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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
Alternate title:
Cypress swamps
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Davis, Harry Goodwin, 1947-
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English
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vi, 164 leaves : ill. ; 28 cm.

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Birds ( jstor )
Encephalitis ( jstor )
Groundwater ( jstor )
Larvae ( jstor )
Mammals ( jstor )
Sewage ( jstor )
Species ( jstor )
Swamps ( jstor )
Viruses ( jstor )
Wetlands ( jstor )
Arbovirus infections ( fast )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Mosquitoes ( fast )
Sewage lagoons ( fast )
Swamp ecology ( fast )
Florida ( fast )
City of Gainesville ( local )
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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.
General Note:
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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*(- 4-) A4-) )4-) )4-) A41) S*- C C -C -C *- C 0 E-4- E -4- E 4- E -4- E 4(04- (04- 4- (4- (04a 0 oLD0 C\j0 CD0 MCD o





(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





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;




,











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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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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81


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









83

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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
Females
122
Aug. Sept.


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


36


Figure 2.7. A. Ramp-trap in its original form.
B. Removing trapped mosquitoes
from the ramp-trap by aspiration.
C. Removing the trapping
head when not in use.


Table 3-1. Summary of ramp-trap collections for 1974 at S-l.
Month
Apri 1
May
June
July
Sept.
Oct.
Nov.
Total
%
Trap Nights
6
9
8
3
4
7
3
40
Number
of mosquitoes collected
Females:
Aedes fulvus pallens
8
1
9
0.4
Aedes mitchellae
3
2
1
6
0.3
Aedes vexans
1
1
0.0
Aedes canadensis canadensis
1
1
0.0
Aedes infirmatus
9
4
1
14
0.6
Aedes dupreei
1
1
0.0
Aedes atlanti cus
56
76
14
146
6.2
Aedes taeniorh.ynchus
1
1
0.0
Psorophora ciliata
0
Psorophora colurnbiae
1
11
5
5
22
0.9
Psorophora ferox
2
23
4
3
32
1.3
Anopheles crucians
4
20
92
21
8
13
4
162
6.8
Anopheles quadrimaculatus
3
7
10
0.4
Culex (Melanoconion)
20
21
6
142
48
2
239
10.1
Culex territans
5
3
5
5
1
1
1
21
0.9
Culex salinarius
5
5
9
4
1
9
3
36
1.5
Culex niqripalpus
3
5
12
12
60
56
14
162
6.8
Culex restuans
2
2
0.1
Culex pipiens quinquefasciatus
2
1
2
5
0.2
Culiseta melanura
6
134
151
149
30
158
21
649
27.4
Coquillettidia perturbans
1
9
20
5
3
5
1
44
1.9
Uranotaenia sapphirina
1
20
146
46
306
66
6
591
24.9
Uranotaenia lowii
22
3
68
60
31
184
7.8
Unidentified specimens
8
21
2
3
34
1.4
Total females
30
319
604
281
630
425
83
2372
Males
11
45
208
85
80
38
10
477


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


25


Table 3-2. Summary of ramp-trap collections for 1974 at C-1.
Month
May
June
July
Sept.
Oct.
Nov.
Total
%
Trap nights
6
8
3
4
8
3
32
Number
of mosquitoes collected
Females:
Aedes fulvus pallens
0
Aedes mitchellae
1
1
0.1
Aedes vexans
1
ft
1
0.1
Aedes canadensis canadensis
3
9
3
15
0.8
Aedes infirmatus
7
12
1
20
1.1
Aedes dupreei
4
1
5
0.3
Aedes atlanti cus
69
134
50
253
13.7
Aedes taeniorh.ynchus
0
Psorophora ciliata
1
1
2
0.1
Psorophora columbiae
1
5
1
1
1
9
0.5
Psorophora ferox
15
15
6
1
37
2.0
Anopheles crucians
10
45
15
11
12
8
101
5.5
Anopheles quadrimaculatus
1
18
15
2
36
1.9
Culex (Melanoconion)
2
2
134
40
5
183
9.9
Culex territans
1
22
10
7
12
52
2.8
Culex salinarius
2
4
1
3
3
13
0.7
Culex nigripalpus
6
9
1
36
41
11
104
5.6
Culex restuans
0
Culex pipiens quinquefasciatus
1
1
0.1
Culiseta melanura
39
90
69
59
126
32
415
22.4
Coquillettidia perturbans
10
7
2
1
20
1.1
Uranotaenia sapphirina
22
93
30
200
52
6
403
21.8
Uranotaenia lowii
1
11
6
81
37
22
158
8.5
Unidentified specimens
5
11
3
1
1
21
1.1
Total females
200
470
200
542
337
101
1850
Males
58
164
51
204
75
15
567


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.C. Prather, E.C. 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 trapAn 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


21


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-l, 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-l or C-l, and that S-l
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


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 Florida--Their 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.


4 66
^l
j
$
V "V / N
V
/ ,'
*1
-J *
\ >
S< X Hr Vr
> > S' S'- V' V
~S* > S \ / > -
.
M
**** '%w-
i&tL +*
v V Y' v ^
. J
-*
VVWr^- - -
# S' ^ ~ : ^
o y 4 j , *# *->
1* *. i- 2* Si 5> 2^ I " -.
25
v
V


iauie j-iy.
KanK comparison test of the null hypothesis
at S-2 is similar to the number at C-2.
that the number of
female mosquitoes in CDC traps
Species
n y z
Decision
(ct=0.05)
Cone!usion
Floodwater Aedes
21
1
4.15
reject null
hypothesis --
(C-2)>(S-2)
Anopheles crucians
21
14
1.25
accept null
hypothesis
insufficient evidence
disprove (S-2)=(C-2)
to
Culex (Melanoconion)
18
18
4.24
reject null
hypothesis
(S-2)>(C-2)
Culex niqripalpus
13
4
1.38
accept null
hypothesis --
insufficient evidence
disprove (S-2)=(C-2)
to
Culiseta melanura
22
9
0.85
accept nul1
hypothesis --
insufficient evidence
disprove (S-2)=(C-2)
to
Coquillettidia perturbans
20
17
3.12
reject null
hypothesis
(S-2)>(C-2)
Uranotaenia sapphirina
21
21
4.15
reject null
hypothesis --
(S-2)>(C-2)
(S-2)=the number of females of a particular species trapped on a given night at dome S-2.
(C-2)=the number of females of a particular species trapped on a given night at dome C-2.
p= the probability of (S-2)>(C-2).
q=the probability of (S-2)>(C-2).
n=the number of trials (trap nights compared).
y=the number of times the particular species was more numerous in samples from S-2.
z= the test statistic y"np (Mendenhall, 1968).
npq


133
Table 3-26. Summary of truck trap results at Whitney Mobile Home Park
from August 1975 to August 1976 (22 trap nights).
Number of mosquitoes collected
Females
Aecles fulvus 'pallens 1
Aedes infirmatus 2
Aedes atl anti cus 2
Anopheles crucians 47
Anopheles quadrimaculatus 5
Culex (Melalioconion) 32
Culex salinarius 5
Culex nigripalpus 16
Culex pipiis quinquefasciatus 3
Culi seta melanura 26
Coquillettidia perturbans 35
Psorophora ciliata 2
Psorophora columbiae 159
Uranotaenia sapphirina 15
Uranotaenia lowii 1
Unidentified specimens 3
Total females 354
Males 234


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.


107
Table 3-21. Results from CDC traps at S-2 and AC grouped on a
seasonal basis.
AC
S-2
Mar.-May
June-Aug.
Sept.-Nov
Dec.-Feb.
Mar.-May
June-Aug.
Sept.-Nov
Dec.-Feb.
Trap nights
9
32
13
9
Females:
Aedes fulvus pallens
6
7
Aedes mitchellae
Aedes vexans
6
Aedes canadensis canadensis
7
1
5
Aedes infirmatus
8
877
182
5
Aedes dupreei
11
Aedes atlanticus
6
468
34
193
23
Anopheles crucians
268
1815
185
101
169
575
130
46
Anopheles quadrimaculatus
2
1
12
5
Culex (Melanoconion)
79
669
167
3
27
352
113
4
Culex territans
3
1
3
9
1
Culex salinarius
1
29
17
3
1
Culex nigripalpus
3
1458
313
67
1
942
103
14
Culex restuans
1
Culiseta melanura
97
591
76
8
199
822
170
35
Coqui1lettidia perturbans
49
54
11
56
95
34
Mansonia indubitans
1
5
5
Psorophora ciliata
3
7
1
1
Psorphora columbiae
2
Psorphora ferox
3
1
Uranotaenia sapphirina
690
1390
356
12
279
1246
764
8
Uranotaenia lowii
18
3
22
21


Table 3-10. New Jersey Light trap records for S-l.
Date
9/74 10/74 11/74 12/74
1/75 2/75
5/75 6/75
7/75 8/75 9/75
10/75 11/75
12/75 1/75 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76
Totals
Trap nights
3 5 3 9
3 2
1 2
A 5 1
4 3
3 3 3 4 5 4 3 4 3 2
78
Number of
mosquitoes
collected
Females:
Aedes fulvus
pallens
1
1
Aeces mitchellae
1
1
Acjaes vexans
1
1
2
Aeaes canadensis
canadensis
1
1
Aedes infirmatus
1
2
1 1
5
Aedes dunreei
Aedes atlanticus
1
1
12
3 1
18
Aedes soilicitans
Aedes aecypti
Psoropnora
ci1iata
1
1
5
7
Psoropnora
columbiae
Psoroohora
45
4
2
10
8
4
1
2
2
78
ferox
Anopheles
crucians
61
41
9
38 25 28 11 21
82
135 24
144
10
13
6 69 234 16 19 49 98
31
35
1199
Ancpheles
punctipennis
Anopheles
1
1
2
quadrimaculatus
3
5
2
1 1 1
5
1
1
3
3
2
28
Culex
fKelanoconion)
Culex
428
182
7
1
32
8
13
209
218
8
15
4
33
5 57
73
74
52
23
1442
territans
CuTex
1
1
1
1
4
salinarius
Culex
6
5
7
2
11
3
6
18
2
1
14
4 17
22
3
121
niqripalpus
49
263
25
84
9
1
31
20
6
33
8
1
2
25
16
12
15
600
Culex restuans
7
5
6
1
1
20


% of total capture
Culiseta
melanura
Culex
(Mel anoconion)
Culex
territans
Culex
mgripalpus
Floodwater
Aedes
Uranotaenia
sp.
AQP_utl£l£S
crucians
62 L


Table 3-14. Degree of similarity among the three domes, comparing two ata time, based on abundance
data from 1975-1976 New Jersey light trap samples.'
Abundance
(S-1) (S-2) (S-1) (C-l) (S-2) (C-l)
Anopheles crucians
18.8 -
11.1
= 7.7
18.8 -
7.9 =
= 10.9
11.1 -
7.9 =
* 3.2
Anopheles quadrimaculatus
0.3 -
0.1 =
0.2
0.3 -
0.4 =
-0.1
0.1 -
0.4 =
-0.3
Culex (Melanoconion)
14.9 -
7.4 =
= 7.5
14.9 -
15.6
= -0.7
7.4 -
15.6 =
-8.2
Culex territans
0.0 -
0.9 =
-0.9
0.0 -
0.0 =
0.0
0.9 -
0.0 =
0.9
Culex salinarius
1.7 -
0.4 =
1.3
1.7 -
0.1 =
1 .6
0.4 -
0.1 =
0.3
Culex ni gripal pus
3.2 -
5.4 =
-2.2
3.2 -
4.5 =
-1.3
5.4 -
4.5 =
0.9
Culi seta melanura
2.5 -
2.2 =
0.3
2.5 -
3.4 =
-0.9
2.2 -
3.4 =
-1.2
Coquillettidia perturbans
1.8 -
3.8 =
-2.0
1.8 -
1.7 =
0.1
3.8 -
1.7 =
2.1
Mansonia indubitans
1.7 -
2.6 =
-0.9
1.7 -
0.2 =
1.5
2.6 -
0.2 =
2.4
Psorophora columbiae
0.5 -
0.0 =
0.5
0.5 -
0.0 =
0.5
0.0 -
0.0 =
0.0
Uranotaenia sapphirina'
49.0 -
74.2 =
-25.2
49.0 -
25.6 =
23.4
74.2 -
25.6 =
48.6
Uranotaenia lowii
5.1 -
6.6 =
-1.5
5.1 -
0.1 =
5.0
6.6 -
0.1, =
6.5
Index of similarity
2|(S-1)
- (S-2) |= 50.2
S|(S-1)
- (C-l)| = 46.0
Si(S-2) (C--
DI -
The numerical values (females per night) are derived from sampling data taken simultaneously
at all three domes. Data collected on nights when all three traps were not in operation are
omitted from this table and Table 3-15.


Table 3-6. Ramp-trap results from 1975 at S-l.
Month
April
May
June
July
Auqust
Total
%.
Trap nights
1
4
2
4
3
14
Number
of mosquitoes
collected
Females:
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
Aedes infirmatus
Aedes dupreei
Aedes atlanti cus
Aedes taeniorhynchus
5
5
0.9
Psorophora ciliata
Psorophora columbiae
Psorophora ferox
Anopheles crucians
Anopheles quadrimaculatus
3
5
4
1
13
2.2
Culex (Melanoconion)
3
30
37
70
11.9
Culex territans
1
1
1
2
1
6
1.0
Culex salinarius
2
16
8
11
2
39
6.6
Culex niqripalpus
Culex restuans
5
6
11
1.9
Culex pipiens quinquefasciatus
1
1
0.2
Culiseta melanura
38
169
5
10
10
232
39.6
Coquillettidia perturbans
1
1
2
0.3
Uranotaenia sapphirina
35
9
56
72
172
29.3
Uranotaenia lowii
11
20
31
5.3
Unidentified specimens
1
4
5
0.9
Total females
44
233
27
130
153
587
Males
3
2
9
9
23


Table 3-25. Summary of Malaise trap sampling of 1976.
Number of mosquitoes collected
Month
May
June
July
S-2
Aug.
Sept.
Total
May
June
AC
July Auq.
Sept. Total
Females:
Aedes vexans
1
1
Aedes infirmatus
2
1
1
6
Aedes atlanti cus
1
1
2
1
3
1
1
6
Anopheles crucians
3
1
5
1
10
2
1
7
10
Culex (Melanoconion)
2
1
3
1
10
11
Culex territans
5
1
3
9
1
1
Culex niqripalpus
2
2
1
1
Culi seta melanura
1
3
2
1
7
1
7
8
Coquillettidia perturbans
1
1
Psorophora ferox
1
1
Uranotaenia sapphirina
6
9
9
20
6
50
3
1
1
5
Uranotaenia lowii
4
12
6
22
Total females
15
12
25
38
17
107
2
5
34
3
2
46
Males
4
3
4
9
11
31
1
31
1
33
GJ
no


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.


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


Figure 3-18. HAI conversions against WEE.


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.


