot positive/tot positive/tot positive/tot positive/tot positive/tot
Ae. canad 9/9 (1.0) 0/9 (0.0) 4/5 (.80) 1/4 (.25) 13/14 (.93) 1/13 (.08)
Ae. vexans 3/8 (.38) 0/3 (0.0) - - 3/8 (.38) 0/3 (0.0)
Cq. perturb 6/26 (.23) 0/6 (0.0) 1/3 (.33) 0/1 (0.0) 7/29 (.24) 0/7 (0.0)
Cx. salinar 2/32 (.06) 0/2 (0.0) 8/36 (.22) 1/8 (.13) 10/68 (.15) 1/10 (.10)
An. quadr 6/8 (.75) 1/6 (.17) - - 6/8 (.75) 1/6 (.17)
An. punctip 6/6 (1.0) 0/6 (0.0) 3/3 (1.0) 0/3 (0.0) 9/9 (1.0) 0/9 (0.0)
Cs. melan 33/33 (1.0) 31/33 (.94) 27/27 (1.0) 25/27 (.93) 60/60 (1.0) 56/60 (.94)
24 Table 6. Mosquitoes and saliva producing CPE on a BHK monolayer 7 and 14 days post blood feeding on an EEE-viremic chick, 1994 and 1995 combined. day 7 day 14 total
mosq saliva mosq saliva mosq saliva species positive/tot positive/tot positive/tot positive/tot positive/tot positive/tot
Ae. canad 16/16(1.0) 0/16(0.0) 15/16 (.94) 4/15 (.27) 31/32 (.97) 4/31 (.13)
Ae. vexans 8/28 (.29) 0/8 (0.0) 1/5 (.20) 0/1 (0.0) 9/33 (.27) 0/9 (0.0)
Cq. perturb 16/42 (.38) 1/16 (.06) 7/11 (.64) 0/7 (0.0) 23/53 (.43) 1/23 (.04)
Cx. salinar 2/32 (.06) 0/2 (0.0) 8/36 (.22) 1/8 (.13) 10/68 (.15) 1/10 (.10)
An. quadr 15/28 (.54) 2/15 (.13) 5/21 (.24) 0/5 (0.0) 20/49 (.41) 2/20 (.10)
An. punctip 7/7 (1.0) 0/7 (0.0) 3/3 (1.0) 0/3 (0.0) 10/10(1.0) 0/10(0.0)
Cs. melan 33/33 (1.0) 31/33 (.94) 27/27 (1.0) 25/27 (.93) 60/60(1.0) 56/60 (.94)
Discussion
Vectorial capacity (V) measures the rate of parasite inoculation, or the number of new infections disseminated/case/day/vector.
V = _ma2 pn b
-lOge P Where m = vector density in relation to host (number of vectors/person)
a = man-biting rate
p = probability of daily survival
n = days of extrinsic incubation b = proportion exposed becoming infective (vector competence)
The current study examines p, n, and b in the laboratory. Vector competence
(b) was tested by applying to a BHK monolayer the saliva of mosquitoes fed on an
EEE-viremic chick, waiting several days, and recording CPE. This technique is extremely sensitive and may overestimate natural transmission rates. Nevertheless, it
shows if a mosquito can or cannot transmit virus.
25 The current study confirms previous reports of the ability of Cs. melanura to maintain a persistent infection and transmit EEE virus (Chamberlain 1958b, Scott &
Burrage 1984, Scott et al. 1984). Proof of vector competence, coupled with frequent virus isolations (Burbutis & Jobbins 1957; Chamberlain 1960; Chamberlain et al.
1951, 1958, 1969; Feemster et al. 1958; Hayes 1961; Holden et al. 1954; LeDuc et al.
1972; Wallis 1959) and predominant avian feeding (Crans 1964, Edman et al. 1972,
Irby & Apperson 1988, Magnarelli 1977, Means 1968, Nasci 1980, Nasci & Edman
1981, Schober & Collins 1966), reinforce the role of Cs. melanura as the enzootic vector of EEE virus.
Weaver et al. (1988) observed sloughing of midgut epithelium, loss of brush border membrane, and degeneration of basal lamina integrity in Cs. melanura infected with EEE virus. Pathology, mortality, and changes in fecundity due to EEE virus in epidemic vectors remain undocumented and warrant further study (Weaver et al.
1988).
