BEHAVIORAL PHOTOTAXIS OF PREVITELLOGENIC AND ......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

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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

21

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

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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).

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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.

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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

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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

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

th