Recycling treated sewage effluents through cypress swamps

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Title:
Recycling treated sewage effluents through cypress swamps its effects on mosquito populations and arbo-virus implications
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Cypress swamps
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vi, 164 leaves : ill. ; 28 cm.
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Davis, Harry Goodwin, 1947-
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Subjects / Keywords:
Mosquitoes -- Florida   ( lcsh )
Sewage lagoons -- Florida   ( lcsh )
Arbovirus infections -- Florida   ( lcsh )
Swamp ecology -- Florida   ( lcsh )
Arbovirus infections   ( fast )
Mosquitoes   ( fast )
Sewage lagoons   ( fast )
Swamp ecology   ( fast )
Florida   ( fast )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 158-163).
Additional Physical Form:
Also available online.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Harry Goodwin Davis.

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RECYCLING TREATED SEWAGE EFFLUENTS
THROUGH CYPRESS SWAMPS: ITS EFFECTS
ON MOSQUITO POPULATIONS AND ARBO-VIRUS IMPLICATIONS







By

HARRY GOODWIN DAVIS














A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY












UNIVERSITY OF FLORIDA 1978













C4Z
ACKNOWLEDGMENTS
00
L- Several people have helped enormously in the preparation of this

dissertation. Dr. Lewis Berner and Dr. Dave Dame, the Co-Chairmen of

my graduate committee, have been especially generous with their time

and help. My supervisors at the Center for Wetlands, Dr. Howard Odum,

Dr. Kathy Ewel, James Ordway, and Margaret Johnston have been understanding and patient throughout the study.

Dr. Jack Gaskin, of the College of Veterinary Medicine, has

given me advise and support on several occasions. Without help from

Dr. Flora Mae Wellings, Dr. Arthur Lewis, Dr. Sam Mountain, and

Hardrick Gay of the Florida Department of Health Education and Welfare

none of the virus studies would have been possible. Dr. Wellings

allowed me to work at the DHEW lab in Tampa. Mrs. Anna Chang, also

at the Tampa lab, taught me the serological techniques. Working with Mrs. Chang was an enjoyable experience. She is an excellent teacher and a very fine lady. Drs. Gary and Janet Stein, of the biochemistry

department, often let me use their equipment in the preparation of

my serological samples. They also kept me well fed by frequently

inviting me to their excellent parties.

My older brother Lloyd and his wife Sally were always helpful,

and my parents were a constant source of encouragement. My wife

Jeudi, unselfishly worked to support us during my research. Jeudi

also did most of the figures in the dissertation.
ii















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS . . . . . . . . . . . . ii

ABSTRACT . . . . . . . . . . . . . v

INTRODUCTION . . . . . . . . . . . . 1
General Statement .................. . 1
Cypress Domes . . . . . . . . . . . 5
Mosquitoes . . . . . . . . . . . 6
Mosquito Sampling . . . . . . . . . . 10
Mosquito-Borne, Human Diseases . . . . . . . 13

MATERIALS AND METHODS .................. . 17
Site Selection . . . . . . . . . . . 17
Adult Mosquito Sampling .................. 31
Ramp-Traps . . . . . . . . . . . 31
New Jersey Light Traps ................. 37
CDC Light Traps . . . . . . . . . . 37
D-Vac and "Homemade" Suction Device ........... 37
Malaise Trap . . . . . . . . . . . 42
Truck Trap .................. . . 42
Larval Mosquito Sampling .................. 48
Virus Activity . . . . . . . . . . . 55
Sentinel Chickens . . . . . . . . . . 55
Mosquito Pools . . . . . . . . . . 60
Mammal Sampling . . . . . . . . . . . 60
Serology: Hemagglutination Inhibition Test . . . . 63 Serology: Neutralization Test . . . . . . . 64

RESULTS AND DISCUSSION . . . . . . . . . . 67
Adult Mosquito Sampling .................. 67
Ramp-Traps .................. . 67
New Jersey Light Traps ................. 82
CDC Light Traps . . . . . . . . . . . 97
Suction Devices . . . . . . . . . . ...108
Malaise Traps . . . . . . . . . . . 126
Truck Traps . . . . . . . . . . . . 131
Larval Sampling . . . . . . . . . . ...134
Virus Activity . . . . . . . . . . . ... 142
Sentinel Chickens .................. .142
Isolation Attempts from Mosquito Pools . . . . ....152
Serology and Isolation Attempts from Mammal Samples . .152
Summary . . . . ........ ...... . .154


iii










Page

CONCLUSIONS . . . . . . . . . . . . ... 156

RECOMMENDATIONS FOR FUTURE WORK ................ 157

LITERATURE CITED . . . . . . . . . . . . 158

BIOGRAPHICAL SKETCH . . . . . . . . . . . 164










































iv










Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy


RECYCLING TREATED SEWAGE EFFLUENTS
THROUGH CYPRESS SWAMPS: ITS EFFECTS
ON MOSQUITOO POPULATIONS AND ARBO-VIRUS IMPLICATIONS By

Harry Goodwin Davis

June, 1978

Chairman: Dr. Lewis Berner
Co-Chairman: Dr. David Dame
Major Department: Entomology and Nematology

The task of determining the feasibility of using cypress domes as natural filters in purifying and recycling water from secondarily treated sewage effluents was undertaken by the Center for Wetlands at the University of Florida with major funding from the Rockefeller and National Science Foundations.

Certain aspects of the proposed project were of great concern to public health officials. Many thought that the enrichment of stagnant, swamp water would result in drastically increased mosquito populations. To answer this concern, several methods were used to sample mosquitoes at both experimental and control swamps. Also, mosquito-borne encephalitis activity was monitored at these same sites.

Three years of sampling failed to demonstrate any measurable increase in human pest species at the experimental domes. The floodwater species of Aedes atlanticus and Aedes infirmatus declined at these sites. Serological studies, using sentinel birds, indicated that eastern and western equine encephalitis viruses are common in both the experimental and control swamps.



V









This study showed that cypress domes in north central Florida

could be used to recycle treated effluents, without causing significant increases in mosquito populations; however, because of the endemic nature of the viral encephalitis in these habitats, any swamp which is ecologically disturbed should be monitored for changes in mosquito and virus activity.








































vi














INTRODUCTION


General Statement


The state of Florida is presently experiencing rapid growth and development resulting in much of its forested area and natural wetlands being destroyed to accomodate this human expansion. Great expenditures of fossil fuel energy are required to restructure and maintain the environment in an altered state. People are finally beginning to realize that the simplification of complex natural systems often results in unpredictable calamity, and that it is much wiser to conserve natural systems and allow them to function for the benefit of man.

As a means of dealing with some of the problems created by this

rapid growth, the Center for Wetlands was established at the University of Florida in 1973 to study the feasibility of using cypress wetlands in water management, sewage treatment, and conservation. This dissertation is part of the report on the multiphasic study of the use of cypress wetlands in sewage treatment and wastewater reclamation. More specifically, the topic of this discussion is related to some of the public health uncertainties arising from the use of cypress domes in the tertiary treatment of sewage effluents, i.e. the effects of secondarily treated sewage effluents on mosquito populations and mosquito-borne virus activity in north Florida cypress domes. A cypress dome is defined as a stand of cypress, usually pond cypress,


1







2



growing in a wet depression in a flatwoods area. From a distance the stand is dome-shaped with the taller trees in the middle and the smaller, shorter trees at the margins.

The overall project has been supported with funds from the RANN Division of the National Science Foundation and The Rockefeller Foundation. Beginning in 1974, annual progress reports have been published by the Center for Wetlands (Odum et al., 1974, 1975, 1976). By summarizing data collected from all phases of the project, these annual reports have helped in coordinating the overall investigative effort.

Two cypress domes were selected to receive secondarily treated

sewage effluent from a small package treatment plant (capacity: 30,000 gallons per day) operated by the Alachua County Utilities Department. These two experimental domes and several control domes (Fig. 1-1) were monitored simultaneously. The hypothesis being tested was that the vegetation of the cypress domes would remove the dissolved nutrients from the effluent (stimulating the growth of cypress trees which could be periodically harvested) and that in recycling the water to the groundwater aquifer, slow percolation through the geological strata below the domes would filter out potentially pathogenic microbes. It was hoped that such a purification system would be economically competitive with the more standard methods of tertiary treatment.

Of public health concern was the possibility that the altered hydroperiod and increased nutrient loads of the experimental domes would cause a significant increase in mosquito production. There are several species of mosquitoes which develop in great numbers in















Figure 1-1. One of the experimental cypress domes (S-1) receiving
sewage effluent.

























A control cypress dome (C-i) receiving untreated
well water.







4






5



stagnant, polluted water, and some of these species can serve as vectors in the transmission of human disease. For these reasons, mosquito populations were monitored in the domes, and during the last year, the project was expanded to include a study of the mosquito-borne viruses which cause human encephalitis.


Cypress Domes


The pine-palmetto flatwoods of the southern Atlantic and Gulf

Coastal plain are interspersed with thousands of small cypress domes. These dome-like stands are primarily composed of Taxodium distichum var. nutans, Nyssa biflora, Pinus elliottii, Acer rubrum, and Myrica cerifera (Monk and Brown, 1965).

Kurz (1933) has shown that the larger cypress trees in the central portion of a dome are older than the smaller trees at the margins. The germination and early survival of the cypress seedlings is dependent upon a period of dry-down (Demaree, 1932). In an undisturbed dome, the central pond often holds standing water year-round, and thus the only suitable place for cypress regeneration is at the margins.

Understory vegetation within the domes is usually clumped around the expanded bases of the cypress trees or protrudes from dead stumps. The Virginia chainfern (Woodwardia virginica) and fetterbush (Lyonia lucida) are common understory species and are especially abundant at the margins of domes which have been partially drained.

Cypress domes exist at surface depressions which collect rainwater, surface runoff, and groundwater recharge. The standing water of a typical dome is low in dissolved nutrients, as indicated by the frequent







6



presence of Utricularia species. These are submerged, carnivorous plants (Fig. 1-2) which are adapted to aquatic situations containing little dissolved nitrogen and phosphorus. Wharton et al. (1976) have stated that naturally occurring phosphorus in cypress domes is unavailable to plants as it is bound in the clay layers below the standing water as a result of low pH.

Present trends in land use have been to drain wetlands areas in an attempt to replace the cypress with faster growing slash pine. The lowering of water tables by these methods has resulted in an ecological backlash--the occurrence and severity of forest fires has increased.

Wharton et al. (1976) have described the different types of

forested wetlands and have summarized their ecological values. Ewel and Odum (1978) have summarized some of the results which indicate that cypress domes could be used in the tertiary treatment of sewage effluents.


Mosquitoes


Among the hundreds of kinds of mosquitoes, some
representative is capable of living in every conceivable collection of water. Some may live at altitudes of over 400 m while others may live in mines 1000 m or more below the earth's surface. Species range in latitudes northward from the tropics well into the Artic regions and southward to the ends of the continents. A wingless species has been
reported from Antartica. No natural collection of water,
whether fresh, saline, or foul, occurs but that part of it
may be occupied by some mosquito. No forest is so dense, nor area so barren, but that some mosquito may live there.
One may be annoyed by them in the heart of a metropolitan
district or on the most isolated island; he may be attacked on land or on ships at sea, and he may be disturbed at home
or in camp. All in all, this is a remarkably adaptable
group of insects. Horsefall, 1972, p. 7.



























Fig. 1-2 Flowering Utricularia in March at the control swamp
receiving untreated, low nutrient, well water.
Commonly known as bladderwort, this floating aquatic
plant is an indicator of low nutrient situations.













j :ol, f : Av.






















-Z4







9



Because of their involvement in the transmission of human diseases, mosquitoes have received enormous amounts of study. Publications are available which describe the fauna of any geographic region in the United States. The mosquitoes of Florida are covered in detail by King et al. (1960).

Detailed, ecological investigations of the mosquitoes inhabiting cypress swamps have been made in Maryland (Williams, Watts, and Reed, 1971; Saugstad, Dalrymple, and Eldridge, 1972; Joseph and Bickley, 1969; Le Duc et al., 1972; Muul, Johnson, and Harrison, 1975). Similar studies were done in Louisiana cypress swamps (Kissling et al., 1955).

By adding treated effluents to cypress domes, not only are the seasonal fluctuations in water level changed, but also the water chemistry is greatly altered. These complex alterations in chemistry undoubtedly affect changes in the aquatic fauna and flora. It is possible that these ecological disturbances acting independently or jointly, and perhaps synergistically, can result in subtle changes or in drastic differences in mosquito fauna in terms of abundance and diversity.

By the selection of specific oviposition sites the adult females determine where future generations will develop. In some cases it is easy to associate a certain habitat with a particular species. For example, larvae of Culiseta melanura are usually found in freshwater swamps (cedar swamps in the north and cypress swamps in the south). Larvae'of Culex pipiens quinquefasciatus are commonly associated with highly polluted water sources. They develop in great numbers in sewage lagoons. Larvae of Aedes triseriatus develop in water holding, rot







10



holes in trees. Some species of Culex, Uranotaenia, Culiseta, Mansonia, Anopheles, and Coquillettidia deposit their eggs on the surface of relatively permanent bodies of water. The so-called floodwater species of Aedes and Psorophora deposit their eggs on moist surfaces which are subject to periodic inundation. Clements, (1963) points out that various investigations have illustrated a wide range of physical and chemical factors used by different species in discerning oviposition sites; however, in no species has a simple chemical factor been found which typifies the breeding site. In most species it appears that a suitable site is discerned by a combination of particular physical factors and water of appropriate purity or pollution.


Mosquito Sampling


Knight (1964) and Service (1976) have described theoretical methods for calculating the absolute population density of mosquito larvae from certain habitats. These methods, however, are of limited usefulness in a quantitative examination of the entire mosquito fauna from a habitat as heterogeneous as a Florida cypress dome. Larvae are not evenly distributed throughout the habitat, and, because of differences in avoidance behavior, various species are not always captured in numbers proportional to their actual abundance.

Dipping for larvae with a long handled, pint dipper has been the most commonly used method to determine breeding sites and qualitative information on community structure; however, as Wilson and Msangi (1955) have demonstrated, there are frequent inaccuracies associated with trying to extract quantitative information from these kinds of data.











Standard types of adult sampling techniques are no more accurate

than larval surveys and often are more difficult to interpret. Trapping

results vary a great deal from night to night and from one location to

another. Bidlingmayer has discussed this variability and he lists

three major causes for it:

A). Environmental. These factors may be divided somewhat
artificially into positional and meteorological. The former would include distance from the breeding area,
habitat, competing attractants, food sources, reflecting
surfaces, control activities, and predators.
Meteorological factors would include effects of
temperature, humidity, wind, and light intensities.

B). Biological. This category includes inherent behavior
patterns such as the characteristic responses of different species and sexes and the behavior changes during
the life of an individual mosquito according to its
physiological state. The expression of these behavior
patterns is often modified or suppressed by environmental
factors. Actual changes in population size because of
seasonal trends or previous weather patterns may be
included here.

C). Operational. Variation may result from differences
between apparently identical equipment and techniques,
or, at times operational effectiveness of the method
may change. Bidlingmayer, 1967, pp. 200-201.

To insure the accuracy of any mosquito sampling survey Huffaker

and Back (1943) noted that the results from several trapping techniques

should be analyzed.

Adult trapping techniques can be categorized as either attractant

or nonattractant. The nonattractant methods such as sweeping adults

by suction from resting sites or flight intercepting devices supposedly

demonstrate more accurately the true population composition than do the

attractant methods such as light traps or baited traps. The attractant

methods usually yield large samples with a minimum of actual field






12



work; whereas, the more unbiased, nonattractant methods require more time and effort for smaller samples.

The best known and most widely used of the attractant-type

devices is the New Jersey mosquito trap. The development of this trap was described by Headlee (1932) and Mulhern (1934). The function of the trap is based on the knowledge that some mosquitoes are attracted or perhaps disoriented by certain light intensities (Barr, Smith, and Boreham, 1960; Robinson, 1952). A trap of similar design but smaller and more portable was described by Nelson and Chamberlain (1955). This trap was later improved (Sudia and Chamberlain, 1962) and has become known as the CDC1 miniaturelight trap. Both these traps have been extremely popular with mosquito control workers in monitoring fluctuations in population levels.

Nonattractant sampling devices can be used to collect either resting adults or to intercept adults in flight. A suction device such as the commercially available D-Vac2 can be used to collect adults from resting sites. Samples collected with such a device will contain a higher proportion of males and blood-engorged females than samples from attractant-type traps. The nonattractant, flight interception devices can be either stationary or mobile. Malaise traps of various designs (Townes, 1962; Gressitt and Gressitt, 1962; Breeland and Pickard, 1965; Roberts, 1972) and the ramp-trap (Gillies, 1969) are examples of the stationary type. The truck trap described by Bidlingmayer (1966) is a good example of a mobile interception device.

1CDC after the Center for Disease Control in Atlanta, Georgia.

2D-Vac Company, Riverside, California.






13



Mosquito-Borne, Human Diseases


The history and development of Florida have been influenced a great deal by mosquitoes and the diseases they transmit. Epidemics of yellow fever and malaria were common in the 19th Century. In 1901, it was discovered that yellow fever was transmitted from human to human by the mosquito Aedes aegypti, and by 1905 Florida was waging a successful battle against yellow fever. Dengue fever was last noted in epidemic proportions in 1934, and malaria disappeared from Florida after 1948 (Schoonover, 1970).

At the present time in Florida, the only real, mosquito-borne threat to human health is from a family of neurotropic viruses known as togaviruses. Several of these viruses are capable of causing human encephalitis. The togaviruses are classified in groups A and B on the basis of hemagglutination-inhibition reactions. Members within a group will cross react but there is little cross reactivity between groups. Members of the Bunyamwere supergroup are classified according to cross reactivity by complement fixation. Included in group A are eastern equine encephalitis (EEE), western equine encephalitis (WEE), and Venequelan equine encephalitis (VEE). Group B includes St. Louis encephalitis (SLE), the Bunyamwere supergroup includes the California encephalitis (CEV) group of viruses. Specific viral determinations within groups are done by the neutralization test. Strains of all the previous groups of togaviruses have been recorded from Florida.

Venequelan equine encephalitis occurs in epidemic and endemic forms (Gibbs, 1976). Only the endemic form, which apparently causes subclinical infections in man and horses, has been found in Florida;






14



and it seems to be restricted to south Florida (Bigler, 1969). The natural endemic cycle is maintained through transmission by Culex (Melanoconion) species among wild rodent reservoirs (Chamberlain et al., 1964; Bigler, 1969; and Bigler and Hoff, 1975).
Western equine encephalitis also occurs as different antigenic strains (Theiler and Downs, 1973). The strain from areas primarily west of the ississippi River causes clinical illness and fatality in man and horses (McGowan, Bryan, and Gregg, 1973). The strain found east of the Mississippi, along the Atlantic Coast and in Florida appears to be nonpathogenic for man or horses, although at least one equine fatality has been reported from Florida (Jennings, Allen, and Lewis, 1966). The pathogenic strain seems to be restricted by the range of its primary vector, Culex tarsalis (Stark, 1967). Culex tarsalis is primarily an avian blood feeder, with seasonal shifts to a preference for mammals (Tempelis et al., 1965 and 1967, and Tempelis and Washino, 1967). This pattern of behavior is undoubtedly important in respect to epidemiology. Along the Atlantic Coast the natural cycle of the nonpathogenic strain is apparently maintained in a bird to bird transmission by Culiseta melanura (Stark, 1967; Dalrymple et al., 1972; Stamm, 1966).

