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Response of Aedes albopictus (Skuse) (Diptera

Permanent Link: http://ufdc.ufl.edu/UFE0020440/00001

Material Information

Title: Response of Aedes albopictus (Skuse) (Diptera Culicidae) to Residual Bifenthrin on Plant Leaves
Physical Description: 1 online resource (91 p.)
Language: english
Creator: Doyle, Melissa A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aedes, bifenthrin, mosquito, traps
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The Asian tiger mosquito, Aedes albopictus (Skuse), was first introduced into the United States in Harris County, Texas in 1985. This species is known as an aggressive daytime biter. It has invaded 26 states, including Florida. Aedes albopictus larvae are found in tree holes, water-containing plants, and man-made containers, such as birdbaths. It is recommended that residents keep their yards free of water-holding containers to avoid this pest however, heavy infestations of Ae. albopictus may require additional control methods. The objective of my study was to evaluate commercially available control methods against Ae. albopictus in residences in Gainesville, Florida. The control methods studied were the placement of a propane powered trap called the Mosquito Deleto 2500, treatment with the pesticide TalstarOne (active ingredient bifenthrin) by a pest control company, the combination of the trap and pesticide treatment, and a control treatment where no treatments were applied. Further investigation into the efficacy of bifenthrin treatment on different plant leaf types against Ae. albopictus was evaluated. Plants commonly found in Florida residential landscapes were chosen and treated with TalstarOne. The efficacy of bifenthrin-treated leaves was also evaluated against male, female, blood-fed, and gravid Ae. albopictus.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Melissa A Doyle.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kline, Daniel L.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0020440:00001

Permanent Link: http://ufdc.ufl.edu/UFE0020440/00001

Material Information

Title: Response of Aedes albopictus (Skuse) (Diptera Culicidae) to Residual Bifenthrin on Plant Leaves
Physical Description: 1 online resource (91 p.)
Language: english
Creator: Doyle, Melissa A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aedes, bifenthrin, mosquito, traps
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The Asian tiger mosquito, Aedes albopictus (Skuse), was first introduced into the United States in Harris County, Texas in 1985. This species is known as an aggressive daytime biter. It has invaded 26 states, including Florida. Aedes albopictus larvae are found in tree holes, water-containing plants, and man-made containers, such as birdbaths. It is recommended that residents keep their yards free of water-holding containers to avoid this pest however, heavy infestations of Ae. albopictus may require additional control methods. The objective of my study was to evaluate commercially available control methods against Ae. albopictus in residences in Gainesville, Florida. The control methods studied were the placement of a propane powered trap called the Mosquito Deleto 2500, treatment with the pesticide TalstarOne (active ingredient bifenthrin) by a pest control company, the combination of the trap and pesticide treatment, and a control treatment where no treatments were applied. Further investigation into the efficacy of bifenthrin treatment on different plant leaf types against Ae. albopictus was evaluated. Plants commonly found in Florida residential landscapes were chosen and treated with TalstarOne. The efficacy of bifenthrin-treated leaves was also evaluated against male, female, blood-fed, and gravid Ae. albopictus.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Melissa A Doyle.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kline, Daniel L.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0020440:00001


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RESPONSE OF Aedes albopictus (SKUSE) (DIPTERA: CULICIDAE) TO RESIDUAL
BIFENTHRIN ON PLANT LEAVES




















By

MELISSA ANN DOYLE


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE


UNIVERSITY OF FLORIDA

2007




























2007 Melissa Ann Doyle





































To my parents









ACKNOWLEDGMENTS

I would like to thank my advisor Dr. Daniel L. Kline for his guidance and support. I would

also like to thank the other members of my committee, Dr. Sandra Allan and Dr. Phillip

Kaufman, for their encouragement and their editorial help. Special thanks go to the staff at the

Center for Medical, Agriculture and Veterinary Entomology for their camaraderie and support,

especially Erin Vrzal, Aaron Lloyd, Joyce Urban, and David Hoel. I would like to thank Dr.

Donald Hall, Dr. John Capinera, Debbie Hall and Josh Crews for all of their administrative help

and the Department of Entomology for their financial support. I would also like to thank Dr.

Ruide Xue, director of Anastasia Mosquito Control District. I would like to acknowledge the

support of my fellow graduate students and friends, especially Kendra Pesko, Dave Melius, and

Eileen Enrique. I thank the homeowners who allowed me to treat and trap in their yards. I would

especially like to thank my parents Madeline and Paul Doyle for their constant encouragement

and support throughout my academic career.









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ......... ..... .......... ...............................................................................4

L IST O F TA B LE S ......... .... ........................................................................... 7

LIST OF FIGURES .................................. .. ..... ..... ................. .8

CHAPTER

1 LITERATURE REVIEW OF SURVEILLANCE AND CONTROL TECHNIQUES
USED TO CONTROL Aedes albopictus (Skuse) .......................... ......................10

Introduction to A edes albop ictus ..................................................................... .................. 10
E ecology of A edes albop ictus ................................................................. ...........................10
Surveillance Methods for Aedes albopictus ................. ......... .... ................. 13
Control M ethods for Aedes albopictus .................................... ........................................... 15
Research Objectives........... ........ ........... .. .... ..... ................. 18

2 Effect OF COMMERCIAL TRAPS AND PESTICIDE TREATMENT ON Aedes
albopictus (Skuse) POPULATIONS IN SUBURBAN SETTINGS IN NORTH
C E N TR A L FL O R ID A ............................................................................................. 19

Introduction ..... ..................................... ..............................................19
M materials and M methods ........................ .. ........................ .. .... ........ ........ 20
R e su lts ................... .............................................................. ................ 2 6
D discussion .................................... ..................................... ................. 28

3 EFFECT OF PLANT SPECIES AND LEAF texture ON RESPONSE OF FEMALE
Aedes albopictus (Skuse) TO PESTICIDE-TREATED LEAVES.......................................43

In tro d u ctio n .............................................................................. .4 3
M materials and M methods ........................ .. ........................ .. .... ........ ........ 44
R esu lts ................... .............................................................. ................. 4 9
D discussion ................................... ...................................... ................ 50

4 EFFECT OF GENDER AND PHYSIOLOGICAL STATE OF Aedes Albopictus
(Skuse) ON RESPONSE TO PESTICIDE TREATED LEAVES ......................................57

Introduction ..... ..................................... ..............................................57
M materials and M methods ........................ .. ........................ .. .... ........ ........ 58
R results .......... .... ........ .. .. .......... ...... ........................................... ..... 6 1
D discussion .................................... ..................................... ................. 63












5 AREAS FOR FURTHER STUDY AND CONCLUSIONS ................................................69

A areas for F u rth er Stu dy ............................................................................... .....................6 9
C onclu sions.......... ..........................................................75

APPENDIX

A H O M EO W N ER SU RV EY ............................................................................. ............... 76

B PR O PA N E U SA G E TR IA L ................................................................... ...... ....................81

L IST O F R E F E R E N C E S .............................................................................. ...........................84

B IO G R A PH IC A L SK E T C H .............................................................................. .....................91







































6









LIST OF TABLES


Table page

2-1 Composition of mosquito species collected from four yards by one Mosquito Deleto
2500 per yard from August 18, 2005 to October 7, 2005 ............................................34

2-2 Collections of mosquitoes from one Mosquito Deleto 2500 per yard placed in four yards
from August 18, 2005 to October 7, 2005. ............................................. ............... 35

2-3 Average (SE) of total mosquitoes captured from one Mosquito Deleto 2500TM per yard
placed in four yards from August 18, 2005 to October 7, 2005. .....................................36

2-4 Species composition of mosquitoes observed during human landing rate counts in 8
yards participating in trap and pesticide trials in Gainesville, Florida from July 7,
2005 until O ctob er 7, 2 005 ........................................................................ .................. 37

3- 1 Mean percent control (SE) ofAe. albopictus following 1 hour exposure to TalstarOne
treated leaves (a.i. bifenthrin 7.9%) to plant leaves on August 22, 2006 and October
17 2 0 0 6 ............... .. ............ .............. ................................................ 5 4

3-2 Mean percent control (SE) ofAe. albopictus following 24 hours exposure to TalstarOne
treated leaves (a.i. bifenthrin 7.9%) to plant leaves on August 22, 2006 and October
17 2 0 0 6 ............... .. ............ .............. ................................................ 5 5

4-1 Mean percent knock down (SE) ofAe. albopictus following 1 hour exposure to
TalstarOne treated Rhododendron simsii leaves (a.i. bifenthrin 7.9%) .............................66









LIST OF FIGURES


Figure pe

2-1 Location of field sites in the A) University area and B) Devil's Millhopper area ................37

2-2 The Mosquito Deleto 2500 (MD2500) propane-powered trap used as trap treatments in
suburban yards in Gainesville, Florida. ........................................ ......................... 39

2-3 Average number (SE) of landing mosquitoes in the University area for each treatment
from July 8, 2005 until O ctober 7, 2005 ........................................ ........................ 40

2-4 Average number (SE) of landing mosquitoes in the Devil's Millhopper Area for each
treatm ent from July 8, 2005 until October 7, 2005 ....................................... .................41

2-5 Average number (SE) of mosquitoes landing during pre-treatment (N = 6 weeks) and
post-treatment (N = 12 weeks) collection in residential yards from July 8, 2005 until
O ctob er 7 2 0 0 5 ....................................................................... 4 2









Abstract of Thesis Presented to the Graduate School
of the University Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

RESPONSE OF Aedes albopictus (SKUSE) (DIPTERA: CULICIDAE) TO RESIDUAL
BIFENTHRIN ON PLANT LEAVES

By

Melissa Ann Doyle

August 2007


Chair: Daniel L. Kline
Major Department: Entomology and Nematology


The Asian tiger mosquito, Aedes albopictus (Skuse), was first introduced into the United

States in Harris County, Texas in 1985. This species is known as an aggressive daytime biter. It

has invaded 26 states, including Florida. Aedes albopictus larvae are found in tree holes, water-

containing plants, and man-made containers, such as birdbaths. It is recommended that residents

keep their yards free of water-holding containers to avoid this pest however, heavy infestations

ofAe. albopictus may require additional control methods.

The objective of my study was to evaluate commercially available control methods against

Ae. albopictus in residences in Gainesville, Florida. The control methods studied were the

placement of a propane powered trap called the Mosquito Deleto 2500, treatment with the

pesticide TalstarOne (active ingredient bifenthrin) by a pest control company, the combination of

the trap and pesticide treatment, and a control treatment where no treatments were applied.

Further investigation into the efficacy of bifenthrin treatment on different plant leaf types

against Ae. albopictus was evaluated. Plants commonly found in Florida residential landscapes

were chosen and treated with TalstarOne. The efficacy of bifenthrin-treated leaves was also

evaluated against male, female, blood-fed, and gravid Ae. albopictus.









CHAPTER 1
LITERATURE REVIEW OF SURVEILLANCE AND CONTROL TECHNIQUES USED TO
CONTROL Aedes albopictus (Skuse)

Introduction to Aedes albopictus

Aedes albopictus (Skuse) is a container breeding mosquito believed to be indigenous to

southeast Asia (Estrada-Franco and Craig 1995). In the past 20 years this species has spread

rapidly throughout the world and is a major nuisance pest in the United States. The mosquito is

established in 26 states, mainly along the eastern seaboard (CDC 2005). Defining control

techniques for this species is warranted due to its ability to rapidly spread to new areas and its

capacity to vector arboviruses, such as dengue (Estrada-Franco and Craig 1995).

This species is established in Asia, North America, South America, Europe and in Africa

(Gratz 2004, Gubler 2002). Aedes albopictus is an invasive species first introduced into the

continental United States in Harris County, Texas through the used tire trade in 1985 (Sprenger

and Wuithiranyagool 1986). It has displaced other container breeding species such as Aedes

aegypti Linnaeus (O'Meara et al. 1993). Prior to the introduction of Ae. albopictus, the most

common container breeding mosquito species in Florida was Ae. aegypti (O'Meara et al. 1993).

The directors of Florida mosquito control agencies believe that Ae. albopictus is

responsible for many service requests made by residents, second only to salt-marsh mosquito

complaints (Florida Coordinating Council on Mosquito Control 1998). Identifying control and

surveillance techniques for this pest will aid Florida residents and mosquito control personnel.

Ecology of Aedes albopictus

The Asian tiger mosquito, Ae. albopictus, was first described as Culex albopictus by

F.A.A. Skuse (1894) from specimens trapped in Calcutta, India. This species is in the subgenus

Stegomyia and the Scutelleris group (Hawley 1988). This mosquito species is black with a









distinctive, thin stripe of silvery-white scales down the back of the thorax and a completely black

clypeus (Darsie and Ward 2004, Hawley 1988).

Eggs are laid singly on the sides of natural and artificial water containers just above the

water line (Hawley 1988). Gubler and Bhattacharya (1971) found that colony mosquitoes laid an

average of 58.9 eggs per gonotrophic cycle with a range of total egg production of 0 to 784 eggs

over the lifetime. Aedes albopictus have been found to oviposit in natural containers such as tree

holes, bamboo, and water containing plants such as bromeliads (Hawley 1988). Eggs can also be

laid in water-holding artificial containers, such as discarded tires, potted plants, and bird baths

(Kay et al. 1995).

Hatching is stimulated by a combination of the container flooding, the availability of food

and the oxygen content of the water (Hawley 1988). Eggs often require several submersions

prior to hatching (Hawley 1988). The amount of time required for egg hatch varies between

populations, as observed between different field populations and colony mosquitoes (Mogi

1982). Eggs from the urban populations hatched more readily in colony than the eggs obtained

from natural rural-occurring containers, however, both populations displayed sporadic hatching.

Larval development times vary based on temperature, food availability, and crowding.

Development often occurs between 5 and 10 days under field conditions (Hawley 1988). Briegel

and Timmerman (2001) reared Ae. albopictus and found that larval development ranged from 7

days at 32 C to 28 days at 12 C. Food availability is also influenced development time. Mori

(1979) found that larval development was lengthened with lowered food levels and increased

larval density. In addition, adults from these stressed conditions were smaller and had decreased

fecundity.









Adults are often found resting in areas of dense vegetation (Estrada-Franco and Craig

1995, Hawley 1988). Males may spend time resting in leaf ground cover (personal observations

in Florida). Males have been observed to exhibit swarming behavior near hosts and intercept the

females as they approached the host (Gubler and Bhattacharya 1972). Host seeking occurs during

the daylight hours and populations often exhibit two landing rate peaks. In Penang, Malaysia,

human landing rate counts showed peaks just after sunrise and a few hours prior to sunset

(Hassan et al. 1996). Shirai et al. (2002) found that the preferred landing site ofAe. albopictus on

humans were primarily the feet followed by the hands and face.

Oviposition activity in the laboratory has a slight peak in the morning with the majority of

oviposition occurring between noon and darkness (Trexler et al. 1996). Aedes albopictus is not

considered to be a strong flier, often remaining within 100 meters of the larval emergence site

(Estrada-Franco and Craig 1995). However, Ae. albopictus dispersed up to 525 meters in a mark-

release study in Potsoi, Missouri (Niebylski and Craig 1994).

Aedes albopictus feeds on a variety of mammals and birds (Estrada-Franco and Craig

1995). Using ELISA, field captured Ae. albopictus in Potosi, Missouri were found to feed

commonly on mammals such as rabbits, deer, and dogs in addition to humans (Savage et al.

1993). Blood meal analysis performed on mosquitoes collected in central North Carolina

indicated that Ae. albopictus preferred human hosts, but would feed on domestic animals and in

some cases birds (Richards et al. 2006). The blood feeding habits ofAe. albopictus should be

taken into consideration given that it is a possible vector or bridge species for arboviruses.

Vector competence of Aedes albopictus The anthropophilic and opportunistic nature of

Ae. albopictus makes it a candidate to vector many pathogens. Its vector competence inside and

outside the laboratory has been reviewed extensively (Gratz 2004, Mitchell 1991, Shroyer 1986).









The most common disease-causing agents associated with Ae. albopictus are the four serotypes

of dengue virus (Estrada-Franco and Craig 1995). Aedes albopictus is considered to be the

secondary vector in areas also inhabited by Ae. aegypti. (Gratz 2004).

Several viruses have been isolated from North American populations ofAe. albopictus,

including La Crosse virus (Gerhardt et al. 2001), West Nile virus (Holick et al. 2002, Sardelis et

al. 2002), and Eastern Equine Encephalitis virus (Mitchell 1991). Aedes albopictus is also

capable of vectoring several parasites and pathogens of veterinary importance including

Dirofilaria immitis (Leidy) (Konishi 1989), Potsoi virus, and Cache Valley virus (Mitchell et al.

1998).

Surveillance Methods for Aedes albopictus

Population surveillance ofAe. albopictus can be accomplished using adult traps, larval

sampling and human landing rate counts (Florida Coordinating Council on Mosquito Control

1998). These methods are often combined in studies and in surveillance programs to provide

accurate estimations ofAe. albopictus populations.

Trapping methods

Many different types of mosquito traps are available to mosquito control organizations for

monitoring adult and larval populations. Traps can consist of a simple box that attracts resting

mosquitoes to a wide variety of electrically powered traps that use baits to lure and fans to

capture mosquitoes (Service 1993). Light traps are a common technique in any surveillance

operation. The New Jersey light trap has been used for many years to monitor mosquito

populations, however, it requires a permanent light source and collects many non-target insects.

Another commonly used trap for surveillance is the Centers for Disease Control (CDC) light

trap. It is highly portable and uses batteries to power a fan and light source. It also

accommodates a variety of attractants such as 1-octen-3-ol (octenol) or CO2 (Service 1993).









Traps can be modified to increase the number of mosquitoes captured and be more

attractive to specific species through the use of attractants and visual modifications (Service

1993). The most commonly used attractants are carbon dioxide and octenol. The Fay-Prince trap

is designed with contrasting panels of black and white to be more attractive to Ae. aegypti (Fay

and Prince 1970). Additional studies by Jensen et al. (1994) compared the numbers ofAe.

aegypti and Ae. albopictus females captured by CDC light traps, a bi-directional Fay trap, an

omni-directional trap (two Fay traps fastened together), and a duplex cone trap. The omni-

directional trap collected the most Ae. albopictus. In addition, the numbers ofAe. aegypti and Ae.

albopictus captured by each trap were significantly different, suggesting that the two species are

attracted to different visual cues.

Oviposition traps are another tool used in monitoring the presence of female Ae. albopictus

(Florida Coordinating Council on Mosquito Control 1998). These traps are containers that are

placed in the field and examined for eggs. Traps are often small containers painted black, such as

a cup (Gottfried et al. 2002) or glass jar (Holck et al. 1988), filled with water and have a wooden

paddle or paper offered for oviposition (Service, 1993, Thavara et al. 1989). Additional types of

oviposition traps include bamboo containers (Amersinghe and Alagoda 1984), modified tires

(Pena et al. 2004), tin cans (Swanson et al. 2000) or plastic jars (Thavara et al. 1989).

The water used in the oviposition traps may affect the attractiveness of the trap to female

Ae. albopictus. In a field study conducted near a tire disposal yard in Baton Rouge, Louisiana,

Holck et al. (1988) reported that Ae. albopictus oviposited more in jars with a hay infusion than

in jars containing either a leaf litter infusion, distilled water or a mixture of 1% fish oil and

water. In addition to the water infusions used in the ovitrap, care should be taken in the









placement of the ovitrap when targeting Ae. albopictus. In southern Brazil Sant'ana et al. (2006)

found that Ae. albopictus were more likely to lay eggs in traps surrounded by shrubbery.

Human landing rate counts are another effective tool for monitoring pest mosquitoes,

especially Ae. albopictus (Florida Coordinating Council on Mosquito Control 1998). Landing

rate counts measure the number of mosquitoes landing on a person during a period of time

(Florida Coordinating Council on Mosquito Control 1998). It is recommended that the same

person perform landing rate counts due to the differing attractiveness of people to mosquitoes.

Shirai et al. (2002) reported that Ae. albopictus prefers to land near the feet.

Control Methods for Aedes albopictus

Managing mosquito populations is a complex task often requiring the combination of

several different techniques (Florida Coordinating Council on Mosquito Control 1998). Various

techniques can be chosen based the life stage that is targeted. Organized mosquito control

involves the use of integrated pest management (IPM) practices to reduce mosquito populations

(Florida Coordinating Council on Mosquito Control 1998). An IPM program incorporates

multiple methods such as habitat alteration, source reduction, other cultural controls,biological

controls, and pesticides (Ware 1989).

Larval populations ofAe. albopictus often can be reduced by removing water-holding

containers, treating water containers with larvicides, or adding predacious organisms into the

container. The use of Bacillus linl iiugiel'i\ var. israeliensis (Bti) Berliner was evaluated under

field conditions by Fansiri et al. (2006) and found to be effective in reducing larval populations.

Dieng et al. (2002) evaluated the use of cyclopoid copepods as biological controls in the

laboratory and found them to be most effective against Ae. albopictus during the early mosquito

instars.









Broad spectrum pesticides should be a last resort in any IPM program and can include the

use of larvicides and adulticides (Florida Coordinating Council on Mosquito Control 1998).

Techniques that target specific areas for pesticide application, such as barrier pesticide

applications, can reduce the amount of damage to non-targeted organisms, especially beneficial

insects, by targeting specific areas (Anderson et al. 1991, Perich et al. 1993, Cilek and Hallmon

2006). Relying on the use of residual pesticides in homes has been useful in combating

mosquitoes that transmit malaria and that are found in the home (Kanda et al. 1995, Kerdipule et

al. 1978, WHO 2006). Mosquitoes also rest in natural areas, such as vegetation and in ground

cover, and applying residual pesticides to these areas also shows promise in reducing mosquito

populations (Service 1993).

Early field studies of area mosquito repellency looked at the use of malathion and naled

combined with a masking agent called Deodall and found that human landing rate counts were

reduced 51% (Berry et al. 1965). Barrier pesticide treatment may prove to be quite useful to

mosquito control professionals in a variety of situations. These situations include treatment for

people living near areas that cannot be treated, such as protected natural areas or chemically

sensitive neighbors. Another important use could be during arboviral transmission peaks to

protect people in high activity areas.

Permethrin and malathion applied to foliage has been used as an effective barrier treatment

against the salt marsh mosquitoes, Ochlerotatus sollicitans Walker and Ochlerotatus

taeniorhynchus Wiedemann, in North Carolina (Anderson et al. 1991). Shrubs bordering a park

area were treated with permethrin and malathion. Areas treated with permethrin had reduced

landing rate counts for 24 hours in comparison to non-treated areas. Landing rates were

decreased for 48 hours in malathion-treated areas.









Several pesticides were also effective as a barrier application against the malaria vector

Anopheles albimanus Wiedemann in the Dominican Republic (Perich et al. 1993). The foliage

surrounding three different villages was treated with permethrin, bendiocarb, and malathion.

Landing rates and light trap captures of An. albimanus were reduced in permethrin, bendiocarb,

and malathion treated villages in comparison to villages that did not receive treatment.

The effectiveness of a barrier often depends on how long a pesticide remains on the surface

of the foliage. Cilek and Hallmon (2006) treated wax myrtle, Myrica cerifera, with three

pesticides. Aqua Reslin (20% active ingredient [AI] permethrin + 20% [AI] piperonyl butoxide),

Permanone EC (10% [AI] permethrin), and Suspend SC(4.75% [AI] deltamethrin) applied at the

maximum label rate. The residual effectiveness of each pesticide was evaluated in large screened

cages by creating a foliage barrier surrounding Mosquito Magnet-Experimental traps. Aedes

albopictus and Culex quinquefasciatus Say mosquitoes were released on three contiguous days

per week in the field cages. Bioassays using leaves from the treated plants were performed to

evaluate the residual pesticide on the leaves over time. Deltamethrin provided the largest

reduction in mosquito trap captures (70% to 80%) during the first 4 weeks. Leafbioassays on

deltamethrin treated plants provided 95% knockdown throughout the study. This treatment was

also more effective in knocking down Ae. albopictus in comparison to Cx. quinquefasciatus.

Two pyrethroid insecticides, bifenthrin and lamda-cyhalothrin, were found to significantly

reduce mosquitoes in suburban Kentucky yards (Trout 2006). The treatments were found to be

most effective immediately following the pesticide application. It is important to note that gravid

mosquitoes were not reduced, suggesting that mosquitoes may have different susceptibilities to

pesticides at different physiological states or that they do not rest in the treated areas. Also, the

treatment was more effective at reducing Aedes and Ochlerotatus species than in reducing









Culex species. Further study of this type of pesticide application with other pesticides may show

promise in controlling Ae. albopictus.

Research Objectives

The establishment ofAe. albopictus in Florida has created new challenges for residents and

mosquito control agencies. Since arriving in Florida this species has quickly become a major pest

(Florida Coordinating Council on Mosquito Control 1998) and current control options for this

pest should be evaluated. The goal of my project is to evaluate current Ae. albopictus control

practices, especially the application of pesticides to residential plants and how the residual

pesticide performs in knocking down Ae. albopictus.









CHAPTER 2
EFFECT OF COMMERCIAL TRAPS AND PESTICIDE TREATMENT ON Aedes albopictus
(Skuse) POPULATIONS IN SUBURBAN SETTINGS IN NORTH CENTRAL FLORIDA

Introduction

Organized mosquito control is available in 27 counties in Florida. Residents can also

employ companies to apply additional mosquito control measures, including pesticide treatment.

Use of pesticides by these companies requires specific training and licensing by the state of

Florida (Florida Coordinating Council on Mosquito Control 1998). In addition to hiring a

company, residents may also purchase and apply a wide variety of mosquito control products.

These options include personal repellents, area repellents such as citronella-burning devices,

over-the-counter pesticides applied to the vegetation, mosquito-repelling devices and trapping

devices. As new products and services enter the marketplace it is necessary to evaluate their

efficacy against targeted species.

A wide variety of commercial traps are available for purchase by the homeowner to aid in

curbing adult mosquito populations. Early designs used ultra-violet lighting and electrocution

grids in attempts to attract and kill mosquitoes and other pest insects. These traps are inexpensive

to operate, but require a permanent power source and do not specifically target mosquitoes.

Additionally, Frick and Tallamy (1996) found that the 99.78% of insects attracted to this type of

trap were non-biting aquatic insects.

Current trap designs target pest insects, specifically mosquitoes. Traps powered by

propane generate multiple attractants (C02, heat and water vapor) whose combined effects with

attractants, such as 1-octen-3-ol (octenol), specifically target mosquitoes and other blood feeding

Diptera (Kline 2002). Propane is burned in these traps to both generate CO2 and electricity to

power the fans. The goal of this study was to assess the effectiveness of a representative









commercially available trap and a pesticide treatment in reducing Aedes albopictus (Skuse)

populations in suburban settings.

Materials and Methods

Field Trial Site Selection

Eight homes in Gainesville, Florida were selected based on homeowner reports of

mosquito activity in their yards during the daytime. The eight homes were assigned to two

groups based on the homeowner's willingness to have their property treated with pesticides and

then randomly assigned a treatment within the two groupings. There were 4 treatments evaluated

with 2 homes assigned to each treatment. Sites were visited twice weekly and evaluated for the

presence ofAe. albopictus using oviposition traps, backpack aspirations, human landing rate

counts, and propane trap captures.

The field trial was conducted in two different neighborhoods in Gainesville, Florida

approximately 9 km apart (Figure 2-1). The first neighborhood was located near the University

of Florida and the second area was located in the northwest section of Gainesville near the

Devil's Millhopper State Park. Aedes albopictus were present in all yards during initial landing

rate counts conducted prior to the study. Additionally, homeowners were also surveyed to

evaluate their attitudes toward mosquito control. (Appendix A)

University Area Neighborhood

Residential areas located just north of the University of Florida were lined with old growth

trees including Quercus virginiana Miller (live oak) and Magnolia grandiflora Linnaeus

(southern magnolia). Lot sizes ranged from 1,200 to 1,700 m2. The homes were constructed of

concrete block and one had wooden siding. All yards were substantially shaded by large pine and

hardwood trees. Leaf litter was also present near the foundations of the houses and yards had

sparse to full lawns of Stenotaphrum secundatum Kuntze (St. Augustine grass). Homes were









surrounded by mixed plantings of bushes including Rhododendron species Chamisso (azalea),

Juniperus horizantalus Linnaeus (juniper), and Ilex cornuta Lindley (holly).

Devil's Millhopper Area Neighborhood

The second neighborhood was located east of Devil's Millhopper Geological State Park.

The area is shaded heavily by live oaks and other oak trees. All homes were constructed of

concrete block with wood siding. The yards in this area are minimally landscaped and covered

with leaf litter. Lot sizes ranged from 1,200 to 3,000 m2. Bambusoideae (bamboo),

Rhododendron species, and ferns were present on all four properties. One yard had a full lawn of

St. Augustine grass. One yard was extensively planted with Billbergia spp. Thunberg and

Neoregelia (red fingernail) bromeliads.

Treatments

The treatments included: 1) placement of a commercial mosquito trap sold for residential

mosquito control; 2) the single application of a pesticide typically used for residential mosquito

control applied by a local pest control company; 3) the combination of one trap and a pesticide

treatment; and 4) a control where no reduction programs were implemented. All traps were

placed in the yards and activated when the pesticide treatments were completed on August 18,

2005 and remained in the field for eight weeks until October 7, 2005. Traps were positioned

away from areas of human activity and in the shade.

Traps

The trap used for this study was representative of commercially available mosquito traps

for residential mosquito control and was the Mosquito Deleto 2500TM (MD2500) [Coleman

Wichita, Kansas] propane-powered trap (Figure 2-2). Traps were activated on August 18, 2005.

The trap is approximately 0.9 meters tall and consists of a head unit mounted on metal stand.

This type of trap uses Dynamic Air Flow technology (Coleman 2006).









The metal tubing that attaches the head unit to the stand serves two important functions. It

houses the commercial lure releasing octenol [Coleman Wichita, Kansas] and expels the

attractants (C02, water vapor and octenol). The fan also draws in mosquitoes into a net that is

mounted in a drawer below the fan unit. The drawer automatically closes as it is removed so that

captured mosquitoes that have not died in the net do not escape. The mosquitoes captured in the

net die due to dessication from the constant air flow across the net.

The traps were placed in the back yard, approximately 6 9 m away from the back door of

the home and away from areas of human activity. Nets were changed weekly and captured

mosquitoes were stored in labeled paper containers, frozen and later identified to species using

keys (Darsie and Ward 2004) and counted. The propane tanks were changed every 14 days. The

octenol lures were changed every 30 days

Two sites had one MD2500 placed in their yards (MDTRAP1 and MDTRAP2). The

residence at MDTRAP1 was located in the University area and was rectangular. The back and

side yards were shaded by large, live oak trees. It had a sparse St. Augustine grass lawn. There

was a hedge of holly plants approximately 30 cm away from the home in the front. A large

magnolia tree (approximately 12 meters tall) was also in the front yard. The side yard had a

camellia, Camelia species, near the gate for the back yard.

The second residence (MDTRAP2) was located in the Devil's Millhopper area. The entire

property was extensively shaded with live oak trees and did not have a grass lawn. The ground of

the lot was covered with leaf litter. The areas to the front and sides of the house were extensively

planted with bromeliad plants, including Bilbergia spp. and Neoregelra spp. (red fingernail)

bromeliads. In the back yard there were five fenced areas for the rehabilitation of injured turtles.









Each pen had a small pond, however, no larvae were observed in the ponds over the course of the

study.

Pesticide Treatments

Florida Pest Control Company (Gainesville, Florida) was hired to perform their Pro-Star

Mosquito Reduction Program. Florida Pest Control Company was selected because they have

locations in 16 cities in north and central Florida and are representative of commercially

available mosquito control for residences across the state. The treatment occurred on August 18,

2005. The program included an inspection of the property for mosquito breeding habitat and

treatment with TalstarOne [FMC Corporation, Agricultural Products Group, Philadelphia, PA],

with the active ingredient bifenthrin. The pesticide was applied according to the label directions

(0.33 fluid ounces per gallon of water, A.I. 7.9%) with a B & G hand pump compressor sprayer

[B & G Equipment Company, Jackson, GA].

