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Evaluation of Different Commercial Lures and Horse Odors as an Attractant and Their Abilities to Increase Mosquito Trap ...

Permanent Link: http://ufdc.ufl.edu/UFE0020343/00001

Material Information

Title: 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
Physical Description: 1 online resource (108 p.)
Language: english
Creator: Holton, Aimee Camille
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: horse, lurex, lurex3, mosquito, mosquitoes, octenol
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mosquitoes cause the equine industry untold economic annual losses due to nuisance biting and disease transmission. Mosquito traps have been used to monitor species configuration and seasonal incidence. Horses out compete traps when in close vicinity. A year-long seasonality study was conducted using four Mosquito Magnet? Pro (MMPro) (American Biophysics, Corp., North Kingston, RI) traps and three commercially available lures: Octenol, Lurex, and Lurex?. Trap counts were taken every three or four days while rotating the different lures through a 4 x 4 Latin square every fourteen days. Seasonal population trends, efficacy of the attractants, and temperature and rainfall were evaluated at the end of the trial, which ran from September 2005 through September 2006. Octenol was proven the most efficient at attracting mosquitoes, showing a significant higher total catch number than the other three lures. Significantly more mosquitoes were trapped during the rainy months of September, October, and November of 2005 when compared to the other nine months. A second series of studies were conducted at the unit using the horse as an attractant. Two Mosquito Magnet? Pro (MMPro) (American Biophysics, Corp., North Kingston, RI) traps were baited with samples collected from the skin of two different horses using cotton balls. Traps were run for twenty-four hour intervals, with fresh samples taken and replaced in the traps corresponding to trap changes. Additional samples were taken for analysis using gas chromatography and mass spectrometry (GC/MS) to determine the chemical composition of the horses' skin and dander. Several common skin compounds were found, such as cholesterol and nonanal, however, one compound was found specific to the horse and its origin is still unknown. Mosquito numbers and species composition were evaluated and compared between the two horses. Odors from one horse appeared to repel while the odors from the second horse increased trap counts when compared to the control trap that ran with carbon dioxide alone. To determine if the two horses differed in their ability to attract mosquitoes, a vacuum aspirator was used to collect the mosquitoes that landed on the two different horses for thirty-minute intervals on two consecutive evenings. Species composition and total mosquito numbers were evaluated and correlated to the previous odor study. No significant difference was found between the two horses; but when the horses were tied on the west side of the arena a greater number of mosquitoes were caught for both.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Aimee Camille Holton.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Tenbroeck, Saundra H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0020343:00001

Permanent Link: http://ufdc.ufl.edu/UFE0020343/00001

Material Information

Title: 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
Physical Description: 1 online resource (108 p.)
Language: english
Creator: Holton, Aimee Camille
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: horse, lurex, lurex3, mosquito, mosquitoes, octenol
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mosquitoes cause the equine industry untold economic annual losses due to nuisance biting and disease transmission. Mosquito traps have been used to monitor species configuration and seasonal incidence. Horses out compete traps when in close vicinity. A year-long seasonality study was conducted using four Mosquito Magnet? Pro (MMPro) (American Biophysics, Corp., North Kingston, RI) traps and three commercially available lures: Octenol, Lurex, and Lurex?. Trap counts were taken every three or four days while rotating the different lures through a 4 x 4 Latin square every fourteen days. Seasonal population trends, efficacy of the attractants, and temperature and rainfall were evaluated at the end of the trial, which ran from September 2005 through September 2006. Octenol was proven the most efficient at attracting mosquitoes, showing a significant higher total catch number than the other three lures. Significantly more mosquitoes were trapped during the rainy months of September, October, and November of 2005 when compared to the other nine months. A second series of studies were conducted at the unit using the horse as an attractant. Two Mosquito Magnet? Pro (MMPro) (American Biophysics, Corp., North Kingston, RI) traps were baited with samples collected from the skin of two different horses using cotton balls. Traps were run for twenty-four hour intervals, with fresh samples taken and replaced in the traps corresponding to trap changes. Additional samples were taken for analysis using gas chromatography and mass spectrometry (GC/MS) to determine the chemical composition of the horses' skin and dander. Several common skin compounds were found, such as cholesterol and nonanal, however, one compound was found specific to the horse and its origin is still unknown. Mosquito numbers and species composition were evaluated and compared between the two horses. Odors from one horse appeared to repel while the odors from the second horse increased trap counts when compared to the control trap that ran with carbon dioxide alone. To determine if the two horses differed in their ability to attract mosquitoes, a vacuum aspirator was used to collect the mosquitoes that landed on the two different horses for thirty-minute intervals on two consecutive evenings. Species composition and total mosquito numbers were evaluated and correlated to the previous odor study. No significant difference was found between the two horses; but when the horses were tied on the west side of the arena a greater number of mosquitoes were caught for both.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Aimee Camille Holton.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Tenbroeck, Saundra H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0020343:00001


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128a2d64092605dd2118b0562cc26a16fe0e3717







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




















By

AIMEE CAMILLE HOLTON


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

UNIVERSITY OF FLORIDA

2007


































2007 Aimee Camille Holton


























To my mom and dad for all of their love and support









ACKNOWLEDGMENTS

My utmost gratitude goes to God, who has blessed me by providing this wonderful chance

and for surrounding me with amazing people to work with throughout my graduate program. I

would like to thank Saundra Tenbroeck for constant encouragement and for the amazing

opportunity. I thank Jerry Hogsette for always being there for assistance and support whenever

the need would arise. I thank Dan Kline for his expertise and willingness to share ideas as well as

for his encouragement. I would also like to thank Ulrich Bernier for being extremely helpful and

for always being there when I needed him. I would like to thank Brian Quinn for running my

samples and for helping me with other laboratory tasks. I would like to thank Joyce Urban and

Aaron Lloyd for their help with the Mosquito Magnet Pro traps as well as with mosquito

identification. My thanks go out to Justin Callaham and all of his crew at the University of

Florida Horse Teaching Unit for their wonderful help and time. I would like to thank Jordan

Barney and Kristin Detwiler for helping me with sample collections when I needed them. I

would also like to thank Kelly Vineyard, Sarah Dilling, Jerome Vickers, and Megan Brew for all

of their support and guidance throughout my program. Huge thanks go to my parents and my

family for believing in me and pushing me when I needed it most. Finally, I thank my husband,

Dustin Law, for loving and helping me and for constantly encouraging me along the way.









TABLE OF CONTENTS

page

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

LIST OF TABLES ............................... ................ .......7

LIST O F FIG U RE S ................................................................. 9

ABSTRACT ................... ............... ......... ... ...... ... .............

CHAPTER

1 L IT E R A TU R E R E V IE W ............................................................................... .................. 13

T ax o n o m y .........................................................................................1 5
L ife C ycle and M orphology .......................................................................... ....................16
F light B behavior and E ecology .................................................................... ........................ 18
H o st L location B eh av ior............................................................................... ..................... 2 0
H o st P referen ce ...................................... ................................................... 2 5
Humans as Attractants .................. ............. ...................... ............ 25
A ttractants from O their H osts................................................................................ ...... ...27

2 SEASONALITY OF MOSQUITOES AT THE UNIVERSITY OF FLORIDA HORSE
TEACHING UNIT IN NORTH CENTRAL FLORIDA USING TRAPS BAITED
W ITH TH REE D IFFEREN T LU RE S............... .....................................................................29

Intro du action ................... .......................................................... ................ 2 9
M materials and M methods ...................................... .. .......... ....... ...... 30
Experimental Design ................................. .. ... ...................3 31
R e su lts ................... ...................3...................2..........
D iscu ssio n ................... ...................3...................4..........
C o n clu sio n ................... ...................4...................0..........

3 STUDIES USING HORSE ODORS TO AUGMENT MOSQUITO TRAP
COLLECTIONS AT THE UNIVERSITY OF FLORIDA HORSE TEACHING UNIT,
G A IN E SV IL L E FL O R ID A ............................................................................................. 63

Intro du action ......... ............................................................................... 6 3
M materials an d M eth o d s ..................................................................................................... 64
Experim mental D design ............................................................... 65
H orse O dor Collection Study ............................................................. 65
H orse O dor Trapping Study ................................................ ............... 66
H orse V acuum ing Study.....................................................68
Results .................. ......................................... 69
H orse O dor Collection Study ............................................................. 69
H orse O dor Trapping Study ................................................ ............... 69









H orse V acuum ing Study ......................................................................... ...................70
D isc u ssio n .................. ...................................................................................................... 7 1
H orse O dor C collection Study ................................................ .............................. 71
H orse O dor Trapping Study .................................................. .............................. 73
H orse V acuum ing Study ......................................................................... ...................75
C onclu sions.......... ..........................................................77

4 CONCLUSIONS AND IMPLICATIONS .................................................. .....................98

APPENDIX ADDITIONAL INFORMATION ABOUT FLORIDA MOSQUITOES ...............100

L IST O F R E F E R E N C E S .................................................................................. ..................... 102

B IO G R A PH IC A L SK E T C H ......................................................................... .. ...................... 108








































6









LIST OF TABLES


Table page

2-1 Four treatment rotation schedule for the MMPro Traps during the 3-Lure seasonality
study at the UF HTU.............. ............................................57

2-2 Mean numbers of total mosquitoes trapped using each attractant + CO2 combination
in the M M Pro trapping study ...................................... ........................................... 58

2-3 Mean numbers of total mosquitoes trapped in each trap location in the MMPro
trap p in g stu d y ........................................................................ 5 8

2-4 Mean numbers of total mosquitoes trapped for each month during the MMPro
trap p in g stu d y ........................................................................ 5 9

2-5 Mean numbers standardd error) of mosquito species captured for each attractant +
CO2 combination for the total MMPro trapping study ....................................................60

2-6 Mean numbers standardd error) of mosquito species captured for each trap location
for the total M M Pro trapping study ............................................................................61

2-7 Total Mosquito Species Count and percent of total count of mosquito species trapped
by MMPro traps in the 3-Lure seasonality study at the UF HTU...................................62

3-1 Compounds found on Steiner, Equus caballus, from samples collected for analysis
by gas chromatography and mass spectrometry. .................................... .................93

3-2 Comparison of compounds found on the dander of two horses.................. ...............94

3-3 Mean numbers (+standard deviation) of mosquitoes captured per trapping interval
using the odors from Lodi in the MMPro traps. ..................................... ............... 95

3-4 Mean numbers (+standard deviation) of mosquitoes captured per trapping interval
using the odors from Steiner in the M M Pro traps................................... ............... 95

3-5 Total mosquito species and percent of total mosquitoes trapped using the odors from
Lodi in the MMPro traps during the horse odor trapping studies...................................96

3-6 Total mosquito species count and percent of total mosquitoes trapped using the odors
from Steiner in the MMPro traps during the horse odor trapping studies .......................96

3-7 Mean numbers (+standard deviation) of mosquitoes captured per trapping interval
for the vacuum aspirator study conducted in October 2006. ...........................................97

3-8 Total mosquito species count and percent of total mosquitoes trapped using the
vacuum aspirator on Lodi in October 2006 at the UF HTU. ...........................................97









3-9 Total mosquito species count and percent of total mosquitoes trapped using the
vacuum aspirator on Steiner in October 2006 at the UF HTU. .......................................97

A Classification of the fam ily Culicidae........................................................... .... ......... 100

A-2 List of m mosquitoes in Florida ....................................................................... 101









LIST OF FIGURES


Figure pe

2-1 Mosquito Magnet Pro (American Biophysics Corporation, East Greenwich, RI)
m mosquito trap. .............................................................................42

2-2 Aerial Photograph of the UF HTU showing the location of the 4 MMPro traps used
in the 3-L ure Seasonality study ............................................................................ .... ... 43

2-3 Comparison of all four lure combinations and total mosquito composition trapped
during the 3-lure seasonality ............................ ..................................... ............... 44

2-4 Total Mosquito counts as related to months during the 3-Lure seasonality study............45

2-5 Total mosquito count from the MMPro traps related to rainfall in centimeters in the
3-Lure seasonality study ................................. ... ... .......................46

2-6 Total rainfall in centimeters measured before the 3-Lure seasonality study ...................47

2-7 Minimum and Maximum temperatures recorded using the meteorological station
during the 3-lure seasonality study .............................................................................. 48

2-8 Total Mosquito Species composition of the 3-Lure seasonality study. ..........................49

2-9 Total numbers ofMansonia spp. females trapped by the MMPro traps during the 3-
Lure seasonality study .................. ......................................... .. .......... 50

2-10 Total numbers of Anopheles crucians trapped by the MMPro traps during the 3-Lure
seasonality study ................................................................. ....... .........51

2-11 Total numbers of Coquillettidiaperturbans trapped by the MMPro traps during the
3-Lure seasonality study ................................. ... ... .......................52

2-12 Total numbers of Culex erraticus trapped by the MMPro traps during the 3-Lure
seasonality stu dy ........................................................................... 53

2-13 Total numbers of Culex nigripalpus trapped by the MMPro traps during the 3-Lure
seasonality stu dy ........................................................................... 54

2-14 Total numbers of Culex salinarius trapped by the MMPro traps during the 3-Lure
seasonality stu dy ........................................................................... 55

2-15 Total numbers of Anopheles quadrimaculatus trapped by the MMPro traps during
the 3-L ure seasonality study ..................................................................... ...................56

3-1 Steiner, sorrel quarter horse gelding used for odor collections and mosquito trapping
stu dies ......................................................... ...................................79









3-2 Lodi, black quarter horse mare used in the odor collections and mosquito trapping
stu dies ......................................................... ...................................80

3-3 Illustration of hypersensitivity found on Lodi, black quarter horse mare used for odor
collections and m osquito trapping studies ....................................................................... 81

3-4 Method of collecting horse odors from different locations on the body using cotton
balls to collect horse odor for mosquito trapping studies. ............................................82

3-5 Aerial view of the University of Florida HTU showing the location of the two
M M P ro trap s. ........................................................... ................. 83

3-6 Portable vacuum aspirator (DC Insect Vac. BioQuip, Rancho, Dominguez, CA) and
technique of aspirating mosquitoes off of the horses used for horse vacuuming
stu dies ......................................................... ...................................84

3-7 Chromatogram from the analysis of extracts from collected hair from "Steiner,"
Equus caballus at the University of Florida HTU.............. ............................................85

3-8 Chromatograms illustrating differences in peaks and abundances of compounds from
the chest hair from Steiner (top), to that of Lodi (bottom). ..............................................86

3-9 Chromatograms comparing the dander from Steiner (top), to that of Lodi (bottom)........87

3-10 Total mosquito species composition for horse odor trapping study using samples
from Lodi in the MMPro traps from May 2006 until October 2006 at the UF HTU. .......88

3-11 Total mosquito species composition for horse odor study using samples from Steiner
in the MMPro traps from May 2006 until October 2006 in trapping study at the UF
H T U ..............................................................................................89

3-12 Total mosquitoes trapped using the horse odors in the MMPro traps; samples from
L odi and Steiner .............................................................................90

3-13 Mosquito species comparison (represented as a percent of the total mosquitoes
collected) aspirated from Lodi during the horse vacuuming study conducted at the
U F H T U ........................................................................................ 9 1

3-14 Mosquito species comparison (represented as a percent of the total mosquitoes
collected) aspirated from Steiner during horse vacuuming study conducted October
2 006 .............. ....................... ................................................ ...... 92









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

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

By

Aimee Camille Holton

August 2007

Chair: Saundra TenBroeck
Major: Animal Sciences

Mosquitoes have a significant economic impact on the equine industry from nuisance

biting and the potential for pathogen transmission resulting in debilitating diseases. Traps are

effective tools for monitoring species composition but little is known about the efficiency of

commercial traps and mosquito attractants in the presence of a horse. Horses readily attract

mosquitoes, but the role of horse produced volatile chemicals for attraction is undefined. Studies

were conducted at the University of Florida Horse Teaching Unit to determine if mosquito trap

collections could be increased using commercial lures or odors collected from horse. A year

long seasonality study was conducted using four Mosquito Magnet Pro (MMPro) (American

Biophysics, Corp., North Kingston, RI) traps and three commercially available lures: octenol,

Lurex, and Lurex3. Trap counts were taken every 3-4 d while rotating the lures through a 4 x 4

Latin square every fourteen days. Seasonal population trends and efficacy of the lures were

evaluated. Temperature and rainfall were recorded throughout the study, which ran from

September 2005 through September 2006. The species trapped in greatest numbers was

Mansonia spp., followed by Anopheles crucians (Wiedemann), and Coquillettidiaperturbans

(Walker). The fall months of September, October, and November of 2005 yielded significantly









greater (P<0.05) numbers of mosquitoes compared to the other seasons, including the summer

months. Octenol baited traps caught significantly more mosquitoes compared to the traps baited

with other commercial lures, which in some cases caught fewer mosquitoes compared to the

control traps with CO2 alone. Lurex and Lurex3 did not differ at any time during any season in

the study.