The range of eastern equine encephalitis activity overlaps the
distribution of its enzootic vector, Culiseta melanura. The severity of this disease for both humans and equines is very great, with fatality rates as high as 50% in humans (McGowan, Bryan, and Gregg, 1973). A high percentage of those who are clinically ill and recover are left with permanent neurological damage. Fortunately this disease is very







15



rare in humans; from 1955 through 1976 only 135 human cases were reported to the Center for Disease Control (CDC) in Atlanta (McGowan, Bryan, and Gregg, 1973 and CDC miscellaneous publication, 1977). There are few human cases because Culiseta melanura breeds primarily in fresh water swamps and feeds almost exclusively on birds. Other species such as Aedes sollicitans, the salt marsh mosquito, are suspected of transmitting sylvan EEE to humans and horses in eastern and gulf coastal areas (Stark, 1967). Bigler et al. (1976) has summarized the endemic nature of EEE in Florida.

St. Louis encephalitis is the most widespread of the North

American encephalitides. Theiler and Downs (1973) list the chief vectors as Culex tarsalis in rural areas and Culex pipiens quniquefasciatus in urban areas. Dow et al. (1964) isolated SLE virus from several pools of Culex nigripalpus during the 1962 epidemic in the Tampa Bay area of Florida. Of the 2,349 human cases reported to CDC from 1955 through 1971, 178 ended in fatality (McGowan, Bryan, and Gregg, 1973). The biological mechanism for the maintenance of SLE virus in nature is not well understood.

The California encephalitis virus complex consists of at least twelve related strains. Three of these are known to produce clinical infection in humans (California, LaCrosse, and Tahyna) (Henderson and Coleman, 1971). From Florida, two nonpathogenic strains, keystone and trivittatus, have been isolated from pools of primarily Aedes atlanticus and Aedes infirmatus (Taylor et al., 1971 and Wellings, Lewis,and Pierce, 1972). Small mammals are apparently the wild reservoirs of these viruses. Taylor et al. (1971) have implicated







16



cotton rats and Jennings et al. (1968) and Taylor et al. (1971) have implicated rabbits as the natural hosts.














MATERIALS AND METHODS


Site Selection


In 1973, sites were selected to receive sewage effluent. The two cypress -domes chosen are located in a pine plantation, owned by Owens-Illinois, Inc., two miles northwest of Gainesville, Florida, just east of U.S. Highway 441 (Fig. 2-1). Secondarily treated effluent was supplied (as detailed below) to the experimental swamps from a county-operated treatment plant serving the residents of Whitney Mobile Home Park (WMHP).

In December 1973, before sewage was added to any of the domes,

a forest fire swept through much of the pine plantation. The fire killed the young pines, cleared the understory vegetation in the experimental cypress domes, and killed some of the older trees in these domes.

Despite the fire damage the project was continued, and in April 1974, effluent was added to the first dome, S-1. At the same time, groundwater from a deep well was added to a control dome, C-1. In December 1974, effluent was added for the first time to S-2, a second experimental dome. Another control dome in the same area, C-2, had been extensively drained in the past (Fig. 2-2), and much of the year it was completely dry. The fluctuating water level at C-2 represented a situation similar to that at S-1, S-2, and C-1 previous to the addition of effluent or groundwater. All these domes are represented in Figure 2-3.

17
























Figure 2-1. Location of the monitored cypress domes
with respect to the city of Gainesville,
Florida. Also, the approximate locations
of the sentinel chicken pens which were
placed in and around Gainesville.






19















0123 (Miles) (5,-1 S-2
C-1, C-2








0 Gainesville









, Sentinel Chicken Sites



























Figure 2-2. Control dome C-2. Located in an area which has
been extensively drained, this dome is dry
much of the year. The material in the foreground is accumulated cypress litter.











21



































































414;"; ~~~ i lb 's~~





:77;





22


















C-2
S-2 l -0 500 1000 /
(Feet)




~~f ---------oe,1 Sevwage
Effluernt Pipeline

.

7 Sewage
Treatment Plant Fig. 2-3

The Whitney Mobile Home Park site, demonstrating the location and relative size of the two experimental domes and two of the control domes.







23



Also, a large, undisturbed cypress dome (Fig. 2-4) on the University of Florida's property in the Austin Cary Forest was studied as a control. This dome is located approximately three miles northeast of Gainesville, off State Road 24 (Fig. 2-1).

Table 2-1 summarizes features of the cypress domes selected and studied.

After initial uncertainties and surface overflows at S-l, it was decided that one inch per week would be the loading rate for S-l, S-2, and C-l. At first the effluent was pumped directly from the treatment plant. However, the treatment plant was inefficient at removing suspended solids and, as a result, S-l (the first dome to receive effluent) received large amounts of organic flocculent which floated on the surface in a solid mat. To avoid this problem, the effluent was taken from an oxidation lagoon, which allowed the suspended solids to settle out.

Brezonik (1974) demonstrated several significant differences

in the standing water of the sewage domes as compared to the standing water of undisturbed domes. For example, water from the undisturbed, Austin Cary dome was low in dissolved solids (specific conductance < 100 P mhos/cm), low in pH (ca. 4.5) and very soft (Ca and Mg in the range of 1-2 mg/l each). Samples from S-l showed higher values of dissolved solids (115-500 p mhos/cm), pH values near neutrality, and moderate levels of hardness (Ca 7.8-18.5 mg/l, Mg 11.4-20.6 mg/l). Total phosphorus and nitrogen were greater in samples from S-1 than from AC, and a major portion of the total nitrogen and phosphorus in samples at S-1 was soluble inorganic forms. Most of the nitrogen and phosphorus in samples from AC was bound in organic forms. The N/p



























Fig. 2-4. Photographs taken within the large control
swamp in the Austin Cary Forest.









25



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(aa l F- C LI) V)


























Figure 2-5. Photos taken at S-1 to demonstrate the openness
of the dome and the extensive cover of duckweed.
The picture at the lower left was taken at the margin of the dome where invading cattails and
dog fennel are abundant.









28

















17,:



4 tqWW,





























Figure 2-6. Dome S-1, characterized by the extensive invasion
of cattails and dog fennel.









30












i ~

":a F ~























U \
": n, Bi~ II" X r a rP

v ~it E
1 ,, ,I I
11 w,;

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31


ratios were low in S-i compared to AC. As Brezonik (1974) stated,this is a reflection of the low N/p ratios found in sewage, and perhaps in part a result of the denitrifying conditions of the anaerobic environment created at S-l.

Immediately following the introduction of effluent at S-1 and S-2, duckweed covers (Fig. 2-5) at both domes became permanently established. The duckweed was composed of two species, identified by Ewel (1976) as Lemna perpusilla and Spirodela oligorhiza. The floating fern, Azolla caroliniana was also present. A cover of duckweed and fern was likewise formed at C-1, but it soon disappeared. The duckweed covers at the sewage domes effectively blocked sunlight from reaching the water and created anaerobic conditions. Species of Utricularia, which were common in the control swamps (Fig. 1-2) containing year-round standing water, were completely inhibited in the sewage domes.


Adult Mosquito Sampling


Ramp-Traps


Eight ramp-traps similar to those described by Gillies (1969)

were constructed in the early spring of 1974 (Fig. 2-7A). Four of these traps were placed equidistant from each other around the perimeter at S-1 and C-1 with the ramp openings facing the center of each dome. Collections were started in April at S-1 and in May at C-1. Samples were collected after 24 hour trapping periods. When not in use, the collection boxes were removed from the ramps (Fig. 2-7C). Trapped mosquitoes were aspirated from the collecting boxes at the end of the















Figure 2.7. A. Ramp-trap in its original form.


























B. Removing trapped mosquitoes C. Removing the trapping
from the ramp-trap by aspiration. head when not in use.







33







34


ramps (Fig. 2-7B) and killed with ethyl acetate. Identifications were made in the laboratory using keys written by King et al. (1960), Stojanovich (1960), and Carpenter and LaCasse (1955).

The ramp traps were also loaned to another investigator, Walt Jetter, who was sampling insects other than mosquitoes. Because Jetter was having difficulty removing his samples from the collecting boxes by aspiration, it was agreed that he could modify the collecting boxes. In July and August of 1974, the modifications were done. The collecting boxes were covered in plastic and a funnel was placed at the bottom in such a way that a cyanide killing jar could be attached (Fig. 2-8). This modification saved time in removing captured insects; however, it had some disadvantages. The plastic used to cover the collecting heads had to be repaired and replaced frequently, and whenever it rained (which was often in the summer) the killing jars would flood with water. This made the mosquito identifications much more difficult.

To cooperate with other investigators on the project, the arrangement of traps was changed in the Winter of 1974-75. Instead of four traps at two domes, two traps were positioned at each of the following domes: S-l, S-2, C-l, and AC. One trap was placed at the perimeter and one in the center of each of these domes. Samples were again collected on a 24 hour basis. In August 1975, sampling by this method was discontinued.

























Figure 2-8. The modified ramp-trap. The collecting head was
modified so that trapped insects would drop into
a killing jar attached to the lower funnel.







36






37


New Jersey Light Traps


By the end of the summer of 1974, boardwalks had been built to the center of domes S-i and C-i, and electric power was supplied to each of these domes. Sampling with New Jersey light traps (Fig. 2-9A) was started in September 1974, at these domes and in the nearby trailer park. In May 1975, similar collections were begun at S-2.

Each trap was located at approximately the center of its respective dome. The traps were activated before sundown and run simultaneously overnight. The mosquitoes were removed from cyanide killing jars and taken to the laboratory for identification. CDC Light Traps


Starting in April 1976, two CDC traps (Fig. 2-9B) were operated

simultaneously in each of three different domes, S-2, AC, and C-2, with the traps presumed to be placed far enough apart to avoid interference with each other. Unlike the New Jersey traps which were stationary at the centers of the domes, the CDC traps were placed in different locations on different nights. These traps were powered by six volt, gell-cell batteries.


D-Vac and "Homemade" Suction Device


In August 1975, a large, gasoline-powered D-Vac (Fig. 2-10) was

obtained to collect resting mosquitoes from vegetation. Although this methodof sampling produced good results, it had many drawbacks. The machine was heavy, awkward, and the engine had to be run at slow speeds to avoid strong suction. Frequently at the slower speeds the engine stalled, and the entire sample escaped.













Figure 2-9. A. New Jersey light trap.

























B. CDC portable light trap.







39





























Figure 2-10. D-Vac suction device used to collect daytime
samples of resting adults.









41


I, ,
i:tJ~ "'
"'r~i~














iii ~




























B



'"lx~

Ej"r;rfZ







42



After repeated engine trouble with the D-Vac in the spring of 1976, a "homemade" suction device was assembled (Fig. 2-11). It was constructed with 14-inch-diameter galvanized pipe, a 12 volt motor with a fan mounted at one end, and a collecting bag attached to intercept specimens which passed through a series of reducers. Starting in July 1976, short interval samples were taken at S-2, AC, and C-2. Intervals were timed with a stop watch. This "homemade" device proved to be as awkward and even more exhausting to use than the D-Vac and, in September 1976, suction-type sampling was discontinued.


Malaise Traps


In 1975, four tent-type malaise traps were purchased (Fig. 2-12A). The traps were supported by aluminum frames, the canpies were of green nylon, and the screen baffles were made of saran. The total interception area of the baffles was approximately 80 square feet per trap. The collecting head was made of aluminum and plexiglass (Fig. 2-128).

Two of these traps were permanently placed at AC and two at S-2 in May 1976. In all cases the traps were erected near the swamp margins where mosquito breeding was assumed to be concentrated.

Trap collections were removed every two weeks and examined for mosquitoes and tabanids. A strip of plastic impregnated with dichlorvos was used as a killing agent. Truck Trap


A truck trap (Fig. 2-13) similar to the one described by Bidlingmayer (1966) was used to sample mosquitoes active in the trailer park adjacent

























Figure 2-11. Homemade suction device used to sample daytime
resting adults.







44






j QI



r 5 "*







.. t a~













I~ ~ ~ ~ 4 .. 4. II i














Figure 2-12. A. Malaise trap, a flight interception device.
















B. A close-up of the trapping chamber above
the interception baffles of the malaise
trap.







46





















i. z P,~4j.







47


































Fig. 2-13 The truck track, a fljoit interception device.









to the experimental swamps. It was mounted on a station wagon, and collections were made by driving slowly (15-20 miles per hour) along a constant route through the park. The route was selected so that each street was sampled twice. Sampling began in August 1975 and concluded in September 1976.


Larval Mosquito Sampling


In July and August 1975, four transects were set up in domes C-l,

S-l, and S-2. Two transects were sampled at AC. The transects extended in straight lines from the center of each dome to the margins along the cardinal compass bearings. At AC the transects were along 00 and 900 bearings. A small nylon rope marked in 0.1 meter units was attached to stakes and trees and extended from the center of the domes along each transect. Random sampling points along the transects were selected from a random number table. One dip was taken at each point. The water depth and number of mosquito larvae taken were recorded. Larvae were taken to the laboratory for identification.

Throughout the investigation, qualitative dipping was done to

determine the presence and distribution of the different larval species. This dipping was concentrated at shallow marginal pools (Fig. 2-14), around emergent vegetation, at marginal seepage holes (Fig. 2-15B, C), and from stump and tree holes (Fig. 2-15A, 2-16A, B).

In February 1977, sampling was done along a 90 degree (east) transect at AC running from the center of the dome to the margin (approximately 410 feet). Ten non-random dips were taken every 16 feet (approximated by pacing). Specific habitats were sampled which, through

























Figure 2-14. Examples of shallow, temporary pools at the margin
of the Austin Cary control dome.







50




t Atib ju



56 !7" i il

















i ii ii













Figure 2-15. A. Dipping for mosquito larvae from stump hole covered with emergent vegetation.

























B. A seepage hole at the C. Dipping for larvae
margin of dome S-2. from a seepage hole.












52








T~a~C~npt ~:~ fiii f I 18;

4 ~ 11~811~~ it
3
i





































r

























;e dL


















Figure 2-16. A. A stump hole at C-l.






















B. A tree hole at C-I.








54




i~T
i~ i





r( i 'I rl i: p I

;




,











I I iQ
wi i
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55


experience, could be identified as being probable breeding sites. A similar transect (90 degree, 190 feet) was sampled at S-2.


Virus Activity

Sentinel Chickens


Viral transmission studies were done using white leghorn, sentinel chickens, approximately 12-18 months old, as the experimental animals. Chicken pens were constructed with plywood as demonstrated in Figure 2-178. In February 1976, five of the finished pens (Fig. 2-17A) were placed in each of domes S-1, S-2, C-l, and AC. The pens were distributed from the center of each dome to the margins, with at least two pens at the margins. Each pen was numbered and held two chickens (one of which was marked so that the two could be distinguished). Plastic water dishes and feeders were installed in all the pens.

Before being placed in their pens, the birds were bled to determine any previous contact with encephalitis. After this initial bleeding, the birds were bled (Fig. 2-18) on a regular basis every three weeks. Three milliliters of blood were taken from a wing vein using a 23 gauge needle. The blood was allowed to clot and held on a slant at ambient temperature for one to two hours. The samples were then refrigerated overnight at about 50C. The next day serum was removed from the clots by low speed centrifugation (ca. 2000 rpm for 5 minutes). The serum samples were then stored frozen at approximately -150C in stoppered test tubes until serological tests could be done to determine the presence or absence of antibodies against the different encephalitide viruses.















Figure 2-17. A. A sentinel chicken cage at the margin of S-1.
























B. Construction of the sentinel cages.







57



























Figure 2-18. Taking a blood sample from a sentinel chicken.








59





60


Once a bird was shown to have developed antibodies against one of the encephalitis viruses, that bird was replaced. New birds were always pre-bled to determine previous infection.

In June 1976, ten more pens (20 more chickens) were added to the sentinal bird study. Five were placed at C-2, and the others were scattered throughout the City of Gainesville (Fig. 2-1).


Mosquito Pools


In July, August, and September of 1976, mosquitoes were collected alive with CDC light traps at S-2 and C-2. They were taken to the USDA, Insects Affecting Man, Laboratory in Gainesville where they were sorted and pooled in a walk-in cold room. The pools were sealed in test tubes and stored over liquid nitrogen at approximately -900C. Later the samples were transported on dry ice to Tampa for viral isolation attempts.

Dr. Arthur Lewis of the Epidemiology Research Center of the State Health and Rehabilitative Services Department did the isolations using the suckling mouse technique described by Sudia and Chamberlain (1974). Viral identifications were based on serum neutralization results. Mammal Sampling


Periodically mammals were trapped at C-1, C-2, S-1, and S-2. Larger mammals (Racoons, bobcat and opossums) were anesthetized by intramuscular injection of ketamine hydrochloride at 8-10 mg/kg of body weight as recommended by Bigler and Hoff (1975). Three milliliters of blood were taken from the larger mammals either by cardiac puncture or from an arm vein (Fig. 2-19). The smaller mammals (rice rats, cotton



















Figure 2-19. Taking blood samples from small mammals.







62






63



rats, and cotton mice) were anesthetized with ether and bled from the orbital sinus (Fig. 2-19). Only about 0.2 ml of blood was taken from the small mammals and this was diluted 1:5 by volume in field diluent as recommended by Sudia, Lord, and Hayes (1970).

The animals were toe-clipped (small mammals) or ear-tagged (large mammals) and released in the same area where they had been caught.

Serum from the mammal samples was collected and stored as previously described for the chicken samples. Serology: Hemagglutination Inhibition Test


The sera from the sentinel chickens and mammals were screened for viral antibodies using the hemagglutination inhibition test (HAI) (Clark and Casals, 1958). The mammal samples were extracted with acetone to remove lipids, which might interfere as non-specific inhibitors of hemagglutination. The chicken samples were treated with protamine sulfate and then acetone extracted to remove non-specific inhibitors. Non-specific agglutinins were removed by goose red-blood cell absorption.

Viral antigens and goose red-blood cells were prepared for this

study by the Epidemiology Research Center in Tampa, and the serological tests were conducted at the Tampa laboratory by the author. The chicken sera were tested against Eastern Equine Encephalitis (EEE), Western Equine Encephalitis (WEE), and St. Louis Encephalitis (SLE). Mammal samples were tested against EEE, WEE, and SLE, and in some cases against California Encephalitis (CEV) and Venezuelan Encephalitis (VEE).

Preliminary titrations were done to determine the dilution of antigens which would give 4-8 hemagglutinating units in each test. Back





64


titrations were carried out on the day of the test to insure that 4-8 units had been used in each test. Serum controls were done to make sure that serum agglutinins had been removed by absorption with the goose red-blood cells previous to the test.

The tests were conducted in microtitter plates (Fig. 2-20). Serial, two-fold dilutions of the serum samples were made with wire loops (Fig. 2-20). A dilution of 1:640 was the highest dilution of serum tested. Antibody titers were designated as the highest dilution to give complete hemagglutination inhibition. A titer of at least 1:20 was considered positive. Occasionally a titer of 1:10 with partial inhibition beyond this dilution was considered positive. Serology: Neutralization Test


Samples, which were positive for HAI antibodies, were then tested for the more specific neutralizing antibodies. Serum neutralization tests were done using the constant serum-varying virus titration method (Dulbecco and Ginsberg, 1973). African Green Monkey Kidney Cells (BGM) in tissue culture tubes were used as a susceptible indicator system. Neutralization indices were determined by the ReedMuench method (Lennette, 1969). A reduction index of at least 1.7 logs was considered positive.
























Figure 2-20. Dilution of serum samples was done in microtiter
plates. The lower photo is a sideways shot of a finished test plate. The next to last row (C-2,
M4; the marked bird in pen no. 4 at dome C-2)
shows a positive antibody titer against eastern
equine encephalitis. In this case complete hemagglutination inhibition is present at a
serum dilution as high as 1:320.