The four houses were sprayed in vertical swaths ensuring that the entire outside of the

residences were coated with the pesticide. Some swaths were performed over the surrounding

vegetation as the technician moved around the home, however, it was not as systematic as the

house treatment. If there was mixed pesticide remaining in the sprayer after the house was

completely coated, the technician would apply the remainder on the vegetation surrounding the

residence.

Pesticide-treated sites (TALl and TAL2) were shaded by large live oak trees. Both homes

had sparse grass lawns and the lots were rectangular shaped. The yard of the home treated in the

University Area contained many ornamental plants. There was an extensive collection of potted

orchids and cacti. Discarded planting pots often held water and were found with larvae

developing. There were approximately 20 red fingernail bromeliad plants planted beneath an oak









tree that were checked weekly for larvae. A bird bath that consistently contained larvae was also

present in this yard.

The lot of the pesticide-treated home in the Devil's Millhopper Area had a St. Augustine

lawn in both the front and backyard. There were no ornamental plants in pots. Occasionally,

discarded oil and coffee grounds containers could be found near the side door of the residence,

however, no larvae were observed developing in these containers. Bamboo plants, Bambusoideae

species, grew on the left side of the house and a low hedge of juniper bushes was located in the

front of the residence.

MD2500 and Pesticide Treatment

Two homes had a MD2500 placed in the back yard and received the Pro-Star Mosquito

Reduction Program. The traps and pesticide treatment were applied on August 18, 2005. Sites

that were treated with the pesticide and had a trap placed on the property (TALTRP1 and

TALTRP2) were also shaded by tall oak trees.

The residence located in the University Area (TALTRP1) had two loblolly pines in the

front yard, several azalea bushes in the back yard, juniper shrubs along the front of the house and

a groups of ferns in the side yard. TALTRP1 also had a sparse lawn of St. Augustine grass.

Occasionally, a wheel barrow would collect water and developing larvae were observed.

The yard of the home located in the Devil's Millhopper area (TALTRP2) was covered in

leaf litter. There were several bamboo plants and azalea bushes in the yard. No containers were

observed to hold water through the course of the study at this residence.

Control Treatments

Two residences did not receive mosquito reduction treatments. Control sites (CON1 and

CON2) were both shaded by large live oak trees, and did not have grass lawns. Both residences

had leaf litter surrounding the foundations of the homes. Both homes also had a number of









Rhododendron species bushes along the perimeter of the property. The homes were both located

on cul de sacs and had irregularly shaped lots with the houses placed in the center of the lot.

Mosquito Sampling

The presence of Ae. albopictus was evaluated using oviposition jars, backpack aspirations,

and human landing rate counts. Four oviposition traps were placed around each yard on August

18, 2005 and were in the field until October 7, 2005. The oviposition trap consisted of 250 ml

glass canning jars painted black and filled with approximately 125 ml of water. Seed germination

paper (ca. 15 x 30 cm) was placed so that the striations of the paper were vertical inside the trap

as an oviposition substrate. The papers were changed weekly. Water level within the jars was

monitored and refilled if the level dropped dramatically.

Backpack aspirations of the yards were performed beginning August 18, 2005 and weekly

thereafter until October 7, 2005 with a modified CDC Backpack Aspirator Model 1412 [John

Hock, Gainesville, FL]. Aspirations were performed around the vegetation surrounding the

foundations of the houses after landing rate counts were performed between 1:00 PM and 4:00

PM. The area surrounding vegetation near the residence was swept with the aspirator in a vertical

manner. The mouth of the aspirator was placed 0.3 meters away from the base of plants and

shrubs. The process took between 10 and 15 minutes depending on the size of the home and the

amount of surrounding vegetation. Collection cups were labeled with house location, covered,

and brought to the lab for identification. Mosquitoes were immediately identified.

Landing rate counts were performed by the same observer in the same four locations

around the yard twice per week between 1:00 PM and 4:00 PM from July 7, 2005 until October

7, 2005. Locations were in shaded areas that were likely to be frequented by humans. The

observer stood with the right calf exposed for three minutes. All landing mosquitoes were









aspirated by a mouth aspirator, counted and identified at the end of the three minutes, and

released.

Statistical Analysis

Mosquito monitoring results and trap captures were analyzed using Statistical Analysis

Software (SAS Institute 2004). Trap capture data were analyzed by ANOVA with means

separation by Bonferroni-Dunn test (a = 0.05). Landing rate counts and trap captures before and

after treatment were compared using t-test analysis. Landing rate counts were also transformed

(log(x+l)) and analyzed by PROC GLIMMIX to examine treatment and site effects on landing

rates.

Results

Collections from MD2500

Mosquito traps constituted part of the treatment program and reflect the composition of

mosquito populations in the yard. A total of 7,625 mosquitoes were trapped from the four yards

(Table 2-1). The eight species captured included Ae. albopictus, Anopheles quadrimaculatus

Say, Coquilletidiaperturbans (Walker), Culex nigripalpus Theobold, Ochlerotatus atlanticus

Dyar and Knab, Psorophora columbiae Dyar and Knab, and Wyeomyia mitchellii Theobold.

Mosquitoes from the genus Mansonia were also captured but were unidentifiable to species due

to loss of scales and desiccation. There were also 81 mosquitoes unidentifiable specimens. The

most prevelant species in descending order were Mansonia species, Cx. nigripalpus, Ae.

albopictus, Ps. ferox, Ps. columbiae, An. quadrimaculatus, unidentifiable specimens, Cq.

perturbans, Oc. atlanticus, and Wy. mitchellii (Table 2-1). Regardless of pesticide application,

the composition of species in trap collections was similar at all sites over the trapping season and

Ae. albopictus was present at all sites (Table 2-2). Aedes albopictus was the third or fourth most

common species at all sites.









Weekly averages of total trap captures for the yards including traps (MD25001 and

MD25002) and pesticide treatment and traps (TALTRP1 and TALTRP2) are included in Table

2-3. Comparison of means of the number of mosquitoes collected each week by analysis of

variance showed significant differences between the sites (F = 3.30, df = 3, P = 0.03).

Significantly more mosquitoes were captured in the trap at MD25002 than in at traps MD25001,

TALTRP1, and TALTRP2 (ANOVA, a = 0.05). Since there were significant differences

between sites, data from the two sites were not combined for each treatment.

There were four trap failures. The trap at MDTRAP2 failed on August 19, 2005. The trap

at TALTRP1 failed on August 24, 2005 and on September 23, 2005 while the trap was not

running at the time of collection, the propane tank must have just emptied because there were

mosquitoes remaining in the net. The trap at TALTRP2 failed on September 29, 2005 and on

October 7, 2005.

Oviposition Trap

No eggs were recovered from the oviposition traps due to the invasion of the traps by

predators (frogs, lizards, and spiders), the oviposition paper being removed, the traps being

knocked over, or snails eating the oviposition paper. Also, after the second week in the field the

traps were refilled weekly with new tap water. Oviposition traps remained in the field until

October 7, 2005.

Backpack Aspirations

One mosquito was recovered from the backpack aspirations on August 29, 2005 at TALl.

The mosquito could not be identified to species.

Human Landing Rate

A total of 884 landing mosquitoes were collected and identified during the trial (Table 2-

4). The five species included Ae. albopictus (90.2%), Culex species (0.3%), Oc. atlanticus









(2.4%), Ps. ferox (6.0%), and Wy. mitchellii (1.1%). The majority of mosquitoes observed

landing were Ae. albopictus, and included 714 females and 83 males (Table 2-4).

The weekly average landing rate counts for each treatment, including the control

treatments, for the University area (Figure 2-3) and for the Devil's Millhopper area (Figure 2-4)

indicated a general trend of reduction after the trap:pesticide treatment was initiated. Analysis

performed using PROC GLIMMIX identified area (F = 18.49, df = 3, P < 0.0001) effects to be

significant, therefore each site was considered a separate treatment for analysis (F = 33.10, df

=7, P <0.0001) and treatment effects were significant. Therefore, the data were grouped by the

two neighborhoods and considered separately by pre- and post-trap:treatment initiation for

further analysis.

Comparisons were made between the landing rate counts before and after treatments at

all sites in each neighborhoods using t-tests (Figure 2-5). At the sites in the university area,

CON1 (t = 4.17, df = 16, P = 0.0003), MD25001 (t = 3.33, df = 16, P = 0.002), TALl (t = 4.66,

df = 16, P = 0.0001) and TALTRP1 (t= 4.77, df = 16, P = 0.0001), showed significant

differences in landing rate counts before and after treatment. In the Devil's Millhopper area, only

the treated sites, MD25002 (t = 3.99, df = 16, P = 0.0005), TAL2 (t = 2.32, df = 16, P = 0.017)

and TALTRP2 (t = 2.76, df = 16, P = 0.006), showed significant different. CON2 did not

receive a treatment and no significant difference was found in the landing rate counts before and

after treatments were applied (t = 0.89, df = 16, P = 0.192).

Discussion

Aedes albopictus is a nuisance mosquito found in north central Florida yards (Hoel et al.

2007) and its diurnal activity (Estrada-Franco and Craig 1995) often conflicts with human

activity in yards. The mosquito species composition of the sites in this study are similar to the

findings of other studies performed in the area (Campbell 2003, Hoel et al. 2007). The aggressive









nature of host-seeking Ae. albopictus necessitates the establishment of control protocols aimed

specifically towards the reduction of these populations, even when populations are at moderate

levels as observed in this study. While Ae. albopictus are generalist feeders, a significant

proportion may feed on humans further enhancing the concern about this species as a nuisance

vector species (Richard et al. 2006b). The evaluation of control technique efficacy against Ae.

albopictus is warranted due to its ability to vector arboviruses (Gratz 2004, Mitchell 1991,

Shroyer 1986) and transmit other pathogens (Konishi 1989, Mitchell et al. 1998).

Different mosquito control techniques such as, the placement of commercially available

traps or the application of pesticide treatments to vegetation, have been evaluated against Ae.

albopictus. A propane powered trap, the Mosquito MagnetTM Pro [American Biophysics

Corporation, East Greenwich, Rhode Island] has been shown to capture Ae. albopictus in

Gainesville, FL (Hoel et al. 2007). Bifenthrin treatments applied to the vegetation in suburban

yards in both Kentucky and Australia were also found to reduce landing Ae. albopictus (Trout

2006, Standfast et al. 2003). The effect of a combination of trap and pesticide application is

unknown and was the object of this study.

The treatments evaluated in this study reduced populations of landing mosquitoes in the

Devil's Millhopper area, where there were no-area wide control measures offered by the local

mosquito control agency. Landing rates following the trap:pesticide treatment initiation were

significantly lower than pre-treatment levels at all treatment sites in the Devil's Millhopper area.

The lack of significant difference in the numbers of landing mosquitoes between the pre-

treatment and post-treatment periods at the control site indicates that the natural population

levels remained constant through the study. Thus, it appears that the population reductions noted

for all treatments were likely the results of control measures.









The homes utilized in the University Area, however, are within the jurisdiction of the

local mosquito control agency. There were significant differences between pre-treatment and

post-treatment collections at all sites evaluated in this neighborhood, including the control site. It

is possible some other factors, such as localized weather conditions, contributed to the reduction

in landing rate counts observed in the University Area. It is more likely that the observed

reductions in landing counts were the result of organized mosquito control operations.

Propane traps clearly offer homeowners an option to reduce Ae. albopictus and other

mosquito species on their properties. The clear preference ofAe. albopictus for residential

propane traps over conventional surveillance traps further supports the utility of these traps as a

potential control option (Hoel 2005). In the current study, traps baited with octenol were

effective in reducing numbers of landing Ae. albopictus at the site MD25001. Recent research

indicates that the addition of lactic acid lures to the traps emitting CO2 and octenol enhances

collections ofAe. albopictus (Hoel et al 2007). Thus it seems advisable to add lactic acid lures to

residential traps to further enhance reduction ofAe. albopictus.

Males swarm around potential hosts while seeking females (Gubler and Bhattacharya

1972). The trap may attract males seeking females, possibly due to the host-like cues provided

by the trap. Therefore, propane-powered traps may attract and capture both male and female Ae.

albopictus potentially reducing mating in localized populations. Hoel et al. (2007) reported a

significantly higher capture rate ofAe. albopictus males than males of any other species. In the

present study, males of other mosquito species were not captured. Male Ae. albopictus

constituted 0.1 % of the total number of mosquitoes captured and 2.0 % of all capture Ae.

albopictus were male and these findings agree with the Hoel et al. (2007) study (Table 2-1).

Also, male Ae. albopictus were often found resting on the trap. The male mosquitoes would









alight and display swarming behavior when the trap was approached. Swarming behavior near

the trap did not, however, lead to landing counts. Male Ae. albopictus have been observed

swarming during landing rate counts in other studies (Hoel et al. 2007). In this study, males were

observed swarming for approximately 30 seconds, then land on the leg and wait for females to

fly near. After the females arrived near the leg, the males would often alight and swarm around

the leg if they evaded aspiration. The males were not observed to mate the females while they

were walking around on the leg. Mated pairs that met around the leg, flew to ground leaf litter,

landed, and then moved under the leaves. The mated pairs were not observed once they moved

beneath the leaf litter.

Resting sites for mosquitoes often consist of vegetation, earth or rocks, or human

habitats, and animal shelters (Clements 1992). Male Ae. albopictus are not active constantly

during daylight and likely seek resting sites in shaded dark areas, such as parts of the traps.The

trap itself may offer resting or host seeking males an additional harborage area. On September 2,

2005 the trap was tipped on its side for some repairs and male Ae. albopictus were observed to

fly out of the hollow piping that connects the trap to the rolling stand. It is possible that male

mosquitoes commonly rest in this area of the trap.

The traps were placed 6 9 m away from the residence, as recommended by the

manufacturer directions. The placement was beyond the area treated with bifenthrin. Further

trials where a trap is placed closer to or within pesticide treatment may show differences in trap

captures between the two treatments.

Trap failures on August 19, August 24, and September 29 were probably due to not being

properly attached to the propane tank. The failure on October 7 may have been due to a problem

with the propane tank because the trap was running at the previous landing rate count, the tank









was full when it was found not running and would not restart with the same tank in the field. A

propane usage trial performed after the field study (Appendix B) to see how the tanks would last.

These traps ran for an average of 17.2 days and two traps failed on 5 occasions due to being

incorrectly attached to the propane tank.

The application of pesticides has been found to reduce the number of landing mosquitoes

in suburban settings (Anderson et al. 1991, Cilek and Hallmon 2006, Trout 2006). At homes

treated with bifenthrin human landing rate counts were reduced after pesticide application,

however, the pesticide application was focused on the home versus the vegetation. During the

pesticide application the technician indicated that it was important to coat the mosquito

harborage areas and it was important to treat darker corners. The label for TalstarOne permits

the use of the pesticide on buildings and mosquitoes are included in the list of targeted pests for

this type of application. The label also allows for vegetation to be treated to target mosquitoes.

At TALTRP1, the technician had nearly half of the one gallon of mixed chemical left

after treating the residence and used the remainder on a seven foot high azalea bush. Landing

rate counts were high near this bush prior to the application, but following the treatment the

landing rate counts dropped considerably in comparison to the landing rate counts near untreated

vegetation. Analysis of the landing rate counts close to and distant from the treatment were not

significant. Despite a lack of statistical difference between the two areas, further evaluation of

the number of landing mosquito species in the proximity of treatment should be undertaken to

determine if there is a localized reduction.

Treating for Ae. albopictus requires an integrated pest management approach. While the

Mosquito Deleto 2500 does not appear to capture large numbers ofAe. albopictus, it catches

many other pest species that also would be a problem for homeowners. Altering the way the









pesticide is applied by focusing the treatment on the vegetation and increasing the attractiveness

of the trap by adding lactic acid lures may improve these treatments against Ae. albopictus.

Further investigation of modified versions of these techniques may prove helpful in reducing

landing by Ae. albopictus and other pest species.









Table 2-1. Composition of mosquito species collected from four yards by one Mosquito Deleto
2500TM per yard from August 18, 2005 to October 7, 2005.


Aedes albopictus female
Aedes albopictus -male
Anopheles quadrimaculatus
Coquilletidia perturbans
Culex nigripalpus
Mansonia species
Ochlerotatus atlanticus
Psorophora columbiae
Psorophoraferox
Wyeomyia mitchellii
Unidentifiable specimens
TOTAL


Total number of mosquitoes (%)
448 (5.8)
9 (0.1)
107 (1.4)
79 (1.0)
859 (11.3)
5359 (70.3)
29 (0.4)
207 (2.7)
420 (5.5)
27 (0.4)
81 (1.1)
7625


All traps were operated from August 18, 2005 until October 7, 2005 and placed 6-9 m away from
areas of human activity. MD2500 at TALTRP1 failed on August 24, 2005. On September 23,
2005 the trap was not running, however live mosquitoes remained in the net. The MD2500 at
TALTRP 2 failed on Spetember 29, 2005 and on October 7, 2005.









Table 2-2. Collections of mosquitoes from one Mosquito Deleto 2500TM per yard placed in four
yards from August 18, 2005 to October 7, 2005.


Species
Aedes albopictus female
Aedes albopictus male
Anopheles quadrimaculatus
Coquileitidia perturbans
Culex nigripalpus
Mansonia species
Ochlerotatus atlanticus
Psorophora columbiae
Psorophoraferox
Wyeomyia mitchellii
Unidentifiable specimens
TOTAL


Traps
MD25001 MD25002
61 181


1
5
2
195
1021
12
1
37
0
4


4
48
26
263
2195
12
75
62
26
56


Pesticide + Traps
TALTRP1 TALTRP2
116 90
1 3
5 49
12 39
92 309
766 1377
4 1
2 129
221 100
0 1
5 16


1339 2948 1224 2114


All traps were operated placed 6-9 m away from areas of human activity. Bifenthrin applied at
TALTRP1 and TALTRAP2 on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of
water to the residences and surrounding vegetation. MD2500 at TALTRP1 failed on August 24,
2005. On September 23, 2005 the trap was not running, however live mosquitoes remained in the
net. The MD2500 at TALTRP 2 failed on September 29, 2005 and on October 7, 2005.









Table 2-3. Average (SE) of total mosquitoes captured from one Mosquito Deleto 2500TM per
yard placed in four yards from August 18, 2005 to October 7, 2005.
Traps Pesticide + Traps
MD25001 MD25002 TALTRP1 TALTRP2
Weekly Averages of Totals 132.9 (44.7)a 307.0 (53.8)b 204.4 (45.2)a 204.4 (45.2)ab

All traps were operated placed 6-9 m away from areas of human activity. Bifenthrin applied at
TALTRP1 and TALTRAP2 on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of
water with mixture sprayed on home and surrounding vegetation. MD2500 at TALTRP1 failed
on August 24, 2005. On September 23, 2005 the trap was not running, however live mosquitoes
remained in the net. The MD2500 at TALTRP 2 failed on September 29, 2005 and on October 7,
2005.ANOVA analysis showed significant difference between mosquitoes capture (ANOVA, F
= 3.30, df= 3, P = 0.03).










Table 2-4. Species composition of mosquitoes observed during human landing rate counts in 8 yards participating in trap and pesticide
trials in Gainesville, Florida from July 7, 2005 until October 7, 2005.


Control Trap Only Pesticide Only Trap + Pesticide
Species CON1 CON2 MD25001 MD25002 TALl TAL2 TRPTAL1 TRPTAL2 TOTAL (%)
Aedesalbopictus female 61 104 59 200 53 31 91 115 714(80.8)
Aedes albopictus male 27 5 4 8 12 0 17 10 83 (9.4)
Culex species 1 0 0 1 0 0 0 1 3 (0.3)
Ochlerotatus atlanticus 0 0 0 7 0 8 1 5 21(2.4)
Psorophoraferox 0 0 0 20 4 13 10 6 53 (6.0)
Wyeomyia mitchellii 0 0 0 8 0 1 0 1 10(1.1)
TOTAL (% of total) 89(10.1) 109 (12.3) 63 (7.1) 244 (27.6) 69(7.8) 53 (6.0) 119(13.4) 138 (15.6) 884

Landing rate counts were performed twice weekly at each home. Mosquitoes landing on the right exposed calf of the observer were
counted and aspirated for 3 minutes for a total of 12 minutes per yard. All traps were operated from August 18, 2005 until October
S 7,2005 and placed 6-9 m away from areas of human activity. Bifenthrin applied at TALl, TAL 2, TALTRP1 and TALTRAP2 on
August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water. MD2500 at TALTRP1 failed on August 24, 2005. On September
23, 2005 the trap was not running, however live mosquitoes remained in the net. The MD2500 at TALTRP 2 failed on September 29,
2005 and on October 7, 2005.


























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Figure 2-1. Location of field sites in A) University area and B) Devil's Millhopper area

neighborhood in Gainesville, Florida.


L


',- -- Fairbanks
z


I













































Figure 2-2. The Mosquito Deleto 2500TM (MD2500) propane-powered trap used as trap
treatments in suburban yards in Gainesville, Florida.















39





















.E
(5



E)
E
S- t TAL1
._ MD25001
UTALTRP1
SCON1



E


-1



0
8-Jul- 22- 1- 10- 12- 15- 19- 22- 24- 2- 7- 12- 21- 23- 28- 30- 2-Oct- 7-Oct-
05 Jul-05 Aug- Aug- Aug- Aug- Aug- Aug- Aug- Sep S Sep Se Sep ep SSep- Sep- 05 05
05 05 05 05 05 05 05 05 05 05 05 05 05 05
Pre-Treatment Post-Treatment
Date



Figure 2-3. Average number (SE) of landing mosquitoes in the University Area for each
treatment from July 8, 2005 until October 7, 2005.


Landing mosquitoes were observed and aspirated for three minutes in 4 different sites around the
homes. MD2500 at TALTRP1 failed on August 24, 2005. On September 23, 2005 the trap was
not running, however live mosquitoes remained in the net. Bifenthrin applied at TALTRP1 and
TALTRAP1 on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water. All traps
were operated from August 18, 2005 until October 7, 2005 and placed 6-9 m away from areas of
human activity. TALl = TalstarOne treatment, MD25001 = Mosquito Deleto 2500TM,
TALTRP1 = TalstarOne and Mosquito Deleto 2500TM CON1 = control yard.























0"5 ITAL2
E OMD25002
SBTALTRP2
4 CON2

a3








05 ul-05 Aug- Aug- Aug- Aug- Aug- Aug- Aug- Sep- Sep- Sep- Sep- Sep- Sep- Sep- 05 05
05 05 05 05 05 05 05 05 05 05 05 05 05 05
Pre-Treatment Post-Treatment
Date

Figure 2-4. Average number (SE) of landing mosquitoes in the Devil's Millhopper Area for each
treatment from July 8, 2005 until October 7, 2005.


Landing mosquitoes were observed and aspirated for three minutes in 4 different sites around the
homes. MD2500 at TALTRP1 failed on August 24, 2005. On September 23, 2005 the trap was
not running, however live mosquitoes remained in the net. Bifenthrin applied at TALTRP1 and
TALTRAPI on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water. All traps
were operated from August 18, 2005 until October 7, 2005 and placed 6-9 m away from areas of
human activity. TALl = TalstarOne treatment, MD25001 = Mosquito Deleto 2500TM,
TALTRP1 = TalstarOne and Mosquito Deleto 2500TM, CON1 = control yard.


































B
6




0 E 4

SPre-treatment
Eg [ Post-treatment



E 15



TAL2 MD25002 TALTRP2 CON2
Treatment



Figure 2-5. Average number (SE) of mosquitoes landing during pre-treatment (N = 6 weeks) and
post-treatment (N = 12 weeks) collection in residential yards from July 8, 2005 until
October 7, 2005. A) University Area Neighborhood B) Devil's Millhopper Area

Landing mosquitoes were observed and aspirated for three minutes in 4 different sites around the homes.
MD2500 at TALTRP 1 failed on August 24, 2005. On September 23, 2005 the trap was not running,
however live mosquitoes remained in the net. Bifenthrin applied at TALTRP1 and TALTRAP 1 on
August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water. All traps were operated from
August 18, 2005 until October 7, 2005 and placed 6-9 m away from areas of human activity. TALI =
TalstarOne treatment, MD25001 = Mosquito Deleto 2500TM, TALTRP 1 = TalstarOne and Mosquito
Deleto 2500TM, CON1 = control yard.


U)
W 4
0

0- 3.5
0
E 3
0-
;5 2.5
F I Pre-treatment
S.- 2 -
-_o
SE 0 Post-treatment
| 1.5

1

-o 0.5
W

> TALl MD25001 TALTRP1 CON1
C









CHAPTER 3
EFFECT OF PLANT SPECIES AND LEAF TEXTURE ON RESPONSE OF FEMALE Aedes
albopictus (Skuse) TO PESTICIDE-TREATED LEAVES

Introduction

Suburban yards contain many types of plants that are potential harborage areas for

mosquitoes. Landscape plants are chosen based on a combination of factors, including aesthetics,

availability, and environmental conditions (Black 2003, Gilman and Brown 2003). Targeting

these mosquito harborage areas with pesticide applications has been found to reduce the number

of mosquitoes in localized areas (Anderson et al. 1991, Cilek and Hallmon 2006, Standfast et al.

2003, Trout 2006). The characteristics of plant leaves, such as the surface tension of the leaf

cuticle, and the overall leaf arrangement are important in the study of residual vegetation sprays.

The treatment of residential landscapes with residual pesticide may reduce the necessity of

applying pesticides added by ground ULV.

Residual pesticides applied to substrates have been used to manage infestations of many

different types of insects including cockroaches (Ali et al. 1993), house flies (Geden et al. 1992),

and mosquitoes (Kerdipibule et al. 1978). The residual effectiveness of DDT, malathion and

chlorpyrifos applied to different wall surfaces against Culexpipiens Linnaeus was evaluated in

Thailand (Kerdpibule et al. 1978). Dursban (chlorpyrifos) was found to kill mosquitoes on eight

wall surface types for 10-30 weeks. Residual malathion treatments were lethal for 10-15 weeks

while the mosquitoes were found to be resistant to the residual DDT treatments.

The physical characteristics, such as surface tension or roughness, of the substrates affect

the manner in which a pesticide can interact with the targeted organism (Kirkwood 1987). The

effectiveness of residual pesticides applied to different plants against Folsomia candida Willem

(Collembola: Isotomidae) was studied (Chowdhury et al. 2001). Sixteen leaf types with different

characteristics, such as age and surface wax-type, were treated under a Potter tower calibrated to









deliver a spray volume equivalent to 1 liter per 10,000 m2 with deltamethrin (25 g liter EC).

Folsomia candida displayed different tolerances on the various plants, suggesting that leaf

surfaces play a role in how pesticides can be transferred to organisms.

Evaluating the effectiveness and longevity of pesticide residues under field conditions is

critical in understanding how well pesticide treatments will control resting mosquitoes, such as

Aedes albopictus (Skuse). The objective of this experiment was to evaluate the residual efficacy

of bifenthrin applied to common landscape plants, in the knock down of Ae. albopictus.

Materials and Methods

Plants were treated on August 22, 2006. Leaves were allowed to dry and removed for the

tests 4 hours post treatment and weekly thereafter for 5 weeks post-treatment. The plants

remained in the field for three additional weeks and the same plants were treated again on

October 17, 2006. Bioassays were performed again using leaves removed 4 hours post treatment

and weekly thereafter for the following five weeks.

Plant Selection

Five plant types were chosen representing two different leaf types, flexible and rigid.

Flexible leaves did not crack or break when bent and did not have shiny surfaces. Rigid plants

cracked or broke when bent and were shiny. All plants are commonly found in yards or in

uncultivated areas in Gainesville, FL. Plants were kept outdoors and watered for one-hour daily

with a drip irrigation system.

Flexible Leaves

Azaleas, Rhododendron x 'Fashion' Linnaeus (family Ericaceae) (Gilman 1999d), are

evergreen shrubs commonly found in yards around Gainesville, Florida (Figure 3-la). The leaves

are small to medium size, approximately 5 cm long, and arranged in an alternate manner. These

shrubs can reach 3 meters in height and have a wide variety of flowers that bloom throughout the









year. Ten plants (eight pesticide-treated and two control) were used for these bioassays and were

approximately 0.5 m tall and purchased at Lowe's Home Improvement Store (Gainesville,

Florida).

Beauty berry plants, Callicarpa americana Loureiro (family Verbenaceae) (Gilman

1999a), are commonly found growing along roadsides and in natural areas in Gainesville, Florida

(Figure 3-1b). Their leaves are broad, between 10 and 20 cm in length. They produce bright

purple berries in the fall and grow 1-2 meters in height. They have long branches with simple

leaf arrangement that have leaves on smaller branches at 7-10 cm intervals. Eight pesticide-

treated and two control plants were obtained from Chiappini Nursery (Melrose, Florida). The

plants used in this study were approximately 0.5 meters tall.

Rigid Leaves

Dwarf buford holly, Ilex cornuta 'Bufordii Nana' Lindley (family Aquifoliaceae)

(Gilman 1999b), is an evergreen shrub that has red berries throughout the seasons. It has stiff,

shiny leaves that are approximately 5 cm long (Figure 3-1c). Berries are toxic to humans but are

edible by birds making it a possible harborage area for mosquitoes that prefer to feed on birds.

The Buford holly plants (eight pesticide-treated and two control) were purchased at Lowe's

Home Improvement Store.

Sand Cordgrass, Spartina bakeri Merrill (family Gramineae) (Gilman 1999e), is found

near wetlands and is popular in landscapes (Figure 3-1d). In addition, plants of the same genus,

Spartinapatens Muhlenberg and Spartina alterniflora Loiseleur-Deslongchamps, are found in

Florida marsh lands (FDEP 2004). Spartina bakeri and other members of the genus could offer

harborage areas for salt marsh mosquitoes, such as Ochlerotatus taeniorynchus Wiedemann and

Ochlerotatus sollicitans Walker, in coastal areas. The leaves are extremely narrow and blades

can be up to a meter in length. The plants used in the study (eight pesticide-treated and two









control) were approximately 1 meter tall. These plants also were purchased from Chiappini

Nursery.

The southern magnolia, Magnolia grandiflora Linnaeus (family Magnoliaceae) (Gilman

and Watson 2006), was chosen because it is common in landscapes in Gainesville, Florida. It has

large oval leaves (10 to 20 cm long) that are rigid and have brown hairs on the underside (Figure

3-le). They can grow up to 27 meters high. The trees can provide harborage for mosquitoes near

the ground if the lower branches are not pruned and mature trees could also provide harborage

areas for mosquitoes found in tree canopies. The Magnolia grandiflora used in this test (eight

pesticide-treated and two control) were approximately 0.75 to 1.0 m tall. The magnolia trees

were purchased at Chiappini Nursery.

Pesticide Application

TalstarOne [FMC Corporation, Agricultural Products Group, Philadelphia, PA], active

ingredient bifenthrin, was chosen for the pesticide treatment. The pesticide was applied

according to the label directions (10 ml per 3.8 L of water, A.I. 7.9%) with a B & G hand pump

compressor sprayer [B & G Equipment Company, Jackson, GA]. Plants were moved

approximately 280 m to a treatment area away from the holding area. Control plants remained in

the holding area during treatment. The plants were placed into 8 groups so that each group

contained one plant of each type and each plant was treated with approximately 0.6 liters of the

mixture. Each group of 8 plants was treated with a separate pesticide mixture.

The pesticide was applied to the plants on August 22, 2006. Each plant was treated to

runoff. The applicator wand was held approximately 0.3 m away from the plant leaves while

three initial vertical swaths were applied. The wand was then placed within the foliage and three

vertical swaths were slowly made from the bottom to the top of the plant. This process was

repeated seven additional times around the circumference of the plant. Plants were allowed to









dry for four hours before being returned to the plant holding area. The plants were left in the

field for three additional weeks and the same plants were retreated on October 17, 2006.

Bioassays were performed as previously described.