In a separate series of trials, horse skin, hair, and dander samples were tested as lures. Two

Mosquito Magnet Pro (MMPro) (American Biophysics, Corp., North Kingston, RI) traps were

baited with binary combinations of carbon dioxide and samples collected from the skin of two

different horses using cotton balls. Traps were operated for twenty-four hour intervals, with new

samples added each interval. Duplicate samples were taken and analyzed for chemical

composition using gas chromatography and mass spectrometry (GC/MS). Compounds found on

the horse included cholesterol, nonanal, and decanal, and unexpectedly, 9-octadecenamide. This

compound seems to be unique to the skin of the horse. Mosquito numbers and species

composition were evaluated and compared between the two horses. Inclusion of emanations

from one horse tended to decrease the number of mosquitoes in the traps while inclusion of

emanations collected from the second horse tended to increase the number of mosquitoes caught

in the trap compared to the control trap, which ran with CO2 alone. However, these differences

were not statistically significant (P<0.05). A final study was conducted to determine if the two

horses differed in their ability to attract mosquitoes. A vacuum aspirator was used to collect the

mosquitoes that landed on the two horses on two consecutive evenings for thirty-minute

intervals. Species composition and total mosquitoes were evaluated. No significant differences

(P<0.05) were found between the two horses and similar species composition was noted.









CHAPTER 1
LITERATURE REVIEW

Mosquitoes have been a menace throughout the ages. Because of their resilience,

persistence, and ability to carry pathogens, these insects are a major entomological concern

worldwide. In spite of attempts to find improved methods of surveillance and control to reduce

disease transmission to humans and livestock, millions of people are infected with mosquito-

borne diseases worldwide each year. The Centers for Disease Control and Prevention (CDC)

estimates 300 to 500 million cases of malaria are reported annually, with over 2 million deaths

per year (over 75% African children). Research is done worldwide in an attempt to prevent and

control the spread of malaria.

The introduction of West Nile virus (WNV) in New York resulted in 62 human cases and 7

deaths (Rose, 2001). This Old World flavivirus is related to the encephalitides, such as Saint

Louis encephalitis (SLE) and Japanese encephalitis (JE). Ninety-eight percent of WNV cases

result in minor symptoms, including fever. However, 2% produce West Nile Neuroinvasive

Disease, which includes meningoencephalitis that can be fatal. In 2003, there were 9,862 human

cases and 5,181 equine cases reported in the United States (Stark and Kazanis, 2003). The

number of human cases reported in 2005 declined to 3000 cases, but there were 170 fatalities

(CDC, 2006). Death rates are even higher in horses. The average mortality rate of infected horses

is around 30% (Porter et al., 2001). It is difficult to diagnosis and supportive care is expensive in

horses.

Since the early 1900s, attempts to reduce Florida mosquito-borne diseases have included

the use of surveillance and chemical control. Concerns with environmental impact, insecticide

resistance in mosquitoes, and health concerns of the human population may limit the use of

chemical control in the future. Therefore, researchers are seeking novel control methods for









mosquitoes (Kline and Mann, 1998). New traps have been developed to provide more accurate

surveillance, as well as to safely and effectively control and limit mosquito numbers in a local

area. Accurate surveillance plays an integral role in predicting future epidemics and disease

transmission in local populations. Mosquito traps are now found in backyards and at livestock

facilities. It is not yet clear if traps can compete against and attract more mosquitoes than a

preferred host that is in proximity to the trap. Despite research done with humans and other

livestock species, little is known about horse and mosquito interactions.

Dilling (2004) found that when a horse was in close proximity trap counts went down and

concluded that traps do not compete well at luring mosquitoes away from the natural host.

Dilling attempted to capture the odors from horses to increase the trap catch of mosquitoes but

was unsuccessful. Mboera and others (1997) baited tents with human odors to increase trap

numbers of Anopheles gambiae Giles. In the 1950s, researchers explored the attraction of

mosquitoes to humans by building robots that mimic humans, including CO2 release and clothing

the robots in fabric soaked with human sweat (Brown et al., 1951). Scientists have used gas

chromatography mass spectrometry to analyze human skin emanations and identify the

compounds from the skin that may function as mosquito attractants (Bernier et al., 2000, 2003).

Livestock species have been studied to identify chemical compounds, other than C02, that can be

used to formulate an insect-attracting lure (Hall et. al., 1984). The volatile compound, 1-octen-3-

ol, was initially identified in the breath of oxen. This compound has been shown to be a potent

mosquito attractant, especially when combined with CO2 (Takken and Kline, 1989). It is

unknown whether horses have chemicals that are odor cues to the mosquito that would be useful

in lures for commercial traps.









This chapter reviews the pertinent background research and details the foundation for the

current exploration into kairomones used by mosquitoes to locate horses for blood meals.

Through the discovery of new kairomones, the development of more efficient traps is possible,

and may eventually allow better methodology to control mosquitoes.

Taxonomy

"Mosquito" is a Spanish word meaning "little fly" and has been used in English since the

late 1500s. Mosquitoes belong to the order Diptera and family Culicidae. The family Culicidae

consists of approximately 3,200 recognized species. There are three subfamilies: Anophelinae,

Culicinae, and Toxorhynchitinae. Most of the differences between these subfamilies are

morphologically apparent in the larval stage. In the larval stage, Anophelinae do not have a

siphon on the eighth segment. This adaptation allows the larvae to be submerged under the water

but still obtain air. Anophelinae lay eggs which float on the surface of the water. The other two

subfamilies, Culicinae and Toxorhynchitinae, have siphons on the eighth segment during the

larval stage and the adult females have palps that are significantly shorter than the proboscis.

Toxorhynchitinae species separate themselves from the Culicinae easily because of their

predaceous larvae and larger sized adults. In addition, they have a uniquely curved proboscis

which has been adapted for feeding only on the nectar of plants (Woodbridge and Walker, 2002).

The three subfamilies separate into thirty-eight genera of mosquitoes worldwide. Thirteen

of these encompass 77 species in Florida alone: Aedes (Meigen), Anopheles (Meigen),

Coquillettidia (Dyar), Culiseta (Felt), Culex (Linnaeus), Deinocerites (Theobald), Mansonia

(Blanchard), Ochlerotatus (Lynch Arribalzaga), Orthopodomyia (Theobald), Psorophora

(Robineau-Desvoidy), Toxorhynchites (Theobald), Uranotaenia (Lynch Arribalzaga), and

Wyeomyia (Theobald) (Public Health Entomology Research and Education Center, 2002).









Life Cycle and Morphology

The mosquito goes through four distinct life stages, similar to other holometabolus insects.

This allows for ample development and separation from the adult female and the young. The four

stages begin with egg, larva, pupa, and then adult. Most mosquitoes have eggs that are found in

various shapes, including ovoid, spherical, rhomboid, elongate, and spindle. Adult female Aedes,

Anopheles, Haemagogus, Ochlerotatus, Psorophora, Toxorhynchites, and Wyeomyia lay their

eggs individually. Other genera, including Culex, Coquillettidia, and Mansonia species, lay their

eggs together in a single clump, forming an egg raft or a submerged cluster which will float on or

near the surface of the water (Darsie, 2006). An average of 75 eggs per ovary develops two to

three days after an adult female mosquito has taken a blood meal. Aedes and Ochlerotatus

species lay their eggs on damp soil that will be flooded by water, which is required for hatching.

On the other hand, Culex, Culiseta, and Anopheles lay their eggs on the surface of the water.

Most eggs will hatch within 48 hours; others have adapted to withstand subzero winter

temperatures before hatching (Harwood and James, 1979).

The next stage in mosquito development is the larval stage, which is an active stage that

requires an aquatic environment for sustaining the larvae. Mosquito larvae have three distinct

body regions: the head, the thorax and the abdomen. Depending on the species, the head is broad

and usually round, with lateral antenna. Toxorhynchites larvae are predaceous and will grasp

their prey; however, most other species have mouthparts that consist of brushes and grinding

structures that filter bacteria and microscopic plants. The next main region is the thorax, which is

broader than the head and flattened. Identification of larval mosquito species is possible with the

help of the number, location, and structure of tiny hair-like projections known as setae that are

found on the different segments of the thorax. The last main distinct body region is known as the

abdomen, which consists of nine segments. The abdomen is elongated and cylindrical, with the









first seven segments similar to each other. The last two segments are modified for specific

functions. The eighth segment, in most species except Anopheles, is the respiratory apparatus,

known as the siphon. The shape, width, and length of this segment are useful tools in identifying

species as well. The last segment is the anal segment. As the mosquitoes pass through the four

different stages of larval development, known as instars, they complete each one by molting and

increasing in size. Depending on environmental factors, including temperature, the average

mosquito species requires 7 days to complete larval development. Larvae can survive in water

temperatures between 17C and 350C in Florida for a period of one to four days. However,

temperatures above or below these will cause an increase in mortality in the population (Nayar,

1968). Following the fourth and last instar, the larva will molt again and become the third

developmental stage, known as the pupal stage (Darsie, 2006).

The third stage in mosquito development is known as the pupal stage, where the juvenile

does not feed and completes development. Pupae are mobile and are often called "tumblers"

because of their jerky movements when they are disturbed. They tumble towards protection,

usually deeper into their temporary home and then they float back up to the surface. The pupa is

comma-shaped and has an outer shell of protection known as chitin. The pupa floats on the water

because of its low density and receives oxygen through two breathing tubes called trumpets

(Darsie, 2006). The sex can be determined by examining the overall size of the pupa along with

the ninth segment of the pupa's abdomen. The male mosquito has a more prominent ninth

segment during this stage of development while the female is larger then the male. The

maturation process into an adult mosquito is completed in the pupal case. When it is finished, the

adult mosquito will split the pupal case and emerge to the surface of the water where it will rest

until its body hardens and dries (Floore, 2003).









The last stage of mosquito development is the adult stage. The body of the adult mosquito

is slender, with three distinct body regions: the head, thorax, and abdomen. Like other insects,

they have six legs, which are thin and narrow. They have two wings, which are long and thin,

with scales. The surface of the body is covered with setae and scales that allow for distinct

markings and colorations, providing characteristics for identification. Females have long,

filamentous antennae that are situated between the eyes on the head, whereas the male has larger,

more hairy antennae which allow for distinction from the female. The proboscis is prominent and

usually projects anteriorly at least two-thirds the length of the abdomen (Woodbridge and

Walker, 2002). Adult mosquitoes of both sexes of most species feed regularly on plant sugars

throughout their lives. Only female mosquitoes feed on hosts for a blood meal, which is essential

for obtaining protein required for egg production. Females feed on cold and warm blooded

animals and birds. Male mosquitoes do not bite, but feed on nectar of flowers or other suitable

sugar sources. Females will also feed on nectar for flight energy. Females of some mosquito

genera, such as Toxorhynchites, feed entirely on plant sugars and do not require a blood meal for

egg development (Woodbridge and Walker, 2002). A combination of different stimuli influence

biting and blood feeding such as carbon dioxide, temperature, moisture, smell, color, and

movement (Floore, 2003). During the summer, adult mosquitoes have a short life span, usually

lasting only a few weeks. However, it has been found that some species can survive through the

winter as adults, therefore increasing their ability to have a longer life span of several months

(Nasci et al., 2001).

Flight Behavior and Ecology

Once the mosquito has emerged from the pupal case, the adult will seek shelter for a rest

period and to allow for hardening of the body. Normally a mosquito will take flight during one or

two periods per day. This flight period depends on whether the specific species is characterized









as being diurnal, nocturnal, and crepuscular. During these periods, both males and females will

take flight without external cues (Woodbridge and Walker, 2002).

Generally, mosquitoes will not travel greater than two kilometers. Yet some mosquitoes

require long distance flights in order to complete their egg-laying mission. The salt-marsh

mosquito, Ochlerotatus taeniorhynchus (Wiedemann) requires long round trips to locate hosts

for blood meals since they emerge in secluded areas where hosts are not readily available. They

have been known to travel long distances with the help of the wind and may be carried hundreds

of kilometers from where they first emerged. Eventually, they make it back to their original

breeding sites for oviposition (Woodbridge and Walker, 2002).

It is possible to categorize mosquito flights into three main categories (Bidlingmayer,

1985). A one-way flight with no return, which usually lacks an objective and does not meet any

need, is known as a migratory flight. Newly emerged mosquitoes will take these flights and

rarely respond to stimuli. The destination is accidental as the mosquito relies on wind conditions

at the time of departure. Direction of migration and the limits of the mosquito's energy bank

reserves control the duration of the flight, as well as meteorological conditions during the flight

(Bidlingmayer, 1985). When the mosquito undergoes a physical stimulus, it will usually respond

by taking an appetential flight. The resting mosquito will begin a flight to satisfy a physical need,

such as taking a blood meal, finding an oviposition site, or moving to a better resting place. The

appropriate sensory organs will be alert during the appetential flight and will be seeking cues that

indicate the presence of the target and the flight will conclude when the objective is located or

until the mosquito's energy reserves are depleted (Bidlingmayer, 1985).

The final flight category is the consumatory flight, the subsequent flight after the female

has located her goal (Haskell, 1966). The consumatory flight is usually direct and brief, since









visual and biochemical cues are lost over long distances. If the cue encountered was olfactory a

direct upwind flight is conducted until other cues, visual perception, movement or thermal,

enable the female to locate her goal more precisely (Gillies and Wilkes, 1972). "Consumatory

flights do not always have to follow appetential flights. An example would be the biting of a host

that enters the daytime resting site of the female mosquito" (Bidlingmayer, 1985).

Other factors affecting the flight of mosquitoes usually involve the weather. The most

influential meteorological factors are light, temperature, humidity, and wind (Day and Curtis,

1989). Nightly variations in wind, rainfall, and relative humidity influence mosquito patterns and

maybe even feeding success. Daily rainfall patterns can potentially determine whether the

mosquito population will continue to build, remain steady, or decline as it relates to feeding and

oviposition behavior, mainly during the rainy season of late summer and early fall in Florida.

Research indicates that most mosquito species possess a bimodal flight activity pattern during

the night, with the larger peak occurring soon after sunset and the smaller peak just prior to dawn

(Schmidt, 2003).

Mosquito activity can be forecasted using the four meteorological factors mentioned

earlier. The Weather Channel has teamed up with the maker of the Mosquito Magnet Pro

(MMPro), American Biophysics, to launch the first ever "Mosquito Activity Forecast" on the

website, www.weather.com. The website link is managed by a team of meteorologists from the

Weather Channel who provide hourly predictions of mosquito activity nationwide. People who

want to participate in outdoor activities in areas inhabited by vector species may be able to better

plan their activities using this valuable tool (Dilling, 2004).

Host Location Behavior

The female mosquito will look for a host from which she can obtain a blood meal one to

three days following emergence. One study indicated host seeking was inhibited for a period of









40 hours following a blood meal for the mosquito An. gambiae (Takken et al., 2001). For over

half the twentieth century, research has been conducted to determine why mosquitoes are

attracted to certain hosts and what odors are responsible for the mosquito's attractive behavior to

a specific host. The principle difference in the orientation of blood-sucking insects, compared

with other insects, lies in the differences in behavioral responses to cues that may be distinct

between long and short range. In 1942 it was demonstrated that unwashed naked children were

preferred by Anopheles spp. over freshly washed naked children. This same group showed that

the presence of dirty human clothing attracted more mosquitoes than an empty hut as the control

(Haddow, 1942). The ability of humans to attract anthropophilic mosquitoes differs among

individuals (Khan et al., 1965). The results of their studies demonstrated that one person was

more attractive to Ae. aegypti than three other people (Khan et al., 1965). Based on these studies

and others, host selection is based on host preference and availability, with a combination of

visual, olfactory, and physical stimuli to help locate the host (Takken, 1991).

Although many mosquito species display visual response to distinct objects at a distance of

up to 19 m (Bidlingmayer and Hem, 1980), olfactory stimuli from host odors are considered to

be the strongest cues for location of hosts for blood meals (Allan et al., 1987 and Bowen, 1991).

Olfactory cues which aid in host-seeking by orienting mosquitoes to a host are known as

kairomones (Howse et al., 1998). Extensive work has been done to determine the mechanism of

mosquito attraction to its host. There is ample evidence that host-seeking by mosquitoes is

mediated by semiochemicals, chemicals which deliver a message, emanating from the host

(Bernier et al., 2003 and Geier et al., 1999). Olfactory cues are detected through an intricate

pathway, beginning with the sensilla located on the antennae and palpi which detect carbon

dioxide (C02). Age and the physiological state of the mosquito determine whether the detection









of olfactory cues results in a behavioral response (Takken, 1996). Volatile chemicals such as

CO2, octenol, and acetone and less volatile substances such as lactic acid and fatty acids are

present on skin as a result of vertebrate metabolism (Sastry et al., 1980). CO2 is universally

attractive to mosquitoes and is probably the best understood of the volatile host cues (Gillies,

1980, Gibson and Torr, 1999). Many researchers believe that volatile compounds act as

attractants that help orient the mosquito towards its host and that CO2 can combine with other

host odors to elicit a synergistic response (Smith et al., 1970, Gillies 1980, Bernier et al., 2003).

