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Behavioral Phototaxis of Previtellogenic and Vitellogenic Mosquitoes to Light Emitting Diodes

Permanent Link: http://ufdc.ufl.edu/UFE0021967/00001

Material Information

Title: Behavioral Phototaxis of Previtellogenic and Vitellogenic Mosquitoes to Light Emitting Diodes
Physical Description: 1 online resource (177 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anopheles, diode, light, mosquito, resting, trap, vision, visualometer, vitellogenesis, wavelength
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: Mosquito wavelength preferences for light emitting diodes (LEDs) were examined using resting boxes and LED equipped light boxes in North Central FL. Wavelength preferences among two physiologically aged mosquitoes were determined using a visualometer (open-port and paired-T configuration). Wavelengths evaluated were blue (470 nm), green (502 nm), red (660 nm) and infrared (IR (860 nm)). Resting boxes fitted with IR LEDs attracted 23% of all mosquitoes recovered from resting boxes. Significantly more Anopheles quadrimaculatus Say females were aspirated from resting boxes fitted with red LEDs than all other treatments. Culex erraticus Dyar and Knab females were recovered in significantly (p = 0.05) higher numbers from resting boxes fitted with blue, green, or red LEDs or the no-light control than with IR LEDs. Approximately 47% of all mosquitoes trapped using LEDs fitted to sticky cards were captured on cards with green LEDs. Significantly more Aedes vexans Meigen females, Cx. nigripalpus Theobald females and Ochlerotatus infirmatus Dyar and Knab females were captured on sticky cards fitted with blue LEDs than those with red or IR LEDs. Blue LED fitted sticky cards captured significantly more Cx. erraticus females than were caught on sticky cards using IR LEDs. In comparisons between previtellogenic and vitellogenic An. quadrimaculatus released into the open-port visualometer, previtellogenic mosquitoes recorded significantly higher contact seconds on red LEDs than did vitellogenic mosquitoes. Vitellogenic mosquitoes were in contact with blue LEDs for a longer period of time that were previtellogenic mosquitoes. In paired-T port comparisons, no significant differences in contact seconds for previtellogenic or vitellogenic An. quadrimaculatus were recorded among blue and red or blue and green LED pairs respectively.
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.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Kaufman, Phillip Edward.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021967:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021967/00001

Material Information

Title: Behavioral Phototaxis of Previtellogenic and Vitellogenic Mosquitoes to Light Emitting Diodes
Physical Description: 1 online resource (177 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anopheles, diode, light, mosquito, resting, trap, vision, visualometer, vitellogenesis, wavelength
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: Mosquito wavelength preferences for light emitting diodes (LEDs) were examined using resting boxes and LED equipped light boxes in North Central FL. Wavelength preferences among two physiologically aged mosquitoes were determined using a visualometer (open-port and paired-T configuration). Wavelengths evaluated were blue (470 nm), green (502 nm), red (660 nm) and infrared (IR (860 nm)). Resting boxes fitted with IR LEDs attracted 23% of all mosquitoes recovered from resting boxes. Significantly more Anopheles quadrimaculatus Say females were aspirated from resting boxes fitted with red LEDs than all other treatments. Culex erraticus Dyar and Knab females were recovered in significantly (p = 0.05) higher numbers from resting boxes fitted with blue, green, or red LEDs or the no-light control than with IR LEDs. Approximately 47% of all mosquitoes trapped using LEDs fitted to sticky cards were captured on cards with green LEDs. Significantly more Aedes vexans Meigen females, Cx. nigripalpus Theobald females and Ochlerotatus infirmatus Dyar and Knab females were captured on sticky cards fitted with blue LEDs than those with red or IR LEDs. Blue LED fitted sticky cards captured significantly more Cx. erraticus females than were caught on sticky cards using IR LEDs. In comparisons between previtellogenic and vitellogenic An. quadrimaculatus released into the open-port visualometer, previtellogenic mosquitoes recorded significantly higher contact seconds on red LEDs than did vitellogenic mosquitoes. Vitellogenic mosquitoes were in contact with blue LEDs for a longer period of time that were previtellogenic mosquitoes. In paired-T port comparisons, no significant differences in contact seconds for previtellogenic or vitellogenic An. quadrimaculatus were recorded among blue and red or blue and green LED pairs respectively.
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.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Kaufman, Phillip Edward.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021967:00001


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BEHAVIORAL PHOTOTAXIS OF PREVITELLOGENIC AND VITELLOGENIC
MOSQUITOES (DIPTERA: CULICIDAE) TO LIGHT EMITTING DIODES





















By

MICHAEL THOMAS BENTLEY


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

2008

































2008 Michael Thomas Bentley

































To my mother, Jill; my father, Mike; and my fiancee, Kristina









ACKNOWLEDGMENTS

I would like to express my sincere gratitude and appreciation to Dr. Phillip Kaufman, my

supervisory committee chair, for investing in me his expertise, guidance and patience. It was a

privilege to be his first master's student, and to share with him the most challenging and

rewarding journey I have experienced. His professional leadership and guidance will be carried

far beyond the field of science.

I would also like to thank my other committee members, Dr. Daniel Kline and Dr. Jerry

Hogsette, of the USDA-ARS, for their added support and assistance. Even with busy schedules,

they always made time to meet for professional or personal matters upon any request. It was a

rewarding and memorable experience to be educated and surrounded by such remarkable

scientists.

I personally would like to thank Dr. Jerry Butler for being my educator, mentor, and friend

through this journey. Entomology was always a love in my life, but he made it a passion. It has

been an honor and a privilege to study under him in science and in life. Using the field as a

classroom, he made learning an adventure rather than a task. I was never made to feel like an

employee, but more as a friend. His respect, curiosity and passion for life have helped shape me

into the scientist I am today. I appreciate all that he has contributed to my career and to my life.

Special thanks go to Dr. Sandra Allan of the USDA-ARS and her staff for their support

and assistance throughout my research. On short notice, she was always able to accommodate

any request without any hesitation. Without her assistance in acquiring mosquitoes from the

USDA-ARS colony, my final project would not have been possible. I owe her a thank you for

investing so much of her time and energy into this research.

I greatly appreciate Dr. Donald Hall for allowing me the opportunity to fund my schooling

by coordinating the Outreach program throughout my education. This has been a wonderful









experience to share my enthusiasm of entomology with so many children. To be an educator is

rewarding within itself, and I am extremely fortunate to have been given the chance to do so.

Thanks go to Dr. Saundra TenBroeck and her staff for their allowing me endless access to

the University of Florida Horse Teaching Unit. This facility was an integral part of my field

research for two years. Thank you for your patience and assistance.

I would like to express appreciation to those residents of the Prairie Oaks subdivision who

participated in my research. With limitless patience, they gladly allowed me free access to trap in

their backyards for two consecutive summers. Their enjoyment and excitement for my projects

made field work that much more enjoyable. Without their cooperation, this research would have

been impossible.

I also would like to thank my lab mates, Peter Obenauer and Jimmy Pitzer, for the great

times I have had while completing this master's degree. Having such good friends to walk the

road with me made these years fly by. Lab work, field work and writing would have been the

most tedious of tasks without their humor to pass the time. I thank them for the help, the laughs

and the memories.

My parents, Mike and Jill, have had a tremendous impact on my life and have made my

educational career possible. Their never ending love and support have carried me through an

extensive journey. Without them, I would not be where I am today. Sacrifice was never a

question when it came to me or my extended education, which is why I share this degree with

them both. I love, admire and appreciate them incredibly.

Most of all, I thank my fiancee Kristina for her never ending patience and love while

earning this degree. For every long night and early morning, she was there to see me through.









Her endless inspiration kept me focused and driven during the hardest of times. I am truly

blessed to have her in my life and love her eternally.










TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IS T O F T A B L E S ............. ..... ............ .........................................................10

LIST OF FIGURES .................................. .. .... ..... ................. 12

A B S T R A C T ......... ....................... ............................................................ 15

CHAPTER

1 LITERATURE REVIEW OF MOSQUITO BIOLOGY, IMPORTANCE AND
SURVEILAN CE..................... ............................ .. .. ....................17

Introdu action to M osquitoes................................................................................... ............ 17
L ife C y cle ..........................................................................17
E g g ................................................................................................. 1 7
L a rv a ........................ .................................................................................................. 1 8
P u p a ....................... .................................................................................................... 1 9
A du lt ......... ........................................................... .......................... 2 0
H a b ita t ........................ .. .................. .................................................................................. 2 1
Medical and Economic Importance ......................................................... ...............25
Vector Surveillance and M monitoring .............................................. ............... 30
M eth o d o lo g y ......................................................................................3 0
Species D diversity ................................. ............................ ..............31
Flight Range and H abits ................................ ........................................... 31
Resting Behavior ......................................................................... 34
P opu lation M on itorin g ............................................................................................... 3 5
M o squ ito A ttractio n ................................................................................................... 3 8

2 RESPONSE OF ADULT MOSQUITOES TO LIGHT EMITTING DIODES PLACED
IN R E STIN G B O X E S ................................................................42

In tro d u ctio n ................... ...................4...................2..........
M materials an d M eth o d s ..................................................................................................... 4 4
R testing B oxes ................................................................... ................... 44
Light Emitting Diodes and Battery Supplies............................................... 45
C D C L ig h t T rap .............................................................................................4 6
Site and R testing B ox Location ................................................................. 46
M eth o d o lo g y ........................................................................................4 7
Statistical A n aly sis ................................................................4 8
R e su lts ................... ...................4...................9..........
D iscu ssio n ................... ...................5...................2..........









3 FIELD RESPONSE OF ADULT MOSQUITOES TO WAVELENGTHS OF LIGHT
E M IT IT IN G D IO D E S ................................................................................. .....................70

In tro du ctio n ................... ...................7...................0..........
M materials and M methods ..................................... ... .. .......... ....... ...... 72
D iode Equipped B oxes .................................................. ........... ..... .............. 72
Light Emitting Diodes and Battery Supplies........................................ ............... 73
S tic k y C a rd s ............................................................................................................... 7 3
C D C L eight T rap ................................................................................. 74
Site and Sticky Card Trap Location ........................................ .......................... 74
M eth o d o lo g y ........................................................................................7 6
Statistical A n aly sis ................................................................77
R e su lts ................... ...................7...................7..........
D iscu ssio n ................... ...................8...................0..........

4 RESPONSES OF PREVITELLOGENIC AND VITELLOGENIC ANOPHELES
QUADRIMACULA TUS TO SELECTED WAVELENGTHS PRODUCED BY LIGHT
EM ITTIN G D IOD E ..................................................................................................... 98

Introduction ......... ......... ............................ ......... ............. ......... 98
M materials and M methods ...............................................................102
V isu alom eter ......... .. ...................................................................................... 102
Light Emitting Diodes ................. ..... .. ......... .......... ... ...... 103
M mosquitoes .........................................103
Open-Port Visualom eter Trials.................................................. 104
Paired-T Port V isualom eter Trials............................................... 105
M methodology ......................................... ...................................................... 105
S statistical A n aly sis .................................................................................................. 10 6
R e su lts .......... ............ ....... .... ........................................................................................... 10 6
O pen-Port V isualom eter ......................................... .............................................106
Paired-T Port Visualometer ............... ......... ...... .........107
D iscu ssio n .................. ....... .. .. ....................................................................................... 10 8

5 THE IMPORTANCE OF MOSQUITO WAVELENGTH PREFERENCE IN
TRAPPING AND POPULATION SAMPLING................... .. .......................................116

APPENDIX

A RESTING BOX AND MODIFIED CDC LIGHT-TRAP CAPTURES OF
M O SQ U ITO E S B Y LO C A TIO N ............................................................................. ........ 122

B STICKY CARD AND MODIFIED CDC LIGHT-TRAP CAPTURES OF
M OSQUITOES BY LOCATION ......................................................................................147

C RESPONSE OF PREVITELLOGENIC AND VITELLOGENIC ANOPHELES
QUADRIMACULATUS TO SELECTED LED WAVELENGTHS USING A
VISUALOMETER IN A PAIR-T AND OPEN-PORT DESIGN .................................157



8









L IST O F R E F E R E N C E S ............................................................................. ..........................163

B IO G R A PH IC A L SK E T C H ......................................................................... .. ...................... 177





















































9









LIST OF TABLES


Table page

2-1 Mean ( SE) numbers of mosquitoes/trap/night attracted to light emitting diodes of
four different wavelengths placed in resting boxes at the University of Florida Horse
Teaching Unit and Prairie Oaks Subdivision from July 2006 Sept. 2007 near
G ain esv ille, F L ............................................................................ 60

2-2 Total number of mosquitoes/trap night for six significant mosquito species captured
at the Horse Teaching Unit and Prairie Oaks Subdivision from July 2006 Sept.
2007 near G ainesville, F L ......................................................................... ...... 6 1

3-1 Mean ( SE) numbers of mosquitoes/trap/night attracted to light emitting diodes
producing four different wavelengths of light during 24 h trapping intervals at the
University of Florida Horse Teaching Unit and Prairie Oaks subdivision in
G ain esv ille, F L ............................................................................ 88

3-2 Number of mosquitoes/trap night for six mosquito species captured a the University
of Florida Horse Teaching Unit and Prairie Oaks subdivision ............... ..... ...........89

4-1 Mean numbers ( SE) of previtellogenic and vitellogenic Anopheles
quadrimaculatus attracted to selected wavelengths of light emitting diodes as
measured by mean contact seconds using an open port visualometer.............................112

4-2 Mean numbers ( SE) of previtellogenic and vitellogenic Anopheles
quadrimaculatus attracted to paired selected wavelengths of light emitting diodes as
measured by mean contact seconds using a paired-T port visualometer.........................112

A-i Evaluation of resting box catches for mosquito species captured at the Horse
Teaching Unit (HTU) from July 2006 Sept. 2007 near Gainesville, FL ....................122

A-2 Evaluation of resting box catches for mosquito species captured at the Prairie Oaks
(PO) subdivision from August 2006 Sept. 2007 near Gainesville, FL .......................129

A-3 Modified CDC light trap mosquito captures at the Horse Teaching Unit (HTU) from
July 2006 Sept. 2007 near Gainesville, FL. ........................................ ............... 136

A-4 Modified CDC light trap mosquito captures at the Prairie Oaks subdivision (PO)
from July August 2006 and May Sept. 2007 near Gainesville, FL..........................142

B-1 Mosquitoes captured in a modified CDC light trap at the University of Florida Horse
Teaching Unit from July August 2006 and May Sept. 2007 near Gainesville, FL....147

B-2 Mosquitoes captured in a modified CDC light trap at the Prairie Oaks subdivision
from July August 2006 and May Sept. 2007 near Gainesville, FL..........................152









C-1 Evaluation of previtellogenic Anopheles quadrimaculatus attraction to four selected
wavelengths of light emitting diodes using an open-port visualometer. .........................157

C-2 Evaluation of vitellogenic Anopheles quadrimaculatus attraction to four selected
wavelengths of light emitting diodes using an open-port visualometer. .........................158

C-3 Previtellogenic Anopheles quadrimaculatus attraction to 470 nm and 660 nm
wavelengths of light emitting diodes using a paired-T port visualometer......................159

C-4 Vitellogenic Anopheles quadrimaculatus attraction to 470 nm and 660 nm
wavelengths of light emitting diodes using a paired-T port visualometer......................160

C-5 Previtellogenic Anopheles quadrimaculatus attraction to 470 nm and 502 nm
wavelengths of light emitting diodes using a paired-T port visualometer......................161

C-6 Vitellogenic Anopheles quadrimaculatus attraction to 470 nm and 502 nm
wavelengths of light emitting diodes using a paired-T port visualometer......................162









LIST OF FIGURES


Figure page

2-1 Resting boxes used at the University of Florida Horse Teaching Unit and Prairie
Oaks subdivision. A) Rear view of 30 x 30 cm resting box showing protective LED
housing. Exterior of all boxes were made using 1 cm thick exterior grade pine
plywood. The outside of each resting box was painted with two coats of flat black
exterior latex paint, and interiorly with two coats of barn red exterior latex paint.
Diode housing consisted of one 470 ml plastic container attached to the exterior rear
wall of each box by container lid. Container lids were modified with a 0.32 cm hole,
and matched to the 0.32 cm hole on the outside back wall of each resting box. B)
Front inside view of 30 x 30 cm resting box illustrating 5 cm x 5 cm x 29 cm
sections of pine used as inside corner supports. A 0.32 cm hole was drilled through
the back wall of each box to allow for the insertion of a LED. Resting boxes were
painted interiorly with two coats of barn red exterior latex paint..............................63

2-2 Light emitting diode configuration used in resting boxes. A) All round lens LEDs
were 8.6 mm long by 5.0 mm in diameter. Viewing angles were 300 except for IR
(200). After a 180-ohm resistor was soldered to each LED, restricting current flow, a
female 9 volt (V) battery snap connector (270-325) was attached. B) Battery housing
used to supply power to LED configurations for resting boxes. Battery supplies (270-
383) pre-equipped with a complimentary male 9 V connecting site were used, each
with a maximum holding capacity of four AA batteries. Four rechargeable 2500
milliamp hour (mAh) AA batteries were used in all assemblages....................... ..64

2-3 CDC light trap modified by the removal of its incandescent bulb. Modified trap used
a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5 cm diameter
clear plastic cylindrical body. A 36 cm diameter beveled edge aluminum lid was set
approximately 3 cm above the cylinder body creating a downdraft air current. All
traps were set 120 cm above ground using a Shepherd's hook, and collection nets
were attached to the bottom of the trap body. Carbon dioxide was provided from a 9
kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15-
psi single-stage regulator equipped with micro-regulators and an inline filter. ................64

2-4 Aerial view of Horse Teaching Unit location. The unit is located east of 1-75 and
approximately 1.6 km northwest of Paine's Prairie State Preserve, Alachua Co., FL......65

2-5 Aerial view of Prairie Oaks subdivision which was located approximately 4.8 km
southwest of the Horse Teaching Unit, adjacent to the Paine's Prairie Preserve,
A lachua C o., FL .............................................................................65

2-6 Test sites located within the Horse Teaching Unit. Each white rectangle represents a
test site where five boxes were equipped with one of five treatments. Sites are
numerically labeled according to corresponding eastern or western direction. White
arrow designates location of modified CDC trap. ........... .............................................66









2-7 Horse Teaching Unit location; west side test site habitat. ............................. ............... 66

2-8 Horse Teaching Unit location; east side test site habitat. ............. .................................. 67

2-9 Representative of test sites chosen at the Prairie Oaks subdivision. All sites chosen
were consistent in surrounding vegetation, sunlight exposure and moisture
conditions ..................................................................................67

2-11 Resting boxes placed with openings facing west and were spaced approximately four
meters apart and out of direct sunlight. Each site contained five treatments, one of
four LED colors and an unlit control, resulting in a total of five resting boxes per
site, 20 resting boxes per location ....................................................................... 68

2-12 Mean monthly temperatures (C) and precipitation (cm) for the Horse Teaching Unit
(HTU) location near Gainesville, FL, using data retrieved from the National Oceanic
and Atmospheric Administration (NOAA) database. A) Monthly temperature, May -
September 2006 and 2007. B) Monthly precipitation from Jan September 2006 and
2007....... ................................................................ 69

3-1 Four sided, diode-equipped pine boxes, each side measuring 400 cm2. Boxes were
constructed and designed to exteriorly support one 13 x 13 cm sticky card and one
diode treatment per side, yielding a total of four sticky cards and four light
treatm ents per diode box ............................. ...... .... .... ...... .......... .....91

3-2 Sticky cards were constructed from black 28 pt. SBS card stock with calendared
coating (EPA # 057296-WI-001), and coated with 32 UVR soft glue containing UV
inhibitors. Individual sticky cards, originally supplied as 41 x 23 cm boards, were cut
to yield tw o 13 x 13 cm sticky cards.......................................... ............................ 91

3-3 CDC light trap modified by the removal of its incandescent bulb. Modified trap used
a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5 cm diameter
clear plastic cylindrical body. A 36 cm diameter beveled edge aluminum lid was set
approximately 3 cm above the cylinder body creating a downdraft air current. All
traps were set 120 cm above ground using a Shepherd's hook, and collection nets
were attached to the bottom of the trap body. Carbon dioxide was provided from a 9
kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15-
psi single-stage regulator equipped with micro-regulators and an inline filter. ................92

3- 4 Aerial view of Horse Teaching Unit location. The unit is located east of 1-75 and
approximately 1.6 km northwest of Paine's Prairie State Preserve, Alachua Co., FL......93

3-5 Aerial view of Prairie Oaks Subdivision which was located approximately 4.8 km
southwest of the Horse Teaching Unit, adjacent to the Paine's Prairie Preserve,
A lachua C o., FL .............................................................................93

3-6 Representative of test sites chosen at the Prairie Oaks subdivision. All sites chosen
were consistent in surrounding vegetation, sunlight exposure and moisture
conditions ..................................................................................94









3-7 Test sites located within Prairie Oaks subdivision. Each solid white rectangle
represents a test site where one box equipped with one of four diode treatments was
placed. White dashed rectangles identify the location of modified CDC traps ................94

3-8 Test sites located within the University of Florida Horse Teaching Unit. Each white
square represents a test site where one diode box was equipped with one of four
diode treatments. White arrow represents location placement of modified CDC trap. .....95

3-9 University of Florida Horse Teaching Unit location. A.) Southeast side test site
habitat. B.) Northeast side test site habitat. C.) Northwest side test site habitat. D.)
South est side test site habitat. ............................................... .............................. 96

3-10 Mean monthly temperatures (C) and precipitation (cm) for the University of Florida
Horse Teaching Unit (HTU) location near Gainesville, FL using data retrieved from
the National Oceanic and Atmospheric Administration (NOAA) database. A)
Monthly temperature, May September 2006 and 2007. B) Monthly precipitation
from Jan Septem ber 2006 and 2007........................................ ............................ 97

4-1 Pie shaped visualometer with 10 available feeding stations, which can be portioned
off individually or left in an open design. A) Visualometer used in an open design,
with treatments placed at all odd numbered feeding stations. B) Visualometer in
operation showing treatments, set as described above. C) Visualometer used in a
paired-T configuration. ............. ........ ........... ............. 114

4-2 Anopheles quadrimaculatus obtained from the USDA-ARS-CMAVE Gainesville,
FL rearing facility held in an incubator at 26 C and 74% humidity under a 14:10
(L:D) photoperiod. Upon eclosion, adult mosquitoes were fed a 10% sugar solution....114

4-3 Blood feeding Anopheles quadrimaculatus occurred 120 h post-eclosion using a
blood ball. Blood ball's consisted of sausage casing and defribrinated bovine blood.
Adult mosquitoes were allowed to blood feed for 3 h. ................ ..... ..................115









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

BEHAVIORAL PHOTOTAXIS OF PREVITELLOGENIC AND VITELLOGENIC
MOSQUITOES (DIPTERA: CULICIDAE) TO LIGHT EMITTING DIODES
By

Michael Thomas Bentley

May 2008

Chair: Phillip E. Kaufman
Major: Entomology and Nematology

Mosquito wavelength preferences for light emitting diodes (LEDs) were examined using

resting boxes and LED equipped light boxes in North Central FL. Wavelength preferences

among two physiologically aged mosquitoes were determined using a visualometer (open-port

and paired-T configuration). Wavelengths evaluated were blue (470 nm), green (502 nm), red

(660 nm) and infrared (IR (860 nm)).

Resting boxes fitted with IR LEDs attracted 23% of all mosquitoes recovered from resting

boxes. Significantly more Anopheles quadrimaculatus Say females were aspirated from resting

boxes fitted with red LEDs than all other treatments. Culex erraticus Dyar and Knab females

were recovered in significantly (p = 0.05) higher numbers from resting boxes fitted with blue,

green, or red LEDs or the no-light control than with IR LEDs.

Approximately 47% of all mosquitoes trapped using LEDs fitted to sticky cards were

captured on cards with green LEDs. Significantly more Aedes vexans Meigen females, Cx.

nigripalpus Theobald females and Ochlerotatus infirmatus Dyar and Knab females were

captured on sticky cards fitted with blue LEDs than those with red or IR LEDs. Blue LED fitted

sticky cards captured significantly more Cx. erraticus females than were caught on sticky cards

using IR LEDs.









In comparisons between previtellogenic and vitellogenic An. quadrimaculatus released

into the open-port visualometer, previtellogenic mosquitoes recorded significantly higher contact

seconds on red LEDs than did vitellogenic mosquitoes. Vitellogenic mosquitoes were in contact

with blue LEDs for a longer period of time that were previtellogenic mosquitoes. In paired-T

port comparisons, no significant differences in contact seconds for previtellogenic or vitellogenic

An. quadrimaculatus were recorded among blue and red or blue and green LED pairs

respectively.









CHAPTER 1
LITERATURE REVIEW OF MOSQUITO BIOLOGY, IMPORTANCE AND SURVEILLANCE

Introduction to Mosquitoes

In 1877, Patrick Manson was the first to credit mosquitoes with disease transmission after

witnessing the development of Wuchereria bancrofti Cobbold in the mosquito Culexpipiens

quinquefasciatus Say (Chemin 1983). This discovery started what is known today as the Golden

Age of Medical Entomology, and helped mosquitoes gain their fearsome reputation as

transmitting some of the world's deadliest diseases. Currently, mosquitoes are implicated as

vectors of over 200 arboviruses to humans and other animals, such as encephalitis, yellow fever

and dengue (Lehane 2005). Of all known mosquito associated diseases, malaria is considered the

most severe, with over 2 billion people in 100 countries are at risk of infection each year (WHO

2007a).

There are approximately 3,200 recognized species of mosquitoes worldwide, occurring in

every continent with the exception of Antarctica (Lehane 2005). Belonging to the family

Culicidae, mosquitoes are recognized by current culicid classification as having three

subfamilies: Anophelinae, Culicinae, and Toxorhynchitinae (Foster and Walker 2002). A

diverse, highly adaptive and durable lifecycle has allowed mosquitoes to evolve side-by-side

with humans. Whether facing extended periods of drought in an urban setting or surviving

monthly monsoons in tropical forests, mosquitoes have adapted to thrive in many conditions.

Life Cycle

Egg

The holometabolous life cycle of mosquitoes begins with the deposition of an elongate,

ovoid or spindle-shaped egg, measuring approximately 0.4-0.6 mm in length (Forattini et al.

1997). Newly oviposited eggs begin white in color, and darken within 12 to 24 hours depending









upon surrounding moisture conditions (Breeland and Beck 1994). The outermost layer of the egg

shell, the chorion, is comprised of three reinforced layers. These reinforced layers not only

provide safety for the embryo, but also protect against dehydration. The chorion's outer most

layer consists of a network of complex patterns and surface boxes which are unique to each

mosquito species. In anopheline species, for example, the chorion has transparent, air filled

compartments lining either side of the egg that serve as floats following oviposition (Foster and

Walker 2002).

Eggs of some mosquito genera such as Anopheles and Aedes are individually oviposited on

the water's surface. Alternatively, eggs may be glued together to form rafts of up to 150 eggs, as

with Culex. In these conditions, hatch rates depend largely upon temperatures. In optimal

conditions larvae can emerge within 2 or 3 days after the eggs are laid (Stage et al. 1952). In

genera including Aedes, Ochlerotatus and Psorophora, oviposition may take place upon detrital

matter or just above the water line along the insides of containers. Egg hatch usually occurs at

warm temperatures after the eggs have been inundated and microbial activity has caused oxygen

levels in the water to drop (Foster and Walker 2002). If not flooded, Aedes and Ochlerotatus

eggs can survive in a quiescent state and accumulate for several years. Sudden temporary

flooding can allow accumulated eggs to hatch along with recently oviposited eggs, resulting in

mass emergences that can lead to public health threats (Breeland and Beck 1994).

Larva

All mosquito larvae are aquatic, molting through four instars before developing to the

pupal stage. When ideal conditions exist (26-28 C), most mosquito species can complete the

larval stage in five to six days with males usually pupating about 1 day earlier. Even under

optimum conditions, the larval stage for some mosquitoes such as Toxorhynchites or Wyeomyia









often takes as long as 2-3 weeks to complete. In most species, cooler temperatures (< 68 C)

slow the developmental process (Matheson 1944).

Respiration is usually achieved through the siphon or air tube located near the last

abdominal segment (Breeland and Beck 1994). The majority of mosquito larvae are required to

come to the water surface for oxygen. However, the siphons of Coquillettidia and Mansonia

have been modified into a short, heavily sclerotized saw-like box used to pierce and attach to

plant tissues in order to obtain oxygen (Bosak and Crans 2002). Larvae ofAnopheles lack a

siphon and diffuse oxygen through a series of small grouped abdominal plates. This causes the

larvae to lie flat at the surface of the water, a behavior characteristic of all Anopheles species

(Foote and Cook 1959).

Most mosquito larvae are filter feeders, living on a diet comprised of tiny plants, animals,

and organic debris (Stage et al. 1952). Palatal brushes located on the labrum circulate water and

debris over combs and sweepers located on the mandibles and maxillae, respectively. These

mouthparts collect and pack food particles, which are then passed into the pharynx for digestion.

The mouthparts of Toxorhynchites, however, are heavily sclerotized and sharply toothed,

designed for the predation of smaller invertebrates, including other mosquito larvae (Foster and

Walker 2002).

Pupa

The pupa is a non-feeding stage of development in a mosquito's life cycle. Mosquito pupae

are comma-shaped, with the head and thorax fused to form a cephalothorax and the abdomen

curled beneath it (Foster and Walker 2002). Pupae are often called tumblers because of their

quick tumbling-like defensive action in response to any light change in the surrounding

environment (AMCA 2007). Pupae of most species obtain oxygen at the water's surface through

two respiratory tubes, or air trumpets, which protrude from the dorsal mesothorax (Lehane









2005). Coquillettidia and Mansonia pupae remain attached to underwater plant tissues, diffusing

oxygen through a modified air trumpet, detaching just before eclosion (Crans 2004).

The entire pupal stage of most species typically lasts two to three days, depending on

temperature. Optimum temperatures for pupal development in most mosquito species range from

26 to 28 C. Some Culex species can complete the pupal stage in approximately two days during

the warm summer months (AMCA 2007). Other species, including Toxorhynchites and

Wyeomyia, cannot complete development in less than five to six days.

Adult

Emergence of adult mosquitoes is a relatively short process usually requiring no more than

20 minutes to complete. Changes in hormone levels signal the approach of emergence, causing

pupae to remain stationary at the waters surface. The abdomen gradually extends allowing

ingestion of enough air through the respiratory tubes to cause the cephalothorax to split. The

adult mosquito then emerges through this opening. Males tend to emerge before females due to

their shorter pupation periods (Foster and Walker 2002).

Newly emerged adults are capable of short flights within minutes, but must wait for the

cuticle to become fully sclerotized before sustaining longer ones. Some species will never travel

farther than a few hundred feet from their site of emergence, while others migrate 50 miles or

more (Breeland and Beck 1994). Adult mosquitoes are able to survive up to three days on lipid

and glycogen reserves carried over from the larval stage. Males of all species have mouthparts

modified to suck nectar and plant secretions. However the maxillae and mandibles of most

females are specially modified to pierce skin. Both sexes require nutrients from sugars found in

plant nectar and honeydew, but the females of most species are anautogenous, requiring a blood

meal for egg production. Females utilize hemoglobin proteins to synthesize vitellogenin,

stimulate egg growth and successfully oviposit (Lehane 2005). Several autogenous mosquito









species including Toxorhynchites and Culex are capable of oogenesis without taking a blood

meal. This is made possible in Toxorhynchites by synthesis of vitellogenin from proteins

obtained during their predacious larval stage (Klowden 1996).

Habitat

Mosquito habitats are generally classified in terms of a female oviposition preference for

permanent water, flood water, transient water or artificial container and tree-hole environments

(Breeland and Beck 1994). Behavioral differences in oviposition and life cycle development

between individual mosquito species play an important role in determining both larval and adult

habitats. These habitats range from fresh to salt water and can be natural or man made. Given

their weak swimming abilities, mosquito larvae are incapable of surviving in continuous moving

water. As a result, larvae occupy more stagnant water conditions such as pools and seepage areas

(Clements 1992). All mosquito species are grouped into two habitat categories; standing water

and flood water habitats as utilized by immature stages. Within these habitats, certain specific

requirements regarding habitat differentials play a critical role in habitat preference between

mosquito species.

The eggs of most standing water species do not tolerate desiccation. As a result,

oviposition typically takes place directly on the water surface, either singly or as rafts on

stagnant pools of water (Clements 1992). Eggs not tolerant to desiccation must hatch soon after

oviposition, influencing the life stage in which mosquitoes endure potentially fatal environmental

conditions. Most species such as Anopheles and Culex survive such harsh circumstances as

mated females (Crans 2004). One exception is that of Coquillettidiaperturbans Walker.

Overwintering in this species takes place during the larval stage of any instar trapped by the

onset of winter. As a result, cohorts of larvae emerge continuously over the course of the summer

(Bosak and Crans 2002).









Vegetation has a large impact on the habitats of several standing water mosquito species.

For example, Culiseta melanura Coquillett larvae thrive in fresh water swamps sparse in aquatic

foliage, whereas, An. quadrimaculatus Say and An. walker Theobald prefer freshwater bogs and

swamps with abundant aquatic vegetation (Horsfall and Morris 1952, Mahmood and Crans

1998). Mansonia and Coquillettidia species are even more selective, requiring specific aquatic

plants such as water lettuce, water hyacinth and cattails for both oviposition and larval habitat

(Hagmann 1953).

Standing water mosquito species are generally classified into two subgroups; permanent

water species and transient water species. Permanent water genera including Anopheles, Culex,

Coquillettidia, and Mansonia are found in established bodies of water such as marshes, swamps,

springs, ponds and lakes (Bentley and Day 1989). The larvae of these species are usually

restricted to the littoral zone where vegetation provides protection and water movement is at a

minimum (Newkirk 1955). However, the larvae of some Psorophora and Ochlerotatus species

are found throughout swamps and bogs, utilizing thick aquatic foliage or dense tree cover to hide

from predators (Laird 1988).

Transient water mosquito species are found in natural ditches, drainage ditches, borrow

pits, and canals (Crans 2004). In coastal habitats, natural ditches commonly run adjacent to

saltwater marshes, but can contain either fresh or brackish water. Ochlerotatus and Anopheles

are common genera found in these ditches because of the wide variety of aquatic vegetation

(Newkirk 1955). Drainage ditches are man-made habitats commonly found along pastures, at the

bottom of road shoulders, in abandoned fields or in lowland groves. These are common larval

habitats for several fresh water mosquitoes including Culex, Uranotania and Psorophora.

Burrow pits and canals are man-made bodies of water which usually remain undisturbed for









extended periods of time. After becoming overgrown with vegetation, these torpid pools become

productive breeding sites for species of Culex, Coquillettidia, and Mansonia (Hagmann 1953,

Slaff and Crans 1982, Clements 1992).

Floodwater mosquito habitats can be artificial or naturally occurring environments prone to

periodic flooding. These range in size from microhabitats such as tree holes and tires, to larger

isolated bodies of water including ground depressions and tidal pools (Matheson 1944).

Floodwater mosquito species commonly produce several broods annually, surviving harsh

environmental conditions in desiccation resistant eggs (King et al. 1960). Vegetation in and

around these habitats can vary greatly, influencing the species diversity from one habitat to the

next. For example, some Ochlerotatus species only oviposit in water containing the leaf litter of

red maple, Acre rubrum, cattail, or certain sphagnum swamp habitats (Clements 1992).

Wyeomyia species are also highly selective when locating a suitable larval habitat, ovipositing

just above the water line in a specific type of pitcher plant (Istock et al. 1975).

Floodwater mosquito species are classified into two subgroups. The first subgroup includes

non-container habitats such as rain and floodwater pools, mangrove swamps, and salt marshes

(Breeland and Beck 1994). Rain and floodwater pools serve as ideal breeding sites for several

mosquito species, especially those in the Psorophora, Aedes, and Ochlerotatus genera. These

habitats are unique in that they do not support true aquatic vegetation such as aquatic grasses,

often containing only leaves and other detrital matter that have settled to the bottom. Temporary

pools usually evaporate quickly in dry weather. As a result, a number of species in this group

rely on direct sunlight and high daytime temperatures to accelerate larval development before the

habitat dries (Crans 2004).









Mangrove swamp habitats are classified as transitional tidal zones that cycle from low to

high tide. Though mosquito breeding occurs throughout tidal zones, immatures and adults tend to

occur in highest numbers around peak tidal zones (Harwood and Horsfall 1959). Natural plant

and grass cover help to retain moisture, maintaining favorable oviposition conditions.

Ochlerotatus and Anopheles eggs will only hatch after being triggered by the alternate flooding

and drying tidal cycles (Bentley and Day 1989).

Few mosquito species are able to utilize the vast expanses of salt marsh wetlands because

of the unique aquatic vegetation and extremely high saline content. Salt-tolerant herbaceous

plants and grasses dominate these habitats, with sizeable areas often overrun by a single plant

species (Hulsman et al. 1989). Ochlerotatus taeniorhynchus Wiedemann and Oc. sollicitans

Walker are adapted to survive in these harsh conditions, and can take advantage of larval habitats

unsuitable for other floodwater mosquito species. These Ochlerotatus species also share intimate

relationships with the vegetation, breeding only where salt-tolerant plant species occur (Horsfall

and Morris 1952).

The second subgroup of floodwater mosquito habitats includes artificial and natural

containers. Most species in this group deposit eggs in bands just above the water line of these

microhabitats, providing additional substrate as evaporation progresses. Subsequent rainfall

events raise the water level immersing eggs, a requirement the eggs of most species in this group

must meet before hatching (Newkirk 1955). Artificial container habitats are classified as any

human-derived activity that results in a habitat in which mosquitoes can successfully complete a

life cycle. Structures that hold water, such as tin cans, rain barrels and clogged gutters, make

excellent breeding habitats for several species. Discarded tires are considered one of the most

problematic examples of artificial containers. Accumulated rain water and decomposing plant









material mimic natural breeding sites, creating an ideal larval habitat for several medically

important mosquito species (Means 1979). Therefore the practice of importing used tires poses a

health threat by contributing to the introduction of several exotic mosquito species including

Aedes albopictus Skuse and Ochlerotatusjaponicus Theobald (Morris and Robinson 1994,

Andreadis et al. 2001).

Tree hole habitats support an extensive and distinctive mosquito fauna with many species

breeding exclusively in these ecological niches (Breeland and Beck 1994). These isolated

habitats offer a great deal of protection from predators, making them ideal larval habitats for

several mosquito species. However, access to optimal tree hole habitats is not always possible.

Often, entrances to these microenvironments are small or blocked, preventing adult mosquitoes

from landing in order to deposit eggs. Some tree hole mosquito species have developed special

oviposition techniques to overcome these problems. For example, some Toxorhynchites species

are able to propel their eggs through small tree hole openings by flicking their abdomens (Linley

1987). While some Anopheles species oviposit aerially, depositing eggs while hovering above

vertical tree hole openings (Foster and Walker 2002).

Crab hole habitats are limited by the geographical distribution of land crabs in the families

Gecarcinidae and Ocypodidae. These habitats span from Florida and the Bahamas throughout

the northern Caribbean (Belkin and Hogue 1959). Deinocerites species are most noted for

utilizing crab holes as breeding habitats. Though no conclusive data have been published relating

specific Deinocerites species to a particular species of crab, members of the Spanius group have

consistently been trapped in the small burrows of certain fiddler crabs (Adams 1971).

Medical and Economic Importance

Mosquitoes are capable of transmitting hundreds of viruses, protozoans and filarial

nematodes to human beings (Karabatsos 1985). The most threatening diseases include malaria,









filariasis, yellow fever, dengue and the encephalitides (Foote and Cook 1959). These unbiased

diseases affect every culture on almost every continent, often leading to serious illness,

disfigurement and even death (Foster and Walker 2002). Because of this, mosquitoes are

considered to be the deadliest and most important vectors of disease to man (Beerntsen et al.

2000).

In 1877, Dr. Patrick Manson was the first to associate mosquitoes with a human related

illness after observing the development of the filarial worm, Wuchereria bancrofti,in the

mosquito Culexpipiens quinquefaciatus Say (Chernin 1983). His research demonstrated that

certain mosquito species were the intermediate hosts and vectors of lymphatic filariasis, a

parasitic disease caused by microscopic filarial worms (Matheson 1944). More than one billion

people in 80 countries throughout the tropics and sub-tropics of Asia, Africa, the Western Pacific

and South America are at risk for lymphatic filariasis. The equivalent of several billion U.S.

dollars is lost annually to medical costs and decreases in labor productivity resulting from

physical injury and deformities caused by lymphatic filariasis (CDC 2007a).

In 2000, the World Health Organization (WHO) initiated an elimination effort known as

the Global Alliance in hopes of counteracting the growing number of lymphatic filariasis cases.

Initial drug administrations were conducted, treating approximately 25 million people in 12

different at-risk countries. By 2005, over 250 million people in 39 countries were being treated

through mass drug administration. The program triumphed, surpassing all initial expectations

and becoming one of the most successful WHO efforts in history. The Global Alliance is

currently on track to meet their goal of elimination of lymphatic filariasis by 2020 (WHO 2006).

Mosquitoes were first incriminated as vectors of malaria to humans in 1897 by Dr. Ronald

Ross. There are four different species of protests that cause human malaria including Plasmodium









vivax, P. falciparum, P. malariae and P. ovale; P. falciparum being responsible for the most

deaths. These parasites can only be vectored to humans by mosquitoes belonging to the genus

Anopheles (Foote and Cook 1959).

Today, malaria is the recognized as one the world's most lethal diseases, primarily

affecting children and pregnant women. Although forty-one percent of the human population

lives in areas where malaria is transmitted, most cases are reported in parts of Africa (CDC

2007b). In all, 105 countries account for 300 to 500 million clinical cases and more than one

million deaths per year. Throughout the 1950's and 1960's, the WHO initiated a worldwide

malaria eradication program with increasing signs of success. However, the goal of global

eradication has faded over the past few decades because of the rapid increase in drug resistance

by parasites, as well as increasing insecticide resistance in mosquitoes (WHO 2007a).

Yellow fever is a viral hemorrhagic pathogen transmitted to humans by infected

mosquitoes. In 1900, research conducted by Dr. Walter Reed and his associates confirmed

previous experiments of Dr. Carlos Finlay, which pointed to Ae. aegypti Linnaeus as the primary

vector (King et al. 1960). Yellow fever continues to persist, with low levels of infection in most

tropical areas of Africa and the Americas. There are an estimated 200,000 cases of yellow fever

reported annually, 30,000 of which result in death (WHO 2007b).

Yellow Fever displays three distinctly different transmission cycles; sylvatic, intermediate

and urban (Foster and Walker 2002). The sylvatic or jungle cycle occurs in tropical rainforests

where the virus is transmitted to monkeys by zoophilic mosquitoes. Humans are infected when

they enter these regions and are fed on by mosquitoes. This type of cycle tends to be sporadic,

commonly affecting young men working within these enzootic forest areas. Transmission of the

more common intermediate cycle of yellow fever occurs in humid regions of Africa, producing









small localized epidemics in surrounding rural villages. Semi-domestic mosquitoes increase the

rate of contact with man, making this the most common transmission of yellow fever (WHO

2007c).

The urban cycle of yellow fever transmission is found primarily in village settings of

tropical Africa and South America. This cycle results in large explosive epidemics when the

virus is introduced into densely populated areas from rural travelers. Virus outbreaks tend to

spread outwards from one source with transmission by domestic mosquito species, primarily Ae.

aegypti (Foster and Walker 2002).

Dengue or "break-bone" fever is caused by a febrile virus occurring in tropical and

subtropical areas including Southeast Asia, Central America and South America. There are four

closely related, but antigenically distinct, serotypes of Dengue fever referred to as Dengue 1, 2, 3

and 4. In humans, this disease takes on one of two forms; classic dengue fever or the more severe

dengue hemorrhagic fever, also known as dengue shock syndrome (Foster and Walker 2002).

Aedes aegypti is the principle vector of dengue fever, although transmission is possible by other

Aedes species. Like yellow fever, dengue is a disease of monkeys, which serve as reservoirs

between epidemic periods (King et al. 1960).

In 2005, the Center for Disease Control (CDC) considered dengue fever the most

important mosquito-borne viral disease affecting humans. Its global distribution is comparable to

that of malaria, with an estimated 2.5 billion people living in areas at risk for epidemic

transmission. There are an estimated 50 to 100 million cases of dengue fever and several hundred

thousand cases of dengue hemorrhagic fever reported worldwide each year. Approximately 5%

of all cases result in fatalities, with the majority occurring among children and young adults.









Because no vaccine is available, the most successful method of disease suppression is directed

towards vector control (CDC 2007c).

The most important mosquito-borne diseases occurring in the United States are the

encephalitides. The five primary viral agents are West Nile virus (WNV), eastern equine

encephalitis (EEE), western equine encephalitis (WEE), St. Louis encephalitis (SLE) and La

Crosse encephalitis (LAC). Though encephalitides can successfully be vectored to humans and

domestic animals, these are usually dead-end hosts incapable of producing sufficient viremia to

contribute to the transmission cycle. Instead, these encephalitides amplify in hosts such as birds,

chipmunks and tree squirrels. Most human incidences of encephalitis occur in the warmer

months between June and September when mosquitoes tend to be most active. In warmer parts of

the country, where mosquitoes stay active late in the year, cases can occur during the winter

months (CDC 2007d).

Of the five encephalitides occurring in the United States, EEE is regarded as the most

serious because of its high mortality rate. Though it is maintained in birds by Cs. melanura, other

mosquito genera such as Aedes, Coquillettidia and Culex contain capable vectors. Eastern Equine

Encephalitis currently occurs in several localized distributions along the eastern seaboard, the

Gulf Coast and in some inland Midwestern locations of the United States (King et al. 1960).

Approximately 220 confirmed cases were reported in the United States from 1964 to 2004.

Florida sits atop the list of total reported cases, followed by Georgia, Massachusetts and New

Jersey. Though a vaccine is available to protect equines against EEE, no such prophylaxis exists

for humans. Currently, vector control methods such as wide area aerial sprays are utilized for

emergency situations (CDC 2007d).









Vector Surveillance and Monitoring


Methodology

A comprehensive assessment of vector surveillance and monitoring methods has been

extensively covered in Service's (1993) book, Mosquito Ecology Field Sampling Methods.

Information included in the next four paragraphs was included in his literature.

Most trapping methods are often baited with a host, or employ attractants such as carbon

dioxide or various forms of visual stimuli. These traps produce a bias when used in vector

surveillance and monitoring by primarily selecting for unfed, host seeking female mosquitoes.

Although some non-baited traps, such as truck mounted nets, give less biased mosquito

collections, these traps still select for the aerial population which is comprised largely of more

active unfed females.

Collections of resting mosquito populations yield a more accurate representative sample of

a mosquito population given that adults probably spend more time resting than in flight. These

collection methods would not only result in catching unfed host-seeking females, but would also

sample males, and both blood-fed and gravid females. Sampling resting mosquito populations

also yields a broad age structure.

Several non-biased methods exist to sample resting mosquito populations. When targeting

indoor resting mosquito species, including several Anopheles as well as some Culex, aspirators,

resting counts and knock-down sprays are commonly used. Though few mosquito species

commonly rest indoors, those that do are often important vectors of malaria, filariasis and some

arboviruses, making accurate sampling methods of these species a necessity.

Sampling outdoor resting mosquitoes is often more difficult because outdoor populations

are usually distributed over large areas and not concentrated in smaller locations. A better

understanding of the general resting habits of most exophilic species has allowed for the









development of more accurate surveillance methods. When sampling mosquito species known to

rest amongst grassy and shrubby vegetation, such as Psorophora columbiae Dyar and Knab,

aspirators or sweep nets have shown to be successful. However, the utilization of artificial

resting places is often the preferred sampling method, allowing for the attraction of mosquitoes

to a specific site from which they can be conveniently collected.

Species Diversity

Mosquitoes are found on almost every continent of the world. They are capable of

developing in a wide variety of ecological niches ranging from arctic tundra's and barren

mountain ranges to salt marshes and ocean tidal zones. Although the greatest species diversity

occurs in tropical forest environments, mosquitoes can also proliferate in ecologically poor

environments (Foster and Walker 2002).

There are approximately 3,200 known mosquito species worldwide (Day 2005). Within the

United States there are 174 known species and subspecies in 14 genera and 29 subgenera (Darsie

and Ward 2005). Florida, having an ideal subtropical climate in most central to southern regions,

has a unique and diverse fauna of mosquito species unlike most other states in the U. S. At least

11 mosquito species within the general Aedes, Culex and Psorophora are unique to FL.

Additionally, several other mosquitoes native to FL have extremely limited in-state distributions,

but are relatively abundant in other parts of the United States (Breeland 1982). Florida's

mosquito population is comprised of indigenous and introduced species within the genera of

Aedes, Anopheles, Coquillettidia, Culex, Deinocerites, Mansonia, Psorophora, Uranotaenia and

Wyeomyia (Darsie and Ward 2005).

Flight Range and Habits

Mosquito flight is classified in three behavioral categories: migratory, appetential or

consumatory (Bidlingmayer 1994). Migratory flights are only performed by newly emerged adult









mosquitoes. During this time, mosquitoes lack any specific physiological directive, and are

unforced to fulfill any individual needs essential to survival (Provost 1953). Conversely,

appetential flight occurs in response to physiological stimuli in mosquitoes over 24 hr of age.

These physiological stimuli commonly result from a need for blood meals, oviposition sites or

suitable resting locations. While in appetential flight, sensory mechanisms, such as olfaction,

vision, thermal and auditory receptors, are actively used to detect cues indicating the presence of

target physiological stimuli. Appetential flight is terminated and consumatory flight begins when

the target cue is detected. The latter is the time during which a mosquito follows detectable cues

to its desired objective (Haskell 1966). Often direct and brief, consumatory flights may occur

without a preceding appetential flight, given proper circumstances (Bidlingmayer 1994).

Multiple environmental factors such as topography, temperature, humidity and wind must

be considered when discussing appetential flight and dispersal habits of mosquitoes (Stein 1986).

Topography and landscape structures can be important influences on short and long range flight

habits of mosquitoes. Specific landscape formations such as shorelines and rivers have been

shown to significantly affect flight patterns ofAedes taeniorhynchus Wiedemann and other

insects (Provost 1952). Small townships and cultivated areas can also direct mosquito flight

preferences and patterns. The abundant amounts of appetitive stimuli these areas readily provide

can attract several mosquito species, causing them to abandon other natural host seeking flight

patterns (Shura-Bura et al. 1958).

The effects of temperature and humidity are well documented examples of how slight

environmental variations can influence mosquito flight preference. In most species, once

temperatures have risen above the minimum flight threshold, higher temperatures have little

impact on flight (Taylor 1963). Though individual temperature thresholds can vary slightly,









upper and lower temperature thresholds affecting flight hold true for most mosquito species. In a

study conducted by Rowley and Graham (1968a) on the flight performance ofAe. aegypti, upper

and lower temperature flight thresholds were found to be 35 C and 10 C, respectively, while

relative-humidity (RH) values ranging from 30 to 90% showed no significant effects. However,

when surveying Ae. sollicitans Walker and Culexpipiens Linnaeus, Rudolfs (1923, 1925) noted

reductions in total catch rates for both mosquito species on nights where RH levels exceeded

85% and 97%, respectively.

Wind may be the most important and complex of all environmental factors affecting

mosquito flight behavior (Stein 1986). Wind velocity and direction have been shown to

significantly impact flight activity, elevation and direction (Klassen and Hocking 1964, Snow

1976). The slightest air currents are enough to affect mosquito flight activity. In laboratory

experiments, average cruising flight speeds of 1.0 meter per second or less were observed for

some Aedes species (Hocking 1953, Rowley and Graham 1968b, Nayar and Sauerman 1972).

When wind velocities decrease below average flight speeds, mosquitoes are able to fly upwind; a

preference displayed by most species. However, wind velocities greater than average flight

speeds tend to overpower mosquitoes, forcing them to find shelter or submit to a downwind

direction (Kennedy 1939). Flight elevation is also determined by flight direction with respect to

wind velocity. Mosquitoes must make elevation adjustments accordingly to keep ground patterns

used for guidance within their visual limits (Bidlingmayer 1985a,b).

Gender may also play an important role in activity and range of mosquito flight. Males

have been shown to travel shorter distances than females, staying within a few kilometers of their

larval habitat. Studies involving mark-and-recapture methods have been used with great success,

demonstrating this behavior in several mosquito species (Horsfall 1954, Quraishi et al. 1966,









Brust 1980, Weathersbee and Meisch 1990). Schemanchuk et al. (1955) demonstrated that Ae.

flavescens Miller males have a proximate flight range of approximately 1.3 km, with females

averaging 10.6 km in range. Females of several Culex species have lower temperature thresholds

for flight activity than males, resulting in a longer dispersive phase of flight and thus a greater

range (Wellington 1944).

Resting Behavior

Based on observed behaviors, adult mosquitoes are believed to spend more time resting

than in flight. Mosquitoes primarily rest to digest meals, or to find shelter from environmental

conditions or predators. Most adult mosquito species are exophilic, resting exclusively outdoors

in natural shelters, such as animal burrows and tree holes, and amongst vegetation.

Comparatively few adult mosquito species are known to be entirely endophilic, preferring man-

made shelters such as huts or sheds (Service 1993).

Exophilic adult mosquitoes seek shelter in a wide range of habitats including termite

mounds, hollow trees and various types of vegetation. Preferences between these habitats have

been observed in several mosquito species (Service 1993). For example, An. freeborni Aitken

prefer to overwinter in animal burrows over other natural shelters. However, Cx. tarsalis

Coquillett, a species found in similar habitats, prefer overwintering in rock-holes and fissures

amongst vegetation (Harwood 1962). Service (1969) noted several adult Aedes species preferred

to rest primarily amongst vegetation, whereas some Anopheles species were recovered only from

tree trunks.