Petri Dish Preparation

Leaves were picked from each plant every seventh day for 35 days post-treatment and

placed into paper envelopes. Rhododendron x, Callicarpa americana, Ilex cornuta and Magnolia

grandiflora were picked by hand. Spartina bakeri blades were cut using scissors. Leaves were

brought into the lab and placed onto prepared 100 x 15 mm petri dishes [Fisher Scientific

Worldwide, Hampton, New Hampshire]. Disposable latex gloves were worn during the

collection of control leaves and gloves were changed prior to handling treated leaves. Leaves

were fastened to petri dishes prepared by covering the inside of the bottom dish with double-

sided, 48 mm wide indoor / outdoor carpet tape [Henkel Consumer Adhesives, Inc., Avon,

Ohio].

Leaves were placed one at a time so that the upper surface of the leaf was exposed until the

entire surface of the dish was covered. If there were small gaps between the leaves a sterile

cotton ball was brushed over the exposed tape so that fibers attached to the exposed adhesive was

exposed. The dishes with Ilex cornuta and Spartina bakeri leaves had two 2.5 mm wide strips of

single sided tape crosswise over the surface of the leaves to ensure that the leaves remained in

place because these leaves tended to detach over the 24 hour period. A rubber band was placed

on the outside of the dishes securing them together and they were stored on their sides in air tight

containers. The containers were lined with damp paper towels to ensure that adequate moisture

levels, relative humidity between 70 85 %, were maintained for mosquito survival.









Mosquitoes

Nulliparous 5-7 day old female Ae. albopictus were obtained from the USDA, ARS,

CMAVE rearing laboratory colony that was established in 1992 (Hoel 2005). Larvae were fed a

3:2 mixture of beef liver powder to yeast. Adults were provided cotton balls saturated with a

10% sucrose water solution. The colony was kept under 14:10 (L:D) photoperiod at room

temperature (27C to 320C).

Adults were obtained from colony cages with a mechanical aspirator and chilled at 10C for

approximately 5 minutes until they were no longer active. Ten mosquitoes were placed into

individual holding cups and then transferred into the prepared petri dishes. Mortality was

assessed at 1 hour and 24 hours post introduction into the petri dish. Mosquitoes were considered

alive if they were able to fly within the dish. Mosquitoes were rated as knocked down if they

were unable to fly within the dish and included mosquitoes that exhibited twitching limbs but

were unable to stand.

Statistical Analyses

All statistical analyses were performed using the Statistical Analysis System (SAS Institute

2004). 'Treatment data were corrected for control mortality by Abbott's formula (Abbott 1925).

Control mortality was 0.0 % for all plant types except for lex cornuta at day 0 (1.7%). Control

mortality at the 24 hour marks ranged between 1.7% and 11.7% for all plant types. Data were

transformed using arcsine transformation then compared with ANOVA (P < 0.05). N differences

between the two trials were observed. The data were pooled and adata from the 1 hour and 24

hours post introduction time check were analyzed by repeated measures analysis of variance

(PROC GLM, SAS Institute 2004). Differences between plant species were detected by ANOVA

with means separation between plant species identified using Bonferroni-Dunn (a = 0.05).









Results


Laboratory Bioassay Results

One-Hour Exposure of Ae. albopictus

There was no knock down observed on days 28 and 35 and these data are not presented.

Knock down of mosquitoes was observed at one-hour on days 7, 14 and 21 and these data are

presented in Table 3-1. Repeated measures analysis performed found significant differences in

both week effects (F = 489.60; df = 5; P < 0.0001) and between plant types (F = 76.99; df = 4, P

< 0.0001).

Significant differences were observed between the plant species were observed with

ANOVA analysis (F = 8.24; df= 4; P < 0.0001). In the Rhododendron x leaves, significantly

fewer mosquitoes were knocked down on day 7 than on day 0 (t = 7.22; df = 30; P < 0.0001) and

between days 7 and 14 (t = 7.22; df = 30; P < 0.0001) with no observed differences between day

14 and 21. Significantly more mosquitoes were knocked down on day 0 with the Callicarpa

americana than day 7 (t = 10.33; df = 30; P < 0.0001) with no differences between days 7, 14

and 21. A significant difference was observed between days 0 and 7 (t = 5.97; df = 30; P <

0.0001) and between days 7 and 14 with the Ilex cornuta leaves (t = 8.57; df = 30; P < 0.0001),

there was no difference between day 14 and 21. Although few mosquitoes were knocked down

on day 0, significantly more mosquitoes on day 0 than on day 7 with the Spartina bakeri leaves

(t = 7.22; df = 30; P < 0.0004) with no differences between days 7, 14 and 21. On day 0,

significantly more mosquitoes were knocked down with the Magnolia grandiflora leaves

between day 0 and day 7 (t = 25.27; df = 30; P < 0.0001) with no differences between days 7, 14

and 21.









Twenty-four Hours Exposure ofAe. albopictus

Knock down of mosquitoes at 24 hours post introduction to the petri dishes was observed

for 35 days post treatment at the 24 hour point and these data are presented in Table 3-2.

Significant differences were found with repeated measures analysis for both week effects over

the course of the study (F= 289.5; df = 5; P < 0.0001) and plant species effects (F= 281.31; df=

5; P < 0.0001).

ANOVA analysis between the plants found significant differences between the plant types

(a = 0.05; F = 18.78; df = 4; P < 0.0001). Knock down at 24 hours was similar between day 0

and 21 with a decrease between days 21 and 28 (t = 2.61; df= 30; P < 0.011) and days 28 and 35

(t = 4.36; df = 30; P < 0.0001) in the Rhododendron x leaves. With Callicarpa americana leaves

knock down was similar between days 0 and 21, however, between days 21 and 28 there was a

significant decrease in knock down (t = 12.80; df = 30; P < 0.0001). There were significant

differences observed between days 14 and 21 (t = 7.13; df = 30; P < 0.0001) and days 21 and 28

(t = 8.12; df = 30; P < 0.0001) in the Ilex cornuta plants (t = 5.38; df = 30; P < 0.0001). Knock

down with Spartina bakeri leaves decreased significantly between days 0 and 7 (t = 3.00; df =

30; P = 0.0035), days 7 and 14 (t = 4.65; df= 30; P < 0.0001) and between days 14 and 21 (t =

7.13; df = 30; P < 0.0001) with no differences between days 21, 28 and 35. Knock down using

treated Magnolia grandiflora leaves was similar between days 0 and 7 with a significant decline

between days 7 and 14 (t = 2.75; df = 30; P = 0.0071), days 14 and 21 (t = 10.12; df = 30; P <

0.0001) and between days 28 and 35 (t = 2.06; df = 30; P = 0.042).

Discussion

Treating the resting areas of mosquitoes, such as the walls of homes, has been successful in

reducing Anopheline mosquitoes in malaria endemic regions (Kerdipibule 1978) and is a

recommended control technique by WHO to reduce malaria transmission (2006). The use of an









indoor residual pesticide spray was evaluated in India and was found to be effective for 24 weeks

on various wall surfaces (Yadav et al. 2003). Use of residual pesticide applications on other

mosquito resting sites, such as on plants outdoors, has been shown to be effective in managing

pest mosquito populations (Anderson et al. 1991, Cilek and Hallmon 2006, Standfast et al. 2003,

Trout 2006), however, the efficacy of outdoor residual sprays was short-lived due to

precipitation and exposure to sunlight (Kirkwood 1987). It is also possible that the substrate,

such as plant surfaces, impact to the efficacy of the residual pesticide.

The type of leaf surface treated has been shown to affect the way in which a pesticide is

transferred to an organism. Chowdhury et al. (2001) treated 16 different plant types with varying

characteristics and found that the efficacy of residual pesticides against Folsomia candida varied

and that pesticide efficacy was not predictable by an any obvious pattern. In this study, five

different plant species were treated with a residual pesticide and the efficacy of the pesticide

against Ae. albopictus varied between plants.

The plants in this study were categorized into two groups, flexible and rigid, based on

visual appearance and texture of the leaves. The efficacy of the pesticide on the leaf surfaces

varied in both groups. In the flexible plant group, knock down was observed at high levels,

Rhododendron x (90.0%) and Callicarpa americana (97.5%) (Table 3-1). On day 7 the efficacy

of the knock down of mosquitoes exposed to treated Callicarpa americana leaves dropped to

21.3% whereas knock down of mosquitoes on Rhododendron x leaves remained relatively high

at 62.5%. Likewise, similar differences in knock down between species in the rigid leaf plant

group also were observed.

The observed differences could be attributed to the type of cuticle in the leaves or the

ability of the plant to absorb the pesticide. The cuticular wax of the plant is the primary barrier to









pesticides (Ford and Salt 1987). Waxes with high polarity will deter a pesticide from being

absorbed by the plant (Ford and Salt 1987). It is possible that the surface of the Callicarpa

americana plant is more polar than the Rhododendron x, therefore the pesticide is able to dry and

remain on the leaf surface longer than occurs on the cuticles of the Callicarpa americana.

In addition to how the pesticide interacts with the cuticular waxes, the application

techniques used to apply the pesticide may contribute the residual efficacy of a pesticide. Results

using treated leaves of Spartina bakeri also showed reduced levels of residual pesticide one week

after treatment at both 1 hour and 24 hours post-exposure. The differences observed with the

Spartina bakeri compared to the other plants could be attributed to several factors. The most

important factor is that the extreme thinness and arrangement of the Spartina bakeri blades may

have hindered the effective application of pesticide using the hand sprayer. The narrowness of

the blades made it difficult to direct the spray onto the blades and to ensure that the blades were

coated properly with the pesticide. The thinness of the blades also made it difficult to determine

the top side of the leaves for the bioassay. It is possible that the use of a pesticide applicator that

creates a finer mist or fog may increase the effectiveness of the residual application.

Plant growth may have played a role in the decreased control percentage over time. To

limit this possibility, leaves were not taken from areas of new growth for the bioassays. New

leaves were readily identifiable from old leaves due to the way that they budded. Also, the

surface area of the leaves was not visually observed to increase, however, measurements were

not taken to verify this.

In addition to the above mentioned factors, the role of the wax deposits on the cutilcle of

plants plays a role in the efficacy of the residual pesticide (Kirkwood 1987, Ford and Salt 1987).

It is possible to perform tests to determine the types and levels of wax that are present on plant









leaves (Chowdhury et al. 2001), however, this was beyond the scope of this experiment. In the

presented study, differences in the efficacy of residual bifenthrin were observed between

common landscape plants. Determining the types of waxes present on common landscape plants

may lead to improvements in the efficacy and longevity of barrier sprays against mosquitoes,

especially Ae. albopictus.











Table 3- 1 Mean percent control (SE) ofAe. albopictus following 1 hour exposure to TalstarOne
treated leaves (a.i. bifenthrin 7.9%) to plant leaves on August 22, 2006 and October
17, 2006.


Days post-treatment
Plant 0 7 14 21
Rhododendron x 90.0 (4.6)a 62.5 (5.3)b 4.4 (1.8)c 1.3 (1.3)c
Callicarpa americana 97.5 (1.9)a 21.3 (7.1)b 3.1 (1.5)b 0.0 (0.0)b
Ilex cornuta 100.0 (0.0)a 77.5 (7.9)b 7.5 (2.1)c 0.0 (0.0)c
Spartina bakeri 15.6 (6.3)a 2.5 (1.4)b 0.0 (0.0)b 0.0 (0.0)b
Magnolia grandiflora 96.9 (1.5)a 8.8 (3.1)b 2.5 (1.7)b 1.3 (0.9)b

Knock down was not observed on day 28 and day 35 post treatment for any plants. Control
mortality was 0% for all plants at the 1 hour mark, percent control was calculated using Abbott's
formula (Abbott 1925) correction was used to maintain consistency with mortality at the 24 hour
mark. Pesticide applied with B & G hand pump compressor sprayer at the rate of 10 ml to 3.8 L
of water. Plants were allowed to rest in the field for 3 weeks prior to re-treatment. Means within
each row followed by the same letter are not significantly different Bonferroni-Dunn test (P =
0.05).











Table 3-2 Mean percent control (SE) ofAe. albopictus following 24 hours exposure to TalstarOne treated leaves (a.i. bifenthrin 7.9%)
to plant leaves on August 22, 2006 and October 17, 2006.


Days Post-Treatment
Plant 0 7 14 21 28 35
Rhododendronx 100.0 (0.0a) 100.0 (0.0)a 98.7 (0.9)a 94.3 (2.3)a 89.4 (2.7)b 77.7 (2.1)c
Callicarpa americana 100.0 (0.0)a 99.3 (0.0)a 91.1 (5.1)a 90.6 (2.8)a 26.2 (3.7)b 19.4 (2.1)b
Ilexcornuta 100.0 (0.0)a 100.0 (0.0)a 100.0 (0.0)a 91.7 (3.1)b 44.0 (8.3)c 36.4 (4.7)c
Spartina bakeri 99.3 (0.7)a 87.2 (3.5)b 75.0 (7.3)c 25.6 (5.6)d 25.0 (4.7)d 16.3 (1.8)d
Magnolia grandiflora 97.9 (1.5)a 100.0 (0.0)a 90.3 (3.1)b 26.3 (6.0)c 27.6 (6.2)c 16.9 (2.5)d

Leaves were allowed to dry for four hours after the pesticide application, leaves were picked and the bioassays were performed.
Leaves were picked every 7 days following treatment for 35 days. The percent mortality for the control ranged between 1.7% and
11.7%. Pesticide applied with B & G hand pump compressor sprayer at the rate of 10 ml to 3.8 L of water. Plants were allowed to rest
in the field for 3 weeks prior to re-treatment. Means within each row followed by the same letter are not significantly different
Bonferroni-Dunn test (P = 0.05).























b. Callicarpa americana
'---. .. "< S


c. Ilex bujordii


d. Spartina bakeri


e. Magnolia grandiflora
Figure 3-1 Plants used in the bioassays performed August 17, 2006 to October 26, 2006


7*7-)/- J J.- -- IT, -1- I









CHAPTER 4
EFFECT OF GENDER AND PHYSIOLOGICAL STATE OF Aedes Albopictus (Skuse) ON
RESPONSE TO PESTICIDE TREATED LEAVES

Introduction

Various factors contribute to the effectiveness of a residual pesticide application in

targeting an insect, including how the pesticide interacts with the substrate and how the insect

comes into contact with the residual pesticide. In addition to these factors, the physiological state

of the insect may also influence how the insect is able to metabolize pesticides. Hadaway and

Barlow (1956) applied DDT directly to the thorax of Aedes aegypti Linnaeus and Anopheles

stephensi Liston of both sexes, different ages, and blood-fed females and found that both species

were less susceptible to pesticides following a blood meal or sugar meal. Susceptibility to the

pesticides returned to pre-feeding levels after oviposition and digestion occurred due the

dehydrochlorination of DDT. Halliday and Feyreisen (1987) hypothesized that the increased

tolerance to DDT was due to increases of dehydrochlorinases in the 48 hours post-blood meal

ingestion.

Additionally, the way in which a mosquito encounters the pesticide may influence the

toxicity of how the pesticide is metabolized by the mosquito. Xue and Barnard (2003) evaluated

boric acid baits against gravid, blood-fed and parous Aedes albopictus (Skuse), Anopheles.

quadrimaculatus Say, and Culex nigripalpus Theobold in the laboratory. The baits were offered

to the mosquitoes mixed with 10% sucrose water. Males were more susceptible to the baits than

females. The boric acid baits were also more toxic to male and female Ae. albopictus and Cx.

nigripalpus than to An. quadrimaculatus. The baits also caused mortality in blood-fed, gravid

and parous mosquitoes, suggesting that the toxicity of the baits is unaffected by the physiological

state of the mosquitoes.









Mosquitoes exist in nature in different physiological states that may be affected by

pesticides and other treatments differently. It is important to evaluate pesticide treatments against

different physiological states to ensure that the label rates are effective against all mosquitoes.

The objective of this study is to evaluate the impact of sex and nutritional state on the efficacy of

residual bifenthrin on plant leaves.

Materials and Methods

Plant Selection

Rhododendron simsii Planch (family Ericaceae) (Gilman 1999c) were the plants chosen

for these bioassays. Plants were purchased from Harmony Gate Nursery in Gainesville, Florida.

This plant was chosen for several reasons. First, mosquitoes were observed to land readily at

landing rate sites located near azalea plants during field work completed in 2005. Second,

azaleas had high knock down rates in the leaf type bioassays after 1 hour and 24 hours of

exposure in comparison to other leaf types. Also, Rhododendron simsii can grow over 3 meters

high and make a good plant barrier candidate. While treated Ilex cornuta Lindley leaves were

found to result in the highest knock down rates in those assays, their leaves are stiff and require

additional manipulations to keep attached to the Petri dishes.

Pesticide Application

Pesticide application occurred on January 27, 2007. TalstarOne [FMC Corporation,

Agricultural Products Group, Philadelphia, PA], active ingredient bifenthrin, was chosen for the

pesticide treatment. The pesticide was applied according to the label directions (0.01 L per 3.8 L

of water, A.I. 7.9%) with a B & G hand pump compressor sprayer [B & G Equipment Company,

Jackson, GA]. Plants were moved to a treatment area 280 m away from the holding area and

control plants remained in the holding area during treatment. The plants were placed into 5









groups so that each group contained 7 plants. Each group was treated with a separate mixture of

the pesticide.

Pesticide was applied to the plants so that each plant was treated until the leaves appeared

to be coated with the pesticide. The applicator wand was held approximately 0.3 m away from

the plant leaves while three initial vertical swaths were made. The wand was then placed within

the foliage and three vertical swaths were slowly made from the bottom to the top of the plant.

This process was repeated 7 additional times around the circumference of the plant. Plants were

allowed to dry before being returned to the plant holding area.

Petri Dish Preparation

Leaves were picked from each plant 4 hours post-treatment and once a week thereafter for

6 weeks post-treatment and placed into paper envelopes. The envelopes were placed into the

freezer because adequate numbers of gravid mosquitoes were not available on the day of initial

treatment. Leaves were removed from the freezer 24 hours prior to the day on which the

bioassays were performed and allowed to completely thaw. Leaves were placed onto prepared

100 x 15 mm petri dishes [Fisher Scientific Worldwide, Hampton, New Hampshire]. To prevent

cross-contamination with the pesticide, disposable latex gloves were worn during the collection

of control leaves and gloves were changed prior to handling treated leaves. Petri dishes were

prepared by covering the inside of the bottom dish with double-sided, 48 mm wide indoor /

outdoor carpet tape [Henkel Consumer Adhesives, Inc., Avon, Ohio]. Leaves were placed on the

taped dish one at a time so that the top of the leaf was exposed until the entire surface of the dish

was covered. If there were small gaps between the leaves a sterile cotton ball was brush over the

exposed tape so that no adhesive was exposed

Leaves were placed one at a time so that the upper surface of the leaf was exposed until the

entire surface of the dish was covered. Small gaps of exposed tape were eliminated by swiping a









sterile cotton ball over the exposed tape so that fibers attached to the exposed adhesive. A rubber

band was placed on the outside of the dishes securing them together and they were stored on

their sides in airtight containers. The containers were lined with damp paper towels to ensure that

adequate moisture levels (relative humidity between 70 85 %) were maintained for mosquito

survival.

Mosquitoes

Ten to twelve day old adult female, male, gravid and blood-fed Ae. albopictus mosquitoes

were obtained from the USDA, ARS, CMAVE rearing laboratory colony that was established in

1992 (Hoel 2005). Larvae were fed a 3:2 mixture of beef liver powder to yeast. Adults were

provided 10% sucrose. The colony was kept under 14:10 (L:D) photoperiod at room temperature

(27C to 320C).

The four types ofAe. albopictus in these bioassays were 8-12 day old males, females,

gravid female, and blood-fed females. Female mosquitoes were offered a blood meal once

mating was observed, usually on day 5 to 7 post-emergence and moved into a separate cage by

aspiration. Defibrinated bovine blood was placed in a membrane, heated in a warm water bath at

40C for 5 to 10 minutes and placed on top of the cage. The membrane was reheated in the warm

water one-hour later for an additional 5 to 10 minutes then replaced on the cage. Blood engorged

females were allowed to rest in the cage 3 5 days until eggs developed. They were not provided

with an oviposition substrate. Mosquitoes for the blood-fed bioassays were offered a blood meal

the morning of the day of the test. Non-blood-fed females and males were held in the colony

until the test date.

Adults were aspirated with a mechanical aspirator and chilled at 1C for approximately 5

minutes on a chill table until they were no longer active. Ten mosquitoes were placed into the

prepared petri dishes and handled as described previously. Knock down was checked at 1 hour









and 24 hours post introduction into the petri dish. Mosquitoes were considered alive if they were

able to fly within the dish. Mosquitoes were rated as knocked down if they were unable to fly

within the dish.

Statistical Analyses

All statistical analyses were performed using the Statistical Analysis System (SAS Institute

2004). Treatment data were corrected for control mortality by Abbott's formula (Abbott 1925)

and transformed using arcsine. After arcsine transformation, data from the 1 hour, 4 hour and 24

hours post-introduction observations were analyzed using repeated measures analysis of variance

(PROC GLM, SAS Institute 2004). Differences between mosquito types were detected by

ANOVA with means separation between gender and physiological states located using

Bonferroni-Dunn t-test analysis (a = 0.05).

Results

Laboratory Bioassay Results

One-hour Exposure of Ae. albopictus

Knock down of mosquitoes was observed at one-hour of exposure on days 0, 7, 14 and 21

and these data are presented in Table 4-1. Knock down was not observed on days 28, 35 and 42

and these data were not presented. Repeated measures analysis performed using PROC GLM

(SAS Institute, Cary, NC) found significant differences between week effects (F = 804.25; df=

2; P < 0.0001) and between the mosquito groups tested (F = 506.93; df = 3, P < 0.0001).

Significant differences were observed between the physiological states and genders tested

(F = 6.40; df = 45; P = 0.0003). A significant reduction in the knock down of males was

observed between days 0 and 7 (t = 12.70; df = 45; P < 0.0001), and again between days 7 and

14 (t = 9.63; df = 45; P < 0.0001) with no difference in knock down observed between days 14

and 21. For female sugar-fed mosquitoes, knock down significantly decreased between days 0









and 7 (t = 32.68; df = 45; P < 0.0001) and again between days 7 and 14 (t = 2.56; df = 45; P =

0.013), however, no differences were observed between days 14 and 21. Significantly more

female gravid mosquitoes were knocked down on day 0 than on day 7 (t = 14.64; df = 30; P <

0.0001) and similarly between days 7 and 14 (t = 12.07; df = 45; P < 0.0001) with no observed

differences between days 14 and 21. A significant decrease in the knock down among blood-fed

females was observed between days 0 and 7 (t = 38.35; df = 45; P < 0.0001) and also between

days 14 and 21 (t= 64.59; df = 45; P < 0.0001).

Four-hour Exposure of Ae. albopictus

Observations of knock down were made at 4 hours on days 7, 14, 21 and 28 and these data

are presented in Table 4-2. There was no knock down observed on days 35 and 42 and these data

were not presented. Repeated measures analysis performed using PROC GLM (SAS Institute

2004) found significant differences between week effects (F = 625.11; df = 2; P < 0.0001) and

between the mosquito groups tested (F = 38.22; df = 3, P < 0.0001).

Overall differences between physiological states and gender groups tested were observed

(F = 23.31; df = 3, P < 0.0001). There were no differences observed in knock down among male

sugar-fed mosquitoes between days 0 and 7 or between days 21 and 28, however, significant

differences were observed between days 7 and 14 (t = 15.81; df = 45; P < 0.0001). Knock down

of females was similar at days 0 and 7 with differences observed in the female sugar-fed

mosquitoes between days 7 and 14 (t =49.17; df = 45; P < 0.0001) and no decreases were

observed between days 14 and 21 or between days 21 and 28. There was a significant difference

in knock down between days 7 and 14 with the female gravid (t = 49.16; df = 45; P < 0.0001),

but no differences were observed between days 0 and 7 and between days 14 and 21. Knock

down of blood-fed females decreased significantly between days 7 and 14 (t = 45.59; df = 45; P

< 0.0001), with no further differences between or between days 14 and 28.









Twenty-four Hours Post Exposure ofAe. albopictus

Observations of knock down were made at 24 hours on days 0, 7, 14, 21, 28, 35 and 42 and

these data are presented in Table 4-3. Repeated measures analysis performed using PROC GLM

(SAS Institute, Cary, NC) found significant differences week effects (F = 1571.98; df = 2; P <

0.0001) and between the mosquito groups tested (F = 37.90; df = 3, P < 0.0001).

ANOVA analysis between the plants found significant differences between the plant types

(F = 37.05; df= 3; P < 0.0001). Differences in knock down were not observed in male, female,

gravid female, or blood-fed female bioassays between days 0, 7, and 14 and the percent knock

down ranged from 97.1 -100%. For sugar-fed males, there were significant differences in knock

down between days 28 and 35 (t = 12.70; df = 45; P < 0.0001), between days 28 and 35 (t =

4.88; df = 45; P < 0.0001), and between days 35 and 42 (t = 6.91; df = 45; P < 0.0001). For

sugar-fed females, significant differences in knock down were observed between days 21 and 28

(t = 22.63; df = 45; P < 0.0001), between days 28 and 35 (t = 8.11; df = 45; P < 0.0001) and

between days 35 and 42 (t = 2.37; df= 45; P = 0.02). Knock down of gravid females decreased

significantly differences from days 14 to 21 (t = 3.66; df = 45; P = 0.0003), from days 21 to 28 (t

= 24.28; df = 45; P < 0.0001), between days 28 to 35 (t = 9.63; df = 45; P < 0.0001) and between

days 35 and 42 (t = 7.28; df = 45; P < 0.0001). Knock down of blood fed females significant

decreased between days 14 and 21 (t= 2.60; df = 45; P = 0.01), days 21 and 28 (t= 29.41; df=

45; P < 0.0001), between days 28 and 35 (t= 10.99; df = 45; P < 0.0001) and between days 35

and 42 (t = 2.68; df = 45; P = 0.008).

Discussion

Mosquitoes in different physiological states have been shown to respond to pesticide

treatments differently (Hardaway and Barlow 1956). Bransy-Williams and Webley (1965)

applied DDT directly to blood-fed Culexpipiensfatigans Wiedemann and observed higher









tolerance to the pesticide than in blood-fed Anopheles gambiae Giles and Ae. aegypti. Halliday

and Feyreisen (1987) found that DDT tolerance increased at 24 hours in Cx. pipiens and

postulated that the increased pesticide tolerance was due to changes in detoxification pathways

activated during blood meal digestion. This increase in tolerance was not observed in Cx. pipiens

treated with cyclodiene and carbamate pesticides.

The above-mentioned studies examined at the effects of pesticide applied directly to

mosquitoes, but how are mosquitoes of different physiological states affected by contact with

pesticides? It is possible that applying the pesticide directly to the insect allows the insect to

process the pesticide easier since it is combined with water. The hydrophillic portions of the

pesticide are combined with water when applied to the insect and perhaps are readily

metabolized due to the pesticide's associations with the water. In the case of these bioassays, the

pesticide was allowed to dry leaving the pesticide able to bond with the insects organs or

hemolymph once the pesticide transfer from the substrate to the insect occurs.

Blood-fed mosquitoes in this study had a slightly higher overall percent knock down.

Previous studies showed higher tolerance to pesticides in blood-fed mosquitoes for 48 hours

post-bloodmeal ingestion at which point the LD50 returned to levels shown in sugar-fed females

(Bransby-Williams and Webley 1965, Halliday and Feyreisen 1987). These conflicting findings

may be due to differences in tolerance levels of the species studied, however, another possibility

may be the differences in experimental design.

Similar studies, examining at the efficacy of residual pesticides, where the insect was

placed in contact with the treated substrate for a period of time and then moved to a holding area

have been conducted (Geden et al. 1992). Exposing the mosquitoes for a period of time mimics

the transient resting behavior of insects and allows for the possibility of recovery from the









pesticide exposure. It is possible that the mosquitoes could recover from the knock down

observed at days 0 and days 7 at the one-hour point negating the effectiveness of the barrier.

Also, performing additional time checks during the first hour of exposure may elicit additional

significant differences between the treatments. Further study of how physiological state impacts

the efficacy of residual pesticide treatments should be undertaken to improve the efficacy of this

control methodology.









Table 4-1 Mean percent knock down (SE) ofAe. albopictus following 1 hour exposure to
TalstarOne treated Rhododendron simsii leaves (a.i. bifenthrin 7.9%).

Days Post-treatment
State 0 7 14 21
Male 100.0 (0.0) a 72.1 (15.3) b 7.1 (8.1)c 5.4 (7.2) c
Female 100.0 (0.0) a 66.3 (11.0) b 4.2 (7.2) c 1.7 (3.8) c
Gravid 100.0 (0.0) a 57.1 (14.6) b 1.7(4.8)c 0.0 (0.0) c
Blooded 100.0 (0.0) a 58.3 (19.5) b 2.9 (7.5) c 1.7 (4.8) c


Knock down was not observed on days 28, 35 and 42 post-treatment for any group and data are
not presented. Control mortality ranged from 0% to 1.2% for all treatments, percent control was
calculated using Abbott's formula (Abbott 1925) correction was used to maintain consistency
with the observations. Pesticide applied with B & G hand pump compressor sprayer at the rate of
10 ml to 3.8 L of water. Means within each row followed by the same letter are not significantly
different (Bonferroni-Dunn test P = 0.05).












Table 4-2 Mean percent knock down (SE) ofAe. albopictus following 4 hours exposure to TalstarOne treated Rhododendron simsii
leaves (a.i. bifenthrin 7.9%).

Days Post-treatment
State 0 7 14 21 28
Male 100.0 (0.0) a 100.0 (0.0) a 60.0 (13.2) b 10.0 (0.0) b 0.4 (2.0) b
Female 100.0 (0.0) a 100.0 (0.0) a 23.8 (2.9) b 0.0 (0.0) b 0.0 (0.0) b
Gravid 100.0 (0.0) a 100.0 (0.0) a 24.42 (3.7)b 0.0 (0.0) b 0.0 (0.0) b
Bloodfed 100.0 (0.0) a 100.0 (0.0) a 32.08 (11.0)b 0.0 (0.0) b 0.0 (0.0) b

Knock down was not observed on days 28, 35 and 42 post-treatment for any group and data are not presented. Control mortality
ranged from 0% to 1.2% for all treatments, percent control was calculated using Abbott's formula (Abbott 1925) correction was used
to maintain consistency with the observations. Pesticide applied with B & G hand pump compressor sprayer at the rate of 10 ml to 3.8
L of water. Means within each row followed by the same letter are not significantly different (Bonferroni-Dunn test P = 0.05).










Table 4-3 Mean percent knock down (SE) ofAe. albopictus following 24 hours exposure to TalstarOne treated Rhododendron simsii
leaves (a.i. bifenthrin 7.9%)

Days Post-treatment
State 0 7 14 21 28 35 42
Male 100.0 (0.0) a 100.0 (0.0) a 100.0 (0.0) a 98.3 (3.8) a 89.6 (9.1) a 82.9 (13.0) b 79.6 ( 2.2) c
Female 100.0 (0.0) a 100.0 (0.0) a 97.1 (5.5) a 95.4 (7.8) a 83.3 (10.1) b 72.5 (16.5) c 58.3 (13.1) d
Gravid 100.0 (0.0) a 100.0 (0.0) a 99.2 (2.8) a 94.6 (9.3) b 73.3 (12.7) c 53.3 (14.9) d 45.4 ( 6.6) e
Bloodfed 100.0 (0.0) a 100.0 (0.0) a 98.7 (4.5) a 96.6 (5.0) b 84.1 (16.9) c 62.1 (26.2) d 56.3 (20.2) e

Knock down was not observed on days 28, 35 and 42 post-treatment for any group and data are not presented. Control mortality
ranged from 0% to 1.2% for all treatments, percent control was calculated using Abbott's formula (Abbott 1925) correction was used
to maintain consistency with the observations. Pesticide applied with B & G hand pump compressor sprayer at the rate of 10 ml to 3.8
L of water. Means within each row followed by the same letter are not significantly different (Bonferroni-Dunn test P = 0.05).