Culex salinarius had a higher engorgement rate (53%) than other potential epidemic species. About 50-60% of females survived for two weeks after imbibing an infectious blood meal. Infection (6 to 22%) and transmission rates (0 to 13%) increased for Cx. salinarius. Davis (1940) found that virus levels in Cx. salinarius tended to decrease 1-7 days post blood feeding. EEE virus isolates have been detected repeatedly in this species (Burbutis & Jobbins 1957, Chamberlain et al. 1958, Muul et al. 1975, Sudia et al. 1968, Wellings et al. 1972). Blood meal identifications suggest that Cx. salinarius is more likely than other potential epidemic species to feed on birds, although it is unclear if this feeding behavior is based on host availability or innate host preference or a combination of the two (Cupp & Stokes 1973, Edman
1974, Hayes 1961b, Murphey et al. 1967, Tempelis 1974). Greater likelihood of avian feeding increases chances that Cx. salinarius will imbibe an infectious blood meal and will play a role in epidemic EEE virus transmission.
Aedes canadensis feeds mainly on mammals, but has been found to feed readily on reptiles, birds, and mammals depending on host availability (Hayes 1961b,
26 LeDuc et al. 1972, Magnarelli 1977, Means 1968). The low engorgement rate (13-
14%) may have been because the host was not attractive, host defensive behavior
(kicking and body shaking), or because the mosquitoes were in captivity. From 40-
80% of infected Ae. canadensis survived 14 days after feeding, with 27% transmitting virus after this EL This two week El is longer than those for Cs. melanura (2-3 days)
(Scott & Burrage 1984) or Ae. triseriatus (9 days) (Whitfield et al. 1971) to transmit in laboratory trials.
Only first generation (spring) Ae. canadensis were collected and tested.
Second generation (mid- to late summer) Ae. canadensis overlap horse and human
EEE cases and could play a role in epidemic transmission. Other mosquitoes have been shown to have seasonal differences in their feeding (Cupp & Stokes 1973,
Edman & Taylor 1968, Ritchie & Rowley 1981); no such change is documented for
Ae. canadensis. Shifts in host feeding, from avian reservoirs to mammals, could enhance the role of Ae. canadensis in epidemic EEE transmission.
Aedes vexans is a predominantly mammalophagic species, but its feeding pattern may be influenced by host availability (Edman 1971, Hayes 1961b, Magnarelli
1977, Means 1968, Murphey et al. 1967). Mosquito nutritional status, host defensive behavior, mosquito age, and being kept in captivity may have influenced female engorgement rate (Hardy 1988).
Probability of daily survival was low for infected Ae. vexans. Infection rates for Ae. vexans were similar from day 7 to 14, but mortality increased from 16 to 88%.
Aedes vexans might not live long enough for salivary infection with EEE virus.
Mosquitoes may have died due to low nutritional reserves or pathology from the virus.
Aedes vexans used in this study developed within 4-5 days in a temporary, grass-infused rainpool. Crowding and poor larval nutrition may enhance mosquito vector competence for arboviruses (Grimstad & Haramis 1984, Takahashi 1976).
However, Ae. vexans, often cited as the most likely epidemic vector of EEE virus
(Chamberlain et al. 1954, Srihongse et al. 1978, Sudia et al. 1968), had one of the lowest transmission rates, despite chick viral titers of 10 -10 virions/ml blood.
27 Aedes vexans readily bites humans and horses and overlaps epidemic EEE cases in Massachusetts (Getting 1941). Although other authors have implicated Ae. vexans in the transmission of EEE virus (Chamberlain et al. 1954, Davis 1940, Ten
Broeck & Merrill 1935), the current study with Massachusetts mosquitoes and a local strain of virus suggests Ae. vexans may be negligible in epidemic transmission.
Although chick viral titer decreased from 1994 to 1995 (107'6'4'3 PFUs), Cq. perturbans did not transmit virus the first year, but females the second year did. For both years combined, females increased in infection rate from day 7 to 14, but mortality increased from around 60-80%.
Coquillettidia perturbans is predominantly mammalophagic, but may feed on passerine and wading birds (Edman 1971, Hayes 1961b, Magnarelli 1977). EEE virus has been isolated repeatedly from this species (Clark et al. 1985, Crans & Schulze
1986, Edman et al. 1993, Howitt et al. 1949, Nasci et al. 1993, Ninivaggi & Guirgis
1994, Srihongse et al. 1978, Sudia et al. 1968). Coquillettidia perturbans is abundant in southeastern Massachusetts from the first to third week of July, overlapping many horse cases. Populations decrease dramatically by mid-August to late September, when most human EEE cases occur in Massachusetts (Edman et al. 1993). However, human cases the subsequent year occur in late July and early August. This may implicate Cq. perturbans, despite the 4% transmission rate, in the transmission of EEE virus to horses and early human cases.