66































f



i1

B :;B








Ic'I












RESULTS AND DISCUSSION


Adult Mosquito Sampling


Ramp Traps


Using no known insect attractants, ramp traps are flight interception devices which capture those mosquitoes whose flight paths are directed toward the ramp openings. Relatively small sample sizes are expected when using such unbaited traps, and this was indeed the case. With four traps per swamp in 1974, the cumulative data in Tables 3-1 and 3-2 are probably reasonably accurate representations of the relative population compositions. Figure 3-1 graphically compares the relative abundance of the seven most common species or species groups from both domes. With the exception of the floodwater Aedes species, community structure was very similar at both domes. ShannonWeaver diversity indices are computed and recorded in Table 3-3. The Shannon-Weaver index, as described by Price (1975), is a measure of both species richness and evenness of abundance. The index for dome C-1 is slightly larger than that for S-1. Since there were two more species captured at S-l than at C-l, the larger index for C-1 represents a slightly more even distribution of species. Only four species were not present at both sites and, where present, each represented only a minor component of the mosquito fauna collected by the ramp traps.



1Aedes species refers to the several species identified.



67









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69






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72


Table 3-3. Shannon-Weaver diversity indices computed from 1974 ramp-trap
data at S-1 and C-1.

S-I C-1

Species Pi Pi(logoPi P Pi(IngePi)

Aedes:
mitchellae .0026 -.0155 .0005 -.0038 vexans .0004 -.0031 .0005 -.0038 infirmatus .0060 -.0307 .0109 -.0493 dupreei .0004 -.0031 .0027 -.0160 atlanticus .0624 -.1731 .1383 -.2736 canadensis .0004 -.0031 .0082 -.0394
taeniorhynchus .0004 -.0031 fulvus pallens .0038 -.0212

Anopheles:
crucians .0693 -.1850 .0552 -.1599 quadrimaculatus .0043 -.0234 .0197 -.0774

Culex:
(Melanoconions) .1022 -.2331 .1001 -.2304 territans .0090 -.0424 .0284 -.1011 salinarius .0154 -.0643 .0071 -.0351 nigripalpus .0693 -.1850 .0569 -.1631 pipiens .0021 -.0129 .0005 -.0038
restuans .0009 -.0063

Culiseta:
melanura .2776 -.3558 .2269 -.3365

Coquillettidia:
perturbans .0188 -.0747 .0109 -.0493

Psorophora:
ciliata .0011 -.0075 columbiae .0094 -.0439 .0049 -.0261 ferox .0137 -.0588 .0202 -.0788

Uranotaenia:
sapphirina .2528 -.3476 .2203 -.3333 lowii .0787 -.2001 .0864 -.2116


H' = EpilogePi = 2.0862 H' = 2.1998

H' = Shannon-Weaver diversity index Pi = the proportion of the ith species in the total sample







73



The four traps at each dome were not equally successful in capturing mosquitoes (Table 3-4). At S-l, the traps at the northern (00) and eastern (900) edges of the dome caught approximately twice as many mosquitoes as the southern (1800) and western (2700) traps. At C-l the eastern and southern traps were most productive. This information indicates the importance of site selection and the uncertainty in sampling and comparing habitats by this method.

Overall abundance of mosquitoes from the two domes is compared in Table 3-5 using a rank comparison test (Mendenhall, 1968). Only samples collected on the same dates are considered. There is insufficient evidence from these data to disprove the null hypothesis that the populations are identical with respect to abundance.

The ramp-trap data for 1974 show no greater mosquito production in an experimental swamp receiving sewage effluent than a control swamp receiving untreated groundwater. Community structure of mosquitoes also appears very similar in both domes. Very few Culex pipiens quinquefasciatus were collected at S-l even though this species has a reputation for developing in great numbers in heavily polluted water.

In July and August 1974, the design of the ramp traps was changed as noted above (Materials and Methods). During the winter of 1975, the traps were rearranged with two traps at S-1, S-2, C-l, and AC. One was placed in the center and another at the margin of each dome.

Data from 1975 cannot be compared with those of the previous year because of the changes in trap design and rearrangement. Numbers of mosquitoes captured in 1975 are summarized in Tables 3-6 through 3-9. By inspection Culex territans appeared to be much more abundant at S-2








74






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76


Table 3-5. Rank comparison test comparing abundance at S-1 and C-I
from ramp-trap data (1974).

Total number of
mosquitoes per four ramp-traps
Trap nights: S-1 C-1

May 13-14 17 10* n=total number of trials 21-22 14 15 (excluding those where
23-24 6 28 (S-1)=(C-I))
27-28 160 99*
30-31 115 99* p= probability of (S-1(C-1) June 3-4 31 41 q= probability of (C-I)>(S-l)
6-7 82 44* y= number of times (S-1)>(C-1)
11-12 33 40 u= np
13-14 43 69 a= npq
17-18 50 61 y= 15
20-21 82 43* n= 30-1=29
24-25 107 127 null hypothesis: p=0.5, q=0.5
27-28 384 209* reject if IZ=Y j> 1.96
July 3-4 131 70* at a=0.05
4-5 209 116* sinceJZJ= 115-14.51 = .267< 1.96
8-9 26 65 1 1.87
Sept.12-13 277 "270* do not reject the null hypothesis
16-17 222 188* at c =0.05
19-20 171 187
23-24 40 101 Oct. 3-4 5 5
7-8 127 129
10-11 48 52
14-15 99 59*
17-18 34 17* 24-25 54 24* 28-29 96 93* Nov. 4-5 66 60*
9-10 17 22 16-17 10 34
(S-1)>(C-1)






77
















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than any of the other domes, and Culex (Melanoconion) spp. were more abundant at C-i. Otherwise there were no noteworthy differences in species composition at the different domes. Culiseta melanura, a medically important species, was abundant in all the domes. It is interesting that AC, a large dome, which at times harbors great numbers of mosquitoes, produced very few in the ramp trap samples. The new trap design and arrangement failed in 1975 to capture many floodwater Aedes species. Field observations have shown these species (especially Aedes atlanticus and Aedes infirmatus) to be concentrated in the dense understory at the margins of the domes. Of the small numbers of these species trapped in 1975, 92.5% (37) were from traps at the margins of the domes and only 7.5% (3) were from traps at the centers.

In 2 years of service, the ramp traps demonstrated that the

most abundant species of adult, female mosquitoes in the cypress domes of this area are species which feed primarily on birds or cold-blooded vertebrates. Culiseta melanura, Culex (Melanoconion) erraticus,1 and Culex nigripalpus were plentiful in all the domes sampled. These species are primarily avian blood feeders, with Culex nigripalpus possibly shifting from birds to mammals at certain times of year (Edman, 1974). Culex territans (common at S-2), Uranotaenia sapphirina, and Uranotoenia lowii are all thought to feed primarily on cold-blooded vertebrates, expecially amphibians. Anopheles crucians, another species abundant in these domes, feeds primarily on mammals, especially rabbits. Several woodland, floodwater species of Aedes and Psorophora were found breeding in large numbers in temporary pools adjacent to the


1This species was assumed to be the most common of the Melanoconion complex.






82


domes and in some cases in the shallow marginal pools of the domes themselves. The adult females of these species are primarily mammalian blood feeders, readily feeding on humans. They rest during daylight hours in the dense, shaded vegetation at the dome margins but bite when disturbed.

The ramp traps have demonstrated that adult, female mosquitoes in swamps receiving sewage effluent are the same species found in a similar swamp receiving untreated groundwater. The added nutrients in the effluent have not been shown to result in an immediate or significant increase in overall mosquito abundance above what could be expected by flooding a similar dome with low nutrient groundwater.


New Jersey Light Traps


New Jersey light traps have been widely used in this country to

monitor mosquito populations for almost 40 years. They act as a biased sampling device, being very selective for positively phototropic species. Unengorged females represent the majority of the usual catch.

These traps have been used at S-l, C-l, WMHP and S-2. Sampling at S-l, C-1, and WMHP began in September 1974 and at S-2 in May 1975. Results from these trapping studies are recorded in Tables 3-10 through 3-13; a graphical representation of monthly totals is expressed in Figure 3-2. It is obvious from Figure 3-2 that sewage dome S-2 was producing larger mosquito samples than either S-1 or C-l, and that S-1 was producing slightly more than C-l.

The degree of similarity among the three domes is calculated on

the basis of female species abundance and community structure (relative abundance of female species) and recorded in Tables 3-14 and 3-15









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respectively. Degree of similarity is based on the amount of differences when comparing two domes at a time. Of course the smallest values represent the greatest similarities. One would expect less difference in a comparison between the two sewage domes than in a comparison between either of the sewage domes with the groundwater, control dome. This was not the case, and on the basis of both abundance and community structure S-l and C-1 were more alike than S-l and S-2. Of the three comparisons, S-2 and C-1 were least similar. In examining Tables 3-14 and 3-15 the only obvious and consistent differences between the sewage domes collectively and the control dome was the greater abundance of the two species of Uranotaenia, especially Uranotaenia sapphirina at the two sewage domes. Starting in June 1975, males of U. sapphirina at the two sewage domes were counted and recorded from the light trap samples; 75.0% (673) of the males at C-l, 83.0% (1,212) at S-l, and 87.0% (3,449) at S-2 were U. sapphirina. It should be obvious at this point that the graphs in Figure 3-2, which demonstrate greater mosquito production at the two sewage domes than at the control dome, are heavily influenced by the sample sizes of U. sapphirina. From both a medical and an economic standpoint this species is of no known importance; and therefore, a more meaningful comparison of the New Jersey light trap data is represented in Figure 3-3, which computes the mean number of adult females per month leaving out U. sapphirina. This information shows that with the exception of U. sapphirina there was very little difference in sample sizes among the three domes, and the sewage domes were not consistently producing larger sample sizes than the control dome.

































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Full Text

PAGE 1

RECYCLING TREATED SEWAGE EFFLUENTS THROUGH CYPRESS SWAMPS: ITS EFFECTS ON MOSQUITO POPULATIONS AND ARBO-VIRUS IMPLICATIONS By HARRY GOODWIN DAVIS A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1978

PAGE 2

ACKNOWLEDGMENTS Several people have helped enormously in the preparation of this dissertation. Dr. Lewis Berner and Dr. Dave Dame, the Co-Chairmen of my graduate committee, have been especially generous with their time and help. My supervisors at the Center for Wetlands, Dr. Howard Odum, Dr. Kathy Ewel James Ordway, and Margaret Johnston have been understanding and patient throughout the study. Dr. Jack Gaskin, of the College of Veterinary Medicine, has given me advise and support on several occasions. Without help from Dr. Flora Mae Wei lings. Dr. Arthur Lewis, Dr. Sam Mountain, and Hardrick Gay of the Florida Department of Health Education and Welfare none of the virus studies would have been possible. Dr. Wellings allowed me to work at the DHEW lab in Tampa. Mrs. Anna Chang, also at the Tampa lab, taught me the serological techniques. Working with Mrs. Chang was an enjoyable experience. She is an excellent teacher and a very fine lady. Drs. Gary and Janet Stein, of the biochemistry department, often let me use their equipment in the preparation of my serological samples. They also kept me well fed by frequently inviting me to their excellent parties. My older brother Lloyd and his wife Sally were always helpful, and my parents were a constant source of encouragement. My wife Jeudi unselfishly worked to support us during my research. Jeudi also did most of the figures in the dissertation. ii

PAGE 3

TABLE OF CONTENTS Page ACKNOWLEDGMENTS ii ABSTRACT v INTRODUCTION 1 General Statement 1 Cypress Domes 5 Mosquitoes 6 Mosquito Sampling 10 Mosquito-Borne, Human Diseases 13 MATERIALS AND METHODS 17 Site Selection 17 Adult Mosquito Sampling 31 Ramp-Traps 31 New Jersey Light Traps 37 CDC Light Traps 37 D-Vac and "Homemade" Suction Device 37 Malaise Trap 42 Truck Trap 42 Larval Mosquito Sampling 48 Virus Activity 55 Sentinel Chickens 55 Mosquito Pools 60 Mammal Sampling 60 Serology: Hemagglutination Inhibition Test 63 Serology: Neutralization Test 64 RESULTS AND DISCUSSION 67 Adult Mosquito Sampling 67 Ramp-Traps 67 New Jersey Light Traps 82 CDC Light Traps 97 Suction Devices 108 Malaise Traps 126 Truck Traps 131 Larval Sampling 134 Virus Activity 142 Sentinel Chickens 142 Isolation Attempts from Mosquito Pools 152 Serology and Isolation Attempts from Mammal Samples .152 Summary 154 iii

PAGE 4

Page CONCLUSIONS 156 RECOMMENDATIONS FOR FUTURE WORK 157 LITERATURE CITED 158 BIOGRAPHICAL SKETCH 164

PAGE 5

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RECYCLING TREATED SEWAGE EFFLUENTS THROUGH CYPRESS SWAMPS: ITS EFFECTS ON [10SQUIT0 POPULATIONS AND ARBO-VIRUS IHPLICATIONS By Harry Goodv;in Davis June, 1978 Chairman: Dr. Lewis Berner Co-Chairman: Dr. David Dame Major Department: Entomology and Nematology The task of determining the feasibility of using cypress domes as natural filters in purifying and recycling v^ater from secondarily treated sewage effluents was undertaken by the Center for Wetlands at the University of Florida with major funding from the Rockefeller and National Science Foundations. Certain aspects of the proposed project were of great concern to public health officials. Many thought that the enrichment of stagnant, sv/amp water would result in drastically increased mosquito populations. To answer this concern, several methods were used to sample mosquitoes at both experimental and control swamps. Also, mosquito-borne encephalitis activity was monitored at these same sites. Three years of sampling failed to demonstrate any measurable increase in human pest species at the experimental domes. The floodwater species of Aedes atlanticus and Aedes infirmatus declined at these sites. Serological studies, using sentinel birds, indicated that eastern and western equine encephalitis viruses are coimon in both the experimental and control swamps. V

PAGE 6

This study showed that cypress domes in north central Florida could be used to recycle treated effluents, without causing significant increases in mosquito populations; however, because of the endemic nature of the viral encephalitis in these habitats, any swamp which is ecologically disturbed should be monitored for changes in mosquito and virus activity. vi

PAGE 7

INTRODUCTION .1 General Statement The state of Florida is presently experiencing rapid growth and development resulting in much of its forested area and natural wetlands being destroyed to accomodate this human expansion. Great expenditures of fossil fuel energy are required to restructure and maintain the environment in an altered state. People are finally beginning to realize that the simplification of complex natural systems often results in unpredictable calamity, and that it is much wiser to conserve natural systems and allow them to function for the benefit of man. As a means of dealing with some of the problems created by this rapid growth, the Center for Wetlands was established at the University of Florida in 1973 to study the feasibility of using cypress wetlands in water management, sewage treatment, and conservation. This dissertation is part of the report on the multiphasic study of the use of cypress wetlands in sewage treatment and wastewater reclamation. More specifically, the topic of this discussion is related to some of the public health uncertainties arising from the use of cypress domes in • the tertiary treatment of sewage effluents, i.e. the effects of secondarily treated sewage effluents on mosquito populations and mosquito-borne virus activity in north Florida cypress domes. A cypress dome is defined as a stand of cypress, usually pond cypress, 1

PAGE 8

2 growing in a wet depression in a flatwoods area. From a distance the stand is dome-shaped with the taller trees in the middle and the smaller, shorter trees at the margins. The overall project has been supported with funds from the RANN Division of the National Science Foundation and The Rockefeller Foundation, Beginning in 1974, annual progress reports have been published by the Center for Wetlands (Odum et al 1974, 1975, 1976). By summarizing data collected from all phases of the project, these annual reports have helped in coordinating the overall investigative effort. Two cypress domes were selected to receive secondarily treated sewage effluent from a small package treatment plant (capacity: 30,000 gallons per day) operated by the Alachua County Utilities Department. These two experimental domes and several control domes (Fig. 1-1) were monitored simultaneously. The hypothesis being tested was that the vegetation of the cypress domes would remove the dissolved nutrients from the effluent (stimulating the growth of cypress trees which could be periodically harvested) and that in recycling the water to the groundwater aquifer, slow percolation through the geological strata below the domes would filter out potentially pathogenic microbes. It was hoped that such a purification system would be economically competitive with the more standard methods of tertiary treatment. Of public health concern was the possibility that the altered hydroperiod and increased nutrient loads of the experimental domes would cause a significant increase in mosquito production. There are several species of mosquitoes which develop in great numbers in

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Figure 1-1. One of the experimental cypress domes (S-1) receiving sewage effluent. A control cypress dome (C-1) receiving untreated well water.

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4 lMi-ii-imiili ja Hill rf'ntTi-ifcuaWf riIWi>lllfclil fof r.juMm <*£5k.

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5 stagnant, polluted water, and some of these species can serve as vectors in the transmission of human disease. For these reasons, mosquito populations were monitored in the domes, and during the last year, the project was expanded to include a study of the mosquito-borne viruses which cause human encephalitis. Cypress Domes The pine-palmetto flatwoods of the southern Atlantic and Gulf Coastal plain are interspersed with thousands of small cypress domes. These dome-like stands are primarily composed of Taxodium distichum var. nutans Nyssa bi flora Pinus ell iottii Acer rubrum and Myrica cerifera (Monk and Brown, 1965). Kurz (1933) has shown that the larger cypress trees in the central portion of a dome are older than the smaller trees at the margins. The germination and early survival of the cypress seedlings is dependent upon a period of dry-down (Demaree, 1932). In an undisturbed dome, the central pond often holds standing water year-round, and thus the only suitable place for cypress regeneration is at the margins. Understory vegetation within the domes is usually clumped around the expanded bases of the cypress trees or protrudes from dead stumps. The Virginia chainfern ( Woodwardia virginica ) and fetterbush ( Lyonia 1 ucida ) are common understory species and are especially abundant at the margins of domes which have been partially drained. Cypress domes exist at surface depressions which collect rainwater, surface runoff, and groundwater recharge. The standing water of a typical dome is low in dissolved nutrients, as indicated by the frequent

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6 presence of Utricularia species. These are submerged, carnivorous plants (Fig. 1-2) which are adapted to aquatic situations containing little dissolved nitrogen and phosphorus. Wharton et al (1976) have stated that naturally occurring phosphorus in cypress domes is unavailable to plants as it is bound in the clay layers below the standing water as a result of low pH. Present trends in land use have been to drain wetlands areas in an attempt to replace the cypress with faster growing slash pine. The lowering of water tables by these methods has resulted in an ecological backlash--the occurrence and severity of forest fires has increased. Wharton et al (1976) have described the different types of forested wetlands and have summarized their ecological values. Ewel and Odum (1978) have summarized some of the results which indicate that cypress domes could be used in the tertiary treatment of sewage effluents. Mosquitoes Among the hundreds of kinds of mosquitoes, some representative is capable of living in every conceivable collection of water. Some may live at altitudes of over 400 m while others may live in mines 1000 m or more below the earth's surface. Species range in latitudes northward from the tropics well into the Artie regions and southward to the ends of the continents. A wingless species has been reported from Antartica. No natural collection of water, whether fresh, saline, or foul, occurs but that part of it may be occupied by some mosquito. No forest is so dense, nor area so barren, but that some mosquito may live there. One may be annoyed by them in the heart of a metropolitan district or on the most isolated island; he may be attacked on land or on ships at sea, and he may be disturbed at home or in camp. All in all, this is a remarkably adaptable group of insects. Horsefall, 1972, p. 7.

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Fig. 1-2 Flowering Utricularia in March at the control swamp receiving untreated, low nutrient, well water. Commonly known as bl adderwort, this floating aquatic plant is an indicator of low nutrient situations.