CHAPTER 5
AREAS FOR FURTHER STUDY AND CONCLUSIONS

Evaluating a mosquito control program in the field requires key elements to create useful

comparisons of treatments. The most important questions for mosquito control treatments are:

which mosquito species is the program targeting, what is the best control program to employ,

and how will the program be evaluated? Once this information has been established, the targeted

mosquito's unique behavior and ecology must be integrated into the management program.

Areas for Further Study

Field Studies

Rigid protocols must be established prior to site selection. In the case of the evaluation of

barrier sprays, the homes should have a defined barrier of continuous vegetation surrounding an

area large enough to allow multiple landing rate count sites. Ideally, the vegetation barrier should

be a single plant species. In addition, the homeowners should be willing to be assigned any

treatment randomly and treatments should be rotated during the study. There must be a

measurable mosquito population existing prior to the study commencing.

It would be interesting to compare residual vegetation sprays at homes with and without a

continuous vegetation border. It is possible that this vegetation treatment technique might be

effective only if there is a true, continuous vegetation barrier. In instances where a continuous

border is absent, additional tools to provide relief from host-seeking mosquitoes will be needed.

Studying the plant:pesticide interaction pesticide is critical in evaluating pesticide

candidates for this treatment. Plants should be evaluated to see if the mosquito will rest on the

plant. Observations of the physical qualities of plants where mosquitoes are found resting should

be taken to see if there are any general qualities to the plants., such as amount of leaves or air









flow through the plant's branches. Also, what role if any does the leaf type play in creating a

mosquito "friendly" plant are the leaves broad, narrow, or does it even matter?

Sampling plants for resting adult mosquitoes should also be evaluated. Conducting

aspirations when the mosquitoes are actually resting in the vegetation is critical. Mosquitoes

often exhibit varied behaviors throughout the day, such as mate seeking, host seeking, and

resting (Hassan et al. 1996). Coordinating aspirations with resting periods in the day should

provide accurate estimations of mosquitoes resting within the plants versus aspirations made

during foraging times.

Careful attention needs to be paid to the aspirator itself. The suction cannot be so strong

that it destroys the insect but must be strong enough to capture the insect. The aspirator should be

easy to carry. It should also be powered adequately by rechargeable, light-weight batteries so that

back up batteries can be brought into the field. An aspirator that is easy to use and carry help

ensure that inaccuracies with aspirations are not due to user fatigue.

Methods used to monitor other insects may prove to be useful in monitoring plants for

mosquitoes. Sticky traps are used to monitor other insects including house flies (Steiner 1999,

Sulaiman et al. 1987) and thrips. This technique, with modifications, may prove useful in

evaluating the mosquitoes resting in plants. An important modification includes hanging the

sticky paper horizontally thereby mimicking leaves. This approach utilizes the mosquito

behavior whereby they rest on the under sides of leaves (personal observations). The colors of

the paper could also be selected to increase attractiveness. Another possible collection method

would be to place small resting boxes within the vegetation. Resting boxes have been shown to

be successful in collecting Anopheline species and may be useful in collecting resting Aedes

albopictus (Skuse) (Harbison et al. 2006).









Further study of barrier sprays against the salt marsh mosquitoes Ochlerotatus

taeniorynchus Wiedemann and Ochlerotatus sollicitans Walker could be successful. Barrier

sprays would be an ideal way to treat for these mosquito species because spraying pesticides near

bodies of water is not permissible. However, spot treatment has very little drift and would be

ideal in this circumstance. Also, the synchronous emergence of Oc. taeniorhynchus and Oc.

solicitans can be predicted based on tide levels allowing for treatment to be preemptively applied

prior to the emergence of the mosquitoes. Newly emerged mosquitoes are believed to rest in

vegetation prior to host-seeking and this would be an ideal time to use a residual spray on the

vegetation.

Barrier treatments may also be useful as a means to reduce arbovirus transmission.

During times of drought mosquito and arbovirus reservoir interactions are increased due to

population concentrations near water sources. Therefore, it may be useful to treat vegetation and

areas surrounding water sources to reduce mosquito / animal reservoir interactions.

Applying pesticides to different plant types may also require specific protocols due to the

varied types and arrangements of leaves. For example, tall grass plants are difficult to spray and

to visually verify that the blades of the plant have been sufficiently treated. However, plants with

horizontal leaves can be treated more thoroughly, because the applicators typically aim the mist

downward. Extremely leafy plants also require care that all of the leaves are coated with

pesticide by ensuring that the wand of the applicator is inserted within the branches of the plant.

Gaps in the treatment could compromise the treatment by providing safe harbors of pesticide free

leaves. Pesticide application methods could be compared to see which is most appropriate for

each plant type.









Multiple traps have been used as barriers to combat mosquitoes and other pests. The use of

traps to remove mosquitoes raises some interesting questions:

Is one trap sufficient to protect a yard?

Does one trap lure mosquitoes from the surrounding area and create a larger problem?

How far are these mosquitoes traveling to get to the trap?

From where are these mosquitoes traveling to the trap?

Are the trap captures an accurate measure of mosquitoes being removed from that

immediate yard?

Have the mosquitoes traveled a long distance to the trap?

In what physiological state are these mosquitoes in?

Are captured females host-seeking females?

Are captured females seeking mates?

If they are host seeking females, is it likely that they will rest in the vegetation on their

way to the trap or will they fly right through the vegetation barrier to find a pesticide free

resting area?

Additional aspirations on the trap itself may also prove useful. The trap stand is made of

hollow tube and I observed mosquitoes flying out of the tube once while making repairs on the

trap. Aspirating the tube may be another option for monitoring mosquito behavior around the

trap.

In surveying the homeowners from the field study (Appendix A), none of them were

willing to pay more than $150.00 for a mosquito control program. The methods utilized in this

study far exceeded this amount. The cost of having Florida Pest Control perform this treatment

from spring to fall would cost approximately $600.00. The traps, which have been discontinued









by Coleman, can be purchased for approximately $350.00. In addition, the propane tanks must be

changed every 14-18 days. Each tank costs approximately $20.00 to refill. Also, the lures cost

around $9.00 and must be replaced monthly. Operation of the trap during the first season would

cost approximately $650.00, however, this cost would be reduced substantially the following

season since the trap can be reused.

Also, homeowners may not be willing to use pesticides for mosquito control around their

homes. One homeowner wrote in on her survey that "I try to avoid pesticides due to having pets

and as a general principle. I would be inclined to use a trap however if it would get rid of the

mosquitoes." Another homeowner wrote about pesticides, "it must be environmentally safe -

would prefer not to have to spray chemicals" but was willing to spray "DEET type of product

when outside" presumably as a personal repellant.

Educating the public about the small amounts of pesticide that are applied may or may

not encourage homeowners to pursue pesticide treatments for mosquitoes. Robert Krieger (2005)

reviews some common misconceptions held by the public about pesticides. He cites

misinformation from media outlets as a major source of misconceptions. Overuse of pesticides,

such as DDT, led to resistance and its subsequent banning in the United States by the EPA

(1972). Despite the discontinued use of DDT in the United States, the WHO (2006) has

reevaluated its stance on the use of DDT as an indoor residual spray. The WHO now

recommends the use of DDT as an indoor residual spray in malaria endemic regions in addition

to the use of insecticide treated bed nets and the use of anti-malarial medications.

The goal of bioassays is to replicate what is seen in the field in a more controlled

environment. If the mosquitoes are resting on the undersides of the leaves, does it make a

difference if the tops of the leaf are used in this bioassay method? Comparisons of bioassays with









the tops and bottoms of the leaves exposed should be conducted. The most important indication

that this could be a factor is that mosquitoes tend to move under the leaves if they are not

securely attached to the bottom. Results of bioassays with fabrics not attached to the petri dish,

documented that the mosquitoes wedged themselves under the fabric to the point that they would

detach their legs.

Another method to consider for running this type of bioassays would be to rest the leaves

in tubes as used by Trout (2006). This may be more effective for several reasons. Using tubes

would allow the mosquitoes to exhibit their natural behavior of going to the underside of the leaf.

Preparing the petri dishes was a time consuming process. Attaching double side tape to the

bottoms so that the entire surface is covered with tape requires several steps. First the tape must

be attached to one side of the tape to a plexiglass surface, cutting around a circular template with

a razor, then the tape is pulled off of the plexiglass and carefully attached the tape to the bottom

of the dish. It is important to do this carefully so that no bubbles appear and create an uneven

surface to attach the leaves. The leaves must also be attached carefully so that they will remain in

place for 24 hours. Attaching the leaves in a way that ensures that the leaves are covering the

entire surface of the taped dish takes between can also be time consuming depending on the size

and shape of the leaves.

The tape also introduces the possibility of unnecessary mortality due to insect sticking to

exposed tape. Swiping the surface of the leaves with a cotton ball can also take time to ensure

that excess cotton is not protruding from the surface. The high number of replications in this type

of experiment would benefit from the time saved by simply placing leaves in tubes verses the

many stepped process of preparing the Petri dishes. Keeping bioassay methods simple would

reduces the possibility of operator error and allow for more accurate measures.









Conclusions

Refining and investigating commercially available mosquito treatments is necessary to

ensure that these techniques are useful in decreasing mosquito populations around the home.

Improving these methods will also create relief for homeowners and allow them to enjoy their

yards and homes during periods of mosquito activity. In addition to reducing nuisance mosquito

problems, these techniques may also be useful in the prevention of arbovirus transmission.









APPENDIX A
HOMEOWNER SURVEY

A preliminary survey was taken to understand the areas of use in each yard and the

homeowners general attitude towards mosquito control and mosquito activity. The homeowners

were asked six questions. Each question had answers provided and allowed for additional

comments.

Question 1: What activities do you do around your home?

Provided with the following options:

a. Gardening how many hours?

b. Grilling- how many hours?

c. Yard/home/car maintenance- how many hours?

d. Playing sports- how many hours?

e. Other- how many hours?

Site 1: 10 hours per week gardening, 1 hour other yard maintenance

Site 2: 7 hours per week gardening, 4 hours other yard maintenance

Site 3: 1 hour gardening, 1 hour grilling, 1 hour other yard maintenance

Site 4: 10 hours per week gardening, 2 hours other yard maintenance

Site 5: 2 hours per week gardening, 10 hours other yard maintenance

Site 6:15 hours per week gardening, 3 hours per week grilling, 1 hour other yard

maintenance

Site 7: 4 hours per week gardening, 4 hours per week grilling, 3 hours other yard

maintenance

Site 8: none

Question 2: How many mosquito bites would it take for you to end your outdoor activity?









Provided with the following options:

a. 1-4

b. 5-10

c. I would not change my activity level based on mosquito bites

Site 1: 5-10 mosquito bites

Site 2: I would not change my activity level based on mosquito bites

Site 3: I would not change my activity level based on mosquito bites

Site 4: 5-10 mosquito bites (Responder wrote in "I may not stay outdoors as long but I'd

never give up doing gardening.")

Site 5: I would not change my activity level based on mosquito bites

Site 6: 5-10 mosquito bites

Site 7: 1-4 mosquito bites

Site 8: 5-10 mosquito bites

Question 3: How many more hours per week would you spend outdoors if there were fewer

mosquitoes?

Site 1: zero

Site 2: blank

Site 3: 2-4 hours

Site 4: "The heat and humidity drives me in over the mosquitoes."

Site 5: zero

Site 6: 20%

Site 7: 2-4 hours

Site 8: 3-5 hours









Question 4: If you find that mosquito activity is unusually high would you?

Provided with the following options:

a. Call the local mosquito control agency for service

b. Hire a private company

c. Purchase pesticide and apply yourself

d. Purchase a trapping device

e. Reduce breeding sources in your yard

f. Stay indoors more

Site 1: Call the local mosquito control agency for service, "Use Off on arms and legs"

Site 2: Reduce breeding sources in yard, "Use more DEET type of product when outside"

Site 3: Stay indoors more

Site 4: Reduce breeding sources in your yard, Stay indoors more

Site 5: Reduce breeding sources in your yard, Stay indoors more

Site 6: Stay indoors more

Site 7: Purchase pesticide and apply yourself, Stay indoors more

Site 8: Call the local mosquito control agency for service, Reduce breeding sources in

your yard, Stay indoors more

Question 5: Do you feel that mosquito activity this summer is:

Provided with the following options:

a. Worse than last year

b. The same as last year

c. Were not living in this home last year

Site 1: The same as last year









Site 2: Worse than last year

Site 3: Worse than last year

Site 4: The same as last year

Site 5: The same as last year

Site 6: The same as last year

Site 7: Worse than last year

Site 8:Were not living in this home last year

Question 6: How much money would you be willing to spend for an effective mosquito

reduction program around your home?

Provided with the following options:

a. $0.00

b. $1.00-50.00

c. $51.00- 150.00

d. $150.00 ormore

Site 1: $1.00 50.00

Site 2: $51.00 150.00 Additional comment: "A year, it must be environmentally safe -

would prefer not to have to spray chemicals

Site 3: $1.00 50.00 Additional comment: "I try to avoid pesticides due to having pets

and as a general principle. I would be inclined to use a trap however if it would get rid of

the mosquitoes."

Site 4: $51.00- 150.00

Site 5: $1.00 50.00

Site 6: $1.00 50.00









* Site 7: $51.00- 150.00

* Site 8: $1.00 50.00









APPENDIX B
PROPANE USAGE TRIAL

Three Mosquito Deleto 2500 traps were run to measure the amount of propane used daily

and to investigate possible sources of error in restarting the trap. Traps were set up without the

octenol lures and nets were not placed onto the collection area so that the fan mechanism could

be observed while it was running. The propane tanks were removed from the trap and weighed at

one hour intervals for the first eight hours. Following the first eight hours, the tanks were

weighed one time per day until the propane ran out of the tank.

Traps were started by holding the starter button for exactly one minute. After one minute

had passed the start button was pushed. If there was no ignition popping sound observed, the

tank was checked to ensure that the trap was securely fastened to the propane tank. The starter

button was held for another minute and the ignition button was pressed again. This process was

repeated, sometimes up to three times, until the ignition sound was heard. Following the ignition

sound, the starter button was depressed and the ignition button was pressed every ten seconds

until the fan was heard to be working. The starter button was held for one minute following the

fan turning on. The process was timed using the second hand on the observers wrist watch. The

process took between three and five minutes. The trap was not restarted during the trial unless

the fan was not running after the tank was weighed.

Traps ran for an average of 18.3 days. The average amount of propane used was 8.75 kg.

Trap one failed on three occasions, trap three failed on two occasions, and trap two did not have

any failures. The two traps experiencing failures ran for two additional days. Failures were

probably due to not securing the propane tank properly to the trap after the weighing (Table A-

1). The components of the trap and the propane tank are sometimes difficult to properly align

due to differences in the tanks and corresponding tank fasteners.









Failures of this nature could be due to the difficulty in ensuring that the trap is fastened

properly. The fan continues to run after the propane tank is detached and there is no way to alert

the user that the trap is not properly attached to the propane tank until the trap stops running. The

trap will fasten to the tank even if it is not properly aligned. In these instances, the attachment

feels like it is aligned and securely attached. Secure alignment can be difficult to achieve and the

directions that accompany the trap should have a note for users to ensure proper seal of the tank

when changing the propane tank. The instructions should also recommend that users check the

trap 20 minutes after changing the tank to ensure that the proper seal was attained in changing

the propane tank.









Table B-1. Amount of propane used and days of operation by 3 Mosquito Deleto 2500TM traps

Trap 1 Trap 2 Trap 3 Average
Start weight (kg) 16.9 17.1 17.6 17.2
End weight (kg) 8.4 8.2 8.7 8.5
Propane used (kg) 8.5 8.9 8.9 8.8
Days operating (kg) 19.0 17 19.0 18.3
Failures 3.0 0.0 2.0 1.7
Propane per day 0.5 0.5 0.5 0.5









LIST OF REFERENCES


Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J. Econ.
Entomol. 18:265-267.

Ali, F.A.F., A. El-Refai, A. Abdel-Rahman, and S.A. Abou-Donia. 1993. Residual
effectiveness of insecticide formulations against German and American cockroach
adults. Int. Pest. Control 35:70-74.

Amerasinghe, F.P., and T.S.B. Alagoda. 1984. Mosquito oviposition in bamboo traps, with
special reference to Aedes albopictus, Aedes novalbopictus and Amigeres subaltus. Insect
Sci. Applic. 5: 493-500.

Anderson, A.L., C.S. Apperson, and R. Knake. 1991. Effectiveness of mist-blower
applications of malathion and permethrin to foliage as barrier sprays for salt
marsh mosquitoes. J. Am. Mosq. Control Assoc. 7: 116-117.

Berry, R., S.R. Joseph, and G.S. Langford. 1965. The question of area mosquito repellency, pp.
190-193. In Proc 52nd Annual Meeting New Jersey Mosquito Extermination Association
24-27 March 1965, New Jersey.

Black, R.J. 2003. Selected Shrubs for North Florida, Circular 500. Institute of Food and
Agricultural Sciences, University of Florida, Gainesville, Florida.

Bransby-Williams, W.R. and C. Webley. 1965. The effects of age and feeding on the
susceptibility of adult female Anopheles gambiae, Aedes aegypti, and Culex pipiens
fatigans. Ann. Trop. Med. Parasitol. 59: 95-98.

Briegel, H., and S.E. Timmerman. 2001. Aedes albopictus (Diptera38: Culicidae)
Physiological aspects of development and reproduction. J. Med. Entomol. 38: 566-571.

Campbell, C.B. 2003. Evaluation of five mosquito traps and a horse for West Nile Vectors on a
north Florida equine facility. M.S. Thesis, University of Florida, Gainesville, Florida.

Centers for Disease Control. 2005. Information on Aedes albopictus
http://www.cdc.gov/ncidod/dvbid/arbor/albopic_new.htm accessed April 5, 2007.

Chowdhury, A.N.U., P.C. Jepson, P.E Howse, and M.G. Ford. 2001. Leaf surfaces and
the bioavailability of pesticide residues. Pest Manag. Sci. 57: 403-412.

Cilek, J.E., and C.F. Hallmon. 2006. Residual effectiveness of pyrethroid-treated foliage
against adult Aedes albopictus and Culex quinquefasciatus in screened field cages.
J. Am. Mosq. Control Assoc. 22: 725-731.

Clements, A.N. 1992. The biology of mosquitoes, Volume 2 sensory reception and behavior.
CABI Publishing, New York, New York.









Coleman Outdoor Company. 2006. Mosquito Deleto 2500TM Active System.
http://www.coleman.com/coleman/colemancom/detail.asp?productid=2920-100 access
April 30, 2007.

Darsie, R.F., and R.A. Ward. 2004. Identification and geographical distribution of the
mosquitoes of north America, North of Mexico. University Press of Florida,
Gainesville, FL.

Dieng, H., M. Boots, N. Tuno, Y. Tsuda, and M. Takagi. 2002. A laboratory and field
evaluation of Macrocyclops distinctus, Megacyclops viridis and Mesocyclops pehpeiensis
as control agents of the dengue vector Aedes albopictus in a peridomestic area in
Nagasaki, Japan. Med. Vet. Entomol. 16: 285-291.

Environmental Protection Agency. 1972. DDT ban takes effect.http://www.epa.gov/
35thanniversary/topics/ddt/01.htm accessed March 25, 2007.

Estrada-Franco, J.G., and B.C. Craig. 1995. Biology, disease, and control of
Aedes albopictus Technical Paper No. 42. Pan American Health Organization.

Fansiri, T., U. Thavara, A. Tawatsin, S. Krasaesub, and R. Sithiprasasna. 2006.
Laboratory and semi-field evaluation of Mosquito Dunks against Aedes aegypti and
Aedes albopictus larvae (Diptera: Culicidae). S.E. Asian J. Trop. Med. Pub. Health 37:
62-66.

Fay, R.W. and W.H. Prince. 1970. A modified visual trap for Aedes aegypti. Mosq.
News 30: 20-23.

Florida Coordinating Council on Mosquito Control 1998. Florida mosquito control: The
state of the mission as defined by mosquito controllers, regulators, and environmental
managers. University of Florida; Vero Beach, Florida.

(FDEP) Florida Department of Environmental Protection. 2004. Salt Marshes.
http://www.dep.state.fl.us/coastal/habitats/saltmarshes.htm accessed April 23, 2007.

Ford, M.G. and D.W. Salt. 1987. Behaviour of insecticide deposits and their transfer from
plant to insect surfaces, pp. 26-81. In H.J. Cottrell (ed.), Pesticides on plant surfaces. John
Wiley & Sons, United Kingdom.

Frick, T.B., and D.W. Tallamy. 1996. Density and diversity of nontarget insects
killed by suburban electric insect traps. Ent. News 107: 77-82.

Geden, C.J., D.A. Rutz, J.G. Scott, and S.J. Long. 1992. Susceptability of house flies
(Diptera: Muscidae) and five pupal parasitoids (Hymenoptera: Pteromalidae) to
abamectin and seven commercial insectides. J. Econ. Entomol. 85: 1241-1246.










Gerhardt, R.R., K.L. Gottfried, C.S. Apperson, B.S. Davis, P.C. Erwin, A.B. Smith,
N.A. Panella, E.E. Powell, and R.S. Nasci. 2001. First Isolation of La Crosse virus
from naturally infected Aedes albopictus. Emerg. Infect. Dis. 7: 807-811.

Gilman, E.F. 1999a. Callicarpa americana fact sheet FPS-90. Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, Florida.

Gilman, E.F. 1999b. Ilex cornuta 'Bordii Nana' fact sheet FPS-263. Institute of Food and
Agricultural Sciences, University of Florida, Gainesville, Florida.

Gilman, E.F. 1999c. Rhododendron simsii fact sheet FPS-507. Institute of Food and
Agricultural Sciences, University of Florida, Gainesville, Florida.

Gilman, E.F. 1999d. Rhododendron x 'fashion' fact sheet FPS-508. Institute of Food and
Agricultural Sciences, University of Florida, Gainesville, Florida.

Gilman, E.F. 1999e. Spartina bakerii fact sheet FPS-554. Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, Florida.

Gilman, E.F. and S.P. Brown. 2003. Florida guide to environmental landscaping. Circular 922
Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida.

Gilman, E.F. and D.G. Watson. 2006. Magnolia grandiflora: Southern magnolia ENH-530
Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida.

Gottfried, K.L., R.R. Gerhardt, R.S. Nasci, M.B. Crabtree, N. Karabatsos, K.L.
Burkhalter, B.S. Davis, N.A. Panella, and D.J. Paulsen. 2002. Temporal abundance,
survival rates, and arbovirus isolation of field-collected containers inhabiting mosquitoes
in eastern Tennesee. J. Am. Mosq. Control Assoc. 18: 164-172.

Gratz, N. 2004. Critical review of the vector status of Aedes albopictus. Med. Vet.
Entomol. 18:215-227.

Gubler, D.J. 2002. The global emergence / resurgence of arboviral diseases as public
health problems. Arch. Med. Res. 33: 330-342.

Gubler, D.J., and N.C. Bhattacharya. 1971. Observations on the reproductive history
of Aedes (Stegomyia) albopictus in the laboratory. Mosq. News 31 356-359.

Gubler, D.J., and N.C. Bhattacharya. 1972. Swarming and mating of Aedes (S.) albopictus
in nature. Mosq. News 32:219 223.

Hadaway, A.B. and F. Barlow. 1956. Effects of age, sex and feeding on the susceptibility of
mosquitoes to insecticides. Ann. Trop. Med. Parasitol. 50: 438-443.









Halliday, W.R., and R. Feyreisen. 1987. Why does DDT toxicity change after a blood meal in
adult female Culexpipiens? Pest. Bioch. Phys. 28: 172-181.

Harbison, J.E., E.M. Mathenge, G.O. Misiani, W.R. Mukabana, and J.F. Day. 2006. A simple
method for sampling indoor resting malaria mosquitoes -Anopheles gambiae and
Anophelesfunestus (Diptera: Culicidae) in Africa. J. Med. Ent. 43: 473-479.

Hassan, A.A., C.R. Adanan, and W.A. Rahman. 1996. Patterns in Aedes albopictus
(Skuse) population density, host-seeking, and oviposition behavior in Penang,
Malaysia. J. Vect. Eco. 21: 17-21.

Hawley, W.A. 1988. The biology of Aedes albopicuts. J. Am. Mosq. Control Assoc. 4: 2-39.

Hoel, D.F. 2005. Response ofAedes albopictus (Diptera: Culicidae) to traps, attractants, and
adulticides in north central Florida. Ph.D. dissertation, University of Florida,
Gainesville, Florida.

Hoel, D.F., D.L. Kline, A. Grant, and S.A. Allan. 2007. Field evaluation of attractant-baited traps
for Aedes albopictus (Skuse). J. Am. Mosq. Control Assoc. 23: 11-17.

Holck, A.R., C.L. Meek, and J.C. Holck. 1988. Attractant enhanced ovitraps for the
surveillance of container breeding mosquitoes.J. Am. Mosq. Control Assoc. 4:97-98.

Holick, J., A. Kyle, W. Ferraro, R.R. Delaney, and M. Iwaseczko. 2002. Discovery of Aedes
albopictus infected with West Nile virus in southeaster Pennsylvania. J Am Mosq
Control 18:131.

Jensen, T., O.R. Willis, T. Fukuda, and D.R. Barnard. 1994. Comparison of Bi-Directional
Fay, Omni-Directional, CDC, and Duplex Cone traps for sampling adult Aedes albopictus
and Aedes aegypti in North Florida. J. Am. Mosq. Control Assoc. 10:74-78.

Kay, B.H., G. Prakash, and R.G. Andre. 1995. Aedes albopictus and other Aedes (Stegomyia)
species in Fiji. J. Am. Mosq. Control Assoc. 11:230-234.

Kanda, T., D. Bunnag, V. Deesin, T. Deesin, S. Leemingsawat, N. Komalamistra, K.
Thimasaran, and S. Sucharit. 1995. Integration of control measures for malaria vectors in
endemic areas of Thailand. S.E. Asian J. Trop. Med. Pub. Hlth. 26: 154-163.

Kerdpibule, V., T. Deesin,and S.Sucharit. 1978. Residual effectiveness of insecticidal deposits
on various wall surfaces. S.E. Asian J. Trop. Med. Pub. Hlth. 9: 423-426.

Kirkwood, R.C. 1987. Uptake and movement of herbicides from plant surfaces and the
effects of formulation and environment upon them, pp. 1-25. In H.J. Cottrell (ed.),
Pesticides on Plant Surfaces. John Wiley & Sons, United Kingdom.









Kline, D.L. 2002. Evaluation of various models of propane-powered mosquito traps. J.
Vect. Ecol. 27: 1-7.

Kline, D.L., G.F. Lemire. 1998. Evaluation of attractant-baited traps/targets for
mosquito management on Key Island, Florida, USA. J. Vect. Ecol. 23: 171-185.

Krieger, R. 2005. Reviewing some origins of pesticide perceptions. Outlooks on Pest
Management. 12: 244-248.

Konishi, E. 1989. Culex tritaeniorhynchus and Aedes albopictus (Diptera: Culicidae) as natural
vectors of Dirofilaria immitis (Spirurida: Filariidae) in Miki City, Japan. J. Med. Ent.
26: 294-300.

Mitchell, C.J. 1991. Vector competence of North and South American strains of Aedes
albopictus for certain arboviruses: a review. J. Am. Mosq. Control Assoc. 7:446-451.

Mitchell, C.J., L.D. Haramis, N. Karabatsos, G.C. Smith, and V.J. Starwalt. 1998. Isolation
of La Crosse, Cache Valley, and Potsoi viruses from Aedes albopictus (Diptera:
Culicidae) collected at used-tires sites in Illinois during (1994-1995). J. Med. Entomol.
35: 573-577.

Mogi, M. 1982. Variation in oviposition, hatch rate and setal morphology in laboratory
strains of Aedes albopictus. Mosq. News 42: 196-200.

Mori, A. 1979. Effects of larval density and on some attributes of immature and adult
Aedes albopictus. Trop. Med. 21: 85-103.

Niebylski, M.L. and C.J. Craig. 1994. Dispersal and survival ofAedes albopictus at
a scrap tire yard in Missouri. J. Am. Mosq. Control Assoc. 10: 339-343.

O'Meara, G.F., A.D. Gettman, L.F. Evans, Jr., and G.A. Curtis. 1993. The spread of Aedes
albopictus in Florida. Am. Entomol. 39: 163-172.

Pena, C.J., G. Gonzalvez, and D.D. Chadee. 2004. A modified tire ovitrap for monitoring
Aedes albopictus in the field. J. Vect. Ecol. 29: 374-375.

Perich, M.J., M.A. Tidwell, S.E. Dobson, M.R. Sardelis, A. Zaglul, and D.C.
Williams. 1993. Barrier spraying to control the malaria vector Anopheles
albimanus: laboratory and field evaluation in the Dominican Republic. Med. Vet.
Entomol. 7: 363-368.

Richards, S.L., L. Ponnusamy, T.R. Unnasch, H.K. Hassan, and C.S. Apperson.
2006. Host-feeding patterns ofAedes albopictus (Diptera:Culicidae) in relation to
availability of human and domestic animals in suburban landscapes of central
North Carolina. J. Med. Entomol. 43: 543-551.









Sant'ana, A.L., R.A. Roque, and A.E. Eiras. 2006. Charecteristics of grass infusions as
oviposition attractants to Aedes (Stegomyia) (Diptera: Culicidae). J. Med. Entomol. 43:
214-220.

Sardelis, M.R., M.J. Turell, M.L. O'Guinn, R.G. Andre, and D.R. Roberts. 2002. Vector
competence fo three North American strains ofAedes albopictus for West Nile virus. J.
Am. Mosq. Control Assoc. 18:284-289.

Savage, H., M. Niebylski, G. Smith, C. Mitchell, and G.B. Craig. 1993. Host-feeding
patterns of Aedes albopictus (Diptera:Culicidae) at a temperate North American
site. J. Med. Entomol. 30: 27-33.

SAS Institute. 2004. SAS 9.1.3 Help and Documentation, Cary, NC.

Service, M.W. 1993. Mosquito Ecology Field Sampling Techniques, Second Edition. Chapman
& Hall.

Shirai, Y., H. Funada, K. Kamimura, T. Seki, and M. Morohashi. 2002. Landing sites on the
human body preferred by Aedes albopictus. J. Am. Mosq. Control Assoc. 18: 97-99.

Shone, S.M., P.N. Ferrao, C.R. Lesser, G.E. Glass, and D.E. Norris. 2003. Evaluation
of carbon dioxide and 1-octen-3-ol baited Centers for Disease Control Fay-Prince traps to
collect Aedes albopictus. J. Am. Mosq. Control Assoc. 19: 445-447.

Shroyer, D.A. 1986. Aedes albopictus and arboviruses: a concise review of the literature. J Am
Mosq Control Assoc 2: 424-428.

Skuse, F. A. A. 1896. The banded mosquito of Bengal. Indian Museum Notes Vol. 3: 20.

Sprenger, D., and T. Wuithiranyagool. 1986. The discovery and distribution of Aedes
albopictus in Harris County, Texas. J. Am. Mosq. Control Assoc. 2: 217-19.

Standfast H., I. Fanning, L. Maloney, D. Purdie, and M. Brown. 2003. Laboratory and field
evaluation of BISTAR 80SC as an effective insecticide harbourage treatment for the
biting midges (Culicoides) and mosquitoes infesting peridomestic situations in an urban
environment. Bull. MCAA Inc. 15: 19-33.

Steiner, M.Y., L.J. Spohr, I. Barchia, and S. Goodwin. 1999. Rapid estimation of numbers of
whiteflies (Hemiptera: Aleurodidae) and thrips (Thysanoptera: Thripidae) on sticky traps.
Aust. J. Entomol. 38: 367.

Sulaiman, S., H. Yunus, and R. Sohadi. 1987. Evaluation of some adhesives for collecting Musca
domestic and Chrysoma megacephala adults or mosquito larvae in sticky traps. Med.
Vet. Entomol. 1: 273-278.









Swanson, J., M. Lancaster, J. Anderson, M. Crandell, L. Harmis, P. Grimstad, and U.
Kitron. 2000. Overwintering and establishment ofAedes albopictus (Diptera: Culicidae)
in an urban La Crosse virus enzootic site in Illinois. J. Med. Entomol. 37: 454-460.