Infection and transmission rates decreased for An. quadrimaculatus. A decrease in infection rate has been shown for Cx. salinarius (Davis 1940), but never
o for An. quadrimaculatus. Chamberlain et al. (1954) fed this species on a 10 suspension of virus, without any transmission. Two out of twenty females from
Georgia imbibing a titer of 10 ' suspension of a New Jersey EEE viral strain transmitted after 10 days (Collins et al. 1965). In the current study, females imbibed titers around 109 PFUs and transmitted at day 7 but not at day 14 of EL Nestling house sparrows {Passer domesticus) inoculated with 103 8 titer of EEE virus manifested 109 titer one day later (Scott et al. 1988). Blood titers had dropped to zero by day 4. If
28 nulliparous An. quadrimaculatus fed on an infected passerine chick, they could play a role in epidemic transmission at least one week after feeding.
Anopheles punctipennis had the lowest engorgement rate (11/150), confirming previous reports of infrequent avian blood feeding (Irby & Apperson 1988, Murphey et al. 1967, Nasci & Edman 1981, Tempelis 1974). Low engorgement also could reflect artificial conditions, such as keeping mosquitoes in a cage or using an exotic host.
Previous laboratory studies indicated that An. punctipennis was unable to transmit EEE virus (Chamberlain et al. 1954, Davis 1940, Merrill et al. 1934, Ten
Broeck & Merrill 1935, Wallis & Main 1974). The current study supports the finding that An. punctipennis is unable to transmit EEE virus, although it had a 100% infection rate. Thirty percent of fed females survived to day 14 with no change in infection rate.
Survival data for this species requires additional investigation because of the low sample size. In addition to its inability to transmit, An. punctipennis infrequently feeds on birds (Murphey et al. 1967, Nasci & Edman 1981, Tempelis 1974), precluding it from imbibing an infectious meal and acting as an epidemic vector.
CPE was not detected from homogenized males of any species, except for Ae. vexans, for which it was detected after 4 days of incubation. This late onset of CPE is consistent with bacterial or fungal contamination, but not with presence of EEE virus, which begins manifesting CPE around 3 days of incubation on a monolayer of BHK cells. Transovarial transmission of EEE virus has not been documented conclusively for either enzootic or epidemic vectors (Hayes et al. 1962, Morris & Srihongse 1978,
Muul et al. 1975, Scott & Weaver 1989). After eclosion, a female must mate, find an acceptable host, feed (and presumably imbibe virus), rest and digest the blood meal, oviposit, and feed again.
How long before a mosquito refeeds after imbibing an infectious blood meal from a bird? The bird may not be the mosquito’s first host, thereby decreasing the critical time before the mosquito can transmit virus to another host. Field parity studies of potential epidemic vectors may clarify and confirm the significance of the current vector competence tests.
29 Conclusions
• Eighty-two percent of colony Cs. melanura engorged, 100% were infected 7 and 14 days post blood feeding, and 93-94% transmitted EEE virus.
• Previous laboratory studies indicated that An. punctipennis, Ae. canadensis, and
Cx. salinarius were unable to transmit EEE virus. The current study supports the
hypothesis that An. punctipennis is unable to transmit virus, but for the first time
Ae. canadensis and Cx. salinarius transmitted EEE virus in the laboratory.
• No evidence of transovarial transmission of EEE virus was detected from field-
caught male mosquitoes of any species.
Summary
Involvement in epidemic transmission
Most likely Rationale Aedes canadensis and 13 and 10% laboratory transmission rate
Culex salinarius Opportunistic avian and mammal feeding Prevalence during human EEE cases
Good probability of daily survival
Likely Coquillettidia perturbans and 4 and 10% laboratory transmission rate
Anopheles quadrimaculatus Tend to feed preferentially on mammals
Cq. perturbans likely vector to horses
and early human cases; abundant.
13% of infected An. quadrimaculatus
transmitted at day 7 El; could transmit to humans and horses soon after imbibing
infectious blood meal from a bird
Unlikely Aedes vexans and No evidence of viral transmission
Anopheles punctipennis Almost exclusive mammal feeding
30 CHAPTER 3
SURVEILLANCE FOR EPIDEMIC VECTORS OF EEE VIRUS
Introduction
CDC light traps, either unbaited or supplemented with CO2, collect Ae. canadensis, Ae. vexans, Cq. perturbans, and Cx. salinarius (Buckley et al. 1994,
Howard et al. 1988, Schreck et al. 1972). Artificial shelters, like resting boxes, are better for collecting Cs. melanura (Edman et al. 1968) and anophelines (Morris 1981).
The current study evaluates specificity, convenience, and expense among CDC light traps, NJ light traps, resting boxes, and a new brand of light trap manufactured by the
American Biophysics® Company of Jamestown, Rhode Island. Trap catches were correlated to human biting collections to assess risk of disease transmission to humans.