90
80
70
60
50
40
30
20
10
112
C-2*-
S-2-
AC-1 -
Apr I
May
June July Aug.
Sept.


Figure 3-
New Jersey light trap samples excluding males and females
of Uranotaenia sappirina.


50


Table 3-10. (continued)
Date
9/74 10/74 11/74 12/74 1/75 2/75 5/75 6/75 7/75 8/75 9
/75
1
O
"'J
cn
cn
12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76
Totals
%
Trap nights
3
5
3
9
3
2
2
4
5
5
3
3
3 3
4
4
4
3
4
3
2
78
Ferales (cont.):
Culex pipiens
quinquefasciatus
1
1
0.0
Culiseta melanura
2
5
1
1
2
1
1
11
6
33
5
10
15
14
14
4
O
L.
12
5
1
145
1.9
Coquillettidia
perturbaos
42
8
4
8
2
14
3
3
6
9
9
21
8
7
4
148
2.0
Mansonia
indubitans
3
3
3
39
10
22
1
2
9
3
95
1.3
Uranotaenia
390
190
12
8
4
33
51
113
322
364
23
181
9
1
1 15
85
46
134
198
510
400
145
3235
43.0
sapphirina
Uranotaenia lowli
53
9
6
5
1
4
37
60
1
17
1
2
10
79
38
21
344
4.6
Unidentified
specimens
1
6
1
1
7
1
1
1
1
1
1
1
1
24
0.3
Total ferales
1076
717
66
158
42
130
81
164
728
873
79
470
37
25
7 107
391
95
243
402
812
561
257
7521
Males
209
123
29
8
2
10
7
55
219
362
12
69
2
4
7
9
4
40
126
435
159
26
1917
oo
-C*


116
July Aug.
April
May
June
Sept.


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 summa
rizes 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 mos
quitoes 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.,
Coqui1lettidia 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
ni gripal pus and Culiseta 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


Table 3-7. Ramp-trap results from 1975 at C-1.
Month
Apri 1
May
June
July
Auqust
Total
%
Trap nights
1
4
2
4
4
15
Number of
mosquitoes
collected
Females:
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans

Aedes canadensis canadensis
2
2
0.5
Aedes infimiatus
3
1
1
5
1.2
Aedes dupreei
Aedes atlanti cus
14
1
2
17
4.1
Aedes taeniorh.ynchus
Psorophora ciliata
Psorophora columbiae
Psorophora ferox
Anopheles crucians
1
13
1
1
16
3.8
Anopheles quadrimaculatus
3
1
6
2
12
2.9
Culex (Melanoconion)
7
25
34
37
103
24.6
Culex territans
1
4
1
6
1.4
Culex salinarius
2
1
2
2
7
1.7
Culex niqripalpus
2
1
4
4
11
2.6
Culex restuans
Culex pipiens quinquefasciatus
Culiseta melanura
61
92
2
17
11
183
43.8
Coquillettidia perturbans
5
1
2
2
10
2.4
Uranotaenia sapphirina
1
7
10
14
10
42
10.0
Uranotaenia lovni
1
2
3
0.7
Unidentified specimens
1
1
0.2
Total females
64
155
42
84
73
418
Males
5
18
5
34
22
84
00


48
to the experimental swamps. It was mounted on a station wagon, and col
lections 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 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) tran
sect at AC running from the center of the dome to the margin (approxi
mately 410 feet). Ten non-random dips were taken every 16 feet (approxi
mated by pacing). Specific habitats were sampled which, through


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-l.
In the-course of larval sampling, Culex pipiens quinquefasciatus
and Culex restuans, two species with reputations for their association
with polluted water, were found at both S-l 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


76
Table 3-5. Rank comparison test comparing abundance at S-l and C-l
from ramp-trap data (1974).
Total number of
mosquitoes per four
ramp-traps
Trap nights:
S-l
C-l
May 13-14
17
10*
21-22
14
15
23-24
6
28
27-28
160
99*
30-31
115
99*
June 3-4
31
41
6-7
82
44*
11-12
33
40
13-14
43
69
17-18
50
61
20-21
82
43*
24-25
107
127
27-28
384
209*
July 3-4
131
70*
4-5
209
116*
8-9
26
65
Sept.12-13
277
'270*
16-17
222
188*
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
n=total number of trials
(excluding those where
(S-l)=(C-l))
P=
q=
y=
u=
0=
y=
probability of (S-l)>(C-l)
probability of (C-l )>(S-1)
number of times (S-l)>(C-1)
np
vnpq
15
n= 30-1=29
null hypothesis: p=0.5, q=0.5
reject if |Z=y^| > 1.96
since |Z| =
115-14.5
I 1.87
at a=0.05
= .267 < 1.96
do not reject the null hypothesis
at a =0.05

(S-l)>(C-1)


141
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 Culi seta 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.
Percentage positive dips
45%
21%
Number of larvae per positive dip
2.09
2.


Mean Number of Adults per Night
1974 1975 1976


HAI Conversions
30
20
10
21 9 29 20 11 4 24
F M A A M Jn' Jl Jl
> cn
Mosquito Abundance
500
400
300
200
100
25 21 10 14 17
S O D Ja F


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 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 when
ever 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 arrange
ment 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 col
lected on a 24 hour basis. In August 1975, sampling by this method
was discontinued.


37
New Jersey Light Traps
By the end of the summer of 1974, boardwalks had been built to
the center of domes S-l and C-l, 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 tnap 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.
CPC 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,
gel 1-cel 1 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.


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-l, C-l, 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


Table 3-12. New Jersey light trap records for WMHP.
Date
9/74
10/74 11/74 12/74
1/75 2/75
i/75 6/75 7/75 8/75 9/75 10/75
11/75 12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76
Totals
%
Trap nights
2
4 3 9
3 1
1 2
5
4 1
4
3 2 3
3 4 4
4 2
4
3 2
73
Number of
mosquitoes collected
Females:
Aedes fulvus
pal lens
Aedes rritchellae
1
1
1
1
4
Aedes vexans
Aedes canadensis
1
1
1
3
canadensis
2.8
Aeoes infirmatus
1
3
1
1
6
Aedes dupreei
Aedes atlanticus
1 2
2
1
6
Aedes soilicitans
1
1
Aedes aeqypti
1
1
t
Psorophora ciliata
1
1
0.1
Psorophora
columbiae
Psorophora ferox
118
3 1
2
50
30 1
4
1
7
3
5 7
232
31
.1
Anopheles
crucians
18
8
12 5
2 3
11
14 3
8
1 1
19 13 4
6 5
24
8 6
171
22
.9
Anopheles
punctipennis
Anopheles
quadrimaculatus
3
1
4
0.5
Cul ex
fFelanoconion)
30
15
1 1
8
17 2
1 1
2
7
4 5
94
12
.6
Culex territans
Culex salinarius
2
1
1
2
6
0
.8
Culex nicripalpus
Culex restuans
1
2 1
1
1
1 1
1 l!
2
1
9
4
1.2
0.5
Culex pipiens
quinquefasciatus
1
1
1
3
0
.4
Culiseta melanura
2 1
2
1
1
3
4 1
15
2
.0


Table 3-4. (continued)
-*
Number
of mosquitoes
collected at traps oriented as
indicated
S-l
C-l
0
90
180
270
0 .
90
180
270
Uranotaenia sapphirina
231
174
93
93
90
38
178
97
Uranotaenia lowii
58
50
36
40
37
17
68
36
Unidentified Specimens
9
15
6
4
2
10
8
1
Total Females
929
975
423
522
516
770
774
357
Males
180
173
53
71
112
211
208
36
cn


Figure 3-4. Comparison of the abundance of the seven most common species
or groups form AC, C-2, and S-2.
% r


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. In-tervals 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 discon
tinued.
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 dich-
lorvos 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-
Dome S-l, characterized by the extensive invasion
of cattails and dog fennel.


44


RU 3 7-8. £,2.8.9.
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 under
standing 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.
i i


8


118


114
June July Aug.
Sept.


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
i i i


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

RU 3 7-8. £,2.8.9.
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 under
standing 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.
i i

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
i i i

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

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 MOSQUITO 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 encepha
litis activity was monitored at these same sites.
Three years of sampling failed to demonstrate any measurable in
crease in human pest species at the experimental domes. The flood-
water 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 wet
lands 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 disserta
tion 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 a 1., 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-l) receiving
sewage effluent.
A control cypress dome (C-l) receiving untreated
well water.


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 depen
dent 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
1ucida) 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 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.

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.

8

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 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 flood-
water 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 dis
cerning 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 informa
tion 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.

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 dif
ferent 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 CDC^ miniature 1 ight trap. Both these traps have been
extremely popular with mosquito control workers in monitoring fluctu
ations 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.

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 suc
cessful 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 -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 trans
mission 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 tarsal is 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
atlantlcus 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-l. At the same time,
groundwater from a deep well was added to a control dome, C-l. 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-l, S-2, and C-l previous to the addition
of effluent or groundwater. All these domes are represented in Figure 2-3.
17

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

19 '
O
0 12 3
C 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 fore
ground is accumulated cypress litter.

21

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.

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 sus
pended 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/1 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/1, Mg 11.4-20.6 mg/1).
Total phosphorus and nitrogen were greater in samples from S-l than
from AC, and a major portion of the total nitrogen and phosphorus in
samples at S-l 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

Table 2-1. Brief descriptions of the cypress domes examined.
Dome
Location
Area*
Description
Understory Veqetation
Hydroloqy
S-l
2 mi. NW of
Gainesvi He,
off Rt. 441
0.5
Experimental dome
receiving sewage
effluent. Badly
burned (1973).
Open canopy.
Extensive duckweed cover
(Figure 2-5), and invasion
by cattails and dog fennel
(Figure 2-6).
Year-round,
standing water.
S-2
2 mi. NW of
Gainesville,
off Rt. 441
1.0
Experimental dome
receiving sewage
effluent. Only
slight fire damage.
Thick canopy.
Partial duckweed cover,
broken at the margins.
Fetterbush and Virginia
chain fern abundant.
Year-round,
standing water.
C-l
2 mi. NW of
Gainesville,
off Rt. 441
0.7
Control dome
receiving well
water. Badly burned.
Open canopy.
Scattered duckweed, more
abundant at the margins.
Bladderwort very abundant
(Figure 1-2).
Year-round,
standing water.
C-2
2 mi. NW of
Gainesville,
off Rt. 441
0.9
Drained, control
dome receiving no
treatment. Not
burned. Thick
canopy.
No duckweed. Fetterbush
abundant. Extensive
accumulations of litter
(Figure 2-2).
Fluctuating water
level. No standing
water much of the year.
AC
3 mi. NE of
Gainesville,
off Rt. 24
4.5
Unburned, undis
turbed, control
dome. Thick
canopy.
Sparse duckweed, extensive
bladderwort, fetterbush,
and ferns.
Fluctuating water
level, but holds
standing water year-round.
*Hectares

Figure 2-5. Photos taken at S-l 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.

r/l
28

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

30

31
ratios were low in S-l 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-l 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 perpusi11a and Spirodela oligorhiza. The
floating fern, Azolla caroliniana was also present. A cover of duck
weed and fern was likewise formed at C-l, 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) con
taining 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-l and C-l with the ramp openings facing the center of each dome.
Collections were started in April at S-l and in May at C-l. 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
from the ramp-trap by aspiration.
C. Removing the trapping
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 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 when
ever 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 arrange
ment 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 col
lected 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-l and C-l, 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 tnap 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.
CPC 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,
gel 1-cel 1 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.

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


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

¡T? 'OT
H l
1: w*
mg* ' i
fii '.iffiVn
* *%e£r

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. In-tervals 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 discon
tinued.
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 dich-
lorvos 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

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.


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

48
to the experimental swamps. It was mounted on a station wagon, and col
lections 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 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) tran
sect at AC running from the center of the dome to the margin (approxi
mately 410 feet). Ten non-random dips were taken every 16 feet (approxi
mated 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

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

52

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


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-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 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 encephalitide viruses.

Figure 2-17.
A. A sentinel chicken cage at the margin of S-l.
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 -90C.
Later the samples were transported on dry ice to Tampa for viral iso
lation 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-l, C-2, S-l, 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 pre
viously 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 ace
tone 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 anti
gens 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 Reed-
Muench 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.

4 66
^l
j
$
V "V / N
V
/ ,'
*1
-J *
\ >
S< X Hr Vr
> > S' S'- V' V
~S* > S \ / > -
.
M
**** '%w-
i&tL +*
v V Y' v ^
. J
-*
VVWr^- - -
# S' ^ ~ : ^
o y 4 j , *# *->
1* *. i- 2* Si 5> 2^ I " -.
25
v
V

RESULTS AND DISCUSSION
Adult Mosquito Sampling
Ramp Traps
Using no known insect attractants, ramp traps are flight inter
ception 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. Shannon-
Weaver 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-l is slightly larger than that for S-l. Since there were two more
species captured at S-l than at C-l, the larger index for C-l repre
sents 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

Table 3-1. Summary of ramp-trap collections for 1974 at S-l.
Month
Apri 1
May
June
July
Sept.
Oct.
Nov.
Total
%
Trap Nights
6
9
8
3
4
7
3
40
Number
of mosquitoes collected
Females:
Aedes fulvus pallens
8
1
9
0.4
Aedes mitchellae
3
2
1
6
0.3
Aedes vexans
1
1
0.0
Aedes canadensis canadensis
1
1
0.0
Aedes infirmatus
9
4
1
14
0.6
Aedes dupreei
1
1
0.0
Aedes atlanti cus
56
76
14
146
6.2
Aedes taeniorh.ynchus
1
1
0.0
Psorophora ciliata
0
Psorophora colurnbiae
1
11
5
5
22
0.9
Psorophora ferox
2
23
4
3
32
1.3
Anopheles crucians
4
20
92
21
8
13
4
162
6.8
Anopheles quadrimaculatus
3
7
10
0.4
Culex (Melanoconion)
20
21
6
142
48
2
239
10.1
Culex territans
5
3
5
5
1
1
1
21
0.9
Culex salinarius
5
5
9
4
1
9
3
36
1.5
Culex niqripalpus
3
5
12
12
60
56
14
162
6.8
Culex restuans
2
2
0.1
Culex pipiens quinquefasciatus
2
1
2
5
0.2
Culiseta melanura
6
134
151
149
30
158
21
649
27.4
Coquillettidia perturbans
1
9
20
5
3
5
1
44
1.9
Uranotaenia sapphirina
1
20
146
46
306
66
6
591
24.9
Uranotaenia lowii
22
3
68
60
31
184
7.8
Unidentified specimens
8
21
2
3
34
1.4
Total females
30
319
604
281
630
425
83
2372
Males
11
45
208
85
80
38
10
477

Table 3-2. Summary of ramp-trap collections for 1974 at C-1.
Month
May
June
July
Sept.
Oct.
Nov.
Total
%
Trap nights
6
8
3
4
8
3
32
Number
of mosquitoes collected
Females:
Aedes fulvus pallens
0
Aedes mitchellae
1
1
0.1
Aedes vexans
1
ft
1
0.1
Aedes canadensis canadensis
3
9
3
15
0.8
Aedes infirmatus
7
12
1
20
1.1
Aedes dupreei
4
1
5
0.3
Aedes atlanti cus
69
134
50
253
13.7
Aedes taeniorh.ynchus
0
Psorophora ciliata
1
1
2
0.1
Psorophora columbiae
1
5
1
1
1
9
0.5
Psorophora ferox
15
15
6
1
37
2.0
Anopheles crucians
10
45
15
11
12
8
101
5.5
Anopheles quadrimaculatus
1
18
15
2
36
1.9
Culex (Melanoconion)
2
2
134
40
5
183
9.9
Culex territans
1
22
10
7
12
52
2.8
Culex salinarius
2
4
1
3
3
13
0.7
Culex nigripalpus
6
9
1
36
41
11
104
5.6
Culex restuans
0
Culex pipiens quinquefasciatus
1
1
0.1
Culiseta melanura
39
90
69
59
126
32
415
22.4
Coquillettidia perturbans
10
7
2
1
20
1.1
Uranotaenia sapphirina
22
93
30
200
52
6
403
21.8
Uranotaenia lowii
1
11
6
81
37
22
158
8.5
Unidentified specimens
5
11
3
1
1
21
1.1
Total females
200
470
200
542
337
101
1850
Males
58
164
51
204
75
15
567

Figure 3-1. The relative abundance of females of the most common species
or groups from 1974 ramp-trap samples at S-l and C-l.