Thavara, U., M. Takagi, Y. Tsuda, and Y. Wada. 1989. Preliminary field experiments on the
oviposition ofAedes albopictus in water with different qualities. Trop. Med. 31: 167-169.

Trexler, J.D., C.S. Apperson, and C. Schal. 1996. Diel oviposition patterns ofAedes
albopictus (Skuse) and Aedes triseriatus (Say) in the laboratory and the field. J. Vect.
Eco. 22: 64-70.

Trout, R.T. 2006. Suppressing peridomestic mosquitoes utilizing residual insecticides on
residential properties. M.S. thesis, University of Kentucky, Lexington.

Ware, G.W. 1989. The pesticide book, 3rd ed. Thomson Publications, Fresno, California.

World Health Organization. 2006. WHO gives indoor use of DDT a clean bill of health
for controlling malaria. Intl. Pest. Cont. 48: 314-315.

Xue, R. and D.R. Barnard. 2003. Boric acid bait kills adult mosquitoes (Diptera:
Culicidae). J. Econ. Entomol. 96: 1559-1562.

Yadav, R.S., H.C. Srivastava, T. Adak, N. Nanda, B.R. Thapar, C.S. Pant, M. Zaim, and
S.K. Subbarao. 2003. House-scale evaluation of bifenthrin indoor residual
spraying for malaria vector control in India. J. Med. Entomol. 40:58-63.









BIOGRAPHICAL SKETCH

Melissa Ann Doyle was born in Gloucester, Massachusetts to Madeline and Paul Doyle.

The family moved to San Diego, California where Melissa lived until graduating from San

Dieguito High School in Encinitas, California. She attended Northeastern University where she

changed majors nearly every quarter, rowed on the crew team, and worked at the Gillette

Company managing their Lotus Notes database system. She began a bachelors of science degree

in biology at the University of Massachusetts, Boston. She took a general entomology course in

her junior year and found that mosquitoes were fascinating creatures to work with and she

decided to pursue graduate education in entomology. She worked at the Massachusetts

Department of Health's West Nile virus surveillance program. After graduation, Melissa worked

as the biological technician at the Anastasia Mosquito Control District in St. Augustine from

until entering the University of Florida. After graduation she will continue her studies at the

University of Florida





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RESPONSE OF Aedes albopictus (SKUSE) (DIPTERA: CULICIDAE) TO RESIDUAL BIFENTHRIN ON PLANT LEAVES By MELISSA ANN DOYLE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007 1

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2007 Melissa Ann Doyle 2

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To my parents 3

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ACKNOWLEDGMENTS I would like to thank my advisor Dr. Daniel L. Kline for his guidance and support. I would also like to thank the other members of my committee, Dr. Sandra Allan and Dr. Phillip Kaufman, for their encouragement and their editoria l help. Special thanks go to the staff at the Center for Medical, Agriculture and Veterinary Entomology for their camaraderie and support, especially Erin Vrzal, Aaron Lloyd, Joyce Urba n, and David Hoel. I would like to thank Dr. Donald Hall, Dr. John Capinera, Debbie Hall and Jo sh Crews for all of their administrative help and the Department of Entomology for their fina ncial support. I would al so like to thank Dr. Ruide Xue, director of Anastasia Mosquito Co ntrol District. I would like to acknowledge the support of my fellow graduate students and friend s, especially Kendra Pesko, Dave Melius, and Eileen Enrique. I thank the homeowners who allowed me to treat and trap in their yards. I would especially like to thank my parents Madeline and Paul Doyle for their constant encouragement and support throughout my academic career. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 CHAPTER 1 LITERATURE REVIEW OF SURVEI LLANCE AND CONTROL TECHNIQUES USED TO CONTROL Aedes albopictus (Skuse)..................................................................10 Introduction to Aedes albopictus ............................................................................................10 Ecology of Aedes albopictus ..................................................................................................10 Surveillance Methods for Aedes albopictus ...........................................................................13 Control Methods for Aedes albopictus ...................................................................................15 Research Objectives ................................................................................................................18 2 Effect OF COMMERCIAL TRAPS AND PESTICIDE TREATMENT ON Aedes albopictus (Skuse) POPULATIONS IN SUBURBAN SETTINGS IN NORTH CENTRAL FLORIDA............................................................................................................19 Introduction .............................................................................................................................19 Materials and Methods ...........................................................................................................20 Results .....................................................................................................................................26 Discussion ...............................................................................................................................28 3 EFFECT OF PLANT SPECIES AND LEAF texture ON RESPONSE OF FEMALE Aedes albopictus (Skuse) TO PESTICIDE-TREATED LEAVES.........................................43 Introduction .............................................................................................................................43 Materials and Methods ...........................................................................................................44 Results .....................................................................................................................................49 Discussion ...............................................................................................................................50 4 EFFECT OF GENDER AND PHYSIOLOGICAL STATE OF Aedes Albopictus (Skuse) ON RESPONSE TO PESTICIDE TREATED LEAVES.........................................57 Introduction .............................................................................................................................57 Materials and Methods ...........................................................................................................58 Results .....................................................................................................................................61 Discussion ...............................................................................................................................63 5

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5 AREAS FOR FURTHER STUDY AND CONCLUSIONS..................................................69 Areas for Further Study ..........................................................................................................69 Conclusions .............................................................................................................................75 APPENDIX A HOMEOWNER SURVEY.....................................................................................................76 B PROPANE USAGE TRIAL...................................................................................................81 LIST OF REFERENCES ...............................................................................................................84 BIOGRAPHICAL SKETCH .........................................................................................................91 6

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LIST OF TABLES Table page 2-1 Composition of mosquito species collected from four yards by one Mosquito Deleto 2500 per yard from August 18, 2005 to October 7, 2005. .................................................34 2 Collections of mosquitoes from one Mosquito Deleto 2500 per yard placed in four yards from August 18, 2005 to October 7, 2005. ........................................................................35 2-3 Average (SE) of total mosquitoes captu red from one Mosquito Deleto 2500 per yard placed in four yards from August 18, 2005 to October 7, 2005. .......................................36 2-4 Species composition of mosquitoes obser ved during human landi ng rate counts in 8 yards participating in trap and pesticide tr ials in Gainesville, Florida from July 7, 2005 until October 7, 2005 ................................................................................................37 31 Mean percent control (SE) of Ae. albopictus following 1 hour exposure to TalstarOne treated leaves (a.i. bifenthrin 7.9%) to plant leaves on August 22, 2006 and October 17, 2006..............................................................................................................................54 3-2 Mean percent control (SE) of Ae. albopictus following 24 hours exposure to TalstarOne treated leaves (a.i. bifenthrin 7.9%) to plant leaves on August 22, 2006 and October 17, 2006..............................................................................................................................55 4-1 Mean percent knock down (SE) of Ae. albopictus following 1 hour exposure to TalstarOne treated Rhododendron simsii leaves (a.i. bifenthrin 7.9%). ............................66 7

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LIST OF FIGURES Figure page 2-1 Location of field sites in the A) Univ ersity area and B) Devils Millhopper area. .................37 2-2 The Mosquito Deleto 2500 (MD2500) propane-pow ered trap used as trap treatments in suburban yards in Gainesville, Florida. .............................................................................39 2-3 Average number (SE) of landing mosquitoes in the University area for each treatment from July 8, 2005 until October 7, 2005. ...........................................................................40 2-4 Average number (SE) of landing mosquito es in the Devils Millhopper Area for each treatment from July 8, 2005 until October 7, 2005. ...........................................................41 2-5 Average number (SE) of mosquitoes land ing during pre-treatment (N = 6 weeks) and post-treatment (N = 12 weeks) collection in residential yards from July 8, 2005 until October 7, 2005. .................................................................................................................42 8

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Abstract of Thesis Presen ted to the Graduate School of the University Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science RESPONSE OF Aedes albopictus (SKUSE) (DIPTERA: CULICIDAE) TO RESIDUAL BIFENTHRIN ON PLANT LEAVES By Melissa Ann Doyle August 2007 Chair: Daniel L. Kline Major Department: Entomology and Nematology The Asian tiger mosquito, Aedes albopictus (Skuse), was first introduced into the United States in Harris County, Texas in 1985. This specie s is known as an aggressive daytime biter. It has invaded 26 states, including Florida. Aedes albopictus larvae are found in tree holes, watercontaining plants, and man-made c ontainers, such as birdbaths. It is recommended that residents keep their yards free of water-holding containers to avoid this pest howev er, heavy infestations of Ae. albopictus may require additional control methods. The objective of my study was to evaluate commercially available c ontrol methods against Ae. albopictus in residences in Gainesville, Florid a. The control methods studied were the placement of a propane powered trap called th e Mosquito Deleto 2500, treatment with the pesticide TalstarOne (active ingr edient bifenthrin) by a pest control compa ny, the combination of the trap and pesticide treatment, and a control treatment where no treatments were applied. Further investigation into the efficacy of bifenthrin treatment on different plant leaf types against Ae. albopictus was evaluated. Plants commonly fou nd in Florida resi dential landscapes were chosen and treated with TalstarOne. The ef ficacy of bifenthrin-treated leaves was also evaluated against male, female, blood-fed, and gravid Ae. albopictus 9

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CHAPTER 1 LITERATURE REVIEW OF SURVEILLANCE AND CONTROL TECHNIQUES USED TO CONTROL Aedes albopictus (Skuse) Introduction to Aedes albopictus Aedes albopictus (Skuse) is a container breeding mo squito believed to be indigenous to southeast Asia (Estrada-Franco and Craig 1995). In the past 20 y ears this species has spread rapidly throughout the world and is a major nuisan ce pest in the United States. The mosquito is established in 26 states, mainly along the eas tern seaboard (CDC 2005). Defining control techniques for this species is warranted due to it s ability to rapidly spread to new areas and its capacity to vector arboviruses, such as dengue (Estrada-Franco and Craig 1995). This species is established in Asia, North Am erica, South America, Europe and in Africa (Gratz 2004, Gubler 2002). Aedes albopictus is an invasive species first introduced into the continental United States in Harris County, Texa s through the used tire trade in 1985 (Sprenger and Wuithiranyagool 1986). It has displaced other container breeding species such as Aedes aegypti Linnaeus (OMeara et al. 1993) Prior to the introduction of Ae. albopictus, the most common container breeding mosquito species in Florida was Ae. aegypti (OMeara et al. 1993). The directors of Florida mosquito control agencies believe that Ae. albopictus is responsible for many service requests made by residents, second only to salt-marsh mosquito complaints (Florida Coordinating Council on Mosquito Control 1998). Id entifying control and surveillance techniques for this pest will aid Fl orida residents and mosquito control personnel. Ecology of Aedes albopictus The Asian tiger mosquito, Ae. albopictus, was first described as Culex albopictus by F.A.A. Skuse (1894) from specimens trapped in Calcutta, India. Th is species is in the subgenus Stegomyia and the Scutelleris group (H awley 1988). This mosquito species is black with a 10

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distinctive, thin stripe of silv ery-white scales down the back of the thorax and a completely black clypeus (Darsie and Ward 2004, Hawley 1988). Eggs are laid singly on the sides of natural a nd artificial water contai ners just above the water line (Hawley 1988). Gubler and Bhattacharya (1971) found that colony mosquitoes laid an average of 58.9 eggs per gonotrophi c cycle with a range of total egg production of 0 to 784 eggs over the lifetime. Aedes albopictus have been found to oviposit in natural containers such as tree holes, bamboo, and water containing plants such as bromeliads (H awley 1988). Eggs can also be laid in water-holding artificial containers, such as discarded tires, potted plants, and bird baths (Kay et al. 1995). Hatching is stimulated by a combination of th e container flooding, the availability of food and the oxygen content of the water (Hawley 1988). Eggs often require several submersions prior to hatching (Hawley 1988). The amount of time required for egg hatch varies between populations, as observed between different fi eld populations and colony mosquitoes (Mogi 1982). Eggs from the urban populations hatched more readily in colony th an the eggs obtained from natural rural-occurring containers, howev er, both populations displa yed sporadic hatching. Larval development times vary based on te mperature, food availability, and crowding. Development often occurs between 5 and 10 da ys under field conditions (Hawley 1988). Briegel and Timmerman (2001) reared Ae. albopictus and found that larval development ranged from 7 days at 32 o C to 28 days at 12 o C. Food availability is also influenced development time. Mori (1979) found that larval development was length ened with lowered food levels and increased larval density. In addition, adults from these stressed conditions were sm aller and had decreased fecundity. 11

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Adults are often found resting in areas of dense vegetation (Estrada-Franco and Craig 1995, Hawley 1988). Males may spend time resting in leaf ground cover (personal observations in Florida). Males have been obs erved to exhibit swarming behavior near hosts and intercept the females as they approached the host (Gubler a nd Bhattacharya 1972). Host seeking occurs during the daylight hours and populations often exhibit two landing rate peaks. In Penang, Malaysia, human landing rate counts showed peaks just after sunrise and a few hours prior to sunset (Hassan et al. 1996). Shirai et al. (2002) found that the preferred landing site of Ae. albopictus on humans were primarily the feet followed by the hands and face. Oviposition activity in the laborat ory has a slight peak in the morning with the majority of oviposition occurring between noon and darkness (Trexler et al. 1996). Aedes albopictus is not considered to be a strong flier, often remaining within 100 meters of th e larval emergence site (Estrada-Franco and Craig 1995). However, Ae. albopictus dispersed up to 525 meters in a markrelease study in Potsoi, Missouri (Niebylski and Craig 1994). Aedes albopictus feeds on a variety of mammals a nd birds (Estrada-Franco and Craig 1995). Using ELISA, field captured Ae. albopictus in Potosi, Missouri were found to feed commonly on mammals such as rabbits, deer, an d dogs in addition to humans (Savage et al. 1993). Blood meal analysis performed on mosqu itoes collected in central North Carolina indicated that Ae. albopictus preferred human hosts, but would feed on domestic animals and in some cases birds (Richards et al. 2006). The blood feeding habits of Ae. albopictus should be taken into consideration given that it is a possi ble vector or bridge species for arboviruses. Vector competence of Aedes albopictus The anthropophilic and opportunistic nature of Ae. albopictus makes it a candidate to vector many pat hogens. Its vector competence inside and outside the laboratory has been reviewed extensively (Gratz 2004, Mitchell 1991, Shroyer 1986). 12

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The most common disease-causing agents associated with Ae. albopictus are the four serotypes of dengue virus (Estrada-Franco and Craig 1995). Aedes albopictus is considered to be the secondary vector in areas also inhabited by Ae. aegypti (Gratz 2004). Several viruses have been isolated from North American populations of Ae. albopictus including La Crosse virus (Gerha rdt et al. 2001), West Nile virus (Holick et al. 2002, Sardelis et al. 2002), and Eastern Equine Encephalitis virus (Mitchell 1991). Aedes albopictus is also capable of vectoring several parasites and pathogens of ve terinary importance including Dirofilaria immitis (Leidy) (Konishi 1989), Potsoi virus, a nd Cache Valley virus (Mitchell et al. 1998). Surveillance Methods for Aedes albopictus Population surveillance of Ae. albopictus can be accomplished using adult traps, larval sampling and human landing rate counts (Flori da Coordinating Council on Mosquito Control 1998). These methods are often combined in stud ies and in surveillance programs to provide accurate estimations of Ae. albopictus populations. Trapping methods Many different types of mosquito traps are avai lable to mosquito control organizations for monitoring adult and larval populations. Traps can consist of a simple box that attracts resting mosquitoes to a wide variety of electrically pow ered traps that use baits to lure and fans to capture mosquitoes (Service 1993). Light traps are a common technique in any surveillance operation. The New Jersey light trap has been used for many years to monitor mosquito populations, however, it requires a permanent light source and collects many non-target insects. Another commonly used trap for surveillance is the Centers for Disease Control (CDC) light trap. It is highly portable a nd uses batteries to power a fan and light source. It also accommodates a variety of attractants su ch as 1-octen-3-ol (octenol) or CO 2 (Service 1993). 13

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Traps can be modified to increase the number of mosquitoes captured and be more attractive to specific species through the use of attractants and visual modifications (Service 1993). The most commonly used attractants are car bon dioxide and octenol. The Fay-Prince trap is designed with contrasti ng panels of black and white to be more attractive to Ae. aegypti (Fay and Prince 1970). Additional st udies by Jensen et al. (1994) compared the numbers of Ae. aegypti and Ae. albopictus females captured by CDC light traps, a bi-directional Fay trap, an omni-directional trap (two Fay traps fastened together), and a duplex cone trap. The omnidirectional trap collected the most Ae. albopictus In addition, the numbers of Ae. aegypti and Ae. albopictus captured by each trap were significantly di fferent, suggesting that the two species are attracted to different visual cues. Oviposition traps are another tool used in monitoring the presence of female Ae. albopictus (Florida Coordinating Council on Mosquito Contro l 1998). These traps are containers that are placed in the field and examined for eggs. Traps ar e often small containers painted black, such as a cup (Gottfried et al. 2002) or glass jar (Holck et al. 1988), fi lled with water and have a wooden paddle or paper offered for oviposition (Service, 1993, Thavara et al. 1989). Additional types of oviposition traps include bamboo containers (A mersinghe and Alagoda 1984), modified tires (Pena et al. 2004), tin cans (S wanson et al. 2000) or plastic jars (Thavara et al. 1989). The water used in the oviposition traps may affect the attractiveness of the trap to female Ae. albopictus. In a field study conducted near a tire di sposal yard in Baton Rouge, Louisiana, Holck et al. (1988) reported that Ae. albopictus oviposited more in jars with a hay infusion than in jars containing either a leaf litter infusion, distilled water or a mixture of 1% fish oil and water. In addition to the wate r infusions used in the ovitrap, care should be taken in the 14

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placement of the ovitrap when targeting Ae. albopictus. In southern Brazil Santana et al. (2006) found that Ae. albopictus were more likely to lay eggs in traps surrounded by shrubbery. Human landing rate counts are another effec tive tool for monitoring pest mosquitoes, especially Ae. albopictus (Florida Coordinating Council on Mosquito Control 1998). Landing rate counts measure the number of mosquito es landing on a person during a period of time (Florida Coordinating Council on Mosquito Contro l 1998). It is recommended that the same person perform landing rate counts due to the di ffering attractiveness of people to mosquitoes. Shirai et al. (2002) reported that Ae. albopictus prefers to land near the feet. Control Methods for Aedes albopictus Managing mosquito populations is a comple x task often requiring the combination of several different techniques (F lorida Coordinating Council on Mosquito Control 1998). Various techniques can be chosen based the life stage that is targeted. Orga nized mosquito control involves the use of integrated pest management (IPM) practices to redu ce mosquito populations (Florida Coordinating Council on Mosquito Co ntrol 1998). An IPM program incorporates multiple methods such as habitat alteration, so urce reduction, other cultural controls,biological controls, and pesticides (Ware 1989). Larval populations of Ae. albopictus often can be reduced by removing water-holding containers, treating water containe rs with larvicides, or adding predacious organisms into the container. The use of Bacillus thuringiensis var. israeliensis ( Bti ) Berliner was evaluated under field conditions by Fansiri et al. (2006) and found to be effective in reducing larval populations. Dieng et al. (2002) evaluated the use of cyclopoid copepods as biological controls in the laboratory and found them to be most effective against Ae. albopictus during the early mosquito instars. 15

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Broad spectrum pesticides should be a last resort in any IP M program and can include the use of larvicides and adulti cides (Florida Coordinating C ouncil on Mosquito Control 1998). Techniques that target specific areas for pes ticide application, such as barrier pesticide applications, can reduce the amount of damage to non-targeted organisms, especially beneficial insects, by targeting specific areas (Anderson et al. 1991, Perich et al 1993, Cilek and Hallmon 2006). Relying on the use of residual pesticides in homes has been useful in combating mosquitoes that transmit malaria and that are found in the home (Kanda et al. 1995, Kerdipule et al. 1978, WHO 2006). Mosquitoes also rest in natural areas, such as vegetation and in ground cover, and applying residual pestic ides to these areas also shows promise in reducing mosquito populations (Service 1993). Early field studies of area mosquito repellen cy looked at the use of malathion and naled combined with a masking agent called Deodall an d found that human landi ng rate counts were reduced 51% (Berry et al. 1965). Barrier pesticide treatment may prove to be quite useful to mosquito control professionals in a variety of s ituations. These situations include treatment for people living near areas that ca nnot be treated, such as protecte d natural areas or chemically sensitive neighbors. Another important use co uld be during arboviral transmission peaks to protect people in high activity areas. Permethrin and malathion applied to foliage has been used as an effective barrier treatment against the salt marsh mosquitoes, Ochlerotatus sollicitans Walker and Ochlerotatus taeniorhynchus Wiedemann, in North Carolina (Anderson et al. 1991). Shrubs bordering a park area were treated with permethrin and malathi on. Areas treated with permethrin had reduced landing rate counts for 24 hours in comparison to non-treated areas. Landing rates were decreased for 48 hours in ma lathion-treated areas. 16

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Several pesticides were also effective as a barrier application agai nst the malaria vector Anopheles albimanus Wiedemann in the Dominican Republic (Perich et al. 1993). The foliage surrounding three different villages was treate d with permethrin, bendiocarb, and malathion. Landing rates and light trap captures of An. albimanus were reduced in permethrin, bendiocarb, and malathion treated villages in comparison to villages that did not receive treatment. The effectiveness of a barrier often depends on how long a pesticide remains on the surface of the foliage. Cilek and Hallm on (2006) treated wax myrtle, Myrica cerifera with three pesticides. Aqua Reslin (20% ac tive ingredient [AI] permethrin + 20% [AI] piperonyl butoxide), Permanone EC (10% [AI] permethrin), and Susp end SC(4.75% [AI] deltamethrin) applied at the maximum label rate. The residual effectiveness of each pesticide was evaluated in large screened cages by creating a foliage barrier surroundi ng Mosquito Magnet-Experimental traps. Aedes albopictus and Culex quinquefasciatus Say mosquitoes were releas ed on three contiguous days per week in the field cages. Bioassays using leav es from the treated plants were performed to evaluate the residual pesticid e on the leaves over time. De ltamethrin provided the largest reduction in mosquito trap captures (70% to 80% ) during the first 4 weeks. Leaf bioassays on deltamethrin treated plants provided 95% knockd own throughout the study. This treatment was also more effective in knocking down Ae. albopictus in comparison to Cx. quinquefasciatus. Two pyrethroid insecticides, bifenthrin and la mda-cyhalothrin, were found to significantly reduce mosquitoes in suburban Kentucky yards (Trout 2006). The treatments were found to be most effective immediately following the pesticide application. It is import ant to note that gravid mosquitoes were not reduced, suggesting that mos quitoes may have different susceptibilities to pesticides at different physiologi cal states or that th ey do not rest in the treated areas. Also, the treatement was more effective at reducing Aedes and Ochlerotatus species than in reducing 17

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Culex species. Further study of this type of pesticide application w ith other pesticides may show promise in controlling Ae. albopictus Research Objectives The establishment of Ae. albopictus in Florida has created new challenges for residents and mosquito control agencies. Since arriving in Florid a this species has quickly become a major pest (Florida Coordinating Council on Mosquito Contro l 1998) and current cont rol options for this pest should be evaluated. The goal of my project is to evaluate current Ae. albopictus control practices, especially the application of pesticides to residen tial plants and how the residual pesticide performs in knocking down Ae. albopictus 18

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CHAPTER 2 EFFECT OF COMMERCIAL TRAPS AND PESTICIDE TREATMENT ON Aedes albopictus (Skuse) POPULATIONS IN SUBURBAN SE TTINGS IN NORTH CENTRAL FLORIDA Introduction Organized mosquito control is available in 27 counties in Florida. Residents can also employ companies to apply additional mosquito c ontrol measures, including pesticide treatment. Use of pesticides by these companies requires specific training and licensing by the state of Florida (Florida Coordinating Council on Mosqui to Control 1998). In addition to hiring a company, residents may also purchase and apply a wide variety of mosquito control products. These options include personal re pellents, area repellents such as citronella-burning devices, over-the-counter pesticides applied to the ve getation, mosquito-repelling devices and trapping devices. As new products and services enter the marketplace it is necessary to evaluate their efficacy against targeted species. A wide variety of commercial traps are ava ilable for purchase by the homeowner to aid in curbing adult mosquito populations. Early designs used ultra-violet light ing and electrocution grids in attempts to attract and kill mosquitoes and other pest inse cts. These traps are inexpensive to operate, but require a permanent power sour ce and do not specifically target mosquitoes. Additionally, Frick and Tallamy ( 1996) found that the 99.78% of insect s attracted to this type of trap were non-biti ng aquatic insects. Current trap designs target pest insects, specifically mosquitoes. Traps powered by propane generate multiple attractants (CO 2 heat and water vapor) whos e combined effects with attractants, such as 1-octen-3-ol (octenol), specifically target mosquitoes and other blood feeding Diptera (Kline 2002). Propane is burned in these traps to both generate CO 2 and electricity to power the fans. The goal of this study was to assess the effectiveness of a representative 19

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commercially available trap and a pesticide treatment in reducing Aedes albopictus (Skuse) populations in suburban settings. Materials and Methods Field Trial Site Selection Eight homes in Gainesville, Florida were selected based on ho meowner reports of mosquito activity in their yards during the da ytime. The eight homes were assigned to two groups based on the homeowners willingness to have their property treated with pesticides and then randomly assigned a treatm ent within the two groupings. There were 4 treatments evaluated with 2 homes assigned to each treatment. Sites we re visited twice weekly and evaluated for the presence of Ae. albopictus using oviposition traps, backpack aspirations, human landing rate counts, and propane trap captures. The field trial was conducted in two different neighborhoods in Gainesville, Florida approximately 9 km apart (Figure 2-1). The firs t neighborhood was located near the University of Florida and the second area was located in the northwest section of Gainesville near the Devils Millhopper State Park. Aedes albopictus were present in all yards during initial landing rate counts conducted prior to the study. Add itionally, homeowners were also surveyed to evaluate their attitudes toward mosquito control. (Appendix A) University Area Neighborhood Residential areas located just north of the Univ ersity of Florida were lined with old growth trees including Quercus virginiana Miller (live oak) and Magnolia grandiflora Linnaeus (southern magnolia). Lot size s ranged from 1,200 to 1,700 m 2 The homes were constructed of concrete block and one had wooden siding. All yards were substantially shaded by large pine and hardwood trees. Leaf litter was also present near the foundations of the houses and yards had sparse to full lawns of Stenotaphrum secundatum Kuntze (St. Augustine grass). Homes were 20

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surrounded by mixed plantings of bushes including Rhododendron species Chamisso (azalea), Juniperus horizantalus Linnaeus (juniper), and Ilex cornuta Lindley (holly). Devils Millhopper Area Neighborhood The second neighborhood was located east of De vils Millhopper Geological State Park. The area is shaded heavily by live oaks and othe r oak trees. All homes were constructed of concrete block with wood siding. The yards in th is area are minimally landscaped and covered with leaf litter. Lot sizes ranged from 1,200 to 3,000 m 2 Bambusoideae (bamboo), Rhododendron species, and ferns were present on all four properties. On e yard had a full lawn of St. Augustine grass. One yard was extensively planted with Billbergia spp. Thunberg and Neoregelia (red fingernail) bromeliads. Treatments The treatments included: 1) placement of a comm ercial mosquito trap sold for residential mosquito control; 2) the single a pplication of a pesticide typically used for residential mosquito control applied by a local pest control company; 3) the combination of one trap and a pesticide treatment; and 4) a control where no reduction programs were implemented. All traps were placed in the yards and activated when the pesticide treatments were completed on August 18, 2005 and remained in the field for eight w eeks until October 7, 2005. Traps were positioned away from areas of human activity and in the shade. Traps The trap used for this study was representa tive of commercially available mosquito traps for residential mosquito control and was the Mosquito Deleto 2500 (MD2500) [Coleman Wichita, Kansas] propane-powered trap (Figur e 2-2). Traps were activated on August 18, 2005. The trap is approximately 0.9 meters tall and consists of a head unit mounted on metal stand. This type of trap uses Dynamic Air Flow technology (Coleman 2006). 21

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The metal tubing that attaches the head unit to the stand serves two important functions. It houses the commercial lure releasing octenol [Coleman Wichita, Kansas] and expels the attractants (CO 2 water vapor and octenol). The fan also dr aws in mosquitoes into a net that is mounted in a drawer below the fan unit. The drawer automatically closes as it is removed so that captured mosquitoes that have not died in the net do not escape. The mosquitoes captured in the net die due to dessication from the c onstant air flow across the net. The traps were placed in the back yard, approxi mately 6 9 m away from the back door of the home and away from areas of human activ ity. Nets were changed weekly and captured mosquitoes were stored in labeled paper containers, frozen and la ter identified to species using keys (Darsie and Ward 2004) a nd counted. The propane tanks were changed every 14 days. The octenol lures were changed every 30 days Two sites had one MD2500 placed in thei r yards (MDTRAP1 and MDTRAP2). The residence at MDTRAP1 was located in the Univer sity area and was rectangular. The back and side yards were shaded by large, live oak trees. It had a sparse St. A ugustine grass lawn. There was a hedge of holly plants approximately 30 cm away from the home in the front. A large magnolia tree (approximately 12 meters tall) was also in the front yard. The side yard had a camellia, Camelia species, near the gate for the back yard. The second residence (MDTRAP2) was located in the Devils Millhopper area. The entire property was extensively shaded with live oak tr ees and did not have a grass lawn. The ground of the lot was covered with leaf litter. The areas to the front a nd sides of the house were extensively planted with bromeliad plants, including Bilbergia spp. and Neoregelra spp. (red fingernail) bromeliads. In the back yard there were five fenced areas for the rehabilitation of injured turtles. 22

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Each pen had a small pond, however, no larvae were observed in the ponds over the course of the study. Pesticide Treatments Florida Pest Control Company (Gainesville, Fl orida) was hired to perform their Pro-Star Mosquito Reduction Program. Florida Pest Cont rol Company was selected because they have locations in 16 cities in north and central Florida and are representative of commercially available mosquito control for residences across the state. The treatment occurred on August 18, 2005. The program included an inspection of the property for mosquito breeding habitat and treatment with TalstarOne [FMC Corporation, Agricultural Pr oducts Group, Philadelphia, PA], with the active ingredient bifenthrin. The pestic ide was applied according to the label directions (0.33 fluid ounces per gallon of water, A.I. 7.9%) with a B & G hand pump compressor sprayer [B & G Equipment Company, Jackson, GA]. The four houses were sprayed in vertical swat hs ensuring that the entire outside of the residences were coated with the pesticide. Some swaths we re performed over the surrounding vegetation as the technician moved around the home however, it was not as systematic as the house treatment. If there was mixed pesticide remaining in the sprayer after the house was completely coated, the technician would appl y the remainder on the vegetation surrounding the residence. Pesticide-treated sites (TAL1 and TAL2) were shaded by large live oak trees. Both homes had sparse grass lawns and the lots were rectangular shaped. The yard of the home treated in the University Area contained many ornamental plants There was an extensiv e collection of potted orchids and cacti. Discarded planting pots of ten held water and were found with larvae developing. There were approximate ly 20 red fingernail bromeliad plants planted beneath an oak 23

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tree that were checked weekly for larvae. A bird bath that consistently contained larvae was also present in this yard. The lot of the pesticide-treated home in th e Devils Millhopper Area had a St. Augustine lawn in both the front and backyard. There were no ornamental plants in pots. Occasionally, discarded oil and coffee grounds containers could be found near the side door of the residence, however, no larvae were observed developing in these containers. Bamboo plants, Bambusoideae species, grew on the left side of the house and a low hedge of juniper bushes was located in the front of the residence. MD2500 and Pesticide Treatment Two homes had a MD2500 placed in the back ya rd and received the Pro-Star Mosquito Reduction Program. The traps and pesticide tr eatment were applied on August 18, 2005. Sites that were treated with the pesticide and had a trap placed on the property (TALTRP1 and TALTRP2) were also shaded by tall oak trees. The residence located in the University Ar ea (TALTRP1) had two l oblolly pines in the front yard, several azalea bushes in the back yard juniper shrubs along the front of the house and a groups of ferns in the side yard. TALTRP1 al so had a sparse lawn of St. Augustine grass. Occasionally, a wheel barrow would collect wa ter and developing larvae were observed. The yard of the home located in the Dev ils Millhopper area (TALTRP2) was covered in leaf litter. There were several bamboo plants a nd azalea bushes in the yard. No containers were observed to hold water through the course of the study at this residence. Control Treatments Two residences did not receive mosquito reduction treatments. Control sites (CON1 and CON2) were both shaded by large live oak trees, a nd did not have grass la wns. Both residences had leaf litter surrounding the foundations of the homes. Both homes also had a number of 24

