BEHAVIORAL PHOTOTAXIS OF PREVITELLOGENIC AND …ufdcimages.uflib.ufl.edu/UF/E0/02/19/67/00001/bentley_m.pdfbehavioral phototaxis of previtellogenic and vitellogenic mosquitoes (diptera:

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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

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© 2008 Michael Thomas Bentley

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

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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

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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 fiancée 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.

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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.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...............................................................................................................4 

LIST OF TABLES.........................................................................................................................10 

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

ABSTRACT...................................................................................................................................15

CHAPTER

1 LITERATURE REVIEW OF MOSQUITO BIOLOGY, IMPORTANCE AND SURVEILANCE.....................................................................................................................17 

Introduction to Mosquitoes.....................................................................................................17 Life Cycle ...............................................................................................................................17 

Egg...................................................................................................................................17 Larva................................................................................................................................18 Pupa .................................................................................................................................19 Adult ................................................................................................................................20 

Habitat.....................................................................................................................................21 Medical and Economic Importance ........................................................................................25 Vector Surveillance and Monitoring ......................................................................................30 

Methodology....................................................................................................................30 Species Diversity .............................................................................................................31 Flight Range and Habits ..................................................................................................31 Resting Behavior .............................................................................................................34 Population Monitoring.....................................................................................................35 Mosquito Attraction.........................................................................................................38 

2 RESPONSE OF ADULT MOSQUITOES TO LIGHT EMITTING DIODES PLACED IN RESTING BOXES ............................................................................................................42 

Introduction.............................................................................................................................42 Materials and Methods ...........................................................................................................44 

Resting Boxes..................................................................................................................44 Light Emitting Diodes and Battery Supplies...................................................................45 CDC Light Trap...............................................................................................................46 Site and Resting Box Location ........................................................................................46 Methodology....................................................................................................................47 Statistical Analysis ..........................................................................................................48 

Results.....................................................................................................................................49 Discussion...............................................................................................................................52 

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3 FIELD RESPONSE OF ADULT MOSQUITOES TO WAVELENGTHS OF LIGHT EMITITING DIODES............................................................................................................70 

Introduction.............................................................................................................................70 Materials and Methods ...........................................................................................................72 

Diode Equipped Boxes ....................................................................................................72 Light Emitting Diodes and Battery Supplies...................................................................73 Sticky Cards.....................................................................................................................73 CDC Light Trap...............................................................................................................74 Site and Sticky Card Trap Location ................................................................................74 Methodology....................................................................................................................76 Statistical Analysis ..........................................................................................................77 

Results.....................................................................................................................................77 Discussion...............................................................................................................................80 

4 RESPONSES OF PREVITELLOGENIC AND VITELLOGENIC ANOPHELES QUADRIMACULATUS TO SELECTED WAVELENGTHS PRODUCED BY LIGHT EMITTING DIODE................................................................................................................98 

Introduction.............................................................................................................................98 Materials and Methods .........................................................................................................102 

Visualometer..................................................................................................................102 Light Emitting Diodes ...................................................................................................103 Mosquitoes ....................................................................................................................103 Open-Port Visualometer Trials......................................................................................104 Paired-T Port Visualometer Trials.................................................................................105 Methodology..................................................................................................................105 Statistical Analysis ........................................................................................................106 

Results...................................................................................................................................106 Open-Port Visualometer ................................................................................................106 Paired-T Port Visualometer...........................................................................................107 

Discussion.............................................................................................................................108 

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

APPENDIX

A RESTING BOX AND MODIFIED CDC LIGHT-TRAP CAPTURES OF MOSQUITOES BY LOCATION.........................................................................................122 

B STICKY CARD AND MODIFIED CDC LIGHT-TRAP CAPTURES OF MOSQUITOES 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 

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LIST OF REFERENCES.............................................................................................................163 

BIOGRAPHICAL SKETCH .......................................................................................................177 

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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 Gainesville, FL...................................................................................................................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 Gainesville, FL..................................................................................................61 

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...................................................................................................................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-1 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 

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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 

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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 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 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 I-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, Alachua Co., 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 

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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 treatments 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 two 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 I-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, Alachua Co., 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 

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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.) Southwest 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 – September 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 ºC 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 occured 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 

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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.

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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.

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CHAPTER 1 LITERATURE REVIEW OF MOSQUITO BIOLOGY, IMPORTANCE AND SURVEILANCE

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 Culex pipiens

quinquefasciatus Say (Chernin 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

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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 ºC), 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

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

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 of Anopheles 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

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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 ºC. 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

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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 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 emerge continuously over the course of the summer

(Bosak and Crans 2002).

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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. 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 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

23

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).

24

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

25

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 Ochlerotatus japonicus 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,

26

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 Culex pipiens 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 protists that cause human malaria including Plasmodium

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 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

28

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.

29

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).

30

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

31

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 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 (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

32

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 of Aedes 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,

33

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 of Ae. aegypti, upper

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

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

34

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

flavescens Müller 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

35

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. walkeri 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,

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 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

37

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

38

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

39

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

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 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 of Ae. 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 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

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 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 of Ae. 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).

42

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.

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 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

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 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 (¼ 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 two coats of flat black exterior latex paint, and interiors with

two coats of barn red exterior latex paint. A 0.64 cm (¼ 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

45

Lake City, UT), protecting the battery supply and LED wiring. A 0.64 cm (¼ in) hole drilled

through the container lids to correspond to the 0.64 cm (¼ 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 2-1b).

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 Plexiglas™ 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 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 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 30o except for IR 860 (20o).

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

46

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 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 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

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 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

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 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 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 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

49

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

Test (α = 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

50

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.

51

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).

52

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.

53

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

54

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)

55

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, CO2, 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

56

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.

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 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 (CO2) 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

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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, Quiñones 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

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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.

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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.