New Jersey (NJ) and CDC light traps historically were used by mosquito control projects to monitor the relative densities and changing population patterns of
Culiseta melanura, the enzootic vector of EEE virus (Headlee 1932, Matsumoto &
Maxfield 1985, Sudia & Chamberlain 1962). The Massachusetts Department of
Health uses unbaited CDC light traps within red maple/white cedar swamps and adjacent lowlands to collect Cs. melanura for EEE virus isolation (D. Buckley,
Massachusetts Department of Health). Standard procedure since 1991 has been to use
CC>2-baited CDC light traps once an EEE virus isolation is made. CDC traps underrepresent mammalophagic species, which commute from woods to unforested areas to feed
Aedes canadensis, Ae. vexans, Cq. perturbans, An. quadrimaculatus. An. punctipennis, and Cx. salinarius rest in shaded woods during the daytime but commute to unforested habitats around sunset or early evening. They feed predominantly on large mammals, including humans and livestock (Edman et al. 1993, Hayes 1962,
LaSalle & Dakin 1982, Weathersbee & Meisch 1990). All but Ae. canadensis host seek and oviposit in open fields or suburban areas during the evening. Aedes
31 canadensis feed on a variety of smaller hosts (both avian and mammalian) along the forest ecotone and oviposit in woodland pools. Because mosquito control projects generally suspend light traps within Cs. melanura habitat, traps underestimate commuter species that may be involved in the transmission of virus from avian reservoirs to humans and horses.
Materials and Methods Site Selection
Bristol and Plymouth Counties in southeastern Massachusetts account for
30,000 acres of the Hockomock Swamp, a red maple-white cedar freshwater swamp in the Taunton River Basin, enzootic for EEE virus and Cs. melanura (A. Decastro,
Bristol County Mosquito Control). Four sites were selected in towns along the
Hockomock margin (Raynham, Taunton, Easton, and East Bridgewater) with histories of light trap collections of potential vector species (A. Decastro, Bristol County
Mosquito Control). Selected sites were near vernal pools, forested swamps, cattail marshes, or abandoned tires, which provided breeding habitats for some or all of the six potential epidemic vectors. Each site was also within 500 meters of people’s homes or horse farms. Forested, sandy swamps held white cedar (Chamaecyparis thyoides), swamp oak (Quercus bicolor), maples (Acer spp.), alders (Alnus spp.), willows (Salix spp.), and poison ivy {Rhus radicans).
Experimental Design Trapping methods included CDC miniature light traps (Chamberlain & Sudia
1962), NJ light traps (Headlee 1932), resting boxes (Edman et al. 1968), human biting collections (Service 1993), and American Biophysics® (ABC) light traps. ABC traps are similar to CDC traps. They can be programmed to have either a steady or flickering light source and can be supplemented with CO2 or octenol or both (B.
Wigton, American Biophysics Company).
32 Along the margin of each site, the following traps were suspended 1.5 m above the ground at sunset and emptied after sunrise: CDC light trap, NJ light trap, ABC- steady light, ABC-flicker light, ABC-flicker+C02, ABC-flicker+octenol, ABC- flicker+C02+octenol. Traps were placed 3.5 meters from one another in a randomized design; positions were changed nightly so that no trap would operate consistently at a particularly productive spot. Traps were compared for about 1-2 weeks per site, from
June to September in 1994 and 1995. Nightly temperatures and rainfall were recorded from a local weather station.
Octenol was emitted at 0.5 mg/h by octenol packets suspended on the light trap. Octenol was contained in a glass vial within a permeable membrane; crushing the vial released octenol. Carbon dioxide was delivered either as dry ice (1994) or emitted at 400-500 ml/min from a C02 tank (1995).
Human biting collections were made on one individual stationed 15 meters from the nearest light trap. Biting collections were made from sunset to two hours after sunset, from June to September in 1994 and 1995. Mosquitoes that alighted on the subject were collected with a hand-held aspirator or aerial net.
In August three resting boxes were placed in the forest, facing the forest margin. Resting boxes were emptied by 10 a.m. every morning using a backpack battery-powered aspirator (Morris 1981).
Data Analysis Mosquitoes were returned to the laboratory, killed at -20° C, and females were identified to species. Nightly means and species means were calculated for each sampling method. All methods were compared with one another using a Friedman’s
Two Way Nonparametric Analysis of Variance (a = 0.05) to determine if there was any difference among methods (NH Analytical Software 1985). If difference was detected, methods were compared pairwise using the Wilcoxon Signed Rank Test to identify which method, if any, was significantly better. If results from the Wilcoxon test found that the biting collection and another method were not significantly
33 different, they were compared using an Unweighted Least Squares Linear Regression to calculate the probability of the second method to estimate or predict the value of a nightly biting collection. If any trap experienced mechanical failure, corresponding trap data for that night were not used in statistical analysis.