Gillies (1980) also found that CO2 and whole-body odors have an orienting effect when

presented singly and an enhanced effect when presented together. Kline and Mann (1998)

showed that traps baited with CO2 capture 8-30 times more mosquitoes than traps without CO2.

Around the attractant plume any insect with a flight speed less than approximately 3.5 m/sec will

be captured (Kline, 1999). High and low release rates (200 and 500 cc/min) of CO2 are utilized

during catches (Kline, 1994). It has been shown that CO2 does not help mosquitoes discriminate

different hosts (Mboera and Takken, 1997), but may actually function as a primer to activate

mosquitoes and make them more receptive to other host odors. (Dekker et al., 2005).

In 1984, Hall et al., through studying the attractiveness of oxen to Tsetse flies in Africa,

identified octenol from the ox. Field tests have demonstrated that octenol serves as a powerful

attractant for certain species of mosquitoes and flies (Kline, 1994). Thus, 1-octen-3-ol (octenol)

is another olfactory attractant documented as an effective mosquito attractant (Dilling, 2004).

This volatile compound has been isolated from many natural sources, mainly plants and fungi

(Dijkstra and Wiken, 1976). Chemically speaking, octenol is an 8-carbon mono-unsaturated

alcohol that has an asymmetric center and therefore two optical isomers with a terminal double

bond (Kline, 1994). This allows for different effects by the different isomers. At the present time,









manufacturers of mosquito control equipment such as Mosquito Magnet Pro, (American

Biophysics Corporation) recommend octenol as supplementary bait to mosquito traps. Kline

(1994) suggested that even though octenol was effective at attracting some species, it is not

correct to say that the compound will successfully attract all mosquito species (Kline, 1994). The

natural release rate of octenol by oxen is 0.043 mg/h (Hall et al., 1984). In studies conducted in

1994, the release rate used was 4 or 40 mg/h (Kline, 1994). Octenol is now available in slow

release packets, which have a release rate of 0.5 mg/hour (Kline, 1999).

Another volatile chemical believed to play a role in host location is lactic acid, which is a

by-product of anaerobic metabolism, common to all mammals. The acidity of freshly secreted

sweat is due to the production and secretion of lactic acid by the eccrine sweat glands (Thurmon

and Ottenstein, 1952). In one study, both fresh and incubated human sweat was used to catch the

malaria mosquito, Anopheles gambiae. They found incubated sweat was more effective than

fresh sweat in catching this particular species. They noted a decrease in lactic acid concentration

associated with the two day old sweat, and concluded that lactic acid may not play a role in

attracting An. gambiae (Braks and Takken, 1998). An. gambiae seem to be attracted to volatiles

of Limburger cheese, which to a human, resemble human foot odor (De Jong and Knols, 1995).

Skin emanations are important because odors from live hosts have been shown to be more

attractive than any combination of these chemicals provided in a warm, humid airstream

(Woodbridge and Walker, 2002). Some researchers believe continuous bacterial action in

secretions on the human skin results in volatiles that function as kairomones for mosquitoes

(Braks and Takken, 1998).

American Biophysics Corporation has been working on additional compounds that have

proven useful in trapping mosquitoes. Lurex and Lurex 3, lactic acid based compounds, when









combined with C02, are thought to be effective in increasing trap catches ofAedes albopictus

(Skuse) (McKenzie et al., 2004).

Visual attraction is also a key component in host location. Both male and female adult

mosquitoes have two compound eyes and two ocelli. Compound eyes are suited for navigation

and sensing movement, patterns, contrast, and color, while ocelli are believed to sense light

levels, and possibly polarized light (Allan et al., 1987). The compound eyes have relatively poor

resolution but overall high light sensitivity (Muir et al., 1992). It has been reported that diurnal

species respond to visual characteristics of hosts such as color, brightness, pattern, and

movement (Allan et al., 1987). It may be concluded that in a human dwelling, when given the

choice, the host seeking Mansonia mosquitoes are more attracted to the blue and red spectra than

the white, yellow, green, and black (Bhuyan and Das, 1985). Movement may also play a role in

host location by mosquitoes and a consistently small but positive attraction to movement has

been affirmed (Wood and Wright, 1968). Within one meter of the host, convective heat and

humidity become the main attractants rather than chemical or visual stimuli (Woodbridge and

Walker, 2002).

Physical stimuli such as temperature and humidity are also attractive to mosquitoes. With

the help of a clothed human robot, Brown (1951) was able to show that mosquitoes landed three

times as often on the clothed robot when the "skin" temperature was body temperature (980F)

than when the surface temperature was lower, around 50-650F (Brown, 1951). In addition,

Brown noticed that moisture coming off of the robot's clothing increased the landing numbers

two to four times, but only at temperatures above 600F. Despite extensive work completed thus

far, it has yet to be determined what causes mosquitoes to locate and feed on a host. It has been









concluded on several occasions that a combination of olfactory, visual and physical stimuli are

attractive factors.

Host Preference

As with host-seeking behavior, host preference varies widely among different genera of

mosquitoes and this preference may change within genera depending on a geographic location.

Some species may feed almost entirely on one host while others who are more opportunitistic

may feed on two or three different vertebrate classes. Culex genus prefers to feed on avian

species, but if the population of birds is insufficient or unavailable, they will happily feed on

mammals (Braverman et al., 1991). Species in Florida such as Aedes, Anopheles, Coquillettidia,

Mansonia, and Psorophora prefer to feed on mammals. Mosquitoes can be found in high

numbers most of the year in Florida on livestock facilities. Unfortunately, some mosquito species

are competent disease vectors which threaten livestock industries year round.

A number of mosquito species are known to feed on horses. Aedes and Ochlerotatus

species have been found to be readily attracted to horses (Loftin et al., 1997) as well as

Psorophora columbiae (Dyar and Knab) (Kuntz et al., 1982). Anopheles spp., Coquillettidia

spp., Culex spp., Culiseta spp., and Mansonia spp. are also equine feeders (Constantini et al.,

1998, Kuntz et al., 1982, and Loftin et al., 1997). Culex nigripalpus Theobald and Culiseta

melanura (Coquillett) have been shown to be vectors of WNV and Eastern Equine Encephalitis,

respectively (Darsie, 2006). Concerned horse owners seek methods to minimize the exposure of

animals to mosquitoes.

Humans as Attractants

Researchers are examining the attraction of the natural host as a way to draw the

mosquitoes to traps and away from the host. Entomologists have used host-baited traps since the

early 1900s for collecting Anopheline mosquitoes during malaria investigations (Mitchell et al.,









1985). In the mid-1960s, researchers were able to determine that people had varying levels of

attractiveness by using Ae. aegypti (Linnaeus) and measuring bloodfeeding and probing

responses (Khan et al., 1965). Human bait catches have been reported as the standard and most

useful method for collecting host-seeking anthropophilic mosquitoes (Service, 1993). Kline

(1994) stated there is a need for new, safe and effective ways to kill and control pest and vector

mosquito species and to deter blood seeking mosquitoes from feeding on animals and humans.

The use of chemical insecticides and topical repellents faces increased restrictions due to

environmental concerns and mosquito resistance.

Several researchers have stated that a difference exists between individuals (Haddow,

1942, Khan et al., 1965, Schreck et al., 1990, Canyon et al., 1998) Therefore, it has been

established that human skin odor contains volatile chemical substances that increase mosquito

attraction in the laboratory (Schreck et al., 1981, Eiras and Jepson 1991, 1994) and in the field

(Gillies and Wilkes, 1972). Once it was determined that differences among people existed,

attempts were made to identify those differences through biological and chemical methods.

Samples collected from people in various locations were typically analyzed by mass

spectrometric detection, e.g. GC/MS, whether the emphasis is on skin emanations, breath, urine,

blood, oral cavity, or the total composite of emanations from an entire person (Bernier et al.,

2006). Volatile substances produced by human skin have been shown to act as either attractants

or repellents. Many of the volatiles responsible for these actions are found in sweat (Takken,

1991). Smallegange reported that carboxylic acids make up an important part of human sweat

(Smallegange et al., 2005). Laboratory studies aimed at elucidating the compounds constituting

human-produced odor blends that mosquitoes use for host location have yielded several active

mixtures and individual substances (Smallegange et al., 2005). In their study, An. gambiae rely









on the combination of ammonia, lactic acid, and carboxylic acids in its orientation to human

hosts, different from the information reported concerning the Ae. aegypti (Smallegange et al.,

2005). Humans seem to have uniquely high levels of lactic acid on their skin compared to other

animals (Dekker, et al., 2002). Lactic acid is known to play an important role in the host-seeking

behavior of another anthropophilic mosquito species, Ae. aegypti (Acree et al., 1968). Ammonia

was also identified as an attractant for Ae. aegypti. It is not attractive when tested alone, but it

enhances the attractiveness of lactic acid. Fatty acids of chain length C1-C3, C5-C8, or C-13-C-

18 had the same effect when mixed with lactic acid. Experiments with An. gambiae females that

were done in Y-tube olfactometers showed that the synergistic effect could also be achieved

when combining ammonia and lactic acid with only one of the short-chain carboxylic acids that

was present in their mixture: hexanoic acid (Smallegange et al., 2005). Others reported an

attractiveness of an unsaturated carboxylic acid, 7-octenoic acid, which is a human-specific

component, secreted from the aprocrine sweat glands in the axillary regions (Smallegange et al.,

2005). Braks concluded that the kairomones to which An. gambiae responds were also present in

fresh sweat but that the quantity or quality of the attractive volatiles was enhanced strongly

during incubation. Skin microorganisms are presumed to break down sweat-borne compounds

into smaller, more volatile components. Furthermore, they noted that the preference for the

incubated sweat decreased after twenty minutes of exposure in the olfactometer. The combined

results led them to suggest that the components responsible for the preference of An. gambiae for

incubated sweat to fresh sweat are highly volatile (Braks et al., 2001).

Attractants from Other Hosts

In addition to human odors, samples from other mammals have been used to attract

mosquitoes and have shown high success rates. Researchers found an increase in trap numbers

when the attractant used was collected from mice using a closed-air system, without the help of









other attractants (McCall et al., 1996). Another study in Israel, led by Braverman in 1991, used

several different animals as bait in traps to catch Culexpipiens (Linnaeus). Sheep, chickens,

calves, and turkeys were the most successful baits. The calf tended to increase the total trap

number when compared to the other three animals.

Birds, such as sentinel chickens, are commonly used for attracting mosquitoes, especially

for surveillance techniques and to predict disease outbreak (Day and Stark, 1996). Cotton swabs

coated with crow uropygial gland secretions caused a significant increase in trap counts when

compared to a clean cotton swab, leading researchers to continue work with other avian species

(Russell and Hunter, 2005).

Previous experiments attempting to use horse odors have been conducted. Several studies

were conducted using a vacuum aspirator to vacuum the odors directly from the horse's body,

which were then fed into a trap in an attempt to increase trap counts. Very little difference was

seen when horse odors were collected by this method. When a horse is present near a trap, trap

counts decrease and the mosquitoes go to the horse (Dilling, 2004). Dekker and fellow

researchers measured the amounts of L-Lactic acid in skin-rubbing extracts from humans

compared with twelve other mammals and chickens, including horses. A greater amount was

found on humans compared to horses (Dekker, 2002). Further research needs to be completed

with the horse to determine if odors exist that have the capability to attract mosquitoes. Traps

baited with horse odor could conceivably protect the horses nearby.









CHAPTER 2
SEASONALITY OF MOSQUITOES AT THE UNIVERSITY OF FLORIDA HORSE
TEACHING UNIT IN NORTH CENTRAL FLORIDA USING TRAPS BAITED WITH
THREE DIFFERENT LURES.

Introduction

Florida's equine industry is affected by mosquitoes which have a direct impact on

the management of horse farms. Although nuisance biting, disease transmission and their

economic effects have not been studied in horses, mosquitoes have been found to have a

negative correlation in other livestock species, specifically dairy cattle. Reduced milk

yield, lower weight gain, and a compromised immune system leading to an increase in

disease from stress caused by insects have been noted (Steelman, 1979 and Byford et al.,

1992). Since the arrival of the West Nile Virus (WNV) in the United States in 1999,

Florida's equine industry has lost millions of dollars through disease prevention, health

care, and overall morbidity of infected horses (Porter et al., 2003).

Mosquito trapping is an effective surveillance tool used to study local mosquito

populations. Trapping allows for the determination of potential disease vectors, the

prediction of disease transmission, and the study of behavior and other patterns. It is

helpful to know the population seasonality trends, which results in more efficient

mosquito control, subsequently paving the way for more accurate protective measures

which could be implemented on horse farms. Because trapping mosquitoes has become

so important to their control, extensive research has been performed to improve the

ability of the traps to work more effectively (Dilling, 2004).

Unfortunately, traps may be less successful in many instances on horse farms

because the horse becomes a competitor against the trap. In this case, the mosquito

prefers the horse over the trap, causing a decrease in mosquitoes trapped and an increase









of mosquitoes that are present on the horses (Dilling, 2004). Dilling determined that the

traps commonly used to study populations of mosquitoes, namely the Center for Disease

Control (CDC) trap model 1012 (John W. Hock Company, Gainesville, FL) and the

Mosquito Magnet Pro (MMPro) (American Biophysics Corp., North Kingston, RI),

shown in Figure 2-1, were effective at trapping mosquitoes as long as a natural host was

not placed in a competitive situation. Many traps use stimuli to mimic host-preference

qualities that attract mosquitoes, such as heat, carbon dioxide, kairomones, and moisture

(Kline and Mann, 1998). It has been thought that adding odors that mimic the scent of the

natural host directly to the trap would improve the efficacy of the trap. Octenol,

discovered in 1984 by Hall et al., has been successful in the past at increasing numbers of

certain species captured. Other odors have been isolated as attractants and are also used

in mosquito traps. Lurex, a human-based lactic acid compound and Lurex3, a lactic acid +

ammonia compound designed for use in the MMPro, are two such products that were

used in this study as attractive baits that may potentially enable mosquito traps to out-

compete the natural host.

There is a definite need for volatile baits (odors) that can make mosquito traps

more competitive with or out-compete the natural host. Therefore, the main objectives of

this study were 1) to conduct competitive trapping studies using three different lures,

octenol, Lurex and Lurex3; and 2) to evaluate the total mosquito population profiles

caught when traps are baited with volatile odors (lures).

Materials and Methods

The lure trapping studies were conducted at the University of Florida Horse

Teaching Unit (HTU) in Gainesville, Florida. The 60-acre facility houses approximately

45 quarter horses used for breeding, teaching, and training, from weanlings to retired age.









The MMPro trap was used for the study. It is a self-powered mosquito trap that

uses propane as an energy source. Upon combustion, the propane catalytically converts to

produce C02, heat and moisture, which act as attractants, and electricity to power the

fans. MMPros may be used with or without an additional lure. When a mosquito nears

the base of the outflow of CO2 and heat, a fan which creates a counter flow current

vacuums the mosquitoes into a collection net where they die of dehydration. The MMPro,

with its patented technology, is made of stainless steel with a PVC outer covering and

stands about 40 inches tall (Figure 2-1).

During the study, a weather station was utilized to measure the minimum and

maximum air temperatures (C) and total rainfall (cm). The station is located just north of

the large pond in the west portion of the HTU. The wooden post used to hold the weather

station stands 1.5 meters tall, with a permanent rain gauge mounted on the top. Located

just below the rain gauge is a waterproof thermometer.

Experimental Design

The study began September 2, 2005, and was completed September 26, 2006.

MMPro traps were placed in 4 predetermined locations at the HTU (Fig. 2-2). Traps

remained at their designated locations for the entire project. Treatments in combination

with carbon dioxide (CO2) included three different baits: Octenol (Treatment #1), Lurex

(Treatment #2) and Lurex3 (Treatment #3), plus a control treatment of CO2 alone

(Treatment # 4). Using a predetermined schedule, treatments were rotated through the

four traps in a 4x4 Latin square design and replicated 9 times (Table 2-1). Each Latin

square was completed in 8 weeks. Treatments were rotated and the lures were replaced

every 14 days. The MMPros' 9-kg propane tanks were changed approximately every 19

days. The nylon collection nets were changed two times a week, on Tuesday and Friday









afternoons around 4 pm. The minimum and maximum air temperatures were recorded, as

well as the total rainfall for the past 3- or 4-day trapping period. Mosquitoes were stored

in a freezer at -250C until counting and identification were completed. The species data

was combined for the total study to analyze the seasonality trends at the HTU.