Environmental factors such as sunlight and relative humidity also play a critical role in the

resting habits of many exophilic mosquitoes. Service (1971) noted a significant difference in the

distribution of mosquitoes found resting among vegetation exposed to sunlight. Direct sunlight

exposure caused populations to converge in more shaded regions of vegetation. In Florida, Day









et al. (1990) found that Cx. nigripalpus Theobald moved deeper into the center of wooded

hammocks towards thicker vegetation in response to negative changes in relative humidity.

Similarly, An. walker Theobald are generally found solely amongst vegetation in cooler seasons,

but are present in covered structures during hot, dry summers (Snow and Smith 1956).

Population Monitoring

Most mosquito species are either nocturnal or crepuscular, remaining relatively inactive

during daylight hours. Sampling these outdoor populations is often difficult, as they are

commonly distributed over wide areas of open vegetation (Crans 2004). In an attempt to

overcome these difficulties and eliminate biases brought on by baited trapping systems, special

monitoring methods were developed with the goal of naturally attracting mosquitoes to specific

sites from which they can be conveniently collected (Crans 1989). These monitoring methods

include several forms of artificial resting boxes, gravid traps and sticky traps.

Earth-lined box traps were the first artificial resting places successfully used to study and

sample exophilic mosquito species (Russell and Santiago 1934). Since then numerous artificial

resting shelters varying in shape and size have been developed and tested. Rolled up mattresses

have also been shown to act as viable artificial resting boxes when sampling for exophilic

mosquitoes (Khan 1964). Some artificial resting places such as keg shelters, box shelters, cloth

shelters, dustbin bags and pipe traps have been shown to target specific exophilic mosquito

species.

While sampling exophilic mosquitoes in Tennessee, Smith (1942) showed that An.

quadrimaculatus Say preferred empty nail kegs when turned on their side capturing as many as

1,100 Anopheles adults in a single keg. Several mosquito genera including Anopheles, Culiseta,

Culex, Aedes as well as the species Cq. perturbans and Ur. sapphirina Sacken were found to

prefer a wide range of box shelters (Goodwin 1942, Burbutis and Jobbins 1958, Gusciora 1961,









Pletsch 1970, McNelly and Crans 1989, Anderson et al. 1990, Crans 1989, Harbison et al. 2006).

Over a 44-night trapping period, Service (1986) caught primarily Ae. caspius Pallas and Culex

quinquefasciatus Say using plastic trash bags. When sampling with self constructed pipe traps,

Nelson (1980) collected more Cx. tarsalis mosquitoes than any other species.

Gravid traps are designed to mimic natural oviposition sites of most mosquito species.

These sites are often dark, and consequently, sheltered from direct sunlight. Therefore, trap color

can influence trap preference, significantly impacting mosquito captures. Belton (1967)

identified preferences for illumination and substrate contrast of possible mosquito oviposition

sites using four artificial pools. Two pools were interiorly lined with translucent film, and two

with black polyethylene film. White reflectors and 40-watt cool white fluorescent lamps were set

on timers, and used to illuminate one translucent lined pool and one black lined pool. Belton

(1967) observed that no mosquito eggs were recovered from illuminated pools. Also,

significantly more mosquito eggs were recovered from pools lined with black than those with

translucent lining. Laing (1964) observed similar results in a comparable study, recovering fewer

mosquito eggs from translucent polyethylene pools or white painted pools. Results from Belton

(1967) and Laing (1964) demonstrated a significant preference for dark, unlit mosquito

oviposition sites when given a choice. These findings suggest little or no preference for light

when searching for possible oviposition sites.

Allan and Kline (2004) observed that infusion pan color significantly affected mosquito

capture while evaluating mosquito gravid traps for collection of Culex mosquitoes in Florida.

When comparing white, tan, light olive green and black pans, significantly greater numbers of

gravid Culex mosquitoes were captured with traps using black or green pans than those with tan

or white pans. Similar observations by Kline et al. (2006) concluded that altering infusion pan









color could have significantly increased trap totals when evaluating the efficacy of the Gravid

Trap (John Hock Company) against three other trap designs. The findings of Allan and Kline

(2004) and Kline et al. (2006) support observations of Belton (1967) and Laing (1964),

demonstrating a strong affinity for gravid mosquitoes to dark surfaces or oviposition sites.

Another effective method of population monitoring is the use of sticky traps. Sticky traps

are grouped into two categories; attractant and non-attractant. Attractant sticky traps are those

used in conjunction with bait animals (Disney 1966), carbon dioxide (Gillies and Snow 1967) or

traps constructed with a specific shape or color that would enhance attractiveness of the trap

(Allan and Stoffolano 1986a). Non-attractant traps are designed with the intention of functioning

independent of bias that might positively or negatively influence the attractiveness of the trap.

Sticky trap adhesives come in a wide variety of compounds, and can be used to capture

many different insects. Various greases and oils are common adhesives but have not shown to be

as effective as resins, usually trapping only small insects. Tree banding resins are of the most

efficient adhesives for catching a wide variety of different sized insects, though they can be

difficult to work with when attempting to remove and identify a catch (Service 1993). Common

application techniques when working with adhesives in regards to mosquito population

monitoring include mesh screens (Gordon and Gerberg 1945), nets (Provost 1960) or sticky

cards (Beck and Turner 1985).

Designed to survey flying insect populations, sticky cards have been utilized for the study

of many insects including house flies (Hogsette et al. 1993, Kaufman et al. 2001, Geden 2005,

Beresford and Stucliffe 2006), whiteflies (Haynes et al. 1986) and aphids (Rohitha and

Stevenson 1987). Though they have been recommended as reliable monitoring tools for more

than 30 years (Haynes et al. 1986), sticky cards have not been widely used in mosquito









population monitoring. Lack of use could be attributed to common difficulties encountered when

working with adhesives.

Achieving the appropriate viscosity and tackiness of adhesives is an important, yet

challenging, task in regards to sticky cards. High temperatures and fluctuating humidity levels

may cause thinner adhesives to become viseus, losing their effectiveness. However, adhesives

that are too thick allow alighting mosquitoes to land and escape, commonly only trapping those

that are forcibly blown on to a treated surface by wind (Service 1993).

Mosquito Attraction

As previously discussed, females of almost every mosquito species are anautogenous,

requiring a vertebrate blood meal to initiate egg development. To obtain this blood meal, female

mosquitoes utilize a variety of olfactory, physical and visual cues during host location. Visual

and physical stimuli including variations in skin temperature and color as well as host odor

provide the necessary information required for most mosquitoes to successfully locate and

identify their hosts (Constantini 1996). Though extensive work has been conducted to determine

the mechanism of mosquito attraction to its host, the effect of odor on mosquito behavior is still

poorly understood (Clements 1999).

The attractiveness of human odors to Ae. aegypti and An. quadrimaculatus was first

demonstrated in 1947 using a dual-port olfactometer (Willis and Roth 1952). Khan et al. (1965)

noted individual variations in host attractiveness when a feeding preference for one person over

three others was shown by Ae. aegypti. This variance was attributed to dissimilar levels of lactic

acid produced by human hosts. Male hosts exhibited higher lactic acid levels, thus accounting for

greater attractiveness than female hosts (Acree et al. 1968). Several other volatiles including

carbon dioxide (CO2) and 1-octen-3-ol (octenol) have been used more recently as successful









adult mosquito attractants (Kline et al. 1990, Kline et al. 1991, Kline and Lemire 1995, Burkett

et al. 2001).

Reeves (1951) was the first to demonstrate the attractiveness of CO2 to female mosquitoes

in field studies. Carbon dioxide is one of the most frequently utilized, and most accepted, volatile

attractants used to trap adult mosquitoes. Commonly found in two forms, CO2 can be added to

traps as a compressed gas or a solid (dry ice) (Kline et al. 1991). Though dry ice is relatively

inexpensive and lightweight, compressed gas cylinders are often the preferred method of

dispensing CO2 with the advantage of regulating the discharge rate (Service 1993). This can be

an important consideration when trapping different mosquito species whose level of

attractiveness varies according to the CO2 emission rate (Reeves 1953, Gillies and Wilkes 1974,

Mboera et al. 1997, Dekker and Takken 1998). Regulating discharge rates can also be crucial

when using CO2 in conjunction with other volatiles. Kline et al. (1990) found that octenol

emissions of 2.3 mg/hr with a CO2 release rate of 200 ml/min have a greater potential as a

mosquito attractant than CO2 alone. Multiple studies testing the attractiveness of octenol when

used in conjunction with regulated release rates of CO2 have produced similar results (Takken

and Kline 1989, Van Essen et al. 1994, Burkett et al. 2001).

Visual stimuli such as movement, light wavelength and intensity, color, shape, pattern, and

contrast also play an important role in host location and identification by adult female

mosquitoes (Bidlingmayer 1994). In some Aedes species, detection of movement is important for

host location (Sippell and Brown 1953). Other species may rely on contrasting or low intensity

colors such as blue, black and red as primary host location stimuli (Browne and Bennett 1981).

Visual attraction traps based on contrast, movement, color and pattern have not been widely used









to collect mosquitoes. The Fay-Prince trap is one exception, utilizing a contrasting black and

white pattern, but is often baited with CO2 to increase its efficacy (Service 1993).

Artificial, reflected and filtered lights have been incorporated in the design of existing

efficient traps to increase their efficacy for mosquito research and surveillance with great success

(Barr et al. 1963, Service 1976, Ali et al. 1989, Burkett and Butler 2005, Hoel 2005). Ali et al.

(1989) were able to demonstrate that both Culex and Psorophora spp. showed a higher

preference for light color rather than intensity when trapping in the field. Similarly Burkett and

Butler (2005) showed that not only light source, but specific light wavelengths played an

important role in host attraction. In laboratory trials, Ae. albopictus, An. quadrimaculatus and

Cx. nigripalpus all displayed preferences for specific wavelengths of light.

Physical stimuli used in host location include radiant and convective heat, moisture, sound

and surface structure (Laarman 1955). Peterson and Brown (1951) used heated billiard balls to

demonstrate the affinity ofAe. aegypti to convective heat as opposed to radiant heat. Mosquitoes

attempted to feed on the heated billiard balls until a window of crystalline thallium bromoiodide

was inserted between the ball and mosquitoes. This window allowed the passage of radiant heat

while blocking the convective heat, confirming the attraction to convective heat. While trapping

in Florida, Kline and Lemire (1995) observed similar results, noting an increase in total captures

of Oc. taeniorhynchus Wiedemann after adding heat to traps.

Moisture is commonly used in conjunction with other stimuli to increase the overall

attractiveness of some traps. Khan et al. (1966) found that moisture, when combined with CO2

and heat, mimicked vertebrate breath, significantly increasing overall catch rates ofAe. aegypti.

In laboratory studies, Brown et al. (1951) found that moist surfaces are more attractive to Aedes

mosquitoes than dry surfaces. Similarly, field studies showed that adding moisture to traps









significantly increased catch rates ofAedes species, suggesting most Aedes species utilize

moisture over other sensory cues (Brown 1951).

Mosquitoes are sensitive to sound frequencies and respond to those ranging from

frequencies of 250 to 1,500 Hz (Kahn et al. 1945). Kahn and Offenhauser (1949) reported that

when the wing beat sound of a single female An. albimanus Wiedemann were repeatedly played

at 5 s intervals, significantly larger numbers of male An. albimanus were trapped than when no

sound was played. In laboratory experiments, Ikeshoji (1981, 1982, 1985) found that sound

attracted males ofAe. aegypti, Ae. albopictus, Cx. pipiens and An. stephensi Liston. It was also

noted that while utilizing acoustic removal equipment in cages, insemination rates of female Ae.

aegypti and An. stephensi decreased by 30% and 20% respectively. However, under field

conditions traps utilizing sound are of little use, because males respond over very short distances,

regardless of its intensity or frequency (Service 1993).









CHAPTER 2
RESPONSE OF ADULT MOSQUITOES TO LIGHT EMITTING DIODES PLACED IN
RESTING BOXES

Introduction

Since the early 1900's, the effectiveness of techniques to attract and track the movements

of hematophagous insects has continued to improve (Crans 1989). Adequate and reliable

population sampling is often seen as the most important and most difficult step in ecological

studies. There are two main types of population sampling: active and passive. Active sampling

involves manually locating and capturing insects with devices such as sweep nets or aspirators.

With passive sampling, insects are collected and monitored using stationary traps such as resting

boxes or sticky cards (Holck and Meek 1991). Additionally, adult mosquito populations are

passively sampled using active traps (New Jersey Light Trap, CDC) (Service 1976). These traps

are frequently supplemented with attractants such as lactic acid, carbon dioxide and/or various

wavelengths of light to enhance mosquito captures. Lactic acid and carbon dioxide exploit

olfactory cues by effectively mimicking host associated volatiles, while the manipulation of light

(wavelength, frequency and intensity) acts as a visual attractant.

Behaviorally, most mosquito species are either nocturnal or crepuscular, remaining

relatively inactive during daylight hours. Sampling outdoor populations is often difficult,

because they can be commonly distributed over wide areas of open vegetation (Crans 1989). To

overcome these difficulties and eliminate the biases brought on by baited trapping systems,

special monitoring methods were developed with the goal of passively attracting mosquitoes to

specific sites from which they can be conveniently collected (Crans 1995). Mosquitoes often rest

or seek shelter in naturally protected sites such as ground burrows, dense vegetation and tree

holes (Crans 1989). The capitalization of this natural phenomenon has allowed researchers to

effectively sample mosquitoes during inactive hours using artificial resting boxes.









Man-made resting structures have been used as adult mosquito sampling tools since the

early days of malaria control when several malaria vectors were observed congregating in

diurnal resting places (Boyd 1930). Old nail kegs turned on their sides were the first of these

structures used to sample resting populations of several mosquito species. After reporting that

nail kegs were not successful in collecting Anopheles quadrimaculatus Say in Georgia, Goodwin

(1942) began experimenting with several different variations in size and color of artificial resting

structures. He found that 1ft3 (30 cm3) wooden boxes, when left open at one end, attracted large

numbers of An. quadrimaculatus adults. Further experiments showed that mean catches of An.

quadrimaculatus were higher when boxes were painted red inside compared with those painted

white, yellow, blue, black or green. A red interior also allowed for easier distinction of

mosquitoes from other background colors. In addition, boxes facing towards the rising sun

caught significantly fewer adult mosquitoes than those facing away from the sun. Goodwin

(1942) concluded that the best shelter was a 1 ft3 wooden box painted dull black on the outside,

red inside and positioned on the ground in a sheltered position, preferably not facing east

(Service 1993).

Today, Goodwin's resting box design is commonly used in adult population monitoring for

several medically important mosquito species. When compared to light traps, Goodwin boxes

were more effective at capturing and measuring population changes in An. freeborni Aitken and

Culex tarsalis Coquillett (Bradley 1943, Hayes et al. 1958, Loomis and Sherman 1959).

Similarly, Gusciora (1961) demonstrated the utility of 1 ft3 resting boxes more so than light-traps

as arboviral surveillance tools for multiple mosquito species in attempting to monitor Culiseta

melanura Coquillett populations for the New Jersey State Department of Health Arbovirus

Surveillance Program. In trapping comparison studies, Gusciora (1961) caught 13,240









mosquitoes in Goodwin box shelters but only 6,260 in CDC light-traps. In addition to the

aforementioned species, adults of An. crucians Wiedemann, An. punctipennis Say, Cx. salinarius

Coquillett, Cx. restuans Theobald, Cx. pipiens Linnaeus, Aedes canadensis Theobald, Ae.

sollicitans Walker, Coquillettidia perturbans Walker and Uranotaenia sapphirina Sacken were

all effectively trapped in Goodwin resting boxes (Service 1993).

Adjustments and advancements in population monitoring procedures involving resting

boxes have led to the modern methods used in today's vector surveillance programs (Crans

1995). Although vector surveillance methods involving both insect wavelength preferences and

resting behavior have been studied extensively, the combination of the two has not yet been

evaluated. The objective of my research was to evaluate the attractiveness of resting boxes fitted

internally with light emitting diodes (LEDs) of selected wavelengths to field populations of

mosquitoes. Wavelengths used in this study were selected based on capture rates and preferences

observed for several mosquito genera, including Aedes, Anopheles, Culex and Psorophora

(Burkett et al. 1998, Burkett and Butler 2005, Hoel 2005).

Materials and Methods

Resting Boxes

Resting boxes with four sides, a back wall and an open front were constructed using the

specifications of a standard 30 x 30 x 30cm resting box, as described by Crans (1995). The four

sides and back wall of all boxes was made from 0.64 cm (14 in) thick exterior grade pine lumber

plywood, while 5 x 5 x 29 cm sections of pine were affixed as inside joint supports (Figure 2-la).

Box exteriors were painted with two coats of flat black exterior latex paint, and interiors with

two coats of barn red exterior latex paint. A 0.64 cm (/4 in) hole was drilled through the back

center wall of each box to allow for the insertion of a LED. The exterior surface of the rear wall

of each box was fitted with a 6.5 x 9 cm, 470 ml plastic screw cap vial (Thornton Plastics, Salt









Lake City, UT), protecting the battery supply and LED wiring. A 0.64 cm (/4 in) hole drilled

through the container lids to correspond to the 0.64 cm (14 in) diameter hole in the back wall of

each resting box. Lids were secured to the back wall, allowing for easy attachment and

detachment of containers to resting boxes (Figure 2-lb).

Mosquitoes were removed from resting boxes using a mechanical aspirator between 1000

and 1300 hours. A 41 x 41 cm section of 0.33 cm thick PlexiglasTM was used to cover the box

opening and prevent the escape of mosquitoes while they were mechanically aspirated. A 15-cm-

diameter hole made in the center of the PlexiglasTM was fitted with a stocking net to allow for

aspirator access.

Light Emitting Diodes and Battery Supplies

All LEDs were obtained from Digi-Key Corporation (Thief River Falls, MN). Diodes, part

number and millicandela (mcd) rating, as described in Hoel (2005), were blue (P466-ND, 470

nm, 650 mcd), green (67-1755-ND, 502 nm, 1,500 mcd), red (67-1611-ND, 660 nm, 1,800 mcd)

and infrared (LN77L-ND, 860 nm). Because infrared radiation is not visible to humans, infrared

diodes are not mcd-rated. Round lens LEDs were 8.6 mm long by 5.0 mm in diameter. Viewing

angles were 300 except for IR 860 (200).

All materials used in the construction of battery supplies were obtained from an electrical

supply company such as RadioShack (Gainesville, FL). A 180-ohm resistor was soldered to all

LEDs, to restrict current flow and prevent mechanical failure of LEDs as a result of

overworking. A female 9 volt (V) battery snap connector (270-325) was soldered to each

modified LED (Figure 2-2a). Battery supplies (270-383) pre-equipped with a complimentary

male 9 V connecting site were used, each with a maximum holding capacity of four AA

batteries. Four rechargeable 2500 milliamp hour (mAh) AA batteries were used in all battery









assemblages (Figure 2-2b). The 9 V connectors permitted a reliable, but temporary, connection

to each battery supply.

CDC Light Trap

Three modified CDC light traps (model 512, John W. Hock Company, Gainesville, FL)

were used to provide representative data on background mosquito populations at two study

locations. As described in Hoel (2005), each CDC light trap used a 6 V DC motor and 4-blade

fan to draw flying insects through an 8.5-cm-diameter clear plastic cylindrical body (Fig. 2-3).

The incandescent bulb was removed from each trap. A 36-cm-diameter beveled edge aluminum

lid was set approximately 3 cm above the cylindrical body creating an increase in air current

flow into the trap. All traps were set 120 cm above ground using a Shepherd's hook with

collection nets attached to the outflow of the trap. Carbon dioxide was provided from a 9 kg

compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15-psi single-stage

regulator equipped with an inline micro-regulator (# 007) and an inline filter (Clarke Mosquito

Control, Roselle, IL). Flow rates were confirmed using a Gilmont Accuiicl' flowmeter (Gilmont

Instrument Company, Barrington IL.). Carbon dioxide was delivered to the trap through a 2 m

long, 6.4 mm outer diameter clear plastic Tygon tubing (Saint-Gobain Performance Plastic,

Akron, OH). Power was provided by a 6 V, 12 ampere-hour (A-h), rechargeable gel cell battery

(Battery Wholesale Distributors, Georgetown, TX).

Site and Resting Box Location

Field trials were conducted at the University of Florida Horse Teaching Unit (HTU) and

the Prairie Oaks subdivision (PO), Gainesville, FL. Both locations were similar, rural

environments previously shown to have productive mosquito breeding habitats (J. F. Butler

personal observation, Holton 2007). The HTU is an equine breeding and training facility housing

an average of 50 horses yearly. The facility consists of 24 hectares, which includes 2.4 hectares









of wetlands and a 0.2 hectare pond. The HTU is located in the southwestern section of

Gainesville, east of 1-75, and is closely bordered on three sides by the Paine's Prairie State

Preserve (Figure 2-4). The PO is a rural subdivision with 18 loosely spaced residential units

located approximately 4 km west of the HTU, adjacent to the Paine's Prairie State Preserve

(Figure 2-5). Both locations are surrounded by a mix of hardwood and pine forest with minimal

undergrowth.

Sites (east 1, 2 and west 1, 2) chosen at the HTU were divided and named according to

corresponding cardinal direction (Figure 2-6). The east side of the HTU differed in both

humidity levels and vegetation from the west side, resulting in a difference in environments

between the east and west side test sites. Test sites chosen on the west side of the HTU were

located in a low-lying depression commonly found to hold water, surrounded by moderate tree

cover and undergrowth, resulting in higher sustained humidity levels (Figure 2-7). The test sites

selected from the east side of the HTU were on a more elevated, drier terrain surrounded by thin

pine forests and adjacent to several homes (Figure 2-8). All residential test sites chosen at the PO

were consistent in surrounding vegetation, sunlight exposure and humidity conditions (Figure 2-

9). Among the 18 Prairie Oaks residences, boxes were located in the rear section of four

backyards, which were spaced approximately three residential units apart (Figure 2-10).

Temperature and humidity conditions at both locations were obtained from online NOAA

databases.

Methodology

A trial began by placing five resting boxes at each test site in a staggered line, out of direct

sunlight and approximately 4 m apart with open ends facing west. CDC light-traps were attached

to Shepherd's hooks with collection nets fitted to the outflow of the trap. After resting boxes and

CDC traps operated in the field for 24 h (one trap night), mosquitoes were aspirated from boxes









and CDC catch bags were changed. Mosquitoes recovered from traps were brought back to the

laboratory where they were counted and identified. CDC traps were serviced daily with batteries

and catch bags changed every 24 h. Carbon dioxide tanks were changed approximately every 10

days or as needed.

Resting box sampling at the HTU occurred from 21 July 14 August 2006 resulting in 20

trap nights, and from 05 May 26 September 2007 resulting in 140 trap nights. Trapping at the

PO occurred from 18 August 27 September 2006 resulting in 17 trap nights, and from 05 May

- 26 September resulting in 140 trap nights. One modified CDC light-trap was operational at the

HTU from 21 July 14 August 2006 resulting in 20 trap nights, and from 05 May 26

September 2007 resulting in 140 trap nights. Of these 160 trap nights, traps operated without

malfunction for 146 trap nights. Trapping at the PO with two CDC traps occurred from 18

August 27 September 2006 resulting in 34 trap nights, and from 5 May 26 September

resulting in 280 trap nights. Traps were operated successfully for 302 of these 314 trap nights.

When trapping nights were not continuous, existing mosquitoes were removed from

resting boxes 24 h prior to subsequent collection. Mosquitoes retrieved from CDC trap catch

bags and resting boxes were identified by sex and species using the dichotomous keys ofDarsie

and Morris (2003) and Darsie and Ward (2005). Identification data were logged into a IS'

Excel 2007 spreadsheet.

Statistical Analysis

Mosquito preference for LED wavelengths was evaluated using a multi-factorial ANOVA

(SAS Institute 2001). For analysis, all data were normalized using the SQRT (n+1)

transformation, however actual values are given in text and tables. The model included the fixed

effects location, site and LED treatment, the interaction term, location*LED treatment and the

random effect, trial. In instances where either the interaction term or the trial effect was









significant, the data were analyzed separately by location or trial (year). Tukey's Standardized

Test (a = 0.05) was used to separate treatment means.

Results

In total, in 160 trap nights at the HTU location, 1,885 mosquitoes were recovered from

resting boxes. In 157 trap nights at the PO location, there were 5,272 mosquitoes recovered from

resting boxes. Anopheles quadrimaculatus females, Cq. perturbans males, Cq. perturbans

females, Cx. erraticus males, Cx. erraticus females, Cx. nigripalpus females, Cx. salinarius

males, Cx. salinarius females and Mansonia titillans Walker females were collected in large

enough numbers to analyze statistically (Table 2-1). Mosquitoes collected, but excluded from

analysis because of low numbers or little medical importance included An. crucians, An.

quadrimaculatus males, Ochlerotatus infirmatus Dyar and Knab, Oc. triseriatus Say,

Uranotaenia lowii Theobald, Ur. sapphirina (Appendices A-1, A-2).

Diode wavelength preference was observed among An. quadrimaculatus and in Cx.

erraticus females in 2007 (Table 2-1). Significantly more An. quadrimaculatus females were

aspirated from resting boxes fitted with red and IR LEDs than from those with blue or green

LEDs or the no-light control (F = 2.47; df= 4, 6315; P -0.0429).

The trial effect was significant for Cx. erraticus males and females (F = 2.4; df = 4, 1126;

P -0.0476). During the 2006 trapping period, one trial was run at the HTU and PO locations. For

the 2006 trapping period, no preferences were observed among treatments. However at the HTU

location, significantly higher numbers of mosquitoes were aspirated from resting boxes at the

east-2 trapping site than at the three other trapping sites (F = 22.56; df = 3, 727; P = < 0.0001).

During the 2007 trapping period, significantly more Cx. erraticus females were aspirated from

resting boxes fitted with blue, green, red LEDs and the no-light control than those with IR LEDs

(F = 8.41; df = 4, 5577; P =< 0.0001). Significantly more Cx. erraticus females were captured









from the west-1 trapping site of the HTU location than from all other sites at both the HTU or

PO location (F = 14.47; df = 7, 5577; P = < 0.0001) (Figure 2-6, 2-10).

Data for Cx. erraticus males, Cq. perturbans males and Ma. titillans females were also

analyzed separately by trial (year) (Table 2-1). During the 2006 trapping period, significantly

more Cx. erraticus males were captured from resting boxes placed at the EAST-2 trapping site at

the HTU location (F = 4.84; df= 3, 727; P -0.0024), while Cq. perturbans males were aspirated

in significantly higher numbers from resting boxes placed at the west-1 and west-2 trapping sites

at the HTU location (F = 32.60; df = 3, 1126; P =< 0.0001) (Figure 2-6). During the 2007

trapping period, significantly more Cx. erraticus males were aspirated from resting boxes placed

at the PO location than from those at the HTU location (F = 8.01; df = 1, 5577; P -0.0047).

Numerically, more male Cx. erraticus (25%) were aspirated from resting boxes without LEDs

than from those with LEDs. No significant differences in LED wavelength preference were

observed for Cq. perturbans males, but 33% were aspirated from resting boxes fitted with blue

LEDs. No significant differences were observed among Ma. titillans females for the 2006 or

2007 trapping periods.

Although no significant differences in LED wavelength preference were observed among

Cx. nigripalpus females, Cx salinarius males or Cx salinarius females, dissimilarities in

mosquito captures among treatments were noted. More than 37% of Cx salinarius males and

females were collected from resting boxes fitted with green LEDs. Culex nigripalpus females

were aspirated in highest numbers from resting boxes affixed with blue (24%) LEDs, whereas

resting boxes with red LEDs (7%) captured the fewest females.









Approximately 100,653 female mosquitoes, including 24 mosquito species from six

genera, were trapped over 448 trap nights (Appendix A-3, A-4). Mean mosquito captures per

trap night of the six mosquito species shown in Table 2-1 are presented in Table 2-2.

During this study, approximately 55% (64,893) of all mosquitoes trapped were captured at

the HTU sites using one CDC trap (34% of trap nights). Proportionality in mosquito capture rates

between trapping the 2006 and 2007 trapping periods also differed. During the 2006 trapping

period at the HTU location, considerably more Cq. perturbans and Cx. erraticus females were

trapped than in the 2007 trapping period. In 2006, an average of 1,400 Cq. perturbans females

per trap night were captured compared with an average of 45 per trap night during 2007.

Similarly, during the 2006 trapping period Cx. erraticus averaged 10 times more mosquitoes

than during the corresponding 2007 trapping period (September). Conversely, Cx. nigripalpus

capture increased during the 2007 trapping period. Approximately one mosquito was captured

per trap night during the 2006 trapping period, whereas in 2007 an average of 657 mosquitoes

were captured per trap night.

Average monthly temperatures for August (27 C) and September (25 C) remained

relatively similar between the 2006 and 2007 trapping periods, differing by no more than 0.7 C

for either monthly average (Figure 2-12a, b). However, average precipitation levels for August

and September of 2006 and 2007 were quite different. In 2006, an average of 7 cm of rainfall

was recorded in August compared with approximately 17 cm in 2007. Similarly, less than 8 cm

of rainfall were recorded for September in 2006, with approximately 9 cm recorded in 2007. The

highest average precipitation levels for 2007 occurred in July (22.6 cm), while lowest

precipitation levels occurred in May (1.9 cm) (Figure 2-12a, b).









Discussion

In this study, LED color (wavelength) choices were blue (460 nm), green (502 nm), red

(660 nm) and IR (860 nm). Blue, at 460 nm, registers at the higher end of the purple-blue range

of the visible light spectrum. However, 502 nm falls at the lower transition point between blue

and green, while 660 nm registers near the lower end of the red-yellow light spectrum. Infrared

wavelength is not detectable by the human eye, registering above the visible spectrum at 860 nm.

For additional information concerning the visible light spectrum, see Ando and Thomas (1996).

Wavelengths selected for in this study were selected based on capture rates and preferences

observed for several mosquito genera, including Aedes, Anopheles, Coquillettidia, Culex and

Psorophora (Burkett et al. 1998, Burkett and Butler 2005, Hoel 2005). Burkett et al. (1998)

recorded higher captures of An. crucians and Cx. nigripalpus using CDC light-traps fitted with

green light than when using IR LEDs. Additionally, Hoel (2005) observed trapping significantly

more Cq. perturbans when using CDC light-traps supplemented with CO2, and modified with

blue LEDs (470 nm) that standard CDC light-traps using incandescent bulbs.

Using the Goodwin (1942) style resting boxes in southern New Jersey, Burbutis and

Jobbins (1958) and Crans (1995) trapped similar mosquito species, including An.

quadrimaculatus, Cs. melanura, Cx. restuans, Cx. salinarius, Cq. perturbans and Ur. sapphirina.

Collections of Cs. melanura and An. quadrimaculatus significantly exceeded those of all other

mosquito species in both studies. Our results agree with these studies in terms of species

diversity, because we collected similar mosquito species, such as An. quadrimaculatus, Cx.

salinarius, Cx. territans and Cq. perturbans. However, we recovered no Cs. melanura from

resting boxes or CO2 baited traps, although Cs. melanura have been reported in this area of

Florida (Burkett et al. 1998). This difference may result from habitat variations or seasonal

emergence patterns exhibited in Florida.









Resting boxes were located in similar hardwood hammock habitats at both locations. Due

to habitat variation, mosquito species not commonly recovered from these environments were

likely excluded from trapping results. Additionally, trapping periods only occurred for three

months during 2006, and five months during 2007. The bias resulting from only utilizing one

habitat during a narrow time period could explain the lack of Cs. melanura among resting box

captures (Crans 1995).

We found that Cx. erraticus males and females were recovered from resting boxes in

higher numbers (48% and 42% respectively) than all other mosquito species. Approximately

26% of male Cx. erraticus were recovered from resting boxes fitted with IR LEDs, and 23% of

females were recovered from boxes left dark. High numbers of Cx. erraticus were anticipated as

this species is commonly captured in light traps (Ali et al. 1989, Cupp et al. 2003, Rodrigues and

Maruniack 2006). Ali et al. (1989) captured numerous Cx. erraticus in Florida while utilizing

New Jersey light traps fitted with white, yellow, orange, blue, green or red incandescent bulbs.

These results suggest the presence of light may impact trapping results for Cx. erraticus.

Similarly, the addition of selected wavelengths to resting boxes may increase the attractiveness

of these boxes to Cx. erraticus.

When testing mosquito wavelength preference with filtered light using a visualometer,

Burkett and Butler (2005) observed significantly longer feeding periods for An. quadrimaculatus

on artificial hosts illuminated with black (no light) or white light compared with other

wavelengths ranging in 50 nm increments from 350 750 nm. Feeding times on artificial hosts

illuminated with filtered light at 350 nm (purple) were significantly shorter than all other feeding

times recorded. These observations were similar to our results where significantly more An.

quadrimaculatus were aspirated from resting boxes fitted with red LEDs (high end of the light









spectrum) than blue or green LEDs. Burkett and Butler's (2005) results and our findings suggest

that lower wavelengths (< 660 nm) are less desirable to An. quadrimaculatus than are

wavelengths higher in the light spectrum (> 660 nm). Therefore, the addition of 660 nm LEDs to

resting boxes may enhance efficacy of sampling An. quadrimaculatus populations.

Overall, more mosquitoes (male and female) were recovered from resting boxes fitted with

IR LEDs (23%) than all other treatments. Resting boxes left dark captured 22% of mosquitoes,

while the fewest mosquitoes were recovered from boxes affixed with red (20%), green (17.6%)

and blue (16.7%) LEDs. Our results suggest general mosquito preference for wavelength

spectrums that were longer than shorter. These observations differ from other findings for

photophilic mosquito species trapped at night, such as Cx. erraticus, Cx. nigripalpus and

Psorophora columbiae Dyar and Knab, which suggest preferences for lower wavelengths

(Bargren and Nibley 1956, Ali et al. 1989, Burkett et al. 1998, Burkett and Butler 2005).

Differences in our results may be the product of variations previously unaccounted for in

wavelength attraction between host seeking and resting mosquitoes. Additionally, the use of

narrow wavelengths may have excluded mosquitoes preferring longer or shorter wavelengths

than those selected.

Male mosquitoes comprised approximately 54% (3,853) of all mosquitoes aspirated from

resting boxes. Culex erraticus males (3,455) accounted for almost half of all mosquitoes

captured, while Cx. nigripalpus males (6) were recovered the least. Aspiration totals for other

mosquito species ranged from seven to 218. Though gravid or blood fed females are highly

desired, high captures of males in resting structures are not uncommon, and can be important

(Goodwin 1942, Nelson 1980, Kay 1983, Edman et al. 1997). Goodwin (1942) reported high

captures of An. quadrimaculatus males using empty nail kegs. Additionally, Edman et al. (1997)









observed high numbers of male Ae. aegypti coming to artificial resting boxes placed inside

houses. Effectively sampling male mosquito populations can be an important tool in the

surveillance and modeling of venereally transmitted arboviruses such as St. Louis encephalitis.

Male mosquito population densities can be important indicators of general population fecundity

and reproductive status of a target species. In population modeling, this combination of factors

makes sampling an effective tool in the comprehension of vector potential of a disease

transmitting population (Garrett-Jones 1964).

Expectedly, more mosquitoes were captured in CDC light-traps than resting boxes.

because of the supplement of an artificial host attractant, C02, in the CDC traps. Both modified

CDC light-traps and resting boxes captured similar mosquito species, including Cq. perturbans,

Cx. erraticus, Cx. nigripalpus, Ma. titillans, Ur. lowii and Ur. sapphirina. Adult mosquitoes are

commonly captured when using trap designs that combine light with alternative host stimuli

(Browne and Bennett 1981, Burkett et al. 1998, Hoel 2005). Most mosquito species, such as An.

quadrimaculatus, are endophilic, and are recovered in higher numbers from resting boxes rather

than CDC traps. Endophilic mosquitoes prefer feeding and resting in or near human dwellings.

These species are more often captured in boxes that are designed to mimic their natural resting

behaviors, rather than target their host seeking behaviors. Therefore, trapping systems must be

chosen based accordingly to the desired species. This further illustrates the physiological and

behavioral differences among mosquito species, and the effects of those differences on trap bias.

Some mosquito species, such as Ps. ciliata and Ps. columbiae were captured in the CDC

light-traps, but not in resting boxes. While some Psorophora are often recovered from resting

boxes, Ps. ciliata and Ps. columbiae are known to frequent light traps in Florida (Ali et al. 1989,

Burkett et al. 1998). The occurrence of both mosquito species in light traps, but not in lit or dark









resting boxes suggests a phototactic relationship. As both mosquitoes are pest species to humans,

this negative association may warrant the integration of LEDs in various repellant applications.

Additionally, light intensity may impact the entry into resting boxes fitted with LEDs. Many

mosquito species are known to exhibit positive phototaxis to light sources, with attraction levels

directly correlating to light intensity (Service 1993). Using light traps, Gaydecki (1984) observed

that smaller insects including mosquitoes became disoriented near light sources. Ali et al. (1989)

demonstrated similar results, trapping significantly more mosquitoes in light traps with lower

intensities.

Male mosquitoes represented less than one percent of all CDC light-trap captures. This

contrasts with 54% of total males recovered from resting boxes during this study. These results

are likely due to the supplement of CO2 as an additional host attractant to the CDC traps.

Because this volatile is utilized as a host attractant, the detection of this gas serves very little

physiological purpose to male mosquitoes. However, female mosquitoes in search of a blood

meal must be able to detect, recognize and locate this compound to obtain nutrients necessary for

vitellogenesis.

Mean mosquito capture per trap night from modified CDC light-traps for Cx. nigripalpus

differed greatly between the 2006 and 2007 trapping periods. Mosquito capture rates at the HTU

and PO locations were approximately one mosquito per trap night in 2006, compared with 657

Cx. nigripalpus per trap night in the respective 2007 trapping period. This dramatic population

increase may have been due to the mosquitoes' seasonal and spatial distribution in response to

wetting and drying conditions, as discussed in Day and Curtis (1994). During the 2007 trapping

period, periodic rains, followed by sufficient drying periods, provided the ideal environmental

conditions for Cx. nigripalpus to exceed average population densities.









Super-bright LEDs have demonstrated superior effectiveness as light sources for various

trap designs. Given their intensity, small size, efficiency and minimal power usage, LEDs make

optimal light sources for civilian or military field applications where access to target sites and/or

transport of equipment are minimal. While their intensity is superior to other compact light

sources, LEDs used in this study only offer a 300 viewing angle. This has little effect on insects

from long distances, but significantly restricts the peripheral visibility of emitted light when

insects are not in line with the targeted LED emission. However, the ability to operate for

extended periods of time on power sources as small as a watch battery eliminates the necessity to

regularly exchange and maintain larger, more cumbersome batteries. Their demonstrated

effectiveness in our resting boxes for attracting mosquitoes without the aid of supplemental host

attractants further eliminates the need for dry ice or heavy tanks (C02) or noxious chemicals

(lactic acid, octenol). Durability of the LED-based equipment also helps to reduce otherwise

necessary and time-consuming field maintenance. By offering extended operating time with

minimal power consumption, field durability and the ability to eliminate the need for

burdensome equipment, LEDs remove restrictions previously set on trap designs.

The addition of LEDs to resting boxes in this study has demonstrated increased

attractiveness for certain mosquito species, while decreasing attractiveness to others. Relevance

of these findings could lead to future civilian or military applications as mosquito repellant

devices. Based on the "push-pull" premise, resting boxes or mechanical adult mosquito traps

could be placed at a considerable distance from a home or military box, and fitted with LEDs

found to be attractive to target mosquito species. Light emitting diodes with wavelengths known

to be undesirable to these species would then be affixed to the desired building. This









combination of attractive and repellant stimulants enhances the effects of each, leading to

improved repellent devices for medically important mosquitoes.

The "push-pull" principle could also be applied to sticky-card traps. Sticky-card traps are

simple, inexpensive and versatile, allowing them to be utilized in multiple trap designs. By

utilizing reflective or colored surfaces to enhance attractiveness, fitting LEDs of preferred

wavelengths to sticky-card traps may increase the effectiveness of these traps in locations where

space and equipment limitations are important. Light emitting diodes with non-preferred

wavelengths affixed to areas of interest would help to repel mosquitoes, while increasing the

attraction of sticky-traps fitted with LEDs of preferred wavelengths. This modified trap design

has promising military and civilian applications.

Additional applications of this research could involve the integration of interior pesticide

applications to LED fitted resting boxes. These spray applications have been demonstrated as

possible control measures for Anopheles species in domestically-placed resting boxes such as

huts or tents (Smith et al. 1966, Quifiones and Suarez 1990). The combination of enhanced

attractiveness to illuminated resting boxes and knock down sprays could serve as an efficient

control method for several medically important mosquito species.

Previous to this study, trapping involving the inclusion of LEDs in resting boxes has not

been conducted. The findings of this research demonstrate the need for further investigation into

the combination of mosquito wavelength attraction and artificial resting boxes. Several mosquito

species recovered from resting boxes fitted with LEDs were previously thought to have little

affinity to light. Based on these results and observations from past research, variations in light

intensity might also significantly impact the attractiveness of resting boxes to mosquitoes.

Additionally, population sampling for those mosquito species may be improved or refined with









the addition of LEDs to resting boxes. Continued research into wavelength frequency may offer

further insight into the attractiveness of some mosquito species to resting boxes fitted with

LEDs.









Table 2-1. Mean ( SE) numbers of mosquitoes/trap/night attracted to light emitting diodes of four different wavelengths placed in
resting boxes at the University of Florida Horse Teaching Unit and Prairie Oaks Subdivision from July 2006 Sept. 2007
near Gainesville, FL.


Species
An. quadrimaculatus

Cq. perturbans 2006 o

Cq. perturbans 2007 o

Cq. perturbans 9

Cx. erraticus 2006 0'

Cx. erraticus 2007 g'

Cx. erraticus 2006

o Cx. erraticus 2007

Cx. nigripalpus

Cx. salinarius g

Cx. salinarius

Ma. titillans 2


TN
1,268

148

320

788

288

1,120

148

1,120

468

388

468

148


Blue
0.004 (+0.003)b

0.372 (0.052)

0.003 (+0.003)

0.023 (+0.006)

2.154 (0.333)

0.022 (+0.005)

2.851 (+0.558)

0.113 (0.012)a

0.017 (+0.011)

0.005 (+0.004)

0.006 (+0.004)

0.014 (0.014)


Green
0.013 (+0.007)b

0.291 (+0.052)

0.006 (+0.004)

0.022 (+0.007)

2.452 (0.396)

0.028 (+0.006)

2.980 (+0.546)

0.087 (+0.011)a

0.017 (+0.010)

0.028 (0.015)

0.009 (+0.005)

0.027 (+0.013)


Diode Wavelength
Red
0.032 (0.013)a

0.250 (0.050)

0.009 (0.005)

0.037 (0.008)

3.009 (0.453)

0.024 (0.005)

3.223 (0.616)

0.083 (0.010)a

0.004 (0.003)

0.005 (0.004)

0.002 (0.002)

0.020 (0.012)


IR
0.007 (+0.005)b

0.230 (+0.050)

0.003 (+0.003)

0.030 (+0.007)

3.868 (+0.617)

0.021 (+0.005)

3.967 (+0.710)

0.038 (+0.007)b

0.009(0.007)

0.015 (+0.009)

0.004 (+0.003)

0.027 (+0.014)


No Light
0.015 (+0.005)b

0.270 (0.063)

0.006 (0.004)

0.028 (0.008)

3.154 (0.501)

0.032 (0.007)

3.932 (0.740)

0.104 (0.012)a

<0.001 (<0.001)

0.015 (0.009)

0.006 (0.004)

0.027 (0.014)


Note: Blue LED = 470 nm, Green LED = 502 nm, IR


860 nm, Red LED = 660 nm and No Light indicates no LED treatment. An


Anopheles; Cq. = Coquillettidia; Cx. = Culex; Ma. = Mansonia. TN = TN = number of trap nights were total mosquito species capture
= >1 per 20 day trapping period. Means within rows followed by the same letter were not significantly different (P< 0.05, Tukey's
standardized test [SAS Institute 2001]). An. quadrimaculatus (F4, 6315 = 2.47; P =0.0429); Cx. erraticus 2007 (F4, 5577 = 8.41; P =<
0.0001).











Table 2-2. Total number of mosquitoes/trap night for six significant mosquito species captured at the Horse Teaching Unit and Prairie


Oaks Subdivision from July 2006 Sept. 2007 near


Gainesville, FL.


Total Mosquitoes/Trap/Trap Night
Date Location TN An. quadrimaculatus Cq. perturbans Cx. erraticus


7/21/06 8/14/06 HTU
8/18/06 9/27/06 PO
5/5/07 5/24/07 HTU
PO
5/25/07 6/13/07 HTU
PO
6/14/07 7/6/07 HTU
PO
7/7/07 7/28/07 HTU
PO
7/29/07 8/17/07 HTU
PO
8/18/07 9/6/07 HTU
PO
9/7/07 9/26/07 HTU
PO


CDC
1.56
2.10
0.63
0.32
0.05
0.03
<0.01
<0.01
0.17
0.03
<0.01
<0.01
0.26
<0.01
0.67
0.03


RB
0.15
3.85
0.10
0.10
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.15
<0.01
0.15
<0.01


CDC
1,391.88
73.77
45.38
53.87
11.40
21.74
15.95
23.74
11.94
10.70
12.89
4.53
14.21
5.95
30.56
10.83


RB
0.60
3.30
0.85
0.30
<0.01
<0.01
0.05
<0.01
0.15
0.05
0.05
<0.01
0.05
<0.01
0.05
<0.01


CDC
154.38
216.47
5.56
3.76
2.95
2.46
3.60
1.31
0.72
0.51
3.37
1.00
4.89
0.68
7.28
1.51


RB
9.90
115.55
3.00
3.45
0.60
2.45
1.05
1.00
0.65
1.30
1.40
1.15
3.10
0.90
2.05
1.70


Cx. nigripalpus
CDC RB
1.13 <0.01
<0.01 0.65
<0.01 0.15
<0.01 0.05
<0.01 <0.01
0.15 <0.01
3.85 0.05
9.36 0.15
0.67 0.05
2.16 <0.01
95.53 <0.01
30.13 <0.01
301.53 <0.01
39.79 <0.01
657.94 <0.01
214.60 <0.01









Table 2-2. Continued.


Total Mosquitoes/Trap/Trap Night


Date


Location TN Cx. salinarius


7/21/06 8/14/06 HTU
8/18/06 9/27/06 PO
5/5/07 5/24/07 HTU
PO
5/25/07 6/13/07 HTU
PO
6/14/07 7/6/07 HTU
PO
7/7/07 7/28/07 HTU
PO
7/29/07 8/17/07 HTU
PO
8/18/07 9/6/07 HTU
SPO
9/7/07 9/26/07 HTU
PO

Note: An. =Anopheles; Cq.


CDC
1.88
5.20
1.81
1.29
1.45
0.67
4.25
2.97
1.11
0.24
21.32
3.20
33.95
1.13
15.11
2.34


Coquillettidia; Cx.


RB
<0.01
0.10
0.10
0.10
0.25
<0.01
0.05
0.05
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01


Ma. titillans
CDC RB
531.75 0.10
7.50 0.75
0.60 <0.01
0.29 <0.01
0.60 <0.01
<0.01 <0.01
1.90 <0.01
<0.01 <0.01
6.44 <0.01
0.16 <0.01
14.84 <0.01
0.13 <0.01
15.42 <0.01
0.08 <0.01
44.72 <0.01
0.11 <0.01


Culex; Ma. = Mansonia. CDC = Modified CDC light-trap; RB = resting box. HTU


= One modified CDC trap + CO2 (250 ml/min); PO = Two modified CDC traps + CO2 (250 ml/min). TN = number of trap nights
CDC traps were in operation. When TN < 20 (HTU) or TN < 40 (PO), traps had malfunctioned. Trap nights for all RB trapping
periods = 20.Total mosquitoes/trap/trap night = total mosquitoes captured/ # of trap nights.























-- .*.. *, .,'i. 1 ,l",. A ^.e^^^iB:r`aagasassB B

Figure 2-1. Resting boxes used at the University of Florida Horse Teaching Unit and Prairie
Oaks subdivision. A) Rear view of 30 x 30 cm resting box showing protective LED
housing. Exterior of all boxes were made using 1 cm thick exterior grade pine
plywood. The outside of each resting box was painted with two coats of flat black
exterior latex paint, and interiorly with two coats of barn red exterior latex paint.
Diode housing consisted of one 470 ml plastic container attached to the exterior rear
wall of each box by container lid. Container lids were modified with a 0.32 cm hole,
and matched to the 0.32 cm hole on the outside back wall of each resting box. B)
Front inside view of 30 x 30 cm resting box illustrating 5 cm x 5 cm x 29 cm sections
of pine used as inside corner supports. A 0.32 cm hole was drilled through the back
wall of each box to allow for the insertion of a LED. Resting boxes were painted
interiorly with two coats of barn red exterior latex paint.























:iA c B

Figure 2-2. Light emitting diode configuration used in resting boxes. A) All round lens LEDs
were 8.6 mm long by 5.0 mm in diameter. Viewing angles were 300 except for IR
(200). After a 180-ohm resistor was soldered to each LED, restricting current flow, a
female 9 volt (V) battery snap connector (270-325) was attached. B) Battery housing
used to supply power to LED configurations for resting boxes. Battery supplies (270-
383) pre-equipped with a complimentary male 9 V connecting site were used, each
with a maximum holding capacity of four AA batteries. Four rechargeable 2500
milliamp hour (mAh) AA batteries were used in all assemblages.


.~ b I~rral -: -~
lu ".
:5z
; 1 s


Figure 2-3. CDC light trap modified by the removal of its incandescent bulb. Modified trap
used a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5 cm
diameter clear plastic cylindrical body. A 36 cm diameter beveled edge aluminum lid
was set approximately 3 cm above the cylinder body creating a downdraft air current.
All traps were set 120 cm above ground using a Shepherd's hook, and collection nets
were attached to the bottom of the trap body. Carbon dioxide was provided from a 9
kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15-
psi single-stage regulator equipped with micro-regulators and an inline filter.

























Figure 2-4. Aerial view of Horse Teaching Unit location. The unit is located east of 1-75 and
approximately 1.6 km northwest of Paine's Prairie State Preserve, Alachua Co., FL.


Figure 2-5. Aerial view of Prairie Oaks subdivision which was located approximately 4.8 km
southwest of the Horse Teaching Unit, adjacent to the Paine's Prairie Preserve,
Alachua Co., FL.


IPine's Prn


I Pane' Priri


I Prarie View Su~ B11










































Figure 2-6. Test sites located within the Horse Teaching Unit. Each white rectangle represents a
test site where five boxes were equipped with one of five treatments. Sites are
numerically labeled according to corresponding eastern or western direction. White
arrow designates location of modified CDC trap.


~i--
i
7 -ClS; *
b~ I:
r ~: i,

.~5C

Z.. r.
r
-c.
;t'.i~'~i-'-' :
.ri. 5
-F~~1 r k
;~ .~~;'


Figure 2-7. Horse Teaching Unit location; west side test site habitat.































,-*

J- 4';
-f -


Figure 2-8. Horse Teaching Unit location; east side test site habitat.


:'~J: f.*. -


Figure 2-9. Representative of test sites chosen at the Prairie Oaks subdivision. All sites chosen
were consistent in surrounding vegetation, sunlight exposure and moisture conditions.








67






























Figure 2-10. Test sites located within Prairie Oaks Subdivision. Each solid white rectangle
represents a test site where five boxes were equipped with one of five treatments. White dashed
rectangles identify the location of modified CDC traps.


Figure 2-11. Resting boxes placed with openings facing west and were spaced approximately
four meters apart and out of direct sunlight. Each site contained five treatments, one
of four LED colors and an unlit control, resulting in a total of five resting boxes per
site, 20 resting boxes per location.




















-*-2006
--W-2007


May


June


August September


Temperature


--2006
---2007


Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Precipiation


Figure 2-12. Mean monthly temperatures (C) and precipitation (cm) for the Horse Teaching
Unit (HTU) location near Gainesville, FL, using data retrieved from the National
Oceanic and Atmospheric Administration (NOAA) database. A) Monthly
temperature, May September 2006 and 2007. B) Monthly precipitation from Jan
September 2006 and 2007.









CHAPTER 3
FIELD RESPONSE OF ADULT MOSQUITOES TO WAVELENGTHS OF LIGHT
EMITITING DIODES

Introduction

In Diptera, photon detection is achieved through the ocelli and compound eyes.

Although ocelli are essential for some perceptual functions, such as the entrainment of

circadian rhythms, the compound eyes act as the primary visual organ (Allan et al. 1987).

These organs are responsible for more specialized functions including detection of

movement, patterns, contrast and color. Several laboratory and field studies have been

conducted to determine the behavior of adult Diptera in response to visual stimuli, with

special attention given to the modification of light wavelength and intensity in Culicidae

(Huffaker and Back 1943, Fox 1958, Bidlingmayer 1967, Burkett and Butler 2005).

Early luminous sources used in light traps included paraffin or acetylene lamps

(Husbands 1976). Today, multiple publications detail various light trap designs, light

sources and other factors that influence mosquito trap catch size. Some devices, such as

the New Jersey light-trap, the CDC light-trap and the Encephalitis Virus Surveillance

(EVS) light-trap, employ motorized suction fans to aid in mosquito capture and

containment (Service 1970, Ginsberg 1988, Foley and Bryan 1991). Others, including

chemical light-traps and sticky light-traps, rely on non-mechanical mosquito containment

methods (Service and Highton 1980, Sulaiman 1982).