Results Trap Comparisons of All Species Combined
In 1994 and 1995, a total of 9938 females were collected with nine monitoring methods during 38 trap nights (Table 7). Human biting collections had the greatest nightly mean (124.8) (Figure 1), which accounted for 44.6% of the total trap catch.
Among nine monitoring methods for female mosquitoes, at least one was significantly different from the others (p < 0.05). The CO2 light trap did not differ significantly from biting collection (p = 0.199). A linear regression analysis between biting collection and C02 resulted in a low coefficient of determination (r2 = 0.0456). This means that despite a nearly 20% probability that biting collection and C02 are the same, the low r2 indicates that C02 light trap collections cannot predict human biting risk. However, one can estimate human biting risk (y) with the following linear regression model: y = mx + b or y = (0.3582)x + 108.0
m = slope
x = nightly sample (in this case, from C02 light trap)
b = y intercept
No significant difference was shown between flicker and steady traps (p =
0.181) or between flicker and NJ traps (p = 0.100). The CDC light trap collected an average of 24.2 females per night, significantly better than either flicker or steady traps
(p = 0.003). Table 8 summarizes trap comparisons and corresponding p-values.
The addition of octenol to a flicker trap increased average yield from 6.3 to
14.6 females per night; the octenol traps were so variable, however, that this difference
34 was not significant (p = 0.065). The addition of CO2 to a flicker trap significantly increased yield from 6.3 to 73.7 females per night (p < 0.05). The C02 trap caught significantly more females per night than an octenol trap (p = 0.004). The addition of
CO2 and octenol to a flicker trap significantly increased average trap yield over octenol alone (p = 0.006) but not C02 alone (p = 0.394). The C02 and C02+octenol traps were significantly better than an unbaited CDC light trap (p = 0.004), which was significantly better than an octenol trap (p = 0.034).
Resting boxes were used only in August. Collections made from resting boxes were not significantly different from CDC (p = 0.541), CO2 (p = 0.919), or
CC>2+octenol traps (p = 0.059) operated during this same period.
Table 7. Nine monitoring methods for mosquitoes: trap total, nights in use, mean, and percent total yields.
Mosq Biting C02 C02Oct CDC Oct Flicker Resting Steady NJ Aedes1 3294 1390 945 370 362 115 1 8 12 Anophls2 115 98 127 29 17 8 63 1 0 Cs. mela 0 51 50 170 52 27 16 5 1 Cq. pert4 924 659 210 212 41 42 0 17 7 Cx. sal5 85 81 143 73 18 6 1 15 16 others6 20 4 2 17 6 10 0 0 2 total 4438 2283 1477 871 496 208 81 46 38 nights 36 31 27 36 34 33 11 13 11 mean 125 74 55 24 15 6 3 4 4 % total 45 23 15 9 5 2 1 1 0
1 Aedes canadensis, Ae. vexans, Ae. stimulans s.l.,Ae. triseriatus, Ae. abserratus, Ae. cinereus, Ae. aurifer. 2 Anopheles quadrimaculatus. An. punctipennis, An. walkeri. Culiseta melanura 4 Coquillettidia perturbans 5 Culex salinarius 6 Culex restuans, Cx. territans, Uranatoenia sapphirina, Aedes punctor.
35 140
X “3 o o z c E (D o 1 CDC + EE CM c + o CD 1 o AB+C02 <1) CD +
AB+Steady < cc < CD <
Moritonng■ ■_*i._:-- memods.i-■_
Rgure 1. Mean ni^tly catches fa nine monitaing methods fa mosqjtoes
36 Table 8. Pairwise comparisons using the Wilcoxon Signed Rank Test between monitoring methods for all species. If p > 0.05, then difference is not significant; the probability that two methods are the same is > 5%. If p < 0.05, then difference is significant.