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Rhododendron species bushes along the perimeter of th e property. The homes were both located on cul de sacs and had irregularly shaped lots with the houses placed in the center of the lot. Mosquito Sampling The presence of Ae. albopictus was evaluated using oviposition jars, backpack aspirations, and human landing rate counts. Four ovipositi on traps were placed around each yard on August 18, 2005 and were in the field until October 7, 2005. The oviposition trap consisted of 250 ml glass canning jars painted black and filled with approximately 125 ml of water. Seed germination paper (ca. 15 x 30 cm) was placed so that the striat ions of the paper were vertical inside the trap as an oviposition substrate. The papers were ch anged weekly. Water level within the jars was monitored and refilled if the level dropped dramatically. Backpack aspirations of the yards were performed beginning August 18, 2005 and weekly thereafter until October 7, 2005 with a modifi ed CDC Backpack Aspirator Model 1412 [John Hock, Gainesville, FL]. Aspirations were performed around the vegetation surrounding the foundations of the houses after landing rate coun ts were performed between 1:00 PM and 4:00 PM. The area surrounding vegetation near the residence was swept with the aspirator in a vertical manner. The mouth of the aspirator was placed 0.3 meters away from the base of plants and shrubs. The process took between 10 and 15 minu tes depending on the size of the home and the amount of surrounding vegetation. Co llection cups were labeled with house location, covered, and brought to the lab for identification. Mosquitoes were immediately identified. Landing rate counts were performed by the sa me observer in the same four locations around the yard twice per week between 1:00 PM and 4:00 PM from July 7, 2005 until October 7, 2005. Locations were in shaded areas that we re likely to be frequented by humans. The observer stood with the right calf exposed for three minutes. All landing mosquitoes were 25

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aspirated by a mouth aspirator, counted and iden tified at the end of the three minutes, and released. Statistical Analysis Mosquito monitoring results and trap captures were analyzed using Statistical Analysis Software (SAS Institute 2004). Trap capture data were analyzed by ANOVA with means separation by Bonferroni-Dunn test ( = 0.05). Landing rate counts a nd trap captures before and after treatment were compared using t-test anal ysis. Landing rate counts were also transformed (log(x+1)) and analyzed by PROC GLIMMIX to examine treatment and site effects on landing rates. Results Collections from MD2500 Mosquito traps constituted pa rt of the treatment program and reflect the composition of mosquito populations in the yard. A total of 7,625 mosquitoes were trapped from the four yards (Table 2-1). The eight species captured included Ae. albopictus Anopheles quadrimaculatus Say, Coquilletidia perturbans (Walker), Culex nigripalpus Theobold, Ochlerotatus atlanticus Dyar and Knab, Psorophora columbiae Dyar and Knab, and Wyeomyia mitchellii Theobold. Mosquitoes from the genus Mansonia were also captured but were unidentifiable to species due to loss of scales and desiccation. There were also 81 mosquitoes unidentifiable specimens. The most prevelant species in descending order were Mansonia species, Cx. nigripalpus, Ae. albopictus, Ps. ferox Ps. columbiae An. quadrimaculatus, unidentifiable specimens, Cq. perturbans Oc. atlanticus, and Wy. mitchellii (Table 2-1). Regardless of pesticide application, the composition of species in trap collections was similar at all sites over the trapping season and Ae. albopictus was present at all sites (Table 2-2). Aedes albopictus was the third or fourth most common species at all sites. 26

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Weekly averages of total trap captures for the yards including traps (MD25001 and MD25002) and pesticide treatment and traps (TA LTRP1 and TALTRP2) are included in Table 2-3. Comparison of means of the number of mosquitoes collecte d each week by analysis of variance showed significant differences between the sites (F = 3.30, df = 3, P = 0.03). Significantly more mosquitoes were captured in the trap at MD25002 than in at traps MD25001, TALTRP1, and TALTRP2 (ANOVA, = 0.05). Since there were significant differences between sites, data from the two sites were not combined for each treatment. There were four trap failures. The trap at MDTRAP2 failed on August 19, 2005. The trap at TALTRP1 failed on August 24, 2005 and on September 23, 2005 while the trap was not running at the time of collection, the propane tank must have just emptied because there were mosquitoes remaining in the net. The trap at TALTRP2 failed on September 29, 2005 and on October 7, 2005. Oviposition Trap No eggs were recovered from the oviposition traps due to the invasion of the traps by predators (frogs, lizards, and spiders), the ovi position paper being removed, the traps being knocked over, or snails eating the oviposition paper. Also, after the second week in the field the traps were refilled weekly with new tap water. Oviposition traps remained in the field until October 7, 2005. Backpack Aspirations One mosquito was recovered from the backpack aspirations on August 29, 2005 at TAL1. The mosquito could not be identified to species. Human Landing Rate A total of 884 landing mosquitoes were collect ed and identified duri ng the trial (Table 24). The five species included Ae. albopictus (90.2%) Culex species (0.3%), Oc. atlanticus 27

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(2.4%), Ps. ferox (6.0%), and Wy. mitchellii (1.1%). The majority of mosquitoes observed landing were Ae. albopictus, and included 714 females and 83 males (Table 2-4). The weekly average landing rate counts for each treatment, including the control treatments, for the University area (Figure 2-3) and for the Devils Mill hopper area (Figure 2-4) indicated a general trend of reduc tion after the trap:pesticide treatment was initiated. Analysis performed using PROC GLIMMIX identified area ( F = 18.49, df = 3, P < 0.0001) effects to be significant, therefore each site was consid ered a separate treatment for analysis ( F = 33.10, df =7, P <0.0001) and treatment effects were significant. Therefore, the data were grouped by the two neighborhoods and considered separately by preand post-trap:treatment initiation for further analysis. Comparisons were made between the landing ra te counts before and after treatments at all sites in each neighborhoods us ing t-tests (Figure 2-5). At th e sites in the university area, CON1 ( t = 4.17, df = 16, P = 0.0003), MD25001 ( t = 3.33, df = 16, P = 0.002), TAL1 ( t = 4.66, df = 16, P = 0.0001) and TALTRP1 ( t = 4.77, df = 16, P = 0.0001), showed significant differences in landing rate counts before and afte r treatment. In the Devils Millhopper area, only the treated sites, MD25002 (t = 3.99, df = 16, P = 0.0005), TAL2 ( t = 2.32, df = 16, P = 0.017) and TALTRP2 ( t = 2.76, df = 16, P = 0.006), showed significant differents. CON2 did not receive a treatment and no significant difference was found in the landing rate counts before and after treatments were applied ( t = 0.89, df = 16, P = 0.192). Discussion Aedes albopictus is a nuisance mosquito found in north central Florida ya rds (Hoel et al. 2007) and its diurnal activity (Estrada-Franco and Craig 1995) often conflicts with human activity in yards. The mosquito species compositi on of the sites in this study are similar to the findings of other studies performed in the ar ea (Campbell 2003, Hoel et al. 2007). The aggressive 28

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nature of host-seeking Ae. albopictus necessitates the establishmen t of control protocols aimed specifically towards the reduction of these populations, even when popula tions are at moderate levels as observed in this study. While Ae. albopictus are generalist feeders, a significant proportion may feed on humans furt her enhancing the concern about this species as a nuisance vector species (Richard et al. 2006b). The ev aluation of control technique efficacy against Ae. albopictus is warranted due to its ability to v ector arboviruses (Gra tz 2004, Mitchell 1991, Shroyer 1986) and transmit other pathoge ns (Konishi 1989, Mitchell et al. 1998). Different mosquito control techniques such as, the placement of commercially available traps or the application of pesticide treatments to vegetation, have been evaluated against Ae. albopictus. A propane powered trap, the Mosqu ito Magnet Pro [American Biophysics Corporation, East Greenwich, Rhode Island] has been shown to capture Ae. albopictus in Gainesville, FL (Hoel et al. 2007) Bifenthrin treatments applied to the vegetation in suburban yards in both Kentucky and Australia were also found to reduce landing Ae. albopictus (Trout 2006, Standfast et al. 2003). The effect of a combination of trap and pesticide application is unknown and was the object of this study. The treatments evaluated in this study redu ced populations of landing mosquitoes in the Devils Millhopper area, where there were no-area wide control measures offered by the local mosquito control agency. Landing rates following the trap:pesticide treatment initiation were significantly lower than pre-treatment levels at a ll treatment sites in the Devils Millhopper area. The lack of significant difference in the num bers of landing mosquitoes between the pretreatment and post-treatment periods at the cont rol site indicates that the natural population levels remained constant through the study. Thus it appears that the population reductions noted for all treatments were likely th e results of control measures. 29

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The homes utilized in the University Area, however, are within the jurisdiction of the local mosquito control agency. There were sign ificant differences between pre-treatment and post-treatment collections at all sites evaluated in this neighborhood, including the control site. It is possible some other factors, such as localiz ed weather conditions, cont ributed to the reduction in landing rate counts observed in the University Area. It is more likely that the observed reductions in landing counts were the result of organized mosquito control operations. Propane traps clearly offer hom eowners an option to reduce Ae. albopictus and other mosquito species on their properties. Th e clear preference of Ae. albopictus for residential propane traps over conventional surv eillance traps further supports th e utility of these traps as a potential control option (Hoel 2005). In the cu rrent study, traps baited with octenol were effective in reducing numbers of landing Ae. albopictus at the site MD25001. Recent research indicates that the addition of lac tic acid lures to the traps emitting CO 2 and octenol enhances collections of Ae. albopictus (Hoel et al 2007). Thus it seems advi sable to add lactic acid lures to residential traps to furt her enhance reduction of Ae. albopictus Males swarm around potential hosts while s eeking females (Gubler and Bhattacharya 1972). The trap may attract males seeking females, possibly due to the host-like cues provided by the trap. Therefore, propane-powered traps ma y attract and capture both male and female Ae. albopictus potentially reducing mating in localized populations. Hoel et al (2007) reported a significantly higher capture rate of Ae. albopictus males than males of any other species. In the present study, males of other mosqu ito species were not captured. Male Ae. albopictus constituted 0.1 % of the tota l number of mosquitoes captu red and 2.0 % of all capture Ae. albopictus were male and these findings agree with the Hoel et al. (200 7) study (Table 2-1). Also, male Ae. albopictus were often found resting on the trap. The male mosquitoes would 30

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alight and display swarming behavior when the trap was approached. Swarming behavior near the trap did not, however, lead to landing counts. Male Ae. albopictus have been observed swarming during landing rate count s in other studies (Hoel et al 2007). In this study, males were observed swarming for approximately 30 seconds, then land on the leg and wait for females to fly near. After the females arrived near the le g, the males would often alight and swarm around the leg if they evaded aspirati on. The males were not observed to mate the females while they were walking around on the leg. Mated pairs that met around the leg, flew to ground leaf litter, landed, and then moved under the leaves. The ma ted pairs were not observed once they moved beneath the leaf litter. Resting sites for mosquitoes often consist of vegetation, earth or rocks, or human habitats, and animal shelters (Clements 1992). Male Ae. albopictus are not active constantly during daylight and likely seek resting sites in sh aded dark areas, such as parts of the traps.The trap itself may offer resting or host seeking male s an additional harborage area. On September 2, 2005 the trap was tipped on its side for some repairs and male Ae. albopictus were observed to fly out of the hollow piping that connects the trap to the rolling stand. It is possible that male mosquitoes commonly rest in this area of the trap. The traps were placed 6 9 m away from the residence, as recommended by the manufacturer directions. The placement was bey ond the area treated with bifenthrin. Further trials where a trap is placed clos er to or within pesticide treatment may show differences in trap captures between the two treatments. Trap failures on August 19, August 24, and Septem ber 29 were probably due to not being properly attached to the propane tank. The failure on October 7 may have been due to a problem with the propane tank because the trap was runni ng at the previous landing rate count, the tank 31

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was full when it was found not running and would not restart with the same tank in the field. A propane usage trial performed after the field stu dy (Appendix B) to see how the tanks would last. These traps ran for an average of 17.2 days a nd two traps failed on 5 occasions due to being incorrectly attached to the propane tank. The application of pesticides has been found to reduce the number of landing mosquitoes in suburban settings (Anderson et al. 1991, Cilek and Hallmon 2006, Trout 2006). At homes treated with bifenthrin human landing rate counts were reduced after pesticide application, however, the pesticide applica tion was focused on the home vers us the vegetation. During the pesticide application the techni cian indicated that it was impo rtant to coat the mosquito harborage areas and it was important to treat da rker corners. The label for TalstarOne permits the use of the pesticide on buildings and mosquitoes are included in the list of targeted pests for this type of application. The label also allows fo r vegetation to be treated to target mosquitoes. At TALTRP1, the technician had nearly half of the one gallon of mixed chemical left after treating the residence and used the rema inder on a seven foot high azalea bush. Landing rate counts were high near this bush prior to the application, but following the treatment the landing rate counts dropped considerably in comp arison to the landing rate counts near untreated vegetation. Analysis of the landing rate counts close to and distant from the treatment were not significant. Despite a lack of st atistical difference between the tw o areas, further evaluation of the number of landing mosquito species in the pr oximity of treatment should be undertaken to determine if there is a localized reduction. Treating for Ae. albopictus requires an integrated pest management approach. While the Mosquito Deleto 2500 does not appear to capture large numbers of Ae. albopictus, it catches many other pest species that also would be a problem for homeowners. Altering the way the 32

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pesticide is applied by focusing the treatment on the vegetation and increa sing the attractiveness of the trap by adding lactic acid lure s may improve these treatments against Ae. albopictus. Further investigation of modifi ed versions of these techniques may prove helpful in reducing landing by Ae. albopictus and other pest species. 33

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Table 2-1. Composition of mosquito species collect ed from four yards by one Mosquito Deleto 2500 per yard from August 18, 2005 to October 7, 2005. Total number of mosquitoes (%) Aedes albopictus female 448 (5.8) Aedes albopictus male 9 (0.1) Anopheles quadrimaculatus 107 (1.4) Coquilletidia perturbans 79 (1.0) Culex nigripalpus 859 (11.3) Mansonia species 5359 (70.3) Ochlerotatus atlanticus 29 (0.4) Psorophora columbiae 207 (2.7) Psorophora ferox 420 (5.5) Wyeomyia mitchellii 27 (0.4) Unidentifiable specimens 81 (1.1) TOTAL 7625 All traps were operated from August 18, 2005 until October 7, 2005 and placed 6-9 m away from areas of human activity. MD2500 at TALTRP1 failed on August 24, 2005. On September 23, 2005 the trap was not running, however live mos quitoes remained in the net. The MD2500 at TALTRP 2 failed on Spetember 29, 2005 and on October 7, 2005. 34

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Table 2. Collections of mosquitoes from one Mo squito Deleto 2500 per yard placed in four yards from August 18, 2005 to October 7, 2005. Traps Pesticide + Traps Species MD25001 MD25002 TALTRP1 TALTRP2 Aedes albopictus female 61 181 116 90 Aedes albopictus male 1 4 1 3 Anopheles quadrimaculatus 5 48 5 49 Coquileitidia perturbans 2 26 12 39 Culex nigripalpus 195 263 92 309 Mansonia species 1021 2195 766 1377 Ochlerotatus atlanticus 12 12 4 1 Psorophora columbiae 1 75 2 129 Psorophora ferox 37 62 221 100 Wyeomyia mitchellii 0 26 0 1 Unidentifiable specimens 4 56 5 16 TOTAL 1339 2948 1224 2114 All traps were operated placed 6-9 m away from areas of human activity. Bifenthrin applied at TALTRP1 and TALTRAP2 on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water to the residences a nd surrounding vegetation. MD2500 at TALTRP1 failed on August 24, 2005. On September 23, 2005 the trap was not running, however live mosquitoes remained in the net. The MD2500 at TALTRP 2 failed on September 29, 2005 and on October 7, 2005. 35

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36 Table 2-3. Average (SE) of total mosquitoes captured from one Mosquito Deleto 2500 per yard placed in four yards fr om August 18, 2005 to October 7, 2005. Traps Pesticide + Traps MD25001 MD25002 TALTRP1 TALTRP2 Weekly Averages of Totals 132.9 (44.7)a 307.0 (53.8)b 204.4 (45.2)a 204.4 (45.2)ab All traps were operated placed 6-9 m away from areas of human activity. Bifenthrin applied at TALTRP1 and TALTRAP2 on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water with mixture sprayed on home and surro unding vegetation. MD2500 at TALTRP1 failed on August 24, 2005. On September 23, 2005 the trap was not running, however live mosquitoes remained in the net. The MD2500 at TALTRP 2 failed on September 29, 2005 and on October 7, 2005.ANOVA analysis showed significant difference between mosquitoes capture (ANOVA, F = 3.30, df = 3, P = 0.03).

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37 Table 2-4. Species composition of mosquitoes observed during human landing rate counts in 8 yards participating in trap and pes ticide trials in Gainesville, Florida from July 7, 2005 until October 7, 2005. Control Trap Only Pesticide Only Trap + Pesticide Species CON1 CON2 MD25001 MD25002 TAL1 TAL2 TRPTAL1 TRPTAL2 TOTAL (%) Aedes albopictus female 61 104 59 200 53 31 91 115 714 (80.8) Aedes albopictus male 27 5 4 8 12 0 17 10 83 (9.4) Culex species 1 0 0 1 0 0 0 1 3 (0.3) Ochlerotatus atlanticus 0 0 0 7 0 8 1 5 21 (2.4) Psorophora ferox 0 0 0 20 4 13 10 6 53 (6.0) Wyeomyia mitchellii 0 0 0 8 0 1 0 1 10 (1.1) TOTAL (% of total) 89 (10.1) 109 (12.3) 63 (7.1) 244 (27.6) 69 (7.8) 53 (6.0) 119 (13.4) 138 (15.6) 884 Landing rate counts were performed twice weekly at each home. Mosquitoes landing on the right exposed calf of the observer were counted and aspirated for 3 minutes for a total of 12 minutes per yard. All traps were operated from August 18, 2005 until Octo ber 7,2005 and placed 6-9 m away from areas of human activity. Bife nthrin applied at TAL1, TAL 2, TALTRP1 and TALTRAP2 on August 18, 2005 at the rate 10 ml of Tals tarOne per 3.8 L of water. MD2500 at TA LTRP1 failed on August 24, 2005. On September 23, 2005 the trap was not running, however live mosquitoes remain ed in the net. The MD2500 at TALTRP 2 failed on September 29, 2005 and on October 7, 2005.

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b. a Figure 2-1. Location of field si tes in A) University area and B) Devils Millhopper area neighborhood in Gaines ville, Florida. 38

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Figure 2-2. The Mosquito Deleto 2500 (MD2500) propane-powered trap used as trap treatments in suburban yards in Gainesville, Florida. 39

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0 1 2 3 4 5 6 8-Jul05 22Jul-05 1Aug05 10Aug05 12Aug05 15Aug05 19Aug05 22Aug05 24Aug05 2Sep05 7Sep05 12Sep05 21Sep05 23Sep05 28Sep05 30Sep05 2-Oct05 7-Oct05 Pre-Treatment Post-Treatment DateAverage number of landing mosquitoes per 3 min per site TAL1 MD25001 TALTRP1 CON1 Figure 2-3. Average number (SE) of landing mo squitoes in the University Area for each treatment from July 8, 2005 until October 7, 2005. Landing mosquitoes were observed and aspirated fo r three minutes in 4 different sites around the homes. MD2500 at TALTRP1 failed on August 24, 2005. On September 23, 2005 the trap was not running, however live mosquitoes remained in the net. Bifenthrin applied at TALTRP1 and TALTRAP1 on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water. All traps were operated from August 18, 2005 until Octobe r 7, 2005 and placed 6-9 m away from areas of human activity. TAL1 = TalstarOne trea tment, MD25001 = Mosquito Deleto 2500 TALTRP1 = TalstarOne and Mosquito Deleto 2500 CON1 = control yard. 40

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0 1 2 3 4 5 6 7 8 9 8-Jul05 22Jul-05 1Aug05 10Aug05 12Aug05 15Aug05 19Aug05 22Aug05 24Aug05 2Sep05 7Sep05 12Sep05 21Sep05 23Sep05 28Sep05 30Sep05 2-Oct05 7-Oct05 Pre-Treatment Post-Treatment DateAverage number of landing mosquitoes per 3 minutes TAL2 MD25002 TALTRP2 CON2 Figure 2-4. Average number (SE) of landing mos quitoes in the Devils Millhopper Area for each treatment from July 8, 2005 until October 7, 2005. Landing mosquitoes were observed and aspirated fo r three minutes in 4 different sites around the homes. MD2500 at TALTRP1 failed on August 24, 2005. On September 23, 2005 the trap was not running, however live mosquitoes remained in the net. Bifenthrin applied at TALTRP1 and TALTRAP1 on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water. All traps were operated from August 18, 2005 until Octobe r 7, 2005 and placed 6-9 m away from areas of human activity. TAL1 = TalstarOne trea tment, MD25001 = Mosquito Deleto 2500 TALTRP1 = TalstarOne and Mosquito Deleto 2500 CON1 = control yard. 41

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A 0 0.5 1 1.5 2 2.5 3 3.5 4 TAL1MD25001TALTRP1CON1Average (SE) number of landing mosquitoes per 3 minutes Pre-treatment Post-treatment B 0 1 2 3 4 5 6 TAL2MD25002TALTRP2CON2 TreatmentAverage (SE) number of landing mosquitoes per 3 minutes Pre-treatment Post-treatment Figure 2-5. Average number (SE) of mosquitoes landing during pre-treatment (N = 6 weeks) and post-treatment (N = 12 weeks) collection in residential yards from July 8, 2005 until October 7, 2005. A) University Area Neighborhood B) Devils Millhopper Area Landing mosquitoes were observed and aspirated for three minutes in 4 different sites around the homes. MD2500 at TALTRP1 failed on August 24, 2005. On September 23, 2005 the trap was not running, however live mosquitoes remained in the net. Bifenthrin applied at TALTRP1 and TALTRAP1 on August 18, 2005 at the rate 10 ml of TalstarOne per 3.8 L of water. All traps were operated from August 18, 2005 until October 7, 2 005 and placed 6-9 m away from areas of human activity. TAL1 = TalstarOne treatment, MD25001 = Mosquito Deleto 2500 TALTRP1 = TalstarOne and Mosquito Deleto 2500 CON1 = control yard. 42

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CHAPTER 3 EFFECT OF PLANT SPECIES AND LEA F TEXTURE ON RESPONSE OF FEMALE Aedes albopictus (Skuse) TO PESTICIDE-TREATED LEAVES Introduction Suburban yards contain many types of plants that are potential harborage areas for mosquitoes. Landscape plants are chosen based on a combination of factors, including aesthetics, availability, and environmental conditions (Black 2003, Gilman and Brown 2003). Targeting these mosquito harborage areas with pesticide applications ha s been found to reduce the number of mosquitoes in local ized areas (Anderson et al. 1991, Cilek and Hallmon 2006, Standfast et al. 2003, Trout 2006). The characteristics of plant leaves, such as the surface tension of the leaf cuticle, and the overall leaf arra ngement are important in the st udy of residual vegetation sprays. The treatment of residential landscapes with residual pesticide may reduce the necessity of applying pesticides added by ground ULV. Residual pesticides applied to substrates have been used to manage infestations of many different types of insects including cockroaches (Ali et al. 1993), house flie s (Geden et al. 1992), and mosquitoes (Kerdipibule et al. 1978). The residual effectiveness of DDT, malathion and chlorpyrifos applied to diffe rent wall surfaces against Culex pipiens Linnaeus was evaluated in Thailand (Kerdpibule et al. 1978). Dursban (chlor pyrifos) was found to kill mosquitoes on eight wall surface types for 10-30 weeks. Residual malathion treatments were lethal for 10-15 weeks while the mosquitoes were found to be re sistant to the residual DDT treatments. The physical characteristics, such as surface te nsion or roughness, of the substrates affect the manner in which a pesticide can interact with the targeted orga nism (Kirkwood 1987). The effectiveness of residual pesticides applied to different plants against Folsomia candida Willem (Collembola: Isotomidae) was studied (Chowdhury et al. 2001). Sixteen leaf types with different characteristics, such as age and surface wax-t ype, were treated under a Po tter tower calibrated to 43

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deliver a spray volume equiva lent to 1 liter per 10,000 m 2 with deltamethrin (25 g liter EC). Folsomia candida displayed different tolerances on the various plants, suggesting that leaf surfaces play a role in how pesticides can be transferred to organisms. Evaluating the effectiveness and longevity of pesticide residues under field conditions is critical in understanding how well pesticide trea tments will control resting mosquitoes, such as Aedes albopictus (Skuse) The objective of this experiment was to evaluate the residual efficacy of bifenthrin applied to common la ndscape plants, in the knock down of Ae. albopictus Materials and Methods Plants were treated on August 22, 2006. Leaves were allowed to dry and removed for the tests 4 hours post treatment and weekly thereafter for 5 weeks post-treatment. The plants remained in the field for three additional w eeks and the same plants were treated again on October 17, 2006. Bioassays were performed again using leaves removed 4 hours post treatment and weekly thereafter for the following five weeks. Plant Selection Five plant types were chosen representing tw o different leaf types, flexible and rigid. Flexible leaves did not crack or break when be nt and did not have shiny surfaces. Rigid plants cracked or broke when bent and were shiny. A ll plants are commonly fo und in yards or in uncultivated areas in Gainesville, FL. Plants were kept outdoors and watered for one-hour daily with a drip irrigation system. Flexible Leaves Azaleas, Rhododendron x Fashion Linnaeus (family Ericaceae) (Gilman 1999d), are evergreen shrubs commonly found in yards around Ga inesville, Florida (Figure 3-1a). The leaves are small to medium size, approximately 5 cm l ong, and arranged in an alternate manner. These shrubs can reach 3 meters in height and have a wide variety of flowers that bloom throughout the 44

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year. Ten plants (eight pesticide-treated and two control) were used for these bioassays and were approximately 0.5 m tall and purchased at Lowe s Home Improvement Store (Gainesville, Florida). Beauty berry plants, Callicarpa americana Loureiro (family Verbenaceae) (Gilman 1999a), are commonly found growing along roadsides a nd in natural areas in Gainesville, Florida (Figure 3-1b). Their leaves ar e broad, between 10 and 20 cm in length. They produce bright purple berries in the fall and gr ow 1-2 meters in height. They have long branches with simple leaf arrangement that have leaves on smaller br anches at 7-10 cm intervals. Eight pesticidetreated and two control plants were obtained from Chiappini Nursery (Melrose, Florida). The plants used in this study were approximately 0.5 meters tall. Rigid Leaves Dwarf buford holly, Ilex cornuta Bufordii Nana Lindley (family Aquifoliaceae ) (Gilman 1999b), is an evergreen shrub that has re d berries throughout the seasons. It has stiff, shiny leaves that are approximately 5 cm long (F igure 3-1c). Berries are toxic to humans but are edible by birds making it a possible harborage area for mosquitoes that pref er to feed on birds. The Buford holly plants (eight pesticide-treate d and two control) were purchased at Lowes Home Improvement Store. Sand Cordgrass, Spartina bakeri Merrill (family Gramineae ) (Gilman 1999e), is found near wetlands and is popular in landscapes (Figure 3-1d). In a ddition, plants of the same genus, Spartina patens Muhlenberg and Spartina alterniflora Loiseleur-Deslongchamps, are found in Florida marsh lands (FDEP 2004). Spartina bakeri and other members of the genus could offer harborage areas for salt marsh mosquitoes, such as Ochlerotatus taeniorynchus Wiedemann and Ochlerotatus sollicitans Walker, in coastal areas. The leaves are extremely narrow and blades can be up to a meter in length. The plants used in the study (eight pe sticide-treated and two 45

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control) were approximately 1 meter tall. Thes e plants also were purchased from Chiappini Nursery. The southern magnolia, Magnolia grandiflora Linnaeus (family Magnoliaceae) (Gilman and Watson 2006), was chosen because it is common in landscapes in Gainesville, Florida. It has large oval leaves (10 to 20 cm long) that are ri gid and have brown hairs on the underside (Figure 3-1e). They can grow up to 27 meters high. The tr ees can provide harborage for mosquitoes near the ground if the lower branches are not pruned and mature trees could also provide harborage areas for mosquitoes found in tree canopies. The Magnolia grandiflora used in this test (eight pesticide-treated and two control) were appr oximately 0.75 to 1.0 m ta ll. The magnolia trees were purchased at Chiappini Nursery. Pesticide Application TalstarOne [FMC Corporati on, Agricultural Products Group, Philadelphia, PA], active ingredient bifenthrin, was chosen for the pe sticide treatment. The pesticide was applied according to the label directions (10 ml per 3.8 L of water, A.I. 7.9%) with a B & G hand pump compressor sprayer [B & G Equipment Comp any, Jackson, GA]. Plants were moved approximately 280 m to a treatment area away from the holding area. Control plants remained in the holding area during treatment. The plants were placed into 8 groups so that each group contained one plant of each type and each plant was treated with approximately 0.6 liters of the mixture. Each group of 8 plants was treat ed with a separate pesticide mixture. The pesticide was applied to the plants on August 22, 2006. Each plant was treated to runoff. The applicator wand was held approxima tely 0.3 m away from the plant leaves while three initial vertical sw aths were applied. The wand was then placed within the foliage and three vertical swaths were slowly made from the bo ttom to the top of the plant. This process was repeated seven additional times around the circumfe rence of the plant. Plants were allowed to 46

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dry for four hours before being returned to the plant holding area. The plants were left in the field for three additional weeks and the same plants were retreated on October 17, 2006. Bioassays were performed as previously described. Petri Dish Preparation Leaves were picked from each plant every seventh day for 35 days post-treatment and placed into paper envelopes. Rhododendron x Callicarpa americana Ilex cornuta and Magnolia grandiflora were picked by hand. Spartina bakeri blades were cut using scissors. Leaves were brought into the lab and placed onto prepared 100 x 15 mm petri dishes [Fisher Scientific Worldwide, Hampton, New Hampshire]. Dispos able latex gloves were worn during the collection of control leaves and gloves were ch anged prior to handling treated leaves. Leaves were fastened to petri dishes prepared by cove ring the inside of the bottom dish with doublesided, 48 mm wide indoor / outdoor carpet tape [Henkel Consumer Adhesives, Inc., Avon, Ohio]. Leaves were placed one at a time so that the upper surface of the leaf was exposed until the entire surface of the dish was covered. If there were small gaps between the leaves a sterile cotton ball was brushed over the exposed tape so that fibers attached to th e exposed adhesive was exposed. The dishes with Ilex cornuta and Spartina bakeri leaves had two 2.5 mm wide strips of single sided tape crosswise over the surface of the l eaves to ensure that the leaves remained in place because these leaves tended to detach over the 24 hour period. A rubber band was placed on the outside of the dishes securing them together and they were stored on their sides in air tight containers. The containers were lined with damp paper towels to ensure that adequate moisture levels, relative humidity between 70 85 %, were maintained for mosquito survival. 47