Data were analyzed by General Linear Model (GLM) after transformation by log (n

+ 1) and the means were separated by Duncan's Multiple Range Test (SAS 2006). The

significance interval was set at P < 0.05. Standard error was calculated from the means

using SSPS.

Results

Traps baited with treatment #1 (Octenol + CO2) captured significantly more

mosquitoes than traps baited with the other three treatments (Table 2-2). Mean numbers

of mosquitoes captured by traps baited with treatments #2 (Lurex3 + CO2) and #3 (Lurex

+ CO2) were not significantly different from each other, but were significantly lower than

those captured by control traps CO2 alone. Thus the addition of the two Lurex baits to

CO2 actually reduced the numbers of mosquitoes captured (Table 2-2). The mean number

of mosquitoes captured by the trap at location 1 (Fig. 2-2) was significantly higher than

that captured by the trap at location 4, no matter which attractant combination was used.

Numbers of mosquitoes captured were greatest at location 1 and decreased from locations

2 through 4 (Table 2-3). There was no significant difference between the mean numbers

of mosquitoes captured daily during the two collection intervals (nets were collected on

Tuesday and Fridays). Significantly higher numbers of mosquitoes were trapped during

the month of September (2005) than during the other 12 months of the study (Table 2-4,

range of means SE). The mean numbers of mosquitoes captured in October and

November were significantly higher than the remaining ten months of the study. There









was no significant difference in the mean numbers of mosquitoes captured between the

remaining months (Table 2-4). The mean differences between the individual mosquito

species to the four attractant combinations are shown in Table 2-5 and the mean

differences of the individual mosquito species trapped in each of the four locations are

shown in Table 2-6.

Figure 2-3 compares the monthly total numbers of mosquitoes trapped with each

attractant combination. Octenol was the most attractive for the majority of the 13 months,

except for February, April, and August, where the control was more effective.

A total of 71, 850 mosquitoes were trapped during this study combined (Table 2-7). Data

were plotted to show the seasonality differences and mosquito population fluctuations

that occurred at the HTU (Figure 2-4). The highest numbers of mosquitoes were caught

during the months of October, November, and December of 2005, but populations

decreased during the months of February, March, and June (Fig. 2-4). An increase in

mosquito population numbers was seen approximately 2-3 weeks following large

rainfalls in October and November (Fig. 2-5). But the same occurrence failed to happen

during December of 2005. Mosquito populations were increasing during rain events in

June and July, but populations did not peak until late July or early August (Figure 2-5).

Average rainfall measured during the study was lower than the previous three years at the

HTU (Figure 2-6). Minimum and maximum temperatures recorded at the HTU

throughout the study (Figure 2-7) further help to explain the trends of mosquito

populations.

Figure 2-8 illustrates the total mosquito species composition for the study. The

most prominent species caught was Mansonia spp. females (50.6 %) Ma. titillans









(Walker); Ma. dyari (Belkin, Heinemann and Page), with Mansonia spp. males (4.7%)

combined for a total Mansonia spp. trapped at 55.1%, followed by Anopheles crucians

(Wiedemann) (19.6 %) and Coquillettidia perturbans (Walker) (14.5%). To a lesser

extent, Culex erraticus (Dyar and Knab) (3.4%), Cx. nigripalpus (Theobald) (2.9 %), Cx.

salinarius (2.2 %), Anopheles quadrimaculatus (Say) (1.7 %) were trapped during the

study. Psorophora columbiae (Dyar and Knab), Uranotaenia sapphirina (Osten Sacken),

and Ochlerotatus infirmatus (Dyar and Knab) combined for just over 1% of the total

collection. Ma. spp. females, An. crucians, Cq. perturbans, Cx. erraticus, Cx.

nigripalpus, Cx. salinarius, and An. quadrimaculatus were trapped in numbers >1,000

over the course of the 13 months and individual seasonality trends are shown in Figures

2-9 through 2-15.

Discussion

During this 3-Lure study, octenol combined with CO2 was found to be significantly

more effective than Lurex, Lurex3, and CO2 alone at increasing mosquito trap counts on a

north central Florida horse farm. In past studies, octenol has proven to be a more

effective bait at increasing mosquito trap numbers in the northern states, with Lurex3

being more efficient at trapping Aedes albopictus (Skuse) in the southern states

(American Biophysics Corp., 2004). Researchers from American Biophysics found that

Ae. albopictus, which is very difficult to catch (Jensen, et al., 1994), was more attracted

to MMPro traps when they were baited with Lurex3 instead of octenol.1 The reverse was

found in this study with octenol out-trapping Mansonia spp. females, An. crucians, Cq.

perturbans, and Cx. nigripalpus when compared to the other lures, including Lurex3. No



1 McKenzie KE, Bedard SD. 2004. Article retrieved November 2006.









Aedes spp. were captured in this study, which may be a reason for the lower total catch

for traps baited with Lurex3. The human-skin-based lures, Lurex and Lurex3, should be

recommended for use in environments where Aedes spp., such as Ae. albopictus are

abundant.

Kline et al. (1990) found that there was a highly significant positive response to

CO2 by all species except Cx. erraticus and An. quadrimaculatus while working in the

phosphate mine pits in central Florida. Both Cq. perturbans and Ma. spp. showed a

significant synergistic enhancement in catch with octenol- supplemented CO2 when

compared with CO2 alone. This disagreed with findings by Kline and Mann in 1998,

where Cq. perturbans was equally attracted to octenol + CO2 and CO2 alone. Kline

(1994) found that the addition of octenol to C02-baited traps caused a decrease in Culex

species trapped, which disagrees with my study. At the HTU, traps baited with the

combination of octenol and CO2 captured significantly more Cx. nigripalpus than traps

baited with other lures. However, with Cx. erraticus there were no significant differences

between the numbers captured by traps baited with any of the 4 attractant combinations

(Table 2-5). With Cx. salinarius, there was no difference between traps baited with

octenol or with CO2 alone. An. quadrimaculatus was more attracted to Lurex and Octenol

when combined with CO2 compared to CO2 alone and Lurex3, where there was no

difference (Table 2-5). Both of the aforementioned studies by Kline et al. made use of

traps different than the MMPros, which may have a direct correlation to the differences in

performance with the different lures.

For some reason, the Mansonia spp. males were attracted to and captured in the

MMPro traps. There was no significant difference between octenol baited traps, CO2









alone, or Lurex3 baited traps. Perhaps Ma. males related the other 3 lures with areas

frequented by Ma. spp. females. Instances of Mansonia males frequenting traps and being

captured in the MMPros have not been reported in the literature.

Octenol combined with CO2 was found to be the most effective at trapping several

key species, including Mansonia spp., An. crucians, Cq. perturbans, and Cx. nigripalpus.

Cq. perturbans and Cx. nigripalpus are known vectors for equine diseases (Woodbridge

and Walker, 2002), while Mansonia has not yet been ruled out as a competent vector for

WNV (Darsie, 2006). Under conditions similar to those at the HTU, octenol + CO2 could

be used in MMPro traps to increase trap counts of these species.

A significant difference was noted in the four different trap locations for the study

(Table 2-3). Trap #1 trapped significantly more mosquitoes than the other three traps

used in the study. Trap #1 was located on the southern most part of the farm, near the

covered riding arena and a wet, marshy area that always contained water and downed

trees. Trap #2, just south of the large swamp, caught the most following trap #1, as the

swamp provided a consistent breeding ground throughout the study. Trap #3 was just

north of the swamp, however, was most near the small paddocks that always contained

horses, thus presenting a constant natural host for the trap to compete against throughout

the study. Therefore, trap #3 location trapped the third most mosquitoes. Finally, trap #4

was at the northern most part of the farm, near the feeding barn. This presented natural

competitors as well as a constant supply of dust, which caused the trap to clog and

malfunction several times. Subsequently, trap #4 was down more than any other trap

during the study. A 4 x 4 Latin square was instituted in order to control for location

differences. This step allowed the four attractant combinations to be rotated randomly









throughout the farm, eliminating a constant location for a single lure. Despite the trap

randomization, trap #1 was closest to the marsh, where Ma. spp. and Cq. perturbans, both

tree hole breeders. This would allow a constant supply of mosquitoes, especially since

these species were trapped the most abundantly during the study. Trap location

differences during my study disagreed from what Dilling (2004) discovered, who's traps

were in similar locations. She found that trap #2 and trap #3 caught significantly more

mosquitoes than did trap #1 and trap #4.

Campbell (2003) stated that mosquitoes are found in north central Florida 12

months a year, but they are present in much more significant numbers during the warm,

wet seasons of summer and fall; this is similar to the results in my study. Mosquito

populations differed throughout the entire study, but were at their highest during the rainy

fall months of September, October, and November, 2005, and again in the late summer

months of July, August, and September of 2006 (Figure 2-4). During these peak periods

of production, the temperature was also ideal for larval development, rarely dropping

below 55F (Figure 2-7). Lower populations were seen during the cool, dry months of

February and March, as well as the dry, late spring months of May and June. The latter

half of 2005 experienced normal rainfall, resulting in peak mosquito population numbers

2-3 weeks following a major rain event, observed in October and November. According

to a 30 year study conducted by the University of Utah's Department of Meteorology,

Gainesville's normal yearly rainfall is 51.81 inches, which is close to the rainfall

measured during my study. However, the time of year when the rainfall should have been

collected (the summer months of June, July, and August) resulted in decreased numbers

for the second half of the study. This agrees with several other studies conducted at the









UF HTU, including those of Campbell (2003) and Dilling (2004). Both stated that

temperature and rainfall appear to be major factors affecting mosquito seasonality trends.

This trend continued until the nights stayed fairly cool, below 550F, around mid-

December. As a result of low temperatures and lower rainfall, the mosquito population

numbers did not reach a peak for the remainder of the study. A major rain event occurred

in late July, but the mosquito numbers never again reached the previous numbers from

earlier in the study. The temperature in Gainesville, Florida, does fluctuate in the winter

months, and becomes steady during the summer months, as noted by other research

conducted in Florida by Campbell (2003) and Dilling (2004). Because of the differences

in temperature during the cool season months, the mosquito populations are never able to

rise until the spring time due to constant change during the night time, with sudden drops

below 550F. During my study, Gainesville experienced lower rainfall than in previous

years (Figure 2-6), resulting in decreased mosquito populations. In addition, the

decreased rainfall caused certain mosquito species breeding sites to go dry, causing a

shift in mosquito species complex. Dilling (2004) found the Culex species to be in greater

numbers through the majority of her study, because numerous hurricanes made landfall

and caused flooded conditions. Culex spp. were not as prevalent in my study (Figure 2-

12, 2-13, 2-14) because of less rainfall (Figure 2-5).

Similar population trends were found throughout this study, closely following

Dilling (2004) despite differences in trap and baits. During the seasonality study An.

crucians and Cx. erraticus were trapped all 13 months. An. crucians and Cx. erraticus

peaked during the cool months and remained steady for the remainder of the trial. This

closely followed previous work by Dilling (2004). Mansonia spp. was present the entire









study except for February. Cq. perturbans, was not present until mid-March, where it

began to rise steadily and remained in high numbers for the rest of the study. The

Mansonia species and Cq. perturbans have the ability to pierce and attach to the roots of

aquatic plants because of the presence of their attenuated siphon which allows them to

withstand longer periods of drought and develop in pools with less water (Darsie, 2006).

This adaptation allowed both species to maintain higher population numbers through the

drought experienced during my study. One species, Ps. columbiae, was not noticed in

large numbers until June, along with An. quadrimaculatus, which disagrees with Dilling

(2004), who found Ps. columbiae in greater numbers during October and An.

quadrimaculatus during November and December. Probably because of climatic

differences such as lower rainfall compared to previous years, several species including

Cx. nigripalpus, Ps. columbiae, and An. quadrimaculatus were not collected in all 12

months or in such high numbers as with Dilling in 2004.

In the future, attempts would be made to keep the MMPro traps in an operational

state by tending to them with regular maintenance, and keeping them clean and running

properly. Several times the traps, especially the trap at position #4, stopped working

because of dust. Furthermore, I would recommend adjusting the CO2 output of the

MMPro trap to a level that more closely matches that of the horse, its main competition at

the HTU. Horses expire approximately 2000 cc/min, (Pelletier and Leith, 1995) or > 4

times the amount that the MMPro trap releases. This fact could increase the chances that

the trap could be beneficial on a horse farm, if in fact olfactory cues are the main

attractant for mosquitoes.









It is important to incorporate mosquito surveillance with effective trapping methods

in order to achieve maximum control of disease. It is crucial to combine the technique of

surveillance with effective lures, such as octenol combined with C02 and to monitor the

environmental and meteorological factors which could potentially influence the mosquito

populations.

Conclusion

This study further supported the fact that CO2 is an effective lure for increasing trap

counts. In addition, when combined with octenol, the trap counts can be increased even

further. Lurex and Lurex3 significantly suppressed trap counts when combined with CO2.

Under similar metrological conditions, octenol + CO2 are the most effective attractant

combination for trapping certain species of mosquitoes in the MMPro, especially the

Mansonia spp. If this species becomes a competent WNV vector, octenol + CO2 could be

used to trap this species. This attractant combination could be useful on livestock

facilities to lower the total mosquito population that would have access to the animals,

especially the Mansonia spp. and Cq. perturbans. Under different meteorological

conditions, such as a cooler climate, a different mosquito species complex trend might be

found and a different lure might be required if trapping is to be effective.

During this study conducted in Gainesville, Florida, 71, 850 mosquitoes were

trapped, comprising a total of 10 different species. The most prominent genus trapped

was Mansonia, followed by An. crucians and Cq. perturbans. The Mansonia genus has

not yet been ruled out as a competent WNV in Florida. Possibly due to the lower rainfall,

Cx. nigripalpus was in lower than expected numbers, indicating that the threat of the

WNV virus at the HTU was low during the study. Because of the lack of rainfall in the

warm summer months, smaller population peaks were seen in July and August, contrary









to previous studies conducted at the HTU. Instead, the large peaks were seen in the late

fall month of November, with relatively stable trap counts in December. Mosquitoes were

trapped all thirteen months, with the lowest numbers occurring in the cold winter months

of January and February. Data from the study suggests that rainfall has a huge impact on

mosquito populations by pausing larval development and preventing them from

continuing into the pupal stage. Furthermore, low nighttime temperatures also had an

adverse effect on population numbers, inhibiting larval development. Generally, 2-3

weeks following a heavy rainfall, mosquito populations increased. In my study

temperature and rainfall appear to play major roles in the production of mosquito

populations.




































Figure 2-1. Mosquito Magnet Pro (American Biophysics Corporation, East Greenwich,
RI) mosquito trap.









































Figure 2-2. Aerial Photograph of the UF HTU showing the location of the 4 MMPro traps
used in the 3-Lure Seasonality study conducted from September 2005 -
September 2006.












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1000

0 0
9 10 11 12 1 2 3 4 5 6 7 8 9
Month

U Lurex Lurex3 U Control U Octenol

Figure 2-3. Comparison of all four lure combinations and total mosquito composition
trapped during the 3-lure seasonality study conducted from September 2005
until September 2006. Note: Month number equals corresponding calendar
date (i.e. 1 = January).












8000

S7000
t 6000
| 5000
S4000
0
S3000
2000
t 1000


' N ZNV <


\ Mo
Month


% a I\ % C)


Figure 2-4. Total Mosquito counts as related to months during the 3-Lure seasonality study conducted at the UF HTU from September
2005 September 2006. Note: Month number equals corresponding calendar date (i.e. 1 = January).











12 8000

7000
10
6000 =
8
5000

6 4000
3000
4-
2000

1000

0 0


Month
Rainfall (cm) # of mosquitoes


Figure 2-5. Total mosquito count from the MMPro traps related to rainfall in centimeters in the 3-Lure seasonality study conducted at
the UF HTU from September 2005 through September 2006. Note: Month number equals corresponding calendar date (i.e.
1 = January).















II,.. ..1


... Il


I


OI \ C bV (\ + ^ \ V x b\ Io (q\ +
Month
Figure 2-6. Total rainfall in centimeters measured before the 3-Lure seasonality study at the UF HTU from September 2003 through
August 2005. Note: Month number equals corresponding calendar date (i.e. 1 = January).


14
12


S4
S10
a 6














50.00


40.00 /V -VV

30.00

S20.00

S10.00

0.00

-10.00

00 -20.00
Month


Max Temp (C) Min Temp (C)
Figure 2-7. Minimum and Maximum temperatures recorded using the meteorological station during the 3-lure seasonality study
conducted at the UF HTU from September 2005 through September 2006. Note: Month number equals corresponding
calendar date (i.e. 1 = January).