Comparison of methods p-value
Significant difference present Trap 1 > Trap 2 CDC vs. Flicker 0.004 Flicker+C02 vs. Flicker 0.000 Flicker+C02 vs. Flicker+octenol 0.004 Flicker+CC>2+octenol vs. Flicker+octenol 0.006 Biting collection vs. Resting box 0.006 Biting collection vs. CDC 0.003 Biting collection vs. Flicker+CC>2+octenol 0.011 CDC vs. ABC+steady 0.003 Flicker+C02 vs. CDC 0.004 Flicker+CC>2+octenol vs. CDC 0.004
No significant difference Flicker vs. Steady 0.181 Flicker vs. NJ 0.100 Flicker vs. Flicker+octenol 0.065 Flicker+C02 vs. Biting collection 0.199 Flicker+CC>2 vs. Flicker+CC>2+octenol 0.394 Resting box vs. Flicker+CC>2 0.919 Resting box vs. Flicker+C02+octenol 0.059 Resting box vs. CDC 0.541
37 Trap Specificity
Monitoring methods for female Aedes species were significantly different from one another (p < 0.05) (Table 9). A nightly mean of 91.5 females were collected from human bait. There was no significant difference between biting collection and flicker+C02 (p = 0.451) or flicker+C02+octenol (p = 0.594). Linear regression analysis of biting collection, flicker+C02, and flicker+C02+octenol for female Aedes species resulted in the following estimation models:
Biting collection vs. flicker+C02 y = (0.6814)x + 39.0 r2 = 0.3098
Biting collection vs. flicker+C02+octenol y = (0.4121)x + 17.1 r2 = 0.2473
Biting collections were significantly better than CDC (p = 0.01), flicker+octenol (p = 0.001), and flicker (p < 0.05). The addition of octenol to an ABC flicker light trap did not significantly increase average yield (p = 0.008). A combination of C02 and octenol significantly increased yield (p = 0.002) over octenol alone but had no significant effect (p = 0.677) over C02 alone.
Monitoring methods for female Cq. perturbans were significantly different from one another (p < 0.05) (Table 10). A nightly mean of 25.7 female Cq. perturbans were collected from human bait. Biting collections correlated best with flicker+C02
(p = 0.217). Linear regression analysis of Cq. perturbans biting collection against flicker+C02 resulted in the following estimation model:
y = (0.9408)x + 7.2 r2 = 0.4864
Biting collections were significantly higher than CDC (p = 0.025), flicker+C02+octenol (p = 0.029), flicker (p = 0.012), and flicker+octenol (p < 0.05).
The addition of octenol to an ABC flicker light trap did not significantly affect trap catch (p = 0.422). The addition of C02 to an ABC flicker light trap significantly increased trap catch (p < 0.05). A combination of C02 and octenol significantly increased ABC trap catch over octenol alone (p = 0.019) but had no significant effect over C02 alone (p = 0.278).
38 Flicker+C02+octenol traps collected an average of 5.3 female Cx. salinarius per night, the highest for nine monitoring methods (p = 0.006) (Table 11). Biting collections were not significantly different from CDC (p = 0.215), C02 (p = 0.246), or
C02+octenol (p = 0.256). Biting collection was significantly different from NJ (p =
0.020). Linear regression analysis between Cx. salinarius biting collection and CDC,
C02, and C02+octenol resulted in these estimation models:
Biting collection vs. flicker+C02+octenol y = (0.2805)x + 2.7 r2 = 0.1731 Biting collection vs. CDC y = (0.0507)x + 4.1 r2 = 0.0014
Biting collection vs. flicker+C02 y = (-0.0296)x + 5.9 r2 = 0.0007
The addition of C02 (p = 0.016) and C02+octenol (p = 0.001) significantly increased trap catch over ABC light trap alone. Octenol by itself did not significantly increase trap catches by ABC flicker light traps (p = 0.406). Traps baited with only
C02 collected significantly greater numbers than only octenol (p = 0.004). A combination of octenol and C02 did not significantly increase yield over C02 alone (p
= 0.007) but did significantly increase yield over octenol alone (p = 0.004).
An average of 5.7 female Anopheles were collected nightly from resting boxes, the highest average out of eight monitoring methods (Table 12). An average of 4.7 females were collected from C02+octenol, but resting boxes were significantly better
(p = 0.008). A linear regression model was fitted for biting collection against
Co2+octenol, resting boxes, and C02, resulting in these estimation models:
Biting collection vs. resting box y = (0.802)x - 3.8 r2 = 0.8264
Biting collection vs. flicker+C02+octenol y = (0.41 l)x + 0.9 r2 = 0.6417
Biting collection vs. flicker+C02 y = (0.554)x + 1.1 r2 = 0.6018
Resting boxes were significantly better than biting collection (p = 0.004), CDC traps (p = 0.004), and octenol (p = 0.004). The addition of octenol, C02, and
C02+octenol to ABC light traps did not significantly increase collections by flicker traps (all p < 0.05). Trap catch from flicker+C02 was not significantly different from
39 flicker+octenol (p = 0.004). A combination of CO2 and octenol significantly increased trap catch over octenol alone (p = 0.156), but had no significant effect over
CO2 alone (p = 0.094).
Monitoring methods for female Cs. melanura were significantly different from one another (p < 0.05). CDC traps collected an average of 4.7 female Cs. melanura per night (Table 13). CDC traps were significantly better than flicker+CC>2+octenol (p
= 0.015), flicker+C02 (p < 0.05), flicker+octenol (p = 0.001), resting boxes (p =
0.055), and flicker (p < 0.05). No female Cs. melanura were collected from human bait; therefore, no regression analysis was performed for biting risk for this species.