%
of Total
Capture
r\j rv> gj
o Ui o
-1--
Culiseta
mela nura
Uranotaenia
sapphirina
Culex
(Melanoconion)
Uranotaenia
Ipwii
Floodwater
Aedes species
Anopheles
X'Xv/Xv'v
m
Yfs,
cruc ians
Culex
'mmm
niqropal pus
U

72
Table 3-3. Shannon-Weaver diversity indices computed from 1974 ramp-trap
data at S-l and C-1.
Species
Pi
S-l
Pj(logcPi)
EM
C-1
Pi (logePjJ
Aedes:
mitchellae
.0026
-.0155
.0005
-.0038
vexans
.0004
-.0031
.0005
-.0038
infirmatus
.0060
-.0307
.0109
-.0493
dupreei
.0004
-.0031
.0027
-.0160
at!anti cus
.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
niqripalpus
.0693
-.1850
.0569
-.1631
pipiens
.0021
-.0129
.0005
-.0038
restuans
.0009
-.0063
Culiseta:
melanura
.2776
-.3558
.2269
-.3365
Coqui11ettidia:
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
1 owi i
.0787
-.2001
.0864
-.2116
H' =
^PilogePi = 2.0862
H' =
2.1998
H' = Shannon-Weaver diversity index
p.j = the proportion of the it*1 species in the total sample

73
The four traps at each dome were not equally successful in cap
turing mosquitoes (Table 3-4). At S-l, 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-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 insuffi
cient 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-l, 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

Table 3-4. Summary of ramp-trap orientation and performance at S-l and C-l for 1974.
Number
of mosquitoes
collected at
traps
oriented as
indicated
S-l
C-l
Females:
0
O
O
cn
co
o
0
270
0
O
O
o
180
270
Aedes fulvus pallens
5
2
1
1
Aedes mitchellae
3
2
1
1
Aedes vexans
1
1
Aedes canadensis canadensis
1
14
1
Aedes infirmatus
6
7
1
3
11
5
1
Aedes dupreei
1
3
2
Aedes atlanticus
82
41
11
12
26
178
39
10
Aedes taeniorhynchus
1
Psorophora ciliata
2
Psorophora columbiae
4
9
9
2
6
3
Psorophora ferox
15
3
4
10
4
34
10
Anopheles crucians
32
59
18
53
37
20
30
14
Anopheles quadrimaculatus
5
2
3
9
22
5
Culex (Melanoconion)
28
91
81
39
51
15
94
23
Culex territans
4
5
12
3
40
4
5
Culex salinarius
12
11
7
6
3
4
4
2
Culex nigripalpus
62
72
12
16
38
19
25
22
Culex restuans
Culex pipiens quinquefasciatus
1
2
1
1
1
Culiseta melanura
184
246
78
141
100
135
93
87
Coquillettidia perturbans
14
6
9
15
1
10
6
3

Table 3-4. (continued)
-*
Number
of mosquitoes
collected at traps oriented as
indicated
S-l
C-l
0
90
180
270
0 .
90
180
270
Uranotaenia sapphirina
231
174
93
93
90
38
178
97
Uranotaenia lowii
58
50
36
40
37
17
68
36
Unidentified Specimens
9
15
6
4
2
10
8
1
Total Females
929
975
423
522
516
770
774
357
Males
180
173
53
71
112
211
208
36
cn

76
Table 3-5. Rank comparison test comparing abundance at S-l and C-l
from ramp-trap data (1974).
Total number of
mosquitoes per four
ramp-traps
Trap nights:
S-l
C-l
May 13-14
17
10*
21-22
14
15
23-24
6
28
27-28
160
99*
30-31
115
99*
June 3-4
31
41
6-7
82
44*
11-12
33
40
13-14
43
69
17-18
50
61
20-21
82
43*
24-25
107
127
27-28
384
209*
July 3-4
131
70*
4-5
209
116*
8-9
26
65
Sept.12-13
277
'270*
16-17
222
188*
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
n=total number of trials
(excluding those where
(S-l)=(C-l))
P=
q=
y=
u=
0=
y=
probability of (S-l)>(C-l)
probability of (C-l )>(S-1)
number of times (S-l)>(C-1)
np
vnpq
15
n= 30-1=29
null hypothesis: p=0.5, q=0.5
reject if |Z=y^| > 1.96
since |Z| =
115-14.5
I 1.87
at a=0.05
= .267 < 1.96
do not reject the null hypothesis
at a =0.05

(S-l)>(C-1)

Table 3-6. Ramp-trap results from 1975 at S-l.
Month
April
May
June
July
Auqust
Total
%.
Trap nights
1
4
2
4
3
14
Number
of mosquitoes
collected
Females:
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
Aedes infirmatus
Aedes dupreei
Aedes atlanti cus
Aedes taeniorhynchus
5
5
0.9
Psorophora ciliata
Psorophora columbiae
Psorophora ferox
Anopheles crucians
Anopheles quadrimaculatus
3
5
4
1
13
2.2
Culex (Melanoconion)
3
30
37
70
11.9
Culex territans
1
1
1
2
1
6
1.0
Culex salinarius
2
16
8
11
2
39
6.6
Culex niqripalpus
Culex restuans
5
6
11
1.9
Culex pipiens quinquefasciatus
1
1
0.2
Culiseta melanura
38
169
5
10
10
232
39.6
Coquillettidia perturbans
1
1
2
0.3
Uranotaenia sapphirina
35
9
56
72
172
29.3
Uranotaenia lowii
11
20
31
5.3
Unidentified specimens
1
4
5
0.9
Total females
44
233
27
130
153
587
Males
3
2
9
9
23

Table 3-7. Ramp-trap results from 1975 at C-1.
Month
Apri 1
May
June
July
Auqust
Total
%
Trap nights
1
4
2
4
4
15
Number of
mosquitoes
collected
Females:
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans

Aedes canadensis canadensis
2
2
0.5
Aedes infimiatus
3
1
1
5
1.2
Aedes dupreei
Aedes atlanti cus
14
1
2
17
4.1
Aedes taeniorh.ynchus
Psorophora ciliata
Psorophora columbiae
Psorophora ferox
Anopheles crucians
1
13
1
1
16
3.8
Anopheles quadrimaculatus
3
1
6
2
12
2.9
Culex (Melanoconion)
7
25
34
37
103
24.6
Culex territans
1
4
1
6
1.4
Culex salinarius
2
1
2
2
7
1.7
Culex niqripalpus
2
1
4
4
11
2.6
Culex restuans
Culex pipiens quinquefasciatus
Culiseta melanura
61
92
2
17
11
183
43.8
Coquillettidia perturbans
5
1
2
2
10
2.4
Uranotaenia sapphirina
1
7
10
14
10
42
10.0
Uranotaenia lovni
1
2
3
0.7
Unidentified specimens
1
1
0.2
Total females
64
155
42
84
73
418
Males
5
18
5
34
22
84
00

Table 3-8. Ramp-trap results from 1975 at S-2.
Month
Apri 1
May
June July
Auqust
Total
%
Trap nights
1
4
2 5
4
16
Number of mosquitoes
collected
Females:
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
2
*
2
0.6
Aedes infirmatus
1
1
0.3
Aedes dupreei
Aedes at!anti cus
7
7
2.3
Aedes taeniorhynchus
Psorophora ciliata
Psorophora columbiae
Psorophora ferox
Anopheles crucians
Anopheles quadrimaculatus
10
16
1 2
29
9.4
Culex (Melanoconion)
1
2 4
7
14
4.5
Culex territans
22
50
6 7
6
91
29.5
Culex salinarius
9
1 2
12
3.9
Culex niqripalpus
1 3
6
10
3.2
Culex restuans
Culex pipiens quinquefasciatus
Culi seta melanura
11
36
6
6
6
65
21.1
Coquillettidia perturbans
2
2
0.6
Uranotaenia sapphirina
5
14
9
22
16
66
21.4
Uranotaenia lowii
1
5
1
7
2.3
Unidentified specimens
2
2
0.6
Total females
48
138
28
52
42
308
Males
14
18
5
18
3
58

Table 3-9. Ramp-trap results from 1975 at AC.
Month
Apri 1
May
June
July
Auqust
Total
%
Trap nights
1
4
2
5
4
16
Number of
mosquitoes
collected
Females:
Aedes fulvus pal lens
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
Aedes infirmatus
Aedes dupreei
Aedes atlanti cus
1
1
1.4
Aedes taeniorhynchus
Psorophora ciliata
Psorophora columbiae
Psorophora ferox
Anopheles crucians
Anopheles quadrimaculatus
2
o
O
2
3
15
20.3
Culex (Melanoconion)
3
9
12
16.2
Culex territans
Culex salinarius
1
1
1 .4
Culex niqripalpus
3
5
8
10.8
Culex restuans
Culex pipiens quinquefasciatus
Culi seta melanura
8
5
3
6
22
29.7
Coquillettidia perturbans
4
4
5.4
Uranotaenia sapphirina
Uranotaenia lowii
2
2
2
4
10
13.5
Unidentified specimens
1
1
1.4
Total females
12
20
0
13
29
74
Males
6
1
4
11
oo
o

81
than any of the other domes, and Culex (Melanoconion) spp. were more
abundant at C-l. 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 ni gripal pus 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
]This 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-l, 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-l or C-l, and that S-l
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

Table 3-10. New Jersey Light trap records for S-l.
Date
9/74 10/74 11/74 12/74
1/75 2/75
5/75 6/75
7/75 8/75 9/75
10/75 11/75
12/75 1/75 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76
Totals
Trap nights
3 5 3 9
3 2
1 2
A 5 1
4 3
3 3 3 4 5 4 3 4 3 2
78
Number of
mosquitoes
collected
Females:
Aedes fulvus
pallens
1
1
Aeces mitchellae
1
1
Acjaes vexans
1
1
2
Aeaes canadensis
canadensis
1
1
Aedes infirmatus
1
2
1 1
5
Aedes dunreei
Aedes atlanticus
1
1
12
3 1
18
Aedes soilicitans
Aedes aecypti
Psoropnora
ci1iata
1
1
5
7
Psoropnora
columbiae
Psoroohora
45
4
2
10
8
4
1
2
2
78
ferox
Anopheles
crucians
61
41
9
38 25 28 11 21
82
135 24
144
10
13
6 69 234 16 19 49 98
31
35
1199
Ancpheles
punctipennis
Anopheles
1
1
2
quadrimaculatus
3
5
2
1 1 1
5
1
1
3
3
2
28
Culex
fKelanoconion)
Culex
428
182
7
1
32
8
13
209
218
8
15
4
33
5 57
73
74
52
23
1442
territans
CuTex
1
1
1
1
4
salinarius
Culex
6
5
7
2
11
3
6
18
2
1
14
4 17
22
3
121
niqripalpus
49
263
25
84
9
1
31
20
6
33
8
1
2
25
16
12
15
600
Culex restuans
7
5
6
1
1
20

Table 3-10. (continued)
Date
9/74 10/74 11/74 12/74 1/75 2/75 5/75 6/75 7/75 8/75 9
/75
1
O
"'J
cn
cn
12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76
Totals
%
Trap nights
3
5
3
9
3
2
2
4
5
5
3
3
3 3
4
4
4
3
4
3
2
78
Ferales (cont.):
Culex pipiens
quinquefasciatus
1
1
0.0
Culiseta melanura
2
5
1
1
2
1
1
11
6
33
5
10
15
14
14
4
O
L.
12
5
1
145
1.9
Coquillettidia
perturbaos
42
8
4
8
2
14
3
3
6
9
9
21
8
7
4
148
2.0
Mansonia
indubitans
3
3
3
39
10
22
1
2
9
3
95
1.3
Uranotaenia
390
190
12
8
4
33
51
113
322
364
23
181
9
1
1 15
85
46
134
198
510
400
145
3235
43.0
sapphirina
Uranotaenia lowli
53
9
6
5
1
4
37
60
1
17
1
2
10
79
38
21
344
4.6
Unidentified
specimens
1
6
1
1
7
1
1
1
1
1
1
1
1
24
0.3
Total ferales
1076
717
66
158
42
130
81
164
728
873
79
470
37
25
7 107
391
95
243
402
812
561
257
7521
Males
209
123
29
8
2
10
7
55
219
362
12
69
2
4
7
9
4
40
126
435
159
26
1917
oo
-C*