Cx. salinarius
2.2%
Cx. nigripalpus
2.9%
Cx. erraticus 3. 4".
Ma. spp males
4.7%




Cq. perturbans
14.5%


An.
quadrimaculatus
1.7%


Ps. columbiae
0.26%


Oc. Infirmatus
0.05%

Ur. sapphirina
0.014".,


An. crucians
19.6%


Figure 2-8. Total Mosquito Species composition of the 3-Lure seasonality study conducted at the
UF HTU conducted from September 2005 through September 2006.


Ma. spp. females
50.6%













S7000

6000

5000

4000

3000 -

2000

( 1000 -
ooo-




Month


Figure 2-9. Total numbers ofMansonia spp. females trapped by the MMPro traps during the 3-Lure seasonality study conducted at the
UF HTU through September 2005 through September 2006. Note: Month number equals corresponding calendar date (i.e.
1 = January).














1800


1600

S1400
1200

1000 -

S800 -

S600
400

200
0


Month

Figure 2-10. Total numbers of Anopheles crucians trapped by the MMPro traps during the 3-Lure seasonality study conducted at the
UF HTU through September 2005 through September 2006. Note: Month number equals corresponding calendar date (i.e.
1 = January).












1400

1200

c 1000 -

S 800 -

600

t 400

200

c 0
0 \ K ^\ ^ \ ( ^ 0I O)
Month

Figure 2-11. Total numbers of Coquillettidiaperturbans trapped by the MMPro traps during the 3-Lure seasonality study conducted at
the UF HTU through September 2005 through September 2006. Note: Month number equals corresponding calendar date
(i.e. 1 = January).













450


400 -
350
S300
250 -
200
150
100
50 -



Month
Figure 2-12. Total numbers of Culex erraticus trapped by the MMPro traps during the 3-Lure seasonality study conducted at the UF
HTU through September 2005 through September 2006. Note: Month number equals corresponding calendar date (i.e. 1
January).
















a 350

I 300

250

t 200

150 -

100

50 -

0

Month

Figure 2-13. Total numbers of Culex nigripalpus trapped by the MMPro traps during the 3-Lure seasonality study conducted at the UF
HTU through September 2005 through September 2006. Note: Month number equals corresponding calendar date (i.e. 1 =
January).












450


400 -

S350

S300

S250

S 200


100 -

50
0



Month




Figure 2-14. Total numbers of Culex salinarius trapped by the MMPro traps during the 3-Lure seasonality study conducted at the UF
HTU through September 2005 through September 2006. Note: Month number equals corresponding calendar date (i.e. 1 =
January).















400

350
300
250

200
150
100

50
0


,o 2N, 2v \ "V ^ ) t K 0

Month

Figure 2-15. Total numbers of Anopheles quadrimaculatus trapped by the MMPro traps during the 3-Lure seasonality study conducted
at the UF HTU through September 2005 through September 2006. Note: Month number equals corresponding calendar
date (i.e. 1 = January).


I _--_t


N'\,^A/\










Table 2-1. Four treatment rotation schedule for the MMPro Traps during the 3-Lure seasonality
study at the UF HTU.


Date
09/02/05
09/09/05
09/16/05
09/23/05
09/30/05
10/07/05
10/14/05
10/21/05
10/28/05
11/04/05
11/11/05
11/18/05
11/25/05
12/02/05
12/16/05
12/30/05
01/13/06
01/27/06
02/10/06
02/24/06
03/10/06
03/28/06
04/11/06
04/25/06
05/09/06
05/23/06
06/06/06
06/20/06
07/04/06
07/18/06
08/01/06
08/15/06
08/29/06
09/12/06


Trap #1
Lurex
Lurex3
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
Lurex3
Octenol
CO2 alone
Lurex
Lurex3
Octenol
CO2 alone
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex


Trap #2
CO2 alone
Lurex
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
CO2 alone
Lurex
Lurex3
Octenol
CO2 alone
Lurex
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol


Trap #3
Octenol
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Octenol
CO2 alone
Lurex
Lurex3
Octenol
CO2 alone
Lurex
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3


Trap #4
Lurex3
Octenol
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
Lurex3
Octenol
CO2 alone
Lurex
Lurex3
Octenol
CO2 alone
Lurex
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone
Lurex3
Octenol
Lurex
CO2 alone









Table 2-2. Mean numbers of total mosquitoes trapped using each attractant + CO2 combination
in the MMPro trapping study conducted from September 2005 through September
2006.
Attractant Mean difference trapped n Total trapped
(Std. err)
Octenol 330.42 (37.098)a 99 32,373
CO2 alone (control) 210.81 (28.674)b 100 21,167
Lurex3 160.83 (+31.161)bc 113 11,865
Lurex 131.86 (25.103)c 90 15,435
Note: Means for attractant combination which are followed by the same number are not
significantly different (P< 0.05) and n= number of observations.


Table 2-3. Mean numbers of total mosquitoes trapped in each trap location in the MMPro
trapping study conducted from September 2005 through September 2006.
Trap location Mean difference trapped n Total trapped
(Std. err)
1 259.41 (+34.817)a 104 27, 260
2 216.08 (24.005)ab 105 22, 688
3 194.66 (30.787)ab 104 20,243
4 156.43 (35.4599)b 89 10, 289
Note: Means for each trap location which are followed by the same number are not significantly
different (P< 0.05) and n= number of observations.









Table 2-4. Mean numbers of total mosquitoes trapped for each month during the MMPro
trapping study from September 2005 through September 2006.
Month Mean difference n Total trapped
trapped ( Std. err)
September 2005 795. 13 (+111.2)a 31 24, 649

October 2005 437.81 (68.74)b 27 11,821

November 2005 430.89 (55.88)b 35 15,081

December 2005 105.37 (18.57)c 35 3,688

January 2006 121.19 (35.75)c 32 4,318

February 2006 35.59 (9.758)c 32 1, 139

March 2006 71.92 (13.85)c 26 1,870

April 2006 95.53 (14.63)c 32 3,057

May 2006 118.19 (16.98)c 32 3,782

June 2006 66.64 (10.30)c 33 2,470

July 2006 164.88 (30.70)c 25 4, 122

August 2006 136.10 (+21.02)c 31 4,219

September 2006 139.65 (24.10)c 31 4,329

Note: Means for each month which are followed by the same number are not significantly
different (P< 0.05) and n= number of observations.









Table 2-5. Mean numbers standardd error) of mosquito species captured for each attractant +
CO2 combination for the total MMPro trapping study conducted September 2005


through


September 2006.


Species Caught Lurex Lurex3 Control Octenol
Mean (Std. err) Mean (Std. err) Mean (Std. err) Mean (Std. err)
N= 97 N= 105 N= 104 N= 87

Mansonia spp. 52.68 (13.06)b 60.67(+10.89)b 91.99(+18.91)b 154.46(+29.22)a
(females)
An. crucians 18.20(2.40)c 26.15(60)bc 34.64(4.86)b 60.73(+9.62)a

Cq. perturbans 8.41(1.50)c 9.50(1.58)c 27.94(4.77)b 57.56(+8.99)a

Mansonia spp. 4.55(+1.22)b 9.31(+1.88)a 9.94(2.23)a 12.08(2.35)a
(males)
Cx. erraticus 4.64(+0.96)a 4.29(+0.96)a 5.86(1.07)a 9.49(+3.62)a

Cx. nigripalpus 4.57(+1.06)bc 2.90(+0.65)c 5.54(1.14)b 7.48(1.59)a

Cx. salinarius 1.81(0.38)b 3.96(0.63)ab 4.54(+0.84)a 5.03(0.94)a

An. 3.29(0.66)ab 2.73(0.60)b 1.91(0.45)b 4.65(+0.76)a
quadrimaculatus
Note: Means for each species which are followed by the same number are not significantly
different (P< 0.05) and n= number of observations. Species with total trapped numbers > 1,000
are included in table.









Table 2-6. Mean numbers standardd error) of mosquito species captured for each trap location
for the total MMPro trapping study conducted September 2005 through September
2006.
Species Caught Trap #1 Trap #2 Trap#3 Trap#4
Mean (Std. err) Mean (Std. err) Mean (Std. err) Mean (Std. err)
N= 107 N= 105 N= 104 N= 87

Mansonia spp. 95.11(+22.74)a 77.89(13.49)a 116.43(+24.14)a 66.15(+13.45)b
(females)
An. crucians 60.15(+9.79)a 42.33(4.56)b 20.83(2.46)c 11.94(+1.67)c

Cq. perturbans 28.50(+5.04)b 41.06(7.87)a 13.67(2.76)c 18.46(+4.39)bc

Mansonia spp. 5.82(1.53)ab 9.20(1.80)b 11.43(2.34)a 9.80(2.25)ab
(males)
Cx. erraticus 11.13(+3.22)a 4.35(+0.99)b 5.79(1.53)b 2.20(+0.72)b

Cx. nigripalpus 6.75) +1.20)a 5.88(1.37)ab 3.66(+0.91)b 3.85(1.03)b

Cx. salinarius 5.14(+0.88)a 5.39(+0.89)a 2.72(+0.46)b 1.78(+0.46)b

An. 3.91(+0.69)a 3.34(+0.63)a 2.30(+0.54)a 2.94(+0.62)a
quadrimaculatus
Note: Means for each species which are followed by the same number are not significantly
different (P< 0.05) and n= number of observations. Species with total trapped numbers over
1,000 are included in table.









Table 2-7. Total Mosquito Species Count and percent of total count of mosquito species trapped
by MMPro traps in the 3-Lure seasonality study at the UF HTU conducted from
September 2005 through September 2006.
Mosquito Species Total Count Percent of Total

Mansonia spp. 36217 50.6
Anopheles crucians 14071 19.6
Coquillettidia perturbans 10392 14.5
Mansonia spp. males 3361 4.7
Culex erraticus 2433 3.4
Culex nigripalpus 2055 2.9
Culex salininarius 1554 2.2
Anopheles quadrimaculatus 1264 1.7
Psorophora columbiae 184 0.26
Ochlerotatus infirmatus 39 0.05
Uranotaenia sapphirina 10 0.014
71580









CHAPTER 3
STUDIES USING HORSE ODORS TO AUGMENT MOSQUITO TRAP COLLECTIONS AT
THE UNIVERSITY OF FLORIDA HORSE TEACHING UNIT, GAINESVILLE, FLORIDA

Introduction

Mosquito traps may be capable of reducing the numbers of mosquitoes near a trap, but

their primary usage is for surveillance. Traps are used to catalog species composition and

estimate the density of mosquitoes in an area. Various trap styles have been developed, spanning

a variety of species specificity and trapping efficiencies of each species (Kline, 1999). Some of

these traps are designed to mimic the natural host, usually through the release of host-seeking

cues that may consist of carbon dioxide (C02), heat, moisture, and odors (Kline and Mann,

1998). An obstacle that helps render these traps as less effective tools for mosquito control is that

they do not compete well against a natural host when the host is near the trap(s). Presumably,

this is because the odor profile of the host elicits greater attraction in mosquitoes than CO2 alone

or any attractant that is comprised of simple blends.

In the past, it was found that significant differences occur between species composition and

total numbers of mosquitoes collected from mosquito traps compared to those vacuumed from a

horse (Campbell, 2003). This line of research has been continued in this chapter through

experiments that were designed and conducted to compare simultaneously the mosquitoes

captured in traps with those captured near horses. Furthermore, the addition of horse odors to the

trap was examined to determine if this bait could produce the collection of the same numbers and

species composition of mosquitoes which would normally be attracted to the horse.

Dilling (2004) performed several studies evaluating the use of horse odors to augment

mosquito trap collections. In one study, a horse was placed in a feeding slip with a Mosquito

Magnet Pro (MMPro) trap directly next to the stall. When the horse was nearby, the numbers of

trapped mosquitoes declined due to a preference of the mosquitoes for the live horse. In another









study, the entire body of a horse was vacuumed using a modified hand-held vacuum. Exhausted

horse volatiles from the vacuum were passed through a PVC pipe and fed into the CO2 flow of a

nearby CDC 1012 trap. Trap catch numbers were not significantly increased by inclusion of the

odors. It is possible that critical volatiles may have adhered to the inside of the PVC pipe, the

flow through the pipe was insufficient to deliver a minimum threshold level of attractants to

impart an effect, or volatiles other than CO2 from breath are missing as are non-chemical cues

such as heat, moisture, and visual ones.

Alternative sampling methods to identify horse odors may need to be developed and

explored as was done to identify human odors that attract mosquitoes (Bernier et al., 2000).

Some of this development involves collecting samples, analyzing and testing the biological

efficacy of the samples to attract mosquitoes, followed by chemical sampling to identify the

attractants that were involved.

The objectives of the studies in this chapter were to 1) Collect hair and dander samples

from different locations on a horse and identify the chemical composition of compounds present

in these samples using gas chromatography and mass spectrometry; 2) determine if a correlation

is present between the ability of two different horses to attract insects and the chemical

differences from the skin of these two different horses; 3) determine the ability of horse odor

samples to attract and collect mosquitoes in a MMPro; and 4) determine if mosquito species

composition and relative numbers of collected mosquitoes in the vacuum aspiration of two

different horses compared to the composition and numbers of mosquitoes caught in traps

augmented with collected horse odors.

Materials and Methods

Three studies were conducted at the University of Florida Horse Teaching Unit (HTU) in

Gainesville, Florida. The teaching unit houses approximately 40 quarter horses varying in age









and sex on 60 acres of land. Two horses used for these experiments were an 8-year old quarter

horse gelding named "Steiner" (Figure 3-1) and a 4-year old quarter horse mare named "Lodi"

(Figure 3-2). Two MMPro traps and two portable vacuum aspirators were used to perform these

studies.

Experimental Design

Horse Odor Collection Study

Sample odors were collected from two horses over the period of May 20 June 30, 2006.

Steiner, the eight-year old Quarter Horse gelding, was chosen to represent the animals that would

be found on horse farms throughout Florida and Lodi, the four-year old black Quarter Horse

mare, was chosen because of her hypersensitivity to insects such as stable flies and Culicoides

spp. Bites from these insects resulted in loss of hair on her chest, ears, and tail (Figure 3-3).

Horses were observed at dusk to determine areas where mosquitoes landed. The sites that

resulted in the greatest mosquito landings were used as the locations where samples were to be

taken and analyzed. Horse odors were collected from the face, barrel/dorsal side of the abdomen,

and the legs using a cotton ball of a 2" diameter. The cotton ball was rubbed in the preferred area

~ (5 in2) for 5 min to ensure ample sample extraction (Figure 3-4). Hair was also removed from

the chest of the horse with a sharp sterilized knife, using a soft, downward motion to cut hair

close to the skin to scrape the dander into a clean glass vial. Mane samples were collected using

scissors, with small samples taken from the underside of the mane. Samples were transported to

the laboratory after collection and extracted immediately upon return to the laboratory to

minimize loss of volatile compounds.

Hair samples from various locations on Steiner and Lodi were extracted with ImL hexane

and the dander samples from these horses were extracted in 250 iL. Extracts were analyzed by

gas chromatography/mass spectrometry (GC/MS) on a ThermoFinnigan Trace Single









Quadrupole GC/MS system (Thermoquest Finnigan, San Jose, CA). This system consists of a

GC oven, a split/splitless injection port equipped with a Programmable Temperature Vaporizer

(PTV) injector.

Injections were performed manually, using 1 [L of sample with the GC injection port set

at 35C prior to injection. Upon injection, the sample was loaded onto the GC column while the

temperature of the injection port was ramped balistically at 14.5 C/s to 240 C and held there for

1.0 min. Following this loading phase, the injection port is set to clean by a second ballistic

ramp at 14.5 C/s to 240 C, and held at that temperature for 3 min. The GC oven was held at

35C for 6 min after injection and then ramped to 2500C at 10 oC/min, and held at that

temperature for 25 min. The injector split was 12:1 (mL/min flow) and the carrier gas was high

purity helium set to maintain a constant flow of 1.20 mL/min. Samples were injected onto a 30

mm x 0.25 mm i.d. DB-5ms column with a stationary phase film thickness of 0.25[im. The

transfer line into the mass spectrometer was held at 2600C throughout the analysis. The mass

spectrometer was tuned and calibrated with perfluorotributylamine (PFTBA) prior to the

acquisition of data. Additionally, control hexane samples were injected prior to analysis of

extracted samples, and a standard hydrocarbon mixture was injected once per day to obtain

retention index data to assist in compound identification.

The mass spectrometer was operated in electron ionization (El) mode, with an average of

70 eV electrons. The ion source was set at 200 C, the emission current was 350 [IA, and the

detector was set at 350 V; the detector dynode was held in the off position until 3.0 min into the

analysis. A scan rate of 0.5 s per full scan was used cover the scan range of m/z 35-565.