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8

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9 Because of their involvement in the transmission of human diseases, mosquitoes have received enormous amounts of study. Publications are available which describe the fauna of any geographic region in the United States. The mosquitoes of Florida are covered in detail by King et al (1960). Detailed, ecological investigations of the mosquitoes inhabiting cypress swamps have been made in Maryland (Williams, Watts, and Reed, 1971; Saugstad, Dalrymple, and Eldridge, 1972; Joseph and Bickley, 1969; Le Due et al 1972; Muul Johnson, and Harrison, 1975). Similar studies were done in Louisiana cypress swamps (Kissling et al 1955). By adding treated effluents to cypress domes, not only are the seasonal fluctuations in water level changed, but also the water chemistry is greatly altered. These complex alterations in chemistry undoubtedly affect changes in the aquatic fauna and flora. It is possible that these ecological disturbances acting independently or jointly, and perhaps synergistical ly, can result in subtle changes or in drastic differences in mosquito fauna in terms of abundance and diversity. By the selection of specific oviposition sites the adult females determine where future generations will develop. In some cases it is easy to associate a certain habitat with a particular species. For example, larvae of Culiseta melanura are usually found in freshwater swamps (cedar swamps in the north and cypress swamps in the south). Larvae of Culex pipiens quinquefasciatus are commonly associated with highly polluted water sources. They develop in great numbers in sewage lagoons. Larvae of Aedes triseriatus develop in water holding, rot

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10 holes in trees. Some species of Culex, Uranotaenia Culiseta Mansonia Anopheles and Coqui Hettidia deposit their eggs on the surface of relatively permanent bodies of water. The so-called floodwater species of Aedes and Psorophora deposit their eggs on moist surfaces which are subject to periodic inundation. Clements, (1963) points out that various investigations have illustrated a wide range of physical and chemical factors used by different species in discerning oviposition sites; however, in no species has a simple chemical factor been found which typifies the breeding site. In most species it appears that a suitable site is discerned by a combination of particular physical factors and water of appropriate purity or pollution. Mosquito Sampling Knight (1964) and Service (1976) have described theoretical methods for calculating the absolute population density of mosquito larvae from certain habitats. These methods, however, are of limited usefulness in a quantitative examination of the entire mosquito fauna from a habitat as heterogeneous as a Florida cypress dome. Larvae are not evenly distributed throughout the habitat, and, because of differences in avoidance behavior, various species are not always captured in numbers proportional to their actual abundance. Dipping for larvae with a long handled, pint dipper has been the most commonly used method to determine breeding sites and qualitative information on community structure; however, as Wilson and Msangi (1955) have demonstrated, there are frequent inaccuracies associated with trying to extract quantitative information from these kinds of data.

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11 standard types of adult sampling techniques are no more accurate than larval surveys and often are more difficult to interpret. Trapping results vary a great deal from night to night and from one location to another. Bidlingmayer has discussed this variability and he lists three major causes for it: A) Environmental. These factors may be divided somewhat artificially into positional and meteorological. The former would include distance from the breeding area, habitat, competing attractants, food sources, reflecting surfaces, control activities, and predators. Meteorological factors would include effects of temperature, humidity, wind, and light intensities. B) Biological. This category includes inherent behavior patterns such as the characteristic responses of different species and sexes and the behavior changes during the life of an individual mosquito according to its physiological state. The expression of these behavior patterns is often modified or suppressed by environmental factors. Actual changes in population size because of seasonal trends or previous weather patterns may be included here. C) Operational. Variation may result from differences between apparently identical equipment and techniques, or, at times operational effectiveness of the method may change. Bidlingmayer, 1967, pp. 200-201. To insure the accuracy of any mosquito sampling survey Huffaker and Back (1943) noted that the results from several trapping techniques should be analyzed. Adult trapping techniques can be categorized as either attractant or nonattractant. The nonattractant methods such as sweeping adults by suction from resting sites or flight intercepting devices supposedly demonstrate more accurately the true population composition than do the attractant methods such as light traps or baited traps. The attractant methods usually yield large samples with a minimum of actual field

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12 work; whereas, the more unbiased, nonattractant methods require more time and effort for smaller samples. The best known and most widely used of the attractant-type devices is the New Jersey mosquito trap. The development of this trap was described by Headlee (1932) and Mulhern (1934). The function of the trap is based on the knowledge that some mosquitoes are attracted or perhaps disoriented by certain light intensities (Barr, Smith, and Boreham, 1960; Robinson, 1952). A trap of similar design but smaller and more portable was described by Nelson and Chamberlain (1955). This trap was later improved (Sudia and Chamberlain, 1962) and has become known as the CDC^ miniature light trap. Both these traps have been extremely popular with mosquito control workers in monitoring fluctuations in population levels. Nonattractant sampling devices can be used to collect either resting adults or to intercept adults in flight. A suction device such as the 2 commercially available D-Vac can be used to collect adults from resting sites. Samples collected with such a device will contain a higher proportion of males and blood-engorged females than samples from attractant-type traps. The nonattractant, flight interception devices can be either stationary or mobile. Malaise traps of various designs (Townes, 1962; Gressitt and Gressitt, 1962; Breeland and Pickard, 1965; Roberts, 1972) and the ramp-trap (Gillies, 1969) are examples of the stationary type. The truck trap described by Bidlingmayer (1966) is a good example of a mobile interception device. ^CDC after the Center for Disease Control in Atlanta, Georgia. 2 D-Vac Company, Riverside, California.

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13 Mosquito-Borne, Human Diseases The history and development of Florida have been influenced a great deal by mosquitoes and the diseases they transmit. Epidemics of yellow fever and malaria were common in the 19th Century. In 1901, it was discovered that yellow fever was transmitted from human to human by the mosquito Aedes aegypti and by 1905 Florida was waging a successful battle against yellow fever. Dengue fever was last noted in epidemic proportions in 1934, and malaria disappeared from Florida after 1948 (Schoonover, 1970). At the present time in Florida, the only real, mosquito-borne threat to human health is from a family of neurotropic viruses known as togaviruses. Several of these viruses are capable of causing human encephalitis. The togaviruses are classified in groups A and B on the basis of hemagglutination-inhibition reactions. Members within a group will cross react but there is little cross reactivity between groups. Members of the Bunyamwere supergroup are classified according to cross reactivity by complement fixation. Included in group A are eastern equine encephalitis (FEE), western equine encephalitis (WEE), and Venequelan equine encephalitis (VEE). Group B includes St. Louis encephalitis (SLE), the Bunyamwere supergroup includes the California encephalitis (CEV) group of viruses. Specific viral determinations within groups are done by the neutralization test. Strains of all the previous groups of togaviruses have been recorded from Florida. Venequelan equine encephalitis occurs in epidemic and endemic forms (Gibbs, 1976). Only the endemic form, which apparently causes subclinical infections in man and horses, has been found in Florida;

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14 and it seems to be restricted to south Florida (Bigler, 1969). The natural endemic cycle is maintained through transmission by Culex ( Melanoconion ) species among wild rodent reservoirs (Chamberlain et al 1964; Bigler, 1969; and Bigler and Hoff, 1975). Western equine encephalitis also occurs as different antigenic strains (Theiler and Downs, 1973). The strain from areas primarily west of the -Mississippi River causes clinical illness and fatality in man and horses (McGowan, Bryan, and Gregg, 1973). The strain found east of the Mississippi, along the Atlantic Coast and in Florida appears to be nonpathogenic for man or horses, although at least one equine fatality has been reported from Florida (Jennings, Allen, and Lewis, 1966) The pathogenic strain seems to be restricted by the range of its primary vector, Culex tarsalis (Stark, 1967). Culex tarsalis is primarily an avian blood feeder, with seasonal shifts to a preference for mammals (Tempelis et al., 1965 and 1967, and Tempelis and Washino, 1967) This pattern of behavior is undoubtedly important in respect to epidemiology. Along the Atlantic Coast the natural cycle of the nonpathogenic strain is apparently maintained in a bird to bird transmission by Culiseta melanura (Stark, 1967; Dalrymple et al 1972; Stamm, 1966). The range of eastern equine encephalitis activity overlaps the distribution of its enzootic vector, Culiseta melanura The severity of this disease for both humans and equines is very great, with fatality rates as high as 50% in humans (McGowan, Bryan, and Gregg, 1973). A high percentage of those who are clinically ill and recover are left with permanent neurological damage. Fortunately this disease is very

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15 rare in humans; from 1955 through 1976 only 135 human cases were reported to the Center for Disease Control (CDC) in Atlanta (McGowan, Bryan, and Gregg, 1973 and CDC miscellaneous publication, 1977). There are few human cases because Culiseta melanura breeds primarily in fresh water swamps and feeds almost exclusively on birds. Other species such as Aedes sollicitans the salt marsh mosquito, are suspected of transmittingsylvan EEE to humans and horses in eastern and gulf coastal areas (Stark, 1967). Bigler et al (1976) has summarized the endemic nature of EEE in Florida. St. Louis encephalitis is the most widespread of the North American encephal itides. Theiler and Downs (1973) list the chief vectors as Culex tarsalis in rural areas and Culex pi pi ens guniguefasciatus in urban areas. Dow et al (1964) isolated SLE virus from several pools of Culex niqripalpus during the 1962 epidemic in the Tampa Bay area of Florida. Of the 2,349 human cases reported to CDC from 1955 through 1971, 178 ended in fatality (McGowan, Bryan, and Gregg, 1973). The biological mechanism for the maintenance of SLE virus in nature is not well understood. The California encephalitis virus complex consists of at least twelve related strains. Three of these are known to produce clinical infection in humans (California, LaCrosse, and Tahyna) (Henderson and Coleman, 1971). From Florida, two nonpathogenic strains, keystone and trivittatus, have been isolated from pools of primarily Aedes atlanticus and Aedes infirmatus (Taylor et al 1971 andWellings, Lewis, and Pierce, 1972). Small mammals are apparently the wild reservoirs of these viruses. Taylor et al (1971) have implicated

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16 cotton rats and Jennings et al (1968) and Taylor et al (1971) have implicated rabbits as the natural hosts.

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MATERIALS AND METHODS Site Selection In 1973, sites were selected to receive sewage effluent. The two cypress -domes chosen are located in a pine plantation, owned by Owens-Illinois, Inc., two miles northwest of Gainesville, Florida, just east of U.S. Highway 441 (Fig. 2-1). Secondarily treated effluent was supplied (as detailed below) to the experimental swamps from a county-operated treatment plant serving the residents of Whitney Mobile Home Park (WMHP). In December 1973, before sewage was added to any of the domes, a forest fire swept through much of the pine plantation. The fire killed the young pines, cleared the understory vegetation in the experimental cypress domes, and killed some of the older trees in these domes Despite the fire damage the project was continued, and in April 1974, effluent was added to the first dome, S-1. At the same time, groundwater from a deep well was added to a control dome, C-1. In December 1974, effluent was added for the first time to S-2, a second experimental dome. Another control dome in the same area, C-2, had been extensively drained in the past (Fig. 2-2), and much of the year it was completely dry. The fluctuating water level at C-2 represented a situation similar to that at S-1, S-2, and C-1 previous to the addition of effluent or groundwater. All these domes are represented in Figure 2-3. 17

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Figure 2-1. Location of the monitored cypress domes with respect to the city of Gainesville, Florida. Also, the approximate locations of the sentinel chicken pens which were placed in and around Gainesville.

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19 Sentinel Chicken Sites

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Figure 2-2. Control dome C-2. Located in an area which has been extensively drained, this dome is dry much of the year. The material in the foreground is accumulated cypress litter.

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22 Fig. 2-3 The Whitney Mobile Home Park site, demonstrating the location and relative size of the two experimental domes and two of the control domes.

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23 Also, a large, undisturbed cypress dome (Fig. 2-4) on the University of Florida's property in the Austin Gary Forest was studied as a control. This dome is located approximately three miles northeast of Gainesville, off State Road 24 (Fig. 2-1). Table 2-1 summarizes features of the cypress domes selected and studied. After i^nitial uncertainties and surface overflows at S-1 it was decided that one inch per week would be the loading rate for S-1, S-2, and C-1. At first the effluent was pumped directly from the treatment plant. However, the treatment plant was inefficient at removing suspended solids and, as a result, S-1 (the first dome to receive effluent) received large amounts of organic flocculent which floated on the surface in a solid mat. To avoid this problem, the effluent was taken from an oxidation lagoon, which allowed the suspended solids to settle out. Brezonik (1974) demonstrated several significant differences in the standing water of the sewage domes as compared to the standing water of undisturbed domes. For example, water from the undisturbed, Austin Gary dome was low in dissolved solids (specific conductance < 100 y mhos/cm), low in pH (ca. 4.5) and very soft (Ga and Mg in the range of 1-2 mg/1 each). Samples from S-1 showed higher values of dissolved solids (115-500 y mhos/cm), pH values near neutrality, and moderate levels of hardness (Ga 7.8-18.5 mg/1, Mg 11.4-20.6 mg/1). Total phosphorus and nitrogen were greater in samples from S-1 than from AG, and a major portion of the total nitrogen and phosphorus in samples at S-1 was soluble inorganic forms. Most of the nitrogen and phosphorus in samples from AG was bound in organic forms. The N/p

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Fig. 2-4. Photographs taken within the large control swamp in the Austin Gary Forest.

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26 TO c 3 c >— o CD 1 C7) >^ J_ C (O t. •!d) SOJ 0) -o ^ QJ (/) >, • 4-> C 4-> --> X5 c Sro I— s0) Ol (U 2 +-' 42 0 0) +-> 4-> -M in o ^ +-> > to •* (O •> fO CD fO -O 2 -O 2 XI 2 cox: E (-> 2 c c c •ro •13 3 CD 3 Ol +J 3 +-> -Q Ol o c o c o c E (0 c t. -rSr3 • 3 "T1 T3 1 -o 1 -o -(-> 1— s_ +-> r— T3 SC ic sc O Ol cu U OJ C (O (C ro ta (C +-> 3 > (TJ QJ -tj> Ol -M a> +J 1— O) (O r— Ol 4-> >1/1 >1/1 >in u. ,— 2 U_ 1 — 1/5 C r— 0 OJ S-IC O) 01 I/) C > n3 S> fO CU S_ To O > M C •.• •r— cr> o •!cn +-> •> "O o o cn t. C T3 O) "D "O S_ -rrtJ O) (U E "O <0 > "O O) 2 fO T3 Ol E c 2 E OJ XJ 3 j: a to 2a)c-Q o 3^— ^^(OfO 3 o un 1/1 lo u +-> -a 1 r— I 3 ^ E OICM-r-CM -a+->(/lSTD > fO ro 3 OJ CD •r-OJ+JOl r— SS OICTIOOI +J-i<:+-''r4-> +-> •!-rio +-> ro fO X Li_ >,U. fO SO) ^ o LU X3 — Q_ J3 U. O 00 +-> > E QJ 3 CD 3 iQJ +-> +-> XI S_ XI QJ > •i X SCO n3 4-> QJ QJ E +J 1/1 -P >^ QJ C i+4-> O) SU_ OJ 0 o QJ -C QJ (-> QJ M4-> > X (/) QJ LU E 2 +-> M OvJ -a 0 CM -t-> to i~ 1 Ol •P— 1 u S. O 1— QJ --> CVJ 3 0 to 2 2 +J n3 X3 2 E E SQJ E QJ SSto QJ S(J CO 3 SQJ QJ QJ -a -O 3 -a E 3 1/1 T3 E O Ol -a E 3 CD S-0 3 to -r3 0 n3 (O "O -Q 1 — Ll_ o XI 0 UO. I— E iC CQ ~ 2: fO ro JD ro Q) QJ QJ CD E Q) >, E QJ 0 Oil— 0 01 >, E •a to -a • 2 (o-^ o to 1— 2 E da QJ CO ro r — QJ 0 >) Q) to to >) ra to QJ Q. E +j CTl Q. +-> S0 0 E C71 • r— 0 E CJ1 • •1 — E -0 QJ E 4->--E QJ E 4-> <+ro E •1E (O E •1E •r— > o) -o •r— > QJ +-> 0 s•r3 Q) S•13 ^ sQJ QJ 1— C E QJ QJ r— CD 0 +-> Q. 0 <4SQ) CL 0 E X QJ 43 Q. X QJ r— x: 0 LU SQJ XJ 0 LU SQJ W1 0 OJ c $0 3 E 1 0 -M to SCD 0 *r0 ^ >. (-) E T3 i. QJ •— c •rE 4-) ^ 2 X> >i 0 > 3 c 0 to Q. o •r— -C 0 •CDDQ 0 QJ +-> 1— 0 x: E E E X) ro a QJ QJ • > • QJ i. 0 >> E a >i •.Sc -M QJ Q. SQJ • Q. O) QJ E rro E 0 3 X3 QJ 0 0 -M QJ ro QJ SE XI tE E QJ ro Q. S0 S3 ro E 3 0 ro S2 0 Q -a +J XI 0 ID -t- -0 0 Ln o MQJ I — O r— ^ <— ^ z > • to +-> • QJ q; •1c E •-tro >4CSJ O O I 4QJ 1 — O r— >=Jr— ^ z > • 1/1 4-> • QJ q: •rE E •-i*ro 4C\J tiJ O I 1/1 1^ O 14QJ 1 — O r1— ':JZ > • to +J • QJ q: •rE E 4ro MCVI O I O (71 Lf> 0 MQ) tQ) 0 t— 0 r— r— CM 3: ^ LU 2: > 2: > • VI 4-> 1/1 +J • QJ q: QJ Cd •>C E E •-i+E r1ro ro 4CM t3 0 CO CJ 0 CM I O 10 OJ i. to U

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Figure 2-5. Photos taken at S-1 to demonstrate the openness of the dome and the extensive cover of duckweed The picture at the lower left was taken at the margin of the dome where invading cattails and dog fennel are abundant.

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Figure 2-6. Dome S-1, characterized by the extensive invasion of cattails and dog fennel.

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30

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31 ratios were low in S-1 compared to AC. As Brezonik (1974) stated, this is a reflection of the low N/p ratios found in sewage, and perhaps in part a result of the denitrifying conditions of the anaerobic environment created at S-1. Immediately following the introduction of effluent at S-1 and S-2, duckweed covers (Fig. 2-5) at both domes became permanently established. The duckv/eed was composed of two species, identified by Ewel (1976) as Lemna perpusilla and Spirodela oligorhiza The floating fern, Azolla caroliniana was also present. A cover of duckweed and fern was likewise formed at C-1 but it soon disappeared. The duckweed covers at the sewage domes effectively blocked sunlight from reaching the water and created anaerobic conditions. Species of Utricularia which were common in the control swamps (Fig. 1-2) containing year-round standing water, were completely inhibited in the sewage domes. Adult Mosquito Sampling Ramp-Traps Eight ramp-traps similar to those described by Gillies (1969) were constructed in the early spring of 1974 (Fig. 2-7A). Four of these traps were placed equidistant from each other around the perimeter at S-1 and C-1 with the ramp openings facing the center of each dome. Collections were started in April at S-1 and in May at C-1. Samples were collected after 24 hour trapping periods. When not in use, the collection boxes were removed from the ramps (Fig. 2-7C). Trapped mosquitoes were aspirated from the collecting boxes at the end of the

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Figure 2.7. A. Ramptrap in its original form. Removing trapped mosquitoes from the ramptrap by aspiration. C. Removing the trapping head when not in use.

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33

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ramps (Fig. 2-7B) and killed with ethyl acetate. Identifications were made in the laboratory using keys written by King et al (1960), Stojanovich (1960), and Carpenter and LaCasse (1955). The ramp traps were also loaned to another investigator, Walt Jetter, who was sampling insects other than mosquitoes. Because Jetter was having difficulty removing his samples from the collecting boxes by asp-iration, it was agreed that he could modify the collecting boxes. In July and August of 1974, the modifications were done. The collecting boxes were covered in plastic and a funnel was placed at the bottom in such a way that a cyanide killing jar could be attached. (Fig. 2-8). This modification saved time in removing captured insects; however, it had some disadvantages. The plastic used to cover the collecting heads had to be repaired and replaced frequently, and whenever it rained (which was often in the summer) the killing jars would flood with water. This made the mosquito identifications much more difficult. To cooperate with other investigators on the project, the arrangement of traps was changed in the Winter of 1974-75. Instead of four traps at two domes, two traps were positioned at each of the following domes: S-1, S-2, C-1, and AC. One trap was placed at the perimeter and one in the center of each of these domes. Samples were again collected on a 24 hour basis. In August 1975, sampling by this method was discontinued.