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Mosquitoes Nulliparous 5-7 day old female Ae. albopictus were obtained from the USDA, ARS, CMAVE rearing laboratory colony that was established in 1992 (H oel 2005). Larvae were fed a 3:2 mixture of beef liver powder to yeast. Adults were provide d cotton balls saturated with a 10% sucrose water solution. The colony was kept under 14:10 (L:D) photoperiod at room temperature (27 o C to 32 o C). Adults were obtained from colony cages with a mechanical aspirator and chilled at 1 o C for approximately 5 minutes until they were no longer active. Ten mosquitoes were placed into individual holding cups and th en transferred into the prepar ed petri dishes. Mortality was assessed at 1 hour and 24 hours pos t introduction into the petri dis h. Mosquitoes were considered alive if they were able to fly within the dis h. Mosquitoes were rated as knocked down if they were unable to fly within the dish and include d mosquitoes that exhi bited twitching limbs but were unable to stand. Statistical Analyses All statistical analyses were performed using the Statistical Analysis System (SAS Institute 2004). 'Treatment data were corrected for c ontrol mortality by Abbott's formula (Abbott 1925). Control mortality was 0.0 % for all plant types except for Ilex cornuta at day 0 (1.7%). Control mortality at the 24 hour marks ranged between 1. 7% and 11.7% for all plant types. Data were transformed using arcsine transformation then compared with ANOVA ( P < 0.05). N differences between the two trials were observed. The data were pooled and adata from the 1 hour and 24 hours post introduction time check were analyzed by repeated measures analysis of variance (PROC GLM, SAS Institute 2004). Differences be tween plant species were detected by ANOVA with means separation between plant species identified usi ng Bonferroni-Dunn ( = 0.05). 48

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Results Laboratory Bioassay Results One-Hour Exposure of Ae. albopictus There was no knock down observed on days 28 and 35 and these data are not presented. Knock down of mosquitoes was observed at one -hour on days 7, 14 and 21 and these data are presented in Table 3-1. Repeated measures anal ysis performed found significant differences in both week effects ( F = 489.60; df = 5; P < 0.0001) and between plant types ( F = 76.99; df = 4, P < 0.0001). Significant differences were observed between the plant species were observed with ANOVA analysis ( F = 8.24; df = 4; P < 0.0001). In the Rhododendron x leaves, significantly fewer mosquitoes were knocked down on day 7 than on day 0 ( t = 7.22; df = 30; P < 0.0001) and between days 7 and 14 ( t = 7.22; df = 30; P < 0.0001) with no observed differences between day 14 and 21. Significantly more mosquitoes were knocked down on day 0 with the Callicarpa americana than day 7 ( t = 10.33; df = 30; P < 0.0001) with no differences between days 7, 14 and 21. A significant difference was observed between days 0 and 7 (t = 5.97; df = 30; P < 0.0001) and between days 7 and 14 with the Ilex cornuta leaves ( t = 8.57; df = 30; P < 0.0001), there was no difference between day 14 and 21. Although few mosquitoes were knocked down on day 0, significantly more mosquitoes on day 0 than on day 7 with the Spartina bakeri leaves ( t = 7.22; df = 30; P < 0.0004) with no differences between days 7, 14 and 21. On day 0, significantly more mosquitoes were knocked down with the Magnolia grandiflora leaves between day 0 and day 7 ( t = 25.27; df = 30; P < 0.0001) with no differences between days 7, 14 and 21. 49

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Twenty-four Hours Exposure of Ae. albopictus Knock down of mosquitoes at 24 hours post in troduction to the petri dishes was observed for 35 days post treatment at the 24 hour point and these data are presented in Table 3-2. Significant differences were found with repeated measures analys is for both week effects over the course of the study ( F = 289.5; df = 5; P < 0.0001) and plant species effects ( F = 281.31; df = 5; P < 0.0001). ANOVA analysis between the plants found signi ficant differences between the plant types ( = 0.05; F = 18.78; df = 4; P < 0.0001). Knock down at 24 hours was similar between day 0 and 21 with a decrease between days 21 and 28 ( t = 2.61; df = 30; P < 0.011) and days 28 and 35 ( t = 4.36; df = 30; P < 0.0001) in the Rhododendron x leaves. With Callicarpa americana leaves knock down was similar between days 0 and 21, however, between days 21 and 28 there was a significant decrease in knock down ( t = 12.80; df = 30; P < 0.0001). There were significant differences observed between days 14 and 21 ( t = 7.13; df = 30; P < 0.0001) and days 21 and 28 ( t = 8.12; df = 30; P < 0.0001) in the Ilex cornuta plants ( t = 5.38; df = 30; P < 0.0001). Knock down with Spartina bakeri leaves decreased significantly between days 0 and 7 ( t = 3.00; df = 30; P = 0.0035), days 7 and 14 (t = 4.65; df = 30; P < 0.0001) and between days 14 and 21 ( t = 7.13; df = 30; P < 0.0001) with no differences between days 21, 28 and 35. Knock down using treated Magnolia grandiflora leaves was similar between days 0 and 7 with a significant decline between days 7 and 14 ( t = 2.75; df = 30; P = 0.0071), days 14 and 21 (t = 10.12; df = 30; P < 0.0001) and between days 28 and 35 ( t = 2.06; df = 30; P = 0.042). Discussion Treating the resting areas of mos quitoes, such as the walls of homes, has been successful in reducing Anopheline mosquitoes in malaria en demic regions (Kerdipibule 1978) and is a recommended control technique by WHO to reduce malaria transmission (2006). The use of an 50

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indoor residual pesticide spray was evaluated in India and was found to be effective for 24 weeks on various wall surfaces (Yadav et al. 2003). Use of residual pesticide applications on other mosquito resting sites, such as on plants outdoo rs, has been shown to be effective in managing pest mosquito populations (Anderson et al. 1991, Cilek and Hallmon 2006, Standfast et al. 2003, Trout 2006), however, the efficacy of outdoor residual sprays was short-lived due to precipitation and exposure to sunl ight (Kirkwood 1987). It is also possible that the substrate, such as plant surfaces, impact to the efficacy of the residual pesticide. The type of leaf surface treated has been shown to affect the way in which a pesticide is transferred to an organism. Chowdhury et al. (20 01) treated 16 different plant types with varying characteristics and found that the effi cacy of residual pesticides against Folsomia candida varied and that pesticide efficacy was not predictable by an any obvious pattern. In this study, five different plant species were treated with a residual pesticide and the efficacy of the pesticide against Ae. albopictus varied between plants. The plants in this study were categorized into two groups, flexible and rigid, based on visual appearance and texture of the leaves. The efficacy of the pesticid e on the leaf surfaces varied in both groups. In the flexible plan t group, knock down was observed at high levels, Rhododendron x (90.0%) and Callicarpa americana (97.5%) (Table 3-1). On day 7 the efficacy of the knock down of mosquitoes exposed to treated Callicarpa americana leaves dropped to 21.3% whereas knock down of mosquitoes on Rhododendron x leaves remained relatively high at 62.5%. Likewise, similar differences in knock down between species in the rigid leaf plant group also were observed. The observed differences could be attributed to the type of cuticle in the leaves or the ability of the plant to absorb the pesticide. The cu ticular wax of the plant is the primary barrier to 51

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pesticides (Ford and Salt 1987). Waxes with high polarity will deter a pesticide from being absorbed by the plant (Ford and Salt 1987). It is possible that the surface of the Callicarpa americana plant is more polar than the Rhododendron x therefore the pesticid e is able to dry and remain on the leaf surface longer th an occurs on the cuticles of the Callicarpa americana In addition to how the pesticide interacts with the cuticular waxes, the application techniques used to apply the pesticide may contribu te the residual efficacy of a pesticide. Results using treated leaves of Spartina bakeri also showed reduced levels of residual pes ticide one week after treatment at both 1 hour and 24 hours post-exposure. The differences observed with the Spartina bakeri compared to the other plants could be attributed to several factors. The most important factor is that the extr eme thinness and arrangement of the Spartina bakeri blades may have hindered the effective application of pes ticide using the hand sprayer. The narrowness of the blades made it difficult to direct the spray onto the blades and to ensure that the blades were coated properly with the pesticide. The thinness of the blades also made it difficult to determine the top side of the leaves for the bioassay. It is possible that the use of a pesticide applicator that creates a finer mist or fog may increase th e effectiveness of the residual application. Plant growth may have played a role in th e decreased control percentage over time. To limit this possibility, leaves were not taken fr om areas of new growth for the bioassays. New leaves were readily identifiable from old leaves due to the way that they budded. Also, the surface area of the leaves was not visually obser ved to increase, however, measurements were not taken to verify this. In addition to the above mentioned factors, th e role of the wax depos its on the cutilcle of plants plays a role in the efficacy of the re sidual pesticide (Kirkwood 1987, Ford and Salt 1987). It is possible to perform tests to determine the types and levels of wax that are present on plant 52

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leaves (Chowdhury et al. 2001), how ever, this was beyond the scope of this experiment. In the presented study, differences in the efficacy of residual bifenthrin were observed between common landscape plants. Determining the types of waxes present on common landscape plants may lead to improvements in the efficacy and lo ngevity of barrier sprays against mosquitoes, especially Ae. albopictus 53

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54 Table 31 Mean percent control (SE) of Ae. albopictus following 1 hour exposure to TalstarOne treated leaves (a.i. bifenthrin 7.9%) to plant leaves on August 22, 2006 and October 17, 2006. Days post-treatment Plant 0 7 14 21 Rhododendron x 90.0 (4.6)a 62.5 (5.3)b 4.4 (1.8)c 1.3 (1.3)c Callicarpa americana 97.5 (1.9)a 21.3 (7.1)b 3.1 (1.5)b 0.0 (0.0)b Ilex cornuta 100.0 (0.0)a 77.5 (7.9)b 7.5 (2.1)c 0.0 (0.0)c Spartina bakeri 15.6 (6.3)a 2.5 (1.4)b 0.0 (0.0)b 0.0 (0.0)b Magnolia grandiflora 96.9 (1.5)a 8.8 (3.1)b 2.5 (1.7)b 1.3 (0.9)b Knock down was not observed on day 28 and day 35 post treatment for any plants. Control mortality was 0% for all plants at the 1 hour mark, pe rcent control was calc ulated using Abbotts formula (Abbott 1925) correction was used to mainta in consistency with mo rtality at the 24 hour mark. Pesticide applied with B & G hand pump comp ressor sprayer at the rate of 10 ml to 3.8 L of water. Plants were allowed to rest in the fi eld for 3 weeks prior to re-treatment. Means within each row followed by the same letter are not si gnificantly different Bonferroni-Dunn test (P = 0.05).

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55 Table 3-2 Mean percen t control (SE) of Ae. albopictus following 24 hours exposure to TalstarOne treated leaves (a.i. bifenthrin 7.9%) to plant leaves on Augus t 22, 2006 and October 17, 2006. Days Post-Treatment Plant 0 7 14 21 28 35 Rhododendron x 100.0 (0.0a) 100.0 (0.0)a 98.7 (0.9)a 94.3 (2.3)a 89.4 (2.7)b 77.7 (2.1)c Callicarpa americana 100.0 (0.0)a 99.3 (0.0)a 91.1 (5.1)a 90.6 (2.8)a 26.2 (3.7)b 19.4 (2.1)b Ilex cornuta 100.0 (0.0)a 100.0 (0.0)a 100.0 (0.0)a 91.7 (3.1)b 44.0 (8.3)c 36.4 (4.7)c Spartina bakeri 99.3 (0.7)a 87.2 (3.5)b 75.0 (7.3)c 25.6 (5.6)d 25.0 (4.7)d 16.3 (1.8)d Magnolia grandiflora 97.9 (1.5)a 100.0 (0.0)a 90.3 (3.1)b 26.3 (6.0)c 27.6 (6.2)c 16.9 (2.5)d Leaves were allowed to dry for four hours after the pesticide applicati on, leaves were picked and th e bioassays were performed. Leaves were picked every 7 days followi ng treatment for 35 days. The percent mortality for the control range d between 1.7% and 11.7%. Pesticide applied with B & G hand pump compressor sprayer at the rate of 10 ml to 3.8 L of water. Plants were allowed to rest in the field for 3 weeks prior to re-tre atment. Means within each row followed by the same letter are not significantly differe nt Bonferroni-Dunn test ( P = 0.05).

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a. Rhododendron x Fashion b. Callicarpa americana c. Ilex bufordii d. Spartina bakeri e. Magnolia grandiflora Figure 3-1 Plants used in the bioassays performed August 17, 2006 to October 26, 2006 56

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CHAPTER 4 EFFECT OF GENDER AND PHYSIOLOGICAL STATE OF Aedes Albopictus (Skuse) ON RESPONSE TO PESTICIDE TREATED LEAVES Introduction Various factors contribute to the effectiveness of a residu al pesticide application in targeting an insect, including how the pesticide interacts with the substrate and how the insect comes into contact with the residual pesticide. In addition to these factor s, the physiological state of the insect may also influen ce how the insect is able to me tabolize pesticides. Hadaway and Barlow (1956) applied DDT di rectly to the thorax of Aedes aegypti Linnaeus and Anopheles stephensi Liston of both sexes, different ages, and blood-fed females and found that both species were less susceptible to pestic ides following a blood meal or s ugar meal. Susceptibility to the pesticides returned to pre-feeding levels after oviposition and dige stion occurred due the dehydrochlorination of DDT. Halliday and Feyr eisen (1987) hypothesized that the increased tolerance to DDT was due to in creases of dehydrochlorinases in the 48 hours post-blood meal ingestion. Additionally, the way in which a mosquito en counters the pesticide may influence the toxicity of how the pesticide is metabolized by the mosquito. Xue and Barnard (2003) evaluated boric acid baits against gr avid, blood-fed and parous Aedes albopictus (Skuse), Anopheles. quadrimaculatus Say, and Culex nigripalpus Theobold in the laboratory. The baits were offered to the mosquitoes mixed with 10% sucrose water. Males were more susceptible to the baits than females. The boric acid baits were al so more toxic to male and female Ae. albopictus and Cx. nigripalpus than to An. quadrimaculatus. The baits also caused mort ality in blood-fed, gravid and parous mosquitoes, suggesting that the toxicity of the baits is unaffect ed by the physiological state of the mosquitoes. 57

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Mosquitoes exist in nature in different phys iological states that may be affected by pesticides and other treatments di fferently. It is important to eval uate pesticide treatments against different physiological st ates to ensure that the label rates are effective against all mosquitoes. The objective of this study is to evaluate the im pact of sex and nutritiona l state on the efficacy of residual bifenthrin on plant leaves. Materials and Methods Plant Selection Rhododendron simsii Planch (family Ericaceae ) (Gilman 1999c) were the plants chosen for these bioassays. Plants were purchased from Harmony Gate Nursery in Gainesville, Florida. This plant was chosen for several reasons. First, mosquitoes were observed to land readily at landing rate sites located near azalea plants during field work completed in 2005. Second, azaleas had high knock down rates in the leaf type bioassays after 1 hour and 24 hours of exposure in comparison to other leaf types. Also, Rhododendron simsii can grow over 3 meters high and make a good plant barrier candidate. While treated Ilex cornuta Lindley leaves were found to result in the highest knock down rates in those assays, their leaves are stiff and require additional manipulations to keep attached to the Petri dishes. Pesticide Application Pesticide application occu rred on January 27, 2007. TalstarOne [FMC Corporation, Agricultural Products Group, Philadelphia, PA], ac tive ingredient bifenthr in, was chosen for the pesticide treatment. The pesticide was applied ac cording to the label di rections (0.01 L per 3.8 L of water, A.I. 7.9%) with a B & G hand pump compressor sprayer [B & G Equipment Company, Jackson, GA]. Plants were moved to a treatment area 280 m away from the holding area and control plants remained in the holding area duri ng treatment. The plants were placed into 5 58

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groups so that each group contained 7 plants. Each group was treated with a separate mixture of the pesticide. Pesticide was applied to the plants so that e ach plant was treated until the leaves appeared to be coated with the pesticid e. The applicator wand was held approximately 0.3 m away from the plant leaves while three initial vertical swat hs were made. The wand was then placed within the foliage and three vertical swat hs were slowly made from the bottom to the top of the plant. This process was repeated 7 additional times ar ound the circumference of the plant. Plants were allowed to dry before being returned to the plant holding area. Petri Dish Preparation Leaves were picked from each plant 4 hours post-treatment and once a week thereafter for 6 weeks post-treatment and placed into paper enve lopes. The envelopes were placed into the freezer because adequate numbers of gravid mosquitoes were not available on the day of initial treatment. Leaves were removed from the freezer 24 hours prior to the day on which the bioassays were performed and allowed to complete ly thaw. Leaves were placed onto prepared 100 x 15 mm petri dishes [Fisher Scientific Worl dwide, Hampton, New Hampshire]. To prevent cross-contamination with the pesticide, disposab le latex gloves were wo rn during the collection of control leaves and gloves were changed prio r to handling treated leaves. Petri dishes were prepared by covering the inside of the bottom dish with double-sided, 48 mm wide indoor / outdoor carpet tape [Henkel Consumer Adhesives, Inc., Avon, Ohio]. Leaves were placed on the taped dish one at a time so that the top of the leaf was exposed until the enti re surface of the dish was covered. If there were small gaps between the leaves a sterile cotton ball was brush over the exposed tape so that no adhesive was exposed Leaves were placed one at a time so that the upper surface of the leaf was exposed until the entire surface of the dish was covered. Small gaps of exposed ta pe were eliminated by swiping a 59

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sterile cotton ball over the exposed tape so that fibers attached to the exposed adhesive. A rubber band was placed on the outside of the dishes secu ring them together and they were stored on their sides in airtight c ontainers. The containers we re lined with damp paper towels to ensure that adequate moisture levels (relative humidity between 70 85 %) were maintained for mosquito survival. Mosquitoes Ten to twelve day old adult female, male, gravid and blood-fed Ae. albopictus mosquitoes were obtained from the USDA, ARS, CMAVE reari ng laboratory colony that was established in 1992 (Hoel 2005). Larvae were fed a 3:2 mixture of beef liver powder to yeast. Adults were provided 10% sucrose. The colony was kept un der 14:10 (L:D) photoperiod at room temperature (27 o C to 32 o C). The four types of Ae. albopictus in these bioassays were 812 day old males, females, gravid female, and blood-fed females. Female mosquitoes were offered a blood meal once mating was observed, usually on day 5 to 7 post-em ergence and moved into a separate cage by aspiration. Defibrinated bovine bloo d was placed in a membrane, heated in a warm water bath at 40 o C for 5 to 10 minutes and placed on top of the cage. The membrane was reheated in the warm water one-hour later for an additional 5 to 10 minutes then replaced on the cage. Blood engorged females were allowed to rest in the cage 3 5 days until eggs developed. They were not provided with an oviposition substrate. Mosquitoes for the blood-fed bioassays were offered a blood meal the morning of the day of the test. Non-blood-fe d females and males were held in the colony until the test date. Adults were aspirated with a mechanical aspirator and chilled at 1 o C for approximately 5 minutes on a chill table until they were no longer active. Ten mosquitoes were placed into the prepared petri dishes and handl ed as described previously. Kn ock down was checked at 1 hour 60

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and 24 hours post introduction into th e petri dish. Mosquitoes were c onsidered alive if they were able to fly within the dish. Mosquitoes were rated as knocked down if th ey were unable to fly within the dish. Statistical Analyses All statistical analyses were performed using the Statistical Analysis System (SAS Institute 2004). Treatment data were corrected for contro l mortality by Abbott's formula (Abbott 1925) and transformed using arcsine. After arcsine tr ansformation, data from the 1 hour, 4 hour and 24 hours post-introduction observations we re analyzed using repeated m easures analysis of variance (PROC GLM, SAS Institute 2004). Differences between mosquito types were detected by ANOVA with means separation between gender and physiological states located using Bonferroni-Dunn t-test analysis ( = 0.05). Results Laboratory Bioassay Results One-hour Exposure of Ae. albopictus Knock down of mosquitoes was observed at one-hour of exposure on days 0, 7, 14 and 21 and these data are presented in Table 4-1. Knock down was not observed on days 28, 35 and 42 and these data were not presented. Repeated measures analysis performed using PROC GLM (SAS Institute, Cary, NC) found significan t differences between week effects ( F = 804.25; df = 2; P < 0.0001) and between the mosquito groups tested ( F = 506.93; df = 3, P < 0.0001). Significant differences were observed between the physiological states and genders tested ( F = 6.40; df = 45; P = 0.0003). A significant reduction in the knock down of males was observed between days 0 and 7 (t = 12.70; df = 45; P < 0.0001), and again between days 7 and 14 ( t = 9.63; df = 45; P < 0.0001) with no difference in knock down observed between days 14 and 21. For female sugar-fed mosquitoes, knock down significantly decreased between days 0 61

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and 7 ( t = 32.68; df = 45; P < 0.0001) and again between days 7 and 14 ( t = 2.56; df = 45; P = 0.013), however, no differences were observed between days 14 and 21. Significantly more female gravid mosquitoes were knocked down on day 0 than on day 7 ( t = 14.64; df = 30; P < 0.0001) and similarly between days 7 and 14 ( t = 12.07; df = 45; P < 0.0001) with no observed differences between days 14 and 21. A signifi cant decrease in the knock down among blood-fed females was observed between days 0 and 7 ( t = 38.35; df = 45; P < 0.0001) and also between days 14 and 21 ( t = 64.59; df = 45; P < 0.0001). Four-hour Exposure of Ae. albopictus Observations of knock down were made at 4 hours on days 7, 14, 21 and 28 and these data are presented in Table 4-2. There was no knock down observed on days 35 and 42 and these data were not presented. Repeated measures analysis performed using PROC GLM (SAS Institute 2004) found significant differences between week effects ( F = 625.11; df = 2; P < 0.0001) and between the mosquito groups tested ( F = 38.22; df = 3, P < 0.0001). Overall differences between physiological stat es and gender groups tested were observed ( F = 23.31; df = 3, P < 0.0001). There were no differences observed in knock down among male sugar-fed mosquitoes between days 0 and 7 or between days 21 and 28, however, significant differences were observed between days 7 and 14 ( t = 15.81; df = 45; P < 0.0001). Knock down of females was similar at days 0 and 7 with differences observed in the female sugar-fed mosquitoes between days 7 and 14 ( t =49.17; df = 45; P < 0.0001) and no decreases were observed between days 14 and 21 or between da ys 21 and 28. There was a significant difference in knock down between days 7 and 14 with the female gravid (t = 49.16; df = 45; P < 0.0001), but no differences were observed between days 0 and 7 and between days 14 and 21. Knock down of blood-fed females decreased significantly between days 7 and 14 ( t = 45.59; df = 45; P < 0.0001), with no further differences between or between days 14 and 28. 62

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Twenty-four Hours Post Exposure of Ae. albopictus Observations of knock down were made at 24 hours on days 0, 7, 14, 21, 28, 35 and 42 and these data are presented in Table 4-3. Repeated measures analysis performed using PROC GLM (SAS Institute, Cary, NC) found signi ficant differences week effects ( F = 1571.98; df = 2; P < 0.0001) and between the mosquito groups tested ( F = 37.90; df = 3, P < 0.0001). ANOVA analysis between the plants found signi ficant differences between the plant types ( F = 37.05; df = 3; P < 0.0001). Differences in knock down were not observed in male, female, gravid female, or blood-fed female bioassays between days 0, 7, and 14 and the percent knock down ranged from 97.1 -100%. For sugar-fed males, there were significant differences in knock down between days 28 and 35 ( t = 12.70; df = 45; P < 0.0001), between days 28 and 35 ( t = 4.88; df = 45; P < 0.0001), and between days 35 and 42 ( t = 6.91; df = 45; P < 0.0001). For sugar-fed females, significant differences in knock down were observed between days 21 and 28 ( t = 22.63; df = 45; P < 0.0001), between days 28 and 35 ( t = 8.11; df = 45; P < 0.0001) and between days 35 and 42 ( t = 2.37; df = 45; P = 0.02). Knock down of gravid females decreased significantly differences from days 14 to 21 ( t = 3.66; df = 45; P = 0.0003), from days 21 to 28 ( t = 24.28; df = 45; P < 0.0001), between days 28 to 35 ( t = 9.63; df = 45; P < 0.0001) and between days 35 and 42 ( t = 7.28; df = 45; P < 0.0001). Knock down of blood fed females significant decreased between days 14 and 21 ( t = 2.60; df = 45; P = 0.01), days 21 and 28 (t = 29.41; df = 45; P < 0.0001), between days 28 and 35 ( t = 10.99; df = 45; P < 0.0001) and between days 35 and 42 ( t = 2.68; df = 45; P = 0.008). Discussion Mosquitoes in different physiological states have been shown to respond to pesticide treatments differently (Hardaway and Barl ow 1956). Bransy-Williams and Webley (1965) applied DDT directly to blood-fed Culex pipiens fatigans Wiedemann and observed higher 63

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tolerance to the pesticide than in blood-fed Anopheles gambiae Giles and Ae. aegypti Halliday and Feyreisen (1987) found that DDT to lerance increased at 24 hours in Cx. pipiens and postulated that the increased pesticide tolerance was due to changes in detoxification pathways activated during blood meal digestion. This increase in tolerance was not observed in Cx. pipiens treated with cyclodiene a nd carbamate pesticides. The above-mentioned studies examined at the effects of pesticide applied directly to mosquitoes, but how are mosquitoes of different physiological states affected by contact with pesticides? It is possible that applying the pestic ide directly to the insect allows the insect to process the pesticide easier since it is combin ed with water. The hydr ophillic portions of the pesticide are combined with water when app lied to the insect and perhaps are readily metabolized due to the pesticides associations with the water. In the case of these bioassays, the pesticide was allowed to dry l eaving the pesticide able to bond with the insects organs or hemolymph once the pesticide transfer from the substrate to the insect occurs. Blood-fed mosquitoes in this study had a slightly higher overa ll percent knock down. Previous studies showed higher tolerance to pe sticides in blood-fed mosquitoes for 48 hours post-bloodmeal ingestion at which point the LD 50 returned to levels shown in sugar-fed females (Bransby-Williams and Webley 1965, Halliday a nd Feyreisen 1987). These conflicting findings may be due to differences in tolerance levels of the species studied, however, another possibility may be the differences in experimental design. Similar studies, examinng at the efficacy of residual pesticides, where the insect was placed in contact with the treated substrate for a period of time and then moved to a holding area have been conducted (Geden et al. 1992). Exposi ng the mosquitoes for a period of time mimics the transient resting behavior of insects and allows for the possibility of recovery from the 64

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pesticide exposure. It is possible that the mosquitoes could recover from the knock down observed at days 0 and days 7 at the one-hour point negating th e effectiveness of the barrier. Also, performing additional time checks during th e first hour of exposure may elicit additional significant differences between the treatments. Further study of how physiological state impacts the efficacy of residual pesticide treatments should be undertaken to improve the efficacy of this control methodology. 65

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66 Table 4-1 Mean percen t knock down (SE) of Ae. albopictus following 1 hour exposure to TalstarOne treated Rhododendron simsii leaves (a.i. bifenthrin 7.9%). Days Post-treatment State 0 7 14 21 Male 100.0 (0.0) a 72.1 (15.3) b 7.1 (8.1) c 5.4 (7.2) c Female 100.0 (0.0) a 66.3 (11.0) b 4.2 (7.2) c 1.7 (3.8) c Gravid 100.0 (0.0) a 57.1 (14.6) b 1.7 (4.8) c 0.0 (0.0) c Blooded 100.0 (0.0) a 58.3 (19.5) b 2.9 (7.5) c 1.7 (4.8) c Knock down was not observed on days 28, 35 and 42 post-treatment for any group and data are not presented. Control mortality ranged from 0% to 1.2% for all treatments, percent control was calculated using Abbotts formula (Abbott 1925) co rrection was used to maintain consistency with the observations. Pesticide applied with B & G hand pump compressor sp rayer at the rate of 10 ml to 3.8 L of water. Means within each row fo llowed by the same letter are not significantly different (Bonferroni-Dunn test P = 0.05).

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Table 4-2 Mean percen t knock down (SE) of Ae. albopictus following 4 hours exposure to TalstarOne treated Rhododendron simsii leaves (a.i. bifenthrin 7.9%). Days Post-treatment State 0 7 14 21 28 Male 100.0 (0.0) a 100.0 (0.0) a 60.0 (13.2) b 10.0 (0.0) b 0.4 (2.0) b Female 100.0 (0.0) a 100.0 (0.0) a 23.8 (2.9) b 0.0 (0.0) b 0.0 (0.0) b Gravid 100.0 (0.0) a 100.0 (0.0) a 24.42 (3.7) b 0.0 (0.0) b 0.0 (0.0) b Bloodfed 100.0 (0.0) a 100.0 (0.0) a 32.08 (11.0) b 0.0 (0.0) b 0.0 (0.0) b Knock down was not observed on days 28, 35 and 42 post-treatment fo r any group and data are not presented. Control mortality ranged from 0% to 1.2% for all treatments, percent control was calculated using Abbotts formul a (Abbott 1925) correction was u sed to maintain consistency with the observati ons. Pesticide applied with B & G hand pump compressor sprayer at the rate of 10 ml to 3.8 L of water. Means within each row followed by the same letter are not significantly different (Bonferroni-Dunn test P = 0.05). 67

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68 Table 4-3 Mean percen t knock down (SE) of Ae. albopictus following 24 hours exposure to TalstarOne treated Rhododendron simsii leaves (a.i. bifenthrin 7.9%) Days Post-treatment State 0 7 14 21 28 35 42 Male 100.0 (0.0) a 100.0 (0.0) a 100.0 (0.0) a 98.3 (3.8) a 89.6 ( 9.1) a 82.9 (13.0) b 79.6 ( 2.2) c Female 100.0 (0.0) a 100.0 (0.0) a 97.1 (5.5) a 95.4 (7.8) a 83.3 (10.1) b 72.5 (16.5) c 58.3 (13.1) d Gravid 100.0 (0.0) a 100.0 (0.0) a 99.2 (2.8) a 94.6 (9.3) b 73.3 (12.7) c 53.3 (14.9) d 45.4 ( 6.6) e Bloodfed 100.0 (0.0) a 100.0 (0.0) a 98.7 (4.5) a 96.6 (5.0) b 84.1 (16.9) c 62.1 (26.2) d 56.3 (20.2) e Knock down was not observed on days 28, 35 and 42 post-treatment fo r any group and data are not presented. Control mortality ranged from 0% to 1.2% for all treatments, percent control was calculated using Abbotts formul a (Abbott 1925) correction was u sed to maintain consistency with the observati ons. Pesticide applied with B & G hand pump compressor sprayer at the rate of 10 ml to 3.8 L of water. Means within each row followed by the same letter are not significantly different (Bonferroni-Dunn test P = 0.05).