Horse Odor Trapping Study

The horse odor trapping study was conducted from May 23 September 30, 2006. Two

MMPro traps were operated 4 m apart on the south side of the main body of water, just north of









the large covered arena at the HTU (Figure 3-5). The MMPro is a self-powered mosquito trap

that bums propane catalytically to produce C02, heat, and moisture. The output of combustion

results in enhanced attraction of some insect species. Combustion of propane also produces

electricity to power the fan. MMPros may be operated with additional lures or by simply

releasing the combustion products without added lure. The principle by which the trap collects

insects is patented as "counter flow geometry" where a mosquito may near the base of the

outflow while one fan vacuums the insect into a 2 qt. collection net attached to the bottom of the

fan inside the trap (US Patent: 7074830, Durand et al., 2006). The mosquitoes are stored in the

net after the fan pulls them into the inside of the trap. A window on the front of the trap provides

a view of the collection net within the trap. The trap is constructed with stainless steel with a

PVC outer covering and stands about 40" tall.

Two treatments were tested: horse collected odor + CO2 and CO2 only. Each treatment

employing horse odor was collected every 24 h to minimize volatilization of the compounds

from the sample. Skin extracts of the horses were collected by rubbing a cotton ball 2" diameter

in a localized area (5 in2) for 5 minutes. Cotton balls were rotated to ensure that all surfaces

were covered with the scent and the length of time confirmed the presence of the odors and oils

from the skin on the cotton ball. A latex gloved hand held the cotton balls to minimize

contamination from human skin (Dekker et al., 2002). The cotton ball was placed in a modified

plastic cartridge, designed by American Biophysics to hold the Lurex3 lure. The cartridge was

placed at the bottom of the trap, near the exit. The control MMPro trap was operated with the

normal emission of C02, heat, and water vapor only.

The samples were collected from the same physiological area on both horses every 24 h

and tested in the traps daily for four consecutive days. The cotton balls containing collected









odorants were changed after each collection was made, around 11 am EST. With each change of

cotton, the nylon collection nets were also changed. The propane tanks were changed every 18 d

to ensure an uninterrupted supply of propane during the experiments. Trapped mosquitoes were

stored in a freezer at -250C until counting and identification could be conducted. Data were

analyzed by GLM and means separated by Duncan's Multiple Range Test using SAS 2006 after

transformation by log (n + 1). Standard error of the means was calculated using SAS 2006.

Horse Vacuuming Study

Species composition and the number of mosquitoes that attempted to blood feed from two

different horses were examined on the consecutive nights of September 20 and 21, 2006.

Collection began at 7:30 p.m. (around dusk) both days and concluded one hour later at 8:30 p.m.

Each horse was tied at the south end of the covered arena at the HTU, 61 m apart, one on the far

east and the other on the far west side of the arena. Approximately 150 m from the east side of

the south side of the arena was a retention pond and near the west side (20 m) was a swampy,

wooded area with thick natural vegetation, down trees, and standing water.

Two individuals vacuumed the horses using portable vacuum aspirators simultaneously for

30 min (Figure 3-6). After the first 30 min, the two individuals switched horses and used portable

vacuum aspirators to vacuum the other horse on the opposite side of the arena for the next 30

min. The same two individuals vacuumed each night. Mosquitoes were vacuumed from all body

surfaces of the horses. On night one, individual one vacuumed horse one (Lodi) and individual

two vacuumed horse two (Steiner) for the first 30 min. For the next 30 min, horse one (Lodi) was

vacuumed by individual two and horse 2 (Steiner) was vacuumed by individual one. On night

two, this same collection scheme was repeated; however, the locations of horses were rotated

from left to right side of the arena compared to the previous night's location. Automobile

batteries supplied the power, and the two vehicles were both white to eliminate bias due to









vehicle color. A 1995 white Ford Ranger with a 12-V Interstate battery was parked on the east

side and a 1995 white Lincoln Continental with a 12-V Auto Zone battery was parked on the

west side for both consecutive nights. Mosquitoes were stored in a freezer at -250C until

counting and identification could be completed. Data were analyzed by GLM and means

separated by Duncan's Multiple Range Test using SAS 2006 after transformation by log (n + 1).

Standard error was calculated using SAS 2006.

Results

Horse Odor Collection Study

Figure 3-7 is a chromatogram depicting the compound peaks observed from the analysis of

hair from the horse, Steiner. One of the most abundant compound peaks based on peak area is

that at 13.39 min in the chromatogram. The mass spectrum corresponding to this peak could not

be matched with any of the library mass spectra. Additionally, this chromatogram had significant

peaks at 19.01 (geranylacetone) and at 28.60 (9-octadecenamide). Additional compounds

identified in this examination of Steiner are listed in Table 3-1. There are differences in

compounds on the hair between the horses examined in this study; these are reported in Table 3-

2. The chromatogram in Figure 3-8 demonstrates visually the remarkable differences in

compounds collected from the hair of Steiner and Lodi. Figure 3-9 shows very similar results

from the dander of both horses. It also demonstrates the peak at 13.39 min, an unknown

compound that has not previously been detected nor reported in horse odor, nor other host odor

samples from humans, chickens, or other mammals that have been studied previously (Bernier et.

al., 2000; Bemier, U.R., pers. communication).

Horse Odor Trapping Study

There were a total of 6,282 mosquitoes captured during the five month horse odor study.

The species composition trapped throughout the study on Lodi and Steiner are shown in Figures









3-10 and 3-11, respectively. The three most abundant species collected on both horses was

Coquillettidiaperturbans, the Mansonia spp., and Culex nigripalpus. When odor from Lodi was

collected and added to C02, 1,758 mosquitoes were trapped compared to the control trap with

just CO2 which caught only 1,523 mosquitoes (Figure 3-12). In this same figure, Steiner's odor +

CO2 used in the trap caught only 1,437 mosquitoes, compared to 1,564 mosquitoes for CO2

alone. The mean numbers of mosquitoes trapped using the odors from Lodi and Steiner are in

Tables 3-3 and 3-4, respectively. The breakdown of species composition trapped for both horses

are listed in Tables 3-5 and 3-6, for Lodi and Steiner respectively. There was no significant

difference (P<0.05) between the treatment groups for Steiner or Lodi, including comparisons

against each other, nor was there a significant difference for catches in different locations.

Horse Vacuuming Study

A total of 474 mosquitoes were vacuumed from Steiner on the east side of the arena,

compared to only 411 mosquitoes when he was located on the west side. There were 437

mosquitoes aspirated from Lodi when she was located on the east side compared to 381

mosquitoes from the west side. The mean numbers for each horse and for both sides of the arena

are in Table 3-7. No significant difference (P<0.05) was found in the species composition and

the number of mosquitoes aspirated when mosquitoes were compared between the two horses,

nor was a significant difference in collections found for location in the arena, nor for the

individual person who vacuumed the horse. The total number of mosquitoes vacuumed off of

both horses for both nights was 1,703 with the most prominent species trapped being Mansonia

spp. comprising 86% of those captured, followed by Cq. perturbans at just 8% and Cx. erraticus

in third with just 4% of the total catch. Nearly the same species profile was found for both

horses, with only slight differences in total percentage of species for each horse as illustrated in









Figure 3-13 and 3-14. Table 3-8 and 3-9 illustrate the total mosquito species composition trapped

for each horse.

Discussion

Horse Odor Collection Study

Results from GC/MS analysis of horse hair and dander reveal that the compounds present

on the horses contain some similarities and differences compared to those present on other

mammals, such as humans and bovines. Aldehydes such as nonanal and decanal are common on

the skin and other surfaces of just about all animals and could possibly play a role in insect

attraction to host odors. Another compound class containing members that were present on the

horses was the alcohols and the role of many of these in the host-seeking process remains

unknown, although 1-octen-3-ol is a known mosquito attractant (Takken and Kline, 1989). An

interesting aspect regarding horses as hosts for mosquito blood meals from is that they are

appealing to mammal feeders, such as Cq. perturbans as well as avian feeders, such as those of

the genus Culex. Therefore, examination of horse odors may reveal clues about chemicals

involved in the host-seeking process.

Of notable interest is that the chromatograms of both horses' dander contained a peak at

13.39 min (Figure 3-7, 3-8, and 3-9). The identity of this compound is still under investigation.

Until identification can be made, it will not be possible to determine whether or not it is crucial

to mosquito location of the horses. Despite this optimism, it should not be discounted that other

cues may be vital for host location, such as body temperature, respired C02, production of lactic

acid or even other volatile compounds such as octenol, methane, or excess nitrogen excretion

from bodily fluids such as urination.

The similarities in both horses' dander may explain how the equine species as a whole,

combined with the aforementioned cues, has an increased ability to attract insects. Despite









similarities in chemical composition of the horse dander, the hair samples taken from both horses

revealed a different chemical composition, which could be attributable to the deposition of

exogenous compounds on the outside surface (hair) of the horse. Lodi had several compounds on

her hair that were terpene-based, similar to those found in nature on plants and trees, as well as in

pressure-treated wood, like fence posts. Since Lodi and Steiner were pastured in separate fields

on different ends of the HTU, this may explain the differences in the profile of compounds

present in the extracts of their hair. Both horses were restricted from bathing and excessive

brushing throughout the study in an attempt to minimize contamination by exogenous chemicals

from shampoos and contaminants on the equipment. These precautions were also used during the

horse odor trapping study. Despite these efforts, substances highly likely of exogenous origin

were found in the analysis, such as compounds found in plastic gloves that were worn when

samples were collected to prevent contamination of oils and other compounds present on the

surface of human skin. Hair contaminants found on Steiner during the study are noted when

possible in Table 3-1, Table 3-2 compares compounds that may be unique to a horse or that were

found from the dander of both horses.

All of the samples were similarly for each horse with respect to location on the horse, such

as the face, legs, and the neck. However, it was difficult to control for environmental conditions.

Lodi was kept in a large field during the summer, with greater exposure to the environment

including the sun, dirt, and exogenous chemicals, such as those from vegetation. When samples

were collected from Lodi, it was apparent that they contained sweat and dirt from her habitat.

Steiner was kept in a smaller paddock with greater shading, resulting collections from his hair

and skin that contained less sweat than Lodi.









However, the differences in composition of the samples from each horse could not simply

be attributed to the sweat of one horse versus the lack of such on the other. Bernier (1995)

showed that human sweat was too aqueous, with very low volatile content and contained mostly

water and salts. It could be beneficial to thoroughly analyze the sweat from horses to determine

its chemical make up and then compare it to other mammals, such as the human. Another useful

study that could be valuable to understanding host preference of mosquitoes may be the

comparison of lactic acid content in the sweat of horses as related to others. Since lactic acid has

been a component in trap studies with Aedes spp. mosquitoes, the combination of this compound

with others identified in horse odor could be an important lure for more efficient trapping of

mosquitoes.

Horse Odor Trapping Study

It has been shown that differences exist between individual people and their ability to

attract mosquitoes (Schreck et al., 1990). Therefore it is likely to expect variation in attraction

between individual horses. In addition to exploration of differences that exist between horses

with respect to compounds present on the hair and dander, it is important to find out how a

mosquito trap would fare with respect to collecting mosquitoes in close proximity of a horse.

When comparing trapping of mosquito species for either horse during this study, the

results are essentially identical as seen in Tables 3-5 and 3-6 and Figures 3-10 and 3-11. The

similar species profile of both horses could be due to the season of year when the particular

treatment was run. Cq. perturbans was the most abundant species trapped; this closely follows

the time of year when these mosquitoes show preference for mammals. Mansonia spp. was

trapped effectively throughout this study, as seen in the seasonality study discussed in previous

research by Dilling (2004). Finally, Cx. nigripalpus was the third most abundant species trapped

and its appearance did not occur until mid-June, very similar to the seasonality study.









The GC/MS analysis of collected horse odors demonstrated that there is little difference in

the dander between the two horses in the study and that the only difference between them was on

their hair. It is extremely likely that these differences are due to exogenous chemicals from the

environment.. The difference in the horses themselves, though slight, could have played a role in

the small discrepancies between the total catch numbers for each horse. As previously

mentioned, Dilling (2004) found a slight difference between an appaloosa mare and a paint

gelding and their ability to compete against a trap. Even though there was no significant

difference in that study, the appaloosa mare generally caught more mosquitoes than the paint

gelding. In this study, the mare's odors when combined with CO2 caught more mosquitoes

compared to CO2 alone and compared to the gelding's odors combined with CO2 similar to the

findings of Dilling (2004). In this study, no significant differences were found in mosquito

numbers collected by odors plus CO2 compared to CO2 alone from either horse (Figure 3-12).

Although differences were not found in this study, previous studies indicate that the differences

between a mare and a gelding or a stallion and a gelding could have a direct impact on the

mosquito's host-seeking behavior (Dilling, 2004). It could be very important to study hormone

levels between the female and the male horse; including an intact male horse (the stallion) in

future studies.

One factor that may have impacted the study is the time constraint, as the period ran from

May 2006 until early October 2006. Accordingly, the time and month varied for each set of

treatments, and this may have affected both the species composition as well as the total numbers

of mosquitoes trapped. In addition, this area was under the conditions of a drought during this

five month period, so the total number of mosquitoes present was expected and certainly lower

than the previous year. Therefore, the decrease in total mosquito numbers may confound the









interpretation of the results from this study. An additional factor was that only two MMPro traps

were available for comparison at a time. Subsequently, the individual studies for each horse were

run separately. This means that the numbers of total feeding mosquitoes on a given night during

the study likely differed from one period to another. Furthermore, the traps were placed near trap

#2 of the three-lure study, just south of the large body of water (Figure 3-5). This area contained

varying numbers of nearby horses and these horses fluctuated throughout the entire 13 months all

around the farm. The location of a horse was usually not permanent and the numbers in close

proximity to the test site changed constantly. This fluctuation in natural hosts in the vicinity may

have affected the trap counts.

Future studies involving horse odors combined with CO2 used in traps could be modified

by restricting the time of year by increasing the number of traps used in the comparison, as well

as testing several different horses. In addition, it could be beneficial to increase the CO2 output of

the MMPro trap to more closely follow that of the horse, which is around 2000 cc/min at rest

(Pelletier and Leith, 1995) compared to 200 500 cc/min of CO2 that is emitted from the MMPro

trap (Takken and Kline, 1989).

Horse Vacuuming Study

It has been noted that horses will more proficiently attract mosquitoes when compared to a

mosquito trap using commercial bait when both are in close proximity. Therefore, it is important

to investigate the ability of the factors that result in superior attraction of insects by the horse

whether with olfactory cues or a combination of other factors that trigger the mosquito and affect

host preference. The species composition of vacuumed mosquitoes from both horses closely

followed Dilling's study (2004), again probably due to the low rainfall and warm nights.

There were slight numerical differences of mosquitoes aspirated from the two horses yet

there was no statistical difference noted, as seen in Table 3-7. Contrary to the horse odor trapping









experiment using the two horses, more mosquitoes were aspirated from Steiner on the east side

of the arena when compared to Lodi. This also held true when Steiner stood on the west side of

the arena.

The mosquitoes showed similar increases in activity both nights with respect to the time

frames of collection. The early time from 7:00 7:30 pm had less mosquitoes feeding with a

drastic increase as dusk set in. With this increase in landings and biting, the horses became

agitated and used their tails to swat off mosquitoes. Both horses also stomped and flinched,

which made it more difficult to aspirate mosquitoes and collect them in the container. The

feeding locations were similar for both horses; the smaller Cx. spp. tended to prefer the legs and

near the hooves and came out to feed during the first 30 min period. The feeding location of the

Cx. spp. was similar to what Dilling (2004) found, near the coronet band of one appaloosa mare.

Very little mosquitoes fed on the coronet band in this study; however, it was obvious that the

smaller mosquitoes preferred the legs rather than the other portions of the body. This could be

because Cx. spp. are avian feeders and adapted to feeding in areas of the host with less skin and

muscle tissue, like the legs of the horses which are more similar to the physiological makeup of

birds than the larger areas on the body. The larger species, such as the Cq. perturbans and Ma.

spp., were later feeders and preferred the higher up sections of the body, including the face,

forehead and the top of the hip.

A small difference, though not significant, was noted between the two far sides of the

arena; regardless of which horse was there, a greater number of mosquitoes were vacuumed from

the east side when compared to the west. There was a large retention pond near the east side

which still had some standing water. This may have attributed to the greater number of

mosquitoes nearby, leaving the breeding ground, in search of a blood meal. When the









mosquitoes left their resting site, they may have stopped at the first host they came upon, which

in both cases was the horse on the east side of the arena at the south end.

It was very difficult to draw any conclusions from two nights, so it would be beneficial to

repeat the aspirating experiment an additional number of repetitions throughout the season to

determine if differences really do exist between horses and their ability to attract more

mosquitoes than another.

Conclusions

Mosquitoes affect livestock species and humans all over the world through disease

transmission and resultant morbidity and possibly mortality. When mosquito traps are used on

various livestock facilities, they become less effective when natural hosts are in close proximity.