Diode Wavelength Species TN Blue Green Red IR No Light An. quadrimaculatus ♀ 1,268 0.004 (±0.003)b 0.013 (±0.007)b 0.032 (±0.013)a 0.007 (±0.005)b 0.015 (±0.005)b

Cq. perturbans 2006 ♂ 148 0.372 (±0.052) 0.291 (±0.052) 0.250 (±0.050) 0.230 (±0.050) 0.270 (±0.063)

Cq. perturbans 2007 ♂ 320 0.003 (±0.003) 0.006 (±0.004) 0.009 (±0.005) 0.003 (±0.003) 0.006 (±0.004)

Cq. perturbans ♀ 788 0.023 (±0.006) 0.022 (±0.007) 0.037 (±0.008) 0.030 (±0.007) 0.028 (±0.008)

Cx. erraticus 2006 ♂ 288 2.154 (±0.333) 2.452 (±0.396) 3.009 (±0.453) 3.868 (±0.617) 3.154 (±0.501)

Cx. erraticus 2007 ♂ 1,120 0.022 (±0.005) 0.028 (±0.006) 0.024 (±0.005) 0.021 (±0.005) 0.032 (±0.007)

Cx. erraticus 2006 ♀ 148 2.851 (±0.558) 2.980 (±0.546) 3.223 (±0.616) 3.967 (±0.710) 3.932 (±0.740)

Cx. erraticus 2007 ♀ 1,120 0.113 (±0.012)a 0.087 (±0.011)a 0.083 (±0.010)a 0.038 (±0.007)b 0.104 (±0.012)a

Cx. nigripalpus ♀ 468 0.017 (±0.011) 0.017 (±0.010) 0.004 (±0.003) 0.009(±0.007) <0.001 (<0.001)

Cx. salinarius ♂ 388 0.005 (±0.004) 0.028 (±0.015) 0.005 (±0.004) 0.015 (±0.009) 0.015 (±0.009)

Cx. salinarius ♀ 468 0.006 (±0.004) 0.009 (±0.005) 0.002 (±0.002) 0.004 (±0.003) 0.006 (±0.004)

Ma. titillans ♀ 148 0.014 (±0.014) 0.027 (±0.013) 0.020 (±0.012) 0.027 (±0.014) 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).

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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 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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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/trap/trap night = total mosquitoes captured/ # of trap nights.

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A 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.

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A 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 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 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.

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.

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Figure 2-4. Aerial view of Horse Teaching Unit location. The unit is located east of I-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.

Paine’s Prairie Preserve

HTU

Prairie View Subdivision

Paine’s Prairie Preserve

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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.

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

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.

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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.

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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 Administration (NOAA) database. A) Monthly temperature, May – September 2006 and 2007. B) Monthly precipitation from Jan – September 2006 and 2007.

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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

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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

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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 (¼ 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

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0.64 cm (¼ 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 (¾ in) inner-diameter PVC pipe. PVC pipe sections,

subsequently, were supported by a 120 cm length of 1.27 cm (½ 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 30o except for IR (20o).

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 (¼ in) diameter

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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 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), 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

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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 I-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

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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

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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 MS® 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 (α=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

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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 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).

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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 of Ma. 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-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 all mosquitoes captured (64,893). Combined,

both CDC traps placed at the PO location accounted for only 26,873 mosquitoes

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(Appendices B-1, 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.

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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

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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. 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 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

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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

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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 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 rates for mosquito species.

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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.

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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 (CO2) 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

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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.

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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.

Diode Wavelength

Species TN Blue Green Red IR

Ae. vexans ♀ 640 0.019 (±0.006)a 0.013 (±0.004)ab 0.002 (±0.002)b <0.001 (±<0.001)b

Cq. perturbans ♂ 560 0.014 (±0.006)b 0.043 (±0.010)a 0.011 (±0.006)ab 0.016 (±0.005)b

Cq. perturbans ♀ 720 0.031 (±0.007)ab 0.049 (±0.009)a 0.024 (±0.007)b 0.008 (±0.003)b

Cx. erraticus ♀ 320 0.034 (±0.012)a 0.016 (±0.007)ab 0.013 (±0.006)ab 0.003 (±0.003)b

Cx. nigripalpus ♀ 640 0.019 (±0.007)a 0.016 (±0.005)ab 0.002 (±0.002)b <0.001 (±<0.001)b

Cx. salinarius ♀ 320 0.016 (±0.007) 0.010 (±0.005) <0.001 (±<0.001) <0.001 (±<0.001)

Ma. titillans 2006 ♀ 80 0.313 (±0.068)a 0.363 (±0.110)a 0.100 (±0.038)b 0.050 (±0.025)b

Ma. titillans 2007 ♀ 160 0.006 (±0.006) 0.006 (±0.006) 0.006 (±0.006) <0.001 (±<0.001)

Oc. infirmatus ♀ 720 0.014 (±0.004)a 0.004 (±0.002)ab 0.003 (±0.002)b 0.001 (±0.001)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 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 ♂ (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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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 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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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/trap/trap night = total mosquitoes captured/ # of trap nights.

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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.

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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.

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Figure 3- 4. Aerial view of Horse Teaching Unit location. The unit is located east of I-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.

HTU

Paine’s Prairie Preserve

Prairie View Subdivision

Paine’s Prairie Preserve

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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.

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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.

Southeast Site

Southwest Site

Northeast Site

Northwest Site

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A B

C D 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.

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A

B Figure 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 – September 2006 and 2007.

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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 of Aedes

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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 reenters 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

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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

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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 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 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

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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 4-1a).

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 28° and 32° C. This room was designed to protect against outside

electrical interference and extraneous sources of light. All visualometer surfaces were kept free

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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 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 30o except for IR (20o). 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 ºC 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 ºC and 75% humidity under a 14:10

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(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 4-1a,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

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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

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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

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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

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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.

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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.

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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 180°. 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 30°. 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

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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.

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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 (±0.0526)bNo 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 (±0.0138) Vitellogenic 4 Blue 0.0723 (±0.0158) Red 0.0785 (±0.0209) Blue:Green Previtellogenic 7 Blue 0.0314 (±0.0096) Green 0.0221 (±0.0069) Vitellogenic 7 Blue 0.0346 (±0.0104) 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.

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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.

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C 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 ºC and 74% humidity under a 14:10 (L:D) photoperiod. Upon eclosion, adult mosquitoes were fed a 10% sugar solution.

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Figure 4-3. Blood feeding Anopheles quadrimaculatus occured 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.

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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

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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)

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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

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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 (CO2) 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

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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

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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.

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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 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

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

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

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

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

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

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.

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

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

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

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

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

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

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.