Table 3-11. New Jersey light trap records for C-1.
Date 9/74 10/74 11/74 12/74 1/75 2/75 6/75 7/75 8/75 9/75 10/75 11/75 12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 Totals %
rap niohts
3
5
3
10
3
2
1
4
5
1
4
2
3 3
3
4
4
4
1
4
3
74
'ema 1 es:
Number of mosquitoes
collected
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
1
1
0.3
Aedes infirma tus
1
8
9
Aedes dupreei
Aedes atlanticus
Aedes soilicitans
Aedes aeqypti
3
2
5
'
Psorophora ciliata
Psorophora coiumbiae
1
1
1
3
Psorophora ferox
Anopheles crucians
50
47
3
23
26
26
7
33
57
20
46
5
11
19
64
8
17
26
50
14
552
10.3
Anopheles punctipennis
1
1
0.0
Anopneles quadrimaculatus
6
8
1
1
3
3
2
2
1
1
2
3
1
34
0.6
Culex (Kelanoconion)
294
114
1
19
9
109
50
no
143
7
11
1
46
92
19
50
95
76
50
1296
24
.3
Culex territans
1
1
0.0
Culex salinarius
3
3
2
2
10
0
.2
Culex niqripalpus
58
11
80
13
1
9
29
2
10
2
3
2
1
116
32
11
380
7
.1
Culex restuans
5
2
7
0
.1
Culex pipiens quinquefasciatus
Culiseta nelanura
5
13
18
5
13
6
1
37
4
13
11
10
22
9
17
28
4
216
4
.0
Coauillettidia perturbans
17
5
1
3
1
5
2
14
23
29
2
102
1
.9
i'ansonia indubitans
5
1
1
2
2
11
0
.2
Uranotaenia sapphirina
819
536
14
3
1
19
59
205
287
6
27
4
14
57
26
81
264
132
68
2622
49
.1
Uranotaenia lowii
48
16
1
1
2
1
1
1
2
73
1.4
Jnidentified specimens
1
1
1
4
1
1
2
1
12
0.2
rotal females
1299
751
18
153
59
172
122
364
532
36
149
18
28 0
93
228
81
172
549
355
154
5335
iales
334
342
61
5
6
10
24
128
189
10
80
7
4
2
27
20
43
141
179
106
1718
co
cn

Table 3-12. New Jersey light trap records for WMHP.
Date
9/74
10/74 11/74 12/74
1/75 2/75
i/75 6/75 7/75 8/75 9/75 10/75
11/75 12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76
Totals
%
Trap nights
2
4 3 9
3 1
1 2
5
4 1
4
3 2 3
3 4 4
4 2
4
3 2
73
Number of
mosquitoes collected
Females:
Aedes fulvus
pal lens
Aedes rritchellae
1
1
1
1
4
Aedes vexans
Aedes canadensis
1
1
1
3
canadensis
2.8
Aeoes infirmatus
1
3
1
1
6
Aedes dupreei
Aedes atlanticus
1 2
2
1
6
Aedes soilicitans
1
1
Aedes aeqypti
1
1
t
Psorophora ciliata
1
1
0.1
Psorophora
columbiae
Psorophora ferox
118
3 1
2
50
30 1
4
1
7
3
5 7
232
31
.1
Anopheles
crucians
18
8
12 5
2 3
11
14 3
8
1 1
19 13 4
6 5
24
8 6
171
22
.9
Anopheles
punctipennis
Anopheles
quadrimaculatus
3
1
4
0.5
Cul ex
fFelanoconion)
30
15
1 1
8
17 2
1 1
2
7
4 5
94
12
.6
Culex territans
Culex salinarius
2
1
1
2
6
0
.8
Culex nicripalpus
Culex restuans
1
2 1
1
1
1 1
1 l!
2
1
9
4
1.2
0.5
Culex pipiens
quinquefasciatus
1
1
1
3
0
.4
Culiseta melanura
2 1
2
1
1
3
4 1
15
2
.0

Date 9/74 10/74 11/74 12/74 1/75 2/75 5/75 6/75 7/75 8/75 9/75 10/75 11/75 12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76 Totals %
Trap nights 2 4 3 9 3 1 T ~2 5 4 1 4 3 2 3 3 4 4 4 2 4 3 2 73
Fen.ales (cont.):
Coquillettidia
perturoans 8
Hansonia
ir.dubl tans
Uranotaenia
sapphirina 21
Urar.otaenia
1 owi i 4
Unidentified
specimens
Total females 203
Males 6
3
6 2 2
1 1
30 5 12 20 8
6 113 1
2 2 2
7 1 2 8
1
1 1
10 12 77 80 8
4 5 18 20
1
4 2 14 3
1
1
26 4 3 4 26
18 111
2 15 14 7 6
2 4 7 5 2
2
20 28 30 28 46
2 2 10 10 9
8
3
73
9.8
7
9
99
13.3
1
9
1.2
5
0.7
33
33
746
12
25
156

Table 3-13
New Jersey light trap records for S-2.
Date 5/75 6/75 7/75 8/75 9/75 10/75 11/75 12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76 Totals %
Trap nights
2
3
4
3"
3 3
3
4
4
4
3
4
~T~
2
53
Number of mosquitoes
collected
Fema 1 es:
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans
1
1
Aeoes canadensis canadensis
0
3
Aedes infirma tus
2
1
3
Aedes duoreei
1
1
Aedes atlanticus
Aedes sollicitans
Aeces aeq.ypti
1
2
5
1
2
11
f
Psorophora ciliata
1
1
0
0
Psorophora columpiae
1

1
0
0
Pscrochora ferox
1
1
0
0
Anopheles crucians
31
19
42
35
25
47
1
10 2
59
128
17
19
34
65
35
19
588
9
6
Ancoheles pur.ctipennis
Anopheles quadrimaculatus
1
1
1
1
2
1
7
0
.1
Culex (t-'.elanoconion)
5
23
130
107
4
5
1
3
5
3
9
14
23
26
36
394
6
4
Cuiex temtans
17
5
8
3
1
2
1
5
2
1
3
48
0.8
Culex salinarius
3
6
2
1
7
1
1
21
0
.3
Cuiex niqripalpus
18
108
53
1
20
3
1
8
32
33
12
289
4
.7
Culex restuans
1
1
0.0
Culex pipiens quinquefasciatus
Culi seta melanura
6
3
8
9
1
15
3
12 1
12
10
15
1
2
5
4
7
114
1
.9
Coqui1lettidia perturbaos
34
17
14
1
1
3
31
60
14
16
10
1
202
3
.3
Mansonia indubitans
2
71
16
35
3
1
2
6
2
138
2
.3
Uranotaenia sapphirina
143
276
347
611
29
233
9
3
16
149
131
240
368
517
518
341
3931
64.2
llranotaenia lowii
13
67
42
51
3
29
7
3
2
1
4
40
47
40
349
5
.7
Unidentified specimens
3
2
8
2
1
1
4
3
24
0.4
Total females
255
425
714
953
81
397
23
29 3
94
298
204
334
454
707
688
461
6125
Males
162
1013
573
628
17
137
32
14 1
6
31
171
261
370
811
513
199
4939
co
oo

Figure 3-2. Comparison of New Jersey light trap samples from
four different sites.

Mean Number of Adults per Night
1974 1975 1976

Table 3-14. Degree of similarity among the three domes, comparing two ata time, based on abundance
data from 1975-1976 New Jersey light trap samples.'
Abundance
(S-1) (S-2) (S-1) (C-l) (S-2) (C-l)
Anopheles crucians
18.8 -
11.1
= 7.7
18.8 -
7.9 =
= 10.9
11.1 -
7.9 =
* 3.2
Anopheles quadrimaculatus
0.3 -
0.1 =
0.2
0.3 -
0.4 =
-0.1
0.1 -
0.4 =
-0.3
Culex (Melanoconion)
14.9 -
7.4 =
= 7.5
14.9 -
15.6
= -0.7
7.4 -
15.6 =
-8.2
Culex territans
0.0 -
0.9 =
-0.9
0.0 -
0.0 =
0.0
0.9 -
0.0 =
0.9
Culex salinarius
1.7 -
0.4 =
1.3
1.7 -
0.1 =
1 .6
0.4 -
0.1 =
0.3
Culex ni gripal pus
3.2 -
5.4 =
-2.2
3.2 -
4.5 =
-1.3
5.4 -
4.5 =
0.9
Culi seta melanura
2.5 -
2.2 =
0.3
2.5 -
3.4 =
-0.9
2.2 -
3.4 =
-1.2
Coquillettidia perturbans
1.8 -
3.8 =
-2.0
1.8 -
1.7 =
0.1
3.8 -
1.7 =
2.1
Mansonia indubitans
1.7 -
2.6 =
-0.9
1.7 -
0.2 =
1.5
2.6 -
0.2 =
2.4
Psorophora columbiae
0.5 -
0.0 =
0.5
0.5 -
0.0 =
0.5
0.0 -
0.0 =
0.0
Uranotaenia sapphirina'
49.0 -
74.2 =
-25.2
49.0 -
25.6 =
23.4
74.2 -
25.6 =
48.6
Uranotaenia lowii
5.1 -
6.6 =
-1.5
5.1 -
0.1 =
5.0
6.6 -
0.1, =
6.5
Index of similarity
2|(S-1)
- (S-2) |= 50.2
S|(S-1)
- (C-l)| = 46.0
Si(S-2) (C--
DI -
The numerical values (females per night) are derived from sampling data taken simultaneously
at all three domes. Data collected on nights when all three traps were not in operation are
omitted from this table and Table 3-15.

Table 3-15. Degree of similarity based on relative abundance data from 1975-76 New Jersey light
trap samples.
Relative abundance
(S-l]
1 (S-2)
(S-l)
- (C-
-1).
(S-2)
- (C-l)
Anopheles crucians
18.7 -
9.6 = 9.1
18.7 -
13.1
= 5.6
9.6 -
13.1 = -3.5
Anopheles quadrimaculatus
0.3 -
0.1 = 0.2
0.3 -
0.7 =
-0.4
0.1 -
0.7 = -0.6
Culex (Melanoconion)
14.9 -
6.4 = 8.5
14.9 -
26.0
= -11.1
6.4 -
26.0 = -19.i
Culex territans
0.0 -
0.8 = -0.8
0.0 -
0.0 =
0.0
0.8 -
0.0 = 0.8
Culex salinarius
1.7 -
0.3 = 1.4
1.7 -
0.1 =
1 .6
0.3 -
0.1 =' 0.2
Culex nigripalpus
3.2 -
4.7 = -1.5
3.2 -
7.5 =
-4.3
4.7 -
7.5 = -2.8
Culiseta melanura
2.5 -
1.9 = 0.6
2.5 -
5.6 =
-3.1
1.9 -
5.6 = -3.7
Coquillettidia perturbans
1.8 -
3.3 = -1.5
1.8 -
2.8 =
-1.0
3.3 -
2.8 = 0.5
Mansonia indubitans
1.7 -
2.3 = -0.6
1.7 -
0.4 =
1.3
2.3 -
0.4 = 1.9
Psorophora columbiae
0.5 -
0.0 = 0.5
0.5 -
0.0 =
0.5
0.0 -
0.0 = 0.0
Uranotaenia sapphirina
48.7 -
64.2 = -15.5
48.7 -
42.7 =
6.0
64.2 -
42.7 = 21.5
Uranotaenia lowii
5.1 -
5.7 = -0.6
5.1 -
0.2 =
4.9
5.7 -
0.2 = 5.5
Index of similarity
Z|(S-1)
- (S-2) | = 40.8
Z|(S-l)
- (C-l)| = 39.8
11 (S-2)
- (C-l) | = 60

93
respectively. Degree of similarity is based on the amount of differ
ences 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-l were more alike than S-l and S-2.
Of the three comparisons, S-2 and C-l 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
Uranotaenja sapphirina at the two sewage domes. Starting in June 1975,
males of LL sapphirina at the two sewage domes were counted and re
corded 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 l. 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 l. sapphirina. From both a medical and an economic standpoint
this species is of no known importance; and therefore, a more meaning
ful 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.

Figure 3-
New Jersey light trap samples excluding males and females
of Uranotaenia sappirina.

' 1974 ' 1975 ' 1976
<£>
en

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 Coqui1let-
tidia 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 de
pressions 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-l, S-2, and C-l, differences

97
in mosquito populations at these domes are assumed to be a result of
the differences in water chemistry and altered vegetation patterns.
This of course is itself based on the assumption that mosquito popula
tions 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.
CPC 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 year-
round 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-l 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

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 summa
rizes 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 mos
quitoes 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.,
Coqui1lettidia 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
ni gripal pus and Culiseta 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

Table 3-16. The 1976 CPC light trap results at AC.
Date
4/76
5/76
6/76
7/76
8/76
9/76
Total
Trap nights
8
8
8
6
5
6
41
Number
of mosquitoes
collected
Specimens
per niqht
Females:
Aedes fulvus pallens
1
5
6
A
Aedes mitchellae
Aedes vexans
6
6
Aedes canadensis canadensis
7
7
36
.58
Aedes infirmatus
2
6
707
91
57
125
988
Aedes dupreei
10
1
11
Aedes atlanti cus
4
44
400
5
29
482
/
Psorophora ciliata
3
2
3
8
0
.20
Psorophora columbiae
Psorophora ferox
2
1
3
0
.07
Anopheles crucians
125
112
298
1054
93
70
1752
42
.73
Anopheles punctipennis
Anopheles quadrimaculatus
1
1
0
.02
Culex (Melanoconion)
20
58
351
160
120
47
756
18
.44
Culex territans
1
1
2
0
.05
Culex salinarius
1
1
0
.02
Culex niqripalpus
3
723
520
77
234
1557
37
.89
Culex restuans
Culex pipiens quinquefasciatus
Culi seta melanura
36
57
188
317
12
56
666
16.24
Coquillettidia perturbans
11
25
14
28
4
7
89
2.17
Mansonia indubitans
Uranotaenia sapphirina
152
527
609
411
109
225
2033
49.59
Uranotaenia lowii
5
9
14
0.34
Unidentified specimens
1
17
25
3
3
49
1.20
Total females
346
796
2973
3029
490
797
8431
205.63
Males
127
620
332
1122
145
53
2399
58.51

Table 3-17. The 1976 CPC light trap results at C-2.
Date
4/76
5/76
6/76
7/76
8/76
9/76
10/76
Total
Trap nights
8
8
8
8
6
8
2
48
Number
of mosquitoes
collected
Specimens per night
Females:
Aedes fulvus pallens
Aedes mitchellae
17
13
2
1
33
\
Aedes vexans
1
. 1
Aedes canadensis canadensis
2
4
6
19.
52
Aedes infirmatus
9
27
155
5
2
3
201
Aedes dupreei
4
1
5
Aedes at!anti cus
30
130
307
69
154
1
691
V
Psorophora ciliata
1
1
0
02
Psorophora columbiae
1
1
0
02
Psorophora ferox
3
1
4
0
08
Anopheles crucians
59
42
133
270
76
62
9
651
13
56
Anopheles punctipennis
Anopheles quadrimaculatus
1
6
2
6
15
0
31
Culex (Melanoconion)
2
14
5
7
28
0
.58
Culex territans
1
3
1
5
0
.10
Culex salinarius
1
1
0
.02
Culex niqripalpus
Culex restuans
Culex pipiens quinquefasciatus
1
185
717
378
377
1658
34
.54
Culiseta melanura
38
205
487
919
247
211
11
2118
44
.13
Coquillettidia perturbans
2
24
13
22
15
2
78
1
.63
Mansonia indubitans
1
1
0
.02
Uranotaenia sapphirina
17
16
35
293
285
193
12
851
17
.73
Uranotaenia lowii
2
5
11
18
0.38
Unidentified specimens
8
11
2
10
1
32
0
.67
Total females
125
345
1174
2591
1089
1041
34
6399
133.31
Males
69
18
52
168
102
128
28
565
11
.77