It is important to find ways to improve trapping so that they are more effective in decreasing the

numbers of mosquitoes that are able to feed on animals. When various samples were collected

and analyzed using GC/MS, it was found that horses were very similar to other mammals

including humans and that there could be a compound found on the dander of horses that is

unique to them. This compound, in combination with other host-seeking cues, could be an

important key in the amazing ability of the horse to attract the mosquito. Further analysis will

need to be completed to determine the nature of the compound and the role that it may or may

not play in mosquito attraction. Once this compound has been discovered it could be used with

other cues in mosquito traps in order to increase their ability to collect mosquitoes and other

haematophagous insects.

When odor samples were collected from the horses and used in the mosquito trap, it was

found that the trap count did not exhibit a significant difference between the uses of odors from

either horse. Additional field research is needed to confirm that horse odor can increase a trap

count and that the mosquito is not just feeding opportunistically. Furthermore, a horse exhales









CO2 at a rate of 2000 cc/min at rest, over 4 times the amount that the MMPro trap releases

(Pelletier and Leith, 1995). It could be beneficial to adjust the CO2 output from the MMPro to be

more quantitatively similar to that of the horse.

To further investigate the differences between two horses, mosquitoes were

aspirated off of each horse and the numbers were compared, with no statistically

significant differences observed in the numbers and species aspirated. There appeared to

be a location factor so further research needs to be completed in order to confirm the

differences between horses. A better understanding of horse odors and the role that they

play in host-seeking could lead to more efficient traps designed to compete against

natural hosts.














S--., ------ ---_- -------r









I_ r
IS











Figure 3-1. Steiner, sorrel quarter horse gelding used for odor collections and mosquito trapping
studies.



































Figure 3-2. Lodi, black quarter horse mare used in the odor collections and mosquito trapping
studies.






























Figure 3-3. Illustration of hypersensitivity found on Lodi, black quarter horse mare used for odor
collections and mosquito trapping studies.


































Figure 3-4. Method of collecting horse odors from different locations on the body using cotton
balls to collect horse odor for mosquito trapping studies.








































Figure 3-5. Aerial view of the University of Florida HTU showing the location of the two
MMPro traps used during the horse odor study (yellow) and those used during the
seasonality study (red).











---------------
-----~|tit


-'1.












Figure 3-6. Portable vacuum aspirator (DC Insect Vac. BioQuip, Rancho, Dominguez, CA) and
technique of aspirating mosquitoes off of the horses used for horse vacuuming
studies.
























84
















RT 300- 31 00


2892 NL
2 41E6
TIC F MS
UB-051806-
B


unknown

compound


4 05
5 8633

670
498

738 730

1 882 1043 12 23


15 12

J


20 80


18 68
1883
1746 1971
ALJlJ


27 9128 00
26 02 2741 -

23j54 2478 2522
2208
,itt^


2907
l-4J


4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (mm)


Figure 3-7. Chromatogram from the analysis of extracts from collected hair from "Steiner,"

Equus caballus at the University of Florida HTU.












RT 3 oo-3 1 10






a!i



to([M I.*


T I











d a 10 2 22 2 a 30
1 n'T i 1.


Figure 3-8. Chromatograms illustrating differences in peaks and abundances of compounds from
the chest hair from Steiner (top), to that of Lodi (bottom).
the chest hair from Steiner (top), to that of Lodi (bottom).





























Figure 3-9. Chromatograms comparing the dander from Steiner (top), to that of Lodi (bottom)













Oc. infirmatus Cx. nigripalpus Ma. spp
0.3% 11%
0032.8%
Ps. columbiae
<0.001%






Cq. perturbans
34.8%
Ma. spp males
7.5%
An. crucians
6.3%
Cx. erraticus
An. / Cx. salinarius 0.5%
quadrimaculatus
3.0%
3.6%


Figure 3-10. Total mosquito species composition for horse odor trapping study using samples
from Lodi in the MMPro traps from May 2006 until October 2006 at the UF HTU.

















Cx. nigripalpus
12.2%


Ps. columbiae
<0.01%









Cq. perturbans
38.7%


Ma. spp males
4.6%



An. crucians
9.6%


Cx. erraticus
2.8%
Cx. salinarius
An. 7.6%
quadrimaculatus
3.2%


Figure 3-11. Total mosquito species composition for horse odor study using samples from
Steiner in the MMPro traps from May 2006 until October 2006 in trapping study at
the UF HTU.










2000
1800 -
1600 -
1400
1200
1000 -
800 -
600 -
400
200
0-


175\ .'
1523 ;i 15i,4 ;


* Lodi (C02 only)

* Steiner (C02 only)


E Lodi (odor + C02)

* Steiner (odor + C02)


Figure 3-12. Total mosquitoes trapped using the horse odors in the MMPro traps; samples
from Lodi and Steiner. Note: Totals were not found to be statistically different
(p<0.05), and these are indicated by the same letter.












Cq.
perturbans
19.6% \




Cx. erraticus
3.8%


An. crucians
0.28%


Cq.
perturbans
males
4.0%


f Ma. spp
72.3%


Figure 3-13. Mosquito species comparison (represented as a percent of the total
mosquitoes collected) aspirated from Lodi during the horse vacuuming study
conducted at the UF HTU.











Cq.
perturbans
9.3%


Cq.
perturbans
males
2.2%


Cx. erraticus
4.1%





An. crucians
0.37%







Ma. spp
84.0%


Figure 3-14. Mosquito species comparison (represented as a percent of the total
mosquitoes collected) aspirated from Steiner during horse vacuuming study
conducted October 2006.









Table 3-1. Compounds found on Steiner, Equus caballus, from samples collected for
analysis by gas chromatography and mass spectrometry.
Compound GC (tR) (min)

Unknown 13.44
Nonanal 14.04
Decanal 15.12
Decanal 15.73
Nonanoic acid 15.97
Undecanal 16.64
Undecenal 17.24
Undecanoic acid 18.68
Dodecenal 18.82
Geranylacetone 19.05
Diethyl phthalate, contaminant 20.80
Unsaturated alcohol, or an aldehyde 23.35
Farnesol related compound 23.54
1-heptdecanol 26.01
Tetradecanal 27.91
Dioctyladipate 28.92
Long Chain Hydrocarbon 30.40
Diisooctyl phthalate (contaminant) 30.83
Suspected long chain amide 36.06
Cholesterol 49.30
Cholestanol 49.78









Table 3-2. Comparison of compounds found on the dander of two horses.
Compound GC (tR) (min) Horse'
Terpene, alpha-pinene 10.61 L
2-methylnonane 11.36 S
Cyclosiloxane 11.73 B
Octanal 12.15 B
2-ethyl-l-hexanol 12.64 B
Unknown Compound 13.39 B
Terpene, 4-Carene 13.69 L
2-hydroxyacetophenone 13.84 B
Nonanal 14.00 B
Terpene, p-menth-1-en-4-ol 14.59 L
Benzyl ester of acetic acid 14.96 L
Unsaturated alcohol 15.08 B
Decanal 15.64 B
Terpene or terpene-like, e.g. geraniol or myrcene 15.91 L
Terpene or terpene-like, e.g. geraniol or myrcene 16.28 B
Unsaturated aldehyde 16.48 B
Undecanal 16.60 B
Caryophellene 18.78 L
1-dodecanol 19.34 B
Tridecanal 19.81 B
4-methoxy-6-(2-propenyl)-1,3-benzodioxole 19.96 B
Terpene-related 20.06 L
Aldehyde or unsaturated alcohol 21.05 B
Tridecanol 21.78 B
Pentadecanal 22.22 B
Aldehyde 23.32 B
Alcohol 23.98 B
Hexadecanoic acid 24.75 B
Aldehyde, long chain 25.40 B
Alcohol, long chain 25.99 S
Diisoctyl maleate or related 26.49 S
Dioctyl maleate or related 27.26 B
9-octadecenamide 28.60 B
Key: Lodi (L), Steiner (S) and Both (B).









Table 3-3. Mean numbers (+standard deviation) of mosquitoes captured per trapping interval
using the odors from Lodi in the MMPro traps.
Interval Mean difference trapped ( SD) n
Treatment 109.88 (+ 18.33)a 16
Control 95.20 (+ 15.62)a 16

Note: Means followed by the same number are not significantly different (P< 0.05) and n=
number of observations.


Table 3-4. Mean numbers (+standard deviation) of mosquitoes captured per trapping interval
using the odors from Steiner in the MMPro traps.
Interval Mean difference trapped ( SD) n

Treatment 119.75 (+ 22.75)a 12
Control 130.33 (+ 26.07)a 12

Note: Means followed by the same number are not significantly different (P< 0.05) and n=
number of observations









Table 3-5. Total mosquito species and percent of total mosquitoes trapped using the odors from
Lodi in the MMPro traps during the horse odor trapping studies.
Mosquito Species Total trapped Percent of total
(%)


Coquillettidia perturbans
Mansonia spp.
Culex nigripalpus
Mansonia males
Anopheles crucians
Anopheles quadrimaculatus
Culex salinarius
Culex erraticus
Ochlerotatus infirmatus
Psorophora columbiae
Total


1143
1076
362
246
207
117
100
16
10
4
3281


34.8
32.8
11.03
7.5
6.3
3.6
3.04
0.49
0.31
0.12
100.0%


Table 3-6. Total mosquito species count and percent of total mosquitoes trapped using the odors
from Steiner in the MMPro traps during the horse odor trapping studies.
Mosquito Species Total trapped Percent of total
(%)


Coquillettidia perturbans
Mansonia spp.
Culex nigripalpus
Anopheles crucians
Culex salinarius
Mansonia males
Anopheles quadrimaculatus
Culex erraticus
Ochlerotatus infirmatus
Psorophora columbiae
Total


1162
636
365
288
229
137
98
83
10
3
3001


38.7
21.2
12.2
9.6
7.6
4.6
3.3
2.8
0.33
0.1
100.0%









Table 3-7. Mean numbers (+standard deviation) of mosquitoes captured per trapping interval for
the vacuum aspirator study conducted in October 2006.
Interval Mean difference trapped ( SD) n
Lodi 204.50 (+ 181.41)a 4
Steiner 221.25 (+ 139.92)a 4
West 198.00 (+ 152.07)a 4
East 227.75 (+ 170.14)a 4

Note: Means followed by the same number are not significantly different (P< 0.05) and n=
number of observations


Table 3-8. Total mosquito species count and percent of total mosquitoes trapped using the
vacuum aspirator on Lodi in October 2006 at the UF HTU.
Mosquito Species Total Count Percent of Total
(%)
Mansonia spp. 687 84.0
Coquillettidia perturbans 76 9.30
Culex erraticus 34 4.15
Culex nigripalpus 18 2.20
Anopheles crucians 3 0.37
Total 818 100.0%



Table 3-9. Total mosquito species count and percent of total mosquitoes trapped using the
vacuum aspirator on Steiner in October 2006 at the UF HTU.
Mosquito species Total count Percent of total
(%)
Mansonia spp. 774 87.6
Coquillettidia perturbans 58 6.60
Culex erraticus 41 4.63
Culex nigripalpus 7 0.79
Anopheles quadrimaculatus 2 0.23
Total 885 100.0%









CHAPTER 4
CONCLUSIONS AND IMPLICATIONS

Mosquito trapping is an effective tool used to monitor species composition in the area and

allows professionals to predict possible disease outbreaks in a population. Many different

commercial traps and lures are available to attract and trap the mosquitoes. It is important to

study the efficacy of these different traps and lures in different situations, some that are effective

on North Florida Horse farms. Disease prevention and a decrease in nuisance biting are crucial

for minimal economic loss to equine owners. Several lures were tested in comparison to a natural

host at the University of Florida HTU. Octenol, Lurex, and Lurex3 were combined with CO2 and

tested against a control trap the Mosquito Magnet Pro trap operated with CO2 alone. It was found

that throughout the study, octenol proved to be the most effective lure when used in close

vicinity of a natural host. There was a significant difference between the three lures tested;

octenol was more effective, followed by the control trap (CO2 alone), with no difference between

Lurex and Lurex3

An additional set of studies examined the odors of a horse as an attractant. Odor samples

from two different horses were collected and used in the MMPro traps. The traps were operated

for 24 h. No significant difference in mosquitoes trapped was found between the odors of the two

horses. An additional study was conducted where the same two horses were vacuumed to

determine if either horse had an increased ability to attract mosquitoes. No significant differences

were found between the two horses.

The series of research studies conducted at the UF HTU have indicated possible directions

for future studies. The horse odor studies answered several questions, yet raised several more. It

was found that the hair and dander of the two horses was very similar chemically. When the









analyzed samples were used in the traps, the results were less clear. It would be beneficial to

continue studying different horses, both for chemical analyses and efficacy in mosquito traps.

In addition to increasing the number of horses in these studies, it would be beneficial to

compare attraction to various locations of the horse, similar to studies in humans. Alternatively,

horse sweat samples could be analyzed and then compared to humans and other mammals. Then

the samples could be used in the mosquito traps and compared to the other horse hair and dander

samples. Other sample collection methods could be explored as well. Actual pieces of hair and

shavings of horse hair could be used in the traps.

More could be done with the horses themselves. In these series of studies, a castrated male

and a female were used, both with different hormonal profiles. A stallion could be used as well

as a gelding and a mare, to compare the different effects of hormones and the ability to attract

mosquitoes. More repetitions of the studies conducted here would support the results and make

the horse a more valuable tool for mosquito surveillance and control.

Mosquito trapping is an effective tool at monitoring local species composition and

predicting potential disease outbreaks. However, when the natural host is in the area, the trap's

effectiveness decreases and other methods must be used. If the horse is in fact effective at

increasing trap numbers, more experiments are needed to refine the use of the horse and horse

odors as trap lures.









APPENDIX
ADDITIONAL INFORMATION ABOUT FLORIDA MOSQUITOES

Table A-1. Classification of the family Culicidae
Tribe Genera


Anopheline
Culicinae Aedeomyiini
Aedini



Culcini
Culisetini
Ficalbiini
Hodgesiini
Mansoniini
Orthopodomyiini
Sabethini




Uranotaeniini
Toxorhynchitinae
The classification of all mosquitoes into 3
based on Knight and Stone (1977).


Anopheles, Bironella, Chagasia
Aedeomyia
Aedes, Ochlerotatus, Verrallina,
Ayurakitia, Armigeres, Eretmapodites,
Haemagogus, Heizmannia, Opifex,
Psorophora, Udaya, Zeugnomyia
Culex, Deinocerites, Galindomyia
Culiseta
Ficalbia, Mimomyia
Hodgesia
Coquillettidia, Mansonia
Orthopodomyia
l,, theI/ie, Wyeomyia, Phoniomyia, Limatus,
Trichoprosopon, .Nh\/ iili iiil. ui i,
Runchomyia, Johnbelkinia, Isostomyia,
Tripteroides, Malaya, Topomyia,
Maorigoeldia
Uranotaenia
Toxorhynchites
subfamilies, 10 tribes of Culicinae, and 38 genera is









Table A-2 List of mosquitoes in Florida
Genus species
Anopheles albimanus, atropos, barberi, bradleyi, crucians, diluvialis,
georgianus, grabhamii, inundatus, maverlius, nyssorhynchus,
perplexens, punctipennis, quadrimaculatus, smaragdinus, walker
Aedes aegypti, albopictus, cinereus, vexans
Ochlerotatus atlanticus, bahamensis, canadensis, dupreei, fulvus pallens,
hendersoni, infirmatus, m it\hei\,ni, mitchellae, scapularis,
sollicitians, stiticus, taeniorhynchus, thelcter, thibaulti, tormentor,
tortilis, triseriatus,
Psorophora ciliata, columbiae, cyanescens, discolor, ferox, horrida, howardii,
johnstonii, ill the ln'\i, pygmaea
Culex atratus, bahamensis, biscaynensis, cedecei, erraticus, iolambdis,
mulrennani, nigripalpus, peccator, pilosus, quinquefasciatus,
restuans, salinarius, tarsalis, territans
Deinocerites cancer
Culiseta inornata, melanura
Coquillettidia perturbans
Mansonia dyari, titillans
Orthopodomyia alba, signifera
Wyeomyia mitchellii, smithii, vanduzeei
Uranotaenia lowii, sapphirina
Toxorhynchites rutilus septentrionalis, rutilis rutilis,
The classification of Florida mosquitoes by Genus species was taken from Richard Darsie, 2006.









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BIOGRAPHICAL SKETCH

Aimee Camille Holton was born in Gainesville, Florida, in 1981 to Quinn and Suzanne

Holton. She has lived in Gainesville, Florida her whole life, attended and graduated from Santa

Fe High School in Alachua, Florida in 2000. She went to Santa Fe Community College and

earned an Associate of Arts degree in 2002, and then she transferred to the University of Florida.