136

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

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

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

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

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

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. = Psorophora; Ur. = Uranotaenia. One modified CDC trap + CO2 (250 ml/min). When N < 20, traps had malfunctioned.

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

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

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

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

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. = Psorophora; Ur. = Uranotaenia. Two modified CDC traps + CO2 (250 ml/min). When N < 40, traps had malfunctioned.

147

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 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

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

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

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

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. = Psorophora; Ur. = Uranotaenia. One modified CDC trap + CO2 (250 ml/min). When N < 20, traps had malfunctioned.

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

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

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

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

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. = Psorophora; Ur. = Uranotaenia. Two modified CDC traps + CO2 (250 ml/min). When N < 40, traps had malfunctioned.

157

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.

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 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.

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.

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 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.

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 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.

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 contact activity on sensors over blue or green diodes. Blue = 470 nm and Green = 502 nm.

163

LIST OF REFERENCES

Adams, A. J. 1971. A revision of the crabhole mosquitoes of the genus Deinocerites. Contrib. of the Amer. Entomol. Inst. 7: 2.

Acree, F., R. B. Turner, H. K. Gouck, M. Beroza and M. Smith. 1968. L-lactic Acid: A mosquito attractant isolated from humans. Sci. 161: 1346-47.

Ali, A., J. K. Nayar, J. W. Knight and B. H. Stanley. 1989. Attraction of Florida mosquitoes (Diptera: Culicidae) to artificial light in the field. Proc. Calif. Mosq. Control Assoc. 57, 82–88.

Allan, S. A. 1994. Physics of mosquito vision – an overview. J. Am. Mosq. Control Assoc. 10: 266-271.

Allan, S. A. and D. Kline. 2004. Evaluation of various attributes of gravid female traps for collection of Culex in Florida. J. Vect. Ecol. 29: 285-294.

Allan, S. A., J. F. Day and J. D. Edman. 1987. Visual ecology of biting flies. Ann. Rev. Entomol. 32: 297-316.

Allan, S. A. and J. G. Stoffolano. 1986a. Effects of background contrast on visual attraction and orientation of Tabanus nigrovittatus (Diptera: Tabanidae). Environ. Entomol. 15: 689-694.

Allan, S. A. and J. G. Stoffolano. 1986b. Effects of hue and intensity on visual attraction of adult Tabanus nigrovittatus (Diptera: Tabanidae). J. Med. Entomol. 23: 83-91.

American Mosquito Control Association. 2007. Mosquito lifecycle. AMCA. Last retrieved March 22, 2007, in http://www.mosquito.org/mosquito-information/lifecycle.aspx.

Anderson, R. A., J. D. Edman and T. W. Scott. 1990. Rubidium and cesium as host blood-markers to study multiple blood feeding by mosquitoes (Diptera: Culicidae). J. Med. Entomol. 27: 999-1001.

Ando, D. J. and M. J. Thomas. 1996. Ultraviolet and visible spectroscopy: analytical chemistry by open learning, 2nd ed. John Wiley & Sons Inc, West Sussex, England.

Andreadis, T. G., J. F. Anderson, L. E. Munstermann, R. J. Wolfe and D. A. Florin. 2001. Discovery, distribution, and abundance of the newly introduced mosquito Ochlerotatus japonicus (Diptera: Culicidae) in Connecticut, USA. J. Med. Entomol. 38: 774-779.

Bargren, W. C. and C. Nibley. 1956. Comparative attractiveness of colored lights of equal intensity to specific species of mosquitoes. 3rd Area Med. Lab, Ft. McPherson, GA.

Barr, A. R., Smith, T. A., Boreham, M. and K. E. White. 1963. Evaluation of some factors affecting the efficiency of light traps on colleting mosquitoes. J. Econ. Entomol. 56: 123-27.

164

Beck, A. F. and E. C. Turner, Jr. 1985. A comparison of five house-fly (Diptera: Muscidae) population monitoring techniques. J. Med. Entomol. 22: 346-348.

Beerntsen, B. T., A. J. James and B. M. Christensen. 2000. Genetics of mosquito vector competence. Microbiol. Molec. Biol. Rev. 64: 115-137.

Belkin, J. N. and C. L. Hogue. 1959. A review of crab hole mosquitoes of the genus Deinocerites (Diptera: Culicidae). Univ. of California Pub. in Entomol. 14: 411-458.

Belton, P. 1967. The effect of illumination and pool brightness on oviposition by Culex restuans (Theo.) in the field. Mosq. News. 27: 66-68.

Bentley, M. D. and J. F. Day. 1989. Chemical ecology and behavioral aspects of mosquito oviposition. Ann. Rev. Entomol. 34: 401-421.

Beresford, D. V. and J. F. Sutcliffe. 2006. Studies on the effectiveness of coroplast sticky traps for sampling stable flies (Diptera: Muscidae), including a comparison to alsynite. J. Econ. Entomol. 99: 1025-1035.

Bidlingmayer, W. L. 1967. A comparison of trapping methods for adult mosquitoes: species response and environmental influence. J. Med. Entomol. 4: 200-220.

Bidlingmayer, W. L. 1985a. The measurement of adult mosquito population changes – some considerations. J. Am. Mosq. Control Assoc. 1: 328-348.

Bidlingmayer, W. L. 1985b. Preliminary study of the effects of wind velocities and wind shadows upon suction trap catches of mosquitoes (Diptera: Culicidae). J. Med. Entomol. 22: 295-302.

Bidlingmayer, W. L. 1994. How mosquitoes see traps: role of visual response. J. Am. Mosq. Control Assoc. 10: 272-279.

Bosak, P. J. and W. J. Crans. 2002. The structure and function of the larval siphon of Coquillettidia perturbans (Walker). J. Am. Mosq. Contr. Assoc. 18: 280-283.

Boyd, M. F. 1930. An introduction to malariology. Harvard University Press, Cambridge, MA.

Bracken, G. K., W. Hanec and A. J. Thorsteinson. 1962. The orientation of horseflies and deerflies (Tabanidae: Diptera). II. The role of some visual factors in the attractiveness of decoy silhouettes. Can. J. Zool. 40: 685-695.

Bradbury, W. C. and G. F. Bennett. 1974. Behavior of adult Simuliidae (Diptera). I. Response to color and shape. Can. J. Zool. 52: 251-259.