Date
4/76
5/76
6/76
7/76
8/76
9/76
10/76 Total
Trap nights
8
8
8
7
6
8
4
49
Number
of mosquitoes
collected
Specimens per night
Females:
Aedes fulvus pal lens
4
3
7
A
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
1
1
4
6
4.
78
Aedes infirmatus
Aedes dupreei
4
1
5
Aedes atlanti cus
6
175
12
22
1
216
/
Psorophora ciliata
1
1
2
0.04
Psorophora columbiae
1
1
2
0
04
Psorophora ferox
1
1
0
02
Anopheles crucians
66
103
122
374
79
115
15
874
17
.84
Anopheles punctipennis
Anopheles quadrimaculatus
4
7
1
3
2
17
0
.35
Culex (Melanoconion)
3
24
105
160
87
111
2
492
10
.04
Culex territans
2
1
3
5
1
12
0.24
Culex salinarius
7
22
15
2
3
49
1
.00
Culex niqripalpus
1
179
605
158
76
27
1046
21
.35
Culex restuans
Culex pipiens quinquefasciatus
1
1
0
.02
Culiseta melanura
98
101
316
399
107
121
49
1191
24
.31
Coquillettidia perturbans
15
41
18
62
15
34
185
3
.78
Mansonia indubitans
5
4
1
10
0
.20
Uranotaenia sapphirina
131
148
379
471
396
733
31
2289
46
.71
Uranotaenia lowii
2
1
2
5
15
17
4
46
0
.94
Unidentified specimens
3
1
2
2
8
0
.16
Total females
325
442
1163
2275
878
1240
136
6459
131
.82
Males
113
227
421
498
515
400
no
2284
46.61

Figure 3-4. Comparison of the abundance of the seven most common species
or groups form AC, C-2, and S-2.
% r

Floodwater
Aedes
Anophe les
crucians
Culex
(Melanocomon)
Average Number per Trap Night
_> r\j w -k
O O O o o
Culex
niqropalpus
Culi seta
melanura
Coquillett idia
perterbans
Uranotaenia
sapphi rina
501 C-2

iauie j-iy.
KanK comparison test of the null hypothesis
at S-2 is similar to the number at C-2.
that the number of
female mosquitoes in CDC traps
Species
n y z
Decision
(ct=0.05)
Cone!usion
Floodwater Aedes
21
1
4.15
reject null
hypothesis --
(C-2)>(S-2)
Anopheles crucians
21
14
1.25
accept null
hypothesis
insufficient evidence
disprove (S-2)=(C-2)
to
Culex (Melanoconion)
18
18
4.24
reject null
hypothesis
(S-2)>(C-2)
Culex niqripalpus
13
4
1.38
accept null
hypothesis --
insufficient evidence
disprove (S-2)=(C-2)
to
Culiseta melanura
22
9
0.85
accept nul1
hypothesis --
insufficient evidence
disprove (S-2)=(C-2)
to
Coquillettidia perturbans
20
17
3.12
reject null
hypothesis
(S-2)>(C-2)
Uranotaenia sapphirina
21
21
4.15
reject null
hypothesis --
(S-2)>(C-2)
(S-2)=the number of females of a particular species trapped on a given night at dome S-2.
(C-2)=the number of females of a particular species trapped on a given night at dome C-2.
p= the probability of (S-2)>(C-2).
q=the probability of (S-2)>(C-2).
n=the number of trials (trap nights compared).
y=the number of times the particular species was more numerous in samples from S-2.
z= the test statistic y"np (Mendenhall, 1968).
npq

event, these latter three species were produced in great numbers at
both domes. The obvious conclusion from these results is that by main
taining 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., Coqui1lettidia perturbans, and
Uranotaenia sapphirina have been created. Three other species,
Culiseta melanura, Culex nigripalpus, 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-l and S-l
from New Jersey traps to results from CDC traps at S-2 and C-2. How
ever, 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 place
ment 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
Coquillettidia perturbans, in the winter. Adults of many species
of Culex, Culiseta melanura, Anopheles crucians, and Uranotaenia
sapphirina survive the winter as adults in this part of Florida.

Table 3-20. Results from S-2 in 1976 using New Jersey and CPC light traps.
CDC trap results New Jersey light trap results
(S-2, 1976) (S-2, 1976)
Trap nights
Total
Relative Abundance (%)
Total
Relative Abundance (%)
49
20.
Females:
Aedes fulvus pal lens
7
0.11
0
Aedes vexans
0
1
0.04
Aedes canadensis canadensis
6
0.09
0
Aedes infirmatus
5
0.07
0
Aedes atlanticus
216
3.34
3
0.11
Anopheles crucians
874
13.53
189
6.64
Anopheles quadrimaculatus
17
0.26
3
0.11
Culex (Melanoconion)
492
7.62
111
3.90
Culex territans
12
0.19
11
0.39
Culex salinarius
49
0.76
10
0.35
Culex niqripalpus
1046
16.19
85
2.98
Culex restuans
1
0.02
0
Culiseta melanura
1191
18.44
34
1.19
Coquillettidia perturbans
185
2.86
132
4.63
Mansonia indubitans
10
0.15
11
0.39
Psorophora ciliata
2
0.03
0
Psorophora columbiae
2
0.03
0
Psorophora ferox
1
0.02
0
Uranotaenia sapphirina
2289
35.43
2115
74.26
Uranotaenia lowii
46
0.71
134
4.71
Unidentified specimens
8
0.12
9
0.32
Total females
6459
2848
M a 1 es
2284
2325
Total (males and females)
8743
5173
Females per night
131.8
142.4
Total per night
178.4
258.7

107
Table 3-21. Results from CDC traps at S-2 and AC grouped on a
seasonal basis.
AC
S-2
Mar.-May
June-Aug.
Sept.-Nov
Dec.-Feb.
Mar.-May
June-Aug.
Sept.-Nov
Dec.-Feb.
Trap nights
9
32
13
9
Females:
Aedes fulvus pallens
6
7
Aedes mitchellae
Aedes vexans
6
Aedes canadensis canadensis
7
1
5
Aedes infirmatus
8
877
182
5
Aedes dupreei
11
Aedes atlanticus
6
468
34
193
23
Anopheles crucians
268
1815
185
101
169
575
130
46
Anopheles quadrimaculatus
2
1
12
5
Culex (Melanoconion)
79
669
167
3
27
352
113
4
Culex territans
3
1
3
9
1
Culex salinarius
1
29
17
3
1
Culex nigripalpus
3
1458
313
67
1
942
103
14
Culex restuans
1
Culiseta melanura
97
591
76
8
199
822
170
35
Coqui1lettidia perturbans
49
54
11
56
95
34
Mansonia indubitans
1
5
5
Psorophora ciliata
3
7
1
1
Psorphora columbiae
2
Psorphora ferox
3
1
Uranotaenia sapphirina
690
1390
356
12
279
1246
764
8
Uranotaenia lowii
18
3
22
21

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 Coquillettidia perturbans, all species had one summer peak
of abundance; C. perturbans had two seasonal peaks, one in May and one
in July.
Suction Devices
Suction devices such as the commercially available D-Vac have been
used by mosquito investigators to sample resting adult mosquito popula
tions 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-l, C-l and WMHP. In the
smaller domes (S-l, S-2, and C-l) 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

Figure 3-5. Aedes atlanticus, abundance in CDC light trap
collections.

no
Apn I
May June July Aug. Sept.

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

90
80
70
60
50
40
30
20
10
112
C-2*-
S-2-
AC-1 -
Apr I
May
June July Aug.
Sept.

Figure 3-7.
Anopheles crucians, abundance in CDC light trap
collections.

114
June July Aug.
Sept.

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

116
July Aug.
April
May
June
Sept.

Figure 3-9. Culex nigripalpus, abundance in CDC light trap
collections.

118

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

120
June July Aug.
Sept.

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

Females
122
Aug. Sept.

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

124

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

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 popu
lations 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.

Table 3-23. Summary of homemade suction device sampling for 1976.
-
S-2
C-2
AC
Percent
Percent
Percent
relative
relative
relative
Total abundance
Total abundance
Total abundance
Aedes fulvus pallens
2
0.6
1
0.2
0
Aedes infirmatus
6
1.9
17
3.8
3
0.8
Aedes atlanticus
23
7.1
185
40.9
23
6.1
Anopheles crucians
9
2.8
23
5.1
26
Culex (Melanoconion)
48
14.9
5
1.1
43
11.3
Culex territans
38
11.8
45
10.0
18
4.7
Culex nigripalpus
18
5.6
50
11.0
35
9.2
Culiseta melanura
114
35.3
94
20.7
71
18.7
Coquillettidia perturbans
1
0.3
0
4
1.1
Psorophora ferox
2
0.6
0
3
0.8
Uranotaenia sapphirina
53
16.4
32
7.1
133
35.1
Uranotaenia lowii
9
2.8
0
20
5.3
Males
619
382
407
Sample time (min.)
27.8
28.0
32.8
Mosquitoes per minute
33.8
29.8
24.0

Figure 3-13. Relative abundance of females of the seven most common
species or groups from the homemade suction device.

% of total capture
Culiseta
melanura
Culex
(Mel anoconion)
Culex
territans
Culex
mgripalpus
Floodwater
Aedes
Uranotaenia
sp.
AQP_utl£l£S
crucians
62 L

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

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 Coquillettidia perturbans ranking third. It should be noted that
Culiseta melanura was not restricted to the cypress domes in its
nighttime flight activity.

Table 3-25. Summary of Malaise trap sampling of 1976.
Number of mosquitoes collected
Month
May
June
July
S-2
Aug.
Sept.
Total
May
June
AC
July Auq.
Sept. Total
Females:
Aedes vexans
1
1
Aedes infirmatus
2
1
1
6
Aedes atlanti cus
1
1
2
1
3
1
1
6
Anopheles crucians
3
1
5
1
10
2
1
7
10
Culex (Melanoconion)
2
1
3
1
10
11
Culex territans
5
1
3
9
1
1
Culex niqripalpus
2
2
1
1
Culi seta melanura
1
3
2
1
7
1
7
8
Coquillettidia perturbans
1
1
Psorophora ferox
1
1
Uranotaenia sapphirina
6
9
9
20
6
50
3
1
1
5
Uranotaenia lowii
4
12
6
22
Total females
15
12
25
38
17
107
2
5
34
3
2
46
Males
4
3
4
9
11
31
1
31
1
33
GJ
no

133
Table 3-26. Summary of truck trap results at Whitney Mobile Home Park
from August 1975 to August 1976 (22 trap nights).
Number of mosquitoes collected
Females
Aecles fulvus 'pallens 1
Aedes infirmatus 2
Aedes atl anti cus 2
Anopheles crucians 47
Anopheles quadrimaculatus 5
Culex (Melalioconion) 32
Culex salinarius 5
Culex nigripalpus 16
Culex pipiis quinquefasciatus 3
Culi seta melanura 26
Coquillettidia perturbans 35
Psorophora ciliata 2
Psorophora columbiae 159
Uranotaenia sapphirina 15
Uranotaenia lowii 1
Unidentified specimens 3
Total females 354
Males 234

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 manage
ment 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-l and S-2 resulted in
the almost immediate formation of duckweed covers at both domes. The
central portion of the duckweed mat at S-l 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

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 vegeta
tion. There was much more open water around the margins of S-2 than
at S-l. The difference in duckweed concentrations at the two domes
was probably a result of the open canopy at S-l, which allowed a great
deal of sunlight to reach the water's surfaced 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-l. 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-l, S-2, C-l, and AC. Results are given
in Figures 3-14 and 3-15. The data recorded in these figures indicate
that at S-l 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-l, 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.

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

Distance from Center of Dome (Meters)
Depth of Water (Centimeters)

Figure 3-15.
A. Larval distribution (histogram) and water depth (dashed line) from the
center to the martin at AC.
B. Larval distribution (histogram) and water depth (dashed line) from the
center to the margin at S-2.

Mosquito Larvae per
0 5 10 15 20 25 30 35 40 45 50 55 50 65 70 75 30
Distance from Center of Dome (Meters)
CO
LQ
Depth of Water (Centemet ers)

140
Culi seta 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-l.
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 Culiseta 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-l.
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

141
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 Culi seta 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.
Percentage positive dips
45%
21%
Number of larvae per positive dip
2.09
2.

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-l.
In the-course of larval sampling, Culex pipiens quinquefasciatus
and Culex restuans, two species with reputations for their association
with polluted water, were found at both S-l 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

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
Gainesvilie.
Between October 21 and December 10, one of the chickens at S-l
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-l, C-2)
in a 2 x 2 contingency table (Mendenhall, 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

Figure 3-16.
Eastern and western equine encephalitis activity (HAI
conversions) and mosquito activity (CDC light trap
records). p

HAI Conversions
30
20
10
21 9 29 20 11 4 24
F M A A M Jn' Jl Jl
> cn
Mosquito Abundance
500
400
300
200
100
25 21 10 14 17
S O D Ja F

Figure 3-17. HAI conversions against EEE. "T" represents levels of virus
activity in the sentinels which were placed in and around
Gainesville (Fig. 2-1).

Conversions
T
50-
0-
C-2
50-
Feb MarAprAprMayJun Jul Jul Aug Sep Sep Oct Dec Jan Feb
25 21 9 29 20 11 4 24 15 4 25 21 10 14 17

Figure 3-18. HAI conversions against WEE.

Conversions
T
50
0

Table 3-28. Comparison of viral activity observations at the experimental and control sites.
The fractions represent positive serological conversions divided by total (positive
plus negative) results.
Comparison
S-l
S-2
(S+l)+(S+2)
C-l
C-2
AC
(C-l)+(C-2)+(AC)
d.f.
x2
A
44
Z5F
39
328
1
3.22
B
28
125
16
133
14
136
6
69
19
123
4
11.10*
C
16
133
14
136
6
69
19
123
3
2.48
*At a-0.05, the null hypothesis that all domes are similar is rejected.

151
activity between individual domes. By removing S-l 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-l.
Why there was more virus activity in sentinels at S-l 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, i£ 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 Culiseta melanura is present at
S-l, 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-l 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-l 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-l, 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

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-l, C-l, 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

153
Table 3-31.
Summary of virus
mosquitoes.
isolation attempts from pooled
Species
Number Total
of number
pools in pools
Number
of
isolations
Culex ni gripal pus
4
261
1*
Culex (Melanoconion)
2
46
0
Coquillettidia 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 ni gripal pus 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 pal ustris (rice rat)
12
0
0
Didel phis marsupialis (opposum)
6
0
0
Procyon lotor (raccoon)
2
0
0
Lynx rufus (bobcat)
1
0
0

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 Culiseta
melanura, Culex nigripalpus, Culex (Melanoconion) spp., Anopheles
crucians, Uranotaenia sapphirina, and U. 1owii 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 Coqui1lettidia
perturbans have become more numerous as a result of the effluent. At
the same time, species of Aedes and Psorophora have declined at the

155
sewage domes. These trends can be attributed to the transition from
fluctuating water levels to permanent standing water at the experimental
domes.
Culex pipiens 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 re
ceiving 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-l 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-l; more
investigation is needed to determine the significance, if any, of this
one case.