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Figure 2-8. The modified ramp-trap. The collecting head was modified so that trapped insects would drop into a killing jar attached to the lower funnel.

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37 New Jersey Light Traps By the end of the summer of 1974, boardwalks had been built to the center of domes S-1 and C-1 and electric power was supplied to each of these domes. Sampling with New Jersey light traps (Fig. 2-9A) was started in September 1974, at these domes and in the nearby trailer park. In May 1975, similar collections were begun at S-2. Each tr-ap was located at approximately the center of its respective dome. The traps were activated before sundown and run simultaneously overnight. The mosquitoes were removed from cyanide killing jars and taken to the laboratory for identification. CDC Light Traps Starting in April 1976, two CDC traps (Fig. 2-9B) were operated simultaneously in each of three different domes, S-2, AC, and C-2, with the traps presumed to be placed far enough apart to avoid interference with each other. Unlike the New Jersey traps which were stationary at the centers of the domes, the CDC traps were placed in different locations on different nights. These traps were powered by six volt, gell-cell batteries. D-Vac and "Homemade" Suction Device In August 1975, a large, gasoline-powered D-Vac (Fig. 2-10) was obtained to collect resting mosquitoes from vegetation. Although this method, of sampling produced good results, it had many drawbacks. The machine was heavy, awkward, and the engine had to be run at slow speeds to avoid strong suction. Frequently at the slower speeds the engine stalled, and the entire sample escaped.

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Figure 2-9. A. New Jersey light trap. B. CDC portable light trap.

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39

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Figure 2-10. D-Vac suction device used to collect daytime samples of resting adults.

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42 After repeated engine trouble with the D-Vac in the spring of 1976, a "homemade" suction device was assembled (Fig. 2-11). It was constructed with 14-inch-diameter galvanized pipe, a 12 volt motor with a fan mounted at one end, and a collecting bag attached to intercept specimens which passed through a series of reducers. Starting in July 1976, short interval samples were taken at S-2, AC, and C-2. Intervals were timed with a stop watch. This "homemade" device proved to be as awkward and even more exhausting to use than the D-Vac and, in September 1976, suction-type sampling was discontinued. Malaise Traps In 1975, four tent-type malaise traps were purchased (Fig. 2-12A). The traps were supported by aluminum frames, the canpies were of green nylon, and the screen baffles were made of saran. The total interception area of the baffles was approximately 80 square feet per trap. The collecting head was made of aluminum and plexiglass (Fig. 2-12B). Two of these traps were permanently placed at AC and two at S-2 in May 1976. In all cases the traps were erected near the swamp margins where mosquito breeding was assumed to be concentrated. Trap collections were removed every two weeks and examined for mosquitoes and tabanids. A strip of plastic impregnated with dichlorvos was used as a killing agent. Truck Trap A truck trap (Fig. 2-13) similar to the one described by Bidlingmayer (1966) was used to sample mosquitoes active in the trailer park adjacent

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Figure 2-11. Homemade suction device used to sample dayti resting adults.

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44

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Figure 2-12. A. Malaise trap, a flight interception device. B. A close-up of the trapping chamber above the interception baffles of the malaise trap.

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46

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47 Fig. 2-13 The truck, trap, a fUgat interception device.

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48 to the experimental sv/amps. It was mounted on a station wagon, and collections were made by driving slowly (15-20 miles per hour) along a constant route through the park. The route was selected so that each street was sampled twice. Sampling began in August 1975 and concluded in September 1976. Larval Mosquito Sampling In July and August 1975, four transects were set up in domes C-1 S-1, and S-2. Two transects were sampled at AC. The transects extended in straight lines from the center of each dome to the margins along the cardinal compass bearings. At AC the transects were along 0 and 90 bearings. A small nylon rope marked in 0.1 meter units was attached to stakes and trees and extended from the center of the domes along each transect. Random sampling points along the transects were selected from a random number table. One dip was taken at each point. The water depth and number of mosquito larvae taken were recorded. Larvae were taken to the laboratory for identification. Throughout the investigation, qualitative dipping was done to determine the presence and distribution of the different larval species. This dipping was concentrated at shallow marginal pools (Fig. 2-14), around emergent vegetation, at marginal seepage holes (Fig. 2-15B, C), and from stump and tree holes (Fig. 2-15A, 2-16A, B). In February 1977, sampling was done along a 90 degree (east) transect at AC running from the center of the dome to the margin (approximately 410 feet). Ten non-random dips were taken every 16 feet (approximated by pacing). Specific habitats were sampled which, through

PAGE 55

Figure 2-14. Examples of shallow, temporary pools at the margin of the Austin Gary control dome.

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50

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Figure 2-15. A. Dipping for mosquito larvae from stump hole covered with emergent vegetation. A seepage hole at the C. Dipping for larvae margin of dome S-2. from a seepage hole.

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52

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Figure 2-16. A, A stump hole at C-1. B. A tree hole at C-1

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55 experience, could be identified as being probable breeding sites, A similar transect (90 degree, 190 feet) was sampled at S-2. Virus Activity Sentinel Chickens Viral transmission studies were done using white leghorn, sentinel chickens, approximately 12-18 months old, as the experimental animals. Chicken pens were constructed with plywood as demonstrated in Figure 2-17B, In February 1976, five of the finished pens (Fig. 2-17A) were placed in each of domes S-1, S-2, C-1, and AC. The pens were distributed from the center of each dome to the margins, with at least two pens at the margins. Each pen was numbered and held two chickens (one of which was marked so that the two could be distinguished). Plastic water dishes and feeders were installed in all the pens. Before being placed in their pens, the birds were bled to determine any previous contact with encephalitis. After this initial bleeding, the birds were bled (Fig. 2-18) on a regular basis every three weeks. Three milliliters of blood were taken from a wing vein using a 23 gauge needle. The blood was allowed to clot and held on a slant at ambient temperature for one to two hours. The samples were then refrigerated overnight at about 5C. The next day serum was removed from the clots by low speed centrifugation (ca. 2000 rpm for 5 minutes). The serum samples were then stored frozen at approximately -15C in stoppered test tubes until serological tests could be done to determine the presence or absence of antibodies against the different encephal i tide viruses.

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Figure 2-17. A. A sentinel chicken cage at the margin of S-1. B. Construction of the sentinel cages.

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57

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Figure 2-18. Taking a blood sample from a sentinel chicken.

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59

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60 Once a bird was shown to have developed antibodies against one of the encephalitis viruses, that bird was replaced. New birds were always pre-bled to determine previous infection. In June 1976, ten more pens (20 more chickens) were added to the sentinal bird study. Five were placed at C-2, and the others were scattered throughout the City of Gainesville (Fig. 2-1). Mosquito Pools In July, August, and September of 1976, mosquitoes were collected alive with CDC light traps at S-2 and C-2. They were taken to the USDA, Insects Affecting Man, Laboratory in Gainesville where they were sorted and pooled in a walk-in cold room. The pools were sealed in test tubes and stored over liquid nitrogen at approximately -90C. Later the samples were transported on dry ice to Tampa for viral isolation attempts. Dr. Arthur Lewis of the Epidemiology Research Center of the State Health and Rehabilitative Services Department did the isolations using the suckling mouse technique described by Sudia and Chamberlain (1974). Viral identifications were based on serum neutralization results. Mammal Sampling Periodically mammals were trapped at C-1 C-2, S-1, and S-2. Larger mammals (Racoons, bobcat and opossums) were anesthetized by intramuscular injection of ketamine hydrochloride at 8-10 mg/kg of body weight as recommended by Bigler and Hoff (1975). Three milliliters of blood were taken from the larger mammals either by cardiac puncture or from an arm vein (Fig. 2-19), The smaller mammals (rice rats, cotton

PAGE 67

Figure 2-19. Taking blood samples from small mammals.

PAGE 69

63 rats, and cotton mice) were anesthetized with ether and bled from the orbital sinus (Fig. 2-19). Only about 0.2 ml of blood was taken from the small mammals and this was diluted 1:5 by volume in field diluent as recommended by Sudia, Lord, and Hayes (1970). The animals were toe-clipped (small mammals) or ear-tagged (large mammals) and released in the same area where they had been caught. Serum f-rom the mammal samples was collected and stored as previously described for the chicken samples. Serology: Hemagglutination Inhibition Test The sera from the sentinel chickens and mammals were screened for viral antibodies using the hemagglutination inhibition test (HAI) (Clark and Casals, 1958). The mammal samples were extracted with acetone to remove lipids, which might interfere as non-specific inhibitors of hemagglutination. The chicken samples were treated with protamine sulfate and then acetone extracted to remove non-specific inhibitors. Non-specific agglutinins were removed by goose red-blood cell absorption. Viral antigens and goose red-blood cells were prepared for this study by the Epidemiology Research Center in Tampa, and the serological tests were conducted at the Tampa laboratory by the author. The chicken sera were tested against Eastern Equine Encephalitis (EEE), Western Equine Encephalitis (WEE), and St. Louis Encephalitis (SLE). Mammal samples were tested against EEE, WEE, and SLE, and in some cases against California Encephalitis (CEV) and Venezuelan Encephalitis (VEE). Preliminary titrations were done to determine the dilution of antigens which would give 4-8 hemagglutinating units in each test. Back

PAGE 70

64 titrations were carried out on the day of the test to insure that 4-8 units had been used in each test. Serum controls were done to make sure that serum agglutinins had been removed by absorption with the goose red-blood cells previous to the test. The tests were conducted in microtitter plates (Fig. 2-20). Serial, two-fold dilutions of the serum samples were made with wire loops (Fig. 2-20). A dilution of 1:640 was the highest dilution of serum tested. Antibody titers were designated as the highest dilution to give complete hemagglutination inhibition. A titer of at least 1:20 was considered positive. Occasionally a titer of 1:10 with partial inhibition beyond this dilution was considered positive. Serology: Neutralization Test Samples, which were positive for HAI antibodies, were then tested for the more specific neutralizing antibodies. Serum neutralization tests were done using the constant serum-varying virus titration method (Dulbecco and Ginsberg, 1973). African Green Monkey Kidney Cells (BGM) in tissue culture tubes were used as a susceptible indicator system. Neutralization indices were determined by the ReedMuench method (Lennette, 1969). A reduction index of at least 1.7 logs was considered positive.

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Figure 2-20. Dilution of serum samples was done in microti ter plates. The lower photo is a sideways shot of a finished test plate. The next to last row (C-2, M4; the marked bird in pen no. 4 at dome C-2) shows a positive antibody titer against eastern equine encephalitis. In this case complete hemagglutination inhibition is present at a serum dilution as high as 1:320.

PAGE 72

1^ It ^ 1.t'^ il ^

PAGE 73

RESULTS AND DISCUSSION Adult Mosquito Sampling Ramp Traps Using no known insect attractants, ramp traps are flight interception devices which capture those mosquitoes whose flight paths are directed toward the ramp openings. Relatively small sample sizes are expected when using such unbaited traps, and this was indeed the case. With four traps per swamp in 1974, the cumulative data in Tables 3-1 and 3-2 are probably reasonably accurate representations of the relative population compositions. Figure 3-1 graphically compares the relative abundance of the seven most common species or species groups from both domes. With the exception of the floodwater Aedes species^ community structure was very similar at both domes. ShannonWeaver diversity indices are computed and recorded in Table 3-3. The Shannon-Weaver index, as described by Price (1975), is a measure of both species richness and evenness of abundance. The index for dome C-1 is slightly larger than that for S-1 Since there were two more species captured at S-1 than at C-1, the larger index for C-1 represents a slightly more even distribution of species. Only four species were not present at both sites and, where present, each represented only a minor component of the mosquito fauna collected by the ramp traps. Aedes species refers to the several species identified. 67

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68 Q. O o •a"^OOOOVOOCMO 000000U30 \0 1 — 1 — ^ 1 — r— CTi ro CO^ I— CT^LnCOi— CM i-DO OOi— UDOO cn OD O CM CM CM 00 CO T3 OJ -(-> u o o O) "o u +j Q (/I OJ OJ t/1 o 4-> cr >to r— o E o S CO c: 10 0) T3 O) CO CM CM CMO CTii — VOCMCMlD v£) 1 — ro CM ro 1— CM r— CM I— CO CO CO f— CTi CM 1— ^ Ln CO 00 CM I— I— O CM CTi IX) CM cn CO LO f— Ln 00 r— r— aCM r— cn o CM (A CO LO c E c ra X re •r>, ••o SSo "O S"o O) n re o (_) u Ms:3 (j cr ro re 03 QJ SSito to o o o CU cu +-> -C -£Z Q. Q. Q. I— CM CO CO If) o CO CO to o >X3 CO O to CO Lf) cn CM o CO tn LD CM IT) in CO CO C3~i o CM vo I— :3 o to to to ZJ Q. c: 1— re ire 4-) re Q. •1 — c: •r— S•r— SScr Ol re 4-> to c: X X X 0) 0) (U 3 o o o to 3 +-> re u to re QJ c: •rCT to c OJ Q. c re re ic 3 -)-> i•rre re re 0) +j to o o 3 c c cre re 3 o SiO o ro cDLn^d-CM CM cn tn toco cm r— ^ r— O CM I— UO CM ^ CM CM CO u 0) Q. to cu c (U o CM CO ^ CM CO o CO f— LD CO CM CO o o CO CO VO ID CO CO CM ^ CO o o to CM cn LD ro O r— 00 r0) to re o)

PAGE 75

4jr CD • • I/) •r— c/)
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    PAGE 76

    C/1 ^o) O r— Q(/) E O) "3 I — l/l fO E Q. 0) n3 i+S-t-J 4I O Q. E O) 03 O Sc fa ^ o C 4+-> m ta CL I— 3 QJ O SCT) Ol ^ sho I rn OJ sOl •r-

    PAGE 77

    71 o U) (J sndiedojbiu J3;eMpoo) j I MO I (uoiuoDoueis^Aj) euijiL|ddes Biuae^ouejfi ejnu eiauu

    PAGE 78

    72 Table 3-3. Shannon-Weaver diversity indices computed from 1974 ramp-trap data at S-1 and C-1 S-1 C-1 Species Pi Pi(^9QPi) ^ Pi(lngePi) Aedes : mitchel lae .0026 -.0155 .0005 .0038 vexans .0004 -.0031 .0005 -.0038 infi rmatus .0060 -.0307 .0109 -.0493 dupreei .0004 -.0031 .0027 -.0160 at 1 ann cus 1 /i 1 1 383 -.2736 canauen sis UUU4 C\f\ O 1 0082 -.0394 tden 1 urnyncnus UUU4 -. UUJ 1 Tu 1 vub pail ens UUoo nolo Anopheles: cruci ans 0693 -.1850 0552 -.1599 quadrimaculatus .0043 -.0234 .0197 -.0774 Cul ex : (Melanoconions) .1022 -.2331 .1001 -.2304 territans .0090 -.0424 .0284 -.1011 od 1 1 ndr 1 us • U 1 d4 AC /I O -. 0643 .0071 -.0351 II 1 y n pa i pus uoy J 1 o£r A .0569 -.1631 P 1 p 1 ens UU^: 1 m o n -.0129 .0005 -.0038 restuans .0009 -.0063 Cul iseta : iiie 1 an ura 077C .CI lb o c c o -.3558 .2269 -.3365 Coquillettidia: perturbans .0188 -.0747 .0109 -.0493 Psorophora: cil iata .0011 -.0075 columbiae .0094 -.0439 .0049 -.0261 ferox .0137 -.0588 .0202 -.0788 Uranotaenia: sapphirina .2528 -.3476 .2203 -.3333 1 owi i .0787 -.2001 .0864 -.2116 H' = ip-log^p. = 2.0862 H' = 2.1998 H' = Shannon-Weaver diversity index P^ = the proportion of the ith species in the total sample

    PAGE 79

    73 The four traps at each dome were not equally successful in capturing mosquitoes (Table 3-4). At S-1, the traps at the northern (0) and eastern (90) edges of the dome caught approximately twice as many mosquitoes as the southern (180) and western (270) traps. At C-1 the eastern and southern traps were most productive. This information indicates the importance of site selection and the uncertainty in sampling andcomparing habitats by this method. Overall abundance of mosquitoes from the two domes is compared in Table 3-5 using a rank comparison test (Mendenhall, 1968). Only samples collected on the same dates are considered. There is insufficient evidence from these data to disprove the null hypothesis that the populations are identical with respect to abundance. The ramp-trap data for 1974 show no greater mosquito production in an experimental swamp receiving sewage effluent than a control swamp receiving untreated groundwater. Community structure of mosquitoes also appears very similar in both domes. Very few Culex pipiens quinquefasciatus were collected at S-1 even though this species has a reputation for developing in great numbers in heavily polluted water. In July and August 1974, the design of the ramp traps was changed as noted above (Materials and Methods). During the winter of 1975, the traps were rearranged with two traps at S-1, S-2, C-1, and AC. One was placed in the center and another at the margin of each dome. Data from 1975 cannot be compared with those of the previous year because of the changes in trap design and rearrangement. Numbers of mosquitoes captured in 1975 are summarized in Tables 3-6 through 3-9. By inspection Culex territans appeared to be much more abundant at S-2

    PAGE 80

    74 So a. ra 5+J I Q. E ra i4O ^ ra E E :3 00 I CO CD ra cu 0 +j n3 CO CJ to 0 o <^ CTi d CU s_ o o 0 o (_) +J CU ra 0 cu o O (J CO (T3 1/1 E cu So r— O +-> <+1 — SZ5 cu CJ o c CO O o CD a E 4O o Scu +-> JD o ra E O +-> :3 sr I— o • 1 — ID C\J CTl vt 1— CO CO OO 1— CM CO 1 — r~. 1 — 1 — CO to c 3 T3 cu > o CO (O •r— s_ (0 -M X 4d, cu sz +-> 4E > o -a to to to to to (/) to (/) cu cu cu cu cu cu cu CU r— T3 o o -o (t) ^ So cu +-> to cu -a OJ CM CO O c^ CM o CTl ^ ^iuo o CO So C) cu ra -Q E :3 o CJ CO s_ o a. o s_ o CO CX CO So d o So to Cl. O OO O CM CM CM CTl CO CO CO CO en Ln tn CM CO CO un c\) CM CM CM Ln CTl CM Ln o en 1— ^ .— r— CO CO CO Ln CO en CM C£> LO en i — — CXD CM I— 1— CO t--~ CM 1— CTl CM CM CO ^ OvJ CM 1— CM r— IJD +-) ra CO CO cn Ln CO o o 00 CM CO cn CO to CJ to 3 CO c +J 03 ra E •M to C/) i. CT) to Q. +-> cu cu cu CO •1 — CU fO cu 4-1 CO s_ d +-> cu CU cu x: X X X X X CO d cx cu cu cu cu
    PAGE 81

    ; — f*^ *-,o r— CO CO QJ Q 4-" fO CD O C\J "O c 1 <_5 (D o O CO t J "D cn 00 — £Z OJ o 0 o ( — ) to CO Q. LO fO S+-' +-> o o I J CnJ C\J a LO Ol 4-> O O) — — o o o CO 00 OO o CO LO ^ (/) 0) o r— -M 1 cr o in o ^ o Lf) CO O 3^ 1^ E r — i+O sJ2 E < r— CO cn O CO to CO CO CTl c •rlA sC (U E 'r— Q. s o (T3 o cu l>1 Sp (U ro CO •r— X) O) Ol +-> o O Ulc: c ns (D Ol CO JS_ X3 na OJ ID •1 — +j c o to ZD (—