Bradley, G. H. 1943. Determination of densities of populations of Anopheles quadrimaculatus on the wing. Proc. New Jers. Mosq. Exterm. Assoc. 30: 22-27.

165

Breeland, S. G. 1982. Bibliography and notes on Florida mosquitoes with limited distribution in the United States. Mosq. Systematics. 14: 53-72.

Breeland, S. G. and E. Beck. 1994. Public health pest control applicator training manual. U.S. EPA Pub.

Brett, G. A. 1938. On the relative attractiveness to Aedes aegypti of certain coloured cloths. Trans. R. Soc. Trop. Med. Hyg. 32: 113-124.

Brown, A. W. A. 1951. Studies of the responses of the female Aedes mosquito. Part IV. Field experiments on Canadian species. Bull. Entomol. Res. 42: 575-582.

Brown, A. W. A., D. S. Sarkaria and R. P. Thompson. 1951. Studies on the responses of the female Aedes mosquito. Part I. The search for attractant vapours. Bull. Entomol. Res. 42: 105-114.

Brown, M. R., R. Graf and K. Swiderek. 1995. Structure and function of mosquito gonadotropins. In Molecular mechanisms of insect metamorphosis and diapause. Industrial Publishing and Consulting. Tokyo. pp. 229-238.

Browne, S. M. and G. F. Bennett. 1980. Color and shape as mediators of host-seeking responses of simuliids and tabanids (Diptera) in the Tantramar marshes, New Brunswick, Canada. J. Med. Entomol. 17: 58-62.

Browne, S. M. and G. F. Bennett. 1981. Response of mosquitoes (Diptera: Culicidae) to visual stimuli. J. Med. Entomol. 18: 505-521.

Brust, R. A. 1980. Dispersal behavior of adult Aedes sticticus and Aedes vexans (Diptera: Culicidae) in Manitoba. Can. Entomol. 112: 31-42.

Burbutis, P. P. and D. M. Jobbins. 1958. Studies on the use of a diurnal resting box for the collection of Culiseta melanura (Coquillett). Bull. Brooklyn Entomol. Soc. 53: 53-58.

Burkett, D. A and J. F. Butler. 2005. Laboratory evaluation of colored light as an attractant for female Aedes aegypti, Aedes albopictus, Anopheles quadrimaculatus and Culex nigripalpus. Fla. Entomol. 88: 383-389.

Burkett, D. A., J. F. Butler and D. L. Kline. 1998. Field evaluation of colored light-emitting diodes as attractants for woodland mosquitoes and other Diptera in north central Florida. J. Am. Mosq. Control Assoc. 14: 186-195.

Burkett, D. A., W. J. Lee, K. W. Lee, H. C. Kim, H. I. Lee, J. S. Lee, E. H. Shin, R. A. Wirtz, H. E. Cho, D. M. Claborn, R. E. Coleman and T. A. Klein. 2001. Light, carbon dioxide, and octenol-baited mosquito trap and host-seeking activity evaluations for mosquitoes in a malarious area of the Republic of Korea. J. Am. Mosq. Control Assoc. 17: 196-205.

166

Centers for Disease Control and Prevention (CDC). 2007a. Filariasis Fact Sheet. Last retrieved March 16, 2007, from http://www.cdc.gov/ncidod/dpd/parasites/lymphaticfilariasis/factsht_lymphatic_filar.htm.

Centers for Disease Control and Prevention (CDC). 2007b. Malaria Fact Sheet. Last retrieved March 20, 2007, from http://www.cdc.gov/malaria/facts.htm.

Centers for Disease Control and Prevention (CDC). 2007c. Dengue and Dengue Hemorrhagic Fever Fact Sheet. Last retrieved April 2, 2007, from http://www.cdc.gov/ncidod/dvbid.dengue/facts.htm.

Centers for Disease Control and Prevention (CDC). 2007d. Information on Arboviral Encephalitides. Last retrieved April 2, 2007, from http://www.cdc.gov/ncidod/dvbid/arbo/arbdet.htm.

Chernin, E. 1983. Sir Patrick Manson’s studies on the transmission and biology of filariasis. Rev. of Infect. Dis. 5: 148-168.

Clements, A. N. 1956. Hormonal control of ovary development in mosquitoes. J. Exp. Biol. 33: 211-223.

Clements, A. N. 1992. The biology of mosquitoes, Vol. 1. Development, nutrition and reproduction. Chapman and Hall, New York, NY.

Clements, A. N. 1999. The biology of mosquitoes, Vol. 2. Sensory reception and behavior. CABI Publishing, New York, NY.

Constantini, C. 1996. Introduction III: odors for host-finding mosquitoes. Ciba Foundation Symposia. 200: 124-131.

Coon, B. R. 2006. Field and laboratory studies of Culex erraticus (Diptera: Culicidae) ability to detect hosts, habitat identification and attempts at colonization. Ph.D. dissertation, University of Florida, Gainesville.

Crans, W. J. 1989. Resting boxes as mosquito surveillance tools, pp. 53-57. In Proceedings of the 82nd Ann. Meet. New Jers. Mosq. Control Assoc.

Crans, W. J. 1995. Resting boxes as mosquito surveillance tools. Proc. New Jers. Mosq. Control Assoc. 82: 53-56.

Crans, W. J. 2004. A classification system for mosquito life cycles: life cycle types for mosquitoes of the northeastern United States. J. Vector Ecol. 29: 1-10.

Cupp, E. W., K. Klingler, H. K. Hassan, L. M. Viguers and T. R. Unnasch. 2003. Transmission of eastern equine encephalomyelitis virus in central Alabama. Am. J. Trop. Med. Hyg. 68: 495-500.

167

Darsie, R. F., and C. D. Morris. 2003. Keys to the adult females and fourth instar larvae of the mosquitoes of Florida (Diptera, Culicidae), vol. 1. E. O. Painter Printing Company, Delon Springs, FL.

Darsie, R. F. and R. A. Ward. 2005. Identification and geographical distribution of the mosquitoes of North America, north of Mexico. University Press of Florida, Gainesville, FL.