Deviations in light intensity can significantly influence the numbers and species of

mosquitoes caught in light-traps (Service 1993). Although mosquitoes may initially

exhibit positive phototaxis to light-traps, negative phototaxis occurs at certain distances

and is dependent upon light intensity. Headlee (1937) first demonstrated the impact of

varying light intensities on catch size after noting that significant quantities of mosquitoes









were attracted to within a certain proximity of traps but were not being caught. These

proximate mosquitoes were only captured after the addition of a motorized fan to traps.

The effect of light intensity on mosquito catch has been extensively investigated and

similar results have been repeatedly produced (Barr et al. 1963, Reisen and Pfuntner

1987, Ali et al. 1989).

Variations in wavelength also impact mosquito catch rates in light-traps.

Importantly, not all mosquito species respond equally to dissimilarities in wavelength. In

laboratory studies, Gjullin et al. (1973) demonstrated that male Culex tarsalis Coquillett,

Cx. quinquefasciatus Say and Aedes sierrensis Ludlow prefer ceramic-dipped red bulbs

over similar green, blue, orange or white incandescent bulbs. Similarly, Ali et al. (1989)

found that field populations of Culx and Psorophora display wavelength preference.

Higher proportions of Cx. nigripalpus Theobald, Cx. erraticus Dyar and Knab, Ps.

columbiae Dyar and Knab and Ps. ciliata Fabricius were collected with New Jersey light-

traps modified with incandescent blue lights than did traps modified with yellow, orange,

green, red or white lights.

Much of what is known today concerning the affinity of Diptera to different

wavelengths of light can be credited to studies in which scientifically poor light sources

were used (Brett 1938, Bracken et al. 1962, Bradbury and Bennett 1974, Browne and

Bennett 1980, 1981, Allan and Stoffolano 1986b). The recent development of super-

bright light emitting diodes (LEDs) has allowed for the isolation of specific wavelengths

permitting researchers to refine techniques to more effectively attract mosquitoes using a

more precise light sources. When used in Center for Disease Control (CDC) traps, these

highly efficient, low cost LEDs have a greater intensity and have a significantly lower









energy requirement than existing incandescent bulbs (Burkett et al. 1998). Little

information exists describing the attractiveness of LEDs to different mosquito species.

Additionally, knowledge of wavelength preferences of mosquito species in suburban and

rural habitats of Florida is limited.

Therefore, the objective of this study was to determine the response of adult

mosquitoes to four selected wavelengths of light from LEDs placed in suburban and rural

habitats. Studies were conducted during 2006 and 2007 in Gainesville, FL. Light emitting

diode wavelengths selected were blue (460 nm), green (502 nm), red (640 nm) and IR

(860 nm). Blue, at 460 nm, registers at the higher end of the purple-blue range of the

visible light spectrum. However, 502 nm falls at the lower transition point between blue

and green, while 640 nm registers near the lower end of the red-yellow light spectrum.

Infrared wavelength is not detectable by the human eye, registering above the visible

spectrum at 860 nm. For additional information concerning the visible light spectrum, see

Ando and Thomas (1996). Wavelengths used in this study were selected based on capture

rates and preferences observed for several mosquito genera, including Aedes, Anopheles,

Culex and Psorophora (Burkett et al. 1998, Burkett and Butler 2005, Hoel 2005).

Materials and Methods

Diode Equipped Boxes

Diode equipped boxes with four sides and an open top and bottom were constructed

from 0.64 cm (14 in) thick exterior-grade pine lumber plywood. Each of the four sides

measured 20 x 20 cm. Boxes were constructed and designed to support one 13 x 13 cm

sticky card with one diode centered per vertical side, yielding a total of four sticky cards

and four light treatments per box. Each light treatment corresponded to one of four

colored diodes; blue (470 nm), green (502 nm), red (660 nm) or infrared (860 nm). A









0.64 cm (/4 in) diameter hole was drilled in the center of each outward facing surface of

the boxes to allow for insertion of the diode. The outside surface of each diode box was

painted with two coats of flat black exterior latex paint. Boxes were held above ground

by a 90-cm length of 1.9 cm (3% in) inner-diameter PVC pipe. PVC pipe sections,

subsequently, were supported by a 120 cm length of 1.27 cm (/2 in) diameter steel rod

(Figure 3-1).

Light Emitting Diodes and Battery Supplies

All LEDs were obtained from Digi-Key Corporation (Thief River Falls, MN).

Diodes, part number and millicandela (mcd) rating, as described in Hoel (2005), were

blue (P466-ND, 470 nm, 650 mcd), green (67-1755-ND, 502 nm, 1,500 mcd), red (67-

1611-ND, 660 nm, 1,800 mcd) and infrared (LN77L-ND, 860 nm). Because infrared

radiation is not visible to humans, infrared diodes are not mcd-rated. Round lens LEDs

were 8.6 mm long by 5.0 mm in diameter. Viewing angles were 300 except for IR (200).

A 180-ohm resistor was soldered to all LEDs, restricting current flow to prevent

mechanical failure. Power was provided by a 6 v, 12 ampere-hour (A-h), rechargeable gel

cell battery which was changed every 24 48 h (Battery Wholesale Distributors,

Georgetown, TX) (Figure 3-1).

Sticky Cards

Sticky cards (Atlantic Paste & Glue Corporation, Brooklyn, NY) were made from

black 28 pt. SBS card stock (EPA # 057296-WI-001), and coated with 32 UVR soft glue

with UV inhibitors. Black sticky cards were selected to reduce variability of reflected

light caused by LEDs. Individual sticky cards, originally supplied as 41 x 23 cm boards,

were cut to yield two 13 x 13 cm sticky cards for field use. A 0.64 cm (14 in) diameter









hole was drilled into the center of each sticky card to allow for insertion of a diode

(Figure 3-2).

CDC Light Trap

Three modified CDC light traps (model 512, John W. Hock Company, Gainesville,

FL) were used to provide a representative background mosquito population at two study

locations. As described in Hoel (2005), each CDC light trap used a 6 V DC motor and 4-

blade fan to draw flying insects through an 8.5 cm diameter clear plastic cylindrical body

(Figure 3-3). The incandescent bulb was removed from each trap. A 36-cm diameter

beveled edge aluminum lid was set approximately 3 cm above the cylindrical body

increasing the downdraft caused by the fan. All traps were set 120 cm above ground

using a Shepherd's hook, and collection nets were attached to the bottom of the trap

body.

Carbon dioxide was provided from a 9 kg compressed gas cylinder, and delivered

to traps through a 2 m long, 6.4 mm outer diameter clear plastic Tygon tubing (Saint-

Gobain Performance Plastic, Akron, OH). A flow rate of 250 mL/min was achieved by

using a 15-psi single-stage regulator equipped with an inline micro-regulator (# 007) and

an inline filter (Clarke Mosquito Control, Roselle, IL). Flow rates were confirmed using a

Gilmont Accuiicl' flowmeter (Gilmont Instrument Company, Barrington IL.). Carbon

dioxide tanks were changed approximately every 10 days or as needed. Power was

provided by a 6 V, 12 ampere-hour (A-h), rechargeable gel cell battery changed every 24

- 48 h (Battery Wholesale Distributors, Georgetown, TX).

Site and Sticky Card Trap Location

Field trials were conducted at the University of Florida Horse Teaching Unit

(HTU) and the Prairie Oaks subdivision (PO), Gainesville, FL. Both locations were









similar environments, previously shown to have productive mosquito breeding sites (J. F.

Butler personal observation, Holton 2007). The HTU is a rural equine breeding and

training facility housing an average of 50 horses yearly. The HTU is an equine breeding

and training facility housing an average of 50 horses yearly. The facility consists of 24

hectares, which includes 2.4 hectares of wetlands and a 0.2 hectare pond. The HTU is

located in the southwestern section of Gainesville, east of 1-75, and is closely bordered on

three sides by the Paine's Prairie State Preserve (Figure 3-4). The PO is a rural

subdivision with 18 loosely spaced residential units located approximately 4 km west of

the HTU, adjacent to the Paine's Prairie State Preserve (Figure 3-5). Both locations are

surrounded by a mix of hardwood and pine forest with minimal undergrowth.

Diode equipped boxes were placed at four different sites. Glue boards were

attached to the outside of the four walls so the holes in the walls and glue boards were in

alignment. Light emitting diodes were inserted into the holes from the inside of the boxes

so the LED protrudes through the glue board. The outward facing side of boxes were

fitted with one of four colored light treatments and four sticky cards. This resulted in a

total of four boxes at the HTU and four boxes at the PO. All residential test sites utilized

at the PO were consistent in surrounding vegetation, sunlight exposure and moisture

conditions (Figure 3-6). Among the 18 PO residences, boxes were located in the rear

section of four backyards, spaced approximately three houses apart (Figure 3-7).

Sites chosen at the HTU were divided and named according to the corresponding

cardinal direction (Figure 3-8). Differences in surrounding vegetation were noted in all

sites, with differences in humidity assumed. Both northeast and southeast sites were

similar in fauna, and were located within 30 yards of residential units. However, the









southeastern site was separated from the residential units by a thin stretch of mixed pine

forest while the northeastern site was not (Figure 3-9a, b). The northwestern site was

adjacent to the quarter hectare pond, containing a mixture of aquatic and terrestrial

vegetation (Figure 3-9c). The southwestern site was moderately shaded, surrounded by

inconsistent ground cover and mixed hardwood forest (Figure 3-9d). Temperature and

humidity conditions at both locations were obtained from online NOAA databases.

Methodology

To begin a trial, diode equipped boxes were placed at four sites, with the outward

facing side of boxes fitted with one of four colored light treatments and four sticky cards.

CDC light-traps were hung from Shepherd's hooks, with collection nets attached to the

outflow of the trap. After diode equipped boxes and CDC traps operated in the field for

24 h (one trap night), sticky cards were collected and CDC catch bags were changed.

Mosquitoes recovered from traps were brought back to the laboratory where they were

counted and identified. CDC light traps were serviced daily with batteries and catch bags

changed every 24 h. Carbon dioxide tanks were changed approximately every 10 days or

as needed.

Sticky card trapping at the HTU occurred from 16 Aug. 27 Sept. 2006 resulting in

20 trap nights, and from 5 May 13 Sept. 2007 resulting in 120 trap nights. Sticky card

trapping at the PO took place from 5 May 13 Sept. 2007 resulting in 120 trap nights.

One modified CDC light-trap was operational at the HTU from July 21 August 16,

2006 resulting in 20 trap nights, and from 5 May 13 Sept. 2007 resulting in 120 trap

nights. In 2006, at the HTU, CDC trapping (July 21 August 16, 2006) took place prior

to, but not during the 2006 sticky card trapping period (16 Aug. 27 Sept. 2006). Since

relative mosquito species composition of the HTU is known, these previously run CDC









data (July 21 August 16, 2006) were used to represent mosquito population data for the

2006 sticky card trial (16 Aug. 27 Sept. 2006). Trapping at the PO with two CDC traps

took place from 5 May 13 Sept. 2007 resulting in 240 trap nights. At the PO, trapping

intervals for the two CDC traps and the sticky traps were identical in 2007. Mosquitoes

captured from both sticky card traps and CDC light-traps were identified to sex and

species using the dichotomous keys of Darsie and Morris (2003) and Darsie and Ward

(2005). Identification data were logged into a IS' Excel 2007 spreadsheet.

Statistical Analysis

Mosquito preference for diode wavelengths was evaluated using a multi-factorial

ANOVA (SAS Institute 2001). For analysis, all data were normalized using the SQRT

(n+1) transformation, however actual values are given in text and tables. The model

included the fixed effects of location, site and diode treatment, the interaction term,

location*diode treatment and the random effect, trial. In instances where either the

interaction term or the trial effect was significant, the data were analyzed separately by

location or trial (year). Tukey's Standardized Test (a=0.05) was used to separate

treatment means.

Results

In 140 trap nights at the HTU and PO, 452 mosquitoes, including 29 mosquito

species from seven genera, were captured on sticky cards. Aedes vexans Meigen females,

Cq. perturbans males, Cq. perturbans females, Cx. erraticus females, Cx. nigripalpus

females, Cx. salinarius females, Mansonia titillans Walker females and Oc. infirmatus

females were collected in numbers high enough to analyze (Table 3-1). Mosquitoes

excluded from analysis due to low numbers or little medical importance included Ae.

albopictus Skuse, An. crucians Wiedemann, An. quadrimaculatus Say, Oc. canadensis









Theobald, Oc. infirmatus Dyar and Knab, Oc. sollicitans Walker, Oc. taeniorhynchus

Wiedemann, Oc. triseriatus Say, Ps. ciliata, Ps. columbiae, Ps. ferox Humboldt, Ur.

lowii Theobald and Ur. sapphirina Sacken (Appendix B-l).

Significantly more Ae. vexans females, Cx. nigripalpus females and Oc. infirmatus

females were captured on sticky cards fitted with blue diodes (F = 4.00; df = 3, 2544; P =

0.0074) than those with red or IR diodes (F = 4.66; df = 3, 2544; P = 0.0030; F = 3.49; df

= 3, 2864; P = 0.0150, respectively) (Table 3-1). Numerically, sticky cards affixed with

IR diodes caught the fewest female Ae. vexans, Cx. nigripalpus and Oc. infirmatus.

Only one trial was completed during the 2006 trapping period. Because mosquito

population densities differed between the 2006 and 2007 trapping periods, dissimilarities

between multiple mosquito species were observed. Among Coquillettidia males and

females and Oc. infirmatus females, significantly more mosquitoes were captured during

trial one in 2006 than all 2007 trials (F = 3.86; df = 3, 2226; P = 0.0091, F = 6.19; df= 3,

2864; P < 0.0003, F = 3.49; df = 3, 2864; P = 0.0150, respectively).

Significantly more Cq. perturbans males were captured on sticky cards containing

green diodes than those with the blue or IR diodes (F = 3.86; df= 3, 2226; P = 0.0091).

Numerically, the greatest numbers of males were counted on sticky cards affixed with

green diodes, with the fewest on sticky cards with blue diodes. Significantly more Cq.

perturbans female mosquitoes were captured on sticky cards with green diodes than on

sticky cards fitted with red or IR diodes (F = 4.66; df = 3, 2864; P = 0.0003).

Numerically, sticky cards fitted with IR diodes captured the fewest Cq. perturbans

females (Table 3-1).









Preferences between diode treatments were observed for multiple Culex species.

Blue diode fitted sticky cards captured significantly more Cx. erraticus females than were

caught on sticky cards using IR diode treatments (F = 2.96; df = 3; P = 0.0309). There

was a significant interaction between diode treatment and location (F = 2.81, df= 3,

1267; P = 0.0381), therefore the p-values for diode effects were determined using the

interaction error term. However, no significant differences in diode preference were

observed (Table 3-1).

Data for Ma. titillans were analyzed separately by trial (year). During the 2006

trapping period, significantly more Ma. titillans females were captured at the HTU

location on sticky cards fitted with either blue or green LEDs than those with red or IR

LEDs (F = 6.22; df = 3; P = 0.0003). Numerically, the total HTU capture ofMa. titillans

females was lowest with IR diodes (Table 3-1). Also, considerably more females were

captured at the northwest trapping site at the HTU than from any other HTU trapping

sites (F = 5.41; df = 3, 313; P = 0.0012).

Approximately 91,766 female mosquitoes were captured using modified CDC

light-traps in 140 trap nights at the HTU location (one CDC trap), and 240 trap nights at

the PO location (two CDC traps). Mean numbers of mosquitoes captured per trap night of

the seven mosquito species analyzed from sticky card collections are presented in Table

3-2. Overall, 29 species from 8 genera were captured (Appendices B-l). The only species

captured with CDC traps but not on sticky cards was Cx. quinquefasciatus Say.

With 2/3 more trap nights and two operational CDC traps at the PO, the HTU

location accounted for more than 70% of all mosquitoes captured (64,893). Combined,

both CDC traps placed at the PO location accounted for only 26,873 mosquitoes









(Appendices B-l, B-2). Mean numbers of mosquitoes captured per trap night in modified

CDC light-traps greatly differed for several mosquito species between the 2006 to 2007

trapping periods. During the 2006 trapping period at the HTU location, approximately

1,400 Cq. perturbans females were captured, compared with an average of 73 females

during the corresponding 2007 trapping period (September) (Table 3-2). Average capture

of Cx. erraticus and Cx. nigripalpus also varied between 2006 and 2007 trapping periods.

Culex erraticus capture at the HTU and PO locations during 2006 was over 10 times

higher than during the corresponding 2007 trapping period (September). Conversely, Cx.

nigripalpus capture at the HTU and PO locations were approximately one mosquito per

trap night in 2006, compared with 657 mosquitoes per trap night in the respective 2007

trapping period (Table 3-2).

Average monthly temperatures for August (27 C) and September (25 C) remained

relatively similar between the 2006 and 2007 trapping periods, differing by no more than

0.7 C for either monthly average (Figure 3-10a, b). However, in 2006, an average of 7

cm of rainfall was recorded in August 2006 compared with approximately 17 cm during

the same period in 2007. Similarly, less than 8 cm of rainfall were recorded for

September in 2006, with approximately 9 cm were recorded in September of 2007. The

highest average precipitation for 2007 occurred in July (22.6 cm), and lowest average

precipitation occurred in May (1.9 cm) (Figure 3-10a, b).

Discussion

Using New Jersey traps fitted with colored lamps of equal intensity, Bargren and

Nibley (1956) observed that Ae. vexans and Cx. salinarius demonstrated higher attraction

to blue (peak at 447 nm) lamps than to yellow (peak at 570 nm) or white lamps (peak at

649 nm). However, a wavelength preference for Cx. nigripalpus was not observed.









Burkett et al. (1998) demonstrated mixed results when comparing total captures of Cx.

nigripalpus with CDC light-traps fitted with IR (940 50 nm), red (613 50 nm), orange

(605 50 nm), yellow (587 50 nm), green (567 50 nm), blue (450 50 nm), white or

no-light wavelength treatments. Mosquitoes were captured in high numbers with blue,

green and orange wavelength treatments, resulting in no clear wavelength preference

between those spectral ranges.

Our findings agree with Bargren and Nibley's (1956) observations where

considerably more Ae. vexans mosquitoes were captured on sticky cards fitted with blue

diode treatments. However, we observed no significant differences in wavelength

preference for Cx. salinarius. In contrast to Burkett et al. (1998) observations,

considerably more Cx. nigripalpus were captured on sticky cards fitted with blue diodes

than those with red diodes. These results suggest a spectral sensitivity for Cx. nigripalpus

females at the higher end of the blue spectrum (> 450 nm), with little sensitivity for

wavelengths in the lower end of the red spectrum (< 640 nm).

While testing filtered light of known wavelengths to equate host preference with

landing rates of Cq. perturbans, Browne and Bennett (1981) determined that shorter,

blue-green wavelengths (400-600 nm) attracted significantly more mosquitoes than did

longer wavelengths (> 800 nm). Ali et al. (1989) observed a similar light preference for

Cq. perturbans while assessing multiple wavelengths with varying intensities, reporting

the greatest attraction to blue-green wavelengths (430 550 nm). These results were

comparable to our observations that significantly more Cq. perturbans were captured on

sticky cards affixed with lower spectrum green diodes (502 nm), while fewer were

captured on sticky cards fitted with higher spectrum IR (860 nm) diodes. Through the use









of more scientifically exact LEDs, our results demonstrated a stronger preference for Cq.

perturbans to green wavelengths (502 nm) than blue (470 nm), suggesting wavelength

attraction nearer to the green range of the blue-green spectrum (> 500 nm).

Ali et al. (1989) reported higher capture rates for Cx. erraticus when using blue

colored bulbs (430 490 nm) compared with red colored bulbs (620 720 nm) of similar

intensity. We captured significantly more Cx. erraticus females on sticky cards affixed

with blue diodes than with sticky cards fitted with IR diodes. However, we observed no

significant preferences between blue, green or red diodes. Therefore, wavelength

preferences for Cx. erraticus range in the upper blue band of the spectrum (< 470 nm),

with little preference for wavelengths higher in the visual spectrum (> 620 nm).

Significantly more Ma. titillans females were captured at the HTU location on

sticky cards affixed with blue or green diodes than those with red or IR diodes. Burkett et

al. (1998) observed similar preferences with Ma. dyari Belkin, capturing mosquitoes

using CDC light-traps fitted with either yellow or green LEDs. These wavelengths fall

within the 500-600 nm range, which is consistent with most known mosquito wavelength

spectral sensitivities (Allan 1994).

Approximately 23% (105) of all mosquitoes captured on sticky cards (451) were

males. Coquillettidiaperturbans represented the majority of males captured with 52

mosquitoes, but no male Ae. albopictus, Ps. columbiae or Ur. lowii were trapped. The

number of male mosquitoes captured for other species ranged from one to 13.

Effectively sampling male mosquito populations can be an important tool in the

surveillance of transovarially transmitted arboviruses such as La Crosse virus. Male

mosquito population densities in combination with female population densities can be









important indicators of general population fecundity and reproductive status of a target

species. In population modeling, this combination of factors makes age-grading a

possible tool in the comprehension of vector potential of a disease transmitting

population (Garrett-Jones 1964).

Among mosquito species captured on sticky cards, the five most common species

were Cq. perturbans (132), Ma. titillans (78), Ur. sapphirina (56), Ur. lowii (37) and Cx.

nigripalpus (36). Least common mosquitoes captured included Cx. territans (7), An.

crucians (3), Ps. columbiae (3), An. quadrimaculatus (2), and Ae. albopictus (1),

respectively. This sticky card trapping system measured mosquito preference to

wavelengths of light in the absence of alternative host stimuli. Those species captured on

sticky cards in highest numbers are species commonly observed using trap designs that

combine light attraction with alternative host stimluli (Browne and Bennett 1981, Burkett

et al. 1998, Hoel 2005). Our results demonstrate that light detection may be more

significant in host location for those mosquito species than for species captured in fewer

numbers. Mosquito species such as An. quadrimaculatus and Ae. albopictus, not captured

in high numbers on sticky cards, are species known to utilize light sources far less in host

location. Anopheles quadrimaculatus are known to prefer dark unlit surfaces, and

subsequently, are commonly captured in high numbers using dark colored resting boxes

(Goodwin 1942, Crans 1989, Irby and Apperson 1992). Aedes albopictus, a diurnal

feeding mosquito, commonly utilizes movement and/or background contrasts as primary

host cues, rather than light (Sippel and Brown 1953, Gillett 1972, Allan et al. 1987).

Overall, capture of mosquitoes on sticky cards was greatest with green LEDs (198

mosquitoes), followed by the blue (159 mosquitoes), red (60 mosquitoes) and IR (35









mosquitoes) LEDs. Mosquito wavelength preference has been shown to be in the blue-

green range (400 600 nm), with diminishing attraction as wavelengths increase in

length (> 600 nm) (Ali et al. 1989, Burket et al. 1998). Similarly, our results

demonstrated that mosquitoes exhibited a preference for the blue (470 nm) and the green

(502 nm) LEDs, with strongest preferences observed with the green diodes. While these

findings do not exclude the possible effectiveness of wavelengths in the higher blue

spectral range (> 470 nm), wavelengths in the lower green spectrum (502 nm) result in

higher mosquito attraction.

Modified CDC traps captured many more mosquitoes than did sticky cards. These

results were anticipated because of the supplement of an artificial host attractant, CO2 in

the CDC traps. Both trapping systems captured several similar mosquito species,

including Cq. perturbans and Cx. nigripalpus. Comparable to results discussed

previously, both mosquito species are commonly captured when using trap designs that

combine light with alternative host stimulation (Browne and Bennett 1981, Burkett et al.

1998, Hoel 2005). Sticky trap results further illustrate the importance of light alone in

host location for these species. Mosquito species primarily captured in modified CDC

traps, such as An. crucians and An. quadrimaculatus, are not generally observed

frequenting light-traps (Irby and Apperson 1992). However, it is important to note that

some mosquito species known to frequent light traps were only captured in high numbers

using the baited CDC trap. Ali et al. (1989) captured high numbers ofAe. vexans in light

traps, independent of other host stimulants. These results indicate the incorporation of

light into baited traps may significantly increase capture rates for mosquito species.









Mean mosquito capture per trap night from modified CDC traps for Cx. nigripalpus

differed greatly between the 2006 and 2007 trapping periods. Mosquito capture at the

HTU and PO locations were approximately one per trap night in 2006, compared to 657

Cx. nigripalpus per trap night in the respective 2007 trapping period. This dramatic

population increase may have been due to the mosquitoes' seasonal and spatial

distribution, as discussed in Day and Curtis (1994). Culex nigripalpus display an annual

population increase that coincides with Florida's summer and autumn rainy seasons,

beginning in June or July. Under normal rainy season conditions, Cx. nigripalpus can

extend their flight range beyond their breeding and resting areas. While experiencing

drought, however, populations concentrate as ground and vegetation in open areas dries.

Once drought is broken by one or more heavy rains (>5 cm), adult mosquitoes thrive. The

more frequent and rhythmic the rains, the more populations flourish. Increased

population densities such as these become a public nuisance, and provide great cause for

public health concern, given that Cx. nigripalpus is an effective vector of St. Louis

encephalitis virus and West Nile virus (Day and Curtis 1994).

Weather conditions necessary for Cx. nigripalpus to experience such dramatic

population increases occurred during the 2007 trapping period. A severe drought early in

the year caused ground water to dry, eliminating most mosquito habitats. Dry conditions

were followed by several 5 9 cm rains in June and July, occurring during optimal

periods for Cx. nigripalpus population development. These periodic rains, followed by

ample drying periods, provided the ideal environmental conditions for Cx. nigripalpus to

suddenly exceed average population densities.









Much of the early work about mosquito wavelength attraction involved the use of

imperfect light sources, such as filtered light or painted bulbs, which were only able to

generate ranges of wavelengths instead of exact wavelengths. While earlier research

provided valuable knowledge, the lack of specific wavelength data left a serious void in a

science where mosquito control/research operations are based largely on types and

numbers of mosquitoes captured in light-baited traps (Burkett and Butler 2005). The

results of this study suggest that, in the absence of alternative host-stimuli, wavelengths

in the lower green (502 nm) spectral range would be optimal for targeting a broad range

of mosquito species. Additionally, the use of LEDs as opposed to wavelength filters or

colored bulbs provides a more precise and efficient wavelength delivery system when

attempting to attract and capture spectrally sensitive insects.

The utilization of LEDs in combination with sticky cards has demonstrated the

superior effectiveness of LEDs in attracting a variety of mosquito species, as well as

capturing males and females. Given their accuracy in exact wavelength achievement,

small size and minimal power usage, light emitting diodes can be used as light sources

for various trap designs where access and equipment to targeted sites are minimal. The

ability of LEDs to operate for extended periods of time with minimal power consumption

allows these light sources to be added to virtually any trap design, with little modification

or additional equipment. Their demonstrated effectiveness for attracting mosquitoes

without the aid of supplemental host attractants further eliminates the need and costs of

heavy tanks (C02) or noxious chemicals (lactic acid, octenol). Durability of the minimal-

LED based equipment required also helps to reduce otherwise necessary and time-

consuming field maintenance. By offering extended operating time with minimal power









consumption, field durability and the ability to eliminate the need for burdensome

equipment, LEDs are removing restrictions previously set on trap designs where

equipment or field conditions were major limiting factors.

The results of this research warrant serious considerations into other aspects of

mosquito wavelength attraction. These findings demonstrate that the use of only light in a

trapping system without additional host based attractants (CO2, octenol and lactic acid)

can effectively capture mosquitoes. While differing exact wavelengths influence

mosquito preference, manipulation of wavelength frequency or intensity may also

enhance capture rates for specific mosquito species. Using poor light sources, past studies

demonstrated that these factors can significantly impact mosquito preferences to light.

With the development of LEDs capable of achieving precise wavelengths, future research

in this field will be able to further refine the knowledge of factors affecting mosquito

behavior in response to light.









Table 3-1. Mean ( SE) numbers of mosquitoes/trap/night attracted to light emitting diodes producing four different wavelengths of
light during 24 h trapping intervals at the University of Florida Horse Teaching Unit and Prairie Oaks subdivision in
Gainesville, FL.


Species
Ae. vexans


Cq. perturbans o

Cq. perturbans

Cx. erraticus

Cx. nigripalpus

Cx. salinarius
Ma. titillans 2006

Ma. titillans 2007

Oc. infirmatus


Note: Blue diode


TN Blue
640 0.019 (0.006)a

560 0.014 (0.006)b

720 0.031 (0.007)ab

320 0.034 (0.012)a

640 0.019 (0.007)a

320 0.016 (0.007)

80 0.313 (0.068)a

160 0.006 (0.006)

720 0.014 (0.004)a


470 nm, Green diode


Diode Wavelength
Green Red
0.013 (0.004)ab 0.002 (0.002)b


0.043 (0.010)a

0.049 (0.009)a


0.011 (0.006)ab

0.024 (0.007)b


0.016 (0.007)ab 0.013 (0.006)ab

0.016 (10.005)ab 0.002 (10.002)b


0.010 (0.005)

0.363 (0.110)a

0.006 (0.006)


<0.001 (<0.001)

0.100 (0.038)b

0.006 (0.006)


0.004 (0.002)ab 0.003 (0.002)b


502 nm, IR = 860 nm and Red diode = 660 nm.


<0.001 (<0.001)b

0.016 (0.005)b

0.008 (0.003)b

0.003 (0.003)b

<0.001 (<0.001)b

<0.001 (+<0.001)

0.050 (0.025)b

<0.001 (+<0.001)

0.001 (0.001)b


Ae. = Aedes; Cq. = Coquillettidia; Cx.


Culex; Ma. = Mansonia; Oc. = Ochlerotatus. TN = number of trap nights were total mosquito species capture = >1 per 20 day trapping
period. Means within rows followed by the same letter were not significantly different (P< 0.05, Tukey's standardized test [SAS
Institute 2001]). Ae. vexans (F = 4.00; df= 3, 2544; P = 0.0074); Cq. perturbans o (F = 3.86; df= 3, 2226; P = 0.0091), Cq.
perturbans Y (F = 6.19; df= 3, 2864; P = 0.0003); Cx. erraticus Y (F = 2.80; df= 3, 1261; P = 0.0386); Cx. nigripalpus Y (F = 4.66;
df = 3, 2544; P = 0.0030); Ma. titillans 2006 Y (F = 6.18; df= 3, 313; P = 0.0004); Oc. infirmatus Y (F = 3.49; df = 3, 2864; P =
0.0150).










Table 3-2. Number of mosquitoes/trap night for six mosquito species captured a the University of Florida Horse Teaching Unit and
Prairie Oaks subdivision.
Total Mosquitoes/Trap/Trap Night


Date


Location TN


7/21/06 8/16/06 HTU
8/16/06 9/27/06 PO
5/5/07 6/5/07 HTU
PO
6/6/07 6/25/07 HTU
PO
6/26/07 7/15/07 HTU
PO
7/16/07 8/4/07 HTU
PO
8/5/07 8/24/07 HTU
PO
8/25/07 9/13/07 HTU
PO


Ae. vexans


CDC
1.56

6.47
6.11
5.44
9.68
8.79
13.50
2.35
7.64
27.05
25.75
42.11
6.63


SC

<0.01
0.10
<0.01
0.10
0.05
0.20
0.05
0.10
0.05
0.40
<0.01
<0.01
0.05


Ca. perturbans


CDC
1,391.88

47.33
51.69
22.63
21.88
14.21
18.35
9.24
8.82
12.32
5.88
18.11
7.95


SC

2.15
0.60
0.20
0.45
0.25
0.05
0.20
0.05
<0.01
<0.01
0.05
<0.01
<0.01


Cx. erraticus


CDC
154.38

5.47
3.50
5.50
1.26
1.63
0.78
0.41
0.42
4.21
0.98
5.42
1.03


SC

0.70
0.15
0.00
<0.01
<0.01
0.10
<0.01
<0.01
<0.01
0.10
<0.01
<0.01
<0.01


Cx. nigripalpus
CDC SC
1.13


<0.01
0.03
1.56
1.85
3.26
9.33
1.00
3.00
113.32
32.75
667.58
140.82


<0.01
0.15
<0.01
0.10
<0.01
<0.01
0.05
<0.01
<0.01
<0.01
0.10
0.10
0.15












Table 3-2. Continued.


Total Mosquitoes/Trap/Trap Night


Date


Location TN Ma. titi


7/21/06 8/16/06 HTU
8/16/06 9/27/06 PO
5/5/07 6/5/07 HTU
PO
6/6/07 6/25/07 HTU
PO
6/26/07 7/15/07 HTU
PO
7/16/07 8/4/07 HTU
PO
8/5/07 8/24/07 HTU
PO
8/25/07 9/13/07 HTU
PO


Note: An. =Anopheles; Cq.
= One modified CDC trap +


CDC
1.88

2.87
0.31
1.31
<0.01
3.00
0.03
6.88
0.15
15.63
0.15
22.11
0.13


llans Oc. infirmatus
SC CDC SC
531.75
3.30 <0.01
<0.01 <0.01 0.05
<0.01 5.83 <0.01
<0.01 2.69 0.05
<0.01 11.12 <0.01
<0.01 5.32 0.10
<0.01 21.33 0.05
<0.01 1.24 0.05
<0.01 10.91 <0.01
0.50 16.79 0.25
0.10 17.35 0.20
<0.01 33.84 <0.01
<0.01 5.92 0.05


= Coquillettidia; Cx. = Culex; Ma. = Mansonia. CDC = Modified CDC light-trap; RB = resting box. HTU
CO2 (250 ml/min); PO = Two modified CDC traps + CO2 (250 ml/min). TN = number of trap nights


CDC traps were in operation. When TN < 20 (HTU) or TN < 40 (PO), traps had malfunctioned. Trap nights for all RB trapping
periods = 20.Total mosquitoes/trap/trap night = total mosquitoes captured/ # of trap nights.































Figure 3-1. Four sided, diode-equipped pine boxes, each side measuring 400 cm2. Boxes were
constructed and designed to exteriorly support one 13 x 13 cm sticky card and one
diode treatment per side, yielding a total of four sticky cards and four light treatments
per diode box.


Figure 3-2. Sticky cards were constructed from black 28 pt. SBS card stock with calendared
coating (EPA # 057296-WI-001), and coated with 32 UVR soft glue containing UV
inhibitors. Individual sticky cards, originally supplied as 41 x 23 cm boards, were cut
to yield two 13 x 13 cm sticky cards.


























fo- .;
*3-' .i'

,*' ''A
' -. -
u 4~


Figure 3-3. CDC light trap modified by the removal of its incandescent bulb. Modified trap
used a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5 cm
diameter clear plastic cylindrical body. A 36 cm diameter beveled edge aluminum lid
was set approximately 3 cm above the cylinder body creating a downdraft air current.
All traps were set 120 cm above ground using a Shepherd's hook, and collection nets
were attached to the bottom of the trap body. Carbon dioxide was provided from a 9
kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15-
psi single-stage regulator equipped with micro-regulators and an inline filter.

























Figure 3- 4. Aerial view of Horse Teaching Unit location. The unit is located east of 1-75 and
approximately 1.6 km northwest of Paine's Prairie State Preserve, Alachua Co., FL.


Figure 3-5. Aerial view of Prairie Oaks Subdivision which was located approximately 4.8 km
southwest of the Horse Teaching Unit, adjacent to the Paine's Prairie Preserve,
Alachua Co., FL.


I Pines Paire


I Paine's Prairi


I ririe View SuBI























~~*F; )i -i
~n~~L~~~S.~F*5!
~c' ~YI~Y. Jl~tJI i~Jl~t-~l .
-- I~-~ r~~r- i


Figure 3-6. Representative of test sites chosen at the Prairie Oaks subdivision. All sites chosen
were consistent in surrounding vegetation, sunlight exposure and moisture conditions.


Figure 3-7. Test sites located within Prairie Oaks subdivision. Each solid white rectangle
represents a test site where one box equipped with one of four diode treatments was
placed. White dashed rectangles identify the location of modified CDC traps.

































Figure 3-8. Test sites located within the University of Florida Horse Teaching Unit. Each white
square represents a test site where one diode box was equipped with one of four diode
treatments. White arrow represents location placement of modified CDC trap.























I- IMAMsi s .:- -. A il B
















Figure 3-9. University of Florida Horse Teaching Unit location. A.) Southeast side test site
habitat. B.) Northeast side test site habitat. C.) Northwest side test site habitat. D.)
Southwest side test site habitat.


















-*-2006
--W-2007


August September


Temperature


--2006
---2007


Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Precipiation


Figure 3-10. Mean monthly temperatures (OC) and precipitation (cm) for the University of
Florida Horse Teaching Unit (HTU) location near Gainesville, FL using data
retrieved from the National Oceanic and Atmospheric Administration (NOAA)
database. A) Monthly temperature, May September 2006 and 2007. B) Monthly
precipitation from Jan September 2006 and 2007.


25

20

U 15

10

5

0


May


20

E 15

10

5

0











CHAPTER 4
RESPONSES OF PREVITELLOGENIC AND VITELLOGENIC ANOPHELES
QUADRIMACULATUS TO SELECTED WAVELENGTHS PRODUCED BY LIGHT
EMITTING DIODE

Introduction

Physiological stage, in regards to female hematophagous Culicidae, is the course of

development through which the ovaries mature. In anautogenous female mosquitoes,

development can be classified into three main phases: previtellogenic, vitellogenic, and

postvitellogenic. Each phase has important impacts on behavior including feeding, host seeking

and oviposition (Klowden 1997).

From eclosion to just preceding the first blood meal, female mosquitoes are considered

previtellogenic. During this phase the fat bodies become capable of intense synthesis of yolk

protein precursors (Lehane 2005). During the early previtellogenic phase, egg follicles remain in

a quiescent or "resting" stage until a blood meal is taken (Clements 1992). Several instinctive

behaviors of the female such as a reduction in female receptivity to males are affected because of

increased levels of Juvenile Hormone III (JH III) (Klowden 1997). Meola and Petralia (1980)

also showed that altering levels of JH III resulted in a significant impact on the biting behavior of

Culex mosquitoes.

The second, and least understood, phase is the vitellogenic phase. Considerable

information on the hormonal sequence that occurs during this phase remains unclear. Clements

(1956) and Gillett (1956) were able to definitively establish that there was a hormonal

significance throughout oogenesis. Based on this principle, Hagedorn et al. (1979) made the

important observation that ovaries of adult female mosquitoes produced ecdysteroids. This

eventually led to the isolation of several ecdysteroidogenic hormones from the head ofAedes









aegypti Linnaeus, most notably the ovarian ecdysteroidogenic hormone I (OEH) (Borovsky and

Thomas 1985, Whisenton et al. 1987). It is OEH that is believed to be the key factor in the

vitellogenic phase of oogenesis (Klowden 1997).

The vitellogenic phase is initiated by the ingestion of a blood meal. This results in the

release of OEH from the brain, stimulating the ovaries to produce ecdysteroids (Brown et al.

1995). These ecdysteroids immediately react with the fat bodies, resulting in the activation of

vitellogenin genes. Oocytes take up the vitellogenin through the hemolymph, completing

oogenesis. All eggs develop through this process synchronously, usually completing the phase in

2-5 days at favorable temperatures (Foster and Walker 2002).

Once oogenesis is complete, the female mosquito enters the postvitellogenic phase. During

this phase, hormonal reactions halt vitellogenin production, and inhibit the development of

secondary egg follicles until after oviposition has taken place. These hormones also impact the

female's actions leading to behavioral changes that increase the chances for survival of her

progeny. Once oviposition has occurred, the mosquito renters the previtellogenic stage.

Following a subsequent blood meal, a new cycle of oogenetic events begins and the cycle repeats

(Klowden 1997).

Arthropod-borne pathogens, such as those causing malaria, dengue and yellow fever,

require an incubation period within the insect vector before they can be successfully transmitted

(Lehane 1985). Additionally, only specific physiological stages of a female mosquito have the

capacity for disease transmission to humans. This combination of factors makes age-grading a

valuable tool in the comprehension of vector potential.

In epidemiological investigations, age grading allows scientists to estimate the probability

of a single mosquito surviving for one day. This key element is used in equations to estimate the









vector potential of a disease transmitting population (Garrett-Jones 1964). Additionally, age

grading also plays a pivotal role when monitoring vector control operations. When examining

diseases such as malaria, a reduction in the lifespan of a female mosquito has a much larger

impact in transmission rates than a reduction in the overall mosquito population (Wu and Lehane

1999). It is with this knowledge that modelers are able to predict future malaria epidemics, or

possible high incidence seasons.

With both the development of insecticide resistance in multiple anopheline species

(Metcalf 1989), and the devastating resurgence of malaria worldwide (Rogoff 1985), Anopheles

quadrimaculatus Say stands as a potential health threat to Florida's population. It is this intimate

relationship with malaria, coupled with their abundance in Florida that makes An.

quadrimaculatus an excellent target species in this study.

Vision plays a significant role in all major activities of an adult mosquito's life including

mating, dispersal, appetitive flight, as well as nutrient location (sugars), host location, resting,

and oviposition (Allan et al. 1987). Nielson and Haeger (1960) and Gatehouse (1972)

demonstrated that mating swarms were located by female mosquitoes using visual markers such

as corners of buildings or human observers. The structure of the swarms appeared to be

dependent upon the characteristics of the markers.

Artificial, reflected and filtered lights have been incorporated in the design of existing

efficient traps to increase their efficacy for mosquito research and surveillance with great success

(Barr et al. 1963, Service 1976, Ali et al. 1989, Burkett and Butler 2005). Ali et al. (1989) were

able to demonstrate that both Culex and Psorophora spp. showed a higher preference to light

source as opposed to light intensity when trapping in the field. Similarly Burkett and Butler

(2005) showed that not only light source, but specific light wavelengths played an important role









in host attraction. In laboratory trials, Aedes albopictus Skuse, An. quadrimaculatus and Culex

nigripalpus Theobald all showed preferences for specific wavelengths of light.

Age may also significantly impact mosquito preferences to light. Nielsen and Nielsen

(1953) observed that female Ae. taeniorhynchus Wiedemann demonstrated light preferences

approximately seven days post emergence. Following this period, mosquitoes displayed a cyclic

light preference about every fifth day. Similarly, age-influenced photophilic behavior has been

observed among field collections ofAe. taeniorhynchus (Provost 1952). However, field collected

female Ae. taeniorhynchus were noted responding to light at five days post emergence. Male

mosquitoes only exhibited preferences to light during the first three days post emergence.

Though much research has been done in regards to a female mosquito's attraction to

different wavelengths, past research has mostly focused on one physiological stage of

mosquitoes, the previtellogenic stage. When working with either colonized or wild adult females

in laboratory conditions, few researchers have worked with anything other than previtellogenic

females. Similarly, in the field, researchers have based most findings on the assumption that

female mosquitoes attracted to modified light traps were previtellogenic, host seeking

mosquitoes. Past research has been scant in regards to finding any direct link between

physiological stage and feeding patterns. However, Mogi et al. (1995) demonstrated a possible

link between ovarian development of An. subpictus Grassi and feeding habits. This assumption

was based upon a significantly higher catch rate of parous females in light traps (86.6%) than

that from cattle-baited collections (69.6%). These results were unique given that baited trapping

systems such as light traps commonly capture host seeking females (Browne and Bennett 1981,

Ali et al. 1989, Burkett et al. 1998). A direct reference to the possible application of these

findings to malaria epidemiology was also made. It is this assumption upon which my hypothesis









is based. The objective of my research was to identify preferences of two physiological stages

(previtellogenic and vitellogenic) of An. quadrimaculatus to four wavelengths (Infrared (IR),

Blue, Green and Red) in a visualometer using an open-port design. To further assess preferences

for particular wavelengths, diodes showing the highest and lowest responses were evaluated in a

visualometer using a paired-T port design.

Materials and Methods

Visualometer

The visualometer is a modified version of the olfactometer (Burkett and Butler 2005),

originally designed and built by Dr. Jerry F. Butler at the University of Florida, to evaluate

mosquito responses to different olfactory host stimuli. The visualometer, as previously described

by Hoel (2005), was modified to measure responses to visual, as opposed to olfactory (Coon

2006), stimuli. The pie shaped visualometer has 10 individually numbered sensor ports, modified

from existing olfactometer feeding stations, which can be portioned off or left in an open design.

All ports were equipped with an electrical amplification box, artificial host sensor, airflow intake

and outflow ports, and CO2 circulation system (Figure 4-la).

Attractiveness was measured as the amount of time a mosquito completes an electric

circuit positioned over a stimulus (specified wavelength). The circuit is complete when an

attracted mosquito makes contact with the sensor in an attempt to reach the artificial host stimuli.

Contact activity on artificial sensors was measured, recorded and logged over an 8 h period using

a computer. Attractiveness was quantified in contact seconds to make standardized comparisons

and measurements possible.

The visualometer was enclosed in a Faraday cage room (Lindgren Enclosures, Model no.

18-3/5-1), maintained between 280 and 320 C. This room was designed to protect against outside

electrical interference and extraneous sources of light. All visualometer surfaces were kept free









of direct human exposure, and those surfaces exposed to mosquitoes were disposable or cleaned

with soapy water before trials. (Figure 4-la, b). With the exception of LEDs, trials were run in

the dark.

Light Emitting Diodes

Light emitting diodes (LED) of four wavelengths were evaluated for their attractiveness to

two physiological stages (previtellogenic and vitellogenic) of An. quadrimaculatus in a

visualometer. All LEDs were obtained from Digi-Key Corporation (Thief River Falls, MN).

Diodes, part number and millicandela (mcd) rating, as described in Hoel (2005), were blue

(P466-ND, 470 nm, 650 mcd), green (67-1755-ND, 502 nm, 1,500 mcd), red (67-1611-ND, 660

nm, 1,800 mcd) and infrared (LN77L-ND, 860 nm). Because infrared radiation is not visible to

humans, infrared diodes are not mcd-rated. A stimulus (LED) not connected to a power source

was used as a control. Round lens LEDs were 8.6 mm long by 5.0 mm in diameter. Viewing

angles were 300 except for IR (200). A 180-ohm resistor was soldered to all LEDs, restricting

current flow to prevent mechanical failure. Power was provided by a 6 v, 12 ampere-hour (A-h),

rechargeable gel cell battery changed every 24 48 h (Battery Wholesale Distributors,

Georgetown, TX). Placement of all LEDs was completely randomized before each trial, in an

attempt to eliminate interactions between wavelengths.

Mosquitoes

Anopheles quadrimaculatus were obtained from the USDA-ARS-CMAVE Gainesville, FL

rearing facility. Rearing room conditions were maintained between 27 o and 32 C and

approximately 50 60% RH. Adults were held under a 14:10 (L:D) photoperiod.

Between 1,000 and 1,500 An. quadrimaculatus pupae were obtained weekly at

approximately 12 h pre-eclosion. Pupae were taken to the University of Florida Veterinary

Entomology Laboratory, and held in an incubator at 26 C and 75% humidity under a 14:10









(L:D) photoperiod. Upon eclosion, adult mosquitoes were fed a 10% sucrose solution for 72 h

(Figure 4-2). At 72 h post-eclosion, 150 previtellogenic females were mechanically aspirated

from the holding cage and released into the visualometer. Newly released mosquitoes were

allowed ten minutes to adjust to light and temperature conditions within the visualometer before

sensors were activated, and a trial was initiated.

Remaining mosquitoes were held for an additional 48 h in the incubator and allowed to

feed on a 10% sugar solution before being allowed to blood-feed. Bloodfeeding took place 120 h

post-eclosion using a suspended sausage casing that held warmed defribrinated bovine blood

(Figure 4-3). Adult mosquitoes were allowed to bloodfeed for 3 h before the sausage casing was

removed and discarded. At 144 h post-eclosion, 150 vitellogenic mosquitoes were mechanically

aspirated from the holding cage, and used in a new visualometer trial as previously described.

Open-Port Visualometer Trials

The visualometer was first used in an open design incorporating all treatments. This design

allowed mosquitoes to freely move between the four LED treatments and unlit treatment that

were affixed to five of the 10 sensor ports. One LED or an unlit LED was placed in a vertical

arrangement at each odd numbered sensor port. Even numbered ports were equipped with

sensors, but were unlit. Treatment placement was completely randomized before each trial

(Figure 4-la,b).

A minimum of 15 previtellogenic and 15 vitellogenic trials were conducted. Successful

trials were trials where the average contact seconds were within +50% of the group mean (Hoel

2005). Trials with contact second averages outside this range either suffered from equipment

malfunction (faulty sensor, low humidity) or poor mosquito quality.

Based on data collected from visualometer trials, two pairs of diodes were selected for

subsequent study. The one pair was selected based upon significant differences in recorded levels









of mosquito activity between physiological stages. The second pair was selected based upon

highest and lowest recorded levels of mosquito activity between LED treatments, within both

physiological stages.

Paired-T Port Visualometer Trials

Using plastic dividers, the visualometer was divided into five equal arenas, each containing

two sensor ports (Coon 2006). This arrangement allowed for completion of five replications per.

trial. For each trial, the arena contained one diode treatment (either blue/red or blue/green)

positioned in a vertical arrangement. Each arena contained airflow intake and outflow ports,

mosquito insertion hole and two diode insertion points with paired sensors. Thirty mosquitoes

were released in each arena, totaling 150 mosquitoes used per trial (Figure 4-1c). All other

visualometer setup and sterilization procedures were completed as previously described.

A minimum of three previtellogenic and three vitellogenic trials were conducted. For

blue/green diode treatment trials, seven previtellogenic and eight vitellogenic trials were

completed to achieve ten replications. For the blue/red diode treatment trials, four previtellogenic

and six vitellogenic trials were required to achieve ten replications.

Methodology

Before each trial, all visualometer surfaces not disposable were cleaned with soapy water

kept free of direct human exposure. For open-port trials, the four LED treatments and unlit LED

were randomly affixed to five odd numbered sensor ports. Then, 150 female mosquitoes (72 h

post-eclosion for previtellogenic mosquitoes, 144 h post eclosion for vitellogenic mosquitoes)

were mechanically aspirated from the holding cage and released into the visualometer. Finally,

power to LEDs was connected, the faraday cage was sealed, and an eight-hour trial was initiated.

For paired-T port trials, plastic dividers were used to divide the visualometer into five

equal arenas, each containing two sensor ports. All other visualometer setup and sterilization









procedures were completed as described above. For previtellogenic trials, thirty mosquitoes (72 h

post-eclosion) were released in each arena, totaling 150 mosquitoes used per trial (Figure 4-1c).

For vitellogenic trials, thirty mosquitoes (144 h post-eclosion) were released in each arena,

totaling 150 mosquitoes used per trial, and used as previously described.

Statistical Analysis

As previously described in Coon (2006), the Medusa 2.1.2 software designed by N.

Hostettler in Gainesville, FL, was used to analyze the cumulative contact seconds of An.

quadrimaculatus at each sensor port per eight hour trial. All data were normalized using the

SQRT (n+1) transformation but actual values are shown in text and tables. Eight previtellogenic

and eight vitellogenic open-port trials were selected from a pool of 31 trials. Selected trials were

found to be within 50% of the group mean (Hoel 2005). Data collected from selected open-port

trials were evaluated using a multi-factorial ANOVA (SAS Institute 2001). The model included

the fixed effect of diode treatment (wavelength). For paired-T trials, a one tailed t-test was used

to evaluate significant differences between means.

Results

Open-Port Visualometer

Amongst previtellogenic An. quadrimaculatus released in the open-port visualometer,

there were no significant differences in mosquito contact seconds between mosquitoes exposed

to the four LED wavelengths. However, mosquito contact seconds were recorded most

frequently on green LEDs (0.2514 s), followed by red (0.2189 s), control (0.1855 s), IR (0.1622

s) and blue LEDs (0.0996 s) (Table 4-1). Results for individual trials, as well as 50% ranges

can be found in Appendix C-1.

Similarly, among vitellogenic An. quadrimaculatus released in the open-port visualometer

there were no significant differences in mosquito contact seconds when mosquitoes were









exposed to the four LED wavelengths. However, mosquito contact seconds (cs) were recorded

most frequently on blue LEDs (0.1612 cs), followed by red (0.1496 cs), control (0.1397 cs), IR

(0.1255 cs) and green LEDs (0.0804 cs) (Table 4-1). Results for individual trials, as well as +

50% ranges can be found in Appendix C-2.

In comparisons within wavelengths, significant differences in mosquito contact seconds

were observed between previtellogenic and vitellogenic An. quadrimaculatus examined in the

open-port visualometer. Significantly higher activity was recorded with previtellogenic

mosquitoes (0.2189 cs) than with vitellogenic mosquitoes (0.1486 cs) with red LEDs (F = 98.08;

df = 1, 2; P = 0.0100). Inversely, vitellogenic mosquitoes were in contact with blue LEDs

(0.1428 cs) for a longer period of time than were previtellogenic mosquitoes (0.0656 cs) (F =

111.24; df = 1, 2; P = 0.0089) (Table 4-1).

Because of increased contact seconds for previtellogenic and vitellogenic An.

quadrimaculatus, certain LED wavelengths were selected for additional analysis using a paired-

T port visualometer system. The blue/red diode treatments were selected based upon significant

differences in recorded levels of mosquito activity between physiological stages. The blue/green

diode pair was selected based upon highest and lowest recorded levels of mosquito activity

between LED treatments, within each physiological stage.

Paired-T Port Visualometer

No significant differences in mosquito contact seconds were observed among

previtellogenic or vitellogenic An. quadrimaculatus exposed to blue and red LEDs. For

previtellogenic An. quadrimaculatus, sensors recorded mosquito contact over blue LEDs (1.0334

cs) more frequently than over red LEDs (1.0207 cs). However, vitellogenic An. quadrimaculatus

contacted sensors over red LEDs (1.0377 cs) more frequently than blue LEDs (1.0351 cs) (Table









4-2). Results for all previtellogenic and vitellogenic individual replications can be found in

Appendix C-3 and C-4, respectively.