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.
Mosquitoes 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 experi
mental 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-l is
of interest; however, its importance is undetermined.
156

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 ef
fectiveness of different mosquito control strategies as applied at these
experimental domes. In conjunction with a knowledge of larval distri
bution 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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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.

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, C.D., and T.W. Brown. 1965. Ecological consideration of cypress
heads in north central Florida. Amer. Midi. Natur. 74:126-140.
Mulhern, T.D. 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 swamp: I. population dynamics
and habitat distribution of potential mosquito vectors. Amer. J.
Epidemiol. 96:114-122.

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.
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PHS, CDC, Atlanta, Ga. 65 pp.
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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
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Townes, H.K. 1962. Design for a malaise trap. Proc. Entomol. Soc.
Wash. 64:253-262.

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 Florida--Their 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.

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 under
graduate 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

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.
L. Berner, Chairman
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.
xS^-c/ $
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
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
( L:Vi
TJ. Walkpr
Professo/ 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.
\
v
X.
Professor of Entomology and Nematology
/, a t.
-c
I.L. Nation
/../.ClIj-L,
-Li.
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.
& U).
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
Philosophy.
June 1978
Dean, Graduate School



Figure 3-1. The relative abundance of females of the most common species
or groups from 1974 ramp-trap samples at S-l and C-l.


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 Culiseta
melanura, Culex nigripalpus, Culex (Melanoconion) spp., Anopheles
crucians, Uranotaenia sapphirina, and U. 1owii 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 Coqui1lettidia
perturbans have become more numerous as a result of the effluent. At
the same time, species of Aedes and Psorophora have declined at the


81
than any of the other domes, and Culex (Melanoconion) spp. were more
abundant at C-l. 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 ni gripal pus 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
]This species was assumed to be the most common of the Melanoconion
complex.


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 under
graduate 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


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 encephalitides:
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.


Table 3-17. The 1976 CPC light trap results at C-2.
Date
4/76
5/76
6/76
7/76
8/76
9/76
10/76
Total
Trap nights
8
8
8
8
6
8
2
48
Number
of mosquitoes
collected
Specimens per night
Females:
Aedes fulvus pallens
Aedes mitchellae
17
13
2
1
33
\
Aedes vexans
1
. 1
Aedes canadensis canadensis
2
4
6
19.
52
Aedes infirmatus
9
27
155
5
2
3
201
Aedes dupreei
4
1
5
Aedes at!anti cus
30
130
307
69
154
1
691
V
Psorophora ciliata
1
1
0
02
Psorophora columbiae
1
1
0
02
Psorophora ferox
3
1
4
0
08
Anopheles crucians
59
42
133
270
76
62
9
651
13
56
Anopheles punctipennis
Anopheles quadrimaculatus
1
6
2
6
15
0
31
Culex (Melanoconion)
2
14
5
7
28
0
.58
Culex territans
1
3
1
5
0
.10
Culex salinarius
1
1
0
.02
Culex niqripalpus
Culex restuans
Culex pipiens quinquefasciatus
1
185
717
378
377
1658
34
.54
Culiseta melanura
38
205
487
919
247
211
11
2118
44
.13
Coquillettidia perturbans
2
24
13
22
15
2
78
1
.63
Mansonia indubitans
1
1
0
.02
Uranotaenia sapphirina
17
16
35
293
285
193
12
851
17
.73
Uranotaenia lowii
2
5
11
18
0.38
Unidentified specimens
8
11
2
10
1
32
0
.67
Total females
125
345
1174
2591
1089
1041
34
6399
133.31
Males
69
18
52
168
102
128
28
565
11
.77


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


Figure 2-5. Photos taken at S-l 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.


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


57


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 suc
cessful 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;


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.
Mosquitoes 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 experi
mental 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-l is
of interest; however, its importance is undetermined.
156


event, these latter three species were produced in great numbers at
both domes. The obvious conclusion from these results is that by main
taining 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., Coqui1lettidia perturbans, and
Uranotaenia sapphirina have been created. Three other species,
Culiseta melanura, Culex nigripalpus, 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-l and S-l
from New Jersey traps to results from CDC traps at S-2 and C-2. How
ever, 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 place
ment 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
Coquillettidia perturbans, in the winter. Adults of many species
of Culex, Culiseta melanura, Anopheles crucians, and Uranotaenia
sapphirina survive the winter as adults in this part of Florida.


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 1 ight trap. Both these traps have been
extremely popular with mosquito control workers in monitoring fluctu
ations 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.


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 Coquillettidia perturbans ranking third. It should be noted that
Culiseta melanura was not restricted to the cypress domes in its
nighttime flight activity.


Table 3-9. Ramp-trap results from 1975 at AC.
Month
Apri 1
May
June
July
Auqust
Total
%
Trap nights
1
4
2
5
4
16
Number of
mosquitoes
collected
Females:
Aedes fulvus pal lens
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
Aedes infirmatus
Aedes dupreei
Aedes atlanti cus
1
1
1.4
Aedes taeniorhynchus
Psorophora ciliata
Psorophora columbiae
Psorophora ferox
Anopheles crucians
Anopheles quadrimaculatus
2
o
O
2
3
15
20.3
Culex (Melanoconion)
3
9
12
16.2
Culex territans
Culex salinarius
1
1
1 .4
Culex niqripalpus
3
5
8
10.8
Culex restuans
Culex pipiens quinquefasciatus
Culi seta melanura
8
5
3
6
22
29.7
Coquillettidia perturbans
4
4
5.4
Uranotaenia sapphirina
Uranotaenia lowii
2
2
2
4
10
13.5
Unidentified specimens
1
1
1.4
Total females
12
20
0
13
29
74
Males
6
1
4
11
oo
o




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


124


Figure 3-5. Aedes atlanticus, abundance in CDC light trap
collections.


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 vegeta
tion. There was much more open water around the margins of S-2 than
at S-l. The difference in duckweed concentrations at the two domes
was probably a result of the open canopy at S-l, which allowed a great
deal of sunlight to reach the water's surfaced 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-l. 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-l, S-2, C-l, and AC. Results are given
in Figures 3-14 and 3-15. The data recorded in these figures indicate
that at S-l 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-l, 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.


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.


33


72
Table 3-3. Shannon-Weaver diversity indices computed from 1974 ramp-trap
data at S-l and C-1.
Species
Pi
S-l
Pj(logcPi)
EM
C-1
Pi (logePjJ
Aedes:
mitchellae
.0026
-.0155
.0005
-.0038
vexans
.0004
-.0031
.0005
-.0038
infirmatus
.0060
-.0307
.0109
-.0493
dupreei
.0004
-.0031
.0027
-.0160
at!anti cus
.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
niqripalpus
.0693
-.1850
.0569
-.1631
pipiens
.0021
-.0129
.0005
-.0038
restuans
.0009
-.0063
Culiseta:
melanura
.2776
-.3558
.2269
-.3365
Coqui11ettidia:
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
1 owi i
.0787
-.2001
.0864
-.2116
H' =
^PilogePi = 2.0862
H' =
2.1998
H' = Shannon-Weaver diversity index
p.j = the proportion of the it*1 species in the total sample


no
Apn I
May June July Aug. Sept.


155
sewage domes. These trends can be attributed to the transition from
fluctuating water levels to permanent standing water at the experimental
domes.
Culex pipiens 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 re
ceiving 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-l 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-l; more
investigation is needed to determine the significance, if any, of this
one case.


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 dif
ferent 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


Figure 3-13. Relative abundance of females of the seven most common
species or groups from the homemade suction device.


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


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




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


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


Table 2-1. Brief descriptions of the cypress domes examined.
Dome
Location
Area*
Description
Understory Veqetation
Hydroloqy
S-l
2 mi. NW of
Gainesvi He,
off Rt. 441
0.5
Experimental dome
receiving sewage
effluent. Badly
burned (1973).
Open canopy.
Extensive duckweed cover
(Figure 2-5), and invasion
by cattails and dog fennel
(Figure 2-6).
Year-round,
standing water.
S-2
2 mi. NW of
Gainesville,
off Rt. 441
1.0
Experimental dome
receiving sewage
effluent. Only
slight fire damage.
Thick canopy.
Partial duckweed cover,
broken at the margins.
Fetterbush and Virginia
chain fern abundant.
Year-round,
standing water.
C-l
2 mi. NW of
Gainesville,
off Rt. 441
0.7
Control dome
receiving well
water. Badly burned.
Open canopy.
Scattered duckweed, more
abundant at the margins.
Bladderwort very abundant
(Figure 1-2).
Year-round,
standing water.
C-2
2 mi. NW of
Gainesville,
off Rt. 441
0.9
Drained, control
dome receiving no
treatment. Not
burned. Thick
canopy.
No duckweed. Fetterbush
abundant. Extensive
accumulations of litter
(Figure 2-2).
Fluctuating water
level. No standing
water much of the year.
AC
3 mi. NE of
Gainesville,
off Rt. 24
4.5
Unburned, undis
turbed, control
dome. Thick
canopy.
Sparse duckweed, extensive
bladderwort, fetterbush,
and ferns.
Fluctuating water
level, but holds
standing water year-round.
*Hectares


%
of Total
Capture
r\j rv> gj
o Ui o
-1--
Culiseta
mela nura
Uranotaenia
sapphirina
Culex
(Melanoconion)
Uranotaenia
Ipwii
Floodwater
Aedes species
Anopheles
X'Xv/Xv'v
m
Yfs,
cruc ians
Culex
'mmm
niqropal pus
U


Distance from Center of Dome (Meters)
Depth of Water (Centimeters)


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 sus
pended 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/1 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/1, Mg 11.4-20.6 mg/1).
Total phosphorus and nitrogen were greater in samples from S-l than
from AC, and a major portion of the total nitrogen and phosphorus in
samples at S-l was soluble inorganic forms. Most of the nitrogen and
phosphorus in samples from AC was bound in organic forms. The N/p


Table 3-13
New Jersey light trap records for S-2.
Date 5/75 6/75 7/75 8/75 9/75 10/75 11/75 12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76 Totals %
Trap nights
2
3
4
3"
3 3
3
4
4
4
3
4
~T~
2
53
Number of mosquitoes
collected
Fema 1 es:
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans
1
1
Aeoes canadensis canadensis
0
3
Aedes infirma tus
2
1
3
Aedes duoreei
1
1
Aedes atlanticus
Aedes sollicitans
Aeces aeq.ypti
1
2
5
1
2
11
f
Psorophora ciliata
1
1
0
0
Psorophora columpiae
1

1
0
0
Pscrochora ferox
1
1
0
0
Anopheles crucians
31
19
42
35
25
47
1
10 2
59
128
17
19
34
65
35
19
588
9
6
Ancoheles pur.ctipennis
Anopheles quadrimaculatus
1
1
1
1
2
1
7
0
.1
Culex (t-'.elanoconion)
5
23
130
107
4
5
1
3
5
3
9
14
23
26
36
394
6
4
Cuiex temtans
17
5
8
3
1
2
1
5
2
1
3
48
0.8
Culex salinarius
3
6
2
1
7
1
1
21
0
.3
Cuiex niqripalpus
18
108
53
1
20
3
1
8
32
33
12
289
4
.7
Culex restuans
1
1
0.0
Culex pipiens quinquefasciatus
Culi seta melanura
6
3
8
9
1
15
3
12 1
12
10
15
1
2
5
4
7
114
1
.9
Coqui1lettidia perturbaos
34
17
14
1
1
3
31
60
14
16
10
1
202
3
.3
Mansonia indubitans
2
71
16
35
3
1
2
6
2
138
2
.3
Uranotaenia sapphirina
143
276
347
611
29
233
9
3
16
149
131
240
368
517
518
341
3931
64.2
llranotaenia lowii
13
67
42
51
3
29
7
3
2
1
4
40
47
40
349
5
.7
Unidentified specimens
3
2
8
2
1
1
4
3
24
0.4
Total females
255
425
714
953
81
397
23
29 3
94
298
204
334
454
707
688
461
6125
Males
162
1013
573
628
17
137
32
14 1
6
31
171
261
370
811
513
199
4939
co
oo


151
activity between individual domes. By removing S-l 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-l.
Why there was more virus activity in sentinels at S-l 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, i£ 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 Culiseta melanura is present at
S-l, 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-l 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-l 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-l, 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


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 tarsal is 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
atlantlcus 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


31
ratios were low in S-l 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-l 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 perpusi11a and Spirodela oligorhiza. The
floating fern, Azolla caroliniana was also present. A cover of duck
weed and fern was likewise formed at C-l, 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) con
taining 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-l and C-l with the ramp openings facing the center of each dome.
Collections were started in April at S-l and in May at C-l. 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


120
June July Aug.
Sept.


Table 3-16. The 1976 CPC light trap results at AC.
Date
4/76
5/76
6/76
7/76
8/76
9/76
Total
Trap nights
8
8
8
6
5
6
41
Number
of mosquitoes
collected
Specimens
per niqht
Females:
Aedes fulvus pallens
1
5
6
A
Aedes mitchellae
Aedes vexans
6
6
Aedes canadensis canadensis
7
7
36
.58
Aedes infirmatus
2
6
707
91
57
125
988
Aedes dupreei
10
1
11
Aedes atlanti cus
4
44
400
5
29
482
/
Psorophora ciliata
3
2
3
8
0
.20
Psorophora columbiae
Psorophora ferox
2
1
3
0
.07
Anopheles crucians
125
112
298
1054
93
70
1752
42
.73
Anopheles punctipennis
Anopheles quadrimaculatus
1
1
0
.02
Culex (Melanoconion)
20
58
351
160
120
47
756
18
.44
Culex territans
1
1
2
0
.05
Culex salinarius
1
1
0
.02
Culex niqripalpus
3
723
520
77
234
1557
37
.89
Culex restuans
Culex pipiens quinquefasciatus
Culi seta melanura
36
57
188
317
12
56
666
16.24
Coquillettidia perturbans
11
25
14
28
4
7
89
2.17
Mansonia indubitans
Uranotaenia sapphirina
152
527
609
411
109
225
2033
49.59
Uranotaenia lowii
5
9
14
0.34
Unidentified specimens
1
17
25
3
3
49
1.20
Total females
346
796
2973
3029
490
797
8431
205.63
Males
127
620
332
1122
145
53
2399
58.51


Table 3-20. Results from S-2 in 1976 using New Jersey and CPC light traps.
CDC trap results New Jersey light trap results
(S-2, 1976) (S-2, 1976)
Trap nights
Total
Relative Abundance (%)
Total
Relative Abundance (%)
49
20.
Females:
Aedes fulvus pal lens
7
0.11
0
Aedes vexans
0
1
0.04
Aedes canadensis canadensis
6
0.09
0
Aedes infirmatus
5
0.07
0
Aedes atlanticus
216
3.34
3
0.11
Anopheles crucians
874
13.53
189
6.64
Anopheles quadrimaculatus
17
0.26
3
0.11
Culex (Melanoconion)
492
7.62
111
3.90
Culex territans
12
0.19
11
0.39
Culex salinarius
49
0.76
10
0.35
Culex niqripalpus
1046
16.19
85
2.98
Culex restuans
1
0.02
0
Culiseta melanura
1191
18.44
34
1.19
Coquillettidia perturbans
185
2.86
132
4.63
Mansonia indubitans
10
0.15
11
0.39
Psorophora ciliata
2
0.03
0
Psorophora columbiae
2
0.03
0
Psorophora ferox
1
0.02
0
Uranotaenia sapphirina
2289
35.43
2115
74.26
Uranotaenia lowii
46
0.71
134
4.71
Unidentified specimens
8
0.12
9
0.32
Total females
6459
2848
M a 1 es
2284
2325
Total (males and females)
8743
5173
Females per night
131.8
142.4
Total per night
178.4
258.7


Figure 3-15.
A. Larval distribution (histogram) and water depth (dashed line) from the
center to the martin at AC.
B. Larval distribution (histogram) and water depth (dashed line) from the
center to the margin at S-2.