Day, J. F., G. A. Curtis and J. D. Edman. 1990. Rainfall-directed oviposition behavior of Culex nigripalpus (Diptera: Culicidae) and its influence on St. ouis encephalitis virus transmission in Indian River County, Florida. J. Med. Entomol. 27: 43-50.

Day, J. F. and G. A. Curtis. 1994. When it rains, they soar – and that makes Culex nigripalpus a dangerous mosquito. Am. Entomol. 40: 162-167.

Day, J. F. 2005. Host-seeking strategies of mosquito disease vectors. J. Am. Mosq. Control Assoc. 21: 17-22.

Dekker, T. and W. Takken. 1998. Differential responses of mosquito sibling species Anopheles arabiensis and An. quadriannulatus to carbon dioxide, a man or a calf. Med. Vet. Entomol. 12: 136-140.

Disney, R. L. 1966. A trap for Phlebotomine sandflies attracted to rats. Bull. Ent. Res. 56: 445-451.

Edman, J., K. Pattamaporn, K. Linthicum and T. Scott. 1997. Attractant resting boxes for rapid collection and surveillance of Aedes aegypti (L.) inside houses. J. Am. Mosq. Control Assoc. 13: 24-27.

Foley, D. H. and J. H. Bryan. 1991. Anopheles annulipes Walker (Diptera: Culicidae) at Griffith, New South Wales. 2. Biology and behavior of two sibling species. J. Aust. Ent. Soc. 30: 113-118.

Foote, R. H. and D. R. Cook. 1959. Mosquitoes of medical importance. Agriculture Handbook No. 152. Agricultural Research Service, USDA.

Forattini, O. P., M. A. M. Sallum, G. R. A. M. Marques and D. C. Flores. 1997. Description of the eggs of Anopheles (Kerteszia) Laneanus and Anopheles (Nyssorhynchus) Antunesi (Diptera: Culicidae) by scanning electron microscopy. J. Am. Mosq. Control Assoc. 13: 368-374.

Foster, W. A. and E. D. Walker. 2002. Mosquitoes (Culicidae), pp. 203-262. In G. Mullen and L. Durden (eds.), Medical and veterinary entomology. Academic Press. San Diego, California.

Fox, I. 1958. The mosquitoes of the international airport, Isla Verde, Puerto Rico, as shown by light traps. Mosq. News. 18: 117-124.

168

Gatehouse, A. G. 1972. Some responses of tsetse flies to visual and olfactory stimuli. Nat. New Biol. 236: 63-64.

Garret-Jones, C. 1964. The human blood index of malaria vectors in relation to epidemiological assessment. Bull. WHO. 30: 240-261.

Gaydecki, P. A. 1984. A quantification of the behaviourial dynamics of certain Lepidoptera in response to light. Ph.D. dissertation, Cransfield Institute of Technology, Ecological Physics Research Group, Bedford, England.

Geden, C. J. 2005. Methods for monitoring outdoor populations of house flies, Musca domestica L. (Diptera: Muscidae). J. Vector Ecol. 30: 244-250.

Gillies, M. T. and W. F. Snow. 1967. A CO2-baited sticky trap for mosquitoes. Trans. R. Soc. Trop. Med. Hyg. 61: 20.

Gillies, M. T. and T. J. Wilkes. 1974. The range of attraction of birds as baits for some West African mosquitoes (Diptera: Culicidae). Bull. Ent. Res. 63: 573-581.

Gillett, J. D. 1956. Initiation and promotion of ovarian development in the mosquito Aedes (Stegomyia) aegypti (Linnaeus). Ann. Trop. Med. Parasitol. 50: 375-380.

Gillett, J. D. 1972. The mosquito: its life, activities, and impact on human affairs. Doubleday, New York, NY.

Ginsberg, H. S. 1988. Survivorship of mosquitoes (Diptera: Culicidae) captured in CDC miniature light traps. Proc. Ann. Mtg. New Jers. Mosq. Control Assoc. 75: 86-92.

Gjullin, C. M., D. G. Brandl and J. J. O’Grady. 1973. The effect of colored lights and other factors on the numbers of Culex pipiens quinquefasciatus, C. tarsalis and Aedes sierrensis entering light traps. Mosq. News. 33: 67-71.

Goodwin, M. N. 1942. Studies on artificial resting places of Anopheles quadrimaculatus Say. J. Natl. Malar. Soc. 1:93-99.

Gordon, S. W., R. F. Tammeriello, K. J. Linthicum, R. A. Wirtz and J. P. Digoutte. 1991. Feeding patterns of mosquitoes collected in the Senegal river basin. J. Am. Mosq. Control. Assoc. 7: 424-432.

Gordon, W. M. and E. J. Gerberg. 1945. A directional mosquito barrier trap. J. Natn. Malar. Soc. 4: 123-125.

Gusciora, W. R. 1961. The resting box technique for sampling of Culiseta melanura (Coquillett). Proc. New Jers. Mosq. Exterm. Assoc. 48: 122-125.

Hagedorn, H. H., J. P. Shapiro and K. Hanaoka. 1979. Ovarian ecdysone secretion is controlled by a brain hormone in an adult mosquito. Nat. 282: 92-94.

169

Harbison, J. E., E. M. Mathenge, G. O. Misiani, W. R. Mukabana and J. F. Day. 2006. A simple method for sampling indoor-resting malaria mosquitoes Anopheles gambiae and Anopheles funestus (Diptera: Culicidae) in Africa. J. Med. Entomol. 43: 473-479.

Harwood, R. F. 1962. Trapping overwintering adults of the mosquitoes Culex tarsalis and Anopheles freeborni. Mosq. News. 22: 26-31.

Harwood, R. F. and W. R. Horsfall. 1959. Development, structure and function of coverings of eggs of floodwater mosquitoes. III Functions of coverings. Ann. Entomol. Soc. Am. 52: 113-116.

Haskell, P. T. 1966. Flight behavior. Symposium: Insect Behavior. Roy. Entomol. Soc. London. 3: 29-45.

Hayes, R. O., R. E. Bellamy, W. C. Reeves and M. J. Willis. 1958. Comparison of four sampling methods for measurement of Culex tarsalis adult populations. Mosquito News. 20: 174-178.

Haynes, K. F., T. A. Miller, M. P. Parrella and J. T. Trumble. 1986. Monitoring insecticide resistance with yellow sticky cards. Cal. Ag. November-December: 11-12.