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    76 Table 3-5. Rank comparison test comparing abundance at S-1 and C-1 from ramp-trap data (1974). Total number of mosquitoes per four ramp-traps Trap nights: S-1 C-1 May 13-14 17 10* n=total number of trials 21-22 14 15 (excluding those where 23-24 6 28 (S-1)=(C-1)) 27-28 160 99* 30-31 115 99* p= probability of (S-1>(C-1) June 3-4 31 41 qprobability of (C-1 )>(S-1 ) 6-7 82 44* y= number of times (S-1)>(C-1) 11-12 33 40 P= np 13-14 43 69 a= "/npq 17-18 50 61 y= 15 20-21 82 43* n= 30-1=29 24-25 107 127 null hypothesis: p= ^0.5, q=0.5 27-28 384 reject if |Z=J^|>_ 1.96 July 3-4 131 it a=0.05 4-5 209 1 1 fi* since |Z| = 15-14.5 = .267 < 1.96 8-9 ?fi 1.87 Sept. 12-13 ?77 L. / U do not reject the null hypothesis 16-17 ??? 1 RR* 1 oo at a =0.05 19-20 1 71 1 o / 23-24 40 1 01 Oct. 3-4 5 5 7-8 127 129 10-11 48 52 14-15 99 59* 17-18 34 17* 24-25 54 24* 28-29 96 93* Nov. 4-5 66 60*9-10 17 22 16-17 10 34 (S-1)>(C-1)

    PAGE 83

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    PAGE 85

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    80 3 I/) Ol iQ. ra i~ +-) I Q. B ra CTl ro (U 3^ I/) CD O o cu o +J >Jun -rCT O 3 I. Q. CO CM ^ CO un o 1— o CO C\J CVJ t3 1+O $X5 re 1/1 c o re to +-> X +J 3 0) x: Mi > cn •r— i/) re o c— CM CM 10 o to to c M re re re 1+JD re cu Sc (L) 3 3 3 re a +-) re •1 — t/1 re o c s4-> -Q c E to •rre QJ ^ *rre E X re sz I/) 3 3 SCL Q. •( — 3 o •r— z. o to 3 Cl. cr 3 Q, iU o to C re re o o E +-> •r— SSsto _a) 1r— CT to Cl. -M c o o o to c sCL 4-> re re Cl Q. Q. OJ QJ 4-> +-> o O O X X X X X X 1/5 •ro o SSSd Cl OJ <_) o ZD (J OJ OJ to -o OJ c OJ cn CM fO r— O 1,0 CM CM to o re 1—

    PAGE 87

    81 than any of the other domes, and Culex ( Melanoconion ) spp. were more abundant at C-1. Otherwise there were no noteworthy differences in species composition at the different domes. Culiseta melanura a medically important species, was abundant in all the domes. It is interesting that AC, a large dome, which at times harbors great numbers of mosquitoes, produced very few in the ramp trap samples. The new trap design and arrangement failed in 1975 to capture many floodwater Aedes species. Field observations have shown these species (especially Aedes atlanticus and Aedes infirmatus ) to be concentrated in the dense understory at the margins of the domes. Of the small numbers of these species trapped in 1975, 92.5% (37) were from traps at the margins of the domes and only 7.5% (3) were from traps at the centers. In 2 years of service, the ramp traps demonstrated that the most abundant species of adult, female mosquitoes in the cypress domes of this area are species which feed primarily on birds or cold-blooded vertebrates. Culiseta melanura Culex ( Melanoconion ) erraticus ^ and Culex nigripalpus were plentiful in all the domes sampled. These species are primarily avian blood feeders, with Culex nigripalpus possibly shifting from birds to mammals at certain times of year (Edman, 1974). Culex territans (common at S-2), Uranotaenia sapphirina and Uranotoenia lowii are all thought to feed primarily on cold-blooded vertebrates, expecially amphibians. Anopheles crucians another species abundant in these domes, feeds primarily on mammals, especially rabbits. Several woodland, floodwater species of Aedes and Psorophora were found breeding in large numbers in temporary pools adjacent to the lTh is species was assumed to be the most common of the Melanocon ion complex.

    PAGE 88

    82 domes and in some cases in the shallow marginal pools of the domes themselves. The adult females of these species are primarily mammalian blood feeders, readily feeding on humans. They rest during daylight hours in the dense, shaded vegetation at the dome margins but bite when disturbed. The ramp traps have demonstrated that adult, female mosquitoes in swamps receiving sewage effluent are the same species found in a similar swamp receiving untreated groundwater. The added nutrients in the effluent have not been shown to result in an immediate or significant increase in overall mosquito abundance above what could be expected by flooding a similar dome with low nutrient groundwater. New Jersey Light Traps New Jersey light traps have been widely used in this country to monitor mosquito populations for almost 40 years. They act as a biased sampling device, being very selective for positively phototropic species. Unengorged females represent the majority of the usual catch. These traps have been used at S-1, C-1 WMHP and S-2. Sampling at S-1, C-1, and WMHP began in September 1974 and at S-2 in May 1975. Results from these trapping studies are recorded in Tables 3-10 through 3-13; a graphical representation of monthly totals is expressed in Figure 3-2. It is obvious from Figure 3-2 that sewage dome S-2 was producing larger mosquito samples than either S-1 or C-1, and that S-1 was producing slightly more than C-1. The degree of similarity among the three domes is calculated on the basis of female species abundance and community structure (relative abundance of female species) and recorded in Tables 3-14 and 3-15

    PAGE 89

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    PAGE 90

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    PAGE 91

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    PAGE 93

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    PAGE 94

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    PAGE 95

    to OJ Q. E fO t/J a. ra sM 4-> JZ ai •r^ 10 $Ol (/) "-D 2 •r— cu CO -(-> Mc O 0) s(U O Mco 4rST5 rO Q. SE o O o 4CM CO 0) s-

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    90

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    PAGE 98

    92 o c -a > •r+-) ra 'oj I CM 00 CO I ro I — I in cn o I II o I lO I II o C\J I 00 O u o o I CO o CM o II o I ro o 00 I II I CO I II Ln I Ln CTi o o I— o II II II CO o CM O O I I I ro CO o ro CM o IT) CM II CM Lf> LD II CM O <•£> O U3 I CM CM l~~ 1 oo 1X3 Ln 00 LO CO O en CO Ln O ro Ln o CTl II CD CO II II o 1 1 o LO 1 II (_) II o II II II II II II II II ( — 1 CO UD o Ln vo 00 o CM o 1 CM CM o o O Ln CM o o •=3o 1 1 1 1 CTi 1 1 1 t 1 1 1 OO CO o 1^ CM un CO Ln 1 CO ^ 00 00 o o CO CM ( o Ln 00 Ln o CM Ln 00 Ln Ln LD Ln Ln LD CTl CO o O CD II CM o 1 1 o 1 1 o 1 II II II OO II II II II II II II II II CM CO 1 CO CO CTl CO CO o UO 1 cn LD o o O CO CM o LD Ln 1 1 1 1 1 1 1 1 1 OO CT) CO o Cvj Ln 00 Ln 1— 1 CO CO OO o o CO CM o Ln to 13 c +-> ro re i3 O c +-> to re in ro o ic c E (/> re cu re •r— •r(O •r— c 3 s_ CL +-> E cx •r— so (/I 3 CL 3 •r— 3 3 u o o c C re \a 1 — ro 0 fO o re re re 3 0 to •r— 3 c -t-j re CL o T3 0 E u cr re c •r— ai 1— ro ro s_ sE +-> re •r— •rI/) i/) (U SCD 4-> Sc C 4+-> LO cr +-> ro ro 0 OJ 0) c Q. !-> -C X X to o 0 0 0 X CL Q. OJ a;
    PAGE 99

    93 respectively. Degree of similarity is based on the amount of differences when comparing two domes at a time. Of course the smallest values represent the greatest similarities. One would expect less difference in a comparison between the two sewage domes than in a comparison between either of the sewage domes with the groundwater, control dome. This was not the case, and on the basis of both abundance and community structure S-1 and C-1 were more alike than S-1 and S-2. Of the three comparisons, S-2 and C-1 were least similar. In examining Tables 3-14 and 3-15 the only obvious and consistent differences between the sewage domes collectively and the control dome was the greater abundance of the two species of Uranotaenia especially Uranotaenia sapphirina at the two sewage domes. Starting in June 1975, males of IJ. sapphirina at the two sewage domes were counted and recorded from the light trap samples; 75.0% (673) of the males at C-1, 83.0% (1,212) at S-1, and 87.0% (3,449) at S-2 were U. sapphirina It should be obvious at this point that the graphs in Figure 3-2, which demonstrate greater mosquito production at the two sewage domes than at the control dome, are heavily influenced by the sample sizes of IJ. sapphirina From both a medical and an economic standpoint this species is of no known importance; and therefore, a more meaningful comparison of the New Jersey light trap data is represented in Figure 3-3, which computes the mean number of adult females per month leaving out L[. sapphirina This information shows that with the exception of U. sapphirina there was very little difference in sample sizes among the three domes, and the sewage domes were not consistently producing larger sample sizes than the control dome.

    PAGE 100

    (/) Oi <0 c l|_ O. (t3 +-> to fO •r— r— cu >) (O O) +-> (/) o Sc: m i~ ID 4o 00 CO CP) IZ

    PAGE 101

    95

    PAGE 102

    96 In comparing samples from the domes and the nearby trailer park, there are obvious differences. The light traps indicated fewer mosquitoes active in the trailer park; however, this result was likely affected by the interference of the several other light sources within the park. Two of the most common species in the trailer park samples were the avid, human blood-feeders, Psorophora columbiae and Coquillettidia perturbans C. perturbans was relatively rare in the domes, and probably the domes contribute very little to its abundance in the park. There are several, permanent-water habitats with floating and emergent vegetation which are closer to the park than the domes. These areas are probably responsible for most of the C^. perturbans activity within the park. Psorophora columbiae the most abundant species within the park, was not being produced in the cypress domes at all. Larvae of this species were found developing in great numbers in shallow depressions in the pine plantation surrounding the domes and trailer park. The depressions were created by the mechanical removal of dead trees and the replanting procedures after the 1973 fire. Culex ( Melanoconion ) spp. and Anopheles crucians were abundant at both the park and the domes; however, it has been the author's experience that neither of these groups is of great annoyance, and neither is restricted in its breeding habits to the cypress domes. The impact of the effluent on mosquito populations can be divided into two categories: 1) the effects resulting from changes in water chemistry and altered vegetation (these are responses to increased nutrient loading), and 2) the effects resulting from a change in the normally fluctuating hydroperiod of the domes. Since water levels were maintained relatively constant at S-1 S-2, and C-1 differences

    PAGE 103

    97 in mosquito populations at these domes are assumed to be a result of the differences in water chem-istry and altered vegetation patterns. This of course is itself based on the assumption that mosquito populations at all three domes were very similar previous to treatments. In this respect neither ramp traps nor New Jersey light traps have demonstrated any immediate change which might be of public health significance. CDC Light Traps The CDC light traps operate on the same principle as the New Jersey light traps, but because they are powered by small six-volt batteries, they are much more portable. The CDC traps were used to compare the mosquitoes from S-2 and the two untreated domes, AC and C-2. The AC dome, as previously described, is a large swamp with a yearround accumulation of standing water. During drier periods of the year its margins recede and its standing water is limited to a much smaller central pond. The other control dome, C-2, is very similar in size to S-2. It holds standing water during the wet summer months, but during the. fall and winter it has no standing water. Similar patterns to those of C-2 were undoubtedly true for S-1 and S-2 before the addition of effluent. It has already been pointed out that there was little change in the adult mosquito fauna of S-2 as a result of the increased nutrients added to its water supply; any differences between S-2 and C-2 are therefore attributed to the flooding and maintenance of a relatively constant water level at S-2. This conclusion, again, is based on the untestable assumption that the mosquito fauna at S-2 was

    PAGE 104

    98 similar to that at C-2 before treatment with effluent. Results of the 1976 sampling at AC, C-2, and S-2 with the CDC traps are recorded in Tables 3-16 to 3-18, respectively. Figure 3-4 summarizes the data for the seven most common species in terms of mean abundance of adult females per trap night. There are several points to be made from the data. This sampling technique indicates that mosquitoes were more abundant at AC than at either S-2 or C-2. Results from other trapping devices (ramp traps, malaise traps, and suction devices) have not supported this observation. Unlike light traps which attract and concentrate their catch from a relatively large area, ramp traps, malaise traps, and suction devices sample only a small, prescribed area. The overall results would thus indicate that AC did not have as high a concentration of adult mosquitoes; however, because of its much larger area, it had a greater number of adults during the mosquito season. The CDC traps indicate that AC was producing large numbers of floodwater species as well as those species which develop in more permanent aquatic situations. The really important comparison, showing species differences, is between S-2 and C-2. Figure 3-4 and the rank comparison data in Table 3-19 show that S-2 was producing greater numbers of Culex ( Melanoconion ) spp., Coquillettidia perturbans and Uranotaenia sapphirina than C-2. On the other hand, C-2 produced greater numbers of floodwater Aedes species than S-2. There is the indication that there were more Culex nigri palpus and Cul iseta melanura at C-2 than at S-2 (Fig. 3-4); however, the rank comparison tests of Table 3-19 show insufficient data to confirm this. Anopheles crucians appears to have been more abundant at S-2, but the rank comparison test again fails to confirm it. In any 1

    PAGE 105

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    PAGE 106

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    PAGE 107

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    PAGE 108

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    PAGE 109

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    PAGE 110

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    PAGE 111

    105 event, these latter three species were produced in great numbers at both domes. The obvious conclusion from these results is that by maintaining the water level at S-2 with effluent, egg-laying sites of the woodland floodwater species have been destroyed and new oviposition sites for Culex ( Melanoconion ) spp. Coqui 1 lettidia perturbans and Uranotaenia sapphirina have been created. Three other species, Cul iseta melanura Culex nigri palpus and Anopheles crucians which were probably abundant before the effluent was added, were still abundant after the treatment. Since both CDC light traps and New Jersey light traps function on the same principle, it is tempting to compare results at C-1 and S-1 from New Jersey traps to results from CDC traps at S-2 and C-2. However, caution must be employed when comparing trapping results using two, even slightly different methods. For example, Table 3-20 gives a summary of results at S-2 for the summer of 1976 using New Jersey traps and CDC traps. The differences are striking, and it is not clear whether they are due to differences in trap design or the placement of the traps within the dome. The New Jersey trap was operated only at the center of the dome; whereas, the CDC traps were moved around to sample different locations within the dome. Table 3-21 summarizes the CDC light trap data for S-2 and AC on a seasonal basis. These data were collected from 1974 through 1976, and are useful in revealing the species present in the winter months as adults. The floodwater species are notably absent, as is Coqui 1 lettidia perturbans in the winter. Adults of many species of Culex Culiseta melanura A nopheles crucians and Uranotaenia sapphirina survive the winter as adults in this part of Florida.

    PAGE 112

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    PAGE 113

    Table 3-21. Results from CDC traps at S-2 and AC grouped on a seasonal basis. Trap nights Females: Aedes fulvus pallens Aedes mi tchel lae Aedes vexans Aedes canadensis canadensis Aedes infirmatus Aedes dupreei Aedes atlanticus Anopheles crucians Anopheles quadrimaculatus Culex ( Melanoconion ) Culex territans Culex sal inarius Culex nigri palpus Culex restuans Culiseta melanura Coquillettidia perturbans Mansonia indubitans Psorophora ciliata Psorphora columbiae Psorphora ferox Uranotaenia sapphirina Uranotaenia lowii AC >5 CD AO JD Ol 2: < 1 u_ 1 1 +-> 1 c c Q. 0 >> 0 n3 ~Zi O) eC U1 1 a> +-> 1 il c Q0 ID QJ (U s: T3 (/I Q 7 1 5 5 193 23 169 575 130 46 12 5 27 352 113 4 3 9 1 29 17 3 1 1 942 103 14 1 199 822 170 35 56 95 34 5 5 1 1 2 1 279 1246 764 8 3 22 21

    PAGE 114

    108 Temporal distributions of the eight most common species or species groups are recorded in Figures 3-5 through 3-12. These data are based on results from 1976 comparisons at AC, C-2, and S-2. With the exception of Coqui llettidia perturbans all species had one summer peak of abundance; C. perturbans had two seasonal peaks, one in May and one in July. Suction Dev>ces Suction devices such as the commercially available D-Vac have been used by mosquito investigators to sample resting adult mosquito populations during the daytime. Most other trapping devices depend for success on the mosquitoes' nighttime flight activity. These suction devices have been especially useful to other workers in collecting males and blood-engorged females. Late in the summer of 1975, a D-Vac was obtained and a limited amount of sampling was done at S-2, AC, S-1, C-1 and WMHP. In the smaller domes (S-1, S-2, and C-1) the sampling was done in the thick vegetation at the margins and around cypress trees and hollow stumps and tree bases inside the dome. The sampling at the trailer park was carried out around the edges of trailers and in areas of tall weeds. Results with the D-Vac, recorded in Table 3-22, were very similar to those obtained with the ramp traps. One notable exception was the relative abundance of the floodwater species, Aedes atlanticus in D-Vac samples. The scarcity of this species in 1975 ramp-trap samples indicates that the alteration of the ramp traps after 1974 somehow affected their ability to capture this species. The floodwater species were rarer at AC in the D-Vac samples, presumably because the sampling

    PAGE 115

    Figure 3-5. Aedes atlanticus abundance in CDC light trap col lections

    PAGE 116

    no April May June July Aug. Sept.

    PAGE 117

    Figure 3-6. Aedes infirmatus abundance in CDC light trap collections.

    PAGE 118

    112

    PAGE 119

    Figure 3-7. Anopheles crucians abundance in CDC light trap col lections

    PAGE 120

    114 180' 160140120cn ^1008040' 20 0' C-2 •S-2 •AC-1 April May June July Aug. Sept.

    PAGE 121

    Figure 3-8. Culex ( Melanoconion ) spp., abundance in CDC light trap collections.

    PAGE 122

    April May June July Aug. Sept.

    PAGE 123

    Figure 3-9. Culex m'gn'palpus abundance in CDC light trap collections.

    PAGE 124

    118 April May June July Aug. Sept.

    PAGE 125

    Figure 3-10. Culiseta melanura abundance in CDC light trap collections.

    PAGE 126

    140April May June July Aug. Sept.

    PAGE 127

    Figure 3-11. Coquillettidia perturbans abundance in CDC light trap collections.

    PAGE 128

    I < I 1 it April May June July Aug. Sept.

    PAGE 129

    Figure 3-12. Uranotaenia sapphlrina abundance in CDC light trap collections.