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CHAPTER 5 AREAS FOR FURTHER ST UDY AND CONCLUSIONS Evaluating a mosquito control program in the field requires key elements to create useful comparisons of treatments. The most important que stions for mosquito control treatments are: which mosquito species is the program targeti ng, what is the best control program to employ, and how will the program be evaluated? Once this information has been established, the targeted mosquitos unique behavior and ecology must be integrated into the management program. Areas for Further Study Field Studies Rigid protocols must be establis hed prior to site selection. In the case of the evaluation of barrier sprays, the homes should have a defined barrier of continuous vegetation surrounding an area large enough to allow multiple landing rate count sites. Ideall y, the vegetation barrier should be a single plant species. In addition, the hom eowners should be willing to be assigned any treatment randomly and treatments should be rotated during the study. There must be a measurable mosquito population ex isting prior to the study commencing. It would be interesting to co mpare residual vegetation sprays at homes with and without a continuous vegetation border. It is possible that this vegetation treatment technique might be effective only if there is a true continuous vegetation barrier. In instances where a continuous border is absent, additional tools to provide relie f from host-seeking mosquitoes will be needed. Studying the plant:pesticide interaction pestic ide is critical in evaluating pesticide candidates for this treatment. Plants should be eval uated to see if the mosquito will rest on the plant. Observations of the physical qualities of plants where mosquitoes are found resting should be taken to see if there are any general qualities to th e plants., such as amount of leaves or air 69

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flow through the plants branches. Also, what role if any does the leaf type play in creating a mosquito friendly plant are the leaves broad, narrow, or does it even matter? Sampling plants for resting adult mosquito es should also be evaluated. Conducting aspirations when the mosquitoes are actually resting in the vegetation is critical. Mosquitoes often exhibit varied behaviors throughout the day, such as mate seeking, host seeking, and resting (Hassan et al. 1996). Coordinating aspirations with res ting periods in the day should provide accurate estimations of mosquitoes resting within the plants versus aspirations made during foraging times. Careful attention needs to be paid to the aspi rator itself. The suction cannot be so strong that it destroys the insect but must be strong e nough to capture the insect The aspirator should be easy to carry. It should also be powered adequately by rechargeable, light-weight batteries so that back up batteries can be brought in to the field. An aspirator that is easy to use and carry help ensure that inaccuracies with aspira tions are not due to user fatigue. Methods used to monitor other insects may pr ove to be useful in monitoring plants for mosquitoes. Sticky traps are used to monitor other insects including hou se flies (Steiner 1999, Sulaiman et al. 1987) and thrips. This techni que, with modifications, may prove useful in evaluating the mosquitoes resting in plants. An important modification includes hanging the sticky paper horizontally thereby mimicking leav es. This approach utilizes the mosquito behavior whereby they rest on th e under sides of leav es (personal observati ons). The colors of the paper could also be select ed to increase attrac tiveness. Another possible collection method would be to place small resting boxes within th e vegetation. Resting boxes have been shown to be successful in collecting Anopheline species and may be useful in collecting resting Aedes albopictus (Skuse) (Harbison et al. 2006). 70

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Further study of barrier sprays ag ainst the salt marsh mosquitoes Ochlerotatus taeniorynchus Wiedemann and Ochlerotatus sollicitans Walker could be successful. Barrier sprays would be an ideal way to treat for these mosquito species because spraying pesticides near bodies of water is not permissible. However, spot treatment has very little drift and would be ideal in this circumstance. Al so, the synchronous emergence of Oc. taeniorhynchus and Oc. solicitans can be predicted based on tide levels allowi ng for treatment to be preemptively applied prior to the emergence of the mosquitoes. Newly emerged mosquitoes are believed to rest in vegetation prior to host-seeking and this would be an ideal time to us e a residual spray on the vegetation. Barrier treatments may also be useful as a means to reduce arbovirus transmission. During times of drought mosquito and arbovirus reservoir interactions are increased due to population concentrations near water sources. Theref ore, it may be useful to treat vegetation and areas surrounding water sources to reduce mosquito / anim al reservoir interactions. Applying pesticides to different plant types may also require specific protocols due to the varied types and arrangements of leaves. For example, tall grass plants are difficult to spray and to visually verify that the blades of the plant ha ve been sufficiently treated. However, plants with horizontal leaves can be treated more thoroughly, because the applicators typically aim the mist downward. Extremely leafy plants also require ca re that all of the leaves are coated with pesticide by ensuring that the wand of the applicator is inserted with in the branches of the plant. Gaps in the treatment could compromise the trea tment by providing safe harbors of pesticide free leaves. Pesticide application methods could be compared to see which is most appropriate for each plant type. 71

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Multiple traps have been used as barriers to co mbat mosquitoes and other pests. The use of traps to remove mosquitoes rais es some interesting questions: Is one trap sufficient to protect a yard? Does one trap lure mosquitoes from the surrounding area and create a larger problem? How far are these mosquitoes traveling to get to the trap? From where are these mosquito es traveling to the trap? Are the trap captures an accurate measure of mosquitoes being removed from that immediate yard? Have the mosquitoes traveled a long distance to the trap? In what physiological state are these mosquitoes in? Are captured females host-seeking females? Are captured females seeking mates? If they are host seeking females, is it likely that they will rest in the vegetation on their way to the trap or will they fly right through the vegetation ba rrier to find a pesticide free resting area? Additional aspirations on the trap itself may al so prove useful. The trap stand is made of hollow tube and I observed mosquitoes flying out of the tube once while making repairs on the trap. Aspirating the tube may be another option for monitoring mosquito behavior around the trap. In surveying the homeowners from the fiel d study (Appendix A), none of them were willing to pay more than $150.00 for a mosquito c ontrol program. The methods utilized in this study far exceeded this amount. The cost of having Florida Pest Control perform this treatment from spring to fall would cost approximately $600.00. The traps, which have been discontinued 72

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by Coleman, can be purchased for approximately $350.00. In addition, the propane tanks must be changed every 14-18 days. Each tank costs approx imately $20.00 to refill. Also, the lures cost around $9.00 and must be replaced monthly. Operation of the trap during the first season would cost approximately $650.00, however, this cost would be reduced substantially the following season since the trap can be reused. Also, homeowners may not be willing to use pesticides for mosquito control around their homes. One homeowner wrote in on her survey that I try to avoi d pesticides due to having pets and as a general principle. I would be inclined to use a trap however if it would get rid of the mosquitoes. Another homeowner wrote about pesticides, it must be environmentally safe would prefer not to have to spray chemicals but was willing to spray DEET type of product when outside presumably as a personal repellant. Educating the public about the small amounts of pesticide that are applied may or may not encourage homeowners to purs ue pesticide treatments for mo squitoes. Robert Krieger (2005) reviews some common misconceptions held by the public about pesticides. He cites misinformation from media outlets as a major s ource of misconceptions. Overuse of pesticides, such as DDT, led to resistance and its subse quent banning in the United States by the EPA (1972). Despite the discontinue d use of DDT in the United States, the WHO (2006) has reevaluated its stance on the use of DDT as an indoor residual spray. The WHO now recommends the use of DDT as an indoor residua l spray in malaria endemic regions in addition to the use of insecticide treated bed nets and the use of anti-malarial medications. The goal of bioassays is to replicate what is seen in the field in a more controlled environment. If the mosquitoes are resting on the undersides of the leaves, does it make a difference if the tops of the leaf are used in this bioassay method? Comparisons of bioassays with 73

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the tops and bottoms of the leaves exposed s hould be conducted. The most important indication that this could be a factor is that mosquitoes tend to move under the leaves if they are not securely attached to the bottom. Results of bioass ays with fabrics not attached to the petri dish, documented that the mosquitoes wedged themselves under the fabric to the point that they would detach their legs. Another method to consider for running this type of bioassays would be to rest the leaves in tubes as used by Trout (2006). This may be more effective for several reasons. Using tubes would allow the mosquitoes to exhibit their natura l behavior of going to the underside of the leaf. Preparing the petri dishes was a time consuming process. Attaching double side tape to the bottoms so that the entire surface is covered with tape requires several steps. First the tape must be attached to one side of the tape to a plex iglass surface, cutting around a circular template with a razor, then the tape is pulled off of the plexig lass and carefully attached the tape to the bottom of the dish. It is important to do this carefully so that no bubbles appear and create an uneven surface to attach the leaves. The leaves must also be attached carefully so th at they will remain in place for 24 hours. Attaching the leaves in a way that ensures that the leaves are covering the entire surface of the taped dish takes between can also be time consuming depending on the size and shape of the leaves. The tape also introduces the possibility of unnece ssary mortality due to insect sticking to exposed tape. Swiping the surface of the leaves w ith a cotton ball can also take time to ensure that excess cotton is not protruding from the surface. The high number of replications in this type of experiment would benefit from the time save d by simply placing leaves in tubes verses the many stepped process of prepar ing the Petri dishes. Keeping bioassay methods simple would reduces the possibility of operator erro r and allow for more accurate measures. 74

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Conclusions Refining and investigating commercially availa ble mosquito treatments is necessary to ensure that these techniques are useful in decreasing mosquito populations around the home. Improving these methods will also create relief for homeowners and allow them to enjoy their yards and homes during periods of mosquito acti vity. In addition to re ducing nuisance mosquito problems, these techniques may also be usef ul in the prevention of arbovirus transmission. 75

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APPENDIX A HOMEOWNER SURVEY A preliminary survey was taken to understa nd the areas of use in each yard and the homeowners general attitude towards mosquito control and mosquito activity. The homeowners were asked six questions. Each question had answers provided and allowed for additional comments. Question 1: What activities do you do around your home? Provided with the following options: a. Gardening how many hours? b. Grilling how many hours? c. Yard/home/car maintenance how many hours? d. Playing sports how many hours? e. Other how many hours? Site 1: 10 hours per week garden ing, 1 hour other yard maintenance Site 2: 7 hours per week garden ing, 4 hours other yard maintenance Site 3: 1 hour gardening, 1 hour gril ling, 1 hour other yard maintenance Site 4: 10 hours per week garden ing, 2 hours other yard maintenance Site 5: 2 hours per week gardeni ng, 10 hours other yard maintenance Site 6: 15 hours per week gardening, 3 hours per week grilling, 1 hour other yard maintenance Site 7: 4 hours per week gardening, 4 hours per week grilling, 3 hours other yard maintenance Site 8: none Question 2: How many mosquito bites would it take for you to end your outdoor activity? 76

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Provided with the following options: a. 1-4 b. 5-10 c. I would not change my activity level based on mosquito bites Site 1: 5-10 mosquito bites Site 2: I would not change my ac tivity level based on mosquito bites Site 3: I would not change my ac tivity level based on mosquito bites Site 4: 5-10 mosquito bites (Responder wrote in I may not stay outdoors as long but Id never give up doing gardening.) Site 5: I would not change my ac tivity level based on mosquito bites Site 6: 5-10 mosquito bites Site 7: 1-4 mosquito bites Site 8: 5-10 mosquito bites Question 3: How many more hours per week w ould you spend outdoors if there were fewer mosquitoes? Site 1: zero Site 2: blank Site 3: 2-4 hours Site 4: The heat and humidity drives me in over the mosquitoes. Site 5: zero Site 6: 20% Site 7: 2-4 hours Site 8: 3-5 hours 77

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Question 4: If you find that mosquito activity is unusually high would you? Provided with the following options: a. Call the local mosquito control agency for service b. Hire a private company c. Purchase pesticide and apply yourself d. Purchase a trapping device e. Reduce breeding sources in your yard f. Stay indoors more Site 1: Call the local mosquito control agen cy for service, Use Off on arms and legs Site 2: Reduce breeding sources in yard, Use more DEET t ype of product when outside Site 3: Stay indoors more Site 4: Reduce breeding sources in your yard, Stay indoors more Site 5: Reduce breeding sources in your yard, Stay indoors more Site 6: Stay indoors more Site 7: Purchase pesticide and apply yourself, Stay indoors more Site 8: Call the local mosquito control agen cy for service, Reduce breeding sources in your yard, Stay indoors more Question 5: Do you feel that mo squito activity this summer is: Provided with the following options: a. Worse than last year b. The same as last year c. Were not living in this home last year Site 1: The same as last year 78

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Site 2: Worse than last year Site 3: Worse than last year Site 4: The same as last year Site 5: The same as last year Site 6: The same as last year Site 7: Worse than last year Site 8:Were not living in this home last year Question 6: How much money w ould you be willing to spend for an effective mosquito reduction program around your home? Provided with the following options: a. $0.00 b. $1.00 50.00 c. $51.00 150.00 d. $150.00 or more Site 1: $1.00 50.00 Site 2: $51.00 150.00 Additional comment: A year, it must be environmentally safe would prefer not to have to spray chemicals Site 3: $1.00 50.00 Additional comment: I try to avoid pesticides due to having pets and as a general principle. I would be inclined to use a trap however if it would get rid of the mosquitoes. Site 4: $51.00 150.00 Site 5: $1.00 50.00 Site 6: $1.00 50.00 79

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Site 7: $51.00 150.00 Site 8: $1.00 50.00 80

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APPENDIX B PROPANE USAGE TRIAL Three Mosquito Deleto 2500 traps were run to measure the amount of propane used daily and to investigate possible sources of error in restarting the tra p. Traps were set up without the octenol lures and nets were not placed onto the co llection area so that the fan mechanism could be observed while it was running. The propane tanks were removed from the trap and weighed at one hour intervals for the first eight hours. Following the firs t eight hours, the tanks were weighed one time per day until the propane ran out of the tank. Traps were started by holding the starter button for exactly one minute. After one minute had passed the start button was pushed. If there was no ignition popping sound observed, the tank was checked to ensure that the trap was s ecurely fastened to the propane tank. The starter button was held for another minute and the ignition button was pressed again. This process was repeated, sometimes up to three times, until the ignition sound was heard. Following the ignition sound, the starter button was depressed and the ignition button was pressed every ten seconds until the fan was heard to be working. The star ter button was held for one minute following the fan turning on. The process was timed using th e second hand on the observers wrist watch. The process took between th ree and five minutes. The trap was not restarted during the trial unless the fan was not running afte r the tank was weighed. Traps ran for an average of 18.3 days. The average amount of propane used was 8.75 kg. Trap one failed on three occasions, trap three failed on two occasions, and trap two did not have any failures. The two traps experiencing failures ran for two additional days. Failures were probably due to not securing the propane tank properly to the trap after the weighing (Table A1). The components of the trap and the propane tank are sometimes difficult to properly align due to differences in the tanks and corresponding tank fasteners. 81

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Failures of this nature could be due to the di fficulty in ensuring that the trap is fastened properly. The fan continues to run after the propane tank is detached and there is no way to alert the user that the trap is not properly attached to the propane tank until the trap stops running. The trap will fasten to the tank even if it is not properly aligned. In these instances, the attachment feels like it is aligned and secure ly attached. Secure alignment can be difficult to achieve and the directions that accompany the trap should have a note for users to ensure proper seal of the tank when changing the propane tank. The instructions should also recommend that users check the trap 20 minutes after changing the tank to ensure that the proper seal was attained in changing the propane tank. 82

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Table B-1. Amount of propane used and days of operation by 3 Mosquito Deleto 2500 traps Trap 1 Trap 2 Trap 3 Average Start weight (kg) 16.9 17.1 17.6 17.2 End weight (kg) 8.4 8.2 8.7 8.5 Propane used (kg) 8.5 8.9 8.9 8.8 Days operating (kg) 19.0 17 19.0 18.3 Failures 3.0 0.0 2.0 1.7 Propane per day 0.5 0.5 0.5 0.5 83

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LIST OF REFERENCES Abbott, W.S. 1925. A method of computing the effectiveness of an in secticide. J. Econ. Entomol. 18:265-267. Ali, F.A.F., A. El-Refai, A. Abdel-Ra hman, and S.A. Abou-Donia. 1993. Residual effectiveness of insecticide formulations against German and American cockroach adults. Int. Pest. Control 35:70-74. Amerasinghe, F.P., and T.S.B. Alagoda. 1984. Mos quito oviposition in bamboo traps, with special reference to Aedes albopictus Aedes novalbopictus and Amigeres subaltus Insect Sci. Applic. 5: 493-500. Anderson, A.L., C.S. Apperson, and R. Knake. 1991. Effectiveness of mist-blower applications of malathion and permethrin to foliage as barrier sprays for salt marsh mosquitoes. J. Am. Mosq. Control Assoc. 7: 116-117. Berry, R., S.R. Joseph, and G.S. Langford. 1965. The question of area mosquito repellency, pp. 190-193. In Proc 52 nd Annual Meeting New Jersey Mosquito Extermination Association 24-27 March 1965, New Jersey. Black, R.J. 2003. Selected Shrubs for North Fl orida, Circular 500. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida. Bransby-Williams, W.R. and C. Webley. 1965. The effects of age and feeding on the susceptibility of adult female Anopheles gambiae, Aedes aegypti, and Culex pipiens fatigans Ann. Trop. Med. Parasitol. 59: 95-98. Briegel, H., and S.E. Timmerman. 2001. Aedes albopictus (Diptera38: Culicidae) Physiological aspects of development a nd reproduction. J. Med. Entomol. 38: 566-571. Campbell, C.B. 2003. Evaluation of five mosquito traps and a horse for We st Nile Vectors on a north Florida equine facility. M.S. Thesis, Un iversity of Florida, Gainesville, Florida. Centers for Disease Control. 2005. Information on Aedes albopictus http://www.cdc.gov/ncidod/dvbid/arbor/a lbopic_new.htm accessed April 5, 2007. Chowdhury, A.N.U., P.C. Jepson, P.E Howse, and M.G. Ford. 2001. Leaf surfaces and the bioavailability of pesticide residues. Pest Manag. Sci. 57: 403-412. Cilek, J.E., and C.F. Hallmon. 2006. Residual eff ectiveness of pyrethroid-treated foliage against adult Aedes albopictus and Culex quinquefasciatus in screened field cages. J. Am. Mosq. Control Assoc. 22: 725-731. Clements, A.N. 1992. The biology of mosquitoes, Volume 2 sensory reception and behavior. CABI Publishing, New York, New York. 84

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Coleman Outdoor Company. 2006. Mosquito Deleto 2500 Active System. http://www.coleman.com/coleman/coleman com/detail.asp?product_id=2920-100 access April 30, 2007. Darsie, R.F., and R.A. Ward. 2004. Identifica tion and geographical distribution of the mosquitoes of north America, North of Mexico. University Press of Florida, Gainesville, FL. Dieng, H., M. Boots, N. Tuno, Y. Tsuda, a nd M. Takagi. 2002. A laboratory and field evaluation of Macrocyclops distinctus, Megacyclops viridis and Mesocyclops pehpeiensis as control agents of the dengue vector Aedes albopictus in a peridomestic area in Nagasaki, Japan. Med. Vet. Entomol. 16: 285. Environmental Protection Agency. 1972. DDT ban takes effect.http://www.epa.gov/ 35thanniversary/topics/ddt/01.htm accessed March 25, 2007. Estrada-Franco, J.G., and B.C. Craig. 1995. Biology, disease, and control of Aedes albopictus Technical Paper No. 42. Pan American Health Organization. Fansiri, T., U. Thavara, A. Tawatsi n, S. Krasaesub, and R. Sithiprasasna. 2006. Laboratory and semi-field evalua tion of Mosquito Dunks against Aedes aegypti and Aedes albopictus larvae (Diptera: Culicidae). S.E. Asian J. Trop. Med. Pub. Health 37: 62-66. Fay, R.W. and W.H. Prince. 1970. A modified visual trap for Aedes aegypti Mosq. News 30: 20-23. Florida Coordinating Council on Mosquito C ontrol 1998. Florida mosquito control: The state of the mission as defined by mosquito co ntrollers, regulators, and environmental managers. University of Florida; Vero Beach, Florida. (FDEP) Florida Department of Enviro nmental Protection. 2004. Salt Marshes. http://www.dep.state.fl.us/coastal/habitats/saltmarshes .htm accessed April 23, 2007. Ford, M.G. and D.W. Salt. 1987. Behaviour of in secticide deposits and their transfer from plant to insect surfaces, pp. 2681. In H.J. Cottrell (ed.), Pesticides on plant surfaces. John Wiley & Sons, United Kingdom. Frick, T.B., and D.W. Tallamy. 1996. Density and diversity of nontarget insects killed by suburban electric insect traps. Ent. News 107: 77-82. Geden, C.J., D.A. Rutz, J.G. Scott, and S. J. Long. 1992. Susceptability of house flies (Diptera: Muscidae) and five pupal parasi toids (Hymenoptera: Pteromalidae) to abamectin and seven commercial ins ectides. J. Econ. Entomol. 85: 1241-1246. 85

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Gerhardt, R.R., K.L. Gottfried, C.S. Apperson, B.S. Davis, P.C. Erwin, A.B. Smith, N.A. Panella, E.E. Powell, and R.S. Nasci. 2001. First Isolation of La Crosse virus from naturally infected Aedes albopictus. Emerg. Infect. Dis. 7: 807-811. Gilman, E.F. 1999a. Callicarpa americana fact sheet FPS-90. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida. Gilman, E.F. 1999b. Ilex cornuta Bordii Nana fact sheet FPS-263. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida. Gilman, E.F. 1999c. Rhododendron simsii fact sheet FPS-507. Inst itute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida. Gilman, E.F. 1999d. Rhododendron x fashion fact sheet FPS-508. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida. Gilman, E.F. 1999e. Spartina bakerii fact sheet FPS-554. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida. Gilman, E.F. and S.P. Brown. 2003. Florida guid e to environmental landscaping. Circular 922 Institute of Food and Agricultural Sciences, Un iversity of Florida, Gainesville, Florida. Gilman, E.F. and D.G. Watson. 2006. Magnolia grandiflora : Southern magnolia ENH-530 Institute of Food and Agricultural Sciences, Univ ersity of Florida, Ga inesville, Florida. Gottfried, K.L., R.R. Gerhardt, R.S. Nasci, M.B. Crabtree, N. Karabatsos, K.L. Burkhalter, B.S. Davis, N.A. Panella, a nd D.J. Paulsen. 2002. Temporal abundance, survival rates, and arbovirus isolation of field-collected co ntainers inhabiting mosquitoes in eastern Tennesee. J. Am. Mosq. Control Assoc. 18: 164-172. Gratz, N. 2004. Critical review of the vector status of Aedes albopictus Med. Vet. Entomol. 18:215-227. Gubler, D.J. 2002. The global emergence / resu rgence of arboviral diseases as public health problems. Arch. Med. Res. 33: 330-342. Gubler, D.J., and N.C. Bhattacharya. 1971. Observations on the reproductive history of Aedes ( Stegomyia) albopictus in the laboratory. Mosq. News 31 356-359. Gubler, D.J., and N.C. Bhattacharya. 1972. Swarming and mating of Aedes ( S.) albopictus in nature. Mosq. News 32:219 223. Hadaway, A.B. and F. Barlow. 1956. Effects of age, sex and feeding on the susceptibility of mosquitoes to insecticides. A nn. Trop. Med. Parasitol. 50: 438-443. 86

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Halliday, W.R., and R. Feyreisen. 1987. Why does DDT toxicity change after a blood meal in adult female Culex pipiens ? Pest. Bioch. Phys. 28: 172-181. Harbison, J.E., E.M. Mathenge, G.O. Misiani, W.R. Mukabana, and J.F. Day. 2006. A simple method for sampling indoor resting malaria mosquitoes Anopheles gambiae and Anopheles funestus (Diptera: Culic idae) in Africa. J. Med. Ent. 43: 473-479. Hassan, A.A., C.R. Adanan, and W.A. Rahman. 1996. Patterns in Aedes albopictus (Skuse) population density, host-seeking, and oviposition behavior in Penang, Malaysia. J. Vect. Eco. 21: 17-21. Hawley, W.A. 1988. The biology of Aedes albopicuts. J. Am. Mosq. Control Assoc. 4: 2-39. Hoel, D.F. 2005. Response of Aedes albopictus (Diptera: Culicidae) to traps, attractants, and adulticides in north central Florida. Ph.D dissertation, University of Florida, Gainesville, Florida. Hoel, D.F., D.L. Kline, A. Grant, and S.A. Allan. 2007. Field evaluation of a ttractant-baited traps for Aedes albopictus (Skuse). J. Am. Mosq. Control Assoc. 23: 11-17. Holck, A.R., C.L. Meek, and J.C. Holck. 1988. Attractant enhanced ovitraps for the surveillance of container br eeding mosquitoes.J. Am. Mosq. Control Assoc. 4:97-98. Holick, J., A. Kyle, W. Ferraro, R.R. De laney, and M. Iwaseczko. 2002. Discovery of Aedes albopictus infected with West Nile virus in southeaster Pennsylvania. J Am Mosq Control 18:131. Jensen, T., O.R. Willis, T. Fukuda, and D.R. Barnard. 1994. Comparison of Bi-Directional Fay, Omni-Directional, CDC, and D uplex Cone traps for sampling adult Aedes albopictus and Aedes aegypti in North Florida. J. Am. Mosq. Control Assoc. 10:74-78. Kay, B.H., G. Prakash, and R.G. Andre. 1995. Aedes albopictus and other Aedes ( Stegomyia) species in Fiji. J. Am. Mosq. Control Assoc. 11:230-234. Kanda, T., D. Bunnag, V. Deesin, T. Deesin, S. Leemingsawat, N. Komalamistra, K. Thimasaran, and S. Sucharit. 1995. Integration of control measures for malaria vectors in endemic areas of Thailand. S.E. Asia n J. Trop. Med. Pub. Hlth. 26: 154-163. Kerdpibule, V., T. Deesin,and S. Sucharit. 1978. Residual effectivene ss of insecticidal deposits on various wall surfaces. S.E. Asia n J. Trop. Med. Pub. Hlth. 9: 423-426. Kirkwood, R.C. 1987. Uptake and movement of he rbicides from plant surfaces and the effects of formulation and environment upon them, pp. 1-25. In H.J. Cottrell (ed.), Pesticides on Plant Surfaces. John Wiley & Sons, United Kingdom. 87

PAGE 88

Kline, D.L. 2002. Evaluation of various models of propane-powered mosquito traps. J. Vect. Ecol. 27: 1-7. Kline, D.L., G.F. Lemire. 1998. Evaluation of attractant-baited traps/targets for mosquito management on Key Island, Florida, USA. J. Vect. Ecol. 23: 171-185. Krieger, R. 2005. Reviewing some origins of pesticide perceptions. Outlooks on Pest Management.12: 244-248. Konishi, E. 1989. Culex tritaeniorhynchus and Aedes albopictus (Diptera: Culicidae) as natural vectors of Dirofilaria immitis (Spirurida: Filariidae) in Miki City, Japan. J. Med. Ent. 26: 294-300. Mitchell, C.J. 1991. Vector competence of North and South American strains of Aedes albopictus for certain arboviruses: a review. J. Am. Mosq. Control Assoc. 7:446-451. Mitchell, C.J., L.D. Haramis, N. Karabatsos, G.C. Smith, and V.J. Starwalt. 1998. Isolation of La Crosse, Cache Valley, and Potsoi viruses from Aedes albopictus (Diptera: Culicidae) collected at used-tires sites in Illinois during (1994-1995). J. Med. Entomol. 35: 573-577. Mogi, M. 1982. Variation in ovi position, hatch rate and seta l morphology in laboratory strains of Aedes albopictus Mosq. News 42: 196-200. Mori, A. 1979. Effects of larval density and on some attributes of immature and adult Aedes albopictus Trop. Med. 21: 85-103. Niebylski, M.L. and C.J. Craig. 1994. Dispersal and survival of Aedes albopictus at a scrap tire yard in Missouri. J. Am. Mosq. Control Assoc. 10: 339-343. OMeara, G.F., A.D. Gettman, L.F. Evans, Jr., and G.A. Curtis. 1993. The spread of Aedes albopictus in Florida. Am. Entomol. 39: 163-172. Pena, C.J., G. Gonzalvez, and D.D. Chadee. 2004. A modified tire ovitrap for monitoring Aedes albopictus in the field. J. Vect. Ecol. 29: 374-375. Perich, M.J., M.A. Tidwell, S.E. Dobson, M.R. Sardelis, A. Zaglul, and D.C. Williams. 1993. Barrier spraying to control the malaria vector Anopheles albimanus : laboratory and field ev aluation in the Dominican Republic. Med. Vet. Entomol. 7: 363-368. Richards, S.L., L. Ponnusamy, T.R. U nnasch, H.K. Hassan, and C.S. Apperson. 2006. Host-feeding patterns of Aedes albopictus (Diptera:Culicidae) in relation to availability of human and domestic animals in suburban landscapes of central North Carolina. J. Med. Entomol. 43: 543-551. 88

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Santana, A.L., R.A. Roque, and A.E. Eiras. 2006. Charecteristics of grass infusions as oviposition attractants to Aedes ( Stegomyia) (Diptera: Culicidae). J. Med. Entomol. 43: 214-220. Sardelis, M.R., M.J. Turell, M.L. OGuinn, R. G. Andre, and D.R. Roberts. 2002. Vector competence fo three North American strains of Aedes albopictus for West Nile virus. J. Am. Mosq. Control Assoc. 18:284-289. Savage, H., M. Niebylski, G. Smith, C. Mitchell, and G.B. Craig. 1993. Host-feeding patterns of Aedes albopictus (Diptera:Culicidae) at a temperate North American site. J. Med. Entomol. 30: 27-33. SAS Institute. 2004. SAS 9.1.3 Help and Documentation, Cary, NC. Service, M.W. 1993. Mosquito Ecology Field Sa mpling Techniques, Second Edition. Chapman & Hall. Shirai, Y., H. Funada, K. Kamimura, T. Seki and M. Morohashi. 2002. Landing sites on the human body preferred by Aedes albopictus. J. Am. Mosq. Control Assoc. 18: 97-99. Shone, S.M., P.N. Ferrao, C.R. Lesser, G.E. Glass, and D.E. Norris. 2003. Evaluation of carbon dioxide and 1-octen-3-ol baited Cent ers for Disease Contro l Fay-Prince traps to collect Aedes albopictus J. Am. Mosq. Control Assoc. 19: 445-447. Shroyer, D.A. 1986. Aedes albopictus and arboviruses: a concise review of the literature. J Am Mosq Control Assoc 2: 424-428. Skuse, F. A. A. 1896. The banded mosquito of Bengal. Indian Museum Notes Vol. 3: 20. Sprenger, D., and T. Wuithiranyagool. 1986. The discovery and distribution of Aedes albopictus in Harris County, Texas. J. Am. Mosq. Control Assoc. 2: 217-19. Standfast H., I. Fanning, L. Maloney, D. Pu rdie, and M. Brown. 2003. Laboratory and field evaluation of BISTAR 80SC as an effective insecticide harbourage treatment for the biting midges (Culicoides) and mosquitoes infe sting peridomestic situations in an urban environment. Bull. MCAA Inc. 15: 19-33. Steiner, M.Y., L.J. Spohr, I. Barchia, and S. Goodwin. 1999. Rapid estimation of numbers of whiteflies (Hemiptera: Aleurodidae) and thrips (Thysanoptera: Thripi dae) on sticky traps. Aust. J. Entomol. 38: 367. Sulaiman, S., H. Yunus, and R. Sohadi. 1987. Ev aluation of some adhesives for collecting Musca domestica and Chrysoma megacephala adults or mosquito larvae in sticky traps. Med. Vet. Entomol. 1: 273-278. 89

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Swanson, J., M. Lancaster, J. Anderson, M. Cr andell, L. Harmis, P. Grimstad, and U. Kitron. 2000. Overwintering and establishment of Aedes albopictus (Diptera: Culicidae) in an urban La Crosse virus enzootic site in Illinois. J. Med. Entomol. 37: 454-460. Thavara, U., M. Takagi, Y. Tsuda, and Y. Wada. 1989. Preliminary field experiments on the oviposition of Aedes albopictus in water with different qualities. Trop. Med. 31: 167-169. Trexler, J.D., C.S. Apperson, and C. Schal. 1996. Diel oviposition patterns of Aedes albopictus (Skuse) and Aedes triseriatus (Say) in the laboratory and the field. J. Vect. Eco. 22: 64-70. Trout, R.T. 2006. Suppressing peridomestic mosquitoes utilizing residual insecticides on residential properties. M.S. thesis University of Kentucky, Lexington. Ware, G.W. 1989. The pesticide book, 3 rd ed. Thomson Publications, Fresno, California. World Health Organization. 2006. WHO gives indoor use of DDT a clean bill of health for controlling malaria. Intl. Pest. Cont. 48: 314-315. Xue, R. and D.R. Barnard. 2003. Boric acid bait kills adult mos quitoes (Diptera: Culicidae). J. Econ. Entomol. 96: 1559-1562. Yadav, R.S., H.C. Srivastava, T. Adak, N. Nanda, B.R. Thapar, C.S. Pant, M. Zaim, and S.K. Subbarao. 2003. House-scale evaluati on of bifenthrin indoor residual spraying for malaria vector control in India. J. Med. Entomol. 40:58. 90

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BIOGRAPHICAL SKETCH Melissa Ann Doyle was born in Gloucester, Massachusetts to Madeline and Paul Doyle. The family moved to San Diego, California where Melissa lived until graduating from San Dieguito High School in Encinita s, California. She attended Nort heastern University where she changed majors nearly every quarter, rowed on the crew team, and worked at the Gillette Company managing their Lotus Notes database sy stem. She began a bachelors of science degree in biology at the University of Massachusetts, Boston. She took a general entomology course in her junior year and found that mosquitoes were fascinating creatures to work with and she decided to pursue graduate education in entomology. She worked at the Massachusetts Department of Healths West Nile virus surv eillance program. After graduation, Melissa worked as the biological technician at the Anastasia Mosquito Control District in St. Augustine from until entering the University of Florida. After graduation she will continue her studies at the University of Florida 91


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