Headlee, T. J. 1937. Some facts underlying the attraction of mosquitoes to sources of radiant energy. J. Econ. Entomol. 30: 309-312.

Hocking, B. 1953. The intrinsic range and speed of flight of insects. Trans. R. Entomol. Soc. Lond. 104: 223-345.

Hoel, D. 2005. Response of Aedes albopictus (Diptera: Culicidae) to traps, attractants, and adulticides in north central Florida. Ph.D. dissertation, University of Florida, Gainesville.

Hogsette, J. A, R. D. Jacobs and R. W. Miller. 1993. The sticky card: device for studying the distribution of adult house fly (Diptera: Muscidae) populations in closed poultry houses. J. Econ. Entomol. 86: 450-54.

Holck, A. R. and C. L. Meek. 1991. Comparison of sampling techniques for adult mosquitoes and other Nematocera in open vegetation. J. Ent. Sci. 26: 231-236.

Holton, A. C. 2007. Evaluation of different commercial lures and horse odors as an attractant and their abilities to increase mosquito trap numbers at the University of Florida Horse Teaching Unit. M. S. thesis, University of Florida, Gainesville.

Horsfall, W. R. 1954. A migration of Aedes vexans Meigen. J. Econ. Entomol. 47: 544.

Horsfall, W. R. and A. P. Morris. 1952. Surface conditions limiting larval sites of certain marsh mosquitoes. Am. Entomol. Soc. 45: 492-498.

170

Hulsman, K., P. R. Dale and B. H. Kay. 1989. The runnelling method of habitat modification: An environment-focused tool for salt marsh mosquito management. J. Am. Mosq. Control Assoc. 5: 226-234.

Huffaker, C. B. and R. C. Back. 1943. A study of methods of sampling mosquito populations. J. Econ. Entomol. 36: 561-569.

Husbands, R. C. 1976. Light traps and the significance of collection data. Bull. Soc. Vect. Ecol. 3: 17-26.

Hagmann, L. E. 1953. The biology of Mansonia perturbans (Walker). Proc. NJ Mosq. Exterm. Assoc. 40: 141-149.

Ikeshoji, T. 1981. Acoustic attraction of male mosquitoes in a cage. Jap. J. Sanit. Zool. 32: 7-15.

Ikeshoji, T. 1982. Attractive sounds for autochemosterilization of male mosquitoes. Jap. J. Sanit. Zool. 33: 41-49.

Ikeshoji, T. 1985. Age structure and mating status of male mosquitoes responding to sound. Jap. J. Sanit. Zool. 36: 95-101.

Irby, W. S. and C. S. Apperson. 1992. Spatial and temporal distribution of resting female mosquitoes (Diptera: Culicidae) in the costal plain of North Carolina. J. Med. Entomol. 29: 150-159.

Istock, C. A., S. S. Wasserman and H. Zimmer. 1975. Ecology and evolution of the pitcher plant mosquito, Wyeomyia smithii: 1. Population dynamics and laboratory responses to food and population density. Evolution. 29: 296-312.

Kahn, M. C. and W. Offenhauser. 1949. The first field tests of recorded mosquito sounds used for mosquito destruction. Am. J. Trop. Med. 29: 811-825.

Kahn, M. C., W. Celestin and W. Offenhauser. 1945. Recording of sounds produced by certain disease-carrying mosquitoes. Science 101: 335-336.

Karabatsos, N. 1985. International catalogue of arthropod borne viruses. 3rd. Ed. Am. Soc. for Trop. Med. and Hyg., San Antonio.

Kaufman, P. E., D. A. Rutz and S. Frisch. 2001. Sticky traps for large scale house fly (Diptera: Muscidae) trapping in New York poultry facilities. J. Agric. Urban Entomol. 18: 43-49.

Kay, B. H. 1983. Collection of resting adult mosquitoes at Kowanyama, Northern Queensland and Charleville, South-western Queensland. J. Aust. Ent. Soc. 22: 19-24.

Kennedy, J. S. 1939. The visual responses of flying mosquitoes. Proc. Zool. Soc. Lond. 109: 221-242.

171

Khan, A. A., H. I. Maibach, W. G. Strauss and W. R. Fenley. 1965. Screening humans for degrees of attractiveness to mosquitoes. J. Econ. Entomol. 58: 694-697.

Khan, A. A., H. I. Maibach, W. G. Strauss and W. R. Fenley. 1966. Quantification of effect of several stimuli on the approach of Aedes aegypti. J. Econ. Entomol. 59: 690-694.

Khan, M. K. U. 1964. Rapid and efficient methods for sampling anopheline populations in insecticide treated areas. J. Burmese Med. 12: 130-134.

King, W. V., G. H. Bradley, C. N. Smith and W. C. McDuffie. 1960. Mosquitoes of the Southeastern United States. Agriculture Handbook No. 173. Agriculture Research Service, USDA.

Klassen, W. and B. Hocking. 1964. The influence of deep river valley on the dispersal of Aedes mosquitoes. Bull. Entomol. Res. 55: 289-304.

Kline, D. L. and G. F. Lemire. 1995. Field evaluation of heat as an added attractant to traps baited with carbon dioxide and octenol for Aedes taeniorhynchus. J. Am. Mosq. Control Assoc. 11: 454-456.

Kline, D. L., M. Patnaude and D. R. Barnard. 2006. Efficacy of four trap types for detecting and monitoring Culex spp. In North Central Florida. J. Med. Entomol. 43: 1121-1128.

Kline, D. L., W. Takken, J. R. Wood and D. A. Carlson. 1990. Field studies on the potential of butanone, carbon dioxide, honey extract, 1-octen-3-ol, lactic acid and phenols as attractants for mosquitoes. Med. Vet. Entomol. 4: 383-391.

Kline, D. L., J. R. Wood and J. A. Cornell. 1991. Interactive effects of 1-Octen-3-ol and carbon dioxide on mosquito (Diptera: Culicidae) surveillance and control. J. Med. Entomol. 28: 254-258.

Klowden, M. J. 1996. Vector Behavior. pp 34-50. In B. J. Beaty and W. C. Marquardt (eds.), The biology of disease vectors. Univ. Press of Colorado, Niwot.