In paired studies comparing blue and green LEDs, previtellogenic and vitellogenic An.

quadrimaculatus showed no significant differences in contact seconds. Mosquito contact seconds

for previtellogenic An. quadrimaculatus were nearly equal over blue LEDs (1.0153 cs) and green

LEDs (1.0109 cs). Similar responses were observed with vitellogenic An. quadrimaculatus,

where green LEDs (1.0176 cs) and blue LEDs (1.0168 cs) performed similarly (Table 4-2).

Results for all previtellogenic and vitellogenic individual replications can be found in Appendix

C-5 and C-6, respectively.

Discussion

In comparisons between previtellogenic and vitellogenic An. quadrimaculatus released in

the open port visualometer, previtellogenic mosquitoes were in contact with red diodes

significantly longer than were vitellogenic mosquitoes. However, these results are confounded

when compared with other LEDs. Among previtellogenic An. quadrimaculatus released in the

open-port visualometer, no significant differences in mosquito contact seconds were observed

between the four LED wavelengths. Likewise, no significant differences were observed among

previtellogenic An. quadrimaculatus exposed to blue and red or blue and green LEDs in a paired-

T port visualometer.

Under ideal conditions, previtellogenic mosquitoes at four days post eclosion have

physiologically initiated the host seeking stage (Clements 1992). During this stage, mosquitoes

are known to utilize visual cues such as color (wavelength) to locate hosts in search of a blood

meal (Service 1993). We expected to see attraction to LEDs during this stage. That mosquitoes

did not exhibit higher preference for LEDs than for the unlit control is surprising and suggests

that light alone is a poor attractor or that our experimental design needs to be refined.









Using a visualometer incorporating an artificial host (blood agar/CO2), Burkett and Butler

(2005) exposed previtellogenic An. quadrimaculatus to a white light control, a black control and

filtered light ranging from 350 nm 750 nm (50 nm increments). Significantly longer feeding

times were recorded over black and white controls than all other wavelengths. Additionally, 350

nm wavelengths recorded significantly less feeding time than all other individual wavelengths.

These observations suggest previtellogenic An. quadrimaculatus prefer no light to all other

wavelengths during host location, or when feeding. Our findings differed from Burkett and

Butler (2005) in that mean contact seconds were highest with green diodes than all other

treatments. However, these differences may have occurred because an artificial host was not

used during our visualometer trials. The stimulation of a blood-meal could serve as the precursor

to additional functions in the physiological responses of previtellogenic mosquitoes to different

wavelengths. These unstudied variables warrant additional research in the physiological effects

of a blood meal on previtellogenic mosquitoes.

Vitellogenic An. quadrimaculatus were in contact with blue LEDs significantly longer than

were previtellogenic mosquitoes in the open-port visualometer (Table 4-1). However, among

vitellogenic mosquitoes released in either an open-port or paired-T port visualometer, we

observed no significant differences in wavelength preference among the LEDs.

Based on past literature, vitellogenic An. quadrimaculatus were expected to be repelled by

light (Bradley 1943, Burkett and Butler 2005). Although, no significant differences in

wavelength preference among treatments was observed in visualometer trials, notable differences

were observed in mean mosquito contact seconds among treatments. In all trials, mosquito

contact seconds for vitellogenic An. quadrimaculatus were never higher for the unlit control than

lit LEDs. These findings suggest a possible phototactic association with parous mosquitoes.









Mogi et al. (1995) observed similar results when evaluating the feeding habits of An. subpictus

Grassi using light traps and cattle-bait traps. Significantly more parous An. subpictus were

captured in light traps (86.6%) than cattle-baited samples (69.6%).

When trapping parous mosquitoes, gravid traps utilizing darker, non-reflective water pans

captured significantly more gravid mosquitoes than traps using lighter colored pans (Belton

1967, Laing 1964, Allan and Kline 2004, Kline et al. 2006). Also, Belton (1967) demonstrated

that mosquito preference for oviposition sites is significantly decreased when oviposition sites

are illuminated. However, our observations demonstrated that vitellogenic An. quadrimaculatus

may prefer light in certain wavelengths instead of no light when given a choice. These results

also indicate that fitting LEDs of selected wavelengths to gravid traps may increase their trap

efficacy. Lights used for Belton's (1967) study were cool white lamps with a wide viewable

angle of approximately 1800. These lights uncontrollably illuminated a large area, likely

repelling photophobic mosquitoes in search of darker oviposition sites. Light emitting diodes

used in our study produce a specific wavelength, with a narrow viewable angle of 300. This

allows the delivery of exact wavelengths in one direction with little excess illumination. Utilizing

exact wavelengths would enhance the attraction of gravid traps to specific mosquito species from

longer distances, while the oviposition site of the trap remains unlit. This application could

improve population monitoring methods for medically important species known to exhibit

photophilic behavior, while maintaining dark oviposition sites.

Few significant differences in wavelength preference were observed among previtellogenic

and vitellogenic An. quadrimaculatus. Previtellogenic mosquitoes were in contact with red LEDs

significantly longer than vitellogenic mosquitoes, while vitellogenic mosquitoes contacted blue

LED significantly longer than previtellogenic mosquitoes. These findings demonstrate the effects









of physiological development on mosquito wavelength preference. During the previtellogenic

stage mosquitoes are host seeking, thus utilizing specific visual parameters to locate a blood

meal (Bidlingmayer 1994). However, in the vitellogenic stage, mosquitoes are in search of an

oviposition site and are possibly sensitive to alternative visual cues (Allan and Kline 2004). Our

results offer additional evidence of behavioral differences between reproductive stages.

Our observations merit additional research to fully understand the differences in

wavelength preference between previtellogenic and vitellogenic mosquitoes. The incorporation

of an artificial host into a visualometer would be necessary to evaluate the effects of alternative

host stimuli on previtellogenic mosquitoes in open-port and paired-T port trials. Additionally,

assessing these effects on alternative medically important mosquito species may yield different

results given that wavelength preferences can significantly differ among mosquito species

(Browne and Bennett 1981, Ali et al. 1989, Burkett et al. 1998). Ultimately, these findings need

to be examined in field trials using wild mosquitoes to avoid unnatural behaviors often

experienced with colonized mosquitoes. By affixing preferred diodes to gravid traps, mosquito

captures could be analyzed and compared to visualometer results to further elicit diode

preference, future application and field viability.











Table 4-1. Mean numbers ( SE) of previtellogenic and vitellogenic Anopheles quadrimaculatus
attracted to selected wavelengths of light emitting diodes as measured by mean
contact seconds using an open port visualometer.
Physiological Stages
Diode Treatment n Previtellogenic Vitellogenic
Blue 8 0.0996 (0.0549)b 0.1612 (0.0532)a
Green 8 0.2514 (0.0517) 0.0804 (0.0332)
IR 8 0.1621 (0.0468) 0.1254 (0.0194)
Red 8 0.2189 (0.0632)a 0.1485 (10.0526)b
No Light 8 0.1854 (0.0821) 0.1396 (0.0417)

Note: Blue = 470 nm, Green = 502 nm, IR (Infrared) = 860 nm, Red = 660 nm and No light
constituted an unlit control treatment. Means = total contact seconds per treatment over eight
hour trials (N = 8). 150 previtellogenic or vitellogenic mosquitoes released into an open port
visualometer for each trial. Means within rows followed by the same letter were not significantly
different. ANOVA: Blue (F1,15=111.24; P < 0.009); Red (F1, 15=98.08; P < 0.01)

Table 4-2. Mean numbers ( SE) of previtellogenic and vitellogenic Anopheles quadrimaculatus
attracted to paired selected wavelengths of light emitting diodes as measured by mean
contact seconds using a paired-T port visualometer.
Comparison Stage n Diode Treatment Mean (SEM)
Blue:Red Previtellogenic 4 Blue 0.0692 (0.0179)
Red 0.0427 (10.0138)
Vitellogenic 4 Blue 0.0723 (10.0158)
Red 0.0785 (10.0209)

Blue:Green Previtellogenic 7 Blue 0.0314 (10.0096)
Green 0.0221 (10.0069)
Vitellogenic 7 Blue 0.0346 (10.0104)
Green 0.0362 (0.0108)

Note: Physiological stage of An. quadrimaculatus: Previtellogenic stage = mosquitoes 72 h post
emergence. Vitellogenic stage = mosquitoes bloodfed at 120 h and released into visualometer at
144 h post emergence. Blue diode = 470 nm, Red diode = 660 nm and Green diode = 502 nm.


























A


















B

Figure 4-1. Pie shaped visualometer with 10 available feeding stations, which can be portioned
off individually or left in an open design. A) Visualometer used in an open design,
with treatments placed at all odd numbered feeding stations. B) Visualometer in
operation showing treatments, set as described above. C) Visualometer used in a
paired-T configuration.





























Figure 4-1. Continued.


Figure 4-2. Anopheles quadrimaculatus obtained from the USDA-ARS-CMAVE Gainesville,
FL rearing facility held in an incubator at 26 C and 74% humidity under a 14:10
(L:D) photoperiod. Upon eclosion, adult mosquitoes were fed a 10% sugar solution.
































Figure 4-3. Blood feeding Anopheles quadrimaculatus occurred 120 h post-eclosion using a
blood ball. Blood ball's consisted of sausage casing and defribrinated bovine blood.
Adult mosquitoes were allowed to blood feed for 3 h.











CHAPTER 5
THE IMPORTANCE OF MOSQUITO WAVELENGTH PREFERENCE IN TRAPPING AND
POPULATION SAMPLING

Since the early 1900's, the effectiveness of techniques to attract and track the movements

of hematophagous insects has continued to improve (Crans 1989). Adequate and reliable

population sampling is often seen as the most important and most difficult step in ecological

studies. Most adult mosquito trapping methods utilize attractants, including a live host, olfactory

stimuli (carbon dioxide, octenol, lactic acid) or various forms of visual stimuli (wavelength, light

source, intensity, frequency). These traps produce a bias when used in vector surveillance and

monitoring by primarily selecting for unfed, host-seeking female mosquitoes. Collections of

resting mosquito populations yield a more accurate representative sample of a mosquito

population given that adults probably spend more time resting than in flight. Resting collection

methods not only result in catching unfed host-seeking females, but also both blood-fed and

gravid females as well as males. Sampling resting mosquito populations also yields a broad age

structure.

Several non-biased methods exist for sampling resting mosquito populations. When

targeting indoor resting mosquito species, including several Anopheles and some Culex,

aspirators, resting counts and knock-down sprays are commonly used. Though few mosquito

species commonly rest indoors, those that do are often important vectors of malaria, filariasis and

some arboviruses, making accurate sampling methods of these species a necessity.

Sampling outdoor resting mosquitoes is often more difficult because outdoor populations

are usually distributed over larger areas. A better understanding of the general resting habits of

most exophilic species has allowed for the development of more accurate surveillance methods.

When sampling mosquito species known to rest amongst grassy and shrubby vegetation, such as









Psorophora columbiae Dyar and Knab, aspirators or sweep nets have been shown to be

successful. However, the utilization of artificial resting places is often the preferred sampling

method, allowing for the attraction of mosquitoes to a specific site from which they can be

conveniently collected.

Though biased, modifications and advancements to baited trapping systems continue to

show promise for increasing the efficiency of existing population sampling methods. Artificial,

reflected and filtered lights have been incorporated in the design of existing traps to increase

their effectiveness for mosquito research and surveillance with great success (Barr et al. 1963,

Service 1976, Ali et al. 1989, Burkett and Butler 2005, Hoel 2005). Additionally, the recent

development of super-bright light emitting diodes (LEDs) has allowed for the isolation of

specific wavelengths permitting researchers to refine techniques to more effectively attract

mosquitoes using more precise light sources. When used in Center for Disease Control (CDC)

traps, these highly efficient, low cost LEDs have a greater intensity and have a significantly

lower energy requirement than existing incandescent bulbs (Burkett et al. 1998). However, little

information concerning the attractiveness of LEDs to different mosquito species exists.

Knowledge of mosquito wavelength preferences without the presence of other host attractants is

limited. The objective of this project was to investigate the effects of LEDs of selected

wavelengths on mosquitoes under various behavioral and physiological states. This was

accomplished in the field with resting boxes and sticky cards. Laboratory studies were

conducted with a visualometer using mosquitoes in two stages of ovarian development. This is

the first instance of LEDs being used for this type of research.

Using Goodwin (1942) style resting boxes, wavelength preferences for adult mosquitoes

utilizing resting structures were evaluated in Chapter 2. Light emitting diode color (wavelength)









choices were blue (460 nm), green (502 nm), red (660 nm) and IR (860 nm). Center for Disease

Control (CDC) traps were used to provide representative background mosquito populations.

Results for Chapter 2 showed that certain mosquito species were attracted to, or repelled

by the LEDs, depending on color. Previous to this study, trapping involving the inclusion of

LEDs in resting boxes had not been conducted. The findings of this research demonstrate the

need for further investigation into the combination of mosquito wavelength attraction and

artificial resting boxes. Several mosquito species recovered from resting boxes fitted with LEDs

were previously thought to have little affinity to light. Based on these results and observations

from past research, variations in light intensity might also significantly impact the attractiveness

of resting boxes to mosquitoes. Relevance of these findings could lead to their future

applications in mosquito repellant devices, or to enhance the attractiveness of existing trap

models. Based on the "push-pull" premise, resting boxes or mechanical adult mosquito traps

could be placed at a considerable distance away from a home or military building, and fitted with

LEDs found to be attractive to target mosquito species. Light emitting diodes with wavelengths

known to be undesirable to these species would then be affixed to the desired building. This

combination of attractive and repellant stimulants enhances the effects of each, leading to

improved repellent devices for medically important mosquitoes.

Adult mosquito wavelength preferences were evaluated independently of other

physiological or biological stimuli in Chapter 3. Overall, mosquito capture on sticky cards was

greatest with green diodes (198 mosquitoes), followed by sticky cards with blue (159

mosquitoes), red (60 mosquitoes) and IR (35 mosquitoes) diodes. Past research has identified

mosquito wavelength preference in the blue-green range (400 600 nm), observing diminishing

results as wavelengths increase in length (> 600 nm). Similarly, our results demonstrated that









mosquitoes exhibited a preference for blue (470 nm) and green (502 nm) diodes, but stronger

preferences were observed when using green diodes. While these findings do not discount the

possible effectiveness of wavelengths in the higher blue spectral range (> 470 nm), wavelengths

ranging in the lower green spectrum (502 nm) resulted in higher mosquito capture.

These findings demonstrate that the use of only light in a trapping system without

additional host based attractants (CO2, octenol and lactic acid) can effectively capture

mosquitoes. While differing wavelengths influenced mosquito preference, manipulation of

wavelength frequency or intensity may also enhance capture for specific mosquito species. Their

demonstrated effectiveness for attracting mosquitoes without the aid of supplemental host

attractants further eliminates the need and costs of commonly used alternative host-based

attractants (C02) or noxious chemicals (lactic acid, octenol). Additionally, durability of LED-

based equipment required also helps to reduce otherwise necessary and time-consuming field

maintenance. By offering extended operating time with minimal power consumption, field

durability and the ability to eliminate the need for burdensome equipment, LEDs are removing

restrictions previously set on trap designs where equipment or field conditions were major

limiting factors.

Some mosquito species not captured in high numbers on sticky cards, such as An.

quadrimaculatus, and Ae. albopictus, were species not known to utilize light sources as primary

cues in host location. Therefore, low trap numbers were expected. However, these species were

captured in higher numbers during the resting box study (Chapter 2). An. quadrimaculatus, and

Ae. albopictus are known to prefer dark unlit surfaces, and subsequently, are commonly

recovered in high numbers using dark colored resting boxes (Goodwin 1942, Crans 1989, Irby

and Apperson 1992) Because of this, the presence these species in lit resting boxes was









surprising. The combination of results from the Chapter 2 and the Chapter 3 studies further

shows the large amount of information we have yet to gain concerning mosquito wavelength

preference.

Anopheles quadrimaculatus wavelength preferences between physiological stages

(previtellogenic, vitellogenic) were evaluated in Chapter 4. Blue (460 nm), green (502 nm), red

(660 nm) and IR (860 nm) LEDs were utilized in an open-port visualometer. Due to increased

contact seconds for previtellogenic and vitellogenic An. quadrimaculatus, certain LED

wavelengths were selected for additional analysis in a follow-up study using a paired-T port

visualometer system.

In comparisons between previtellogenic and vitellogenic An. quadrimaculatus released in

the open port visualometer, previtellogenic mosquitoes were in contact with red diodes

significantly longer than were vitellogenic mosquitoes. However, among previtellogenic An.

quadrimaculatus released in the open-port visualometer, no significant differences in mosquito

contact seconds were observed between the four LED wavelengths. Likewise, no significant

differences were observed among previtellogenic An. quadrimaculatus exposed to blue and red

or blue and green LEDs in a paired-T port visualometer. Previtellogenic (host seeking)

mosquitoes were expected to exhibit attraction to LEDs. That mosquitoes did not exhibit higher

preference for LEDs than for the unlit control is surprising and suggests that light alone is a poor

attractant or that our experimental design needs to be refined.

Future trapping applications based on data collected and field observations from Chapters

2, 3 and 4, could be useful in several fields and for multiple purposes. By utilizing the repellency

and attractiveness of specific wavelengths of light in the absence of additional host attractants,

the efficacy of virtually any trapping model can be improved. In fitting LEDs of selected









wavelengths to resting boxes, both the species specificity and the efficiency of adult mosquito

population monitoring can be drastically enhanced. Also, by incorporating LEDs of various

wavelengths in trapping systems designed to attract adult mosquitoes of specific physiological

stages, mosquito captures may be significantly increased.

The results from these studies indicate the need for additional research into mosquito

wavelength preference during multiple physiological stages, and under various biological

conditions. The integration of LEDs into various sampling and trapping systems has

demonstrated great success in impacting trapping numbers for multiple mosquito species. The

need for further species specificity in mosquito population monitoring programs grows as the

demand for more evolved sampling methods increases. Continued research into the effects of

light wavelength, frequency and intensity on individual mosquito species could lead to more

refined trapping methods. The application of this technology would be well received by

governmental agencies, mosquito control programs and homeowner targeted industries.











APPENDIX A
RESTING BOX AND MODIFIED CDC LIGHT-TRAP CAPTURES OF MOSQUITOES BY
LOCATION

Table A-1. Evaluation of resting box catches for mosquito species captured at the Horse
Teaching Unit (HTU) from July 2006 Sept. 2007 near Gainesville, FL.
Diode Wavelength


Species
An. crucians o









An. crucians









An. quadrimaculatus g


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07


Blue
0.050
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.025
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
0.025
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.038
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
0.038
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.050
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001











Table A-1 Continued.

Species
An. quadrimaculatus









Cq. perturbans o









Cq. perturbans









Cx. erraticus 3


Diode Wavelength


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07


Blue
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.513
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.025
0.075
<0.001
<0.001
<0.001
<0.001
<0.001
0.013

2.050
0.100
<0.001
<0.001
0.013
<0.001
0.025
<0.001


Green
<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.338
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
0.013

0.013
0.013
<0.001
<0.001
0.025
0.013
0.013
<0.001

2.813
0.075
0.088
0.013
<0.001
<0.001
<0.001
<0.001


Red
<0.001
0.013
<0.001
<0.001
<0.001
<0.001
0.013
0.025

0.250
0.013
<0.001
0.013
<0.001
<0.001
<0.001
<0.001

0.075
0.038
<0.001
0.013
0.000
<0.001
<0.001
<0.001

3.025
0.025
0.038
<0.001
<0.001
<0.001
<0.001
0.013


IR
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.263
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.025
0.063
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

3.188
0.050
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
0.038
<0.001
<0.001
<0.001
<0.001
<0.001
0.025
0.013

0.288
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.013
0.025
<0.001
<0.001
0.013
<0.001
<0.001
<0.001

2.675
0.063
<0.001
0.013
0.050
0.013
0.000
0.013











Table A-1 Continued.


Diode Wavelength


Species
Cx. erraticus









Cx. nigripalpus o









Cx. nigripalpus









Cx. salinarius g


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07


Blue
0.375
0.100
0.025
0.038
0.050
0.075
0.325
0.213

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
0.013
<0.001
<0.001
<0.001
<0.001

<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
0.688
0.175
0.038
0.038
0.038
0.038
0.138
0.063

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
0.038
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.013
0.025
0.038
<0.001
<0.001
<0.001
<0.001
<0.001


Red
0.488
0.250
0.025
0.113
0.013
0.088
0.025
0.100


<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
0.013
<0.001
<0.001
<0.001

0.013
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
0.450
0.075
0.025
0.013
<0.001
<0.001
0.063
0.000

0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
0.475
0.150
0.038
0.063
0.063
0.150
0.225
0.138

0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.038
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001











Table A-1 Continued.


Diode Wavelength


Species
Cx. salinarius









Cx. territans c









Cx. territans









Ma. titillans g


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07


Blue
<0.001
<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
0.025
<0.001
<0.001
<0.001
<0.001
<0.001

0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
<0.001
0.013
0.038
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
<0.001
<0.001
<0.001
0.013
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
0.025
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
0.025
<0.001
<0.001
<0.001
<0.001
<0.001

0.050
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
<0.001
<0.001
0.013
<0.001
<0.001
<0.001
<0.001
0.000

<0.001
<0.001
0.038
0.025
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001

0.050
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001











Table A-1 Continued.


Diode Wavelength


Species
Ma. titillans








Oc. infirmatus g









Oc. infirmatus 9









Oc. triseriatus cg


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07
- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07


Blue
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001











Table A-1 Continued.


Diode Wavelength


Species
Oc. triseriatus








Ur. lowii g









Ur. lowii









Ur. sapphirina g


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07
-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07


Blue
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.038
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.113
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.063
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.025
0.000
0.000
0.000
0.000
0.000
0.000
0.000

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.063
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001











Table A-1 Continued.


Diode Wavelength


Species
Ur. sapphirina


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07


Blue
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Note: Blue diode = 470 nm, Green diode = 502 nm, Red diode = 660 nm and IR = 860 nm. An.
Anopheles; Cq. = Coquillettidia; Cx. = Culex; Ma. = Mansonia; Oc. = Ochlerotatus; Ur.
Uranotaenia.


No Light
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001












Table A-2. Evaluation of resting box catches for mosquito species captured at the Prairie Oaks
(PO) subdivision from August 2006 Sept. 2007 near Gainesville, FL.
Diode Wavelength


Species
An. crucians c3


An. crucians


An. quadrimaculatus g


Date
8/18/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


- 9/27/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07


Blue
1.000
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.088
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
0.015
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.029
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.353
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.162
0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.191
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001











Table A-2. Continued

Species
An. quadrimaculatus









Cq. perturbans o









Cq. perturbans









Cx. erraticus g


Diode Wavelength


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
-8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
-8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
-8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
-8/17/07
- 9/6/07
9/26/07


Blue
0.191
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.074
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.206
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.118
0.088
0.025
0.013
0.025
0.013
<0.001
0.013


Green
0.235
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.235
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.162
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

4.824
0.113
0.038
0.050
0.013
<0.001
<0.001
<0.001


Red
0.515
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.250
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.235
0.025
<0.001
<0.001
0.013
<0.001
<0.001
<0.001

6.500
0.113
0.088
0.025
0.013
<0.001
<0.001
0.025


IR
0.132
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.191
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.206
0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

9.162
0.163
0.063
<0.001
0.013
<0.001
<0.001
0.013


No Light
0.176
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.250
0.025
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.250
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

7.353
0.125
0.113
0.025
0.013
<0.001
<0.001
0.025











Table A-2. Continued

Species
Cx. erraticus









Cx. nigripalpus o









Cx. nigripalpus









Cx. salinarius g


Diode Wavelength


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07


Blue
4.691
0.150
0.050
0.088
0.150
0.113
0.100
0.113

5.765
<0.001
<0.001
<0.001
0.025
<0.001
<0.001
<0.001

<0.001
0.013
<0.001
0.013
<0.001
<0.001
<0.001
<0.001

0.074
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
5.676
0.225
0.150
0.075
0.050
0.038
0.050
0.100

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.059
<0.001
<0.001
0.013
<0.001
<0.001
<0.001
<0.001

0.074
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
6.441
0.175
0.200
0.038
0.063
0.063
0.000
0.013

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
0.013
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
8.103
0.163
0.063
0.013
0.013
0.013
0.013
0.075

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.059
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.044
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
8.000
0.150
0.150
0.038
0.050
0.063
0.063
0.125

<0.001
<0.001
<0.001
<0.001
0.025
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.029
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001










Table A-2. Continued


Species
Cx. salinarius









Cx. territans g









Cx. territans









Ma. titillans g


Diode Wavelength
Date Blue Green Red IR No Light
7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001
5/5/07 5/24/07 <0.001 <0.001 <0.001 0.013 0.013
5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001
6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 0.013
7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001
8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001


7/21/06 8/14/06 0.029 <0.001 <0.001 <0.001
5/5/07 5/24/07 <0.001 0.050 0.038 0.025
5/25/07 6/13/07 <0.001 <0.001 <0.001 0.025
6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001
7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001
7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001
8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001
9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001

7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001
5/5/07 5/24/07 0.100 0.100 0.038 0.088
5/25/07 6/13/07 <0.001 0.025 0.013 0.038
6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001
7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001
7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001
8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001
9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001

7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001
5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001
5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001
6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001
7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001
7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001
8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001
9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001


<0.001
0.013
0.025
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
0.013
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001










Table A-2. Continued


Species
Ma. titillans


Diode Wavelength
Date Blue Green Red IR No Light
7/21/06 8/14/06 <0.001 0.059 0.044 0.059 0.029
5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001
5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001
6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001
8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001


Oc. infirmatus g 7/21/06 8/14/06 0.029 <0.001 <0.001 <0.001 <0.001
5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001
5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001
6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001
8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001


Oc. infirmatus


7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001
5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001
5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 0.013
6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001
8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001


Oc. triseriatus g 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001
5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001
5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001
6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001
7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001
8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001
9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001











Table A-2. Continued


Diode Wavelength


Species
Oc. triseriatus









Ur. lowii g









Ur. lowii









Ur. sapphirina g


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
-8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
-8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
-8/17/07
- 9/6/07
9/26/07

- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
-8/17/07
- 9/6/07
9/26/07


Blue
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.015
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

0.015
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


No Light
<0001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001

<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001











Table A-2. Continued


Diode Wavelength


Species
Ur. sapphirina


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


- 8/14/06
5/24/07
-6/13/07
- 7/6/07
7/28/07
- 8/17/07
- 9/6/07
9/26/07


Blue
0.015
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Green
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Red
0.015
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


IR
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001


Note: Blue diode = 470 nm, Green diode = 502 nm, Red diode = 660 nm and IR = 860 nm. An.
Anopheles; Cq. = Coquillettidia; Cx. = Culex; Ma. = Mansonia; Oc. = Ochlerotatus; Ur.
Uranotaenia.


No Light
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001












Table A-3. Modified CDC light trap mosquito captures at the Horse Teaching Unit (HTU) from
July 2006 Sept. 2007 near Gainesville, FL.
Species Date Trap Night Total/Trap Night
Ae. albopictus 7/21/06 8/14/06 16 <0.01
5/5/07 5/24/07 16 <0.01
5/25/07 6/13/07 20 <0.01
6/14/07 7/6/07 20 <0.01
7/7/07 7/28/07 18 0.06
7/29/07 8/17/07 19 0.05
8/18/07 9/6/07 19 <0.01
9/7/07 9/26/07 18 <0.01

Ae. vexans 7/21/06 8/14/06 16 <0.01
5/5/07 5/24/07 16 7.13
5/25/07 6/13/07 20 3.10
6/14/07 7/6/07 20 8.70
7/7/07 7/28/07 18 2.89
7/29/07 8/17/07 19 14.89
8/18/07 9/6/07 19 47.26
9/7/07 9/26/07 18 33.28

An. crucians 7/21/06 8/14/06 16 46.19
5/5/07 5/24/07 16 9.56
5/25/07 6/13/07 20 5.05
6/14/07 7/6/07 20 6.50
7/7/07 7/28/07 18 3.61
7/29/07 8/17/07 19 8.05
8/18/07 9/6/07 19 4.89
9/7/07 9/26/07 18 3.00










Table A-3. Continued.
Species
An. quadrimaculatus


Cq. perturbans









Cx. erraticus









Cx. nigripalpus


Date
7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


Trap Night Total/Trap Night
16 1.56
16 0.63
20 0.05
20 <0.01
18 0.17
19 <0.01
19 0.26
18 0.67


1,391.88
45.38
11.40
15.95
11.94
12.89
14.21
30.56

154.38
5.56
2.95
3.60
0.72
3.37
4.89
7.28

1.13
<0.01
<0.01
3.85
0.67
95.53
301.53
657.94










Table A-3. Continued.
Species
Cx. quinquefasciatus


Cx. salinarius









Ma. titillans









Oc. canadensis


Date
7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


Trap Night Total/Trap Night
16 <0.01
16 0.19
20 0.25
20 <0.01
18 <0.01
19 0.26
19 <0.01
18 <0.01


1.88
1.81
1.45
4.25
1.11
21.32
33.95
15.11

531.75
0.60
0.60
1.90
6.44
14.84
15.42
44.72

<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01










Table A-3. Continued.
Species Date
Oc. infirmatus 7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


Trap Night Total/Trap Night
16 <0.01
16 0.50
20 5.25
20 4.80
18 1.44
19 4.74
19 42.58
18 18.06


7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


<0.01
<0.01
<0.01
0.05
<0.01
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
0.05
0.11
<0.01
0.11
<0.01

<0.01
0.06
<0.01
<0.01
<0.01
<0.01
0.11
<0.01


Oc. sollicitans









Oc. taeniorhynchus









Oc. triseriatus










Table A-3. Continued.
Species Date
Ps. ciliata 7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


Trap Night Total/Trap Night
16 <0.01
16 <0.01
20 0.45
20 0.05
18 <0.01
19 <0.01
19 <0.01
18 0.17


7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


16.75
<0.01
<0.01
0.05
0.67
11.53
3.26
7.22

<0.01
<0.01
<0.01
<0.01
<0.01
0.05
<0.01
<0.01

0.13
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01


Ps. columbiae









Ps. ferox









Ur. lowii










Table A-3. Continued.
Species Date Trap Night Total/Trap Night
Ur. sapphirina 7/21/06 8/14/06 16 <0.01
5/5/07 5/24/07 16 <0.01
5/25/07 6/13/07 20 <0.01
6/14/07 7/6/07 20 <0.01
7/7/07 7/28/07 18 <0.01
7/29/07 8/17/07 19 <0.01
8/18/07 9/6/07 19 0.05
9/7/07 9/26/07 18 <0.01


Note: Ae. = Aedes; An. = Anopheles; Cq. = Coquillettidia; Cx. = Culex; Ma. = Mansonia; Oc.
Ochlerotatus; Ps. = Psorophora; Ur. = Uranotaenia. One modified CDC trap + CO2 (250
ml/min). When N < 20, traps had malfunctioned.











Table A-4. Modified CDC light trap mosquito captures at the Prairie Oaks subdivision (PO)
from July August 2006 and May Sept. 2007 near Gainesville, FL.
Species Date Trap Night Total/Trap Night
Ae. albopictus 7/21/06 8/14/06 36 0.13
5/5/07 5/24/07 38 <0.01
5/25/07 6/13/07 39 <0.01
6/14/07 7/6/07 39 0.05
7/7/07 7/28/07 37 0.16
7/29/07 8/17/07 40 1.08
8/18/07 9/6/07 38 0.58
9/7/07 9/26/07 35 0.46


Ae. vexans









An. crucians









An. quadrimaculatus


7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


<0.01
10.50
8.79
13.13
9.92
18.15
14.39
11.97

25.83
17.95
2.15
1.92
2.78
0.88
0.26
0.49

2.10
0.32
0.03
<0.01
0.03
<0.01
<0.01
0.03










Table A-4. Continued.
Species
Cq. perturbans


Cx. erraticus









Cx. nigripalpus









Cx. quinquefasciatus


Date
7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


Trap Night
36
38
39
39
37
40
38
35


Total/Trap Night
73.77
53.84
21.74
23.74
10.70
4.53
5.95
10.83


216.47
3.76
2.46
1.31
0.51
1.00
0.68
1.51

<0.01
<0.01
0.15
9.36
2.16
30.13
39.79
214.60

<0.01
<0.01
<0.01
<0.01
<0.01
0.05
<0.01
<0.01











Table A-4. Continued.


Species
Cx. salinarius


Ma. titillans









Oc. canadensis









Oc. infirmatus


Date
7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -

7/21/06
5/5/07 -
5/25/07
6/14/07
7/7/07 -
7/29/07
8/18/07
9/7/07 -


-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07

-8/14/06
5/24/07
-6/13/07
-7/6/07
7/28/07
-8/17/07
-9/6/07
9/26/07


Trap Night
36
38
39
39
37
40
38
35


Total/Trap Night
5.20
1.29
0.67
2.97
0.24
3.20
1.13
2.34


7.50
0.29
<0.01
<0.01
0.16
0.13
0.08
0.11

<0.01
<0.01
<0.01
0.03
0.03
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.11
0.14










Table A-4. Continued.
Species Date
Oc. sollicitans 7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


Trap Night
36
38
39
39
37
40
38
35


Total/Trap Night
<0.01
5.55
33.64
17.46
14.38
11.25
12.58
7.06


Oc. taeniorhynchus









Oc. triseriatus









Ps. ciliata


7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


<0.01
<0.01
0.03
0.05
<0.01
0.03
<0.01
<0.01

0.77
0.05
<0.01
0.36
0.05
0.08
0.03
<0.01

<0.01
<0.01
0.03
0.03
<0.01
0.03
<0.01
<0.01









Table A-4. Continued.
Species Date
Ps. columbiae 7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


Trap Night
36
38
39
39
37
40
38
35


Total/Trap Night
0.27
<0.01
0.03
0.13
0.16
0.65
<0.01
0.03


Ps. ferox









Ur. lowii


Ur. sapphirina


7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07

7/21/06 8/14/06
5/5/07 5/24/07
5/25/07 6/13/07
6/14/07 7/6/07
7/7/07 7/28/07
7/29/07 8/17/07
8/18/07 9/6/07
9/7/07 9/26/07


Note: Ae. = Aedes; An. Anopheles; Co. = Coquillettidia; Cx. = Culex; Ma. = Mansonia; Oc.
Ochlerotatus; Ps. Psorophora; Ur. Uranotaenia. Two modified CDC traps + CO2 (250
ml/min). When N < 40, traps had malfunctioned.


0.17
<0.01
<0.01
0.31
0.16
1.30
1.42
4.34

0.93
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01

11.20
<0.01
<0.01
0.03
0.05
<0.01
<0.01
<0.01









APPENDIX B
STICKY CARD AND MODIFIED CDC LIGHT-TRAP CAPTURES OF MOSQUITOES BY
LOCATION

Table B-1. Mosquitoes captured in a modified CDC light trap at the University of Florida Horse
Teaching Unit from July August 2006 and May Sept. 2007 near Gainesville, FL.
Species Date Trap Night Total/Trap Night
Ae. albopictus 7/21/06 8/14/06 16 <0.01
5/5/07 6/5/07 15 <0.01
6/6/07 6/25/07 16 <0.01
6/26/07 7/15/07 19 0.05
7/16/07 8/4/07 19 <0.01
8/5/07 8/24/07 19 0.05
8/25/07 9/13/07 19 <0.01


Ae. vexans








An. crucians








An. quadrimaculatus


7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07


<0.01
6.47
5.44
8.79
2.35
27.05
42.11

46.19
9.93
8.75
5.89
4.59
6.74
4.53

1.56
0.67
<0.01
0.11
0.06
<0.01
0.47











Table B-1. Continued.
Species
Cq. perturbans


Cx. erraticus








Cx. nigripalpus








Cx. quinquefasciatus








Cx. salinarius


Date
7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
-8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
-8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
-8/4/07
8/24/07
-9/13/07


Trap Night
16
15
16
19
19
19
19


Total/Trap Night
1,391.88
47.33
22.63
14.21
9.24
12.32
18.11


154.38
5.47
5.50
1.63
0.41
4.21
5.42

1.13
<0.01
1.56
3.26
1.00
113.32
667.58

<0.01
<0.01
0.19
<0.01
<0.01
0.26
<0.01

1.88
1.93
5.25
2.21
0.24
23.58
38.11











Table B-1. Continued.
Species Date
Ma. titillans 7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


Oc. canadensis








Oc. fulvus pallens








Oc. infirmatus








Oc. sollicitans


7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07


Trap Night
16
15
16
19
19
19
19


Total/Trap Night
531.75
2.87
1.31
3.00
6.88
15.63
22.11


<0.01
<0.01
0.06
<0.01
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01

<0.01
0.93
2.69
5.32
1.24
16.79
33.84

<0.01
<0.01
0.07
<0.01
<0.01
<0.01
<0.01











Table B-1. Continued.
Species Date
Oc. taeniorhynchus 7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


Oc. triseriatus








Ps. ciliata








Ps. columbiae








Ps. ferox


7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07


Trap Night
16
15
16
19
19
19
19


Total/Trap Night
<0.01
<0.01
0.06
0.11
<0.01
<0.01
0.11


<0.01
0.07
<0.01
<0.01
<0.01
<0.01
0.11

<0.01
<0.01
0.56
0.05
<0.01
<0.01
0.16

16.75
<0.01
0.06
0.21
1.59
12.53
4.00

<0.01
<0.01
<0.01
<0.01
<0.01
0.05
<0.01











Table B-1.
Species
Ur. lowii


Ur. sapphirina


Continued.
Date
7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


-8/14/06
6/5/07
6/25/07
-7/15/07
-8/4/07
8/24/07
-9/13/07

-8/14/06
6/5/07
6/25/07
-7/15/07
-8/4/07
8/24/07
-9/13/07


Trap Night
16
15
16
19
19
19
19


Total/Trap Night
0.13
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01


<0.01
<0.01
<0.01
<0.01
<0.01
0.05
<0.01


Note: Ae. = Aedes; An. = Anopheles; Cq. = Coquillettidia; Cx. = Culex; Ma. = Mansonia; Oc.
Ochlerotatus; Ps. = Psorophora; Ur. = Uranotaenia. One modified CDC trap + CO2 (250
ml/min). When N < 20, traps had malfunctioned.











Table B-2. Mosquitoes captured in a modified CDC light trap at the Prairie Oaks subdivision
from July August 2006 and May Sept. 2007 near Gainesville, FL.
Species Date Trap Night Total/Trap Night
Ae. albopictus 7/21/06 8/14/06 36 0.13
5/5/07 6/5/07 36 <0.01
6/6/07 6/25/07 34 <0.01
6/26/07 7/15/07 40 0.08
7/16/07 8/4/07 37 0.45
8/5/07 8/24/07 40 1.18
8/25/07 9/13/07 38 0.50


Ae. vexans








An. crucians








An. quadrimaculatus


7/21/06 8/14/06
5/5/07 6/5/07
6/6/07 6/25/07
6/26/07 7/15/07
7/16/07 8/4/07
8/5/07 8/24/07
8/25/07 9/13/07

7/21/06 8/14/06
5/5/07 6/5/07
6/6/07 6/25/07
6/26/07 7/15/07
7/16/07 8/4/07
8/5/07 8/24/07
8/25/07 9/13/07

7/21/06 8/14/06
5/5/07 6/5/07
6/6/07 6/25/07
6/26/07 7/15/07
7/16/07 8/4/07
8/5/07 8/24/07
8/25/07 9/13/07


<0.01
6.11
9.68
13.50
7.64
25.75
6.63

25.83
16.86
2.24
2.68
1.09
0.93
0.24

2.10
0.33
<0.01
0.03
<0.01
<0.01
<0.01










Table B-2. Continued.
Species
Cq. perturbans


Cx. erraticus








Cx. nigripalpus








Cx. quinquefasciatus








Cx. salinarius


Date
7/21/06 8/14/06
5/5/07 6/5/07
6/6/07 6/25/07
6/26/07 -7/15/07
7/16/07 -8/4/07
8/5/07 8/24/07
8/25/07 -9/13/07

7/21/06 8/14/06
5/5/07 6/5/07
6/6/07 6/25/07
6/26/07 7/15/07
7/16/07 -8/4/07
8/5/07 8/24/07
8/25/07 -9/13/07

7/21/06 8/14/06
5/5/07 6/5/07
6/6/07 6/25/07
6/26/07 -7/15/07
7/16/07 -8/4/07
8/5/07 8/24/07
8/25/07 9/13/07

7/21/06 8/14/06
5/5/07 6/5/07
6/6/07 6/25/07
6/26/07 7/15/07
7/16/07 8/4/07
8/5/07 8/24/07
8/25/07 9/13/07

7/21/06 8/14/06
5/5/07 6/5/07
6/6/07 6/25/07
6/26/07 -7/15/07
7/16/07 -8/4/07
8/5/07 8/24/07
8/25/07 -9/13/07


Trap Night Total/Trap Night
36 73.77
36 51.69
34 21.88
40 18.35
37 8.82
40 5.88
38 7.95


216.47
3.50
1.26
0.78
0.42
0.98
1.03

<0.01
0.03
1.85
9.33
3.00
32.75
140.82

<0.01
<0.01
<0.01
<0.01
<0.01
0.05
<0.01

5.20
1.39
1.82
1.75
0.52
3.30
1.71











Table B-2.
Species
Ma. titillans


Oc. canadensis








Oc. fulvus pallens








Oc. infirmatus








Oc. sollicitans


Date
7/21/06
5/5/07 -
6/6/07-
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07-
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07


Trap Night
36
36
34
40
37
40
38


Total/Trap Night
7.50
0.31
<0.01
0.03
0.15
0.15
0.13


<0.01
<0.01
0.03
0.03
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01
0.08
0.03

<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01











Table B-2.
Species
Oc. taeniorhynchus








Oc. triseriatus


Ps. ciliata








Ps. columbiae








Ps. ferox


Date
7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07

7/21/06
5/5/07 -
6/6/07 -
6/26/07
7/16/07
8/5/07 -
8/25/07


-8/14/06
6/5/07
6/25/07
-7/15/07
-8/4/07
8/24/07
-9/13/07

-8/14/06
6/5/07
6/25/07
-7/15/07
-8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
- 8/4/07
8/24/07
-9/13/07

- 8/14/06
6/5/07
6/25/07
-7/15/07
-8/4/07
8/24/07
-9/13/07


Trap Night Total/Trap Night
36 <0.01
36 <0.01
34 0.03
40 0.05
37 <0.01
40 0.03
38 <0.01


0.77
0.06
0.21
0.20
0.12
0.03
<0.01

<0.01
<0.01
0.06
<0.01
<0.01
0.03
<0.01

0.27
<0.01
<0.01
0.25
0.09
0.60
0.03

0.17
<0.01
0.09
0.28
0.58
1.83
2.53










Table B-2.
Species Date Trap Night Total/Trap Night
Ur. lowii 7/21/06 8/14/06 36 0.93
5/5/07 6/5/07 36 <0.01
6/6/07 6/25/07 34 <0.01
6/26/07 7/15/07 40 <0.01
7/16/07 8/4/07 37 <0.01
8/5/07 8/24/07 40 <0.01
8/25/07 9/13/07 38 <0.01

Ur. sapphirina 7/21/06 8/14/06 36 11.20
5/5/07 6/5/07 36 <0.01
6/6/07 6/25/07 34 <0.01
6/26/07 7/15/07 40 0.05
7/16/07 8/4/07 37 0.03
8/5/07 8/24/07 40 <0.01
8/25/07 9/13/07 38 <0.01

Ae. = Aedes; An. = Anopheles; Cq. = Coquillettidia; Cx. = Culex; Ma. = Mansonia; Oc. =
Ochlerotatus; Ps. = Psorophora; Ur. Uranotaenia. Two modified CDC traps + CO2 (250
ml/min). When N < 40, traps had malfunctioned.









APPENDIX C
RESPONSE OF PREVITELLOGENIC AND VITELLOGENIC ANOPHELES
QUADRIMACULATUS TO SELECTED LED WAVELENGTHS USING A
VISUALOMETER IN A PAIR-T AND OPEN-PORT DESIGN

Table C-1. Evaluation of previtellogenic Anopheles quadrimaculatus attraction to four
selected wavelengths of light emitting diodes using an open-port visualometer.
Diode Wavelength
Trial Blue Green Red IR No Light Trial Mean
M020507 <0.0001 0.2588 0.0950 0.1238 0.0275 0.1010
M022007 0.0138 0.3588 0.1100 0.2563 0.0963 0.8350
M022907 0.7625 0.3963 0.0963 0.0963 <0.0001 0.6650
M030607 0.1375 0.3425 0.3713 0.1225 0.2613 1.2350
M041707 0.4600 0.0900 0.2875 0.0400 0.2575 1.1350
M050707 <0.0001 0.3688 0.0813 0.0413 0.7050 1.1963
M061107 <0.0001 0.2763 0.0138 0.3138 0.0950 0.6988
M061207 <0.0001 <0.0001 0.5463 0.0550 0.0550 0.6563
M061907 0.1100 0.1788 0.2450 0.3725 0.0138 0.9200

Note: Means = total contact seconds per treatment over eight hour trials. Trials used were
selected from a pool of 17 open-port visualometer trials with previtellogenic An.
quadrimaculatus. Trials excluded denoted mean contact seconds not within +50% of the
group mean contact seconds. Contact second averages above 50% of total trial means
implied sensor malfunction; contact-second averages below 50% of total trial means
implied poor mosquito quality. Blue = 470 nm, Green = 502 nm, IR = 860 nm, Red = 660
nm and no light constituted an unlit control treatment. Trial means = total contact seconds
per trial.









Table C-2. Evaluation of vitellogenic Anopheles quadrimaculatus attraction to four
selected wavelengths of light emitting diodes using an open-port visualometer.
Diode Wavelength
Trial Blue Green Red IR No Light Trial Mean
M030207 0.1788 0.0413 <0.0001 0.2050 0.0550 0.4800
M032907 0.2463 0.0550 0.1513 0.0838 <0.0010 0.5363
M050507 <0.0001 0.2738 0.0138 0.1225 0.0688 0.4788
M060707 <0.0001 0.1225 <0.0001 0.0275 0.1225 0.4513
M061507 0.1375 <0.0001 0.4000 0.1100 0.0550 0.7025
M062107 0.4388 0.0138 0.2925 0.1663 0.1088 1.0200
M062207 0.2475 <0.0001 0.1000 0.1238 0.2838 0.7550
M062907 0.0413 0.1375 0.2313 0.1650 0.2450 0.8200

Note: Means = total contact seconds per treatment over eight hour trials.Trials used were
selected from a pool of 14 open-port visualometer trials ran with vitellogenic An.
quadrimaculatus. Trials excluded denoted mean contact seconds not within +50% of the
group mean contact seconds. Contact second averages above 50% of total trial means
implied sensor malfunction; contact-second averages below 50% of total trial means
implied poor mosquito quality. Blue = 470 nm, Green = 502 nm, IR = 860 nm, Red = 660
nm and no light constituted an unlit control treatment. Trial means = total contact seconds
per trial.









Table C-3. Previtellogenic Anopheles quadrimaculatus attraction to 470 nm and 660 nm
wavelengths of light emitting diodes using a paired-T port visualometer.
Diode Wavelength
Trial Replication Blue Red
1 1 0.0256 0.0122
2 0.0000 0.0244
3 0.0244 0.0489
4 0.0989 0.0244
5 0.0489 0.0000
2 6 0.1222 0.0244
7 0.0000 0.0611
8 0.0611 0.0489
9 0.2811 0.0600
10 0.2811 0.0000
3 11 0.0611 0.0122
12 0.0489 0.0856
13 0.0000 0.0611
14 0.0489 0.0244
15 0.0489 0.0000
4 16 0.0367 0.0000
17 0.0122 0.0489
18 0.0856 0.0367
19 0.0856 0.2811
20 0.0122 0.0000

Note: Means = total contact seconds per treatment over eight hour trials. Each trial included five
replications. Blue = 470 nm and Red = 660 nm.












Table C-4. Vitellogenic Anopheles quadrimaculatus attraction to 470 nm and 660 nm
wavelengths of light emitting diodes using a paired-T port visualometer.
Diode Wavelength
Trial Replication Blue Red
1 1 0.0367 0.1800
2 0.0122 0.0000
3 0.0611 0.0967
4 0.2789 0.2522
5 0.0733 0.0000
2 6 0.0733 0.0000
7 0.0122 0.1800
8 0.1344 0.0856
9 0.0489 0.0722
10 0.0978 0.0000
3 11 0.1344 0.0000
12 0.0122 0.0122
13 0.0122 0.0489
14 0.0722 0.0000
15 0.0000 0.1344
4 16 0.1344 0.0000
17 0.1344 0.1778
19 0.0122 0.2522
20 0.0367 0.0000

Note: Means = total contact seconds per treatment over eight hour trials. Each trial included five
replications. Replication not included (18) denoted no mosquito contact activity on sensors over
blue or green diodes. Blue = 470 nm and Red = 660 nm.












Table C-5. Previtellogenic Anopheles quadrimaculatus attraction to 470 nm and 502 nm
wavelengths of light emitting diodes using a paired-T port visualometer.


Trial
1





2


Replication
1
2
3
4
5
7
8
9
12
13
14
15
18
19
20
21
23
25
27
28
29
30
31
32
33
34
35


Note: Means = total contact seconds per treatment over eight hour trials. Each trial included five
replications. Replications not included denoted no mosquito contact activity on sensors over blue
or green diodes. Blue = 470 nm and Green = 502 nm.


Diode Wavelength
Blue Green
0.0856 0.0000
0.0000 0.0856
0.0611 0.0489
0.1811 0.0244
0.1788 0.0000
0.0000 0.0244
0.0367 0.0733
0.0367 0.0611
0.0000 0.0122
0.0000 0.0000
0.0367 0.0122
0.0122 0.0000
0.0000 0.0244
0.0000 0.0122
0.0122 0.0000
0.0122 0.0000
0.0122 0.0000
0.0244 0.0000
0.0000 0.0122
0.0489 0.0000
0.0000 0.0122
0.0856 0.0000
0.0122 0.0000
0.0000 0.0122
0.0000 0.1578
0.0000 0.0244
0.0122 0.0000











Table C-6. Vitellogenic Anopheles quadrimaculatus attraction to 470 nm and 502 nm
wavelengths of light emitting diodes using a paired-T port visualometer.


Trial Replication
1 1
2
3
4
5
2 9
10
3 12
13
14
4 16
18
19
5 22
23
24
25
6 27
28
29
30
7 32
33
34
35


Mean Contact Seconds
Blue Green
0.0244 0.0244
0.0000 0.1667
0.0000 0.0856
0.1100 0.0600
0.0489 0.0000
0.2289 0.0000
0.0378 0.0000
0.0000 0.0478
0.0122 0.0244
0.0244 0.0000
0.0122 0.0000
0.0244 0.0122
0.0367 0.0244
0.0000 0.0611
0.0122 0.0367
0.0122 0.0000
0.0122 0.0000
0.0000 0.0122
0.0244 0.0367
0.1100 0.2022
0.0244 0.0000
0.0000 0.1100
0.0000 0.0000
0.0967 0.0000
0.0122 0.0000


Note: Means = total contact seconds per treatment over eight hour trials. Each trial included five
replications. Replications not included denoted no mosquito contact activity on sensors over blue
or green diodes. Blue = 470 nm and Green = 502 nm.











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BIOGRAPHICAL SKETCH

Michael Thomas Bentley was born on October 18th, 1982, in Noblesville, Indiana. He is

the younger of two children, born to Mike and Jill Bentley. He and his family moved to Vero

Beach, FL, where he graduated from Vero Beach High School in 2001. His education continued

at the University of Florida where he got his bachelor's degree in criminology in fall, 2005.

Remaining at the University of Florida, Mr. Bentley was accepted into the entomology graduate

program under Dr. Phillip Kaufman with a specialization in medical and veterinary entomology.

He worked as the Entomology and Nematology department's outreach coordinator while earning

his degree, before graduating with his Master of Science in spring, 2008. Mike will be married to

his fiancee, Kristina Pein, October 17th, 2008, after which he plans to pursue a career in industry.





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1 BEHAVIORAL PHOTOTAXIS OF PR EVITE LLOGENIC AND VITELLOGENIC MOSQUITOES (DIPTERA: CULICIDAE) TO LIGHT EMITTING DIODES By MICHAEL THOMAS BENTLEY 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 2008

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2 2008 Michael Thomas Bentley

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3 To my mother, Jill; my father, Mike; and my fiance, Kristina

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4 ACKNOWLEDGMENTS I would lik e to express my sincere gratitude and appreciation to Dr. Phillip Kaufman, my supervisory committee chair, for investing in me his expertise, guidance and patience. It was a privilege to be his first masters student, and to share with him the most challenging and rewarding journey I have experi enced. His professional leadership and guidance will be carried far beyond the field of science. I would also like to thank my other committee members, Dr. Daniel Kline and Dr. Jerry Hogsette, of the USDA-ARS, for their added sup port and assistance. Even with busy schedules, they always made time to meet for professional or personal matters upon any request. It was a rewarding and memorable experience to be educated and surrounded by such remarkable scientists. I personally would like to thank Dr. Jerry Butl er for being my educat or, mentor, and friend through this journey. Entomology was always a love in my life, but he made it a passion. It has been an honor and a privilege to study under him in science and in life. Using the field as a classroom, he made learning an adventure rather th an a task. I was never made to feel like an employee, but more as a friend. His respect, curios ity and passion for life have helped shape me into the scientist I am today. I appreciate all that he has contributed to my career and to my life. Special thanks go to Dr. Sandr a Allan of the USDA-ARS and her staff for their support and assistance throughout my research. On short notice, she was always able to accommodate any request without any hesita tion. Without her assistance in acquiring mosquitoes from the USDA-ARS colony, my final proj ect would not have been possi ble. I owe her a thank you for investing so much of her time and energy into this research. I greatly appreciate Dr. Donald Hall for allo wing me the opportunity to fund my schooling by coordinating the Outreach program throughout my education. This has been a wonderful

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5 experience to share my enthusiasm of entomology with so many children. To be an educator is rewarding within itself, and I am extremely fortunate to have been given the chance to do so. Thanks go to Dr. Saundra TenBroeck and her st aff for their allowing me endless access to the University of Florida Horse Teaching Unit. This facility was an integral part of my field research for two years. Thank you for your patience and assistance. I would like to express appreciation to those residents of the Prairie Oaks subdivision who participated in my research. With limitless patience, they gladly allowed me free access to trap in their backyards for two consecutive summers. Th eir enjoyment and excite ment for my projects made field work that much more enjoyable. W ithout their cooperation, this research would have been impossible. I also would like to thank my lab mates, Pe ter Obenauer and Jimmy Pitzer, for the great times I have had while completing this masters degree. Having such good friends to walk the road with me made these years fly by. Lab work, field work and writing would have been the most tedious of tasks without their humor to pa ss the time. I thank them for the help, the laughs and the memories. My parents, Mike and Jill, have had a tremendous impact on my life and have made my educational career possible. Thei r never ending love and suppor t have carried me through an extensive journey. Without them, I would not be where I am today. Sacrifice was never a question when it came to me or my extended education, which is why I share this degree with them both. I love, admire and appreciate them incredibly. Most of all, I thank my fiance Kristina fo r her never ending patience and love while earning this degree. For every long night and early morning, she was there to see me through.