    PAGE 130

    124

    PAGE 131

    Table 3-22. Sunmary of D-Vac sampling for 1975. 125 Aedes fulvus pallens 0 Aedes infimiatus 5 Aedes atlanticus 30 Anopheles crucians 2 Anopheles quadrimaculatus 0 Culex (Melanoconion) 15 1 1 Culex nigripalpus 4 Culiseta melanura 12 Psorophora ferox 0 Psorophora columbiae 0 Coquillettidia perturbans 0 Uranotaenia sapphirina 19 Uranotaenia lowii 33 Males 74 Total sample time (min.) 20.5 Mosquitoes per minute 9.5 Number of mosquitoes -J c AC WMHP Ml II 1 I 1 0 0 0 3 13 2 0 41 88 0 1 9 14 27 0 1 0 0 0 34 55 25 0 7 64 18 0 2 21 33 0 55 134 73 0 0 6 0 0 0 0 0 44 0 0 2 0 42 103 75 0 32 25 7 0 253 750 208 0 30.0 63.0 90.0 15.0 16.0 20.2 5.2 3.0

    PAGE 132

    126 omitted the margins where these species are more concentrated. The limited sampling at the trailer park confirmed the New Jersey light trap results that Psorophora columbiae a floodwater species not reproducing in the domes, was abundant in the park. A homemade suction device was used in 1976 to sample at S-2, C-2, and AC, Sample times were recorded accurately with a stopwatch. To avoid picking up large amounts of leaves and other debris, sampling times were limited to approximately three minutes. The results of these collections are recorded in Table 3-23. The relative abundance of the seven most common species or groups are represented in Figure 3-13. Table 3-24 lists the species according to breeding site preferences. It appears from these data that by creating a permanently aquatic habitat at S-2, those mosquitoes that lay their eggs on the water's surface have increased; and the floodwater species, which lay their eggs in shallow depressions and depend for development upon periodic flooding, have decreased. This conclusion is based, of course, on the assumption that S-2 and C-2 had similar mosquito faunas before the effluent was added. The dome at AC was again sampled primarily at the center, and thus the results are assumed to be low for the floodwater species. Otherwise, AC appeared very similar to S-2. Mosquito populations do not appear to have been as highly concentrated at AC as at the other two domes. Malaise Traps Malaise traps of various designs have been recommended by Breeland and Pickard (1965) and Gumstream and Chew (1967) as unbiased sampling devices which can be used effectively to sample mosquito populations.

    PAGE 133

    O) u +-> > c C Tna Ol -!-> -O O ro C 1<— 3 (U Ol X3 Q. Sn3 CM I o OJ o 4> > C C -rra OJ +-> -a O (O c 5r3 O) O) ^ CL. irtJ CM I C/0 M > C TOJ 4-> O rc3 i. r— OJ OJ a. s_ -i-j o 00 •— O UD n CM I— ^ CTi CO CO I— m un ID o CO CM CM CO ui 1— CO I— CO o CO CM CO o CM o CO Cvl ^ CM 00 CM I— O CO O LD f— O O I— CD I— O 1— CM I— r-^ Lf) r— CO CO CM un Lf) o CvJ o CO o 00 CM 00 en CO CM CM CO 1X> CTi .— o 1— 00 CM cn CO tJD ^ I — Ln CO Ln CO CO o U3 O CO 1,0 CM CM lO CO CM CTi CO CO CO ^ 00 1— CO CTi 00 CO cyi CO CM CO CO c CO 3 c +-> (U to o Sc •f— rt3 cu I/) to ro c 3 X (O 3 •r— o CL 3 o Q. +-> O U o C (T3 irrs •r— 3 o (t3 (O ro OJ t/) E 4-) SM Q. TD SC (_) ro r— •r— O) •r— > to SSE 4-J ro to cn +-> Sc: OJ 0) r— ro CD o 1 — fO +-> c: -•-> O ru SlO cu ro CU 3 C i. (U C3. E i/l •1CU +-> O )-> a -11 — 3 a. cr E to ro O 00 s: (U

    PAGE 134

    c o o QJ u o +J > (/) QJ o -o E C c o QJ > +-> a o n3 •+E o QJ E I/) O l/l •1— QJ +J to O QJ OJ Q. o; 1/) CO i. =3

    PAGE 135

    129 rg oj (J ^ 6 < O o CO o rvj O ja"ieMpoo|j sndiedubiu 9UH';iJJ9'i (UOIUODOUei9H) X3|n3

    PAGE 136

    130 Table 3-24. Comparison of S-2 and C-2 mosquito faunas in terms of breeding site preferences. Percentage of total sample that oviposit in habitats of: Permanent water Temporary water S-2 89.2 10.2 C-2 55.1 44.9

    PAGE 137

    131 In 1974, two tent-type malaise traps were stationed in permanent positions at the margins of AC and S-2. Results of collections from these traps are recorded in Table 3-25. The traps caught very few mosquitoes, and catches are listed only to show a comparison of the efficiency of the different trapping devices. Although the traps failed to capture large numbers of mosquitoes, perhaps because of their design, they did capture several other types of flies, including the tabanids, of which the most common species at both domes was Diachlorus ferrugatus (Fabricius). This particular species, which was most abundant in the early spring at both domes, is a severe human pest with a very painful bite. It bites during the daytime and is the most annoying pest in the swamps in the spring. Truck traps Bidlingmayer (1966) has described the truck trap as a non-attractant sampler which very effectively measures nocturnal, mosquito flight activity. One of these devices was used to sample mosquito populations within the trailer park adjacent to the treated domes. The total sample for 22 nights from August 1975 to August 1976 is recorded in Table 3-26. Samples were small since the device was used only for approximately eight minutes per sample (time required to travel over all of the streets in the park twice at 20 mph). The results are consistent with those of the New Jersey light-trap samples for the park. Psorophora columbiae was again the most abundant species with Coquil lettidia perturbans ranking third. It should be noted that Culiseta melanura was not restricted to the cypress domes in its nighttime flight activity.

    PAGE 138

    CO O) u 0) o o 0) o -l-> •I — cr to o 0) ta 4-> CL a; D1 3 E 3 IX) cr Q. ra a s. M v> •r(O r— fC s: O t: ra 3 (/I Lf) CVJ I 00 3 c 3 •-3 03 03 132 1— 3 VO O r— 00 If) X) CO CM CM ro 1— CO CO ^ r— CM cvj o CO CTl CM r— 00 r-. I— 1— CM ro in rc n3 X > I/) U) Ol OJ -o n3 OJ E < Ol (/> c ro O 3 1O to O) 0) x: Q. O 3 (_) X O) 3 <~> io s3 M ito OJ SCL X Q. 3 o Q. i C ra s_ ro o (0 QJ to o 4a; •rro ro E •4-' rO •r— Jrt3 ro ro QJ Q. -•-> +-> to 1 — O O O 3 iC cr o ro ro 3 O to iSO O Cl33 CM o CM 1— in CM o ro U3 to I— O CM 00 (Tt CM 1— CO CTi ^ Ln ^ CM CTl CM CO U3 Ln to QJ ro E fe to ro 0) 4-> O ro 1—

    PAGE 139

    133 Table 3-26. Sunimary of truck trap results at Whitney Mobile Home Park from August 1975 to August 1976 (22 trap nights). Number of mosquitoes collected Females Aecles ful vus pallens Aedes infirmatus Aedes atlanticus 1 2 2 Anopheles crucians 47 Anopheles quadrimaculatus 5 Culex (Melalioconion ) 32 Culex salinarius 5 Culex nigripalpus 16 Culex pipia^s quinquefasci atus 3 Culiseta melanura 26 Coqui 1 lettidia perturbans 35 Psorophora cil iata 2 Psorophora columbiae 1 59 Uranotaenia sapphirina 15 Uranotaenia lowii 1 Unidentified specimens 3 Total females 354 Males 234

    PAGE 140

    134 The overall evidence from the truck-trap samples and other sampling data indicates that the major source of human annoyance in the trailer park was caused by species of mosquitoes which were not reproducing in great numbers in the treated domes. Adding effluent to the nearby swamps has not caused an increase in numbers of pest species within the park. Because of the complaints of park residents, the park management hired the county mosquito control unit to spray in the park one night per week in 1975. In 1976 the park management did its own spraying on a once-a-week basis. Malathion was the insecticide used both years. Because the adult spraying was limited to once a week with no attempt at larval source reduction, the spraying probably had very little effect on adult activity within the park. Larval Sampling Lacking time and manpower, larval sampling was conducted more to determine the actual breeding sites and distribution than to compare quantitative abundance at different domes. The sampling device used was the standard, enamel pint dipper on a long wooden handle. The addition of increased nutrients at S-1 and S-2 resulted in the almost immediate formation of duckweed covers at both domes. The central portion of the duckweed mat at S-1 was several centimeters thick and extended for approximately 30 meters in all directions from the center of the dome. At the dome margins where the water was shallowest and emergent vegetation was most dense, the duckweed was

    PAGE 141

    135 scattered and did not form a solid mat. At S-2 the duckweed cover was extensive; however, the central mat, in some places, was only one plant layer thick with open water being common around emergent vegetation. There was much more open water around the margins of S-2 than at S-1. The difference in duckweed concentrations at the two domes was probably a result of the open canopy at S-1, which allowed a great deal of sunlight to reach the water's surface.^ At S-2 the canopy was denser and provided more shade. The groundwater and control domes were free from complete duckweed covers, although scattered populations of the plant were present at both AC and C-1 The dome at C-2 was completely free of duckweed. These patterns of aquatic vegetation undoubtedly influenced mosquito breeding. In the summer of 1975 larval sampling was done in a random manner along transects to determine the distribution of larvae at S-1, S-2, C-1, and AC. Results are given in Figures 3-14 and 3-15. The data recorded in these figures indicate that at S-1 larval development was restricted by the duckweed cover to the shallow marginal areas of the dome. In three years of periodic, qualitative sampling at S-1, larvae were found within a 30 meter radius of the center only in holow, burned-out stumps (Figures 2-15A and 2-16A) where the duckweed was unable to grow. At all of the other domes, larvae were distributed from center to margins along all transects. In the deeper water toward the middle of the domes, larvae were concentrated around the bases of trees and hidden in smaller, emergent vegetation. Hrees were widely spaced; many had been damaged or killed by the 1973 fire.

    PAGE 142

    Figure 3-14. A. Larval distribution (histogram) and water depth (dashed line) from the center to the margin at S-1. B. Larval distribution (histogram) and water depth (dashed line) from the center to the margin at C-1

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    137 a b Q. 01 > _i CT tn O 2 0 1 0 S-1 •• o 5 10 15 20 25 30 35 40 45 50 C-1 K75 50 h25 0 75 50 25 0 O 5 10 15 20 25 30 35 40 45 50 Distance from Center of Dome (Meters) o -i O n> D <-* 3 n> 1/1

    PAGE 144

    x: 4J O o s-a OJ x: CO T3 CL OJ S0) +J (O 5 o M Q. 3 o c: E to cn • o o I/) •1+-) c E •!O -(J •1S4-> ro 3 £ •rO) i^ 4-) +-> CO •rO -O 4-> 1— t. (O 01 > +-) iC ra Ol E to S• CDCvJ O I -!-> CyO Cd •1+J O C7) •IS4-' re 3 E •rO) s^ +-) +-> CO •rO -o +J r— Sre CL) > -M tc re O) _l o in CO OJ'

    PAGE 145

    139 Depth of Water (Centemeters) O iD iD CM -I I O O o CO If) U o in to o to / o I o / 6 iD O lO m NT o NT in n o in CM o in o in o in d o T O o in in rvj .J. o in \ I I O 00 in O in ID o CD in in / 9 I I O in in o NT in CO o on in o in "T" O 1 m d d in d 1/1 o Q O C U E o u c nj f- d dia J9Cl SBAJP-] o;inbsoiAj

    PAGE 146

    140 Culiseta melanura, Culex territans and Anopheles crucians were always the most common species in larval collections at all domes, and all immature stages (first, second, third, and fourth instar larvae and pupae) of these three species were present during the winter months. Egg rafts of Culiseta melanura were collected even in January 1977, early and late instar larvae of Culiseta melanura Anopheles crucians Culex territans and Culex salinarius were present at S-2 and apparently unaffected by the unusually low temperatures. It should be noted that Aedes canadensis canadensis a species captured only rarely as an adult, was relatively common in wintertime larval collections, especially at AC and C-1. Anopheles crucians was found primarily in open water situations whereas Culiseta melanura was most commonly collected from stump holes within the domes and from shaded or partially hidden seepage holes at the margin (Figures 2-15B and 2-15C). Culex territans was found associated with both Cul iseta melanura and Anopheles crucians Culex salinarius were most easily collected from stump holes within the domes (Figure 2-15A). The two species of Uranotaenia seemed more concentrated where sphagnum moss was present (at the margins). Culex ( Melanoconion ) erraticus was the only Melanoconion species found and it was most common at C-1 In February 1977, one transect at AC and one at S-2 were sampled in a non-random manner (refer to Materials and Methods). Collection results are listed in Table 3-27. The transect at AC was approximately 408 feet long, and at S-2 the transect was very close to 200 feet long. Data from Table 3-27 show that there were more larvae per dip at AC (0.94) than at S-2 (0.57); however, when the numbers of larvae per

    PAGE 147

    Table 3-27. Results from non-random sampling along a transect with a pint dipper at AC (250 dips) and S-2 (120 dips). — Collection sites AC S-2 Number of Anopheles 54 6 Number of Cul iseta melanura 96 42 Number of Culex territans 47 5 Number of Aedes canadensis canadensis 25 0 Number of Aedes vexans 1 0 Number of Culex salinarius 0 2 Number of unidentified early instars 13 13 Number of Larvae per dip 0.94 0.57 Percentage positive dips 45% 21% Number of larvae per positive dip 2.09 2.72

    PAGE 148

    142 positive dip are calculated, figures for S-2 (2.72) are larger than AC (2,09). This indicates that there were more larvae developing at AC than at S-2 and the larvae at AC were more evenly distributed throughout the dome. Larvae at S-2 were concentrated more in stump holes and marginal pools, although not to the extent as already noted for S-1 In thecourse of larval sampling, Culex pipiens quinquefasciatus and Culex restuans two species with reputations for their association with polluted water, were found at both S-1 and S-2, but they never became abundant. Larvae of the floodwater species, Psorophora and Aedes, were found only rarely at the sewage domes and then only at the margins. No specialized larval sampling was done to determine the status of Coqui 1 lettidia perturbans or Mansonia indubitans in the domes. From adult records, one would have to assume that larvae were present in the sewage domes in at least small numbers. Virus Activity Sentinel Chickens Jetter (1975) reported an increase in psychodid fly populations associated with the accidental introduction of organic solids into the domes receiving effluent. In response to the increase in flies, he also reported a large concentration of migratory birds at these domes. These circumstances, coupled with the fact that Culiseta melanura a proven vector of equine encephalitis, was abundant in all domes, led to the inclusion of a surveillance program to determine the status of certain group A and B togaviruses within the domes. Sentinel chicken flocks were established at all the domes being studied and at

    PAGE 149

    143 scattered locations around the City of Gainesville. Hemagglutination inhibition (HAI) followed by serum neutralization tests were used to determine antibody responses in the chickens to eastern equine (EEE), western equine (WEE), and St. Louis (SLE) encephalitis viruses. Antibody responses, as a result of asymptomatic infections with all the viruses, were demonstrated by these serological techniques. The total number of serological conversions as a quantitative measure of virus amplification is plotted versus time in Figure 3-16. This graph very clearly shows that virus amplification was maximal in July, following peak mosquito activity (as recorded with CDC light traps). Figures 3-17 and 3-18 demonstrate the seasonal activity of EEE and WEE viruses at each of the sentinel locations. The figures show that the two viruses were circulating in all the sampled domes, and no virus activity was observed in sentinels placed outside the domes around Gainesville. Between October 21 and December 10, one of the chickens at S-1 developed a very high titer of HAI and neutralizing antibodies against SLE virus. This was the only conversion against this particular virus. When the serological data from the two sewage domes are grouped and compared to the data from the three control domes (AC, C-1, C-2) in a 2 X 2 contingency table (Mendenhal 1 1968), there is insufficient evidence at a=0.05 to indicate more virus activity in the sewage domes than in the control domes (comparison A, Table 3-28). When the domes are compared individually, using a 3 x 5 contingency table (Comparison B, Table 3-28), there is sufficient evidence at a=0.05 to reject the null hypothesis that there was no quantitative difference in virus

    PAGE 150

    ea; ~r Qns 4-> -M > +J •1^ +-> cn (J -rro 1— (/) (_) •rQ -!-> O (O >, x: +-> Q.T0) > O -tc: +-> O) u O) c o •1+-> :3 •!cr 3 QJ cr t/i c o SE Ol +-> -a 00 c O) re 3 T3 I/) (C o c to to ssTD dj OJ S+-> > o CO c u to o O) LxJ u i. I CO (U cn

    PAGE 151

    145

    PAGE 152

    10 =3 S'r— T3 > C O O Sn3 10 a cu c > c: •r— in -i-> -o QJ ai U ro O) • to c ^ C -II— •14-> I CD OJ 01 to •1— £ Uo +-> c O) i•r— > c > o •r— CJ > cu c 1 — 1 •r< O n3 03 1 sCD

    PAGE 153

    147

    PAGE 154

    +-> (/) ra to c: o S> o 00 I CO

    PAGE 155

    149

    PAGE 156

    X CM CM CO O CO CNJ CM I (_) o-i CO CX3 CM cn I o CM CTl CO CM X3 0) o SCM I O CTl to CTl I ro E CO OJ S+J (-> o c 10 ns QJ to r— Ol :3 ro Sto SCL CD •1(U S> SMto O) O C > O -rc o to r03 O) SST Q. E QJ o x: o Qj CM I CO 0) CM + 00 + CM I I c o to Q. E o o 00 CM 00 CM CO CO ID CM to CO CO CO to QJ O -a 4-> to QJ x: +-> o Q. 3 c OJ +-> tn o o II a +->

    PAGE 157

    151 activity between individual domes. By removing S-1 and comparing the remaining domes (Comparison C, Table 3-28), it becomes obvious that the one dome which is statistically different from the others is S-1. Why there was more virus activity in sentinels at S-1 is not at all clear. From an ecological standpoint this dome was stressed more than any of the others. It was the first to receive effluent; and, by accident, it received the largest amounts of organic solids along with the effluent. It was also the dome damaged most by the 1973 fire. However, to say that its elevated levels of virus activity were a result of these ecological disturbances is not necessarily good reasoning. The circulation of EEE and WEE viruses in these cypress swamp habitats appears to be a common and natural occurrence, not the result of an ecological disturbance. Other studies have documented the importance of Culiseta melanura as the avian vector responsible for the transmission of these viruses in nature. Although Cul iseta melanura is present at S-1, its breeding sites have been greatly reduced as a result of the extensive duckweed cover, and there is no evidence that this species is more abundant at S-1 than any of the other domes. It is possible that virus activity at the domes was influenced by the concentration of sentinels. There were more sentinels per area at S-1 than at any of the other sites. Before accepting the view that there was actually more virus circulation in the natural animal populations at S-1, studies need to be done which measure virus activity without the contributing effect of the sentinels themselves. If this arbovirus project were to be continued it would be enlightening to repeat the same sentinel studies using a chicken pen designed to capture the mosquitoes which had fed on the chickens. This would serve two purposes; first, the

    PAGE 158

    152 mosquitoes could be pooled and processed for viral isolations and second, the sentinels themselves would not contribute to the levels of virus already present in natural populations. The natural cycle of SLE virus is poorly understood. Assuming, that the chickens were as sensitive to the presence of SLE as they were to EEE and WEE viruses, this study indicates that SLE is very rare in cypress domes compared to EEE and WEE. The significance, if any, of the one conversion against this virus in the late fall is unknown. Isolation Attempts from Mosquito Pools As time was available, mosquitoes trapped alive with CDC traps were pooled and stored at -70C for viral isolation attempts. Table 3-31 gives a record of the mosquito pools and the results from them. One positive viral isolation was made, and it was identified by Dr. Arthur Lewis as Flanders virus. This virus has been previously isolated from Culiseta melanura Culex pipiens and an ovenbird, all from New York (Theiler and Downs, 1973). In the course of other studies. Dr. Lewis (1976) has also isolated this virus from Culex nigripalpus in Florida. As yet there is no evidence that the virus is pathogenic for man. The lack of viral isolations from this study is not unexpected considering the small number of pools tested. Serology and Isolation Attempts from Mammal Samples Mammals from S-1, C-1, and S-2 were trapped and blood samples taken. The results are recorded in Table 3-32. HAI tests were performed against Venezuelan equine (VEE) and California (CEV) encephalitis