Klowden, M. J. 1997. Endocrine aspects of mosquito reproduction. Archives of Insect Biochem. Physio. 35: 491-512.

Laarman, J. J. 1955. The host-seeking behaviour of the malaria mosquito Anopheles maculipennis atroparvus. Acta Leiden. 25:1-144.

Laing, J. E. 1964. Studies on the bionomics of Culex restuans Theobald (Diptera: Culicidae). M.S. thesis, Carleton University, Ottawa.

Laird, M. 1988. The natural history of larval mosquito habitats. Academic Press, New York, NY.

Lehane M. J., 1985. Determining the age of an insect. Parasitol. Today. 81-85.

172

Lehane, M. J., 2005. Managing the blood meal. pp. 96-97. The Biology of Blood-sucking in Insects, 2nd ed. Cambridge University Press, Cambridge, UK.

Linley, J. R. 1987. Aerial oviposition flight of Toxorhynchites amboinensis (Diptera: Culicidae). J. Med. Entomol. 24: 637-650.

Loomis, E. C and E. J. Sherman. 1959. Comparison of artificial shelters and light traps for measurement of Culex tarsalis and Anopheles freeborni populations. Mosquito News. 19: 232-237.

Matheson, R. 1944. A Handbook of the Mosquitoes of North America, Vol. V. 2ed ed. Comstock Publishing Company, Ithaca, NY.

Mahmood, F. and W. J. Crans. 1998. Effect of temperature on the development of Culiseta melanura (Diptera: Culicidae) and its impact on amplification of eastern equine encephalitis virus in birds. J. Med. Entomol. 35: 1007-10012.

Mboera, L. E. G., B. G. J. Knols, W. Takken and A. Della Torre. 1997. The response of Anopheles gambiae s.l. and A. funestus (DipteraL Culicidae) to tents baited with human odour or carbon dioxide in Tanzania. Bull. Entomol. Research. 2: 173-178.

McNelly, J. and W. J. Crans. 1989. Limitations in the use of resting boxes to assess populations of the mosquito, Culiseta melanura. Proc. New Jers. Mosq. Control Assoc. 69: 32.

Means, R. G. 1979. Mosquitoes of New York. Part 1. The genus Aedes Meigen with identification keys to genera of Culicidae. Bull. No. 430a. NY State Museum, Albany, NY.

Meola, R. W. and R. S. Petralia. 1980. Juvenile hormone induction of biting behavior in Culex mosquitoes. Science. 209: 1548-1550.

Metcalf, R. L. 1989. Insect resistance to insecticides. Pestic. Sci. 26: 333-358.

Mogi, M., I. Miyagi, T. Toma, M. Hasam, K. Abadi and Syafruddin. 1995. Age structure of Anopheles subpictus (Diptera: Culicidae) collected by a light trap in Halmahela, Indonesia. Southeast Asian J. Trop. Med. Pub. Health. 26: 760-766.

Morris, C. D. and J. W. Robinson. 1994. Distribution of mosquito larvae in a waste tire pile in Florida – an initial study. J. Am. Mosq. Control Assoc. 10: 174-180.

Nayar, J. K. and D. M. Sauerman. 1972. Flight performance and fuel utilization as a function of age in female Aedes taeniorhynchus. Israel J. Entomol. 7: 27-35.

Neilson, E. T. and J. S. Haeger. 1960. Swarming and mating in mosquitoes. Misc. Publ. Entomol. Soc. Am. 1: 71-95.

Nielsen, S. A. and A. T. Nielsen. 1953. Field observations on the habits of Aedes taeniorhynchus. Ecol. 34: 141-156.

173

Newkirk, M. R. 1955. On the eggs of some man biting mosquitoes. Ann. Entomol. Soc. Am. 48: 60-66.

Peterson, D. G. and A. W. A. Brown. 1951. Studies of the responses of the female Aedes mosquito. Part III. The response of Aedes aegypti (L.) to a warm body and its radiation. Bull. Entomol. Res. 42:535-541.

Pletsch, D. J. 1970. A collapsible model of the “red box” for measuring mosquito population density. Mosquito News. 30: 646-648.

Provost, M. W. 1952. The dispersal of Aedes taeniorhynchus I. Preliminary studies. Mosq. News. 12: 174-190.

Provost, M. W. 1953. Motives behind mosquito flights. Mosq. News. 13: 106-109.

Provost, M. W. 1960. The dispersal of Aedes taeniorhynchus. III. Study methods for migrating exodus. Mosq. News. 20: 148-161.

Quiñones, M. L. and M. F. Suarez. 1990. Indoor resting heights of some Anophelines in Colombia. J. Am. Mosq. Control Assoc. 6: 602-604.

Quraishi, M. S., M. A. Faghih and N. Esghi. 1966. Flight range, length of gonotrophic cycles, and longevity of P3 2 – labeled Anopheles stephensis mysorensis. J. Econ. Entomol. 59: 50-55.

Reeves, W. C. 1951. Field studies on carbon dioxide as a possible host stimulant to mosquitoes. Proc. Soc. Exp. Biol. Med. 77: 64-66.

Reeves, W. C. 1953. Quantitative field studies on a carbon dioxide chemotropism of mosquitoes. Am. J. Trop. Med. Hyg. 2: 325-331.

Reisen, W. K. and A. R. Pfuntner. 1987. Effectiveness of five methods for sampling adult Culex mosquitoes in rural and urban habitats in San Bernardino County, California. J. Am. Mosq. Control Assoc. 3: 601-606.

Rodrigues, S. C. G. and J. Maruniak. 2006. Blood meal identification from mosquitoes collected at a commercial alligator farm. Am. Mosq. Control Assoc. 22: 557-560.

Rogoff, W. M. 1985. Insects of medical importance. Fundamentals of applied entomology. Macmillan Publishing Company. New York, New York. pp. 659-679.

Rohitha, B. H. and B. E. Stevenson. 1987. An automatic sticky trap for aphids that segregates the catch daily. Bull. Ent. Res. 77: 67-71.

Rowley, W. A. and C. L. Graham. 1968a. The effect of temperature and relative humidity on the flight performance of female Aedes aegypti. J. Insect Physiol. 14: 1251-1257.