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6 Her endless inspiration kept me focused and dr iven during the hardest of times. I am truly blessed to have her in my life and love her eternally.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ........10LIST OF FIGURES.......................................................................................................................12ABSTRACT...................................................................................................................................15 CHAP TER 1 LITERATURE REVIEW OF MOSQUITO BIOLOGY, IMPORTANCE AND SURVEILANCE .....................................................................................................................17Introduction to Mosquitoes.....................................................................................................17Life Cycle...............................................................................................................................17Egg...................................................................................................................................17Larva................................................................................................................................18Pupa.................................................................................................................................19Adult................................................................................................................................20Habitat........................................................................................................................ .............21Medical and Economic Importance........................................................................................ 25Vector Surveillance and Monitoring...................................................................................... 30Methodology....................................................................................................................30Species Diversity.............................................................................................................31Flight Range and Habits.................................................................................................. 31Resting Behavior.............................................................................................................34Population Monitoring..................................................................................................... 35Mosquito Attraction......................................................................................................... 382 RESPONSE OF ADULT MOSQUITOES TO LIGHT EMITTING DIODES PLACED IN RESTING BOXES ............................................................................................................ 42Introduction................................................................................................................... ..........42Materials and Methods...........................................................................................................44Resting Boxes..................................................................................................................44Light Emitting Diodes and Battery Supplies................................................................... 45CDC Light Trap...............................................................................................................46Site and Resting Box Location........................................................................................ 46Methodology....................................................................................................................47Statistical Analysis.......................................................................................................... 48Results.....................................................................................................................................49Discussion...............................................................................................................................52

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8 3 FIELD RESPONSE OF ADULT MOSQUITO E S TO WAVELENGTHS OF LIGHT EMITITING DIODES............................................................................................................70Introduction................................................................................................................... ..........70Materials and Methods...........................................................................................................72Diode Equipped Boxes....................................................................................................72Light Emitting Diodes and Battery Supplies................................................................... 73Sticky Cards.....................................................................................................................73CDC Light Trap...............................................................................................................74Site and Sticky Card Trap Location................................................................................74Methodology....................................................................................................................76Statistical Analysis.......................................................................................................... 77Results.....................................................................................................................................77Discussion...............................................................................................................................804 RESPONSES OF PREVITELL OGENIC AND VITELLOGENIC ANOPHELES QUADRIMACULA TUS TO SELECTED WAVELENGT HS PRODUCED BY LIGHT EMITTING DIODE................................................................................................................ 98Introduction................................................................................................................... ..........98Materials and Methods.........................................................................................................102Visualometer.................................................................................................................. 102Light Emitting Diodes................................................................................................... 103Mosquitoes....................................................................................................................103Open-Port Visualometer Trials...................................................................................... 104Paired-T Port Visualometer Trials................................................................................. 105Methodology..................................................................................................................105Statistical Analysis........................................................................................................ 106Results...................................................................................................................................106Open-Port Visualometer................................................................................................ 106Paired-T Port Visualometer........................................................................................... 107Discussion.............................................................................................................................1085 THE IMPORTANCE OF MOSQUITO WAVE LENGTH PREFERENCE IN TRAPPING AND POPULATION SAMPLING.................................................................. 116 APPENDIX A RESTING BOX AND M ODIFIED CDC LIGHT-TRAP CAPTURES OF MOSQUITOES BY LOCATION ......................................................................................... 122B STICKY CARD AND MODIFIED CD C LIGHT-TRAP CAPTURES OF MOSQUITOES BY LOCATION ......................................................................................... 147C RESPONSE OF PREVITELLOGENIC AND VITELLOGENIC ANOPHELES QUADRIMACULA TUS TO SELECTED LED WAVELENGTHS USING A VISUALOMETER IN A PAIR-T AND OPEN-PORT DESIGN........................................ 157

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9 LIST OF REFERENCES.............................................................................................................163BIOGRAPHICAL SKETCH.......................................................................................................177

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10 LIST OF TABLES Table page 2-1 Mean ( SE) numbers of mosquitoes/trap/night attracted to light emitting diodes of four different waveleng ths placed in resti ng boxes at the University of Florida Horse Teaching Unit and Prairie Oaks Subdivision from July 2006 Sept. 2007 near Gainesville, FL................................................................................................................ ...602-2 Total number of mosquitoes/trap night fo r six significant mosquito species captured at the Horse Teaching Unit and Prairie Oaks Subdivision from July 2006 Sept. 2007 near Gainesville, FL..................................................................................................613-1 Mean ( SE) numbers of mosquitoes/t rap/night attracted to light emitting diodes producing four different wavelengths of light during 24 h trapping intervals at the University of Florida Horse Teaching Unit and Prairie Oaks subdivision in Gainesville, FL................................................................................................................ ...883-2 Number of mosquitoes/trap night for six mosquito species captured a the University of Florida Horse Teaching Unit and Prairie Oaks subdivision.......................................... 894-1 Mean numbers ( SE) of previtellogenic and vitellogenic Anopheles quadrimaculatus attracted to selected wavelengths of light emitting diodes as measured by mean contact seconds using an open port visualometer............................. 1124-2 Mean numbers ( SE) of previtellogenic and vitellogenic Anopheles quadrimaculatus attracted to paired selected wavelengths of light emitting diodes as measured by mean contact seconds us ing a paired-T port visualometer......................... 112A-1 Evaluation of resting box catches for mosquito species captured at the Horse Teaching Unit (HTU) from July 2006 Sept. 2007 near Gainesville, FL...................... 122A-2 Evaluation of resting box catches for mos quito species captured at the Prairie Oaks (PO) subdivision from August 2006 Se pt. 2007 near Gainesville, FL.........................129A-3 Modified CDC light trap mosquito captures at the Horse Teach ing Unit (HTU) from July 2006 Sept. 2007 near Gainesville, FL................................................................... 136A-4 Modified CDC light trap mosquito captures at the Prairie Oa ks subdivision (PO) from July August 2006 and May Sept. 2007 near Gainesville, FL............................ 142B-1 Mosquitoes captured in a modified CDC light trap at the University of Florida Horse Teaching Unit from July August 2006 and May Sept. 2007 near Gainesville, FL.... 147B-2 Mosquitoes captured in a modified CDC li ght trap at the Prai rie Oaks subdivision from July August 2006 and May Sept. 2007 near Gainesville, FL............................ 152

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11 C-1 Evaluation of previtellogenic Anopheles quadrim aculatus attraction to four selected wavelengths of light emitting diodes using an open-port visualometer.......................... 157C-2 Evaluation of vitellogenic Anopheles quadrimaculatus attraction to four selected wavelengths of light emitting diodes using an open-port visualometer.......................... 158C-3 Previtellogenic Anopheles quadrimaculatus attraction to 470 nm and 660 nm wavelengths of light emitting diodes us ing a paired-T port visualometer....................... 159C-4 Vitellogenic Anopheles quadrimaculatus attraction to 470 nm and 660 nm wavelengths of light emitting diodes us ing a paired-T port visualometer....................... 160C-5 Previtellogenic Anopheles quadrimaculatus attraction to 470 nm and 502 nm wavelengths of light emitting diodes us ing a paired-T port visualometer....................... 161C-6 Vitellogenic Anopheles quadrimaculatus attraction to 470 nm and 502 nm wavelengths of light emitting diodes us ing a paired-T port visualometer....................... 162

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12 LIST OF FIGURES Figure page 2-1 Resting boxes used at the University of Florida Horse Teach ing Unit and Prairie Oaks subdivision. A) Rear view of 30 x 30 cm resting box showing protective LED housing. Exterior of all boxes were made using 1 cm thick exterior grade pine plywood. The outside of each resting box was painted with two coats of flat black exterior latex paint, and interiorly with tw o coats of barn red exterior latex paint. Diode housing consisted of one 470 ml plastic container attached to the exterior rear wall of each box by container lid. Container li ds were modified with a 0.32 cm hole, and matched to the 0.32 cm hole on the out side back wall of each resting box. B) Front inside view of 30 x 30 cm res ting box illustrating 5 cm x 5 cm x 29 cm sections of pine used as inside corn er supports. A 0.32 cm hole was drilled through the back wall of each box to allow for th e insertion of a LED. Resting boxes were painted interiorly with two coats of barn red exterior latex paint......................................632-2 Light emitting diode configuration used in resting boxes. A) All round lens LEDs were 8.6 mm long by 5.0 mm in diam eter. Viewing angles were 30o except for IR (20o). After a 180-ohm resistor was soldered to each LED, restricting current flow, a female 9 volt (V) battery snap connector (270-325) was attached. B) Battery housing used to supply power to LED configurations for resti ng boxes. Battery supplies (270383) pre-equipped with a complimentary male 9 V connecting site were used, each with a maximum holding capacity of four AA batteries. Four rechargeable 2500 milliamp hour (mAh) AA batteries were used in all assemblages..................................... 642-3 CDC light trap modified by the removal of its incandescent bulb. Modified trap used a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5 cm diameter clear plastic cylindrical b ody. A 36 cm diameter beveled edge aluminum lid was set approximately 3 cm above the cylinder body creating a downdraft air current. All traps were set 120 cm above ground using a Shepherds hook, and collection nets were attached to the bottom of the tr ap body. Carbon dioxide was provided from a 9 kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15psi single-stage regu lator equipped with micro-regul ators and an inline filter.................642-4 Aerial view of Horse Teaching Unit lo cation. The unit is located east of I-75 and approximately 1.6 km northwest of Paines Prairie State Preserve, Alachua Co., FL...... 652-5 Aerial view of Prairi e Oaks subdivision which was located approximately 4.8 km southwest of the Horse Teaching Unit, adjacent to the Paines Prairie Preserve, Alachua Co., FL.................................................................................................................652-6 Test sites located within the Horse Teach ing Unit. Each white rectangle represents a test site where five boxes were equipped with one of five treatments. Sites are numerically labeled according to correspondi ng eastern or western direction. White arrow designates location of modified CDC trap..............................................................66

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13 2-7 Horse Teaching Unit location; west side test site habitat.................................................. 662-8 Horse Teaching Unit location; east side test site habitat................................................... 672-9 Representative of test sites chosen at the Prairie Oaks subdivision. All sites chosen were consistent in surrounding vegeta tion, sunlight exposure and moisture conditions...........................................................................................................................672-11 Resting boxes placed with openings faci ng west and were spaced approximately four meters apart and out of direct sunlight. E ach site contained five treatments, one of four LED colors and an unlit control, resulting in a total of five resting boxes per site, 20 resting boxes per location...................................................................................... 682-12 Mean monthly temperatures (C) and pr ecipitation (cm) for the Horse Teaching Unit (HTU) location near Gainesville, FL, using data retrieved from the National Oceanic and Atmospheric Administration (NOAA) data base. A) Monthly temperature, May September 2006 and 2007. B) Monthly precipitation from Jan September 2006 and 2007....................................................................................................................................693-1 Four sided, diode-equipped pine boxes, each side measuring 400 cm2. Boxes were constructed and designed to exteriorly support one 13 x 13 cm sticky card and one diode treatment per side, yielding a tota l of four sticky ca rds and four light treatments per diode box.................................................................................................... 913-2 Sticky cards were constructed from black 28 pt. SBS card stock with calendared coating (EPA # 057296-WI-001), and coated with 32 UVR soft glue containing UV inhibitors. Individual sticky car ds, originally supplied as 41 x 23 cm boards, were cut to yield two 13 x 13 cm sticky cards.................................................................................. 913-3 CDC light trap modified by the removal of its incandescent bulb. Modified trap used a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5 cm diameter clear plastic cylindrical b ody. A 36 cm diameter beveled edge aluminum lid was set approximately 3 cm above the cylinder body creating a downdraft air current. All traps were set 120 cm above ground using a Shepherds hook, and collection nets were attached to the bottom of the tr ap body. Carbon dioxide was provided from a 9 kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15psi single-stage regu lator equipped with micro-regul ators and an inline filter.................9234 Aerial view of Horse Teaching Unit lo cation. The unit is located east of I-75 and approximately 1.6 km northwest of Paines Prairie State Preserve, Alachua Co., FL...... 933-5 Aerial view of Prairi e Oaks Subdivision which was located approximately 4.8 km southwest of the Horse Teaching Unit, ad jacent to the Paines Prairie Preserve, Alachua Co., FL.................................................................................................................933-6 Representative of test sites chosen at the Prairie Oaks subdivision. All sites chosen were consistent in surrounding vegeta tion, sunlight exposure and moisture conditions...........................................................................................................................94

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14 3-7 Test sites located within Prairie Oaks subdivision. Each solid white rectangle represents a test site wher e one box equipped with one of four diode treatm ents was placed. White dashed rectangles identify the location of modified CDC traps................. 943-8 Test sites located within the University of Florida Horse Teaching Unit. Each white square represents a test site where on e diode box was equipped with one of four diode treatments. White arrow represents location placement of modified CDC trap...... 953-9 University of Florida Horse Teaching Un it location. A.) Southeast side test site habitat. B.) Northeast side test site habitat. C.) Northwest side test site habitat. D.) Southwest side test site habitat.......................................................................................... 963-10 Mean monthly temperatures (C) and precipitation (cm) for the University of Florida Horse Teaching Unit (HTU) location near Gainesville, FL using data retrieved from the National Oceanic and Atmospheric Administration (NOAA) database. A) Monthly temperature, Ma y September 2006 and 2007. B) Monthly precipitation from Jan September 2006 and 2007................................................................................ 974-1 Pie shaped visualometer with 10 availa ble feeding stations, which can be portioned off individually or left in an open design. A) Visualometer used in an open design, with treatments placed at all odd numbered feeding stations. B) Visualometer in operation showing treatments, set as descri bed above. C) Visualometer used in a paired-T configuration.....................................................................................................1144-2 Anopheles quadrimaculatus obtained from the USDA-ARS-CMAVE Gainesville, FL rearing facility held in an incuba tor at 26 C and 74% humidity under a 14:10 (L:D) photoperiod. Upon eclosion, adult mosquitoes were fed a 10% sugar solution.... 1144-3 Blood feeding Anopheles quadrimaculatus occured 120 h post-eclosion using a blood ball. Blood balls consisted of saus age casing and defribrinated bovine blood. Adult mosquitoes were allowed to blood feed for 3 h..................................................... 115

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15 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science BEHAVIORAL PHOTOTAXIS OF PR EVITELLOGENIC AND VITELLOGENIC MOSQUITOES (DIPTERA: CULICI DAE) TO LIGHT EMITTING DIODES By Michael Thomas Bentley May 2008 Chair: Phillip E. Kaufman Major: Entomology and Nematology Mosquito wavelength preferences for light emitting diodes (LEDs) were examined using resting boxes and LED equipped light boxes in North Central FL. Wavelength preferences among two physiologically aged mosquitoes were determined using a visualometer (open-port and paired-T configuration). Wavelengths evalua ted were blue (470 nm), green (502 nm), red (660 nm) and infrared (IR (860 nm)). Resting boxes fitted with IR LEDs attracted 23% of all mosquitoes recovered from resting boxes. Significantly more Anopheles quadrimaculatus Say females were aspirated from resting boxes fitted with red LEDs th an all other treatments. Culex erraticus Dyar and Knab females were recovered in significantly (p = 0.05) higher numbers from resting boxes fitted with blue, green, or red LEDs or the no-light control than with IR LEDs. Approximately 47% of all mosquitoes trapped using LEDs fitted to sticky cards were captured on cards with green LEDs. Significantly more Aedes vexans Meigen females, Cx. nigripalpus Theobald females and Ochlerotatus infirmatus Dyar and Knab females were captured on sticky cards fitted with blue LEDs than those with red or IR LEDs. Blue LED fitted sticky cards captured significantly more Cx. erraticus females than were caught on sticky cards using IR LEDs.

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16 In comparisons between previtellogenic and vitellogenic An. quadrimaculatus released into the open-port visualometer, previtellogenic mosquitoes record ed significantly higher contact seconds on red LEDs than did vitellogenic mosquito es. Vitellogenic mosquitoes were in contact with blue LEDs for a longer period of time that were previtellogenic mo squitoes. In paired-T port comparisons, no significant differences in contact seconds for previtellogenic or vitellogenic An. quadrimaculatus were recorded among blue and re d or blue and green LED pairs respectively.

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17 CHAPTER 1 LITERATURE REVIEW OF MOSQUITO BI OLOGY, IMP ORTANCE AND SURVEILANCE Introduction to Mosquitoes In 1877, Patrick Manson was the first to credit mosquitoes with disease transm ission after witnessing the development of Wuchereria bancrofti Cobbold in the mosquito Culex pipiens quinquefasciatus Say (Chernin 1983). This discovery star ted what is known today as the Golden Age of Medical Entomology, and helped mosqu itoes gain their fearsome reputation as transmitting some of the worlds deadliest diseas es. Currently, mosquitoes are implicated as vectors of over 200 arboviruses to humans and other animals, such as encephalitis, yellow fever and dengue (Lehane 2005). Of all known mosquito associated diseases, malaria is considered the most severe, with over 2 billion people in 100 coun tries are at risk of in fection each year (WHO 2007a). There are approximately 3,200 recognized specie s of mosquitoes worldwide, occurring in every continent with the exception of Antarctica (Lehane 2005). Belonging to the family Culicidae, mosquitoes are recognized by curre nt culicid classification as having three subfamilies: Anophelinae, Culicinae, and Toxor hynchitinae (Foster and Walker 2002). A diverse, highly adaptive and durable lifecycle ha s allowed mosquitoes to evolve side-by-side with humans. Whether facing exte nded periods of drought in an urban setting or surviving monthly monsoons in tropical forests, mosquitoes have adapted to thrive in many conditions. Life Cycle Egg The holom etabolous life cycle of mosquitoes begins with the deposition of an elongate, ovoid or spindle-shaped egg, meas uring approximately 0.4-0.6 mm in length (Forattini et al. 1997). Newly oviposited eggs begin white in colo r, and darken within 12 to 24 hours depending

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18 upon surrounding moisture conditions (Breeland a nd Beck 1994). The outermost layer of the egg shell, the chorion, is comprised of three reinforced layers. These reinforced layers not only provide safety for the embryo, but also protect against dehydration. The chorions outer most layer consists of a network of complex patte rns and surface boxes which are unique to each mosquito species. In anopheline species, for ex ample, the chorion has transparent, air filled compartments lining either side of the egg that serve as floats following oviposition (Foster and Walker 2002). Eggs of some mosquito genera such as Anopheles and Aedes are individually oviposited on the waters surface. Alternatively, eggs may be glued together to form rafts of up to 150 eggs, as with Culex. In these conditions, hatch rates depend largely upon temperatures. In optimal conditions larvae can emerge within 2 or 3 days after the eggs are laid (Stage et al. 1952). In genera including Aedes, Ochlerotatus and Psorophora oviposition may take place upon detrital matter or just above the water line along the insi des of containers. Egg hatch usually occurs at warm temperatures after the eggs have been inundated and microbial activity has caused oxygen levels in the water to drop (Fos ter and Walker 2002). If not flooded, Aedes and Ochlerotatus eggs can survive in a quiescent state and accu mulate for several years. Sudden temporary flooding can allow accumulated eggs to hatch along with recently oviposited eggs, resulting in mass emergences that can lead to public health threats (Breeland and Beck 1994). Larva All m osquito larvae are aquatic, molting th rough four instars befo re developing to the pupal stage. When ideal conditions exist (26-28 C), most mosquito species can complete the larval stage in five to six days with males usually pupating about 1 da y earlier. Even under optimum conditions, the larval stag e for some mosquitoes such as Toxorhynchites or Wyeomyia

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19 often takes as long as 2-3 weeks to complete. In most species, cooler te mperatures (< 68 C) slow the developmental process (Matheson 1944). Respiration is usually achieved through the siphon or air tube located near the last abdominal segment (Breeland and Beck 1994). The ma jority of mosquito larvae are required to come to the water surface for oxygen. However, the siphons of Coquillettidia and Mansonia have been modified into a shor t, heavily sclero tized saw-like box used to pierce and attach to plant tissues in order to obtain oxyg en (Bosak and Crans 2002). Larvae of Anopheles lack a siphon and diffuse oxygen through a series of sma ll grouped abdominal plates. This causes the larvae to lie flat at the surface of the water, a behavior characteristic of all Anopheles species (Foote and Cook 1959). Most mosquito larvae are filter feeders, living on a diet comprised of tiny plants, animals, and organic debris (Stage et al 1952). Palatal brushes located on the labrum circulate water and debris over combs and sweepers located on the mandibles and ma xillae, respectively. These mouthparts collect and pack food pa rticles, which are then passed into the pharynx for digestion. The mouthparts of Toxorhynchites however, are heavily sclero tized and sharply toothed, designed for the predation of sma ller invertebrates, including othe r mosquito larvae (Foster and Walker 2002). Pupa The pupa is a non-feeding stage of developm ent in a mosquito s life cycle. Mosquito pupae are comma-shaped, with the head and thorax fused to form a cephalothorax and the abdomen curled beneath it (Foster and Walker 2002). Pup ae are often called tumblers because of their quick tumbling-like defensive action in respons e to any light change in the surrounding environment (AMCA 2007). Pupae of most speci es obtain oxygen at the waters surface through two respiratory tubes, or air trumpets, which protrude from the dorsal mesothorax (Lehane

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20 2005). Coquillettidia and Mansonia pupae remain attached to unde rwater plant tissues, diffusing oxygen through a modified air trumpet, detaching just before eclosion (Crans 2004). The entire pupal stage of most species t ypically lasts two to three days, depending on temperature. Optimum temperatures for pupal deve lopment in most mosquito species range from 26 to 28 C. Some Culex species can complete the pupal stag e in approximately two days during the warm summer months (AMC A 2007). Other species, including Toxorhynchites and Wyeomyia cannot complete development in less than five to six days. Adult Em ergence of adult mosquitoes is a relatively short process usually requiring no more than 20 minutes to complete. Changes in hormone leve ls signal the approach of emergence, causing pupae to remain stationary at the waters su rface. The abdomen gra dually extends allowing ingestion of enough air through the respiratory tubes to cause the cephalothorax to split. The adult mosquito then emerges through this openin g. Males tend to emerge before females due to their shorter pupation periods (Foster and Walker 2002). Newly emerged adults are capable of short f lights within minutes, but must wait for the cuticle to become fully sclerotized before sustaining longer ones. Some species will never travel farther than a few hundred feet fr om their site of emergence, wh ile others migrate 50 miles or more (Breeland and Beck 1994). Adult mosquitoes ar e able to survive up to three days on lipid and glycogen reserves carried over from the larval stage. Males of all species have mouthparts modified to suck nectar and plant secretions. However the ma xillae and mandibles of most females are specially modified to pierce skin. Both sexes require nutrients from sugars found in plant nectar and honeydew, but the females of mo st species are anautogenous, requiring a blood meal for egg production. Females utilize hemogl obin proteins to synt hesize vitellogenin, stimulate egg growth and successfully oviposit (Lehane 2005). Several autogenous mosquito

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21 species including Toxorhynchites and Culex are capable of oogene sis without taking a blood meal. This is made possible in Toxorhynchites by synthesis of vitellogenin from proteins obtained during their predacious larval stage (Klowden 1996). Habitat Mosquito habitats are generally classified in terms of a fe male oviposition preference for permanent water, flood water, transient water or artificial container and tree-hole environments (Breeland and Beck 1994). Behavioral differences in oviposition and life cycle development between individual mosquito species play an important role in determini ng both larval and adult habitats. These habitats range from fresh to sa lt water and can be natu ral or man made. Given their weak swimming abilities, mo squito larvae are incapable of surviving in continuous moving water. As a result, larvae occupy more stagnant water conditions such as pools and seepage areas (Clements 1992). All mosquito species are groupe d into two habitat cate gories; standing water and flood water habitats as utilized by immature stages. Within these habitats, certain specific requirements regarding habitat diffe rentials play a critical role in habitat preference between mosquito species. The eggs of most standing water species do not tolerate desiccation. As a result, oviposition typically takes place directly on the water surface, either singly or as rafts on stagnant pools of water (Clements 1992). Eggs not tolerant to desiccati on must hatch soon after oviposition, influencing the life st age in which mosquitoes endure potentially fatal environmental conditions. Most species such as Anopheles and Culex survive such harsh circumstances as mated females (Crans 2004). One exception is that of Coquillettidia perturbans Walker. Overwintering in this species takes place during the larval stage of any instar trapped by the onset of winter. As a result, cohorts of larvae em erge continuously over the course of the summer (Bosak and Crans 2002).

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22 Vegetation has a large impact on the habitats of several standing water mosquito species. For example, Culiseta melanura Coquillett larvae thrive in fres h water swamps sparse in aquatic foliage, whereas, An. quadrimaculatus Say and An. walkeri Theobald prefer freshwater bogs and swamps with abundant aquatic vegetation (Horsfall and Morris 1952, Mahmood and Crans 1998). Mansonia and Coquillettidia species are even more selectiv e, requiring specific aquatic plants such as water lettuce, water hyacinth a nd cattails for both ovipositi on and larval habitat (Hagmann 1953). Standing water mosquito species are generally classified in to two subgroups; permanent water species and transient water species. Permanent water genera including Anopheles, Culex, Coquillettidia, and Mansonia are found in established bodies of water such as marshes, swamps, springs, ponds and lakes (Bentley and Day 1989). The larvae of these species are usually restricted to the littoral zone where vegetation provides protection and water movement is at a minimum (Newkirk 1955). However, the larvae of some Psorophora and Ochlerotatus species are found throughout swamps and bogs, utilizing thick aquatic foliage or dense tree cover to hide from predators (Laird 1988). Transient water mosquito species are found in natural ditches, drainage ditches, borrow pits, and canals (Crans 2004). In coastal habita ts, natural ditches commonly run adjacent to saltwater marshes, but can contain either fresh or brackish water. Ochlerotatus and Anopheles are common genera found in these ditches be cause of the wide variety of aquatic vegetation (Newkirk 1955). Drainage ditches are man-made habitats commonly found along pastures, at the bottom of road shoulders, in abandoned fields or in lowland groves. Th ese are common larval habitats for several fresh water mosquitoes including Culex, Uranotania and Psorophora. Burrow pits and canals are man-made bodies of water which usually remain undisturbed for

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23 extended periods of time. After becoming overgrown with vegetation, these torpid pools become productive breeding si tes for species of Culex, Coquillettidia, and Mansonia (Hagmann 1953, Slaff and Crans 1982, Clements 1992). Floodwater mosquito habitats can be artificial or naturally occurring environments prone to periodic flooding. These range in size from microhabitats such as tree holes and tires, to larger isolated bodies of water in cluding ground depressions and tidal pools (Matheson 1944). Floodwater mosquito species commonly produce several broods annually, surviving harsh environmental conditions in desiccation resistan t eggs (King et al. 1960 ). Vegetation in and around these habitats can vary greatly, influencing the species diversity fr om one habitat to the next. For example, some Ochlerotatus species only oviposit in water containing the leaf litter of red maple, Acre rubrum cattail, or certain sphagnum swamp habitats (Clements 1992). Wyeomyia species are also highly selective when locat ing a suitable larval habitat, ovipositing just above the water line in a specific t ype of pitcher plant (Istock et al. 1975). Floodwater mosquito species ar e classified into two subgr oups. The first subgroup includes non-container habitats such as rain and floodw ater pools, mangrove swamps, and salt marshes (Breeland and Beck 1994). Rain and floodwater pool s serve as ideal breeding sites for several mosquito species, especially those in the Psorophora, Aedes, and Ochlerotatus genera These habitats are unique in that they do not support true aquatic vegeta tion such as aquatic grasses, often containing only leaves and other detrital ma tter that have settled to the bottom. Temporary pools usually evaporate quickly in dry weather. As a result, a number of species in this group rely on direct sunlight and high daytime temperatures to accelerate larval development before the habitat dries (Crans 2004).

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24 Mangrove swamp habitats are classified as tran sitional tidal zones that cycle from low to high tide. Though mosquito breeding occurs throughout tidal zones, immatures and adults tend to occur in highest numbers around peak tidal z ones (Harwood and Horsfall 1959). Natural plant and grass cover help to retain moisture, maintaining favorable oviposition conditions. Ochlerotatus and Anopheles eggs will only hatch after being triggered by the alternate flooding and drying tidal cycles (Bentley and Day 1989). Few mosquito species are able to utilize the vast expanses of salt marsh wetlands because of the unique aquatic vegetation and extremely high saline content. Salt-tolerant herbaceous plants and grasses dominate these habitats, with sizeable areas often overrun by a single plant species (Hulsman et al. 1989). Ochlerotatus taeniorhynchus Wiedemann and Oc. sollicitans Walker are adapted to survive in these harsh cond itions, and can take advantage of larval habitats unsuitable for other floodwater mosquito species. These Ochlerotatus species also share intimate relationships with the vegetation, breeding only where salt-toleran t plant species occur (Horsfall and Morris 1952). The second subgroup of floodwater mosquito ha bitats includes artificial and natural containers. Most species in this group deposit eggs in bands just above the water line of these microhabitats, providing additional substrate as evaporation pr ogresses. Subsequent rainfall events raise the water le vel immersing eggs, a requirement the eggs of most species in this group must meet before hatching (Newkirk 1955). Artific ial container habitats are classified as any human-derived activity that result s in a habitat in which mosquitoes can successfully complete a life cycle. Structures that hold water, such as tin cans, rain barrels and clogged gutters, make excellent breeding habitats for several species. Discarded tires are considered one of the most problematic examples of artificial containers Accumulated rain water and decomposing plant

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25 material mimic natural breeding sites, creating an ideal larval habitat for several medically important mosquito species (Means 1979). Therefor e the practice of importing used tires poses a health threat by contributing to the introduction of several exotic mosquito species including Aedes albopictus Skuse and Ochlerotatus japonicus Theobald (Morris and Robinson 1994, Andreadis et al. 2001). Tree hole habitats support an extensive and distinctive mos quito fauna with many species breeding exclusively in these ecological niches (Breeland and Beck 1994). These isolated habitats offer a great deal of protection from pr edators, making them ideal larval habitats for several mosquito species. However, access to optim al tree hole habitats is not always possible. Often, entrances to these microenvironments are small or blocked, preventing adult mosquitoes from landing in order to deposit eggs. Some tree hole mosquito species have developed special oviposition techniques to overcome th ese problems. For example, some Toxorhynchites species are able to propel their eggs through small tree hole openings by flicking their a bdomens (Linley 1987). While some Anopheles species oviposit aerially, depositing eggs while hovering above vertical tree hole openings (Foster and Walker 2002). Crab hole habitats are limited by the geographical distribution of land crabs in the families Gecarcinidae and Ocypodidae. These habitats span from Florida and the Bahamas throughout the northern Caribbean (Belkin and Hogue 1959). Deinocerites species are most noted for utilizing crab holes as breeding habitats. Though no conclusive data have been published relating specific Deinocerites species to a particular spec ies of crab, members of the Spanius group have consistently been trapped in the small burrows of certain fiddler crabs (Adams 1971). Medical and Economic Importance Mosquitoes are cap able of transmitting hundred s of viruses, protoz oans and filarial nematodes to human beings (Karabatsos 1985). The most threatening diseas es include malaria,

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26 filariasis, yellow fever, dengue and the ence phalitides (Foote and Cook 1959). These unbiased diseases affect every culture on almost every continent, often leadi ng to serious illness, disfigurement and even death (Foster and Walker 2002). Because of this, mosquitoes are considered to be the deadliest and most importa nt vectors of disease to man (Beerntsen et al. 2000). In 1877, Dr. Patrick Manson was the first to as sociate mosquitoes with a human related illness after observing the development of the filarial worm, Wuchereria bancrofti, in the mosquito Culex pipiens quinquefaciatus Say (Chernin 1983). His res earch demonstrated that certain mosquito species were the intermediate hosts and vectors of lymphatic filariasis, a parasitic disease caused by microscopic filarial worms (Mat heson 1944). More than one billion people in 80 countries throughout th e tropics and sub-tropics of As ia, Africa, the Western Pacific and South America are at risk for lymphatic fila riasis. The equivalent of several billion U.S. dollars is lost annually to medical costs and decreases in labor produc tivity resulting from physical injury and deformities caused by lymphatic filariasis (CDC 2007a). In 2000, the World Health Organization (WHO) initiated an elimination effort known as the Global Alliance in hopes of c ounteracting the growing number of lymphatic filariasis cases. Initial drug administrations were conducted, treating approximately 25 million people in 12 different at-risk countries. By 2005, over 250 million people in 39 countries were being treated through mass drug administration. The program triu mphed, surpassing all initial expectations and becoming one of the most successful WHO efforts in history. The Global Alliance is currently on track to meet thei r goal of elimination of lym phatic filariasis by 2020 (WHO 2006). Mosquitoes were first incriminated as vector s of malaria to humans in 1897 by Dr. Ronald Ross. There are four different species of pr otists that cause human malaria including Plasmodium

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27 vivax, P. falciparum, P. malariae and P. ovale; P. falciparum being responsible for the most deaths. These parasites can only be vectored to humans by mos quitoes belonging to the genus Anopheles (Foote and Cook 1959). Today, malaria is the recognized as one the worlds most lethal diseases, primarily affecting children and pregnant women. Althoug h forty-one percent of the human population lives in areas where malaria is transmitted, most cases are reported in parts of Africa (CDC 2007b). In all, 105 countries account for 300 to 500 million clinical cases and more than one million deaths per year. Throughout the 1950s and 1960s, the WHO initiated a worldwide malaria eradication program with increasing si gns of success. However, the goal of global eradication has faded over the past few decades because of the rapid increase in drug resistance by parasites, as well as incr easing insecticide re sistance in mosquitoes (WHO 2007a). Yellow fever is a viral hemorrhagic pathogen transmitted to humans by infected mosquitoes. In 1900, research conducted by Dr. Walter Reed and his a ssociates confirmed previous experiments of Dr. Ca rlos Finlay, which pointed to Ae. aegypti Linnaeus as the primary vector (King et al. 1960). Yellow fe ver continues to persist, with lo w levels of infection in most tropical areas of Africa and the Americas. There are an estimated 200,000 cases of yellow fever reported annually, 30,000 of whic h result in death (WHO 2007b). Yellow Fever displays three dis tinctly different transmission cycles; sylvatic, intermediate and urban (Foster and Walker 2002). The sylvatic or jungle cycle occurs in tropical rainforests where the virus is transmitted to monkeys by zo ophilic mosquitoes. Humans are infected when they enter these regions and are fed on by mosquito es. This type of cycle tends to be sporadic, commonly affecting young men working within these enzootic forest areas. Transmission of the more common intermediate cycle of yellow fever occurs in humid regions of Africa, producing

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28 small localized epidemics in surrounding rural villages. Semi-domestic mosquitoes increase the rate of contact with man, making this the mo st common transmission of yellow fever (WHO 2007c). The urban cycle of yellow fever transmission is found primarily in village settings of tropical Africa and South America. This cycle results in large explosive epidemics when the virus is introduced into densely populated areas from rural travelers. Virus outbreaks tend to spread outwards from one source with transmi ssion by domestic mosquito species, primarily Ae. aegypti (Foster and Walker 2002). Dengue or break-bone fever is caused by a febrile virus occu rring in tropical and subtropical areas including Sout heast Asia, Central America and South America. There are four closely related, but antigenically distinct, seroty pes of Dengue fever referred to as Dengue 1, 2, 3 and 4. In humans, this disease takes on one of two forms; classic dengue fever or the more severe dengue hemorrhagic fever, also known as dengue shock syndrome (Foster and Walker 2002). Aedes aegypti is the principle vector of dengue fever, although transmission is possible by other Aedes species. Like yellow fever, dengue is a di sease of monkeys, whic h serve as reservoirs between epidemic periods (King et al. 1960). In 2005, the Center for Disease Control (C DC) considered dengue fever the most important mosquito-borne viral disease affecting humans. Its global distribu tion is comparable to that of malaria, with an estimated 2.5 billion people living in areas at risk for epidemic transmission. There are an estimated 50 to 100 million cases of dengue fever and several hundred thousand cases of dengue hemorrhagic fever reported worldwide each year. Approximately 5% of all cases result in fataliti es, with the majority occurr ing among children and young adults.

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29 Because no vaccine is available, the most succes sful method of disease suppression is directed towards vector control (CDC 2007c). The most important mosquito-borne diseas es occurring in the United States are the encephalitides. The five primary viral agents are West Nile virus (W NV), eastern equine encephalitis (EEE), western equi ne encephalitis (WEE), St. Loui s encephalitis (SLE) and La Crosse encephalitis (LAC). Though encephalitides can successfully be vectored to humans and domestic animals, these are usually dead-end hos ts incapable of producing sufficient viremia to contribute to the transmission cycl e. Instead, these encephalitides amplify in hosts such as birds, chipmunks and tree squirrels. Most human inci dences of encephalitis occur in the warmer months between June and September when mosquitoes tend to be most active. In warmer parts of the country, where mosquitoes stay active late in the year, cases can occur during the winter months (CDC 2007d). Of the five encephalitides occurring in the United States, EEE is regarded as the most serious because of its high mortality ra te. Though it is maintained in birds by Cs. melanura, other mosquito genera such as Aedes, Coquillettidia and Culex contain capable vectors. Eastern Equine Encephalitis currently occurs in several locali zed distributions along th e eastern seaboard, the Gulf Coast and in some inland Midwestern loca tions of the United States (King et al. 1960). Approximately 220 confirmed cases were repor ted in the United States from 1964 to 2004. Florida sits atop the list of total reported ca ses, followed by Georgia, Massachusetts and New Jersey. Though a vaccine is available to protect equines against EEE, no such prophylaxis exists for humans. Currently, vector cont rol methods such as wide area aerial sprays are utilized for emergency situations (CDC 2007d).

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30 Vector Surveillance and Monitoring Methodology A com prehensive assessment of vector surveillance and monitoring methods has been extensively covered in Services (1993) book, Mosquito Ecology Field Sampling Methods Information included in the next four pa ragraphs was included in his literature. Most trapping methods are often baited with a host, or employ attractants such as carbon dioxide or various forms of visual stimuli. Th ese traps produce a bias when used in vector surveillance and monitoring by primarily selectin g for unfed, host seeking female mosquitoes. Although some non-baited traps, such as truck mounted nets, give less biased mosquito collections, these traps still select for the aerial population which is comprised largely of more active unfed females. Collections of resting mosquito populations yi eld a more accurate representative sample of a mosquito population given that adults probably spend more time resting than in flight. These collection methods would not only result in catching unfed host-seek ing females, but would also sample males, and both blood-fed and gravid fe males. Sampling resting mosquito populations also yields a broad age structure. Several non-biased methods exist to sample resting mosquito populat ions. When targeting indoor resting mosquito species, including several Anopheles as well as some Culex, aspirators, resting counts and knock-down sprays are co mmonly used. Though few mosquito species commonly rest indoors, those that do are often important vectors of malaria, filariasis and some arboviruses, making accurate sampling me thods of these species a necessity. Sampling outdoor resting mosquitoes is ofte n more difficult because outdoor populations are usually distributed over large areas and not concentrated in smaller locations. A better understanding of the general re sting habits of most exophili c species has allowed for the

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31 development of more accurate surveillance met hods. When sampling mosquito species known to rest amongst grassy and shrubby vegetation, such as Psorophora columbiae Dyar and Knab, aspirators or sweep nets have shown to be su ccessful. However, the utilization of artificial resting places is often the preferred sampling me thod, allowing for the attraction of mosquitoes to a specific site from which th ey can be conveniently collected. Species Diversity Mosquitoes are found on alm ost every continent of the world. They are capable of developing in a wide variety of ecological niches ranging from arctic tundras and barren mountain ranges to salt marshes and ocean tidal zones. Although the greatest species diversity occurs in tropical forest environments, mosqu itoes can also proliferate in ecologically poor environments (Foste r and Walker 2002). There are approximately 3,200 known mosquito species worldwide (Day 2005). Within the United States there are 174 known species and subspecies in 14 ge nera and 29 subgenera (Darsie and Ward 2005). Florida, having an ideal subtropical climate in most central to southern regions, has a unique and diverse fauna of mosquito species unlike most other states in the U. S. At least 11 mosquito species within the generas Aedes, Culex and Psorophora are unique to FL. Additionally, several other mosquitoes native to FL have extremely limited in-state distributions, but are relatively abundant in other parts of the United States (Br eeland 1982). Floridas mosquito population is comprised of indigenous and introduced species wi thin the genera of Aedes, Anopheles, Coquillettidia, Culex, Dei nocerites, Mansonia, Psorophora, Uranotaenia and Wyeomyia (Darsie and Ward 2005). Flight Range and Habits Mosquito flight is classified in three beha vioral categories: migr atory, appetential or consum atory (Bidlingmayer 1994). Migratory fli ghts are only performed by newly emerged adult

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32 mosquitoes. During this time, mosquitoes lack any specific physiological directive, and are unforced to fulfill any individual needs essent ial to survival (Provost 1953). Conversely, appetential flight occurs in response to physiological stimuli in mosquitoes over 24 hr of age. These physiological stimuli commonly result from a need for blood meals, oviposition sites or suitable resting locations. While in appetential flight, sensory mechanisms, such as olfaction, vision, thermal and auditory receptors, are actively us ed to detect cues indicating the presence of target physiological stimuli. Appetential flight is terminated and consumatory flight begins when the target cue is detected. The la tter is the time during which a mo squito follows detectable cues to its desired objective (Haskell 1966). Often dir ect and brief, consumatory flights may occur without a preceding appetential flight, give n proper circumstances (Bidlingmayer 1994). Multiple environmental factors such as t opography, temperature, humidity and wind must be considered when discussing appetential flight and dispersal habits of mosquitoes (Stein 1986). Topography and landscape structures can be importa nt influences on short and long range flight habits of mosquitoes. Specific landscape formati ons such as shorelines and rivers have been shown to significantly affect flight patterns of Aedes taeniorhynchus Wiedemann and other insects (Provost 1952). Small townsh ips and cultivated areas can al so direct mosquito flight preferences and patterns. The abundant amounts of appetitive stimu li these areas readily provide can attract several mosquito sp ecies, causing them to abandon ot her natural host seeking flight patterns (Shura-Bura et al. 1958). The effects of temperature and humidity are well documented examples of how slight environmental variations can influence mosquito flight preference. In most species, once temperatures have risen above the minimum fli ght threshold, higher temperatures have little impact on flight (Taylor 1963). Though individual temperature th resholds can vary slightly,

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33 upper and lower temperature thresholds affecting f light hold true for most mosquito species. In a study conducted by Rowley and Graham (1968a) on the flight performance of Ae. aegypti, upper and lower temperature flight thresholds were f ound to be 35 C and 10 C, respectively, while relative-humidity (RH) values ranging from 30 to 90% showed no significa nt effects. However, when surveying Ae. sollicitans Walker and Culex pipiens Linnaeus, Rudolfs (1923, 1925) noted reductions in total catch rates for both mosquito species on nights where RH levels exceeded 85% and 97%, respectively. Wind may be the most important and comple x of all environmental factors affecting mosquito flight behavior (Stein 1986). Wind velocity and direction have been shown to significantly impact flight act ivity, elevation and direction (Klassen and Hocking 1964, Snow 1976). The slightest air currents are enough to affect mosquito flight activity. In laboratory experiments, average cruising flight speeds of 1.0 meter per second or less were observed for some Aedes species (Hocking 1953, Rowley and Gr aham 1968b, Nayar and Sauerman 1972). When wind velocities decrease below average flight speeds, mosquitoes are able to fly upwind; a preference displayed by most species. However, wind velocities greater than average flight speeds tend to overpower mosquitoes, forcing th em to find shelter or submit to a downwind direction (Kennedy 1939). Flight elev ation is also determined by fli ght direction with respect to wind velocity. Mosquitoes must make elevation adjustments accordingly to keep ground patterns used for guidance within their vi sual limits (Bidlingmayer 1985a,b). Gender may also play an important role in activity and range of mo squito flight. Males have been shown to travel shorte r distances than females, staying within a few kilometers of their larval habitat. Studies involving mark-and-recapture methods have been used with great success, demonstrating this behavior in several mos quito species (Horsfall 1954, Quraishi et al. 1966,

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34 Brust 1980, Weathersbee and Meisch 1990). Sche manchuk et al. (1955) demonstrated that Ae. flavescens Mller males have a proximate flight ra nge of approximately 1.3 km, with females averaging 10.6 km in range. Females of several Culex species have lower temperature thresholds for flight activity than males, re sulting in a longer dispersive phase of flight and thus a greater range (Wellington 1944). Resting Behavior Based on observed behaviors, adult mosquitoes are believed to spend m ore time resting than in flight. Mosquitoes primarily rest to dige st meals, or to find shelter from environmental conditions or predators. Most adult mosquito species are exoph ilic, resting exclusively outdoors in natural shelters, such as animal burrows and tree holes, and amongst vegetation. Comparatively few adult mosquito species are k nown to be entirely endophilic, preferring manmade shelters such as huts or sheds (Service 1993). Exophilic adult mosquitoes seek shelter in a wide range of habitats including termite mounds, hollow trees and various ty pes of vegetation. Preferences between these habitats have been observed in several mosquito species (Service 1993). For example, An. freeborni Aitken prefer to overwinter in an imal burrows over other natural shelters. However, Cx. tarsalis Coquillett, a species found in similar habitats, prefer overwintering in rock-holes and fissures amongst vegetation (Harwood 1962). Serv ice (1969) noted several adult Aedes species preferred to rest primarily amongst vegetation, whereas some Anopheles species were recovered only from tree trunks. Environmental factors such as sunlight and relative humidity also play a critical role in the resting habits of many exophilic mosquitoes. Serv ice (1971) noted a signifi cant difference in the distribution of mosquitoes found resting among vege tation exposed to sunlight. Direct sunlight exposure caused populations to converge in more shaded regions of vegetation. In Florida, Day

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35 et al. (1990) found that Cx. nigripalpus Theobald moved deeper in to the center of wooded hammocks towards thicker vegetation in response to negative changes in relative humidity. Similarly, An. walkeri Theobald are generally found solely amongst vegetation in cooler seasons, but are present in covered structures dur ing hot, dry summers (Snow and Smith 1956). Population Monitoring Most m osquito species are either nocturnal or crepuscular, remaining relatively inactive during daylight hours. Sampling these outdoor populations is often difficult, as they are commonly distributed over wide areas of ope n vegetation (Crans 2004). In an attempt to overcome these difficulties and eliminate biases brought on by baited trapping systems, special monitoring methods were developed with the goal of naturally attracting mo squitoes to specific sites from which they can be conveniently co llected (Crans 1989). Th ese monitoring methods include several forms of artificial rest ing boxes, gravid traps and sticky traps. Earth-lined box traps were the first artificial resting places successfully used to study and sample exophilic mosquito species (Russell and Santiago 1934). Since then numerous artificial resting shelters varying in shape and size have been developed and tested. Rolled up mattresses have also been shown to act as viable arti ficial resting boxes when sampling for exophilic mosquitoes (Khan 1964). Some artif icial resting places such as ke g shelters, box shelters, cloth shelters, dustbin bags and pipe traps have been shown to target spec ific exophilic mosquito species. While sampling exophilic mosquitoes in Tennessee, Smith (1942) showed that An. quadrimaculatus Say preferred empty nail kegs when turn ed on their side capturing as many as 1,100 Anopheles adults in a single keg. Several mosquito genera including Anopheles, Culiseta, Culex, Aedes as well as the species Cq. perturbans and Ur. sapphirina Sacken were found to prefer a wide range of box shelters (Goodw in 1942, Burbutis and Jobbins 1958, Gusciora 1961,

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36 Pletsch 1970, McNelly and Crans 1989, Anderson et al. 1990, Crans 1989, Harbison et al. 2006). Over a 44-night trapping period, Service (1986) caught primarily Ae. caspius Pallas and Culex quinquefasciatus Say using plastic trash bags. When samp ling with self constructed pipe traps, Nelson (1980) collected more Cx. tarsalis mosquitoes than any other species. Gravid traps are designed to mimic natural ovi position sites of most mosquito species. These sites are often dark, and cons equently, sheltered from direct sunlight. Therefore, trap color can influence trap preference, significantly impacting mos quito captures. Belton (1967) identified preferences for illumination and substr ate contrast of possibl e mosquito oviposition sites using four artificial pools. Two pools were interiorly lined with tr anslucent film, and two with black polyethylene film. White reflectors and 40-watt cool white fluorescent lamps were set on timers, and used to illuminate one translu cent lined pool and one bl ack lined pool. Belton (1967) observed that no mosquito eggs were recovered from illuminated pools. Also, significantly more mosquito eggs were recovered from pools lined with black than those with translucent lining. Laing (1964) observed similar results in a comparable study, recovering fewer mosquito eggs from translucent polyethylene po ols or white painted pools. Results from Belton (1967) and Laing (1964) demonstrated a sign ificant preference for dark, unlit mosquito oviposition sites when given a choice. These findi ngs suggest little or no preference for light when searching for possible oviposition sites. Allan and Kline (2004) observed that infusion pan color significantly affected mosquito capture while evaluating mosquito gravid traps for collection of Culex mosquitoes in Florida. When comparing white, tan, light olive green and black pans, si gnificantly greater numbers of gravid Culex mosquitoes were captured with traps using black or green pans than those with tan or white pans. Similar observations by Kline et al. (2006) concluded th at altering infusion pan

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37 color could have significantly in creased trap totals when evaluating the efficacy of the Gravid Trap (John Hock Company) agains t three other trap designs. Th e findings of Allan and Kline (2004) and Kline et al. (2006) support observations of Belton (1967) and Laing (1964), demonstrating a strong affinity for gravid mos quitoes to dark surface s or oviposition sites. Another effective method of population monitori ng is the use of sticky traps. Sticky traps are grouped into two categories; attractant and non-attractant. Attractant sticky traps are those used in conjunction with bait animals (Disney 1966), carbon dioxi de (Gillies and Snow 1967) or traps constructed with a specific shape or color that would enhance attractiveness of the trap (Allan and Stoffolano 1986a). Non-at tractant traps are designed w ith the intention of functioning independent of bias that might positively or ne gatively influence the attractiveness of the trap. Sticky trap adhesives come in a wide variety of compounds, and can be used to capture many different insects. Various gr eases and oils are common adhesives but have not shown to be as effective as resins, usually trapping only small insects. Tree banding resins are of the most efficient adhesives for catching a wide variety of different sized insects, though they can be difficult to work with when attempting to re move and identify a catch (Service 1993). Common application techniques when working with a dhesives in regards to mosquito population monitoring include mesh screens (Gordon and Gerberg 1945), nets (Pr ovost 1960) or sticky cards (Beck and Turner 1985). Designed to survey flying inse ct populations, sticky cards ha ve been utilized for the study of many insects including hous e flies (Hogsette et al. 1993, Kaufman et al. 2001, Geden 2005, Beresford and Stucliffe 2006), whiteflies (Ha ynes et al. 1986) and aphids (Rohitha and Stevenson 1987). Though they have been recomme nded as reliable monitoring tools for more than 30 years (Haynes et al. 1986), sticky cards have not been widely used in mosquito

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38 population monitoring. Lack of use could be attributed to common difficulties encountered when working with adhesives. Achieving the appropriate visc osity and tackiness of adhesi ves is an important, yet challenging, task in regards to sticky cards. High temperatures and fluctuating humidity levels may cause thinner adhesives to become viseus, losing their effectiveness. However, adhesives that are too thick allow alighting mosquitoes to land and escape, commonly only trapping those that are forcibly blown on to a treated surface by wind (Service 1993). Mosquito Attraction As previously discussed, fe males of almost every mosquito species are anautogenous, requiring a vertebrate blood meal to initiate egg development. To obtain this blood meal, female mosquitoes utilize a variety of olfactory, physic al and visual cues dur ing host location. Visual and physical stimuli including variations in sk in temperature and color as well as host odor provide the necessary informati on required for most mosquitoes to successfully locate and identify their hosts (Constantini 1996). Though ex tensive work has been conducted to determine the mechanism of mosquito attraction to its host, the effect of odor on mosquito behavior is still poorly understood (Clements 1999). The attractiveness of human odors to Ae. aegypti and An. quadrimaculatus was first demonstrated in 1947 using a dual-port olfactom eter (Willis and Roth 1952). Khan et al. (1965) noted individual variations in host attractivenes s when a feeding preference for one person over three others was shown by Ae. aegypti This variance was attributed to dissimilar levels of lactic acid produced by human hosts. Male hosts exhibited higher lactic acid leve ls, thus accounting for greater attractiveness than fema le hosts (Acree et al. 1968). Se veral other volatiles including carbon dioxide (CO2) and 1-octen-3-ol (octenol) have been used more recently as successful

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39 adult mosquito attractants (Kline et al. 1990, K line et al. 1991, Kline and Lemire 1995, Burkett et al. 2001). Reeves (1951) was the first to demonstrate the attractiveness of CO2 to female mosquitoes in field studies. Carbon dioxide is one of the most frequently utilized, and most accepted, volatile attractants used to trap adult mosqu itoes. Commonly found in two forms, CO2 can be added to traps as a compressed gas or a solid (dry ice) (Kline et al. 1991). Though dry ice is relatively inexpensive and lightweight, compressed gas cy linders are often the preferred method of dispensing CO2 with the advantage of re gulating the discharge rate (Service 1993). This can be an important consideration when trapping di fferent mosquito species whose level of attractiveness varies according to the CO2 emission rate (Reeves 1953, Gillies and Wilkes 1974, Mboera et al. 1997, Dekker and Takken 1998). Regul ating discharge rates can also be crucial when using CO2 in conjunction with other volatiles. Kline et al. (1990) found that octenol emissions of 2.3 mg/hr with a CO2 release rate of 200 ml/min have a greater potential as a mosquito attractant than CO2 alone. Multiple studies testing the attractiveness of octenol when used in conjunction with re gulated release rates of CO2 have produced similar results (Takken and Kline 1989, Van Essen et al 1994, Burkett et al. 2001). Visual stimuli such as movement, light waveleng th and intensity, color, shape, pattern, and contrast also play an important role in host location and identification by adult female mosquitoes (Bidlingmayer 1994). In some Aedes species, detection of movement is important for host location (Sippell and Brown 1953). Other speci es may rely on contrasting or low intensity colors such as blue, black and red as primary host location stimuli (Browne and Bennett 1981). Visual attraction traps based on contrast, movement color and pattern have not been widely used

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40 to collect mosquitoes. The Fay-Prince trap is one exception, utilizing a contrasting black and white pattern, but is often baited with CO2 to increase its efficacy (Service 1993). Artificial, reflected and filter ed lights have been incorporat ed in the design of existing efficient traps to increase their efficacy for mos quito research and surveillance with great success (Barr et al. 1963, Service 1976, Ali et al. 1989, Burkett and Butler 2005, Hoel 2005). Ali et al. (1989) were able to demonstrate that both Culex and Psorophora spp. showed a higher preference for light color rather than intensity wh en trapping in the field. Similarly Burkett and Butler (2005) showed that not only light source, but specific light wa velengths played an important role in host attr action. In laboratory trials, Ae. albopictus, An. quadrimaculatus and Cx. nigripalpus all displayed preferences for specific wavelengths of light. Physical stimuli used in host location include radiant and convectiv e heat, moisture, sound and surface structure (Laarman 1955). Peterson and Brown (1951) used heated billiard balls to demonstrate the affinity of Ae. aegypti to convective heat as opposed to radiant heat. Mosquitoes attempted to feed on the heated billiard balls until a window of crystallin e thallium bromoiodide was inserted between the ball and mosquitoes. Th is window allowed the passage of radiant heat while blocking the convective heat, confirming th e attraction to convective heat. While trapping in Florida, Kline and Lemire (1995) observed similar results, noting an increase in total captures of Oc. taeniorhynchus Wiedemann after adding heat to traps. Moisture is commonly used in conjunction with other stimuli to increase the overall attractiveness of some traps. Khan et al. ( 1966) found that moisture, when combined with CO2 and heat, mimicked vertebrate breath, signi ficantly increasing ove rall catch rates of Ae. aegypti In laboratory studies, Brown et al. (1951) found that moist surfaces are more attractive to Aedes mosquitoes than dry surfaces. Similarly, field studies showed that adding moisture to traps

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41 significantly increased catch rates of Aedes species, suggesting most Aedes species utilize moisture over other sensory cues (Brown 1951). Mosquitoes are sensitive to sound freque ncies and respond to those ranging from frequencies of 250 to 1,500 Hz (Kahn et al. 1945). Kahn and Offenhauser (1949) reported that when the wing beat sound of a single female An. albimanus Wiedemann were repeatedly played at 5 s intervals, significantly larger numbers of male An. albimanus were trapped than when no sound was played. In laboratory experime nts, Ikeshoji (1981, 1982, 1985) found that sound attracted males of Ae. aegypti, Ae. albopictus, Cx. pipiens and An. stephensi Liston. It was also noted that while utilizing acoustic removal equi pment in cages, insemination rates of female Ae. aegypti and An. stephensi decreased by 30% and 20% resp ectively. However, under field conditions traps utilizing sound are of little use, because males respond over very short distances, regardless of its intensity or frequency (Service 1993).