    PAGE 159

    153 Table 3-31. Summary of virus isolation attempts from pooled mosquitoes. Species Number of pools Total number in pools Number of isolations Culex nigripalpus 4 261 1* Culex (Melanoconion) 2 46 0 Coquil lettidia perturbans 2 23 0 Culiseta melanura 4 155 0 Anopheles crucians 4 125 0 Anopheles quadrimaculatus 1 1 0 Aedes atlanticus 2 64 0 *Flanders virus isolated from one pool of 64 Culex nigripalpus trapped on August 25, 1976. Table 3-32. Summary of serological evidence of encephalitis activity in mammals. Species Number bled HA I Isolations Peromyscus gossypinus (cotton mouse) 14 0 0 Sigmodon hispidus (cotton rat) 15 0 0 Oryzomys palustris (rice rat) 12 0 0 Didelphis marsupial is (opposum) 6 0 0 Procyon lotor (raccoon) 2 0 0 Lynx rufus (bobcat) 1 0 0

    PAGE 160

    154 viruses, as well as EEE, WEE, and SLE. Viral isolations were also attempted from some of the samples, but all were negative. The number of samples taken was too small to allow definitive statements about the role of mammals as natural hosts of these viruses at the study sites; however, the indication is clear that mammals play only a small role, if any, in the natural maintenance of EEE and WEE viruses. Summary Two experimental cypress domes receiving sewage effluent and three unpolluted control domes were sampled by several different methods to determine the effects of the effluent on mosquito populations. These domes were also surveyed for mosquito-borne, encephalitis activity. The nearby trailer park was included in the mosquito study, and virus activity was monitored away from the cypress domes in the city of Gainesville. The adult sampling methods have clearly indicated that Cul iseta melanura Cul ex nigripalpus Culex ( Melanoconion ) spp.. Anopheles crucians, Uranotaenia sapphirina and U. 1 owi i are the dominant species developing in the sewage domes. The same species are also abundant in the control domes. Aedes atlanticus and Aedes infirmatus were sometimes abundant in adult samples from the sewage domes; however, larval surveys indicate that these latter species are developing in temporary fresh water pools outside the domes. There is some indication that Uranotaenia sapphirina Culex ( Melanoconion ) spp. and Coqui 1 lettidia perturbans have become more numerous as a result of the effluent. At the same time, species of Aedes and Psorophora have declined at the

    PAGE 161

    155 sewage domes. These trends can be attributed to the transition from fluctuating water levels to permanent standing water at the experimental domes. Culex pi pi ens quinquefasciatus and C^. restuans two species with well-known reputations for proliferating in foul water, have never become abundant at the sewage domes. The mosquito annoyance in the trailer park adjacent to the cypress domes is not a result of experimentation within the domes. The major pest species in the trailer park is Psorophora columbiae a species not reproducing within the domes. For the most part, the mosquitoes developing in the domes receiving effluent are ones which show little feeding preference for humans. For this reason the mosquito fauna from the experimental domes contribute very little to a human pest situation. Both the experimental domes and the control areas produced large numbers of species known to be capable of transmitting encephalitis among wild reservoirs. Serological studies have shown that strains of eastern and western equine encephalitis are common at all of the domes sampled. The experimental dome S-1 demonstrated significantly more virus activity than any of the other domes; however, it is not clear whether this is a true reflection of virus amplification in natural populations or an artifact resulting from the monitoring methods. One positive case of St. Louis encephalitis was encountered at S-1; more investigation is needed to determine the significance, if any, of this one case.

    PAGE 162

    CONCLUSIONS On the basis of results obtained from two experimental sites, it appears that recycling treated sewage effluents through cypress domes caused no immediate or unusual increase in mosquito abundance which would be classified as an economic or public health problem. Culex pipiens quinquefasciatus a pest species which thrives in foul water situations, did not become well established at the experimental domes. Mbsquitoes reproducing in the experimental domes were the same species found in the control domes and, for the most part, are species which show a feeding preference for birds, small mammals, reptiles, and amphibians. The floodwater species of Aedes and Psorophora which are severe human pests, declined at the experimental domes as a result of maintaining relatively constant water levels. Eastern and western equine encephalitis viruses were present in bird populations within the experimental domes, but this is a natural phenomenon also present at all of the control domes. Mosquito control measures directed at lowering vector populations seems inappropriate in these situations considering the absence of human cases and the restriction of virus activity to the domes. No virus activity was recorded outside the domes. The one serological conversion against St. Louis encephalitis from dome S-1 is of interest; however, its importance is undetermined. 156

    PAGE 163

    RECOMMENDATIONS FOR FUTURE WORK In this study the author concentrated on sampling the adult mosquito populations associated with the experimental and control domes. Because of technical problems and a lack of time, no quantitative method was developed for sampling larval populations. As a result, there are no data which correlate larval abundance at the experimental domes with adult abundance. Since the impact of the effluent has a direct effect on larval survival, it would be worthwhile to continue this study, concentrating on the development of techniques which would yield accurate estimates of larval populations. As previously stated it would be enlightening to repeat the arbovirus studies using a sentinel bird pen which trapped those mosquitoes coming to feed. Such a study would more accurately reflect natural viral amplification by removing the contributing effects of the sentinels themselves Future work might logically include studies to determine the effectiveness of different mosquito control strategies as applied at these experimental domes. In conjunction with a knowledge of larval distribution patterns, one method of control worth evaluating would be the mechanical destruction of specific habitats, such as the removal of hollow stumps and marginal, emergent vegetation. 157

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    LITERATURE CITED Barr, A.R., T.A. Smith, and M.M. Boreham. 1960. Light intensity and the attraction of mosquitoes to light traps. J. Econ. Entomol 53:876-880. Bidlingmayer, W.L. 1966. Use of the truck trap for evaluating adult mosquito populations. Mosquito News 26:139-143. Bidlingmayer, W.L. 1967. A comparison of trapping methods for adult mosquitoes: Species response and environmental influence. J. Med. Entomol. 4:200-220. Bigler, W.J. 1969. Venezuelan encephalitis antibody studies in certain Florida wildlife. Bull. Wildl. Dis. Ass. 5:267-270. Bigler, W.J., and G.L. Hoff. 1974. Anesthesia of raccoons with Ketamine hydrochloride. J. Wildl. Manage. 38:364-366. Bigler, W.J., and G.L. Hoff. 1975. Arbovirus surveillance in Florida: Wild vertebrate studies 1965-1974. J. Wildl. Dis. 11:348-356. Bigler, W.J., E.B. Lassing, E.E. Buff, E.G. Prather, E.G. Beck, and G.L. Hoff. 1976. Endemic eastern equine encephalitis in Florida: A twenty-year analysis, 1955-1974. Am. J. Trop. Med. Hyg. 25:884-890. Breeland, S.G., and E. Pickard. 1965. The malaise trap— An efficient and unbiased mosquito collecting device. Mosquito News 25:19-21. Brezonik, P.L. 1974. Water quality and heavy metals. Pages 309-338 in Odum, H.T., K.C. Ewel J.W. Ordway, M.K. Johnston, and W.J. Mitsch, Cypress wetlands for water management, recycling, and conservation, annual report for 1974. Center for Wetlands, University of Florida, Gainesville. Carpenter, S.J., and W.J. LaCasse. 1955. Mosquitoes of North America. University of California Press, Berkeley and Los Angeles. 360pp. Center for Disease Control (DHEW, PHS). 1977. Handout material distributed at the 1977 American mosquito control association meetings in New Orleans, La. Chamberlain, R.W., W. D. Sudia, P.H. Coleman, and T.H. Work. 1964. Venezuelan equine encephalitis virus from south Florida. Science 145:272-274. 158

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    159 Clark, D., and J. Casals. 1958. Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses. Amer. J. Trop. Med. Hyg. 7:561-573. Clements, A.N. 1963. The physiology of mosquitoes. Pergamon Press Inc., New York, N.Y. 393 pp. Dalrymple, J.M., O.P. Young, B.F. Eldridge, and P.K. Russell. 1972. Ecology of arboviruses in a Maryland freshwater swamp. Amer. J. Epidemiol. 96:129-140. Demaree, D. 1932. Submerging experiments with Taxodium Ecology 13:258-262. Dow, R.P., P.H. Coleman, K.E. Meadows, and T.H. Work. 1964. Isolation of St. Louis encephalitis viruses from mosquitoes in the Tampa Bay area of Florida during the epidemic of 1962. Amer. J. Trop. Med. Hyg. 13:462-468. Dulbecco, R.,and H.S. Ginsberg. 1973. Virology. Pages 1010-1449 in Davis, B.D., R. Dulbecco, H.N. Eisen, H.S. Ginsberg, and W.B. Wood, Microbiology. Harper and Row, Hagerstown, Md. Edman, J.D. 1974. Host-feeding patterns of Florida mosquitoes : III Culex ( Culex ) and Culex ( Neoculex ). J. Med. Entomol 11:95-104. Ewel K.C. 1976. Seasonal changes in distribution of water fern and duckweed in cypress domes receiving sewage. Pages 164-170 in Odum, H.T., K.C. Ewel, J.W. Ordway, and M.K. Johnston, Cypress wetlands for water management, recycling and conservation, annual report for 1976. Center for Wetlands, University of Florida, Gainesville. Ewel, K.C, and H.T. Odum. 1978. Cypress domes: Nature's tertiary treatment filter. In press. Gibbs, E.P.J. 1976. Equine viral encephalitis. Equine Vet. J. 8:66-71. Gillies, M.T. 1969. The ramp-trap, an unbaited device for flight studies of mosquitoes. Mosquito News. 29:189-193. Gressitt, J.L., and M.K. Gressitt. 1962. An improved malaise trap. Pacific Insects. 4:87-90. Gunstream, S.E., and R.M. Chew. 1967. A comparison of mosquito collections by malaise and miniature light traps. J. Med. Entomol. 4:495-496. Headlee, T.J. 1932. The development of mechanical equipment for sampling the mosquito fauna and some results of its use. Proc. N.J. Mosquito Exterm. Assoc. 19:106-126.

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    160 Henderson, B.E., and P.H. Coleman. 1971. The growing importance of California arboviruses in the etiology of human disease. Prog. Med. Virol. 13:404-461. Horsefall, W.R. 1972. Mosquitoes: Their bionomics and relation to disease. Hafner Publishing Co. Inc., New York, N.Y. 723 pp. Huffaker, C.B., and R.C. Back. 1943. A study of methods of sampling mosquito populations. J. Econ. Entomol 36:561-569. Jennings, W.L., R.H. Allen, and A.L. Lewis. 1966. Western equine encephalitis in a Florida horse. Amer. J. Trop. Med. Hyg. 15:96-97. Jennings. W.L., A.L. Lewis, G.E. Sather, W.M. Hammon, and J.O. Bond. 1968. California-encephalitis-group viruses in Florida rabbits. Amer. J. Trop. Med. Hyg. 17:781-787. Jetter, W. 1975. Effects of treated sewage on the structure and function of cypress dome consumer communities. Pages 588-610 in Odum, H.T., K.C. Ewel J.W. Ordway, and M.K. Johnston, Cypress wetlands for water management, recycling, and conservation, annual report for 1975. Center for Wetlands, University of Florida, Gainesville. Joseph, S.R., and W.E. Bickley. 1969. Culiseta melanura (Coquillett) on the eastern shore of Maryland, Bulletin A-161. University of Maryland Agricultural Experiment Station, College Park. 84 pp. King, W.V., G.H. Bradley, C.N. Smith, and W.C. McDuffie. 1960. A handbook of the mosquitoes of the southeastern United States. Agriculture handbook no. 173. USDA, U.S. Government Printing Office, Washington D.C. 188 pp. Kissling, R.E., R.W. Chamberlain, D.C. Nelson, and D.D. Stamm. 1955. Studies on the north American arthropod-borne encephal itides : VII equine encephalitis studies in Louisiana. Amer. J. Hyg. 62:233-254. Knight, K.L. 1964. Quantitative methods for mosquito larval surveys. J. Med. Entomol. 1:109-115. Kurz, H, 1933. Cypress domes. Fla. Geol Survey. Ann. Rep. 24:54-65. LeDuc, J.W., W. Suyemoto, B.F. Eldridge, and E.S. Saugstad. 1972. Ecology of arboviruses in a Maryland freshwater swamp: II blood feeding patterns of potential mosquito vectors. Amer. J. Epidemiol 96:123-128. Lennette, E.H., ed. 1969. Computation of 50 per cent end points. Pages 46-51 in Diagnostic procedures for viral and reckettsial infections. American Public Health Association Inc., New York, N.Y.

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    161 Lewis, A.L. 1976. Personal communication. McGowan, J.E., Jr., J. A. Bryan, and M.B. Gregg. 1973. Surveillance of arboviral encephalitis in the United States, 1955-1971. Amer. J. Epidemiol. 97:199-207. Mendenhall, W. 1968. Introduction to probability and statistics. Wadsworth Publishing Co., Inc., Belmont, Cal. 393 pp. Monk, CD., and T.W. Brown. 1965, Ecological consideration of cypress heads in north central Florida. Amer. Midi. Natur. 74:126-140. Mulhern, T.O. 1934. A new development in mosquito traps. Proc. N.J. Mosquito Exterm. Assoc. 21:137-140. Muul, I., B.K. Johnson, and B.A. Harrison. 1975. Ecological studies of Culiseta melanura in relation to eastern and western equine encephalomyelitis viruses on the eastern shore of Maryland. J. Med. Entomol. 11:739-748. Nelson, D.B., and R.W. Chamberlain. 1955. A light trap and mechanical aspirator operating on dry cell batteries. Mosquito News 15:0-0. Odum, H.T., K.C. Ewel J.W. Ordway, M.K. Johnston, and W.J. Mitsch. 1974. Cypress wetlands for water management, recycling, and conservation, first annual report. Center for Wetlands, University of Florida, Gainesville. 947 pp. Odum, H.T., K.C. Ewel, J.W. Ordway and M.K. Johnston. 1975. Cypress wetlands for water management, recycling, and conservation, second annual report. Center for Wetlands, University of Florida, Gainesville. 817 pp. Odum, H.T., K.C. Ewel, J.W. Ordway, and M.K. Johnston. 1976. Cypress wetlands for water management, recycling, and conservation, third annual report. Center for Wetlands, University of Florida, Gainesville. 879 pp. Price, P.W. 1975. Diversity and stability. Pages 372-387 in Insect ecology. John Wiley and Sons, New York, N.Y. Roberts, R.H. 1972. Effectiveness of several types of malaise traps for the collection of tabanidae and culicidae. Mosquito News 32:542-547. Robinson, H.S. 1952. On the behavior of night flying insects in the neighborhood of a bright source of light. Proc. R. Entomol. Soc. Lond. 27:13-21. Saugstad, E.C., J.M. Dalrymple, and B.F. Eldridge. 1972. Ecology of arboviruses in a Maryland freshwater swai)ip: I. population dynamics and habitat distribution of potential mosquito vectors. Amer. J. Epidemiol. 96:114-122.

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    162 Schoonover, R.A., ed. 1970. Florida health notes. 62:171-194. Service, M.W. 1976. Mosquito ecology: Field sampling methods, John Wiley and Sons, New York, N.Y. 583 pp. Stamm, D.D. 1966. Relationships of birds and arboviruses. Auk 83:84-97. Stark, H.E. 1967. Mosquito-borne encephalitis and associated ecological factors with special reference to the southeastern United States. Louisiana State Medical Society 119:257-271. Stojanovich, C.J. 1960. Illustrated key to common mosquitoes of southeastern United States. Cullom and Ghertner Co., Atlanta, Ga. 36 pp. Sudia, W.D., and R.W. Chamberlain. 1962. Battery-operated light trap, an improved model. Mosquito News 22:126-129. Sudia, W.D., and R.W. Chamberlain. 1974. Collection and processing arthropods for arbovirus isolation. USDHEW, PHS, CDC, Atlanta, Ga. 29 pp. Sudia, W.D., R.D. Lord, and R.O. Hayes. 1970. Collection and processing of vertebrate specimens for arbovirus studies. USDHEW, PHS, CDC, Atlanta, Ga. 65 pp. Taylor, D.J., A.L. Lewis, J.D. Edman, and W.L. Jennings. 1971. California group arboviruses in Florida host vector relations. Amer. J. Trop. Med. Hyg. 20:139-145. Tempelis, C.H., D.B. Francy, R.O. Hayes, and M.F. Lofy. 1967. Variations in feeding patterns of seven culicine mosquitoes on vertebrate hosts in Weld and Larimer counties, Colorado. Amer. J. Trop. Med. Hyg. 16:111-119. Tempelis, C.H., W.C. Reeves, R.E. Bellamy, and M.F. Lofy. 1965. A three year study of the feeding, habits of Culex tarsal is in Kern county, California. Amer. J. Trop. Med. Hyg. 14:170-177. Tempelis, C.H., and R.K. Washino. 1967. Host feeding patterns of Culex tarsal is in the Sacramento Valley, California, with notes on other species. J. Med. Entomol 4:315-318. Theiler, M., and W.G. Downs. 1973. The arthropod-borne viruses of vertebrates. Yale University Press, New Haven Conn. 587 pp. Townes, H.K. 1962. Design for a malaise trap. Proc. Entomol. Soc. Wash. 64:253-262.

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    163 Wellings, F.M., A.L. Lewis, and L.V. Pierce. 1972. Agents encountered during arboviral ecological studies: Tampa Bay area, Florida, 1963-1970. Amer. J. Trop. Med. Hyg. 21:201-213. Wharton, C.H., H.T. Odum, K.C. Ewel, M. Duever, A. Lugo, R. Boyt, J. Bartholomew, E. DeBellevue, S. Brown, M. Brown, and L. Duever. 1976. Forested wetlands of Flori da--Thei r management and use. Center for Wetlands, University of Florida, Gainesville. 421 pp. Williams, J.E., D.M. Watts, and T.J. Reed. 1971. Distribution of culicine mosquitoes within the Pocomoke cypress swamp, Maryland. Mosquito News 31 :371-378. Wilson, D.B., and A.S. Msangi. 1955. An estimate of the reliability of dipping for mosquito larvae. The East African Medical Journal. 32:1-3.

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    BIOGRAPHICAL SKETCH Harry Goodwin Davis was born 6 March 1947 at Machias, Maine, He grew up in unorganized territory just across the Penobscot River from East Millinocket, Maine. He is the middle son of Lloyd and Louise Davis. Both his brothers are biologists. Facing the fact that he might never play left field for the Boston Red Sox, he concentrated on his other interests in science. In 1969 he graduated with honors from the University of Maine. As an undergraduate he was elected to Phi Kappa Phi and Alpha Zeta honor societies. After a brief and exhausting experience at teaching high school biology, he was lucky enough to be drafted by the U.S. Army. He spent the next two years at Walter Reed Army Institute of Research as a technician in the department of Virus Diseases. After leaving the Army he entered graduate school in zoology at the University of Minnesota. It was at Minnesota that he developed a keen interest in mosquitoes and another graduate student, Jeudi Olson. He decided to do research on the former and marry the latter. In 1974 he and his wife moved to Gainesville, Florida. He started his research at the University of Florida and his wife, while working for the biochemistry department, started her infamous stray-cat collection. Today both Harry and Jeudi Davis live in Marlborough, New Hampshire, with their fourteen cats and one dog. They both teach at Franklin Pierce College. 164

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    I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Professor of Zoology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. D.A. Dame, Co-Chairman Associate Professor of Entomology and Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Associate Professor of Microbiology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully in scope and quality, as a dissertation for the degree of adequate, Doctor of Philosophy. / A Walkf Professoi 1/1 of Entomology and Nematology

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    I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ^ J.L. Nation -Professor of Entomology and Nematology I cert4fy that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. h U). l>MJl D.W. Hall Assistant Professor of Entomology and Nematology This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Phi losophy June 1978 Dean, Cqi;iege of Agriculture/ Dean, Graduate School