40 Table 9. Nightly means for nine monitoring methods for female Aedes. Species include Aedes canadensis, Ae. vexans, Ae. stimulans s. l.,Ae. triseriatus, Ae. cinereus, Ae. aurifer, Ae. abserratus, and Ae. punctor. Mean values are in descending order. Method Biting co2 C020ct Oct CDC Flicker NJ Steady Rest | Mean 91.5 44.8 35.0 10.7 10.3 3.5 1.1 0.6 0.1
Table 10. Nightly means for eight monitoring methods for female Coquillettidia perturbans. Mean values are in descending order. Method Biting co2 COiOct CDC Flicker Steady Octenol NJ j Mean 25.7 21.3 7.8 5.9 1.3 1.3 1.2 0.6
Table 11. Nightly means for nine monitoring methods for female Culex salinarius. Mean values are in descending order._ Method Octenol o C02Oct n Biting CDC NJ Steady Flicker Resting
Mean 5.3 2.6 2.4 2.0 1.5 1.2 0.5 0.2 0.1
Table 12. Nightly means for eight monitoring methods for female Anopheles quadrimaculatus. An. punctipennis, and An. walked. Mean values are in descending order. Method Resting C02Oct C02 Biting CDC Octenol Flicker Steady
Mean 5.7 4.7 3.2 3.2 0.8 0.5 0.2 0.1 |
Table 13. Nightly means for eight monitoring methods for female Culiseta melanura. Mean values are in descending order. Method CDC C020ct CO; Octenol Resting Flicker Steady NJ
Mean 4.7 1.9 1.7 1.5 1.5 0.8 0.4 0.1
41 Discussion
The CDC light trap collected significantly more females than the ABC trap set for either flicker or steady mode, and was the best trap for Cs. melanura (Table 13)
Mosquito control projects sampling specifically for Cs. melanura for EEE surveillance will not benefit from using ABC light traps, either alone or supplemented with chemical attractants.
ABC light traps with a steady light source were found to be comparable to those with a flickering light source and were not used the second summer (Table 8).
Because flicker and steady were similar, flicker is preferable for mosquito surveillance because it uses less battery power. Flicker also collected greater numbers than NJ or resting boxes. The latter are poor indicators of Aedes abundance but excellent indicators of Anopheles. Size, convenience, and increased trap yield recommend ABC flicker over NJ traps for extended surveillance.
ABC light traps supplemented with CO2 collected an average of 73.7 female mosquitoes per night (Table A). This overall mean was the highest for eight traps and closest to actual human biting numbers. The addition of CO2 significantly increased trap catch over ABC flicker alone for Aedes spp., Cq. perturbans, and Cx. salinarius, but not for Cs. melanura or anophelines. Previous authors have reported the addition of CO2 increased trap catches of Aedes spp., Cq. perturbans, Cx. salinarius, and An. quadrimaculatus (Buckley et al. 1994, Carestia & Savage 1967, Magnarelli 1974,
Newhouse et al. 1966). The addition of CO2 to ABC flicker did increase collections of Cs. melanura, but numbers were still significantly lower than CDC (without CO2).
More anophelines were attracted to flicker+C02 over flicker alone or CDC.
A 5 lb. CO2 tank lasts 40 hours @ 400 ml/min emission and costs $3.00/ tank/month for rental and $5.75/gas (Merriam-Graves Co., Springfield, Massachusetts). This size tank is light enough to carry into the field along with a trap and 6-V gel cell battery. Even though the addition of CO2 did not increase significantly the anopheline catch, it did increase catches of other target mosquitoes.
Specificity, low expense, and ease of use suggest that flicker+CC>2 traps are superior
42 for the full range of potential epidemic vectors of EEE. The current study did not evaluate CDC light traps supplemented with chemical attractants. Subsequent comparisons should be performed between CDC light traps supplemented with C02 and ABC flicker light traps supplemented with C02.
The addition of octenol to an ABC flicker light trap increased average yield from 6.3 to 14.6 females per night (p = 0.065). Despite the high p-value, the increasing trend suggests that octenol still could play a role in mosquito surveillance, especially for mosquitoes with a high affinity for large mammals. Repeated pairwise comparisons also may result in a large standard error. If sample size was low, then calculations may suggest no significant difference between two traps, even if some difference exists. In comparisons with few data points, such as between octenol and flicker, apparent trends may be more important than p-values in evaluating trap efficacy.
Previous authors have noticed an increase in Cq. perturbans, Cx. salinarius, and Aedes spp. with an emission of 3.0 mg/h of octenol to CDC light traps (Kline et al.
1990b, 1991b). The current study used an octenol emission of 0.5 mg/h added to ABC flicker light traps, which may have been inadequate to increase trap catch significantly.