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42 CHAPTER 2 RESPONSE OF ADULT MOSQUITOES TO LIGHT EMITTING DIODES PLACED IN RESTING BOXE S Introduction Since the early 1900s, the effectiveness of t echniques to attract and track the m ovements of hematophagous insects has continued to improve (Crans 1989). Adequate and reliable population sampling is often seen as the most im portant and most difficu lt step in ecological studies. There are two main types of population sampling: active and passive. Active sampling involves manually locating and capturi ng insects with devices such as sweep nets or aspirators. With passive sampling, insects are collected and m onitored using stationary traps such as resting boxes or sticky cards (Holck and Meek 1991). Additionally, adult mosquito populations are passively sampled using active tr aps (New Jersey Light Trap, CDC) (Service 1976). These traps are frequently supplemented with attractants such as lactic acid, carbon dioxide and/or various wavelengths of light to enhance mosquito captu res. Lactic acid and carbon dioxide exploit olfactory cues by effectively mimicking host associ ated volatiles, while the manipulation of light (wavelength, frequency and intensit y) acts as a visual attractant. Behaviorally, most mosquito species are e ither nocturnal or crepuscular, remaining relatively inactive durin g daylight hours. Sampling outdoor populations is often difficult, because they can be commonly distributed over wide areas of open vegetation (Crans 1989). To overcome these difficulties and eliminate the biases brought on by baited trapping systems, special monitoring methods were developed with the goal of passively attracting mosquitoes to specific sites from which they can be convenientl y collected (Crans 1995). Mosquitoes often rest or seek shelter in naturally protected sites such as ground bur rows, dense vegetation and tree holes (Crans 1989). The capitali zation of this natural phenomenon has allowed researchers to effectively sample mosquitoes during in active hours using artif icial resting boxes.

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43 Man-made resting structures have been used as adult mosquito sampling tools since the early days of malaria control when several malaria vectors were observed congregating in diurnal resting places (Boyd 1930). Old nail kegs tu rned on their sides were the first of these structures used to sample resting populations of several mosquito species. After reporting that nail kegs were not successful in collecting Anopheles quadrimaculatus Say in Georgia, Goodwin (1942) began experimenting with seve ral different variations in size and color of ar tificial resting structures. He found that 1ft (30 cm3) wooden boxes, when left open at one end, attracted large numbers of An. quadrimaculatus adults Further experiments showed that mean catches of An. quadrimaculatus were higher when boxes were painted re d inside compared with those painted white, yellow, blue, black or green. A red inte rior also allowed for easier distinction of mosquitoes from other background colors. In addition, boxes facing towards the rising sun caught significantly fewer adult mosquitoes th an those facing away from the sun. Goodwin (1942) concluded that the best shelter was a 1 ft3 wooden box painted dull black on the outside, red inside and positioned on the ground in a sheltered position, preferably not facing east (Service 1993). Today, Goodwins resting box design is commonly used in adult population monitoring for several medically important mosquito species. When compared to light traps, Goodwin boxes were more effective at capturing and measuring population changes in An. freeborni Aitken and Culex tarsalis Coquillett (Bradley 1943, Hayes et al. 1958, Loomis and Sherman 1959). Similarly, Gusciora (1961) dem onstrated the utility of 1 ft3 resting boxes more so than light-traps as arboviral surveillance tools for multiple mosquito species in attempting to monitor Culiseta melanura Coquillett populations for the New Jersey State Department of Health Arbovirus Surveillance Program. In trapping compar ison studies, Gusciora (1961) caught 13,240

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44 mosquitoes in Goodwin box shelters but only 6,260 in CDC light-traps. In addition to the aforementioned species, adults of An. crucians Wiedemann, An. punctipennis Say, Cx. salinarius Coquillett, Cx. restuans Theobald, Cx. pipiens Linnaeus, Aedes canadensis Theobald, Ae. sollicitans Walker, Coquillettidia perturbans Walker and Uranotaenia sapphirina Sacken were all effectively trapped in Goodw in resting boxes (Service 1993). Adjustments and advancements in population monitoring procedures involving resting boxes have led to the modern methods used in todays vector surveillance programs (Crans 1995). Although vector surveillance methods involving both insect wavelength preferences and resting behavior have been studied extensivel y, the combination of the two has not yet been evaluated. The objective of my research was to evaluate the attractiveness of resting boxes fitted internally with light emitting diodes (LEDs) of selected wavelengths to field populations of mosquitoes. Wavelengths used in this study were selected based on capture rates and preferences observed for several mosquito genera, including Aedes, Anopheles, Culex and Psorophora (Burkett et al. 1998, Burkett and Butler 2005, Hoel 2005). Materials and Methods Resting Boxes Resting boxes with four sides, a back wall and an open front were constructed using the specifications of a standard 30 x 30 x 30cm res ting box, as described by Crans (1995). The four sides and back wall of all boxes was made from 0 .64 cm ( in) thick exterior grade pine lumber plywood, while 5 x 5 x 29 cm sections of pine were affixed as inside joint supports (Figure 2-1a). Box exteriors were painted with tw o coats of flat black exterior latex paint, and interiors with two coats of barn red exterior latex paint. A 0.64 cm ( in) hole was drilled through the back center wall of each box to allow for the insertion of a LED. The exte rior surface of the rear wall of each box was fitted with a 6.5 x 9 cm, 470 ml pl astic screw cap vial (T hornton Plastics, Salt

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45 Lake City, UT), protecting the battery suppl y and LED wiring. A 0.64 cm ( in) hole drilled through the container lids to correspond to the 0.6 4 cm ( in) diameter hole in the back wall of each resting box. Lids were secured to the b ack wall, allowing for easy attachment and detachment of containers to resting boxes (Figure 2-1b). Mosquitoes were removed from resting boxes using a mechanical as pirator between 1000 and 1300 hours. A 41 x 41 cm section of 0.33 cm thick Plexiglas was used to cover the box opening and prevent the escape of mosquitoes while they were mechanically aspirated. A 15-cmdiameter hole made in the center of the Plexiglas was fitted with a stocking net to allow for aspirator access. Light Emitting Diodes and Battery Supplies All LEDs were obtained from Digi-Key Corpora tion (Thief River Falls, MN). Diodes, part number and millicandela (mcd) rating, as desc ribed in Hoel (2005), were blue (P466-ND, 470 nm, 650 mcd), green (67-1755-ND, 502 nm, 1,5 00 mcd), red (67-1611-ND, 660 nm, 1,800 mcd) and infrared (LN77L-ND, 860 nm). Because infrared radiation is not visible to humans, infrared diodes are not mcd-rated. Round lens LEDs were 8.6 mm long by 5.0 mm in diameter. Viewing angles were 30o except for IR 860 (20o). All materials used in the construction of batte ry supplies were obtained from an electrical supply company such as RadioShack (Gainesville, FL). A 180-ohm re sistor was soldered to all LEDs, to restrict current flow and prevent mechanical failure of LEDs as a result of overworking. A female 9 volt (V) battery snap connector (270-325) was soldered to each modified LED (Figure 2-2a). Battery supplies (270-383) pre-equipped with a complimentary male 9 V connecting site were used, each w ith a maximum holding capacity of four AA batteries. Four rechargeable 2500 milliamp hour (m Ah) AA batteries were used in all battery

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46 assemblages (Figure 2-2b). The 9 V connectors permitted a reliable, but temporary, connection to each battery supply. CDC Light Trap Three m odified CDC light traps (model 512, Jo hn W. Hock Company, Gainesville, FL) were used to provide representative data on background mosquito populations at two study locations. As described in Hoel (2005), each CD C light trap used a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5-cm-dia meter clear plastic cylin drical body (Fig. 2-3). The incandescent bulb was removed from each tr ap. A 36-cm-diameter beveled edge aluminum lid was set approximately 3 cm above the cylindr ical body creating an increase in air current flow into the trap. All traps were set 120 cm above ground using a Shepherds hook with collection nets attached to th e outflow of the trap. Carbon dioxide was provided from a 9 kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15-psi single-stage regulator equipped with an inline micro-regulator (# 007) and an inline filter (Clarke Mosquito Control, Roselle, IL). Flow rates we re confirmed using a Gilmont Accucal flowmeter (Gilmont Instrument Company, Barrington IL .). Carbon dioxide was delivered to the trap through a 2 m long, 6.4 mm outer diameter clear plastic Tygon tubing (Saint-Gobain Performance Plastic, Akron, OH). Power was provided by a 6 V, 12 ampere -hour (A-h), rechargeable gel cell battery (Battery Wholesale Distributors, Georgetown, TX). Site and Resting Box Location Field trials were conducted at the U nivers ity of Florida Horse Teaching Unit (HTU) and the Prairie Oaks subdivision (PO), Gainesville, FL. Both locations were similar, rural environments previously shown to have producti ve mosquito breeding habitats (J. F. Butler personal observation, Holton 2007). The HTU is an equine breeding and tr aining facility housing an average of 50 horses yearly. The facility cons ists of 24 hectares, whic h includes 2.4 hectares

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47 of wetlands and a 0.2 hectare pond. The HTU is located in the southwestern section of Gainesville, east of I-75, and is closely bordere d on three sides by the Paines Prairie State Preserve (Figure 2-4). The PO is a rural subdiv ision with 18 loosely spaced residential units located approximately 4 km west of the HTU, adjacent to the Paines Prairie State Preserve (Figure 2-5). Both locations are surrounded by a mi x of hardwood and pine forest with minimal undergrowth. Sites (east 1, 2 and west 1, 2) chosen at the HTU were divided and named according to corresponding cardinal direction (Figure 2-6). The east side of the HTU differed in both humidity levels and vegetation from the west si de, resulting in a difference in environments between the east and west side te st sites. Test sites chosen on the west side of the HTU were located in a low-lying depression commonly found to hold water, surrounded by moderate tree cover and undergrowth, resulting in higher sustained humidity levels (Figure 2-7). The test sites selected from the east side of the HTU were on a more elevated, drier te rrain surrounded by thin pine forests and adjacent to several homes (Figure 2-8). All residen tial test sites chosen at the PO were consistent in surrounding vegetation, sun light exposure and humidity conditions (Figure 29). Among the 18 Prairie Oaks residences, boxes we re located in the rear section of four backyards, which were spaced approximately th ree residential units apart (Figure 2-10). Temperature and humidity conditions at both locations were obtained from online NOAA databases. Methodology A trial began by placing five rest ing boxes at each test site in a staggered line, out of direct sunlight and approxim ately 4 m apart with open e nds facing west. CDC light -traps were attached to Shepherds hooks with collection nets fitted to the outflow of the trap. After resting boxes and CDC traps operated in the field for 24 h (one trap night), mosquitoes were aspirated from boxes

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48 and CDC catch bags were changed. Mosquitoes recovered from traps were brought back to the laboratory where they were counted and identified CDC traps were serviced daily with batteries and catch bags changed every 24 h. Carbon dioxid e tanks were changed approximately every 10 days or as needed. Resting box sampling at the HTU occurred from 21 July 14 August 2006 resulting in 20 trap nights, and from 05 May 26 September 2007 resulting in 140 trap nights. Trapping at the PO occurred from 18 August 27 September 2006 re sulting in 17 trap nights, and from 05 May 26 September resulting in 140 trap nights. One modified CDC light-trap was operational at the HTU from 21 July 14 August 2006 resulting in 20 trap nights, and from 05 May 26 September 2007 resulting in 140 tr ap nights. Of these 160 trap nights, traps operated without malfunction for 146 trap nights. Trapping at th e PO with two CDC traps occurred from 18 August 27 September 2006 resulting in 34 trap nights, and from 5 May 26 September resulting in 280 trap nights. Traps were operate d successfully for 302 of these 314 trap nights. When trapping nights were not continuous, ex isting mosquitoes were removed from resting boxes 24 h prior to subsequent collecti on. Mosquitoes retrieved from CDC trap catch bags and resting boxes were identified by sex and species using the dichotomous keys of Darsie and Morris (2003) and Darsie and Ward (2005) Identification data were logged into a MS Excel 2007 spreadsheet. Statistical Analysis Mosquito preference for LED wavelengths was evaluated using a m ulti-factorial ANOVA (SAS Institute 2001). For analysis, all data were normalized using the SQRT (n+1) transformation, however actual values are given in text and tables. The model included the fixed effects location, site and LED treatment, the in teraction term, location*LED treatment and the random effect, trial. In instances where either the interaction term or the trial effect was

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49 significant, the data were analyzed separately by location or trial (year). Tukeys Standardized Test ( = 0.05) was used to se parate treatment means. Results In total, in 160 trap nights at the HTU location, 1,885 mosquitoes were recovered from resting boxes. In 157 trap nights at the PO locat ion, there were 5,272 mosquitoes recovered from resting boxes. Anopheles quadrimaculatus females, Cq. perturbans males, Cq. perturbans females, Cx. erraticus males, Cx. erraticus females, Cx. nigripalpus females, Cx. salinarius males, Cx. salinarius females and Mansonia titillans Walker females were collected in large enough numbers to analyze statistically (Table 2-1). Mosquitoes collected, but excluded from analysis because of low numbers or little medical importance included An. crucians, An. quadrimaculatus males, Ochlerotatus infirmatus Dyar and Knab, Oc. triseriatus Say, Uranotaenia lowii Theobald Ur. sapphirina (Appendices A-1, A-2). Diode wavelength preference was observed among An. quadrimaculatus and in Cx. erraticus females in 2007 (Table 2-1). Significantly more An. quadrimaculatus females were aspirated from resting boxes fitted with red and IR LEDs than from those with blue or green LEDs or the no-light control (F = 2.47; df = 4, 6315; P = 0.0429). The trial effect was significant for Cx. erraticus males and females (F = 2.4; df = 4, 1126; P = 0.0476). During the 2006 trapping period, one trial was run at the HTU and PO locations. For the 2006 trapping period, no preferences were obs erved among treatments. However at the HTU location, significantly higher numbers of mosquito es were aspirated from resting boxes at the east-2 trapping site than at the three ot her trapping sites (F = 22.56; df = 3, 727; P = < 0.0001). During the 2007 trapping period, significantly more Cx. erraticus females were aspirated from resting boxes fitted with blue, green, red LEDs and the no-light cont rol than those with IR LEDs (F = 8.41; df = 4, 5577; P =< 0.0001). Significantly more Cx. erraticus females were captured

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50 from the west-1 trapping site of the HTU locatio n than from all other s ites at both the HTU or PO location (F = 14.47; df = 7, 5577; P = < 0.0001) (Figure 2-6, 2-10). Data for Cx. erraticus males, Cq. perturbans males and Ma. titillans females were also analyzed separately by tria l (year) (Table 2-1). During the 2006 trapping period, s ignificantly more Cx. erraticus males were captured from resting boxes placed at the EAST-2 trapping site at the HTU location (F = 4.84; df = 3, 727; P = 0.0024), while Cq. perturbans males were aspirated in significantly higher numbers from resting boxes placed at the west-1 and west-2 trapping sites at the HTU location (F = 32.60; df = 3, 1126; P =< 0.0001) (Figure 2-6). During the 2007 trapping period, significantly more Cx. erraticus males were aspirated from resting boxes placed at the PO location than from those at the HTU location (F = 8.01; df = 1, 5577; P = 0.0047). Numerically, more male Cx. erraticus (25%) were aspirated from resting boxes without LEDs than from those with LEDs. No significant di fferences in LED wavelength preference were observed for Cq. perturbans males, but 33% were aspirated from resting boxes fitted with blue LEDs. No significant differe nces were observed among Ma. titillans females for the 2006 or 2007 trapping periods. Although no significant differences in LED wavelength preference were observed among Cx. nigripalpus females, Cx salinarius males or Cx salinarius females, dissimilarities in mosquito captures among treatments were noted. More than 37% of C x salinarius males and females were collected from resting boxes fitted with green LEDs. Culex nigripalpus females were aspirated in highest numbers from resti ng boxes affixed with blue (24%) LEDs, whereas resting boxes with red LEDs (7%) captured the fewest females.

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51 Approximately 100,653 female mosquitoes, in cluding 24 mosquito species from six genera, were trapped over 448 trap nights (Appe ndix A-3, A-4). Mean mosquito captures per trap night of the six mosquito species show n in Table 2-1 are presented in Table 2-2. During this study, approximately 55% (64,893) of all mosquitoes trapped were captured at the HTU sites using one CDC trap (34% of trap ni ghts). Proportionality in mosquito capture rates between trapping the 2006 and 2007 trapping periods also differed. During the 2006 trapping period at the HTU location, considerably more Cq. perturbans and Cx. erraticus females were trapped than in the 2007 trapping period. In 2006, an average of 1,400 Cq. perturbans females per trap night were captured compared with an average of 45 per trap night during 2007. Similarly, during the 2006 trapping period Cx. erraticus averaged 10 times more mosquitoes than during the corresponding 2007 tra pping period (September). Conversely, Cx. nigripalpus capture increased during the 2007 trapping period Approximately one mosquito was captured per trap night during the 2006 trapping period, wh ereas in 2007 an average of 657 mosquitoes were captured per trap night. Average monthly temperatures for August (27 C) and September (25 C) remained relatively similar between the 2006 and 2007 trapping periods, differing by no more than 0.7 C for either monthly average (Figure 2-12a, b). Ho wever, average precipitation levels for August and September of 2006 and 2007 were quite different In 2006, an average of 7 cm of rainfall was recorded in August compared with approxima tely 17 cm in 2007. Similarly, less than 8 cm of rainfall were recorded for September in 2006, with approximately 9 cm recorded in 2007. The highest average precipitation levels for 2007 occurred in July (22.6 cm), while lowest precipitation levels occurred in May (1.9 cm) (Figure 2-12a, b).

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52 Discussion In this study, LED color (wavelength) choice s were blue (460 nm ), green (502 nm), red (660 nm) and IR (860 nm). Blue, at 460 nm, registers at the higher end of the purple-blue range of the visible light spectrum. However, 502 nm fa lls at the lower transiti on point between blue and green, while 660 nm registers near the lower end of the red-yellow light spectrum. Infrared wavelength is not detectable by th e human eye, registering above the visible spectrum at 860 nm. For additional information concerning the visi ble light spectrum, see Ando and Thomas (1996). Wavelengths selected for in this study were selected based on capture rates and preferences observed for several mosquito genera, including Aedes, Anopheles, Coquillettidia, Culex and Psorophora (Burkett et al. 1998, Burkett and Butl er 2005, Hoel 2005). Burkett et al. (1998) recorded higher captures of An. crucians and Cx. nigripalpus using CDC light-tra ps fitted with green light than when using IR LEDs. Additiona lly, Hoel (2005) observed trapping significantly more Cq. perturbans when using CDC light-traps supplemented with CO2, and modified with blue LEDs (470 nm) that standard CDC light-traps using incandescent bulbs. Using the Goodwin (1942) style resting boxes in southern New Jersey, Burbutis and Jobbins (1958) and Crans (1995) trappe d similar mosquito species, including An. quadrimaculatus, Cs. melanura, Cx. restuans, Cx. salinarius, Cq. perturbans and Ur. sapphirina Collections of Cs. melanura and An. quadrimaculatus significantly exceeded those of all other mosquito species in both studies. Our results ag ree with these studies in terms of species diversity, because we collected si milar mosquito species, such as An. quadrimaculatus, Cx. salinarius, Cx. territans and Cq. perturbans However, we recovered no Cs. melanura from resting boxes or CO2 baited traps, although Cs. melanura have been reported in this area of Florida (Burkett et al. 1998). This difference ma y result from habitat variations or seasonal emergence patterns exhibited in Florida.

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53 Resting boxes were located in similar hard wood hammock habitats at both locations. Due to habitat variation, mosquito species not comm only recovered from these environments were likely excluded from trapping results. Additiona lly, trapping periods only occurred for three months during 2006, and five months during 2007. The bias resulting from only utilizing one habitat during a narrow time period could explain the lack of Cs. melanura among resting box captures (Crans 1995). We found that Cx. erraticus males and females were recovered from resting boxes in higher numbers (48% and 42% respectively) than all other mosquito species. Approximately 26% of male Cx. erraticus were recovered from resting boxes fitted with IR LEDs, and 23% of females were recovered from boxe s left dark. High numbers of Cx. erraticus were anticipated as this species is commonly captured in light trap s (Ali et al. 1989, Cupp et al. 2003, Rodrigues and Maruniack 2006). Ali et al (1989) captured numerous Cx. erraticus in Florida while utilizing New Jersey light traps fitted with white, yellow, orange, blue, green or red incandescent bulbs. These results suggest the presence of light may impact trapping results for Cx. erraticus. Similarly, the addition of selected wavelengths to resting boxes may increase the attractiveness of these boxes to Cx. erraticus. When testing mosquito wavelength preference with filtered light using a visualometer, Burkett and Butler (2005) observed signi ficantly longer feeding periods for An. quadrimaculatus on artificial hosts illuminated with black (no light) or white light compared with other wavelengths ranging in 50 nm increments from 350 750 nm. Feeding times on artificial hosts illuminated with filtered light at 350 nm (purple) were significantly shorter than all other feeding times recorded. These observations were sim ilar to our results where significantly more An. quadrimaculatus were aspirated from resting boxes fitted with red LEDs (high end of the light

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54 spectrum) than blue or green LEDs. Burkett and Butlers (2005) results and our findings suggest that lower wavelengths (< 660 nm) are less desirable to An. quadrimaculatus than are wavelengths higher in the light spectrum (> 660 nm). Therefore, the a ddition of 660 nm LEDs to resting boxes may enhance efficacy of sampling An. quadrimaculatus populations. Overall, more mosquitoes (male and female) were recovered from resting boxes fitted with IR LEDs (23%) than all other tr eatments. Resting boxes left dark captured 22% of mosquitoes, while the fewest mosquitoes were recovered fr om boxes affixed with red (20%), green (17.6%) and blue (16.7%) LEDs. Our results suggest general mosquito preference for wavelength spectrums that were longer than shorter. These observations differ from other findings for photophilic mosquito species tr apped at night, such as Cx. erraticus, Cx. nigripalpus and Psorophora columbiae Dyar and Knab, which suggest pr eferences for lower wavelengths (Bargren and Nibley 1956, Ali et al. 1989, Bu rkett et al. 1998, Burkett and Butler 2005). Differences in our results may be the product of variations previously unaccounted for in wavelength attraction between host seeking and resting mosquitoes. A dditionally, the use of narrow wavelengths may have excluded mosquitoes preferring longer or shorter wavelengths than those selected. Male mosquitoes comprised approximately 54% (3,853) of all mosquitoes aspirated from resting boxes. Culex erraticus males (3,455) accounted for almost half of all mosquitoes captured, while Cx. nigripalpus males (6) were recovered the least. Aspiration totals for other mosquito species ranged from seven to 218. Though gravid or blood fed females are highly desired, high captures of males in resting stru ctures are not uncommon, and can be important (Goodwin 1942, Nelson 1980, Kay 1983, Edman et al 1997). Goodwin (1942) reported high captures of An. quadrimaculatus males using empty nail kegs. A dditionally, Edman et al. (1997)

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55 observed high numbers of male Ae. aegypti coming to artificial rest ing boxes placed inside houses. Effectively sampling male mosquito popul ations can be an important tool in the surveillance and modeling of vene really transmitted arboviruses su ch as St. Louis encephalitis. Male mosquito population densities can be impor tant indicators of general population fecundity and reproductive status of a targ et species. In population modeli ng, this combination of factors makes sampling an effective tool in the comp rehension of vector po tential of a disease transmitting population (Garrett-Jones 1964). Expectedly, more mosquitoes were captured in CDC light-traps than resting boxes. because of the supplement of an artificial host attractant, CO2, in the CDC traps. Both modified CDC light-traps and resting boxes captur ed similar mosquito species, including Cq. perturbans Cx. erraticus, Cx. nigripalpus, Ma. titillans, Ur. lowii and Ur. sapphirina Adult mosquitoes are commonly captured when using trap designs that combine light with alternative host stimuli (Browne and Bennett 1981, Burkett et al. 1998, Hoel 2005). Most mosquito species, such as An. quadrimaculatus, are endophilic, and are r ecovered in higher numbers fr om resting boxes rather than CDC traps. Endophilic mosquitoes prefer fe eding and resting in or near human dwellings. These species are more often captured in boxes that are designed to mimic their natural resting behaviors, rather than target th eir host seeking behaviors. Ther efore, trapping systems must be chosen based accordingly to the desired species. This further illustrates the physiological and behavioral differences among mosqu ito species, and the effects of those differences on trap bias. Some mosquito species, such as Ps. ciliata and Ps. columbiae were captured in the CDC light-traps, but not in resting boxes. While some Psorophora are often recovered from resting boxes, Ps. ciliata and Ps. columbiae are known to frequent light tr aps in Florida (Ali et al. 1989, Burkett et al. 1998). The occurrence of both mosquito species in light traps, but not in lit or dark

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56 resting boxes suggests a phototactic relationship. As both mosquitoes are pest species to humans, this negative association may warrant the integrat ion of LEDs in various repellant applications. Additionally, light intensity may impact the en try into resting boxes fitted with LEDs. Many mosquito species are known to e xhibit positive phototaxis to light sources, w ith attraction levels directly correlating to light in tensity (Service 1993). Using light traps, Gaydecki (1984) observed that smaller insects including mosquitoes became disoriented near light so urces. Ali et al. (1989) demonstrated similar results, trapping significantl y more mosquitoes in light traps with lower intensities. Male mosquitoes represented less than one pe rcent of all CDC light -trap captures. This contrasts with 54% of total males recovered from resting boxes during this study. These results are likely due to th e supplement of CO2 as an additional host attr actant to the CDC traps. Because this volatile is utilized as a host attrac tant, the detection of this gas serves very little physiological purpose to male mosquitoes. Howeve r, female mosquitoes in search of a blood meal must be able to detect, recognize and loca te this compound to obtain nutrients necessary for vitellogenesis. Mean mosquito capture per trap nigh t from modified CDC light-traps for Cx. nigripalpus differed greatly between the 2006 and 2007 trapping periods. Mosquito capture rates at the HTU and PO locations were approximately one mosqu ito per trap night in 2006, compared with 657 Cx. nigripalpus per trap night in the respective 2007 trapping period. This dramatic population increase may have been due to the mosquitoes seasonal and spatial dist ribution in response to wetting and drying conditions, as discussed in Day and Curtis (1994). During the 2007 trapping period, periodic rains, followed by sufficient drying periods, provided the ideal environmental conditions for Cx. nigripalpus to exceed average population densities.

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57 Super-bright LEDs have demonstrated superior effectiveness as light sources for various trap designs. Given their intensity, small size, efficiency and minimal power usage, LEDs make optimal light sources for civilian or military field applications where access to target sites and/or transport of equipment are minimal. While their intensity is superior to other compact light sources, LEDs used in this study only offer a 30 viewing angle. This has little effect on insects from long distances, but significantly restricts th e peripheral visibility of emitted light when insects are not in line with the targeted LED emission. However, the ability to operate for extended periods of time on power sources as small as a watch battery eliminates the necessity to regularly exchange and maintain larger, more cumbersome batteries. Their demonstrated effectiveness in our resting boxes for attracting mo squitoes without the aid of supplemental host attractants further eliminates the ne ed for dry ice or heavy tanks (CO2) or noxious chemicals (lactic acid, octenol). Durability of the LED-bas ed equipment also helps to reduce otherwise necessary and time-consuming field maintenance. By offering extended operating time with minimal power consumption, field durability an d the ability to eliminate the need for burdensome equipment, LEDs remove restri ctions previously set on trap designs. The addition of LEDs to re sting boxes in this study has demonstrated increased attractiveness for certain mosquito species, whil e decreasing attractivenes s to others. Relevance of these findings could lead to future civilian or military appl ications as mosquito repellant devices. Based on the push-pull premise, res ting boxes or mechanical adult mosquito traps could be placed at a considerable distance fr om a home or military box, and fitted with LEDs found to be attractive to target mosquito spec ies. Light emitting diodes with wavelengths known to be undesirable to these species would then be affixed to the desired building. This

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58 combination of attractive and repellant stimulan ts enhances the effects of each, leading to improved repellent devices for medically important mosquitoes. The push-pull principle could also be applied to sticky-car d traps. Sticky-card traps are simple, inexpensive and versatile, allowing them to be utilized in multiple trap designs. By utilizing reflective or colored surfaces to enhance attractiveness, fitting LEDs of preferred wavelengths to sticky-card traps may increase the effectiveness of these traps in locations where space and equipment limitations are important Light emitting diodes with non-preferred wavelengths affixed to areas of interest would help to repel mosquitoes, while increasing the attraction of sticky-traps fitted with LEDs of preferred wavelengths. This modified trap design has promising military and civilian applications. Additional applications of this research could involve the integration of interior pesticide applications to LED fitted resting boxes. These spray applications have been demonstrated as possible control measures for Anopheles species in domestically-p laced resting boxes such as huts or tents (Smith et al. 1966, Quiones and Suarez 1990). The combination of enhanced attractiveness to illuminated re sting boxes and knock down sprays could serve as an efficient control method for several medically important mosquito species. Previous to this study, trappi ng involving the inclusion of LEDs in resting boxes has not been conducted. The findings of this research dem onstrate the need for further investigation into the combination of mosquito wavelength attraction and artificial resting boxes. Several mosquito species recovered from resting boxes fitted with LEDs were previously thought to have little affinity to light. Based on these results and obser vations from past research, variations in light intensity might also significan tly impact the attractiveness of resting boxes to mosquitoes. Additionally, population sampling for those mosquito species may be improved or refined with

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59 the addition of LEDs to resting boxes. Continued research into wavelength frequency may offer further insight into the attrac tiveness of some mosquito spec ies to resting boxes fitted with LEDs.

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60 Table 2-1. Mean ( SE) numbers of mosquitoes/trap/night attracted to light emitting diodes of four different wavelengths placed in resting boxes at the University of Florida Horse Teaching Unit and Prairie Oaks Subdivi sion from July 2006 Sept. 2007 near Gainesville, FL. Diode Wavelength Species TN Blue Green Red IR No Light An. quadrimaculatus 1,268 0.004 (.003)b 0.013 (.007)b 0.032 ( 0.013)a 0.007 (.005)b 0.015 (.005)b Cq. perturbans 2006 148 0.372 (.052) 0.291 (.052) 0.250 ( 0.050) 0.230 (.050) 0.270 (.063) Cq. perturbans 2007 320 0.003 (.003) 0.006 (.004) 0.009 ( 0.005) 0.003 (.003) 0.006 (.004) Cq. perturbans 788 0.023 (.006) 0.022 (.007) 0.037 ( 0.008) 0.030 (.007) 0.028 (.008) Cx. erraticus 2006 288 2.154 (.333) 2.452 (.396) 3.009 ( 0.453) 3.868 (.617) 3.154 (.501) Cx. erraticus 2007 1,120 0.022 (.005) 0.028 (.006) 0.024 ( 0.005) 0.021 (.005) 0.032 (.007) Cx. erraticus 2006 148 2.851 (.558) 2.980 (.546) 3.223 ( 0.616) 3.967 (.710) 3.932 (.740) Cx. erraticus 2007 1,120 0.113 (.012)a 0.087 (.011)a 0.083 ( 0.010)a 0.038 (.007)b 0.104 (.012)a Cx. nigripalpus 468 0.017 (.011) 0.017 (.010) 0.004 (.0 03) 0.009(.007) <0.001 (<0.001) Cx. salinarius 388 0.005 (.004) 0.028 (.015) 0.005 ( 0.004) 0.015 (.009) 0.015 (.009) Cx. salinarius 468 0.006 (.004) 0.009 (.005) 0.002 ( 0.002) 0.004 (.003) 0.006 (.004) Ma. titillans 148 0.014 (.014) 0.027 (.013) 0.020 ( 0.012) 0.027 (.014) 0.027 (.014) Note: Blue LED = 470 nm, Green LED = 502 nm, IR = 860 nm, Red LED = 660 nm and No Light indicates no LED treatment. An = Anopheles; Cq. = Coquillettidia ; Cx. = Culex; Ma. = Mansonia TN = TN = number of trap nights were total mosquito species capture = 1 per 20 day trapping period. Means within rows followed by the same letter were not significa ntly different (P< 0.05, Tukeys standardized test [SAS Institute 2001]). An. quadrimaculatus (F4, 6315 = 2.47; P = 0.0429); Cx. erraticus 2007 (F4, 5577 = 8.41; P =< 0.0001).

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61 Table 2-2. Total number of mosquitoes/trap night for six signif icant mosquito species captured at the Horse Teaching Unit and Prairie Oaks Subdivision from July 2006 Sept. 2007 near Gainesville, FL. Total Mosquitoes/Trap/Trap Night Date Location TN An. quadrimaculatus Cq. perturbans Cx. erraticus Cx. nigripalpus CDC RB CDC RB CDC RB CDC RB 7/21/06 8/14/06 HTU 16 1.56 0.15 1,391.88 0.60 154.38 9.90 1.13 <0.01 8/18/06 9/27/06 PO 36 2.10 3.85 73.77 3.30 216.47 115.55 <0.01 0.65 5/5/07 5/24/07 HTU 16 0.63 0.10 45.38 0.85 5.56 3.00 <0.01 0.15 PO 38 0.32 0.10 53.87 0.30 3.76 3.45 <0.01 0.05 5/25/07 6/13/07 HTU 20 0.05 <0.01 11.40 <0.01 2.95 0.60 <0.01 <0.01 PO 39 0.03 <0.01 21.74 <0.01 2.46 2.45 0.15 <0.01 6/14/07 7/6/07 HTU 20 <0.01 <0.01 15.95 0.05 3.60 1.05 3.85 0.05 PO 39 <0.01 <0.01 23.74 <0.01 1.31 1.00 9.36 0.15 7/7/07 7/28/07 HTU 18 0.17 <0.01 11.94 0.15 0.72 0.65 0.67 0.05 PO 37 0.03 <0.01 10.70 0.05 0.51 1.30 2.16 <0.01 7/29/07 8/17/07 HTU 19 <0.01 <0.01 12.89 0.05 3.37 1.40 95.53 <0.01 PO 40 <0.01 <0.01 4.53 <0.01 1.00 1.15 30.13 <0.01 8/18/07 9/6/07 HTU 19 0.26 0.15 14.21 0.05 4.89 3.10 301.53 <0.01 PO 38 <0.01 <0.01 5.95 <0.01 0.68 0.90 39.79 <0.01 9/7/07 9/26/07 HTU 18 0.67 0.15 30.56 0.05 7.28 2.05 657.94 <0.01 PO 35 0.03 <0.01 10.83 <0.01 1.51 1.70 214.60 <0.01

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62 Table 2-2. Continued. Total Mosquitoes/Trap/Trap Night Date Location TN Cx. salinarius Ma. titillans CDC RB CDC RB 7/21/06 8/14/06 HTU 16 1.88 <0.01 531.75 0.10 8/18/06 9/27/06 PO 36 5.20 0.10 7.50 0.75 5/5/07 5/24/07 HTU 16 1.81 0.10 0.60 <0.01 PO 38 1.29 0.10 0.29 <0.01 5/25/07 6/13/07 HTU 20 1.45 0.25 0.60 <0.01 PO 39 0.67 <0.01 <0.01 <0.01 6/14/07 7/6/07 HTU 20 4.25 0.05 1.90 <0.01 PO 39 2.97 0.05 <0.01 <0.01 7/7/07 7/28/07 HTU 18 1.11 <0.01 6.44 <0.01 PO 37 0.24 <0.01 0.16 <0.01 7/29/07 8/17/07 HTU 19 21.32 <0.01 14.84 <0.01 PO 40 3.20 <0.01 0.13 <0.01 8/18/07 9/6/07 HTU 19 33.95 <0.01 15.42 <0.01 PO 38 1.13 <0.01 0.08 <0.01 9/7/07 9/26/07 HTU 18 15.11 <0.01 44.72 <0.01 PO 35 2.34 <0.01 0.11 <0.01 Note: An. = Anopheles ; Cq = Coquillettidia ; Cx. = Culex; Ma. = Mansonia CDC = Modified CDC light -trap; RB = resting box. HTU = One modified CDC trap + CO2 (250 ml/min); PO = Two modified CDC traps + CO2 (250 ml/min). TN = number of trap nights CDC traps were in operation. When TN < 20 (HTU) or TN < 40 (PO), traps had malfunctioned. Trap nights for all RB trapping periods = 20.Total mosquitoes/tra p/trap night = total mosquitoes captured/ # of trap nights.

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63 A B Figure 2-1. Resting boxes used at the Universi ty of Florida Horse Teaching Unit and Prairie Oaks subdivision. A) Rear view of 30 x 30 cm resting box showing protective LED housing. Exterior of all boxes were made using 1 cm thick exterior grade pine plywood. The outside of each resting box was painted with two coats of flat black exterior latex paint, and interiorly with tw o coats of barn red exterior latex paint. Diode housing consisted of one 470 ml plastic container attached to the exterior rear wall of each box by container lid. Container li ds were modified with a 0.32 cm hole, and matched to the 0.32 cm hole on the out side back wall of each resting box. B) Front inside view of 30 x 30 cm resting box illustrating 5 cm x 5 cm x 29 cm sections of pine used as inside corner supports. A 0.32 cm hole was drilled through the back wall of each box to allow for the inserti on of a LED. Resting boxes were painted interiorly with two coats of barn red exterior latex paint.

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64 A B Figure 2-2. Light emitting diode configuration used in resti ng boxes. A) All round lens LEDs were 8.6 mm long by 5.0 mm in diam eter. Viewing angles were 30o except for IR (20o). After a 180-ohm resistor was soldered to each LED, restricting current flow, a female 9 volt (V) battery snap connector (270-325) was attached. B) Battery housing used to supply power to LED configurations for resti ng boxes. Battery supplies (270383) pre-equipped with a complimentary male 9 V connecting site were used, each with a maximum holding capacity of four AA batteries. Four rechargeable 2500 milliamp hour (mAh) AA batteries were used in all assemblages. Figure 2-3. CDC light trap modified by the rem oval of its incandescent bulb. Modified trap used a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5 cm diameter clear plastic cylindrical body. A 36 cm diameter beveled edge aluminum lid was set approximately 3 cm above the cy linder body creating a downdraft air current. All traps were set 120 cm above ground usi ng a Shepherds hook, and collection nets were attached to the bottom of the tr ap body. Carbon dioxide was provided from a 9 kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15psi single-stage regu lator equipped with micro-regul ators and an inline filter.

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65 Figure 2-4. Aerial view of Horse Teaching Un it location. The unit is lo cated east of I-75 and approximately 1.6 km northwest of Paines Prairie State Preserve, Alachua Co., FL. Figure 2-5. Aerial view of Prairie Oaks subdivision whic h was located approximately 4.8 km southwest of the Horse Teaching Unit, ad jacent to the Paines Prairie Preserve, Alachua Co., FL. Paines Prairie Preserve HT U Prairie View Subdivision Paines Prairie Preserve

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66 Figure 2-6. Test sites located within the Horse Teaching Unit. Each white rectangle represents a test site where five boxes were equipped with one of five treatments. Sites are numerically labeled according to correspondi ng eastern or western direction. White arrow designates location of modified CDC trap. Figure 2-7. Horse Teaching Unit location; west side test site habitat. East Site 1 East Site 2 West Site 1 West Site 2

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67 Figure 2-8. Horse Teaching Unit locatio n; east side test site habitat. Figure 2-9. Representative of te st sites chosen at the Prairie Oaks subdivision. All sites chosen were consistent in surrounding vegetation, sunlight exposure and moisture conditions.

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68 Figure 2-10. Test sites located within Prairie Oaks Subdivisi on. Each solid white rectangle represents a test site where five boxes were equipped with one of five treatments. White dashed rectangles identify the locati on of modified CDC traps. Figure 2-11. Resting boxes placed with openi ngs facing west and were spaced approximately four meters apart and out of direct sunlight. Each site c ontained five treatments, one of four LED colors and an unlit control, re sulting in a total of five resting boxes per site, 20 resting boxes per location.

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69 A B Figure 2-12. Mean monthly temperatures (C) and precipitation (cm) for the Horse Teaching Unit (HTU) location near Gainesville, FL, using data retrieved from the National Oceanic and Atmospheric Administra tion (NOAA) database. A) Monthly temperature, May September 2006 and 2007. B) Monthly precipitation from Jan September 2006 and 2007.

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70 CHAPTER 3 FIELD RESPONSE OF ADULT MOSQUITO E S TO WAVELENGTHS OF LIGHT EMITITING DIODES Introduction In Diptera, photon detection is achi eved through the ocelli and com pound eyes. Although ocelli are essential fo r some perceptual functions, such as the entrainment of circadian rhythms, the compound eyes act as th e primary visual organ (Allan et al. 1987). These organs are responsible for more sp ecialized functions including detection of movement, patterns, contrast and color. Several laboratory and field studies have been conducted to determine the behavior of adult Di ptera in response to visual stimuli, with special attention given to the modification of light wavelength and intensity in Culicidae (Huffaker and Back 1943, Fox 1958, Bidlingmayer 1967, Burkett and Butler 2005). Early luminous sources used in light trap s included paraffin or acetylene lamps (Husbands 1976). Today, multiple publications detail various light trap designs, light sources and other factors that influence mosquito trap catch size. Some devices, such as the New Jersey light-trap, the CDC light-trap and the Encephalitis Virus Surveillance (EVS) light-trap, employ motorized suction fans to aid in mosquito capture and containment (Service 1970, Ginsberg 1988, Foley and Bryan 1991). Others, including chemical light-traps and stic ky light-traps, rely on non-mech anical mosquito containment methods (Service and Highton 1980, Sulaiman 1982). Deviations in light intensity can significantly influence the numbers and species of mosquitoes caught in light-traps (Servi ce 1993). Although mosquitoes may initially exhibit positive phototaxis to light-traps, nega tive phototaxis occurs at certain distances and is dependent upon light intensity. Headl ee (1937) first demonstrated the impact of varying light intensities on catch size after not ing that significant quan tities of mosquitoes

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71 were attracted to within a certain proximity of traps but were not being caught. These proximate mosquitoes were only captured after the addition of a motorized fan to traps. The effect of light intensity on mosquito ca tch has been extensively investigated and similar results have been repeatedly produ ced (Barr et al. 1963, Reisen and Pfuntner 1987, Ali et al. 1989). Variations in wavelength also impact mosquito catch rates in light-traps. Importantly, not all mosquito species respond equally to dissimilarities in wavelength. In laboratory studies, Gjullin et al. (1973) demonstrated that male Culex tarsalis Coquillett, Cx. quinquefasciatus Say and Aedes sierrensis Ludlow prefer ceramic-dipped red bulbs over similar green, blue, orange or white inca ndescent bulbs. Similarl y, Ali et al. (1989) found that field populations of Culx and Psorophora display wavelength preference. Higher proportions of Cx. nigripalpus Theobald, Cx. erraticus Dyar and Knab, Ps. columbiae Dyar and Knab and Ps. ciliata Fabricius were collected with New Jersey lighttraps modified with incandescent blue lights th an did traps modified with yellow, orange, green, red or white lights. Much of what is known today concerning the affinity of Diptera to different wavelengths of light can be cr edited to studies in which scie ntifically poor light sources were used (Brett 1938, Bracken et al. 1962, Bradbury and Bennett 1974, Browne and Bennett 1980, 1981, Allan and Stoffolano 1986b). The recent development of superbright light emitting diodes (LEDs) has allowed for the isolation of specific wavelengths permitting researchers to refine techniques to more effectively attract mosquitoes using a more precise light sources. When used in Ce nter for Disease Control (CDC) traps, these highly efficient, low cost LEDs have a greate r intensity and have a significantly lower

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72 energy requirement than existing incandescen t bulbs (Burkett et al. 1998). Little information exists describing the attractiveness of LEDs to different mosquito species. Additionally, knowledge of wave length preferences of mosqu ito species in suburban and rural habitats of Florida is limited. Therefore, the objective of this study was to determine the response of adult mosquitoes to four selected wavelengths of light from LEDs placed in suburban and rural habitats. Studies were conducted during 2006 and 2007 in Gainesville, FL. Light emitting diode wavelengths selected were blue (460 nm), green (502 nm), red (640 nm) and IR (860 nm). Blue, at 460 nm, registers at the higher end of the purpl e-blue range of the visible light spectrum. However, 502 nm falls at the lower transition point between blue and green, while 640 nm registers near the lower end of the red-yellow light spectrum. Infrared wavelength is not de tectable by the human eye, re gistering above the visible spectrum at 860 nm. For additional information concerning the visible light spectrum, see Ando and Thomas (1996). Wavelengths used in this study were selected based on capture rates and preferences observed for several mosquito genera, including Aedes, Anopheles, Culex and Psorophora (Burkett et al. 1998, Burkett and Butler 2005, Hoel 2005). Materials and Methods Diode Equipped Boxes Diode equipped boxes with four sides and an open top and bottom were constructed from 0.64 cm ( in) thick exterior-grade pi ne lumber plywood. Each of the four sides measured 20 x 20 cm. Boxes were construc ted and designed to support one 13 x 13 cm sticky card with one diode centere d per vertical side, yielding a total of four sticky cards and four light treatments per box. Each light treatment corresponded to one of four colored diodes; blue (470 nm), green (502 nm), red (660 nm) or infrared (860 nm). A

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73 0.64 cm ( in) diameter hole was drilled in th e center of each outward facing surface of the boxes to allow for insertion of the di ode. The outside surface of each diode box was painted with two coats of flat black exterior latex paint. Boxes were held above ground by a 90-cm length of 1.9 cm ( in) inner-diameter PVC pipe. PVC pipe sections, subsequently, were supported by a 120 cm le ngth of 1.27 cm ( in) diameter steel rod (Figure 3-1). Light Emitting Diodes and Battery Supplies All LEDs were obtained from Digi-Key Corporation (Thief River Falls, MN). Diodes, part number and millicandela (mcd) rating, as described in Hoel (2005), were blue (P466-ND, 470 nm, 650 mcd), green (67-1755-ND, 502 nm, 1,500 mcd), red (671611-ND, 660 nm, 1,800 mcd) and infrared (L N77L-ND, 860 nm). Because infrared radiation is not visible to humans, infrared diodes are not mcd-rated. Round lens LEDs were 8.6 mm long by 5.0 mm in diam eter. Viewing angles were 30o except for IR (20o). A 180-ohm resistor was solder ed to all LEDs, restricting current flow to prevent mechanical failure. Power was provided by a 6 v, 12 ampere-hour (A-h), rechargeable gel cell battery which was changed every 24 48 h (Battery Wholesale Distributors, Georgetown, TX) (Figure 3-1). Sticky Cards Sticky cards (Atlantic Paste & Glue Cor poration, Brooklyn, NY) were made from black 28 pt. SBS card stock (EPA # 057296-WI-001) and coated with 32 UVR soft glue with UV inhibitors. Black sticky cards were se lected to reduce variability of reflected light caused by LEDs. Individua l sticky cards, originally s upplied as 41 x 23 cm boards, were cut to yield two 13 x 13 cm sticky cards for field use. A 0.64 cm ( in) diameter

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74 hole was drilled into the center of each stic ky card to allow for insertion of a diode (Figure 3-2). CDC Light Trap Three m odified CDC light traps (model 512, John W. Hock Company, Gainesville, FL) were used to provide a representative background mos quito population at two study locations. As described in Hoel (2005), each CDC light trap used a 6 V DC motor and 4blade fan to draw flying insects through an 8.5 cm diameter clear pl astic cylindrical body (Figure 3-3). The incandescent bulb was re moved from each trap. A 36-cm diameter beveled edge aluminum lid was set appr oximately 3 cm above the cylindrical body increasing the downdraft caused by the fan. All traps were set 120 cm above ground using a Shepherds hook, and collection nets were attached to th e bottom of the trap body. Carbon dioxide was provided from a 9 kg compressed gas cylinder, and delivered to traps through a 2 m long, 6.4 mm outer diamet er clear plastic Tygon tubing (SaintGobain Performance Plastic, Akron, OH). A fl ow rate of 250 mL/min was achieved by using a 15-psi single-stage re gulator equipped with an inlin e micro-regulator (# 007) and an inline filter (Clarke Mosquito Control, Roselle, IL). Flow rates were confirmed using a Gilmont Accucal flowmeter (Gilmont Instrument Company, Barrington IL.). Carbon dioxide tanks were changed approximately every 10 days or as needed. Power was provided by a 6 V, 12 ampere-hour (A-h), rech argeable gel cell battery changed every 24 48 h (Battery Wholesale Dist ributors, Georgetown, TX). Site and Sticky Card Trap Location Field trials were conducted at the U niv ersity of Florida Horse Teaching Unit (HTU) and the Prairie Oaks subdivision (PO), Gainesville, FL. Both locations were

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75 similar environments, previously shown to ha ve productive mosquito breeding sites (J. F. Butler personal observation, Holton 2007). Th e HTU is a rural equine breeding and training facility housing an average of 50 horses yearly. The HTU is an equine breeding and training facility housing an average of 50 horses yearly. The facili ty consists of 24 hectares, which includes 2.4 hectares of wetlands and a 0.2 hectare pond. The HTU is located in the southwestern section of Gaines ville, east of I-75, and is closely bordered on three sides by the Paines Prairie State Pr eserve (Figure 3-4). The PO is a rural subdivision with 18 loosely spac ed residential units located approximately 4 km west of the HTU, adjacent to the Paines Prairie Stat e Preserve (Figure 3-5). Both locations are surrounded by a mix of hardwood and pine forest with minimal undergrowth. Diode equipped boxes were placed at four different sites. Glue boards were attached to the outside of the four walls so the holes in the walls and glue boards were in alignment. Light emitting diodes were inserted into the holes from the inside of the boxes so the LED protrudes through the glue boar d. The outward facing side of boxes were fitted with one of four colored light treatments and four sticky cards. This resulted in a total of four boxes at the HTU and four boxes at the PO. All re sidential test sites utilized at the PO were consistent in surrounding vegetation, sunlight exposure and moisture conditions (Figure 3-6). Among the 18 PO resi dences, boxes were located in the rear section of four backyards, spaced approxi mately three houses apart (Figure 3-7). Sites chosen at the HTU were divided and named according to the corresponding cardinal direction (Figure 3-8) Differences in surrounding ve getation were noted in all sites, with differences in humidity assu med. Both northeast and southeast sites were similar in fauna, and were located within 30 yards of residentia l units. However, the

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76 southeastern site was separated from the resi dential units by a thin stretch of mixed pine forest while the northeastern site was not (Figure 3-9a, b). The northwestern site was adjacent to the quarter hectare pond, contai ning a mixture of aqua tic and terrestrial vegetation (Figure 3-9c). The southwestern site was moderately shaded, surrounded by inconsistent ground cover and mixed hardw ood forest (Figure 39d). Temperature and humidity conditions at both locations were obtained from online NOAA databases. Methodology To begin a trial, diode equipped boxes were placed at four site s, with the outward facing side o f boxes fitted with one of four co lored light treatments and four sticky cards. CDC light-traps were hung from Shepherds hoo ks, with collection nets attached to the outflow of the trap. After diode equipped boxe s and CDC traps operated in the field for 24 h (one trap night), sticky cards were co llected and CDC catch bags were changed. Mosquitoes recovered from trap s were brought back to the laboratory where they were counted and identified. CDC light traps were se rviced daily with batte ries and catch bags changed every 24 h. Carbon dioxide tanks were changed approximately every 10 days or as needed. Sticky card trapping at the HTU occurred from 16 Aug. 27 Sept. 2006 resulting in 20 trap nights, and from 5 May 13 Sept. 2007 resulting in 120 trap nights. Sticky card trapping at the PO took place from 5 May 13 Sept. 2007 resulting in 120 trap nights. One modified CDC light-trap was operati onal at the HTU from July 21 August 16, 2006 resulting in 20 trap nights, and from 5 May 13 Sept. 2007 resulting in 120 trap nights. In 2006, at the HTU, CDC trapping (July 21 August 16, 2006) took place prior to, but not during the 2006 sticky card trap ping period (16 Aug. 27 Sept. 2006). Since relative mosquito species composition of th e HTU is known, these previously run CDC

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77 data (July 21 August 16, 2006) were used to represent mosquito population data for the 2006 sticky card trial (16 Aug. 27 Sept. 2006). Trapping at the PO with two CDC traps took place from 5 May 13 Sept. 2007 resulting in 240 trap nights. At the PO, trapping intervals for the two CDC traps and the sticky traps were identical in 2007. Mosquitoes captured from both sticky card traps and CDC light-traps were identified to sex and species using the dichotomous keys of Dars ie and Morris (2003) and Darsie and Ward (2005). Identification data were logged into a MS Excel 2007 spreadsheet. Statistical Analysis Mosquito p reference for diode wavelengths was evaluated using a multi-factorial ANOVA (SAS Institute 2001). For analysis, all data were normalized using the SQRT (n+1) transformation, however actual values are given in text and tables. The model included the fixed effects of location, site and diode treatment, the interaction term, location*diode treatment and the random effect trial. In instances where either the interaction term or the trial effect was signifi cant, the data were analyzed separately by location or trial (year). T ukeys Standardized Test ( =0.05) was used to separate treatment means. Results In 140 trap nights at the HTU and PO 452 m osquitoes, including 29 mosquito species from seven genera, were captured on sticky cards. Aedes vexans Meigen females, Cq. perturbans males, Cq. perturbans females, Cx. erraticus females, Cx. nigripalpus females, Cx. salinarius females, Mansonia titillans Walker females and Oc. infirmatus females were collected in numbers high e nough to analyze (Table 3-1). Mosquitoes excluded from analysis due to low number s or little medical importance included Ae. albopictus Skuse, An. crucians Wiedemann An. quadrimaculatus Say, Oc. canadensis

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78 Theobald, Oc. infirmatus Dyar and Knab, Oc. sollicitans Walker Oc. taeniorhynchus Wiedemann Oc. triseriatus Say, Ps. ciliata, Ps. columbiae, Ps. ferox Humboldt Ur. lowii Theobald and Ur. sapphirina Sacken (Appendix B-1). Significantly more Ae. vexans females, Cx. nigripalpus females and Oc. infirmatus females were captured on sticky cards fitted with blue diodes (F = 4.00; df = 3, 2544; P = 0.0074) than those with red or IR diodes (F = 4.66; df = 3, 2544; P = 0.0030; F = 3.49; df = 3, 2864; P = 0.0150, respectively) (Table 3-1). Numerically, sticky cards affixed with IR diodes caught the fewest female Ae. vexans, Cx. nigripalpus and Oc. infirmatus Only one trial was completed during the 2006 trapping period. Because mosquito population densities differed between the 2006 and 2007 trapping periods, dissimilarities between multiple mosquito species were observed. Among Coquillettidia males and females and Oc. infirmatus females, significantly more mo squitoes were captured during trial one in 2006 than all 2007 trials (F = 3.86; df = 3, 2226; P = 0.0091, F = 6.19; df = 3, 2864; P < 0.0003, F = 3.49; df = 3, 2864; P = 0.0150, respectively). Significantly more Cq. perturbans males were captured on sticky cards containing green diodes than those with the blue or IR diodes (F = 3.86; df= 3, 2226; P = 0.0091). Numerically, the greatest numbers of males were counted on sticky cards affixed with green diodes, with the fewest on sticky car ds with blue diodes. Significantly more Cq. perturbans female mosquitoes were captured on s ticky cards with green diodes than on sticky cards fitted with red or IR diodes (F = 4.66; df = 3, 2864; P = 0.0003). Numerically, sticky cards fitted with IR diodes captured the fewest Cq. perturbans females (Table 3-1).