174

Rowley W. A. and C. L. Graham. 1968b. The effect of age on the flight performance of female Aedes aegypti mosquitoes. J. Insect Physol. 14: 719-728

Rudolfs, W. 1923. Observations on the relationship between atmospheric conditions and the behavior of mosquitoes. Bull. N. J. Agric. Exp. Sta. 388.

Rudolfs, W. 1925. Relation between temperature, humidity and activity of house mosquitoes. J. N. Y. Entomol. Soc. 33: 163-169.

Russel, P. F. and D. Santiago. 1934. An earth-lined trap for anopheline mosqutieos. Proc. Ent. Soc. Wash. 36: 1-21.

SAS Institute. 2001. SAS/STAT user’s manual, ver. 8.2. SAS Institute, Cary, N.C.

Schemanchuk, J. A., F. H. Fredeen and A. M. Kristjanson. 1955. Studies on flight range and dispersal habits of Aedes flavescens (Muller) (Diptera: Culicidae) tagged with radio-phosphorus. Can. Entomol. 87: 376-379.

Service, M. W. 1969. Observations on the ecology of some British mosquitoes. Bull. ent. Res. 59: 161-194.

Service, M. W. 1970. A battery-operated light-trap for sampling mosquito populations. Bull. WHO. 43: 635-641.

Service, M. W. 1971. The daytime distribution of mosquitoes resting in vegetation. J. Med. Entomol. 8: 271-278.

Service, M. W. 1976. Mosquito ecology: field sampling methods.: Wiley, New York.

Service, M. W. 1986. The biologies of Aedes caspius (Pallas) and Culex quinquefasciatus (Say) (Diptera: Culicidae) in Dubai. Insect Sci. Applic. 7: 11-18.

Service, M. W. 1993. Ecology field sampling methods, 2nd Ed. Chapman and Hall, Boundary Row, London.

Service, M. W. and R. B. Highton. 1980. A chemical light trap for mosquitoes and other biting insects. J. Med. Entomol. 17: 183-185.

Shura-Bura, B. L., A. D. Shaikov, E. V. Ivanova, A. I. Glazunova, M. S. Mitruiukova and K. G. Fedorova. 1958. The character of disperson from the point of release in certain species of flies of medical importance. Entomol. Obozer. 37: 282-290.

Sippell, W. L. and A. A. Brown. 1953. Studies on the responses of the female Aedes mosquito. Part V. The role of visual factors. Bull. Entomol. Res. 43: 567-574.

Slaff, M. E. and W. J. Crans. 1982. Impounding water as a major producer of Culex salinarius (Diptera: Culicidae) in costal areas of New Jersey. J. Med. Entomol. 19: 185-190.

175

Smith, A., W. O. Obudho and S. Esozed. 1966. Resting patterns of Anopheles gambiae in experimental huts treated with malathion. Royal Society Trop. Med. Hyg. 60: 401–408.

Smith, G. E. 1942. The keg shelter as a diurnal resting place of Anopheles quadrimaculatus. Am. J. Trop. Med. Hyg. 22: 257-269.

Snow, W. F. and E. G. Smith. 1956. Observations on Anopheles walkeri Theobald in the Tennessee Valley. Mosq. News. 16: 294-298.

Snow, W. F. 1976. The direction of flight of mosquitoes (Diptera: Culicidae) near the ground in West Africa savanna in relation to wind direction in the presence and absence of bait. Bull. Entomol. Res. 65: 555-562.

Stage, H. H., C. M. Gjullin and W. W. Yates. 1952. Mosquitoes of northwestern states. U.S. Department of Agriculture. Handout number 46.

Stein, W. 1986. Dispersal of insects of public health importance, pp. 242-252. In W. Danthanarayana (eds.), Insect flight: dispersal and migration. Springer-Verlag, Berlin, Heidelberg.

Sulaiman, S. 1982. The ecology of Aedes cantans (Meigen) and biology of Culex pipiens in hibernation sites in northern England. Ph.D. disertation, University of Liverpool, Liverpool.

Takken, W. and D. L. Kline. 1989. Carbondioxide and 1-octen-3-ol as mosquito attractants. J. Am. Mosq. Control Assoc. 5: 311-316.

Taylor, L. R. 1963. Analysis of the effect of temperature on insects in flight. J. Anim. Ecol. 32: 99-117.

Van Essen, P. H. A., J. A. Kemme, S. A. Ritchie and B. H. Kay. 1994. Differential responses of Aedes and Culex mosquitoes to octenol or light in combination with carbon dioxide in Queensland, Australia. Med. Vet. Entomol. 8: 63-67.

Weathersbee, A. A. III. and M. V. Meisch. 1990. Dispersal of Anopheles quadrimaculatus (Diptera: Culicidae) in Arkansas Ricelands. J. Environ. Entomol. 19: 961-965.

Wellington, W. G. 1944. The effect of ground temperature inversions upon the flight activity of Culex sp. (Diptera: Culicidae). Can. Entomol. 76: 233.

Whisenton, L. R., T. J. Kelly and W. E. Bollenbacher. 1987. Multiple forms of cerebral peptides with steroidogenic functions in pupal and adult brains of the yellow fever mosquito, Aedes aegypti. Molec. Cell Endocrinol. 50: 3-14.

Willis, E. R and L. M. Roth. 1952. Reactions of Aedes aegypti (L.) to carbon dioxide. J. Exp. Zool. 121: 149-179.

World Health Organization (WHO). 2006. Weekly epidemiological record. 81: 221-232.

176

World Health Organization (WHO). 2007a. Malaria Fact Sheet. Last retrieved March 20, 2007, in http://www.searo.who.int/en/Section10/Section21/Section334.htm.

World Health Organization (WHO). 2007b. Yellow Fever Medical Fact Sheet. Last retrieved April 2, 2007, in http://www.who.int/mediacentre/factsheets/fs100/en/.

World Health Organization (WHO). 2007c. Yellow Fever Disease Fact Sheet. Last retrieved April 2, 2007, in http://www.who.int/csr/disease/yellowfev/en/.

Wu, D. and M. J. Lehane. 1999. Pteridine fluorescence for age determination of Anopheles mosquitoes. Med. Vet. Entomol. 13: 48-52.

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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 fiancée, Kristina Pein, October 17th, 2008, after which he plans to pursue a career in industry.