In addition, previous studies were conducted in the Florida Everglades or Arkansas ricefields, where different mosquito species were being sampled. An ABC slow- release packet of octenol costs $4.75 (10 packets for $42.00) and lasts about 2 months at 21° C (70° F). Octenol packets may be inexpensive enough to supplement light traps as mosquito populations increase through the summer. Dry summers in 1994 and 1995 were probably responsible for record low numbers of all recorded species
(Massachusetts Department of Health).
A combination of C02+octenol has been shown to increase Cq. perturbans,
Cx. salinarius, and Aedes spp. (Kline et al. 1990b, Kline et al. 1991b). This combination has yielded ambiguous results with An. quadrimaculatus (Kline et al.
1990a, 1990b; Kline et al. 1991a). In the current study only collections of Cx. salinarius increased with this combination, but even this was not significantly
43 different from CO2 alone. The combination of C02+octenol also was not significantly different from CO2 alone for Anopheles spp., Aedes spp., Cq. perturbans, or Cs. melanura. However, this combination was significantly better over octenol alone for all but Cs. melanura, an omithophagic species with minimal attraction to a volatile derived from cattle.
Mosquito control districts may not experience an appreciable increase in trap catch using CC>2+octenol, If expense and specificity demand either CO2, octenol, or
CC>2+octenol, then CO2 alone provides the best return for the investment and increases collections of Cs. melanura and human-biting mosquitoes.
Resting boxes were best for sampling anophelines, but resting boxes tended to collect engorged or parous females and may give an inaccurate profile of host seeking and human biting risk. Correlating resting box collections of anophelines to human biting risk may be impractical because many mosquitoes sampled by resting boxes have already fed. Mark-recapture studies with nulliparous anophelines collected from resting boxes or C02+octenol light traps may better reflect human biting risk.
Resting boxes placed within hardwood forests have been shown to be effective in collecting blood fed Cs. melanura (Edman et al. 1968). Culiseta melanura rest and feed in wooded swamps and adjacent hardwood forests. In the current study resting boxes were placed along the forest margin, facing out. The position of the traps was unfavorable for resting females.
Previous authors found that CDC traps with CO2 accurately reflected biting risk (Acuff 1976, Slaff et al. 1983). New Jersey traps can underestimate (Slaff et al.
1983) and overestimate (Acuff 1976) biting risk, depending on the species. The current study found NJ traps of little value in predicting human biting risk. Additions of CO2 to ABC light traps better reflected biting risk from Aedes spp. and Cq. perturbans. Additions of C02+octenol better reflected biting risk from Cx. salinarius and anophelines. Biting collections sampled the greatest mean number of host-seeking females, but biting collections are time-intensive, inconvenient, and may pose a health risk to the collector.
44 The linear regression model y = (0.3582)x + 108.0 for predicting overall biting risk from C02 traps is impractical because the low r2 value (0.0456) means that only
4.56% of biting risk is predicted. Individual regression models of Aedes (r2 = 0.3098) and Cq. perturbans (r = 0.4864) collected from C02 traps may estimate human biting risk better than the overall model. Regression analyses of Cx. salinarius (r2 = 0.1731) and anophelines (r2 = 0.6414) collected from C02+octenol traps are better than C02 alone for estimating human biting risk from these species. These equations are most effective when populations are sampled during peak activity; otherwise, they underestimate human biting risk by emphasizing local vagaries in population density instead of overall distribution.
Conclusions
1. CDC light traps are effective for sampling female Culiseta melanura. Changing to an ABC light trap, either by itself or with chemical supplements, will not increase collection efficiency.
2. ABC light traps supplemented with C02 alone collected high numbers of Aedes spp. and Coquillettidia perturbans. These traps best reflected human biting risk. The increase in trap yield and ease of use suggest ABC traps with C02 along margins of wooded swamps would be the most effective at collecting potential epidemic vectors of EEE virus.
3. Either C02 or C02+octenol could be added to light traps to increase collections of
Culex salinarius and Anopheles spp. Budgets may dictate which attractant to use.
4. The addition of octenol alone tended to increase collections with ABC light trap, but this increase was not significant. Supplementing traps with octenol during periods of high activity may increase trap catches of some mammalophagic species. Adding
45 an octenol packet to an already placed light trap requires little effort; octenol use would be determined by expense.
5. Resting boxes were most effective at sampling Anopheles spp., but females were often engorged or parous. Regression analysis to estimate biting risk based on resting box collections may overestimate actual risk.
6. ABC light traps are lighter than CDC or NJ light traps. They also are easier to assemble and dismantle, and have convenient attachments for octenol and CO2 (either as dry ice or from a gas tank).
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