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79 Preferences between diode treatments were observed for multiple Culex species. Blue diode fitted sticky card s captured significantly more C x. erraticus females than were caught on sticky cards using IR diode treatments (F = 2.96; df = 3; P = 0.0309). There was a significant interaction between diode treatment and location (F = 2.81, df = 3, 1267; P = 0.0381), therefore the p-values for diod e effects were determined using the interaction error term. However, no significan t differences in diode preference were observed (Table 3-1). Data for Ma. titillans were analyzed separately by trial (year). During the 2006 trapping period, significantly more Ma. titillans females were captured at the HTU location on sticky cards fitted with either blue or green LEDs than those with red or IR LEDs (F = 6.22; df = 3; P = 0.0003). Numerically, the total HTU capture of Ma. titillans females was lowest with IR diodes (Table 31). Also, considerably more females were captured at the northwest tra pping site at the HTU than from any other HTU trapping sites (F = 5.41; df = 3, 313; P = 0.0012). Approximately 91,766 female mosquitoes were captured using modified CDC light-traps in 140 trap nights at the HTU loca tion (one CDC trap), a nd 240 trap nights at the PO location (two CDC traps). Mean numbers of mosquitoes capture d per trap night of the seven mosquito species analyzed from s ticky card collections are presented in Table 3-2. Overall, 29 species from 8 genera were captured (Appendices B-1). The only species captured with CDC traps but not on sticky cards was Cx. quinquefasciatus Say. With 2/3 more trap nights and two operational CDC traps at the PO, the HTU location accounted for more than 70% of a ll mosquitoes captured (64,893). Combined, both CDC traps placed at the PO loca tion accounted for only 26,873 mosquitoes

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80 (Appendices B-1, B-2). Mean numbers of mosqu itoes captured per trap night in modified CDC light-traps greatly differed for severa l mosquito species between the 2006 to 2007 trapping periods. During the 2006 trapping period at the HTU location, approximately 1,400 Cq. perturbans females were captured, compared with an average of 73 females during the corresponding 2007 trapping period (S eptember) (Table 3-2). Average capture of Cx. erraticus and Cx. nigripalpus also varied between 2006 and 2007 trapping periods. Culex erraticus capture at the HTU and PO lo cations during 2006 was over 10 times higher than during the corre sponding 2007 trapping period (September). Conversely, Cx. nigripalpus capture at the HTU and PO locations were approximately one mosquito per trap night in 2006, compared with 657 mosquitoes per trap night in the respective 2007 trapping period (Table 3-2). Average monthly temperatures for August (27 C) and September (25 C) remained relatively similar between the 2006 and 2007 tr apping periods, differing by no more than 0.7 C for either monthly average (Figure 310a, b). However, in 2006, an average of 7 cm of rainfall was recorded in August 2006 compared with approximately 17 cm during the same period in 2007. Similarly, less than 8 cm of rainfall were recorded for September in 2006, with approximately 9 cm were recorded in September of 2007. The highest average precipitation for 2007 occurred in July (22.6 cm), and lowest average precipitation occurred in May (1.9 cm) (Figure 3-10a, b). Discussion Using New Jersey traps f itted with colore d lamps of equal intensity, Bargren and Nibley (1956) observed that Ae. vexans and Cx. salinarius demonstrated higher attraction to blue (peak at 447 nm) lamps than to yell ow (peak at 570 nm) or white lamps (peak at 649 nm). However, a wavelength preference for Cx. nigripalpus was not observed.

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81 Burkett et al. (1998) demonstrated mixed results when comparing total captures of Cx. nigripalpus with CDC light-traps fitted with IR (940 50 nm), red (613 50 nm), orange (605 50 nm), yellow (587 50 nm), green (567 50 nm), blue (450 50 nm), white or no-light wavelength treatments. Mosquitoes were captured in high numbers with blue, green and orange wavelength treatments, re sulting in no clear wavelength preference between those spectral ranges. Our findings agree with Bargren and Nibleys (1956) observations where considerably more Ae. vexans mosquitoes were captured on sticky cards fitted with blue diode treatments. However, we observed no significant differences in wavelength preference for Cx. salinarius. In contrast to Burkett et al. (1998) observations, considerably more Cx. nigripalpus were captured on sticky card s fitted with blue diodes than those with red diodes. These re sults suggest a spect ral sensitivity for Cx. nigripalpus females at the higher end of the blue spectru m (> 450 nm), with li ttle sensitivity for wavelengths in the lower end of the red spectrum (< 640 nm). While testing filtered light of known wavele ngths to equate host preference with landing rates of Cq. perturbans Browne and Bennett (1981) determined that shorter, blue-green wavelengths (400-600 nm) attracted significantly more mosquitoes than did longer wavelengths (> 800 nm). Ali et al. (1989) observed a similar light preference for Cq. perturbans while assessing multiple wavelengths with varying intensities, reporting the greatest attraction to bl ue-green wavelengths (430 550 nm). These results were comparable to our observations that significantly more Cq. perturbans were captured on sticky cards affixed with lower spectrum green diodes (502 nm), while fewer were captured on sticky cards fitted with higher sp ectrum IR (860 nm) diodes. Through the use

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82 of more scientifically exact LEDs, our results demonstr ated a stronger preference for Cq. perturbans to green wavelengths (502 nm) than blue (470 nm), suggesting wavelength attraction nearer to the green range of the blue-green spectrum (> 500 nm). Ali et al. (1989) reported higher capture rates for Cx. erraticus when using blue colored bulbs (430 490 nm) co mpared with red colored bulb s (620 720 nm) of similar intensity. We captured significantly more Cx. erraticus females on sticky cards affixed with blue diodes than with sticky cards fitted with IR diodes. However, we observed no significant preferences between blue, green or red diodes. Th erefore, wavelength preferences for Cx. erraticus range in the upper blue band of the spectrum (< 470 nm), with little preference for wavelengths higher in the vi sual spectrum (> 620 nm). Significantly more Ma. titillans females were captured at the HTU location on sticky cards affixed with blue or green diodes than those with red or IR diodes. Burkett et al. (1998) observed similar preferences with Ma. dyari Belkin, capturing mosquitoes using CDC light-traps fitted with either ye llow or green LEDs. These wavelengths fall within the 500-600 nm range, which is consistent with most known mosquito wavelength spectral sensitivities (Allan 1994). Approximately 23% (105) of all mosquito es captured on sticky cards (451) were males. Coquillettidia perturbans represented the majority of males captured with 52 mosquitoes, but no male Ae. albopictus, Ps. columbiae or Ur. lowii were trapped. The number of male mosquito es captured for other species ranged from one to 13. Effectively sampling male mosquito populati ons can be an important tool in the surveillance of transovarially transmitted ar boviruses such as La Crosse virus. Male mosquito population densities in combinati on with female population densities can be

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83 important indicators of general population fec undity and reproductive status of a target species. In population modeling, this combination of factors makes age-grading a possible tool in the comprehension of vector potential of a disease transmitting population (Garrett-Jones 1964). Among mosquito species captured on sticky cards, the five most common species were Cq. perturbans (132), Ma. titillans (78), Ur. sapphirina (56), Ur. lowii (37) and Cx. nigripalpus (36). Least common mos quitoes captured included Cx. territans (7), An. crucians (3), Ps. columbiae (3), An. quadrimaculatus (2), and Ae. albopictus (1), respectively. This sticky card trapping sy stem measured mosquito preference to wavelengths of light in the absence of alternative host stimuli. T hose species captured on sticky cards in highest numbers are species commonly observed using trap designs that combine light attraction with alternative host stimluli (B rowne and Bennett 1981, Burkett et al. 1998, Hoel 2005). Our results demonstrate that light detection may be more significant in host location for those mosquito species than for species captured in fewer numbers. Mosquito species such as An. quadrimaculatus and Ae. albopictus, not captured in high numbers on sticky cards, are species kno wn to utilize light sources far less in host location. Anopheles quadrimaculatus are known to prefer da rk unlit surfaces, and subsequently, are commonly captured in high numbers using dark co lored resting boxes (Goodwin 1942, Crans 1989, Irby and Apperson 1992). Aedes albopictus a diurnal feeding mosquito, commonly ut ilizes movement and/or ba ckground contrasts as primary host cues, rather than ligh t (Sippel and Brown 1953, Gillett 1972, Allan et al. 1987). Overall, capture of mosquitoes on sticky cards was greatest with green LEDs (198 mosquitoes), followed by the blue (159 mos quitoes), red (60 mosquitoes) and IR (35

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84 mosquitoes) LEDs. Mosquito wavelength preference has been shown to be in the bluegreen range (400 600 nm), with diminishi ng attraction as wavelengths increase in length (> 600 nm) (Ali et al. 1989, Burket et al. 1998). Similarly, our results demonstrated that mosquitoes exhibited a pr eference for the blue (470 nm) and the green (502 nm) LEDs, with strongest preferences obs erved with the green diodes. While these findings do not exclude the possible effectiv eness of wavelengths in the higher blue spectral range (> 470 nm), wavelengths in th e lower green spectrum (502 nm) result in higher mosquito attraction. Modified CDC traps captured many more mo squitoes than did sticky cards. These results were anticipated because of the suppl ement of an artificial host attractant, CO2 in the CDC traps. Both trapping systems captured several similar mosquito species, including Cq. perturbans and Cx. nigripalpus. Comparable to results discussed previously, both mosquito species are commonl y captured when using trap designs that combine light with alternative host stimulation (Browne and Bennett 1981, Burkett et al. 1998, Hoel 2005). Sticky trap results further i llustrate the importanc e of light alone in host location for these species. Mosquito spec ies primarily captured in modified CDC traps, such as An. crucians and An. quadrimaculatus are not generally observed frequenting light-traps (Irby a nd Apperson 1992). However, it is important to note that some mosquito species known to frequent light traps were only captured in high numbers using the baited CDC trap. Ali et al. (1989) captured high numbers of Ae. vexans in light traps, independent of other host stimulants. These results indicate the incorporation of light into baited traps may significantly increase capture ra tes for mosquito species.

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85 Mean mosquito capture per trap ni ght from modified CDC traps for Cx. nigripalpus differed greatly between the 2006 and 2007 trap ping periods. Mosquito capture at the HTU and PO locations were approximately one per trap night in 2006, compared to 657 Cx. nigripalpus per trap night in the respective 2007 trapping period. This dramatic population increase may have been due to the mosquitoes seasonal and spatial distribution, as discussed in Day and Curtis (1994). Culex nigripalpus display an annual population increase that coincides with Fl oridas summer and autumn rainy seasons, beginning in June or July. Under normal rainy season conditions, Cx. nigripalpus can extend their flight range be yond their breeding and resting areas. While experiencing drought, however, populations concentrate as ground and vegetation in open areas dries. Once drought is broken by one or more heavy ra ins (>5 cm), adult mosquitoes thrive. The more frequent and rhythmic the rains, the more populations flourish. Increased population densities such as these become a public nuisance, and pr ovide great cause for public health concern, given that Cx. nigripalpus is an effective vector of St. Louis encephalitis virus and West Nile virus (Day and Curtis 1994). Weather conditions necessary for Cx. nigripalpus to experience such dramatic population increases occurred during the 2007 tr apping period. A severe drought early in the year caused ground water to dry, elimina ting most mosquito habitats. Dry conditions were followed by several 5 9 cm rains in June and July, occurring during optimal periods for Cx. nigripalpus population development. These periodic rains, followed by ample drying periods, provided the id eal environmental conditions for Cx. nigripalpus to suddenly exceed average population densities.

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86 Much of the early work about mosquito wavelength attraction involved the use of imperfect light sources, such as filtered light or painted bu lbs, which were only able to generate ranges of wavelengths instead of exact wavelengths. While earlier research provided valuable knowledge, the lack of specifi c wavelength data left a serious void in a science where mosquito control/research operations are based largely on types and numbers of mosquitoes captured in light-b aited traps (Burkett and Butler 2005). The results of this study suggest that, in the abse nce of alternative host-stimuli, wavelengths in the lower green (502 nm) spectral range would be optimal for targeting a broad range of mosquito species. Additionally, the use of LEDs as opposed to wavelength filters or colored bulbs provides a more precise and efficient wavelength delivery system when attempting to attract and capture spectrally sensitive insects. The utilization of LEDs in combination with sticky cards has demonstrated the superior effectiveness of LEDs in attracting a variety of mosquito species, as well as capturing males and females. Given their accu racy in exact wavelength achievement, small size and minimal power usage, light em itting diodes can be used as light sources for various trap designs where access and equi pment to targeted sites are minimal. The ability of LEDs to operate for extended peri ods of time with minimal power consumption allows these light sources to be added to virt ually any trap design, with little modification or additional equipment. Their demonstrated effectiveness for attracting mosquitoes without the aid of supplemental host attractants further eliminat es the need and costs of heavy tanks (CO2) or noxious chemicals (lactic acid, octenol). Durability of the minimalLED based equipment required also helps to reduce otherwise necessary and timeconsuming field maintenance. By offering extended operating time with minimal power

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87 consumption, field durability and the ability to eliminate the need for burdensome equipment, LEDs are removing restrictions previously set on trap designs where equipment or field conditions were major limiting factors. The results of this research warrant seri ous considerations in to other aspects of mosquito wavelength attraction. These findings demonstrate that the use of only light in a trapping system without additional host based attractants (CO2, octenol and lactic acid) can effectively capture mosquitoes. While differing exact wavelengths influence mosquito preference, manipulation of wave length frequency or intensity may also enhance capture rates for specific mosquito spec ies. Using poor light sources, past studies demonstrated that these factors can significan tly impact mosquito preferences to light. With the development of LEDs capable of ach ieving precise wavelengt hs, future research in this field will be able to further refine the knowledge of factors affecting mosquito behavior in response to light.

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88 Table 3-1. Mean ( SE) numbers of mosquitoes/trap/night attracted to light emitting diodes producing four different wavelength s of light during 24 h trapping inte rvals at the University of Florida Horse Teaching Unit and Prairi e Oaks subdivision in Gainesville, FL. Diode Wavelength Species TN Blue Green Red IR Ae. vexans 640 0.019 (.006)a 0.013 (.004)ab 0.002 (.002)b <0.001 (<0.001)b Cq. perturbans 560 0.014 (.006)b 0.043 (.010)a 0.011 (.006)ab 0.016 (.005)b Cq. perturbans 720 0.031 (.007)ab 0.049 (.009) a 0.024 (.007)b 0.008 (.003)b Cx. erraticus 320 0.034 (.012)a 0.016 (.007)ab 0.013 (.006)ab 0.003 (.003)b Cx. nigripalpus 640 0.019 (.007)a 0.016 (.005)ab 0.002 (.002)b <0.001 (<0.001)b Cx. salinarius 320 0.016 (.007) 0.010 (.005) < 0.001 (<0.001) <0.001 (<0.001) Ma. titillans 2006 80 0.313 (.068)a 0.363 (.110)a 0.100 (.038)b 0.050 (.025)b Ma. titillans 2007 160 0.006 (.006) 0.006 (.006) 0.006 (.006) <0.001 (<0.001) Oc. infirmatus 720 0.014 (.004)a 0.004 (.002)ab 0.003 (.002)b 0.001 (.001)b Note: Blue diode = 470 nm, Green diode = 502 nm, IR = 860 nm and Red diode = 660 nm. Ae. = Aedes; Cq. = Coquillettidia ; Cx. = Culex; Ma. = Mansonia ; Oc = Ochlerotatus TN = number of trap nights were to tal mosquito species capture = 1 per 20 day trapping period. Means within rows followed by the sa me letter were not signifi cantly different (P< 0.05, Tukeys standardized test [SAS Institute 2001]). Ae. vexans (F = 4.00; df = 3, 2544; P = 0.0074); Cq. perturbans (F = 3.86; df = 3, 2226; P = 0.0091), Cq. perturbans (F = 6.19; df = 3, 2864; P = 0.0003); Cx. erraticus (F = 2.80; df = 3, 1261; P = 0.0386); Cx. nigripalpus (F = 4.66; df = 3, 2544; P = 0.0030); Ma. titillans 2006 (F = 6.18; df = 3, 313; P = 0.0004); Oc. infirmatus (F = 3.49; df = 3, 2864; P = 0.0150).

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89 Table 3-2. Number of mosquitoes /trap night for six mosquito species captured a the University of Florida Horse Teaching Unit a nd Prairie Oaks subdivision. Total Mosquitoes/Trap/Trap Night Date Location TN Ae. vexans Cq. perturbans Cx. erraticus Cx. nigripalpus CDC SC CDC SC CDC SC CDC SC 7/21/06 8/16/06 HTU 16 1.56 1,391.88 154.38 1.13 8/16/06 9/27/06 PO <0.01 2.15 0.70 <0.01 5/5/07 6/5/07 HTU 15 6.47 0.10 47.33 0.60 5.47 0.15 <0.01 0.15 PO 36 6.11 <0.01 51.69 0.20 3.50 0.00 0.03 <0.01 6/6/07 6/25/07 HTU 16 5.44 0.10 22.63 0.45 5.50 <0.01 1.56 0.10 PO 34 9.68 0.05 21.88 0.25 1.26 <0.01 1.85 <0.01 6/26/07 7/15/07 HTU 19 8.79 0.20 14.21 0.05 1.63 0.10 3.26 <0.01 PO 40 13.50 0.05 18.35 0.20 0.78 <0.01 9.33 0.05 7/16/07 8/4/07 HTU 19 2.35 0.10 9.24 0.05 0.41 <0.01 1.00 <0.01 PO 37 7.64 0.05 8.82 <0.01 0.42 <0.01 3.00 <0.01 8/5/07 8/24/07 HTU 19 27.05 0.40 12.32 <0.01 4.21 0.10 113.32 <0.01 PO 40 25.75 <0.01 5.88 0.05 0.98 <0.01 32.75 0.10 8/25/07 9/13/07 HTU 19 42.11 <0.01 18.11 <0.01 5.42 <0.01 667.58 0.10 PO 38 6.63 0.05 7.95 <0.01 1.03 <0.01 140.82 0.15

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90 Table 3-2. Continued. Total Mosquitoes/Trap/Trap Night Date Location TN Ma. titillans Oc. infirmatus CDC SC CDC SC 7/21/06 8/16/06 HTU 16 1.88 531.75 8/16/06 9/27/06 PO 3.30 <0.01 5/5/07 6/5/07 HTU 15 2.87 <0.01 <0.01 0.05 PO 36 0.31 <0.01 5.83 <0.01 6/6/07 6/25/07 HTU 16 1.31 <0.01 2.69 0.05 PO 34 <0.01 <0.01 11.12 <0.01 6/26/07 7/15/07 HTU 19 3.00 <0.01 5.32 0.10 PO 40 0.03 <0.01 21.33 0.05 7/16/07 8/4/07 HTU 19 6.88 <0.01 1.24 0.05 PO 37 0.15 <0.01 10.91 <0.01 8/5/07 8/24/07 HTU 19 15.63 0.50 16.79 0.25 PO 40 0.15 0.10 17.35 0.20 8/25/07 9/13/07 HTU 19 22.11 <0.01 33.84 <0.01 PO 38 0.13 <0.01 5.92 0.05 Note: An. = Anopheles ; Cq = Coquillettidia ; Cx. = Culex; Ma. = Mansonia CDC = Modified CDC light -trap; RB = resting box. HTU = One modified CDC trap + CO2 (250 ml/min); PO = Two modified CDC traps + CO2 (250 ml/min). TN = number of trap nights CDC traps were in operation. When TN < 20 (HTU) or TN < 40 (PO), traps had malfunctioned. Trap nights for all RB trapping periods = 20.Total mosquitoes/tra p/trap night = total mosquitoes captured/ # of trap nights.

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91 Figure 3-1. Four sided, diode-equipped pine boxes, each side measuring 400 cm2. Boxes were constructed and designed to exteriorly support one 13 x 13 cm sticky card and one diode treatment per side, yieldi ng a total of four sticky card s and four light treatments per diode box. Figure 3-2. Sticky cards were constructed from black 28 pt. SBS card stock with calendared coating (EPA # 057296-WI-001), and coated with 32 UVR soft glue containing UV inhibitors. Individual sticky car ds, originally supplied as 41 x 23 cm boards, were cut to yield two 13 x 13 cm sticky cards.

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92 Figure 3-3. CDC light trap modified by the rem oval of its incandescent bulb. Modified trap used a 6 V DC motor and 4-blade fan to draw flying insects through an 8.5 cm diameter clear plastic cylindrical body. A 36 cm diameter beveled edge aluminum lid was set approximately 3 cm above the cy linder body creating a downdraft air current. All traps were set 120 cm above ground usi ng a Shepherds hook, and collection nets were attached to the bottom of the tr ap body. Carbon dioxide was provided from a 9 kg compressed gas cylinder. A flow rate of 250 mL/min was achieved by using a 15psi single-stage regu lator equipped with micro-regul ators and an inline filter.

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93 Figure 34. Aerial view of Horse Teaching Unit location. The unit is lo cated east of I-75 and approximately 1.6 km northwest of Paines Prairie State Preserve, Alachua Co., FL. Figure 3-5. Aerial view of Prairie Oaks Subdivision whic h was located approximately 4.8 km southwest of the Horse Teaching Unit, ad jacent to the Paines Prairie Preserve, Alachua Co., FL. HTU Paines Prairie Preserve Prairie View Subdivision Paines Prairie Preserve

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94 Figure 3-6. Representative of te st sites chosen at the Prairie Oaks subdivision. All sites chosen were consistent in surrounding vegetation, sunlight exposure and moisture conditions. Figure 3-7. Test sites located within Prairie Oaks subdivisio n. Each solid white rectangle represents a test site wher e one box equipped with one of four diode treatments was placed. White dashed rectangles identify the location of modified CDC traps.

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95 Figure 3-8. Test sites located within the Univer sity of Florida Horse Teaching Unit. Each white square represents a test site where one di ode box was equipped with one of four diode treatments. White arrow represents loca tion placement of mo dified CDC trap. Southeast Site Southwest Site Northeast Site Northwest Site

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96 A B C D Figure 3-9. University of Florida Horse Teachin g Unit location. A.) Southeast side test site habitat. B.) Northeast side test site habitat. C.) Northwest side test site habitat. D.) Southwest side test site habitat.

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97 A B Figure 3-10. Mean monthly temperatures (C) and precipitation (cm) for the University of Florida Horse Teaching Unit (HTU) locati on near Gainesville, FL using data retrieved from the National Oceanic and Atmospheric Administration (NOAA) database. A) Monthly temperature, May September 2006 and 2007. B) Monthly precipitation from Jan September 2006 and 2007.

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98 CHAPTER 4 RESPONSES OF PREVITELL OGENIC AND VITELLOGENIC ANOPHELES QUADRIMACULA TUS TO SELECTED WAVELENGT HS PRODUCED BY LIGHT EMITTING DIODE Introduction Physiological stage, in regards to fem ale hematophagous Culicidae, is the course of development through which the ovaries mature. In anautogenous female mosquitoes, development can be classified into three main phases: previtellogeni c, vitellogenic, and postvitellogenic. Each phase has important impacts on behavior including feeding, host seeking and oviposition (Klowden 1997). From eclosion to just preceding the first blood meal, female mosquitoes are considered previtellogenic. During this pha se the fat bodies become capable of intense synthesis of yolk protein precursors (Lehane 2005). During the early previtellogenic phase, egg follicles remain in a quiescent or resting stage until a blood meal is taken (Clements 1992). Several instinctive behaviors of the female such as a reduction in female receptivity to males are affected because of increased levels of Juvenile Hormone III (JH III) (Klowden 1997). Meola and Petralia (1980) also showed that altering levels of JH III resulted in a significant impact on the biting behavior of Culex mosquitoes. The second, and least understood, phase is the vitellogenic phase. Considerable information on the hormonal sequence that occurs during this phase remains unclear. Clements (1956) and Gillett (1956) were able to definitively establish that there was a hormonal significance throughout oogenesis. Ba sed on this principle, Hage dorn et al. (1979) made the important observation that ovaries of adult fe male mosquitoes produced ecdysteroids. This eventually led to the isolation of several ecdysteroidogenic hormones from the head of Aedes

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99 aegypti Linnaeus, most notably the ovarian ecdys teroidogenic hormone I (OEH) (Borovsky and Thomas 1985, Whisenton et al. 1987). It is OEH that is believed to be the key factor in the vitellogenic phase of oogenesis (Klowden 1997). The vitellogenic phase is initiated by the inge stion of a blood meal. This results in the release of OEH from the brain, stimulating the ovaries to produce ecdys teroids (Brown et al. 1995). These ecdysteroids immediately react with th e fat bodies, resulting in the activation of vitellogenin genes. Oocytes take up the v itellogenin through the hemolymph, completing oogenesis. All eggs develop thr ough this process synchronously, us ually completing the phase in 2-5 days at favorable temperat ures (Foster and Walker 2002). Once oogenesis is complete, the female mosqu ito enters the postvitellogenic phase. During this phase, hormonal reactions halt vitellogeni n production, and inhibit the development of secondary egg follicles until after oviposition has taken place. These hormones also impact the females actions leading to behavioral changes that increase the chances for survival of her progeny. Once oviposition has occurred, the mosquito reenters the prev itellogenic stage. Following a subsequent blood meal, a new cycle of oogenetic events begins and the cycle repeats (Klowden 1997). Arthropod-borne pathogens, such as those causing malaria, dengue and yellow fever, require an incubation period within the insect vector before they can be successfully transmitted (Lehane 1985). Additionally, only specific physiological stages of a female mosquito have the capacity for disease transmission to humans. This combination of factors makes age-grading a valuable tool in the compre hension of vector potential. In epidemiological investigations, age grading allows scientists to estimate the probability of a single mosquito surviving for one day. This ke y element is used in equations to estimate the

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100 vector potential of a diseas e transmitting population (Garre tt-Jones 1964). Additionally, age grading also plays a pivotal role when monitori ng vector control operations. When examining diseases such as malaria, a reduction in the lif espan of a female mosquito has a much larger impact in transmission rates than a reduction in the overall mosquito popu lation (Wu and Lehane 1999). It is with this knowledge th at modelers are able to predic t future malaria epidemics, or possible high incidence seasons. With both the development of insecticide resistance in multiple anopheline species (Metcalf 1989), and the devastating resu rgence of malaria wo rldwide (Rogoff 1985), Anopheles quadrimaculatus Say stands as a potential he alth threat to Floridas popu lation. It is this intimate relationship with malaria, coupled with their abundance in Florida that makes An. quadrimaculatus an excellent target species in this study. Vision plays a significant role in all major ac tivities of an adult mosquitos life including mating, dispersal, appetitive fli ght, as well as nutrie nt location (sugars), host location, resting, and oviposition (Allan et al. 1987). Nielson and Haeger (1960) and Gatehouse (1972) demonstrated that mating swarms were located by female mosquitoes using visual markers such as corners of buildings or human observers. The structure of the swar ms appeared to be dependent upon the characteristics of the markers. Artificial, reflected and filter ed lights have been incorporat ed in the design of existing efficient traps to increase their efficacy for mos quito research and surveillance with great success (Barr et al. 1963, Service 1976, Ali et al. 1989, Burk ett and Butler 2005). Ali et al. (1989) were able to demonstrate that both Culex and Psorophora spp. showed a higher preference to light source as opposed to light intensity when tra pping in the field. Similarly Burkett and Butler (2005) showed that not only light source, but specific light wavelengths played an important role

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101 in host attraction. In laboratory trials, Aedes albopictus Skuse, An. quadrimaculatus and Culex nigripalpus Theobald all showed preferences for specific wavelengths of light. Age may also significantly impact mosquito preferences to light. Nielsen and Nielsen (1953) observed that female Ae. taeniorhynchus Wiedemann demonstrated light preferences approximately seven days post emergence. Following this period, mosquitoes displayed a cyclic light preference about every fifth day. Similarl y, age-influenced photophili c behavior has been observed among field collections of Ae. taeniorhynchus (Provost 1952). However, field collected female Ae. taeniorhynchus were noted responding to light at five days post emergence. Male mosquitoes only exhibited prefer ences to light during the first three days post emergence. Though much research has been done in regard s to a female mosquitos attraction to different wavelengths, past re search has mostly focused on one physiological stage of mosquitoes, the previtellogenic stage. When worki ng with either colonized or wild adult females in laboratory conditions, few researchers have worked with anything other than previtellogenic females. Similarly, in the field, researchers have based most findings on the assumption that female mosquitoes attracted to modified li ght traps were previte llogenic, host seeking mosquitoes. Past research has been scant in regards to finding a ny direct link between physiological stage and feeding pa tterns. However, Mogi et al. (1995) demonstrated a possible link between ovarian development of An. subpictus Grassi and feeding ha bits. This assumption was based upon a significantly highe r catch rate of parous females in light traps (86.6%) than that from cattle-baited collections (69.6%). These results were unique given that baited trapping systems such as light traps commonly capture host seeking females (Browne and Bennett 1981, Ali et al. 1989, Burkett et al. 1998). A direct reference to th e possible applic ation of these findings to malaria epidemiology was also made It is this assumption upon which my hypothesis

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102 is based. The objective of my research was to identify preferences of two physiological stages (previtellogenic and vitellogenic) of An. quadrimaculatus to four wavelengths (Infrared (IR), Blue, Green and Red) in a visu alometer using an open-port desi gn. To further assess preferences for particular wavelengths, diode s showing the highest and lowest responses were evaluated in a visualometer using a paired-T port design. Materials and Methods Visualometer The visualom eter is a modified version of the olfactometer (Burkett and Butler 2005), originally designed and built by Dr. Jerry F. Butler at the University of Florida, to evaluate mosquito responses to different olfactory host stim uli. The visualometer, as previously described by Hoel (2005), was modified to measure res ponses to visual, as opposed to olfactory (Coon 2006), stimuli. The pie shaped visualometer has 10 individually numbered sensor ports, modified from existing olfactometer feeding stations, which can be portioned off or left in an open design. All ports were equipped with an electrical amplification box, artifi cial host sensor, airflow intake and outflow ports, and CO2 circulation system (Figure 4-1a). Attractiveness was measured as the amount of time a mosqu ito completes an electric circuit positioned over a stimulus (specified wavelength). The ci rcuit is complete when an attracted mosquito makes contact with the sensor in an attempt to reach the artificial host stimuli. Contact activity on artificial se nsors was measured, recorded a nd logged over an 8 h period using a computer. Attractiveness was qua ntified in contact seconds to make standardized comparisons and measurements possible. The visualometer was enclosed in a Farada y cage room (Lindgren Enclosures, Model no. 18-3/5-1), maintained between 28 and 32 C. This room was designed to protect against outside electrical interference and extran eous sources of light. All visual ometer surfaces were kept free

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103 of direct human exposure, and those surfaces exposed to mosquitoes were disposable or cleaned with soapy water before trials. (Figure 4-1a, b). With the exception of LEDs, trials were run in the dark. Light Emitting Diodes Light em itting diodes (LED) of four wavelengths were evaluated for their attractiveness to two physiological stages (previte llogenic and vitellogenic) of An. quadrimaculatus in a visualometer. All LEDs were obtained from Digi -Key Corporation (Thief River Falls, MN). Diodes, part number and millicandela (mcd) ra ting, as described in Hoel (2005), were blue (P466-ND, 470 nm, 650 mcd), green (67-1755ND, 502 nm, 1,500 mcd), red (67-1611-ND, 660 nm, 1,800 mcd) and infrared (LN77L-ND, 860 nm). B ecause infrared radiati on is not visible to humans, infrared diodes are not mcd-rated. A s timulus (LED) not connected to a power source was used as a control. Round lens LEDs were 8.6 mm long by 5.0 mm in diameter. Viewing angles were 30o except for IR (20o). A 180-ohm resistor was soldered to a ll LEDs, restricting current flow to prevent mechanical failure. Po wer was provided by a 6 v, 12 ampere-hour (A-h), rechargeable gel cell battery changed every 24 48 h (Batte ry Wholesale Distributors, Georgetown, TX). Placement of all LEDs was comp letely randomized before each trial, in an attempt to eliminate interactions between wavelengths. Mosquitoes Anopheles quadrimaculatus were obtained from the USDA-ARS-CMAVE Gainesville, FL rearing facility. Rearing room conditions we re maintained between 27 and 32 C and approximately 50 60% RH. Adults were held under a 14:10 (L:D) photoperiod. Between 1,000 and 1,500 An. quadrimaculatus pupae were obtained weekly at approximately 12 h pre-eclosion. Pupae were take n to the University of Florida Veterinary Entomology Laboratory, and held in an in cubator at 26 C and 75% humidity under a 14:10

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104 (L:D) photoperiod. Upon eclosion, adult mosquito es were fed a 10% sucrose solution for 72 h (Figure 4-2). At 72 h post-eclosion, 150 previtel logenic females were mechanically aspirated from the holding cage and released into the vi sualometer. Newly releas ed mosquitoes were allowed ten minutes to adjust to light and temper ature conditions within th e visualometer before sensors were activated, and a trial was initiated. Remaining mosquitoes were held for an additional 48 h in the incubator and allowed to feed on a 10% sugar solution before being al lowed to blood-feed. Bloodfeeding took place 120 h post-eclosion using a suspended sausage casing that held warmed defribrinated bovine blood (Figure 4-3). Adult mosquitoes were allowed to bloodfeed for 3 h before the sausage casing was removed and discarded. At 144 h post-eclosion, 150 vitellogenic mosquitoes were mechanically aspirated from the holding cage, and used in a new visualometer trial as previously described. Open-Port Visualometer Trials The visualom eter was first used in an open de sign incorporating all treatments. This design allowed mosquitoes to freely move between the four LED treatments and unlit treatment that were affixed to five of the 10 sensor ports. On e LED or an unlit LED was placed in a vertical arrangement at each odd numbered sensor port. Even numbered ports were equipped with sensors, but were unlit. Treatment placement was completely randomized before each trial (Figure 4-1a,b). A minimum of 15 previtellogenic and 15 vite llogenic trials were conducted. Successful trials were trials where the av erage contact seconds were within % of the group mean (Hoel 2005). Trials with contact second averages outside this range e ither suffered from equipment malfunction (faulty sensor, low hum idity) or poor mosquito quality. Based on data collected from visualometer tr ials, two pairs of diode s were selected for subsequent study. The one pair was selected base d upon significant differences in recorded levels

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105 of mosquito activity between physiological stages. The second pair was selected based upon highest and lowest recorded levels of mosqu ito activity between LED treatments, within both physiological stages. Paired-T Port Visualometer Trials Using plastic dividers, the visu alometer was divided into five equal arenas, each containing two sensor ports (Coon 2006). This arrangem ent allo wed for completion of five replications per trial. For each trial, the arena contained one diode treatment (either blue/red or blue/green) positioned in a vertical arrangement. Each aren a contained airflow intake and outflow ports, mosquito insertion hole and two di ode insertion points with paired sensors. Thirty mosquitoes were released in each arena, totaling 150 mosquitoes used per trial (Figure 4-1c). All other visualometer setup and sterilizat ion procedures were completed as previously described. A minimum of three previtell ogenic and three vitellogenic trials were conducted. For blue/green diode treatment tria ls, seven previtellogenic and ei ght vitellogenic trials were completed to achieve ten replications. For the blue /red diode treatment trials, four previtellogenic and six vitellogenic trials were requ ired to achieve ten replications. Methodology Before each trial, all visu alometer surfaces not disposable were cleaned with soapy water kept free of direct human exposure. For open-po rt trials, the four LED treatments and unlit LED were randomly affixed to five odd numbered sensor ports. Then, 150 female mosquitoes (72 h post-eclosion for previtellogenic mosquitoes, 144 h post eclosion for vitellogenic mosquitoes) were mechanically aspirated from the holding cag e and released into the visualometer. Finally, power to LEDs was connected, the faraday cage was sealed, and an eight-hour trial was initiated. For paired-T port trials, plastic dividers were used to divide the visualometer into five equal arenas, each containing two sensor ports. Al l other visualometer setup and sterilization

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106 procedures were completed as described above. For previtellogenic trials, th irty mosquitoes (72 h post-eclosion) were released in each arena, totaling 150 mosquitoes used per trial (Figure 4-1c). For vitellogenic trials, thirty mosquitoes (144 h post-eclosion) were rele ased in each arena, totaling 150 mosquitoes used per trial, and used as previously described. Statistical Analysis As previously described in Coon (2006), the Medusa 2.1.2 software designed by N. Hostettler in Gainesville, FL, was used to analyze the cum ulative contact seconds of An. quadrimaculatus at each sensor port per eight hour trial. All data were normalized using the SQRT (n+1) transformation but actu al values are shown in text a nd tables. Eight previtellogenic and eight vitellogenic open-port tria ls were selected from a pool of 31 trials. Selected trials were found to be within % of the group mean (Hoel 2005). Data coll ected from selected open-port trials were evaluated using a multi-factorial ANOVA (SAS Institute 2001). The model included the fixed effect of diode treatment (wavel ength). For paired-T trials, a one tailed t -test was used to evaluate significant differences between means. Results Open-Port Visualometer Am ongst previtellogenic An. quadrimaculatus released in the open-port visualometer, there were no significant differences in mosqu ito contact seconds betw een mosquitoes exposed to the four LED wavelengths. However, mosquito contact seconds were recorded most frequently on green LEDs (0.2514 s), followe d by red (0.2189 s), cont rol (0.1855 s), IR (0.1622 s) and blue LEDs (0.0996 s) (Table 4-1). Results for i ndividual trials, as well as 50% ranges can be found in Appendix C-1. Similarly, among vitellogenic An. quadrimaculatus released in the open-port visualometer there were no significant differences in mos quito contact seconds when mosquitoes were

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107 exposed to the four LED wavelengths. However, mosquito contact second s (cs) were recorded most frequently on blue LEDs (0.1612 cs), follo wed by red (0.1496 cs), control (0.1397 cs), IR (0.1255 cs) and green LEDs (0.0804 cs) (Table 4-1). Results for individual trials, as well as 50% ranges can be found in Appendix C-2. In comparisons within wavelengths, significant differences in mosquito contact seconds were observed between previt ellogenic and vitellogenic An. quadrimaculatus examined in the open-port visualometer. Significan tly higher activity was recorded with previtellogenic mosquitoes (0.2189 cs) than with vitellogenic mosquitoes (0.1486 cs) with red LEDs (F = 98.08; df = 1, 2; P = 0.0100). Inversely, vitellogenic mosquitoes were in contact with blue LEDs (0.1428 cs) for a longer period of time than were previtellogenic mosquitoes (0.0656 cs) (F = 111.24; df = 1, 2; P = 0.0089) (Table 4-1). Because of increased contact seconds for previtellogenic and vitellogenic An. quadrimaculatus, certain LED wavelengths were selected for additional analysis using a pairedT port visualometer system. The blue/red diode treatments were selected based upon significant differences in recorded levels of mosquito activ ity between physiological stages. The blue/green diode pair was selected based upon highest and lo west recorded levels of mosquito activity between LED treatments, within each physiological stage. Paired-T Port Visualometer No significant differences in m osqu ito contact seconds were observed among previtellogenic or vitellogenic An. quadrimaculatus exposed to blue and red LEDs. For previtellogenic An. quadrimaculatus, sensors recorded mosquito contact over blue LEDs (1.0334 cs) more frequently than over red LEDs (1.0207 cs). However, vitellogenic An. quadrimaculatus contacted sensors over red LEDs (1.0377 cs) more frequently than blue LEDs (1.0351 cs) (Table

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108 4-2). Results for all previtellogenic and vitell ogenic individual replic ations can be found in Appendix C-3 and C-4, respectively. In paired studies comparing blue and gr een LEDs, previtellogenic and vitellogenic An. quadrimaculatus showed no significant differences in c ontact seconds. Mosquito contact seconds for previtellogenic An. quadrimaculatus were nearly equal over blue LEDs (1.0153 cs) and green LEDs (1.0109 cs). Similar responses were observed with vitellogenic An. quadrimaculatus where green LEDs (1.0176 cs) and blue LEDs ( 1.0168 cs) performed sim ilarly (Table 4-2). Results for all previtellogenic and vitellogenic individual replications can be found in Appendix C-5 and C-6, respectively. Discussion In com parisons between previtellogenic and vitellogenic An. quadrimaculatus released in the open port visualometer, previtellogenic mos quitoes were in contact with red diodes significantly longer than were vitellogenic mosquitoes. Howeve r, these results are confounded when compared with other LEDs. Among previtellogenic An. quadrimaculatus released in the open-port visualometer, no significa nt differences in mosquito contact seconds were observed between the four LED wavelengths. Likewise, no significant differenc es were observed among previtellogenic An. quadrimaculatus exposed to blue and red or bl ue and green LEDs in a pairedT port visualometer. Under ideal conditions, previtellogenic mos quitoes at four days post eclosion have physiologically initiated the host seeking stage (Clements 1992). During this stage, mosquitoes are known to utilize visual cues such as color (w avelength) to locate hosts in search of a blood meal (Service 1993). We expected to see attractio n to LEDs during this stage. That mosquitoes did not exhibit higher preference for LEDs than for the unlit control is surprising and suggests that light alone is a poor attractor or that our experimental design needs to be refined.

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109 Using a visualometer incorporating an artificial ho st (blood agar/CO2) Burkett and Butler (2005) exposed previtellogenic An. quadrimaculatus to a white light control, a black control and filtered light ranging from 350 nm 750 nm (50 nm increments). Signif icantly longer feeding times were recorded over black and white contro ls than all other wavelengths. Additionally, 350 nm wavelengths recorded significantly less feed ing time than all other individual wavelengths. These observations sugg est previtellogenic An. quadrimaculatus prefer no light to all other wavelengths during host location, or when feed ing. Our findings differed from Burkett and Butler (2005) in that mean cont act seconds were highest with green diodes than all other treatments. However, these differences may have occurred because an artificial host was not used during our visualometer trials. The stimulation of a blood-m eal could serve as the precursor to additional functions in the phys iological responses of previtell ogenic mosquitoes to different wavelengths. These unstudied variables warrant a dditional research in the physiological effects of a blood meal on previtellogenic mosquitoes. Vitellogenic An. quadrimaculatus were in contact with blue LEDs significantly longer than were previtellogenic mosquitoes in the open-por t visualometer (Table 4-1). However, among vitellogenic mosquitoes released in either an open-port or paired-T port visualometer, we observed no significant differences in wavelength preference among the LEDs. Based on past litera ture, vitellogenic An. quadrimaculatus were expected to be repelled by light (Bradley 1943, Burkett and Butler 2005). Although, no significant differences in wavelength preference among treatment s was observed in visualometer trials, notable differences were observed in mean mosquito contact sec onds among treatments. In all trials, mosquito contact seconds for vitellogenic An. quadrimaculatus were never higher for the unlit control than lit LEDs. These findings suggest a possible photot actic association with parous mosquitoes.

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110 Mogi et al. (1995) observed similar results when evaluating the feeding habits of An. subpictus Grassi using light traps and cattle-bait traps. Significantly more parous An. subpictus were captured in light traps (86.6%) than cattle-baited samples (69.6%). When trapping parous mosquitoes, gravid tr aps utilizing darker, non-reflective water pans captured significantly more gravid mosquitoes than traps using lighter colored pans (Belton 1967, Laing 1964, Allan and Kline 2 004, Kline et al. 2006). Also, Belton (1967) demonstrated that mosquito preference for oviposition sites is significantly decrease d when oviposition sites are illuminated. However, our observati ons demonstrated that vitellogenic An. quadrimaculatus may prefer light in certain wavelengths instead of no light when given a choice. These results also indicate that fitting LEDs of selected wavelengths to grav id traps may increase their trap efficacy. Lights used for Beltons (1967) study were cool white lamps with a wide viewable angle of approximately 180. These lights unc ontrollably illuminated a large area, likely repelling photophobic mosquitoes in search of darker oviposition sites. Light emitting diodes used in our study produce a specific wavelength, with a narrow viewable angle of 30. This allows the delivery of exact wavelengths in one direction with little excess illumination. Utilizing exact wavelengths would enhance th e attraction of gravid traps to specific mosquito species from longer distances, while the oviposition site of th e trap remains unlit. This application could improve population monitoring methods for medi cally important species known to exhibit photophilic behavior, while maintain ing dark oviposition sites. Few significant differences in wavelength preference were observed among previtellogenic and vitellogenic An. quadrimaculatus Previtellogenic mosquitoes we re in contact with red LEDs significantly longer than vitelloge nic mosquitoes, while vitellogen ic mosquitoes contacted blue LED significantly longer than previtellogenic mos quitoes. These findings demonstrate the effects

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111 of physiological development on mosquito wave length preference. During the previtellogenic stage mosquitoes are host seeki ng, thus utilizing specific visual parameters to locate a blood meal (Bidlingmayer 1994). However, in the vitellogenic stage, mos quitoes are in search of an oviposition site and are possibly sensitive to alte rnative visual cues (A llan and Kline 2004). Our results offer additional evidence of behavior al differences between reproductive stages. Our observations merit additional research to fully understand the differences in wavelength preference between previtellogenic a nd vitellogenic mosquitoes. The incorporation of an artificial host into a visual ometer would be necessary to eval uate the effects of alternative host stimuli on previtellogenic mosquitoes in open-port and paired-T port trials. Additionally, assessing these effects on alternative medically important mosquito species may yield different results given that wavelength preferences can significantly differ among mosquito species (Browne and Bennett 1981, Ali et al. 1989, Burkett et al. 1998). Ultimately, these findings need to be examined in field trials using wild mosquitoes to avoid unnatural behaviors often experienced with colonized mosquitoes. By affixi ng preferred diodes to gravid traps, mosquito captures could be analyzed and compared to visualometer results to further elicit diode preference, future applic ation and field viability.

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112 Table 4-1. Mean numbers ( SE) of previtellogenic and vitellogenic Anopheles quadrimaculatus attracted to selected wavelengths of light emitting diodes as measured by mean contact seconds using an open port visualometer. Physiological Stages Diode Treatment n Previtellogenic Vitellogenic Blue 8 0.0996 (.0549)b 0.1612 (.0532)a Green 8 0.2514 (.0517) 0.0804 (.0332) IR 8 0.1621 (.0468) 0.1254 (.0194) Red 8 0.2189 (.0632)a 0.1485 (.0526)b No Light 8 0.1854 (.0821) 0.1396 (.0417) Note: Blue = 470 nm, Green = 502 nm, IR (Infrared) = 860 nm, Red = 660 nm and No light constituted an unlit control treatment. Means = total contact seconds per treatment over eight hour trials (N = 8). 150 previtell ogenic or vitellogenic mosquitoes released into an open port visualometer for each trial. Means within rows followed by the same letter were not significantly different. ANOVA: Blue (F1,15=111.24; P < 0.009); Red (F1, 15=98.08; P < 0.01) Table 4-2. Mean numbers ( SE) of previtellogenic and vitellogenic Anopheles quadrimaculatus attracted to paired selected wavelengths of light emitting diodes as measured by mean contact seconds using a pa ired-T port visualometer. Comparison Stage n Diode Treatment Mean (SEM) Blue:Red Previtellogenic 4 Blue 0.0692 (.0179) Red 0.0427 (.0138) Vitellogenic 4 Blue 0.0723 (.0158) Red 0.0785 (.0209) Blue:Green Previtellogenic 7 Blue 0.0314 (.0096) Green 0.0221 (.0069) Vitellogenic 7 Blue 0.0346 (.0104) Green 0.0362 (.0108) Note: Physiological stage of An. quadrimaculatus : Previtellogenic stage = mosquitoes 72 h post emergence. Vitellogenic stage = mosquitoes bloodfed at 120 h and released into visualometer at 144 h post emergence. Blue diode = 470 nm, Re d diode = 660 nm and Green diode = 502 nm.

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113 A B Figure 4-1. Pie shaped visualometer with 10 av ailable feeding stations which can be portioned off individually or left in an open design. A) Visualometer used in an open design, with treatments placed at all odd numbered feeding stations. B) Visualometer in operation showing treatments, set as descri bed above. C) Visualometer used in a paired-T configuration.