' 1974 ' 1975 ' 1976
<£>
en


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


Table 3-4. Summary of ramp-trap orientation and performance at S-l and C-l for 1974.
Number
of mosquitoes
collected at
traps
oriented as
indicated
S-l
C-l
Females:
0
O
O
cn
co
o
0
270
0
O
O
o
180
270
Aedes fulvus pallens
5
2
1
1
Aedes mitchellae
3
2
1
1
Aedes vexans
1
1
Aedes canadensis canadensis
1
14
1
Aedes infirmatus
6
7
1
3
11
5
1
Aedes dupreei
1
3
2
Aedes atlanticus
82
41
11
12
26
178
39
10
Aedes taeniorhynchus
1
Psorophora ciliata
2
Psorophora columbiae
4
9
9
2
6
3
Psorophora ferox
15
3
4
10
4
34
10
Anopheles crucians
32
59
18
53
37
20
30
14
Anopheles quadrimaculatus
5
2
3
9
22
5
Culex (Melanoconion)
28
91
81
39
51
15
94
23
Culex territans
4
5
12
3
40
4
5
Culex salinarius
12
11
7
6
3
4
4
2
Culex nigripalpus
62
72
12
16
38
19
25
22
Culex restuans
Culex pipiens quinquefasciatus
1
2
1
1
1
Culiseta melanura
184
246
78
141
100
135
93
87
Coquillettidia perturbans
14
6
9
15
1
10
6
3


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 fore
ground is accumulated cypress litter.


140
Culi seta 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-l.
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 Culiseta 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-l.
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


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


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


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 popu
lations 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.


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.


Conversions
T
50-
0-
C-2
50-
Feb MarAprAprMayJun Jul Jul Aug Sep Sep Oct Dec Jan Feb
25 21 9 29 20 11 4 24 15 4 25 21 10 14 17


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


Table 3-23. Summary of homemade suction device sampling for 1976.
-
S-2
C-2
AC
Percent
Percent
Percent
relative
relative
relative
Total abundance
Total abundance
Total abundance
Aedes fulvus pallens
2
0.6
1
0.2
0
Aedes infirmatus
6
1.9
17
3.8
3
0.8
Aedes atlanticus
23
7.1
185
40.9
23
6.1
Anopheles crucians
9
2.8
23
5.1
26
Culex (Melanoconion)
48
14.9
5
1.1
43
11.3
Culex territans
38
11.8
45
10.0
18
4.7
Culex nigripalpus
18
5.6
50
11.0
35
9.2
Culiseta melanura
114
35.3
94
20.7
71
18.7
Coquillettidia perturbans
1
0.3
0
4
1.1
Psorophora ferox
2
0.6
0
3
0.8
Uranotaenia sapphirina
53
16.4
32
7.1
133
35.1
Uranotaenia lowii
9
2.8
0
20
5.3
Males
619
382
407
Sample time (min.)
27.8
28.0
32.8
Mosquitoes per minute
33.8
29.8
24.0


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 manage
ment 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-l and S-2 resulted in
the almost immediate formation of duckweed covers at both domes. The
central portion of the duckweed mat at S-l 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


97
in mosquito populations at these domes are assumed to be a result of
the differences in water chemistry and altered vegetation patterns.
This of course is itself based on the assumption that mosquito popula
tions 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.
CPC 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 year-
round 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-l 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


Floodwater
Aedes
Anophe les
crucians
Culex
(Melanocomon)
Average Number per Trap Night
_> r\j w -k
O O O o o
Culex
niqropalpus
Culi seta
melanura
Coquillett idia
perterbans
Uranotaenia
sapphi rina
501 C-2


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 Coquillettidia perturbans, all species had one summer peak
of abundance; C. perturbans had two seasonal peaks, one in May and one
in July.
Suction Devices
Suction devices such as the commercially available D-Vac have been
used by mosquito investigators to sample resting adult mosquito popula
tions 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-l, C-l and WMHP. In the
smaller domes (S-l, S-2, and C-l) 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


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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.
\
v
X.
Professor of Entomology and Nematology
/, a t.
-c
I.L. Nation
/../.ClIj-L,
-Li.
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.
& U).
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
Philosophy.
June 1978
Dean, Graduate School


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


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 Coqui1let-
tidia 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 de
pressions 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-l, S-2, and C-l, differences


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 trans
mission 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


Figure 3-9. Culex nigripalpus, abundance in CDC light trap
collections.


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 a 1., 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


Date 9/74 10/74 11/74 12/74 1/75 2/75 5/75 6/75 7/75 8/75 9/75 10/75 11/75 12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 9/76 Totals %
Trap nights 2 4 3 9 3 1 T ~2 5 4 1 4 3 2 3 3 4 4 4 2 4 3 2 73
Fen.ales (cont.):
Coquillettidia
perturoans 8
Hansonia
ir.dubl tans
Uranotaenia
sapphirina 21
Urar.otaenia
1 owi i 4
Unidentified
specimens
Total females 203
Males 6
3
6 2 2
1 1
30 5 12 20 8
6 113 1
2 2 2
7 1 2 8
1
1 1
10 12 77 80 8
4 5 18 20
1
4 2 14 3
1
1
26 4 3 4 26
18 111
2 15 14 7 6
2 4 7 5 2
2
20 28 30 28 46
2 2 10 10 9
8
3
73
9.8
7
9
99
13.3
1
9
1.2
5
0.7
33
33
746
12
25
156


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.


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 MOSQUITO 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 encepha
litis activity was monitored at these same sites.
Three years of sampling failed to demonstrate any measurable in
crease in human pest species at the experimental domes. The flood-
water 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


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.


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 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


Table 3-15. Degree of similarity based on relative abundance data from 1975-76 New Jersey light
trap samples.
Relative abundance
(S-l]
1 (S-2)
(S-l)
- (C-
-1).
(S-2)
- (C-l)
Anopheles crucians
18.7 -
9.6 = 9.1
18.7 -
13.1
= 5.6
9.6 -
13.1 = -3.5
Anopheles quadrimaculatus
0.3 -
0.1 = 0.2
0.3 -
0.7 =
-0.4
0.1 -
0.7 = -0.6
Culex (Melanoconion)
14.9 -
6.4 = 8.5
14.9 -
26.0
= -11.1
6.4 -
26.0 = -19.i
Culex territans
0.0 -
0.8 = -0.8
0.0 -
0.0 =
0.0
0.8 -
0.0 = 0.8
Culex salinarius
1.7 -
0.3 = 1.4
1.7 -
0.1 =
1 .6
0.3 -
0.1 =' 0.2
Culex nigripalpus
3.2 -
4.7 = -1.5
3.2 -
7.5 =
-4.3
4.7 -
7.5 = -2.8
Culiseta melanura
2.5 -
1.9 = 0.6
2.5 -
5.6 =
-3.1
1.9 -
5.6 = -3.7
Coquillettidia perturbans
1.8 -
3.3 = -1.5
1.8 -
2.8 =
-1.0
3.3 -
2.8 = 0.5
Mansonia indubitans
1.7 -
2.3 = -0.6
1.7 -
0.4 =
1.3
2.3 -
0.4 = 1.9
Psorophora columbiae
0.5 -
0.0 = 0.5
0.5 -
0.0 =
0.5
0.0 -
0.0 = 0.0
Uranotaenia sapphirina
48.7 -
64.2 = -15.5
48.7 -
42.7 =
6.0
64.2 -
42.7 = 21.5
Uranotaenia lowii
5.1 -
5.7 = -0.6
5.1 -
0.2 =
4.9
5.7 -
0.2 = 5.5
Index of similarity
Z|(S-1)
- (S-2) | = 40.8
Z|(S-l)
- (C-l)| = 39.8
11 (S-2)
- (C-l) | = 60


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
Gainesvilie.
Between October 21 and December 10, one of the chickens at S-l
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-l, C-2)
in a 2 x 2 contingency table (Mendenhall, 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


Table 3-28. Comparison of viral activity observations at the experimental and control sites.
The fractions represent positive serological conversions divided by total (positive
plus negative) results.
Comparison
S-l
S-2
(S+l)+(S+2)
C-l
C-2
AC
(C-l)+(C-2)+(AC)
d.f.
x2
A
44
Z5F
39
328
1
3.22
B
28
125
16
133
14
136
6
69
19
123
4
11.10*
C
16
133
14
136
6
69
19
123
3
2.48
*At a-0.05, the null hypothesis that all domes are similar is rejected.


¡T? 'OT
H l
1: w*
mg* ' i
fii '.iffiVn
* *%e£r


73
The four traps at each dome were not equally successful in cap
turing mosquitoes (Table 3-4). At S-l, 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-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 insuffi
cient 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-l, 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


RESULTS AND DISCUSSION
Adult Mosquito Sampling
Ramp Traps
Using no known insect attractants, ramp traps are flight inter
ception 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. Shannon-
Weaver 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-l is slightly larger than that for S-l. Since there were two more
species captured at S-l than at C-l, the larger index for C-l repre
sents 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




r/l
28


59


Table 3-11. New Jersey light trap records for C-1.
Date 9/74 10/74 11/74 12/74 1/75 2/75 6/75 7/75 8/75 9/75 10/75 11/75 12/75 1/76 2/76 3/76 4/76 5/76 6/76 7/76 8/76 Totals %
rap niohts
3
5
3
10
3
2
1
4
5
1
4
2
3 3
3
4
4
4
1
4
3
74
'ema 1 es:
Number of mosquitoes
collected
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
1
1
0.3
Aedes infirma tus
1
8
9
Aedes dupreei
Aedes atlanticus
Aedes soilicitans
Aedes aeqypti
3
2
5
'
Psorophora ciliata
Psorophora coiumbiae
1
1
1
3
Psorophora ferox
Anopheles crucians
50
47
3
23
26
26
7
33
57
20
46
5
11
19
64
8
17
26
50
14
552
10.3
Anopheles punctipennis
1
1
0.0
Anopneles quadrimaculatus
6
8
1
1
3
3
2
2
1
1
2
3
1
34
0.6
Culex (Kelanoconion)
294
114
1
19
9
109
50
no
143
7
11
1
46
92
19
50
95
76
50
1296
24
.3
Culex territans
1
1
0.0
Culex salinarius
3
3
2
2
10
0
.2
Culex niqripalpus
58
11
80
13
1
9
29
2
10
2
3
2
1
116
32
11
380
7
.1
Culex restuans
5
2
7
0
.1
Culex pipiens quinquefasciatus
Culiseta nelanura
5
13
18
5
13
6
1
37
4
13
11
10
22
9
17
28
4
216
4
.0
Coauillettidia perturbans
17
5
1
3
1
5
2
14
23
29
2
102
1
.9
i'ansonia indubitans
5
1
1
2
2
11
0
.2
Uranotaenia sapphirina
819
536
14
3
1
19
59
205
287
6
27
4
14
57
26
81
264
132
68
2622
49
.1
Uranotaenia lowii
48
16
1
1
2
1
1
1
2
73
1.4
Jnidentified specimens
1
1
1
4
1
1
2
1
12
0.2
rotal females
1299
751
18
153
59
172
122
364
532
36
149
18
28 0
93
228
81
172
549
355
154
5335
iales
334
342
61
5
6
10
24
128
189
10
80
7
4
2
27
20
43
141
179
106
1718
co
cn


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.


93
respectively. Degree of similarity is based on the amount of differ
ences 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-l were more alike than S-l and S-2.
Of the three comparisons, S-2 and C-l 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
Uranotaenja sapphirina at the two sewage domes. Starting in June 1975,
males of LL sapphirina at the two sewage domes were counted and re
corded 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 l. 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 l. sapphirina. From both a medical and an economic standpoint
this species is of no known importance; and therefore, a more meaning
ful 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.


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.
L. Berner, Chairman
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.
xS^-c/ $
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
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
( L:Vi
TJ. Walkpr
Professo/ of Entomology and Nematology


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


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-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 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 encephalitide viruses.


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


Conversions
T
50
0


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


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


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 flood-
water 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 dis
cerning 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 informa
tion 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.


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 ef
fectiveness of different mosquito control strategies as applied at these
experimental domes. In conjunction with a knowledge of larval distri
bution 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


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


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 Reed-
Muench method (Lennette, 1969). A reduction index of at least 1.7
logs was considered positive.


30


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


INTRODUCTION
General Statement
The state of Florida is presently experiencing rapid growth and
development resulting in much of its forested area and natural wet
lands 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 disserta
tion 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


Figure 3-7.
Anopheles crucians, abundance in CDC light trap
collections.


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


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 iso
lation 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-l, C-2, S-l, 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 3-16.
Eastern and western equine encephalitis activity (HAI
conversions) and mosquito activity (CDC light trap
records). p


Figure 3-2. Comparison of New Jersey light trap samples from
four different sites.


Table 3-8. Ramp-trap results from 1975 at S-2.
Month
Apri 1
May
June July
Auqust
Total
%
Trap nights
1
4
2 5
4
16
Number of mosquitoes
collected
Females:
Aedes fulvus pallens
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
2
*
2
0.6
Aedes infirmatus
1
1
0.3
Aedes dupreei
Aedes at!anti cus
7
7
2.3
Aedes taeniorhynchus
Psorophora ciliata
Psorophora columbiae
Psorophora ferox
Anopheles crucians
Anopheles quadrimaculatus
10
16
1 2
29
9.4
Culex (Melanoconion)
1
2 4
7
14
4.5
Culex territans
22
50
6 7
6
91
29.5
Culex salinarius
9
1 2
12
3.9
Culex niqripalpus
1 3
6
10
3.2
Culex restuans
Culex pipiens quinquefasciatus
Culi seta melanura
11
36
6
6
6
65
21.1
Coquillettidia perturbans
2
2
0.6
Uranotaenia sapphirina
5
14
9
22
16
66
21.4
Uranotaenia lowii
1
5
1
7
2.3
Unidentified specimens
2
2
0.6
Total females
48
138
28
52
42
308
Males
14
18
5
18
3
58




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, C.D., and T.W. Brown. 1965. Ecological consideration of cypress
heads in north central Florida. Amer. Midi. Natur. 74:126-140.
Mulhern, T.D. 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 swamp: I. population dynamics
and habitat distribution of potential mosquito vectors. Amer. J.
Epidemiol. 96:114-122.