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114 C Figure 4-1. Continued. Figure 4-2. Anopheles quadrimaculatus obtained from the USDA-ARS-CMAVE Gainesville, FL rearing facility held in an incuba tor at 26 C and 74% humidity under a 14:10 (L:D) photoperiod. Upon eclosion, adult mosquitoes were fed a 10% sugar solution.

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115 Figure 4-3. Blood feeding Anopheles quadrimaculatus occured 120 h post-eclosion using a blood ball. Blood balls consisted of saus age casing and defribrinated bovine blood. Adult mosquitoes were allowed to blood feed for 3 h.

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116 CHAPTER 5 THE IMPORTANCE OF MOSQUITO WAVE LENGT H PREFERENCE IN TRAPPING AND POPULATION SAMPLING Since the early 1900s, the effectiveness of t echniques to attract and track the movements of hematophagous insects has continued to improve (Crans 1989). Adequate and reliable population sampling is often seen as the most im portant and most difficu lt step in ecological studies. Most adult mosquito trapping methods utilize attractants, including a live host, olfactory stimuli (carbon dioxide, octenol, la ctic acid) or various forms of visual stimuli (wavelength, light source, intensity, frequency). These traps produce a bias when used in vector surveillance and monitoring by primarily selecti ng for unfed, host-seeking female mosquitoes. Collections of resting mosquito populations yield a more accurate representative sample of a mosquito population given that adults probably spend more time resting than in flight. Resting collection methods not only result in catching unfed hostseeking females, but also both blood-fed and gravid females as well as males. Sampling restin g mosquito populations al so yields a broad age structure. Several non-biased methods exist for samp ling resting mosquito populations. When targeting indoor resting mosqu ito species, including several Anopheles and some Culex, aspirators, resting counts and knock-down spra ys are commonly used. Though few mosquito species commonly rest indoors, thos e that do are often important vectors of malaria, filariasis and some arboviruses, making accurate sampling methods of these species a necessity. Sampling outdoor resting mosquitoes is ofte n more difficult because outdoor populations are usually distributed over larg er areas. A better understanding of the general resting habits of most exophilic species has allowed for the deve lopment of more accurate surveillance methods. When sampling mosquito species known to rest amongst grassy and shrubby vegetation, such as

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117 Psorophora columbiae Dyar and Knab, aspirators or sw eep nets have been shown to be successful. However, the utiliza tion of artificial resting places is often the preferred sampling method, allowing for the attraction of mosquitoes to a specific site fr om which they can be conveniently collected. Though biased, modifications and advancements to baited trapping systems continue to show promise for increasing the efficiency of existing population sampli ng methods. Artificial, reflected and filtered lights have been incorporat ed in the design of existing traps to increase their effectiveness for mosquito research and surveillance with great success (Barr et al. 1963, Service 1976, Ali et al. 1989, Burkett and Bu tler 2005, Hoel 2005). Additionally, the recent development of super-bright light emitting diode s (LEDs) has allowed for the isolation of specific wavelengths permitting researchers to refine techniques to more effectively attract mosquitoes using more precise light sources. Wh en used in Center for Disease Control (CDC) traps, these highly efficient, low cost LEDs have a greater intensity a nd have a significantly lower energy requirement than existing incandes cent bulbs (Burkett et al. 1998). However, little information concerning the attractiveness of LEDs to different mosquito species exists. Knowledge of mosquito wavelength preferences without the presence of other host attractants is limited. The objective of this project was to i nvestigate the effects of LEDs of selected wavelengths on mosquitoes under various beha vioral and physiological states. This was accomplished in the field with resting boxes a nd sticky cards. Laboratory studies were conducted with a visualometer using mosquitoes in two stages of ovarian development. This is the first instance of LEDs being us ed for this type of research. Using Goodwin (1942) style re sting boxes, wavelength preferences for adult mosquitoes utilizing resting structures were evaluated in Chapter 2. Light emitting diode color (wavelength)

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118 choices were blue (460 nm), green (502 nm), re d (660 nm) and IR (860 nm). Center for Disease Control (CDC) traps were used to provide representative background mosquito populations. Results for Chapter 2 showed that certain mos quito species were attracted to, or repelled by the LEDs, depending on color. Previous to this study, trapping involving the inclusion of LEDs in resting boxes had not been conducted. The findings of this research demonstrate the need for further investigation into the combin ation of mosquito wave length attraction and artificial resting boxes. Several mosquito spec ies recovered from resting boxes fitted with LEDs were previously thought to have little affinity to light. Based on these results and observations from past research, variations in light intensity might also significantly impact the attractiveness of resting boxes to mosquitoes. Relevance of these findings could lead to their future applications in mosquito repellant devices, or to enhance the attractive ness of existing trap models. Based on the push-pull premise, res ting boxes or mechanical adult mosquito traps could be placed at a considerable distance away from a home or military building, and fitted with LEDs found to be attractive to target mosquito species. Light emitting diodes with wavelengths known to be undesirable to these species would th en be affixed to the desired building. This combination of attractive and repellant stimulan ts enhances the effects of each, leading to improved repellent devices for medically important mosquitoes. Adult mosquito wavelength preferences we re evaluated independently of other physiological or biological stimuli in Chapter 3. Overall, mosquito capture on sticky cards was greatest with green diodes (198 mosquitoes), followed by sticky cards with blue (159 mosquitoes), red (60 mosquitoes) and IR (35 mo squitoes) diodes. Past research has identified mosquito wavelength preference in the blue-green range (400 600 nm), observing diminishing results as wavelengths increase in length (> 600 nm). Similarly, our results demonstrated that

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119 mosquitoes exhibited a preference for blue ( 470 nm) and green (502 nm) diodes, but stronger preferences were observed when using green di odes. While these findings do not discount the possible effectiveness of wavelengt hs in the higher blue spectral range (> 470 nm), wavelengths ranging in the lower green sp ectrum (502 nm) resulted in higher mosquito capture. These findings demonstrate that the use of only light in a trapping system without additional host based attractants (CO2, octenol and lactic acid) can effectively capture mosquitoes. While differing wavelengths influe nced mosquito prefer ence, manipulation of wavelength frequency or intensity may also enhan ce capture for specific mosquito species. Their demonstrated effectiveness for attracting mos quitoes without the aid of supplemental host attractants further eliminates the need and costs of commonly used alternative host-based attractants (CO2) or noxious chemicals (lactic acid, oc tenol). Additionally, durability of LEDbased equipment required also helps to reduce otherwise necessary and time-consuming field maintenance. By offering extended operating time with minimal power consumption, field durability and the ability to eliminate the n eed for burdensome equipment, LEDs are removing restrictions previously set on trap designs wh ere equipment or field conditions were major limiting factors. Some mosquito species not captured in high numbers on sticky cards, such as An. quadrimaculatus, and Ae. albopictus, were species not known to uti lize light sources as primary cues in host location. Ther efore, low trap numbers were expe cted. However, these species were captured in higher numbers during th e resting box study (Chapter 2). An. quadrimaculatus and Ae. albopictus are known to prefer dark unlit surfaces and subsequently, are commonly recovered in high numbers using dark co lored resting boxes (Goodwin 1942, Crans 1989, Irby and Apperson 1992) Because of this, the pres ence these species in lit resting boxes was

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120 surprising. The combination of results from th e Chapter 2 and the Chapter 3 studies further shows the large amount of information we have yet to gain concerning mosquito wavelength preference. Anopheles quadrimaculatus wavelength preferences between physiological stages (previtellogenic, vitellogenic) we re evaluated in Chapter 4. Blue (460 nm), green (502 nm), red (660 nm) and IR (860 nm) LEDs were utilized in an open-port visualometer. Due to increased contact seconds for previt ellogenic and vitellogenic An. quadrimaculatus certain LED wavelengths were selected for additional analysis in a follow-up study using a paired-T port visualometer system. In comparisons between previtellogenic and vitellogenic An. quadrimaculatus released in the open port visualometer, previtellogenic mos quitoes were in contact with red diodes significantly longer than were vitellogenic mosquitoes. Ho wever, among previtellogenic An. quadrimaculatus released in the open-port visualometer no significant differences in mosquito contact seconds were observed between the four LED wavele ngths. Likewise, no significant differences were observed among previtellogenic An. quadrimaculatus exposed to blue and red or blue and green LEDs in a paired-T port visualometer. Previtellogenic (host seeking) mosquitoes were expected to e xhibit attraction to LEDs. That mo squitoes did not exhibit higher preference for LEDs than for the unl it control is surprising and sugge sts that light alone is a poor attractant or that our experiment al design needs to be refined. Future trapping applications based on data co llected and field observations from Chapters 2, 3 and 4, could be useful in several fields a nd for multiple purposes. By utilizing the repellency and attractiveness of specific wa velengths of light in the absen ce of additional host attractants, the efficacy of virtually any trapping model can be improved. In fitting LEDs of selected

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121 wavelengths to resting boxes, both the species sp ecificity and the efficiency of adult mosquito population monitoring can be drastically enhanc ed. Also, by incorporating LEDs of various wavelengths in trapping systems designed to attr act adult mosquitoes of specific physiological stages, mosquito captures may be significantly increased. The results from these studies indicate the need for additional research into mosquito wavelength preference during multiple physiologi cal stages, and under various biological conditions. The integration of LEDs into various sampling and trapping systems has demonstrated great success in impacting trapping numbers for multiple mosquito species. The need for further species specificity in mosqu ito population monitoring programs grows as the demand for more evolved sampling methods increas es. Continued research into the effects of light wavelength, frequency and intensity on indi vidual mosquito species could lead to more refined trapping methods. The application of this technology would be well received by governmental agencies, mosquito control pr ograms and homeowner targeted industries.

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122 APPENDIX A RESTING BOX AND MODIFIED CDC LIGHT-TRAP CAPTURES OF MOS QUITOES BY LOCATION Table A-1. Evaluation of res ting box catches for mosquito species captured at the Horse Teaching Unit (HTU) from July 2006 Sept. 2007 near Gainesville, FL. Diode Wavelength Species Date Blue Green Red IR No Light An. crucians 7/21/06 8/14/06 0.050 0.025 0.013 0.025 0.038 5/5/07 5/24/07 <0.001 <0.001 <0.001 0.013 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 An. crucians 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 0.013 0.025 <0.001 0.013 0.025 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 An. quadrimaculatus 7/21/06 8/14/06 0.025 0.013 0.013 0.038 0.050 5/5/07 5/24/07 0.013 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

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123 Table A-1 Continued. Diode Wavelength Species Date Blue Green Red IR No Light An. quadrimaculatus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 0.038 5/5/07 5/24/07 <0.001 0.013 0.013 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 0.013 <0.001 0.025 9/7/07 9/26/07 <0.001 <0.001 0.025 <0.001 0.013 Cq. perturbans 7/21/06 8/14/06 0.513 0.338 0.250 0.263 0.288 5/5/07 5/24/07 <0.001 0.013 0.013 0.013 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 0.013 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 0.013 <0.001 <0.001 <0.001 Cq. perturbans 7/21/06 8/14/06 0.025 0.013 0.075 0.025 0.013 5/5/07 5/24/07 0.075 0.013 0.038 0.063 0.025 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 0.013 <0.001 <0.001 7/7/07 7/28/07 <0.001 0.025 0.000 <0.001 0.013 7/29/07 8/17/07 <0.001 0.013 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 0.013 <0.001 <0.001 <0.001 9/7/07 9/26/07 0.013 <0.001 <0.001 <0.001 <0.001 Cx. erraticus 7/21/06 8/14/06 2.050 2.813 3.025 3.188 2.675 5/5/07 5/24/07 0.100 0.075 0.025 0.050 0.063 5/25/07 6/13/07 <0.001 0.088 0.038 <0.001 <0.001 6/14/07 7/6/07 <0.001 0.013 <0.001 <0.001 0.013 7/7/07 7/28/07 0.013 <0.001 <0.001 <0.001 0.050 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 0.013 8/18/07 9/6/07 0.025 <0.001 <0.001 <0.001 0.000 9/7/07 9/26/07 <0.001 <0.001 0.013 <0.001 0.013

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124 Table A-1 Continued. Diode Wavelength Species Date Blue Green Red IR No Light Cx. erraticus 7/21/06 8/14/06 0.375 0.688 0.488 0.450 0.475 5/5/07 5/24/07 0.100 0.175 0.250 0.075 0.150 5/25/07 6/13/07 0.025 0.038 0.025 0.025 0.038 6/14/07 7/6/07 0.038 0.038 0.113 0.013 0.063 7/7/07 7/28/07 0.050 0.038 0.013 <0.001 0.063 7/29/07 8/17/07 0.075 0.038 0.088 <0.001 0.150 8/18/07 9/6/07 0.325 0.138 0.025 0.063 0.225 9/7/07 9/26/07 0.213 0.063 0.100 0.000 0.138 Cx. nigripalpus 7/21/06 8/14/06 <0.001 <0.001 <0.001 0.013 0.013 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cx. nigripalpus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 0.038 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 0.013 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 0.013 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cx. salinarius 7/21/06 8/14/06 <0.001 0.013 0.013 0.025 0.038 5/5/07 5/24/07 0.013 0.025 0.013 <0.001 <0.001 5/25/07 6/13/07 <0.001 0.038 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

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125 Table A-1 Continued. Diode Wavelength Species Date Blue Green Red IR No Light Cx. salinarius 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 0.013 0.013 <0.001 <0.001 5/25/07 6/13/07 0.013 0.038 <0.001 <0.001 0.013 6/14/07 7/6/07 <0.001 <0.001 <0.001 0.013 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 0.000 Cx. territans 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 0.025 0.038 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 0.025 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cx. territans 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 0.025 <0.001 <0.001 0.025 0.013 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Ma. titillans 7/21/06 8/14/06 0.025 0.025 0.025 0.050 0.050 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

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126 Table A-1 Continued. Diode Wavelength Species Date Blue Green Red IR No Light Ma. titillans 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 0.025 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Oc. infirmatus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Oc. infirmatus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Oc. triseriatus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

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127 Table A-1 Continued. Diode Wavelength Species Date Blue Green Red IR No Light Oc. triseriatus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 0.013 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Ur. lowii 7/21/06 8/14/06 0.038 0.025 <0.001 0.025 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 0.000 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 0.000 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 0.000 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 0.000 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 0.000 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 0.000 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 0.000 <0.001 Ur. lowii 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Ur. sapphirina 7/21/06 8/14/06 0.113 0.063 0.025 0.063 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

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128 Table A-1 Continued. Diode Wavelength Species Date Blue Green Red IR No Light Ur. sapphirina 7/21/06 8/14/06 0.013 0.013 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Note: Blue diode = 470 nm, Green diode = 502 nm, Red diode = 660 nm and IR = 860 nm. An. = Anopheles ; Cq. = Coquillettidia ; Cx. = Culex; Ma. = Mansonia ; Oc = Ochlerotatus; Ur. = Uranotaenia

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129 Table A-2. Evaluation of resting box catches for mosquito species captured at the Prairie Oaks (PO) subdivision from August 2006 Sept. 2007 near Gainesville, FL. Diode Wavelength Species Date Blue Green Red IR No Light An. crucians 8/18/06 9/27/06 1.000 <0.001 0.015 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 0.013 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 An. crucians 7/21/06 8/14/06 <0.001 <0.001 0.029 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 An. quadrimaculatus 7/21/06 8/14/06 <0.001 0.088 0.353 0.162 0.191 5/5/07 5/24/07 <0.001 <0.001 0.013 0.025 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

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130 Table A-2. Continued Diode Wavelength Species Date Blue Green Red IR No Light An. quadrimaculatus 7/21/06 8/14/06 0.191 0.235 0.515 0.132 0.176 5/5/07 5/24/07 <0.001 <0.001 0.013 <0.001 0.013 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cq. perturbans 7/21/06 8/14/06 0.074 0.235 0.250 0.191 0.250 5/5/07 5/24/07 0.013 <0.001 0.013 <0.001 0.025 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cq. perturbans 7/21/06 8/14/06 0.206 0.162 0.235 0.206 0.250 5/5/07 5/24/07 0.013 <0.001 0.025 0.025 0.013 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 0.013 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cx. erraticus 7/21/06 8/14/06 0.118 4.824 6.500 9.162 7.353 5/5/07 5/24/07 0.088 0.113 0.113 0.163 0.125 5/25/07 6/13/07 0.025 0.038 0.088 0.063 0.113 6/14/07 7/6/07 0.013 0.050 0.025 <0.001 0.025 7/7/07 7/28/07 0.025 0.013 0.013 0.013 0.013 7/29/07 8/17/07 0.013 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 0.013 <0.001 0.025 0.013 0.025

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131 Table A-2. Continued Diode Wavelength Species Date Blue Green Red IR No Light Cx. erraticus 7/21/06 8/14/06 4.691 5.676 6.441 8.103 8.000 5/5/07 5/24/07 0.150 0.225 0.175 0.163 0.150 5/25/07 6/13/07 0.050 0.150 0.200 0.063 0.150 6/14/07 7/6/07 0.088 0.075 0.038 0.013 0.038 7/7/07 7/28/07 0.150 0.050 0.063 0.013 0.050 7/29/07 8/17/07 0.113 0.038 0.063 0.013 0.063 8/18/07 9/6/07 0.100 0.050 0.000 0.013 0.063 9/7/07 9/26/07 0.113 0.100 0.013 0.075 0.125 Cx. nigripalpus 7/21/06 8/14/06 5.765 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 0.025 <0.001 <0.001 <0.001 0.025 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cx. nigripalpus 7/21/06 8/14/06 <0.001 0.059 <0.001 0.059 <0.001 5/5/07 5/24/07 0.013 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 0.013 0.013 0.013 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cx. salinarius 7/21/06 8/14/06 0.074 0.074 <0.001 0.044 0.029 5/5/07 5/24/07 0.013 <0.001 <0.001 0.013 0.013 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

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132 Table A-2. Continued Diode Wavelength Species Date Blue Green Red IR No Light Cx. salinarius 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 0.013 0.013 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 0.013 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cx. territans 7/21/06 8/14/06 0.029 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 0.050 0.038 0.025 0.013 5/25/07 6/13/07 <0.001 <0.001 <0.001 0.025 0.025 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Cx. territans 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 0.100 0.100 0.038 0.088 0.013 5/25/07 6/13/07 <0.001 0.025 0.013 0.038 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Ma. titillans 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

PAGE 133

133 Table A-2. Continued Diode Wavelength Species Date Blue Green Red IR No Light Ma. titillans 7/21/06 8/14/06 <0.001 0.059 0.044 0.059 0.029 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Oc. infirmatus 7/21/06 8/14/06 0.029 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Oc. infirmatus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 0.013 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Oc. triseriatus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

PAGE 134

134 Table A-2. Continued Diode Wavelength Species Date Blue Green Red IR No Light Oc. triseriatus 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Ur. lowii 7/21/06 8/14/06 <0.001 <0.001 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Ur. lowii 7/21/06 8/14/06 <0.001 0.015 <0.001 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Ur. sapphirina 7/21/06 8/14/06 <0.001 <0.001 0.015 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001

PAGE 135

135 Table A-2. Continued Diode Wavelength Species Date Blue Green Red IR No Light Ur. sapphirina 7/21/06 8/14/06 0.015 <0.001 0.015 <0.001 <0.001 5/5/07 5/24/07 <0.001 <0.001 <0.001 <0.001 <0.001 5/25/07 6/13/07 <0.001 <0.001 <0.001 <0.001 <0.001 6/14/07 7/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/7/07 7/28/07 <0.001 <0.001 <0.001 <0.001 <0.001 7/29/07 8/17/07 <0.001 <0.001 <0.001 <0.001 <0.001 8/18/07 9/6/07 <0.001 <0.001 <0.001 <0.001 <0.001 9/7/07 9/26/07 <0.001 <0.001 <0.001 <0.001 <0.001 Note: Blue diode = 470 nm, Green diode = 502 nm, Red diode = 660 nm and IR = 860 nm. An. = Anopheles ; Cq. = Coquillettidia ; Cx. = Culex; Ma. = Mansonia ; Oc = Ochlerotatus; Ur. = Uranotaenia .

PAGE 136

136 Table A-3. Modified CDC light trap mosquito captures at the Horse Te aching Unit (HTU) from July 2006 Sept. 2007 near Gainesville, FL. Species Date Trap Night Total/Trap Night Ae. albopictus 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 <0.01 7/7/07 7/28/07 18 0.06 7/29/07 8/17/07 19 0.05 8/18/07 9/6/07 19 <0.01 9/7/07 9/26/07 18 <0.01 Ae. vexans 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 7.13 5/25/07 6/13/07 20 3.10 6/14/07 7/6/07 20 8.70 7/7/07 7/28/07 18 2.89 7/29/07 8/17/07 19 14.89 8/18/07 9/6/07 19 47.26 9/7/07 9/26/07 18 33.28 An. crucians 7/21/06 8/14/06 16 46.19 5/5/07 5/24/07 16 9.56 5/25/07 6/13/07 20 5.05 6/14/07 7/6/07 20 6.50 7/7/07 7/28/07 18 3.61 7/29/07 8/17/07 19 8.05 8/18/07 9/6/07 19 4.89 9/7/07 9/26/07 18 3.00

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137 Table A-3. Continued. Species Date Trap Night Total/Trap Night An. quadrimaculatus 7/21/06 8/14/06 16 1.56 5/5/07 5/24/07 16 0.63 5/25/07 6/13/07 20 0.05 6/14/07 7/6/07 20 <0.01 7/7/07 7/28/07 18 0.17 7/29/07 8/17/07 19 <0.01 8/18/07 9/6/07 19 0.26 9/7/07 9/26/07 18 0.67 Cq. perturbans 7/21/06 8/14/06 16 1,391.88 5/5/07 5/24/07 16 45.38 5/25/07 6/13/07 20 11.40 6/14/07 7/6/07 20 15.95 7/7/07 7/28/07 18 11.94 7/29/07 8/17/07 19 12.89 8/18/07 9/6/07 19 14.21 9/7/07 9/26/07 18 30.56 Cx. erraticus 7/21/06 8/14/06 16 154.38 5/5/07 5/24/07 16 5.56 5/25/07 6/13/07 20 2.95 6/14/07 7/6/07 20 3.60 7/7/07 7/28/07 18 0.72 7/29/07 8/17/07 19 3.37 8/18/07 9/6/07 19 4.89 9/7/07 9/26/07 18 7.28 Cx. nigripalpus 7/21/06 8/14/06 16 1.13 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 3.85 7/7/07 7/28/07 18 0.67 7/29/07 8/17/07 19 95.53 8/18/07 9/6/07 19 301.53 9/7/07 9/26/07 18 657.94

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138 Table A-3. Continued. Species Date Trap Night Total/Trap Night Cx. quinquefasciatus 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 0.19 5/25/07 6/13/07 20 0.25 6/14/07 7/6/07 20 <0.01 7/7/07 7/28/07 18 <0.01 7/29/07 8/17/07 19 0.26 8/18/07 9/6/07 19 <0.01 9/7/07 9/26/07 18 <0.01 Cx. salinarius 7/21/06 8/14/06 16 1.88 5/5/07 5/24/07 16 1.81 5/25/07 6/13/07 20 1.45 6/14/07 7/6/07 20 4.25 7/7/07 7/28/07 18 1.11 7/29/07 8/17/07 19 21.32 8/18/07 9/6/07 19 33.95 9/7/07 9/26/07 18 15.11 Ma. titillans 7/21/06 8/14/06 16 531.75 5/5/07 5/24/07 16 0.60 5/25/07 6/13/07 20 0.60 6/14/07 7/6/07 20 1.90 7/7/07 7/28/07 18 6.44 7/29/07 8/17/07 19 14.84 8/18/07 9/6/07 19 15.42 9/7/07 9/26/07 18 44.72 Oc. canadensis 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 <0.01 7/7/07 7/28/07 18 <0.01 7/29/07 8/17/07 19 <0.01 8/18/07 9/6/07 19 <0.01 9/7/07 9/26/07 18 <0.01

PAGE 139

139 Table A-3. Continued. Species Date Trap Night Total/Trap Night Oc. infirmatus 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 0.50 5/25/07 6/13/07 20 5.25 6/14/07 7/6/07 20 4.80 7/7/07 7/28/07 18 1.44 7/29/07 8/17/07 19 4.74 8/18/07 9/6/07 19 42.58 9/7/07 9/26/07 18 18.06 Oc. sollicitans 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 0.05 7/7/07 7/28/07 18 <0.01 7/29/07 8/17/07 19 <0.01 8/18/07 9/6/07 19 <0.01 9/7/07 9/26/07 18 <0.01 Oc. taeniorhynchus 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 0.05 7/7/07 7/28/07 18 0.11 7/29/07 8/17/07 19 <0.01 8/18/07 9/6/07 19 0.11 9/7/07 9/26/07 18 <0.01 Oc. triseriatus 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 0.06 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 <0.01 7/7/07 7/28/07 18 <0.01 7/29/07 8/17/07 19 <0.01 8/18/07 9/6/07 19 0.11 9/7/07 9/26/07 18 <0.01

PAGE 140

140 Table A-3. Continued. Species Date Trap Night Total/Trap Night Ps. ciliata 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 0.45 6/14/07 7/6/07 20 0.05 7/7/07 7/28/07 18 <0.01 7/29/07 8/17/07 19 <0.01 8/18/07 9/6/07 19 <0.01 9/7/07 9/26/07 18 0.17 Ps. columbiae 7/21/06 8/14/06 16 16.75 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 0.05 7/7/07 7/28/07 18 0.67 7/29/07 8/17/07 19 11.53 8/18/07 9/6/07 19 3.26 9/7/07 9/26/07 18 7.22 Ps. ferox 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 <0.01 7/7/07 7/28/07 18 <0.01 7/29/07 8/17/07 19 0.05 8/18/07 9/6/07 19 <0.01 9/7/07 9/26/07 18 <0.01 Ur. lowii 7/21/06 8/14/06 16 0.13 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 <0.01 7/7/07 7/28/07 18 <0.01 7/29/07 8/17/07 19 <0.01 8/18/07 9/6/07 19 <0.01 9/7/07 9/26/07 18 <0.01

PAGE 141

141 Table A-3. Continued. Species Date Trap Night Total/Trap Night Ur. sapphirina 7/21/06 8/14/06 16 <0.01 5/5/07 5/24/07 16 <0.01 5/25/07 6/13/07 20 <0.01 6/14/07 7/6/07 20 <0.01 7/7/07 7/28/07 18 <0.01 7/29/07 8/17/07 19 <0.01 8/18/07 9/6/07 19 0.05 9/7/07 9/26/07 18 <0.01 Note: Ae. = Aedes; An. = Anopheles ; Cq = Coquillettidia ; Cx. = Culex; Ma. = Mansonia; Oc = Ochlerotatus; Ps. = Psor ophora; Ur. = Uranotaenia One modified CDC trap + CO2 (250 ml/min). When N < 20, traps had malfunctioned.

PAGE 142

142 Table A-4. Modified CDC light trap mosquito captures at the Prairie Oaks subdivision (PO) from July August 2006 and May Sept. 2007 near Gainesville, FL. Species Date Trap Night Total/Trap Night Ae. albopictus 7/21/06 8/14/06 36 0.13 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 0.05 7/7/07 7/28/07 37 0.16 7/29/07 8/17/07 40 1.08 8/18/07 9/6/07 38 0.58 9/7/07 9/26/07 35 0.46 Ae. vexans 7/21/06 8/14/06 36 <0.01 5/5/07 5/24/07 38 10.50 5/25/07 6/13/07 39 8.79 6/14/07 7/6/07 39 13.13 7/7/07 7/28/07 37 9.92 7/29/07 8/17/07 40 18.15 8/18/07 9/6/07 38 14.39 9/7/07 9/26/07 35 11.97 An. crucians 7/21/06 8/14/06 36 25.83 5/5/07 5/24/07 38 17.95 5/25/07 6/13/07 39 2.15 6/14/07 7/6/07 39 1.92 7/7/07 7/28/07 37 2.78 7/29/07 8/17/07 40 0.88 8/18/07 9/6/07 38 0.26 9/7/07 9/26/07 35 0.49 An. quadrimaculatus 7/21/06 8/14/06 36 2.10 5/5/07 5/24/07 38 0.32 5/25/07 6/13/07 39 0.03 6/14/07 7/6/07 39 <0.01 7/7/07 7/28/07 37 0.03 7/29/07 8/17/07 40 <0.01 8/18/07 9/6/07 38 <0.01 9/7/07 9/26/07 35 0.03

PAGE 143

143 Table A-4. Continued. Species Date Trap Night Total/Trap Night Cq. perturbans 7/21/06 8/14/06 36 73.77 5/5/07 5/24/07 38 53.84 5/25/07 6/13/07 39 21.74 6/14/07 7/6/07 39 23.74 7/7/07 7/28/07 37 10.70 7/29/07 8/17/07 40 4.53 8/18/07 9/6/07 38 5.95 9/7/07 9/26/07 35 10.83 Cx. erraticus 7/21/06 8/14/06 36 216.47 5/5/07 5/24/07 38 3.76 5/25/07 6/13/07 39 2.46 6/14/07 7/6/07 39 1.31 7/7/07 7/28/07 37 0.51 7/29/07 8/17/07 40 1.00 8/18/07 9/6/07 38 0.68 9/7/07 9/26/07 35 1.51 Cx. nigripalpus 7/21/06 8/14/06 36 <0.01 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 0.15 6/14/07 7/6/07 39 9.36 7/7/07 7/28/07 37 2.16 7/29/07 8/17/07 40 30.13 8/18/07 9/6/07 38 39.79 9/7/07 9/26/07 35 214.60 Cx. quinquefasciatus 7/21/06 8/14/06 36 <0.01 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 <0.01 7/7/07 7/28/07 37 <0.01 7/29/07 8/17/07 40 0.05 8/18/07 9/6/07 38 <0.01 9/7/07 9/26/07 35 <0.01

PAGE 144

144 Table A-4. Continued. Species Date Trap Night Total/Trap Night Cx. salinarius 7/21/06 8/14/06 36 5.20 5/5/07 5/24/07 38 1.29 5/25/07 6/13/07 39 0.67 6/14/07 7/6/07 39 2.97 7/7/07 7/28/07 37 0.24 7/29/07 8/17/07 40 3.20 8/18/07 9/6/07 38 1.13 9/7/07 9/26/07 35 2.34 Ma. titillans 7/21/06 8/14/06 36 7.50 5/5/07 5/24/07 38 0.29 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 <0.01 7/7/07 7/28/07 37 0.16 7/29/07 8/17/07 40 0.13 8/18/07 9/6/07 38 0.08 9/7/07 9/26/07 35 0.11 Oc. canadensis 7/21/06 8/14/06 36 <0.01 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 0.03 7/7/07 7/28/07 37 0.03 7/29/07 8/17/07 40 <0.01 8/18/07 9/6/07 38 <0.01 9/7/07 9/26/07 35 <0.01 Oc. infirmatus 7/21/06 8/14/06 36 <0.01 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 <0.01 7/7/07 7/28/07 37 <0.01 7/29/07 8/17/07 40 <0.01 8/18/07 9/6/07 38 0.11 9/7/07 9/26/07 35 0.14

PAGE 145

145 Table A-4. Continued. Species Date Trap Night Total/Trap Night Oc. sollicitans 7/21/06 8/14/06 36 <0.01 5/5/07 5/24/07 38 5.55 5/25/07 6/13/07 39 33.64 6/14/07 7/6/07 39 17.46 7/7/07 7/28/07 37 14.38 7/29/07 8/17/07 40 11.25 8/18/07 9/6/07 38 12.58 9/7/07 9/26/07 35 7.06 Oc. taeniorhynchus 7/21/06 8/14/06 36 <0.01 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 0.03 6/14/07 7/6/07 39 0.05 7/7/07 7/28/07 37 <0.01 7/29/07 8/17/07 40 0.03 8/18/07 9/6/07 38 <0.01 9/7/07 9/26/07 35 <0.01 Oc. triseriatus 7/21/06 8/14/06 36 0.77 5/5/07 5/24/07 38 0.05 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 0.36 7/7/07 7/28/07 37 0.05 7/29/07 8/17/07 40 0.08 8/18/07 9/6/07 38 0.03 9/7/07 9/26/07 35 <0.01 Ps. ciliata 7/21/06 8/14/06 36 <0.01 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 0.03 6/14/07 7/6/07 39 0.03 7/7/07 7/28/07 37 <0.01 7/29/07 8/17/07 40 0.03 8/18/07 9/6/07 38 <0.01 9/7/07 9/26/07 35 <0.01

PAGE 146

146 Table A-4. Continued. Species Date Trap Night Total/Trap Night Ps. columbiae 7/21/06 8/14/06 36 0.27 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 0.03 6/14/07 7/6/07 39 0.13 7/7/07 7/28/07 37 0.16 7/29/07 8/17/07 40 0.65 8/18/07 9/6/07 38 <0.01 9/7/07 9/26/07 35 0.03 Ps. ferox 7/21/06 8/14/06 36 0.17 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 0.31 7/7/07 7/28/07 37 0.16 7/29/07 8/17/07 40 1.30 8/18/07 9/6/07 38 1.42 9/7/07 9/26/07 35 4.34 Ur. lowii 7/21/06 8/14/06 36 0.93 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 <0.01 7/7/07 7/28/07 37 <0.01 7/29/07 8/17/07 40 <0.01 8/18/07 9/6/07 38 <0.01 9/7/07 9/26/07 35 <0.01 Ur. sapphirina 7/21/06 8/14/06 36 11.20 5/5/07 5/24/07 38 <0.01 5/25/07 6/13/07 39 <0.01 6/14/07 7/6/07 39 0.03 7/7/07 7/28/07 37 0.05 7/29/07 8/17/07 40 <0.01 8/18/07 9/6/07 38 <0.01 9/7/07 9/26/07 35 <0.01 Note: Ae. = Aedes; An. = Anopheles ; Co = Coquillettidia ; Cx. = Culex; Ma. = Mansonia; Oc = Ochlerotatus; Ps. = Psor ophora; Ur. = Uranotaenia Two modified CDC traps + CO2 (250 ml/min). When N < 40, traps had malfunctioned.

PAGE 147

147 APPENDIX B STICKY CARD AND MODIFIED CDC LIGHT-TRAP CAPTURES OF MOS QUITOES BY LOCATION Table B-1. Mosquitoes captured in a modified CDC light trap at the University of Florida Horse Teaching Unit from July August 2006 and May Sept. 2007 near Gainesville, FL. Species Date Trap Night Total/Trap Night Ae. albopictus 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 <0.01 6/26/07 7/15/07 19 0.05 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 0.05 8/25/07 9/13/07 19 <0.01 Ae. vexans 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 6.47 6/6/07 6/25/07 16 5.44 6/26/07 7/15/07 19 8.79 7/16/07 8/4/07 19 2.35 8/5/07 8/24/07 19 27.05 8/25/07 9/13/07 19 42.11 An. crucians 7/21/06 8/14/06 16 46.19 5/5/07 6/5/07 15 9.93 6/6/07 6/25/07 16 8.75 6/26/07 7/15/07 19 5.89 7/16/07 8/4/07 19 4.59 8/5/07 8/24/07 19 6.74 8/25/07 9/13/07 19 4.53 An. quadrimaculatus 7/21/06 8/14/06 16 1.56 5/5/07 6/5/07 15 0.67 6/6/07 6/25/07 16 <0.01 6/26/07 7/15/07 19 0.11 7/16/07 8/4/07 19 0.06 8/5/07 8/24/07 19 <0.01 8/25/07 9/13/07 19 0.47

PAGE 148

148 Table B-1. Continued. Species Date Trap Night Total/Trap Night Cq. perturbans 7/21/06 8/14/06 16 1,391.88 5/5/07 6/5/07 15 47.33 6/6/07 6/25/07 16 22.63 6/26/07 7/15/07 19 14.21 7/16/07 8/4/07 19 9.24 8/5/07 8/24/07 19 12.32 8/25/07 9/13/07 19 18.11 Cx. erraticus 7/21/06 8/14/06 16 154.38 5/5/07 6/5/07 15 5.47 6/6/07 6/25/07 16 5.50 6/26/07 7/15/07 19 1.63 7/16/07 8/4/07 19 0.41 8/5/07 8/24/07 19 4.21 8/25/07 9/13/07 19 5.42 Cx. nigripalpus 7/21/06 8/14/06 16 1.13 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 1.56 6/26/07 7/15/07 19 3.26 7/16/07 8/4/07 19 1.00 8/5/07 8/24/07 19 113.32 8/25/07 9/13/07 19 667.58 Cx. quinquefasciatus 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 0.19 6/26/07 7/15/07 19 <0.01 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 0.26 8/25/07 9/13/07 19 <0.01 Cx. salinarius 7/21/06 8/14/06 16 1.88 5/5/07 6/5/07 15 1.93 6/6/07 6/25/07 16 5.25 6/26/07 7/15/07 19 2.21 7/16/07 8/4/07 19 0.24 8/5/07 8/24/07 19 23.58 8/25/07 9/13/07 19 38.11

PAGE 149

149 Table B-1. Continued. Species Date Trap Night Total/Trap Night Ma. titillans 7/21/06 8/14/06 16 531.75 5/5/07 6/5/07 15 2.87 6/6/07 6/25/07 16 1.31 6/26/07 7/15/07 19 3.00 7/16/07 8/4/07 19 6.88 8/5/07 8/24/07 19 15.63 8/25/07 9/13/07 19 22.11 Oc. canadensis 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 0.06 6/26/07 7/15/07 19 <0.01 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 <0.01 8/25/07 9/13/07 19 <0.01 Oc. fulvus pallens 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 <0.01 6/26/07 7/15/07 19 <0.01 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 <0.01 8/25/07 9/13/07 19 <0.01 Oc. infirmatus 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 0.93 6/6/07 6/25/07 16 2.69 6/26/07 7/15/07 19 5.32 7/16/07 8/4/07 19 1.24 8/5/07 8/24/07 19 16.79 8/25/07 9/13/07 19 33.84 Oc. sollicitans 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 0.07 6/26/07 7/15/07 19 <0.01 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 <0.01 8/25/07 9/13/07 19 <0.01

PAGE 150

150 Table B-1. Continued. Species Date Trap Night Total/Trap Night Oc. taeniorhynchus 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 0.06 6/26/07 7/15/07 19 0.11 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 <0.01 8/25/07 9/13/07 19 0.11 Oc. triseriatus 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 0.07 6/6/07 6/25/07 16 <0.01 6/26/07 7/15/07 19 <0.01 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 <0.01 8/25/07 9/13/07 19 0.11 Ps. ciliata 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 0.56 6/26/07 7/15/07 19 0.05 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 <0.01 8/25/07 9/13/07 19 0.16 Ps. columbiae 7/21/06 8/14/06 16 16.75 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 0.06 6/26/07 7/15/07 19 0.21 7/16/07 8/4/07 19 1.59 8/5/07 8/24/07 19 12.53 8/25/07 9/13/07 19 4.00 Ps. ferox 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 <0.01 6/26/07 7/15/07 19 <0.01 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 0.05 8/25/07 9/13/07 19 <0.01

PAGE 151

151 Table B-1. Continued. Species Date Trap Night Total/Trap Night Ur. lowii 7/21/06 8/14/06 16 0.13 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 <0.01 6/26/07 7/15/07 19 <0.01 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 <0.01 8/25/07 9/13/07 19 <0.01 Ur. sapphirina 7/21/06 8/14/06 16 <0.01 5/5/07 6/5/07 15 <0.01 6/6/07 6/25/07 16 <0.01 6/26/07 7/15/07 19 <0.01 7/16/07 8/4/07 19 <0.01 8/5/07 8/24/07 19 0.05 8/25/07 9/13/07 19 <0.01 Note: Ae. = Aedes; An. = Anopheles ; Cq = Coquillettidia ; Cx. = Culex; Ma. = Mansonia; Oc = Ochlerotatus; Ps. = Psor ophora; Ur. = Uranotaenia One modified CDC trap + CO2 (250 ml/min). When N < 20, traps had malfunctioned.

PAGE 152

152 Table B-2. Mosquitoes captured in a modified CDC light trap at the Prairie Oaks subdivision from July August 2006 and May Sept. 2007 near Gainesville, FL. Species Date Trap Night Total/Trap Night Ae. albopictus 7/21/06 8/14/06 36 0.13 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 0.08 7/16/07 8/4/07 37 0.45 8/5/07 8/24/07 40 1.18 8/25/07 9/13/07 38 0.50 Ae. vexans 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 6.11 6/6/07 6/25/07 34 9.68 6/26/07 7/15/07 40 13.50 7/16/07 8/4/07 37 7.64 8/5/07 8/24/07 40 25.75 8/25/07 9/13/07 38 6.63 An. crucians 7/21/06 8/14/06 36 25.83 5/5/07 6/5/07 36 16.86 6/6/07 6/25/07 34 2.24 6/26/07 7/15/07 40 2.68 7/16/07 8/4/07 37 1.09 8/5/07 8/24/07 40 0.93 8/25/07 9/13/07 38 0.24 An. quadrimaculatus 7/21/06 8/14/06 36 2.10 5/5/07 6/5/07 36 0.33 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 0.03 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 <0.01 8/25/07 9/13/07 38 <0.01

PAGE 153

153 Table B-2. Continued. Species Date Trap Night Total/Trap Night Cq. perturbans 7/21/06 8/14/06 36 73.77 5/5/07 6/5/07 36 51.69 6/6/07 6/25/07 34 21.88 6/26/07 7/15/07 40 18.35 7/16/07 8/4/07 37 8.82 8/5/07 8/24/07 40 5.88 8/25/07 9/13/07 38 7.95 Cx. erraticus 7/21/06 8/14/06 36 216.47 5/5/07 6/5/07 36 3.50 6/6/07 6/25/07 34 1.26 6/26/07 7/15/07 40 0.78 7/16/07 8/4/07 37 0.42 8/5/07 8/24/07 40 0.98 8/25/07 9/13/07 38 1.03 Cx. nigripalpus 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 0.03 6/6/07 6/25/07 34 1.85 6/26/07 7/15/07 40 9.33 7/16/07 8/4/07 37 3.00 8/5/07 8/24/07 40 32.75 8/25/07 9/13/07 38 140.82 Cx. quinquefasciatus 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 <0.01 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 0.05 8/25/07 9/13/07 38 <0.01 Cx. salinarius 7/21/06 8/14/06 36 5.20 5/5/07 6/5/07 36 1.39 6/6/07 6/25/07 34 1.82 6/26/07 7/15/07 40 1.75 7/16/07 8/4/07 37 0.52 8/5/07 8/24/07 40 3.30 8/25/07 9/13/07 38 1.71

PAGE 154

154 Table B-2. Species Date Trap Night Total/Trap Night Ma. titillans 7/21/06 8/14/06 36 7.50 5/5/07 6/5/07 36 0.31 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 0.03 7/16/07 8/4/07 37 0.15 8/5/07 8/24/07 40 0.15 8/25/07 9/13/07 38 0.13 Oc. canadensis 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 0.03 6/26/07 7/15/07 40 0.03 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 <0.01 8/25/07 9/13/07 38 <0.01 Oc. fulvus pallens 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 <0.01 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 <0.01 8/25/07 9/13/07 38 <0.01 Oc. infirmatus 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 <0.01 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 0.08 8/25/07 9/13/07 38 0.03 Oc. sollicitans 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 <0.01 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 <0.01 8/25/07 9/13/07 38 <0.01

PAGE 155

155 Table B-2. Species Date Trap Night Total/Trap Night Oc. taeniorhynchus 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 0.03 6/26/07 7/15/07 40 0.05 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 0.03 8/25/07 9/13/07 38 <0.01 Oc. triseriatus 7/21/06 8/14/06 36 0.77 5/5/07 6/5/07 36 0.06 6/6/07 6/25/07 34 0.21 6/26/07 7/15/07 40 0.20 7/16/07 8/4/07 37 0.12 8/5/07 8/24/07 40 0.03 8/25/07 9/13/07 38 <0.01 Ps. ciliata 7/21/06 8/14/06 36 <0.01 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 0.06 6/26/07 7/15/07 40 <0.01 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 0.03 8/25/07 9/13/07 38 <0.01 Ps. columbiae 7/21/06 8/14/06 36 0.27 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 0.25 7/16/07 8/4/07 37 0.09 8/5/07 8/24/07 40 0.60 8/25/07 9/13/07 38 0.03 Ps. ferox 7/21/06 8/14/06 36 0.17 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 0.09 6/26/07 7/15/07 40 0.28 7/16/07 8/4/07 37 0.58 8/5/07 8/24/07 40 1.83 8/25/07 9/13/07 38 2.53

PAGE 156

156 Table B-2. Species Date Trap Night Total/Trap Night Ur. lowii 7/21/06 8/14/06 36 0.93 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 <0.01 7/16/07 8/4/07 37 <0.01 8/5/07 8/24/07 40 <0.01 8/25/07 9/13/07 38 <0.01 Ur. sapphirina 7/21/06 8/14/06 36 11.20 5/5/07 6/5/07 36 <0.01 6/6/07 6/25/07 34 <0.01 6/26/07 7/15/07 40 0.05 7/16/07 8/4/07 37 0.03 8/5/07 8/24/07 40 <0.01 8/25/07 9/13/07 38 <0.01 Ae. = Aedes; An. = Anopheles ; Cq = Coquillettidia ; Cx. = Culex ; Ma. = Mansonia; Oc = Ochlerotatus; Ps. = Psor ophora; Ur. = Uranotaenia Two modified CDC traps + CO2 (250 ml/min). When N < 40, traps had malfunctioned.

PAGE 157

157 APPENDIX C RESPONSE OF PREVITELLOGENIC AND VITELLOGENIC ANOPHELES QUADRIMACULA TUS TO SELECTED LED WAVELENGTHS USING A VISUALOMETER IN A PAIR-T AND OPEN-PORT DESIGN Table C-1. Evaluation of previtellogenic Anopheles quadrimaculatus attraction to four selected wavelengths of light emitting diodes using an open-port visualometer. Diode Wavelength Trial Blue Green Red IR No Light Trial Mean M020507 <0.0001 0.2588 0.0950 0.1238 0.0275 0.1010 M022007 0.0138 0.3588 0.1100 0.2563 0.0963 0.8350 M022907 0.7625 0.3963 0.0963 0.0963 <0.0001 0.6650 M030607 0.1375 0.3425 0.3713 0.1225 0.2613 1.2350 M041707 0.4600 0.0900 0.2875 0.0400 0.2575 1.1350 M050707 <0.0001 0.3688 0.0813 0.0413 0.7050 1.1963 M061107 <0.0001 0.2763 0.0138 0.3138 0.0950 0.6988 M061207 <0.0001 <0.0001 0.5463 0.0550 0.0550 0.6563 M061907 0.1100 0.1788 0.2450 0.3725 0.0138 0.9200 Note: Means = total contact seconds per treatment over eight hour trials. Trials used were selected from a pool of 17 open-port visualometer trials with previtellogenic An. quadrimaculatus. Trials excluded denoted mean contac t seconds not within % of the group mean contact seconds. Contact second av erages above 50% of total trial means implied sensor malfunction; contact-second averages below 50% of total trial means implied poor mosquito quality. Blue = 470 nm, Green = 502 nm, IR = 860 nm, Red = 660 nm and no light constituted an unlit control treatment. Trial means = total contact seconds per trial.

PAGE 158

158 Table C-2. Evaluation of vitellogenic Anopheles quadrimaculatus attraction to four selected wavelengths of light emitting diodes using an open-port visualometer. Diode Wavelength Trial Blue Green Red IR No Light Trial Mean M030207 0.1788 0.0413 <0.0001 0.2050 0.0550 0.4800 M032907 0.2463 0.0550 0.1513 0.0838 <0.0010 0.5363 M050507 <0.0001 0.2738 0.0138 0.1225 0.0688 0.4788 M060707 <0.0001 0.1225 <0.0001 0.0275 0.1225 0.4513 M061507 0.1375 <0.0001 0.4000 0.1100 0.0550 0.7025 M062107 0.4388 0.0138 0.2925 0.1663 0.1088 1.0200 M062207 0.2475 <0.0001 0.1000 0.1238 0.2838 0.7550 M062907 0.0413 0.1375 0.2313 0.1650 0.2450 0.8200 Note: Means = total contact s econds per treatment over eight hour trials.Trials used were selected from a pool of 14 open-port visu alometer trials ran with vitellogenic An. quadrimaculatus. Trials excluded denoted mean contac t seconds not within % of the group mean contact seconds. Contact second av erages above 50% of total trial means implied sensor malfunction; contact-second averages below 50% of total trial means implied poor mosquito quality. Blue = 470 nm, Green = 502 nm, IR = 860 nm, Red = 660 nm and no light constituted an unlit control treatment. Trial means = total contact seconds per trial.

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159 Table C-3. Previtellogenic Anopheles quadrimaculatus attraction to 470 nm and 660 nm wavelengths of light emitting diodes using a paired-T port visualometer. Diode Wavelength Trial Replication Blue Red 1 1 0.0256 0.0122 2 0.0000 0.0244 3 0.0244 0.0489 4 0.0989 0.0244 5 0.0489 0.0000 2 6 0.1222 0.0244 7 0.0000 0.0611 8 0.0611 0.0489 9 0.2811 0.0600 10 0.2811 0.0000 3 11 0.0611 0.0122 12 0.0489 0.0856 13 0.0000 0.0611 14 0.0489 0.0244 15 0.0489 0.0000 4 16 0.0367 0.0000 17 0.0122 0.0489 18 0.0856 0.0367 19 0.0856 0.2811 20 0.0122 0.0000 Note: Means = total contact seconds per treatment over eight hour trials. Each trial included five replications. Blue = 470 nm and Red = 660 nm.

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160 Table C-4. Vitellogenic Anopheles quadrimaculatus attraction to 470 nm and 660 nm wavelengths of light emitting diodes using a paired-T port visualometer. Diode Wavelength Trial Replication Blue Red 1 1 0.0367 0.1800 2 0.0122 0.0000 3 0.0611 0.0967 4 0.2789 0.2522 5 0.0733 0.0000 2 6 0.0733 0.0000 7 0.0122 0.1800 8 0.1344 0.0856 9 0.0489 0.0722 10 0.0978 0.0000 3 11 0.1344 0.0000 12 0.0122 0.0122 13 0.0122 0.0489 14 0.0722 0.0000 15 0.0000 0.1344 4 16 0.1344 0.0000 17 0.1344 0.1778 19 0.0122 0.2522 20 0.0367 0.0000 Note: Means = total contact seconds per treatment over eight hour tr ials. Each trial included five replications. Replication not in cluded (18) denoted no mosquito contact activity on sensors over blue or green diodes. Blue = 470 nm and Red = 660 nm.

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161 Table C-5. Previtellogenic Anopheles quadrimaculatus attraction to 470 nm and 502 nm wavelengths of light emitting diodes using a paired-T port visualometer. Diode Wavelength Trial Replication Blue Green 1 1 0.0856 0.0000 2 0.0000 0.0856 3 0.0611 0.0489 4 0.1811 0.0244 5 0.1788 0.0000 2 7 0.0000 0.0244 8 0.0367 0.0733 9 0.0367 0.0611 3 12 0.0000 0.0122 13 0.0000 0.0000 14 0.0367 0.0122 15 0.0122 0.0000 4 18 0.0000 0.0244 19 0.0000 0.0122 20 0.0122 0.0000 5 21 0.0122 0.0000 23 0.0122 0.0000 25 0.0244 0.0000 6 27 0.0000 0.0122 28 0.0489 0.0000 29 0.0000 0.0122 30 0.0856 0.0000 7 31 0.0122 0.0000 32 0.0000 0.0122 33 0.0000 0.1578 34 0.0000 0.0244 35 0.0122 0.0000 Note: Means = total contact seconds per treatment over eight hour tr ials. Each trial included five replications. Replications not included denoted no mosquito cont act activity on sensors over blue or green diodes. Blue = 470 nm and Green = 502 nm.

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162 Table C-6. Vitellogenic Anopheles quadrimaculatus attraction to 470 nm and 502 nm wavelengths of light emitting diodes using a paired-T port visualometer. Mean Contact Seconds Trial Replication Blue Green 1 1 0.0244 0.0244 2 0.0000 0.1667 3 0.0000 0.0856 4 0.1100 0.0600 5 0.0489 0.0000 2 9 0.2289 0.0000 10 0.0378 0.0000 3 12 0.0000 0.0478 13 0.0122 0.0244 14 0.0244 0.0000 4 16 0.0122 0.0000 18 0.0244 0.0122 19 0.0367 0.0244 5 22 0.0000 0.0611 23 0.0122 0.0367 24 0.0122 0.0000 25 0.0122 0.0000 6 27 0.0000 0.0122 28 0.0244 0.0367 29 0.1100 0.2022 30 0.0244 0.0000 7 32 0.0000 0.1100 33 0.0000 0.0000 34 0.0967 0.0000 35 0.0122 0.0000 Note: Means = total contact seconds per treatment over eight hour trials. Each trial included five replications. Replications not included denoted no mosquito cont act activity on sensors over blue or green diodes. Blue = 470 nm and Green = 502 nm.

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177 BIOGRAPHICAL SKETCH Michael Thom as Bentley was born on October 18th, 1982, in Noblesville, Indiana. He is the younger of two children, born to Mike and Jill Bentley. He and his family moved to Vero Beach, FL, where he graduated from Vero B each High School in 2001. His education continued at the University of Florida where he got his bachelors degree in criminology in fall, 2005. Remaining at the University of Florida, Mr. Be ntley was accepted into the entomology graduate program under Dr. Phillip Kaufma n with a specialization in medi cal and veterinary entomology. He worked as the Entomology and Nematology de partments outreach coordinator while earning his degree, before graduating with his Master of Science in spri ng, 2008. Mike will be married to his fiance, Kristina Pein, October 17th, 2008, after which he plans to pursue a career in industry.


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