Date
4/76
5/76
6/76
7/76
8/76
9/76
10/76 Total
Trap nights
8
8
8
7
6
8
4
49
Number
of mosquitoes
collected
Specimens per night
Females:
Aedes fulvus pal lens
4
3
7
A
Aedes mitchellae
Aedes vexans
Aedes canadensis canadensis
1
1
4
6
4.
78
Aedes infirmatus
Aedes dupreei
4
1
5
Aedes atlanti cus
6
175
12
22
1
216
/
Psorophora ciliata
1
1
2
0.04
Psorophora columbiae
1
1
2
0
04
Psorophora ferox
1
1
0
02
Anopheles crucians
66
103
122
374
79
115
15
874
17
.84
Anopheles punctipennis
Anopheles quadrimaculatus
4
7
1
3
2
17
0
.35
Culex (Melanoconion)
3
24
105
160
87
111
2
492
10
.04
Culex territans
2
1
3
5
1
12
0.24
Culex salinarius
7
22
15
2
3
49
1
.00
Culex niqripalpus
1
179
605
158
76
27
1046
21
.35
Culex restuans
Culex pipiens quinquefasciatus
1
1
0
.02
Culiseta melanura
98
101
316
399
107
121
49
1191
24
.31
Coquillettidia perturbans
15
41
18
62
15
34
185
3
.78
Mansonia indubitans
5
4
1
10
0
.20
Uranotaenia sapphirina
131
148
379
471
396
733
31
2289
46
.71
Uranotaenia lowii
2
1
2
5
15
17
4
46
0
.94
Unidentified specimens
3
1
2
2
8
0
.16
Total females
325
442
1163
2275
878
1240
136
6459
131
.82
Males
113
227
421
498
515
400
no
2284
46.61


19 '
O
0 12 3
C Sentinel Chicken Sites


Mosquito Larvae per
0 5 10 15 20 25 30 35 40 45 50 55 50 65 70 75 30
Distance from Center of Dome (Meters)
CO
LQ
Depth of Water (Centemet ers)


Figure 3-17. HAI conversions against EEE. "T" represents levels of virus
activity in the sentinels which were placed in and around
Gainesville (Fig. 2-1).


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


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 depen
dent 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
1ucida) 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



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

PAGE 9

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

PAGE 10

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

PAGE 11

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

PAGE 12

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.

PAGE 13

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.

PAGE 14

8

PAGE 15

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

PAGE 16

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.

PAGE 17

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

PAGE 18

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.

PAGE 19

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;

PAGE 20

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

PAGE 21

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

PAGE 22

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

PAGE 23

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

PAGE 24

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.

PAGE 25

19 Sentinel Chicken Sites

PAGE 26

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.

PAGE 28

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.

PAGE 29

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

PAGE 30

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

PAGE 32

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

PAGE 33

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.

PAGE 35

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

PAGE 36

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

PAGE 38

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.

PAGE 39

33

PAGE 40

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.

PAGE 41

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.

PAGE 43

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.

PAGE 44

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

PAGE 45

39

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

PAGE 48

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

PAGE 49

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

PAGE 53

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

PAGE 54

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

PAGE 59

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

PAGE 61

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.

PAGE 71

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

PAGE 74

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

    77 o in CM I/) 3 3 ^ 3 C7 l/l O E 4O SCM XI CL QJ (/) 3 > 3 tO (/) O) OJ — T3 n3 O) E cC Ol •r(/) C c: X> 0) (J ID SrO +J X CL r— a; 3 +J E > o -o ro (/I l/l -Q n3 E >, 'r3 •1 — o o o o •1 — c (T3 ra Q. Q. to O o QJ isX3 O o Q) to l/l cC Os_ o o So l/l Q_ CT> O CTi 1— r— UD I— o IX) cn 1— CO I— CO CM to O CM CM to m CD o oo CM CM CM o o CO rtD to 3 M n3 ro ro CM CM r— 00 CM o r-. CM to r— tn I — IT) U3 00 ro IT) CO V) u to to c M ro ro (O MXI ro r— Q) 3 3 3 U cr +-) il to to o c S•r— E to •1 — ro QJ •r•r— c to 3 3 SCL Q So l/l 3 CU CT 3 5 u XI o to ro ro 0 3 n3 o ro il ro c to ro to S3 c 4-> ro ro c -a o CJto •r— •r3 Ol QJ ro ro SSM •f— E M •rto to QJ Scr 10 CL -l-> c C QJ O) OJ ro 'r— QJ •r— ro Q) Q) QJ -M to SQ. +-) ro ro Q) QJ Q) +J +J X X X X X X l/l 0 0 Q. a. Q) Q) QJ QJ OJ QJ 3 C O o cr ro 0 0 0 (_3 0 0 13 =3 O to c QJ E •r— o Ol CL to o QJ +-> QJ o •r— c ZD CO CX3 CM m CO cri tn O CTi ro CM CM CO CO ro CM to QJ ro E fe to ro QJ +-> 0 ra (— s:

    PAGE 84

    78 ro +-) O +-> to < o 0) a) c CM 3 CT l/l O o iQ• • • Or^ CM Ln CM CO (O 4to to r— -a fO O) to c rc u a; sQ. 3 "O l/l OJ o OJ to QJ 3 ra -C (0 •r— o -M XI c: (O E X >, 3 o -C s_ S•r'o OJ o u o 4c (0 ro ra x: sz Q. Q. to o o O OJ Ss_ iXJ o o o 0) in to to cC Cl. 00 CT> CO CM r— CM to r-^ uD CM to CM CO to r--. r— .— O CM CO CM ^ r— CM 00 CM ro CO ^ CM CM 00 CO CM o O CD CM CD CO o eg CO 1 — 00 I— ;3CO "^JI— 00 O CM CM CO CM CM CO CO CM I— o CM 10 (J 3 to +J ro ro 4OJ 3 3 O c CT 1/1 re o C c: E to •r— ro ra •1— c to 3 3 Si~ o t/5 3 cr 3 o -a u to C 3 as o S_ (O c to ro S3 c: -t-> ro Cl ro c u cr fO C •r— 3 QJ z. s4-> •r— l/l to (/) c: SQ. +J So. to D 4-> +-> QJ 3 CT O O CM in ID 00 LD I— ^ IS) ID (O c il C 3^ x: c Q. Q O ro O QJ to Q. to (0 ro (/I X> QJ C 0) QJ QJ •rro ro ro E +J +-> •r— QJ O O +j 4c c c ro ro QJ 1 — to $•o (O QJ ID ID •r1— C O ro

    PAGE 85

    79 to CO O CD CO CM p a I/) O) +j CT o 13 : tn o -p 3 CT l/l o E OJ 4C CM O S(U e (T3 Q. to C7> C Q. CM c/i > ui to OJ at r— -O fO CD E < O) to c (U T3 (T3 o CTl cn CM 10 +J OJ s3 o 0) "O OJ to c E •1 — c E X na •r— to 3 0 •r— s0 to 3 i0 T3 u c S•1 — 0 0) 3 n3 0 S0 0 u Hs_ 3 c ro •f— 0 C7 03 C c n3 to (O •r— Ol i1Sto _ai Sto 0 0 0 0) 0) QJ (O +J -C x: +-> to Q. Q. Q. 0) to 0 0 0 X X X . to ^ CM CO CM to 1— 1— IT) CM CM to 3 +J (O •r— u to ItCD 3 cr c 3 cr 1/1 c CM to kO ^ 00 O I — CM CvJ to r~~ to to I— CM Lf) CM to CM ^ I— 00 I— to CD cr> CM CO 00 o to CO CM CO CM CO LO 1— CO 10 CM to c (0 (0 sc: 3 •rto 4-> Sc S•rCU OJ •r— E iCL CL •r•r— 3 a 5 u c fO (0 0 cu to a. 0 to 'a! •r— ro ro to E 4-> •r-a 0) +-> C C ro ro E •r— cu to 0 0 4-> 43 C CT ro ro CO 00 CO f— CO =3O ro

    PAGE 86

    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

    4 (/ C>l m CM ro lA fry CVJ .CM n CM Chi ii) 1— ro r00 en cn >o cn to CM in CM CM a> m — ro CM ro CO CM r-rto ro o eo frCM CSI an •aCO iCM 00 00 m ir> cc CM o CM CM o CM en 00 00 o cn CM CVi ro CM 00 c> CO ^™ CM rCM CO CM m CM 00 00 CM m CM CM vo OO oo \o CM in CO 00 Ot CM •* 01 10 fO Ul (U a) c > o CI >; c <> 3 1/1 0) i/l I/) 0. OJ Ol O) "TS u o u B 0) aj OJ Ol <: •X Lu to c 13 fD U +J +J J c Q. r +-> alO .,T l/l OJ Ol X3 u o QJ Ol o o .c ml :3 Q o o O c ^ o u o u O (/ m CL lA to •rc u a> CI 2 OJ OJ a> L a; XI c c Q. CI o Q. o cr c c c
    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

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    PAGE 96

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    PAGE 97

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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

    101 00 CM 1 l>0 •-> ta to VO +j 3 Lf) l/l Q) Sa la i. -p +j D<_) Q l_) <£> o o o CQ CO o CM O CO O CO 1 — CM CTl CO to • • • o CD O o o CD CVJ o CM CO O CD CD r— IJD CO ^ CTl 1X5 Lf) X3 U CO 0) o o I/) yD O 3 CT O E Mo sco cu JD e 3 CO CO Q. IX} s QJ to to OJ (U r— -O to c X3 c u sQ. "O QJ o CM CM I ^ r— tn ^ UD I— ra u (13 o a o to a. o o (O io x: Q. O So to X o sOl <+so Q. o So to a. CO tn CM CM cr> to I cn I— ^ ^ •=dO I — r— LD CM CO I— CO r-. I CM CD en I— r~. 1 — CO CO CO CM CM CO O VO O LT) CM tn t£) O r— tn uf> CO tn cri ^ 1— CM I CM CM CO CM I— Lf) cn CO 1— ^ CM CO o f— cri CM cn to CO to 00 CO o ^ 00 Lf) ta ta to 03 ^ ta •r— QJ c 3 3 3 •r— o c cr +J to sto cu ro o c •rB t/1 ns QJ ^ •rrtJ •f— to 3 3 S_ a. t-> CL •r— +-) So U) 3 Q. cr 3 •rQ, 3 o o X3 o c to ro JD 03 o ro o (0 in3 c to 3 (/) S3 +-> ra Q c: a a (-) Q. cr •r3 QJ QJ c (0 (0 S+-> •1 — E +j •r— •r— to (/I to Scr to Q. +-> c (U 0) (V QJ QJ fO Q) re QJ QJ +-> to c: SCL +J r— 03 ta OJ OJ QJ QJ c 4J -C JZ X X X X X X ul •1 — o O o Q. cn. Q. QJ QJ QJ QJ QJ QJ •1 — 3 to C c: O o O cr G 03 03 c: c: 3 3 3 3 3 3 3 O 03 s_ S< =C o CD O o CD O CD O 2; ZD ZD CO CM CO CD tn CO ^ CM iXI CM to o CO r— o o ^ o CM :a00 Lf) I— 00 lO LO 00 CM ^ CM CO I— to CM CM ^ CM CvJ Lf) CO CM I— CO 1— o QJ Q. to a QJ QJ XJ to q; 05 E QJ >+(/) 03 QJ 4-) O rq 1—

    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

    106 Up/ (J cu (_ >— .a +j cn [ ^ CM (t! 0) q; u cu OI -(-> To o CM o o I— ^ I— I o cr> Lf) CO CTl OO OO CTl UD O OO O O CXI 1— ^ CO CO CO 1— CM O O CO en CO CO o in o I— 00 CO CM CO o o o 1— CO CM CM CO CO LD CO CM CO ^ CM ^ Ln CO CO 1— r— CM CM CM Ln a •l-> 1/5 (-> CD tM 0) CTl sCL CM s1 4-> 00 o Q O r— CTl ^ I— O O CO CO un CM CM CTl CD CT> CM CD 1— 1^ t— O ID 00 in CO CO CM OO r— CM I — o o o ^ 1 — ooco coo r-~oocDO 00 cm o ooo un o CO CTi r^OCDLDcD ^r^ CMCMCTiCDi — I — LD i—r^i— cTif— '^'^ cnco CM CO 'JO 1 — r— CTl CD CO CM CM CO CO X) i. SMns an CU C 3 +J to 1/1 OJ o fO o Sc +-> c: E •1 — to QJ (D CO CO 1/1 c to 3 a. +-) ns 'ro to 3 a. 3 I/) Q. (/) 4-) o (J o c •r— to C (O 0) c (O 3 fO o S3 +J fO Qro o c o Sc CJ CT (T3 •r— r3 QJ •r> ro n3 i1+-> E +-> •r— X Mto to Q) SCJ) to 4-> o +-> OJ fO c: +-> QJ QJ QJ OJ (O QJ fO CM t> u •r— <13 -M lO S+J 1 CO cn 0) Q) QJ c l/l QJ QJ Q. Q. QJ QJ QJ QJ QJ 3 t/) r— X) TO TO X3 XI O O cr c: CL n3 CU Ta i(— Fe (X3 io Q O So to ex QJ ns E 3 r-— o o (t> Vo a o o to Q. o o So to a a to c OJ E •rO QJ Q. to o QJ c QJ to a; (O E QJ +-> X) c CD 4-) re to CD QJ to QJ i. c: (O QJ E re Q. iQJ E QJ Mes CL to O _ra o QJ o thLl_

    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

    PAGE 143

    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

    PAGE 164

    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

    PAGE 165

    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


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


    153
    Table 3-31.
    Summary of virus
    mosquitoes.
    isolation attempts from pooled
    Species
    Number Total
    of number
    pools in pools
    Number
    of
    isolations
    Culex ni gripal pus
    4
    261
    1*
    Culex (Melanoconion)
    2
    46
    0
    Coquillettidia 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 ni gripal pus 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 pal ustris (rice rat)
    12
    0
    0
    Didel phis marsupialis (opposum)
    6
    0
    0
    Procyon lotor (raccoon)
    2
    0
    0
    Lynx rufus (bobcat)
    1
    0
    0


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


    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 pre
    viously 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 ace
    tone 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 anti
    gens which would give 4-8 hemagglutinating units in each test. Back


    62


    52


    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-l. At the same time,
    groundwater from a deep well was added to a control dome, C-l. 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-l, S-2, and C-l previous to the addition
    of effluent or groundwater. All these domes are represented in Figure 2-3.
    17