She earned a Bachelor of Science degree in animal science with a major in equine industry.

Following graduation in December of 2004, she was accepted into a graduate program under

Saundra TenBroeck with a concentration in equine science management. Throughout the course

of her graduate program, she served as a teaching assistant for equine reproductive management,

sales preparation of thoroughbred yearlings, and psychology and training I and II under the

supervision of Mr. Joel McQuagge. She graduated with a Master of Science degree in animal

science with a minor in veterinary entomology in August of 2007. Aimee plans to pursue a

career as a teacher of biological sciences for secondary students.





PAGE 1

1 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 By AIMEE CAMILLE HOLTON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 2007 Aimee Camille Holton

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3 To my mom and dad for al l of their love and support

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4 ACKNOWLEDGMENTS My utmost gratitude goes to God, who has bl essed me by providing this wonderful chance and for surrounding me with amazing people to wo rk with throughout my graduate program. I would like to thank Saundra Tenbroeck for c onstant encouragement and for the amazing opportunity. I thank Jerry Hogsette for always being there for assistan ce and support whenever the need would arise. I thank Dan Kline for his e xpertise and willingness to share ideas as well as for his encouragement. I would also like to than k Ulrich Bernier for being extremely helpful and for always being there when I needed him. I would like to thank Brian Quinn for running my samples and for helping me with other laboratory tasks. I would like to thank Joyce Urban and Aaron Lloyd for their help with the Mosquito Magnet Pro traps as well as with mosquito identification. My thanks go out to Justin Callah am and all of his crew at the University of Florida Horse Teaching Unit for th eir wonderful help and time. I would like to thank Jordan Barney and Kristin Detwiler for helping me with sample collections when I needed them. I would also like to thank Kelly Vineyard, Sarah Dilling, Jerome Vi ckers, and Megan Brew for all of their support and gu idance throughout my program. Huge thanks go to my parents and my family for believing in me and pushing me when I needed it most. Finally, I thank my husband, Dustin Law, for loving and helping me and for constantly encouraging me along the way.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 LITERATURE REVIEW.......................................................................................................13 Taxonomy....................................................................................................................... ........15 Life Cycle and Morphology....................................................................................................16 Flight Behavior and Ecology..................................................................................................18 Host Location Behavior......................................................................................................... .20 Host Preference................................................................................................................ .......25 Humans as Attractants.......................................................................................................... ..25 Attractants from Other Hosts..................................................................................................27 2 SEASONALITY OF MOSQUITOES AT THE UNIVERSITY OF FLORIDA HORSE TEACHING UNIT IN NORTH CENTRAL FLORIDA USING TRAPS BAITED WITH THREE DIFFERENT LURES....................................................................................29 Introduction................................................................................................................... ..........29 Materials and Methods.......................................................................................................... .30 Experimental Design............................................................................................................ ..31 Results........................................................................................................................ .............32 Discussion..................................................................................................................... ..........34 Conclusion..................................................................................................................... .........40 3 STUDIES USING HORSE ODORS TO AUGMENT MOSQUITO TRAP COLLECTIONS AT THE UNIVERSITY OF FLORIDA HORSE TEACHING UNIT, GAINESVILLE, FLORIDA...................................................................................................63 Introduction................................................................................................................... ..........63 Materials and Methods.......................................................................................................... .64 Experimental Design............................................................................................................ ..65 Horse Odor Collection Study..........................................................................................65 Horse Odor Trapping Study............................................................................................66 Horse Vacuuming Study..................................................................................................68 Results........................................................................................................................ .............69 Horse Odor Collection Study..........................................................................................69 Horse Odor Trapping Study............................................................................................69

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6 Horse Vacuuming Study..................................................................................................70 Discussion..................................................................................................................... ..........71 Horse Odor Collection Study..........................................................................................71 Horse Odor Trapping Study............................................................................................73 Horse Vacuuming Study..................................................................................................75 Conclusions.................................................................................................................... .........77 4 CONCLUSIONS AND IMPLICATIONS.............................................................................98 APPENDIX ADDITIONAL INFORMATION AB OUT FLORIDA MOSQUITOES...............100 LIST OF REFERENCES.............................................................................................................102 BIOGRAPHICAL SKETCH.......................................................................................................108

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7 LIST OF TABLES Table page 2-1 Four treatment rotation schedule for the MMPro Traps during the 3-Lure seasonality study at the UF HTU..........................................................................................................57 2-2 Mean numbers of total mosquitoes trapped using each attractant + CO2 combination in the MMPro trapping study.............................................................................................58 2-3 Mean numbers of total mosquitoes tra pped in each trap location in the MMPro trapping study................................................................................................................. ....58 2-4 Mean numbers of total mosquitoes trapped for each month during the MMPro trapping study................................................................................................................. ....59 2-5 Mean numbers (standard error) of mos quito species captured for each attractant + CO2 combination for the total MMPro trapping study.......................................................60 2-6 Mean numbers (standard error) of mos quito species captured for each trap location for the total MMPro trapping study...................................................................................61 2-7 Total Mosquito Species Count and percent of total count of mosquito species trapped by MMPro traps in the 3-Lure se asonality study at the UF HTU......................................62 3-1 Compounds found on Steiner, Equus caballus from samples collected for analysis by gas chromatography and mass spectrometry................................................................93 3-2 Comparison of compounds f ound on the dander of two horses.........................................94 3-3 Mean numbers (standard deviation) of mosquitoes captured per trapping interval using the odors from Lodi in the MMPro traps.................................................................95 3-4 Mean numbers (standard deviation) of mosquitoes captured per trapping interval using the odors from Stei ner in the MMPro traps..............................................................95 3-5 Total mosquito species and percent of to tal mosquitoes trapped using the odors from Lodi in the MMPro traps during the horse odor trapping studies......................................96 3-6 Total mosquito species count and percent of total mosquitoes trapped using the odors from Steiner in the MMPro traps during the horse odor trapping studies.........................96 3-7 Mean numbers (standard deviation) of mosquitoes captured per trapping interval for the vacuum aspirator st udy conducted in October 2006..............................................97 3-8 Total mosquito species count and percen t of total mosquitoes trapped using the vacuum aspirator on Lodi in October 2006 at the UF HTU..............................................97

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8 3-9 Total mosquito species count and percen t of total mosquitoes trapped using the vacuum aspirator on Steiner in October 2006 at the UF HTU..........................................97 A-1 Classification of th e family Culicidae..............................................................................100 A-2 List of mosquitoes in Florida...........................................................................................101

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9 LIST OF FIGURES Figure page 2-1 Mosquito Magnet Pro (American Biophysics Corporation, East Greenwich, RI) mosquito trap.................................................................................................................. ...42 2-2 Aerial Photograph of the UF HTU showi ng the location of the 4 MMPro traps used in the 3-Lure Seasonality study..........................................................................................43 2-3 Comparison of all four lure combinati ons and total mosquito composition trapped during the 3-lure seasonality..............................................................................................44 2-4 Total Mosquito counts as related to months during the 3-Lure seasonality study.............45 2-5 Total mosquito count from the MMPro traps related to rainfall in centimeters in the 3-Lure seasonality study....................................................................................................46 2-6 Total rainfall in centimeters measured before the 3-Lure seasonality study.....................47 2-7 Minimum and Maximum temperatures re corded using the meteorological station during the 3-lure seasonality study....................................................................................48 2-8 Total Mosquito Species compositi on of the 3-Lure seasonality study..............................49 2-9 Total numbers of Mansonia spp females trapped by the MMPro traps during the 3Lure seasonality study........................................................................................................50 2-10 Total numbers of Anopheles crucians trapped by the MMPro traps during the 3-Lure seasonality study.............................................................................................................. ..51 2-11 Total numbers of Coquillettidia perturbans trapped by the MMPro traps during the 3-Lure seasonality study....................................................................................................52 2-12 Total numbers of Culex erraticus trapped by the MMPro tr aps during the 3-Lure seasonality study.............................................................................................................. ..53 2-13 Total numbers of Culex nigripalpus trapped by the MMPro traps during the 3-Lure seasonality study.............................................................................................................. ..54 2-14 Total numbers of Culex salinarius trapped by the MMPro tr aps during the 3-Lure seasonality study.............................................................................................................. ..55 2-15 Total numbers of Anopheles quadrimaculatus trapped by the MMPro traps during the 3-Lure seasonality study..............................................................................................56 3-1 Steiner, sorrel quarter ho rse gelding used for odor coll ections and mosquito trapping studies........................................................................................................................ ........79

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10 3-2 Lodi, black quarter horse mare used in the odor collections and mosquito trapping studies........................................................................................................................ ........80 3-3 Illustration of hypersensitivity found on Lodi, black quarter horse mare used for odor collections and mosquito trapping studies.........................................................................81 3-4 Method of collecting horse odors from different locations on the body using cotton balls to collect horse odor for mosquito trapping studies..................................................82 3-5 Aerial view of the University of Fl orida HTU showing the location of the two MMPro traps.................................................................................................................... ..83 3-6 Portable vacuum aspirator (DC Insect Vac. BioQuip, Rancho, Dominguez, CA) and technique of aspirating mosquitoes off of the horses used for horse vacuuming studies........................................................................................................................ ........84 3-7 Chromatogram from the analysis of extr acts from collected ha ir from Steiner, Equus caballus at the Univ ersity of Florida HTU..............................................................85 3-8 Chromatograms illustrating differences in peaks and abundances of compounds from the chest hair from Steiner (top), to that of Lodi (bottom)................................................86 3-9 Chromatograms comparing the dander from Steiner (top), to that of Lodi (bottom)........87 3-10 Total mosquito species composition fo r horse odor trapping study using samples from Lodi in the MMPro traps from May 2006 until October 2006 at the UF HTU........88 3-11 Total mosquito species composition for horse odor study using samples from Steiner in the MMPro traps from May 2006 until Oc tober 2006 in trapping study at the UF HTU............................................................................................................................ .......89 3-12 Total mosquitoes trapped using the horse odors in the MMPro traps; samples from Lodi and Steiner............................................................................................................... ..90 3-13 Mosquito species comparison (represented as a percent of the total mosquitoes collected) aspirated from Lodi during the horse vacuuming study conducted at the UF HTU......................................................................................................................... ....91 3-14 Mosquito species comparison (represented as a percent of the total mosquitoes collected) aspirated from Steiner during horse vacu uming study conducted October 2006........................................................................................................................... .........92

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science 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 By Aimee Camille Holton August 2007 Chair: Saundra TenBroeck Major: Animal Sciences Mosquitoes have a signifi cant economic impact on the equine industry from nuisance biting and the potential for pathogen transmission resulting in debilita ting diseases. Traps are effective tools for monitoring species composition but little is known about the efficiency of commercial traps and mosquito attractants in th e presence of a horse. Horses readily attract mosquitoes, but the role of horse produced volat ile chemicals for attraction is undefined. Studies were conducted at the University of Florida Horse Teaching Unit to determine if mosquito trap collections could be increased using commercial lures or odors collected from horse. A year long seasonality study was conducted using four Mosquito Magnet Pro (MMPro) (American Biophysics, Corp., North Kingston, RI) traps and three commercia lly available lures: octenol, Lurex, and Lurex. Trap counts were taken every 3-4 d while rotating the lures through a 4 x 4 Latin square every fourteen days. Seasonal popula tion trends and efficacy of the lures were evaluated. Temperature and rainfall were r ecorded throughout the study, which ran from September 2005 through September 2006. The speci es trapped in greatest numbers was Mansonia spp., followed by Anopheles crucians (Wiedemann), and Coquillettidia perturbans (Walker). The fall months of September, Octobe r, and November of 20 05 yielded significantly

PAGE 12

12 greater ( P < 0.05) numbers of mosquitoes compared to the other seasons, including the summer months. Octenol baited traps caugh t significantly more mosquitoes compared to the traps baited with other commercial lures, which in some cases caught fewer mosquitoes compared to the control traps with CO2 alone. Lurex and Lurex3 did not differ at any time during any season in the study. In a separate series of trials, horse skin, hair and dander samples were tested as lures. Two Mosquito Magnet Pro (MMPro) (American Biophys ics, Corp., North Kingston, RI) traps were baited with binary combinations of carbon dioxide and samples co llected from the skin of two different horses using cotton balls. Traps were oper ated for twenty-four hour intervals, with new samples added each interval. Duplicate sample s were taken and analyzed for chemical composition using gas chromatography and mass spectrometry (GC/MS). Compounds found on the horse included cholesterol, nonanal, and de canal, and unexpectedly, 9-octadecenamide. This compound seems to be unique to the skin of the horse. Mosquito numbers and species composition were evaluated and compared between the two horses. Inclusion of emanations from one horse tended to decrea se the number of mosquitoes in the traps while inclusion of emanations collected from the second horse tend ed to increase the number of mosquitoes caught in the trap compared to the control trap, which ran with CO2 alone. However, these differences were not statistically significant ( P < 0.05). A final study was conducte d to determine if the two horses differed in their ability to attract mosquito es. A vacuum aspirator was used to collect the mosquitoes that landed on the two horses on two consecutive evenings for thirty-minute intervals. Species composition and total mosquito es were evaluated. No significant differences ( P < 0.05) were found between the two horses and similar species composition was noted.

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13 CHAPTER 1 LITERATURE REVIEW Mosquitoes have been a menace throughout the ages. Because of their resilience, persistence, and ability to ca rry pathogens, these insects are a major entomological concern worldwide. In spite of attempts to find improve d methods of surveillanc e and control to reduce disease transmission to humans and livestock, millions of people are infected with mosquitoborne diseases worldwide each year. The Cent ers for Disease Control and Prevention (CDC) estimates 300 to 500 million cases of malaria are reported annually, with over 2 million deaths per year (over 75% African children). Research is done worldwide in an attempt to prevent and control the spread of malaria. The introduction of West Nile virus (WNV) in New York re sulted in 62 human cases and 7 deaths (Rose, 2001). This Old World flavivirus is related to the encepha litides, such as Saint Louis encephalitis (SLE) and Japa nese encephalitis (JE). Ninety -eight percent of WNV cases result in minor symptoms, including fever. However, 2% produce West Nile Neuroinvasive Disease, which includes meningoencephalitis that can be fatal. In 2003, there were 9,862 human cases and 5,181 equine cases reported in the United States (Stark and Kazanis, 2003). The number of human cases reported in 2005 declined to 3000 cases, but there were 170 fatalities (CDC, 2006). Death rates are even higher in horses. The average mort ality rate of infected horses is around 30% (Porter et al., 2001). It is difficult to diagnosis and supportive care is expensive in horses. Since the early 1900s, attempts to reduce Florida mosquito-borne diseases have included the use of surveillance and chemical control. C oncerns with environmental impact, insecticide resistance in mosquitoes, and health concerns of the human population may limit the use of chemical control in the future. Therefore, re searchers are seeking novel control methods for

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14 mosquitoes (Kline and Mann, 1998). New traps have been developed to provide more accurate surveillance, as well as to safely and effectivel y control and limit mosqu ito numbers in a local area. Accurate surveillance plays an integral ro le in predicting future epidemics and disease transmission in local populati ons. Mosquito traps are now found in backyards and at livestock facilities. It is not yet clear if traps can comp ete against and attract mo re mosquitoes than a preferred host that is in proximity to the tr ap. Despite research done with humans and other livestock species, little is known about horse and mosquito interactions. Dilling (2004) found that when a horse was in close proximity trap counts went down and concluded that traps do not comp ete well at luring mosquitoes away from the natural host. Dilling attempted to capture the od ors from horses to increase the trap catch of mosquitoes but was unsuccessful. Mboera and ot hers (1997) baited tents with human odors to increase trap numbers of Anopheles gambiae Giles. In the 1950s, researcher s explored the attraction of mosquitoes to humans by building ro bots that mimic humans, including CO2 release and clothing the robots in fabric soaked with human sweat (B rown et al., 1951). Scientists have used gas chromatography mass spectrometry to analyze human skin emanations and identify the compounds from the skin that may function as mo squito attractants (Bernier et al., 2000, 2003). Livestock species have been studied to identify chemical compounds, other than CO2, that can be used to formulate an insect-a ttracting lure (Hall et. al., 1984) The volatile compound, 1-octen-3ol, was initially identified in the breath of oxen. This compound has been shown to be a potent mosquito attractant, especially when combined with CO2 (Takken and Kline, 1989). It is unknown whether horses have chemicals that are odor cues to the mosquito that would be useful in lures for commercial traps.

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15 This chapter reviews the pertinent background research and details the foundation for the current exploration into kairomones used by mo squitoes to locate horses for blood meals. Through the discovery of new kairomones, the deve lopment of more effici ent traps is possible, and may eventually allow better me thodology to control mosquitoes. Taxonomy Mosquito is a Spanish word meaning little fly and has been used in English since the late 1500s. Mosquitoes belong to the order Diptera and family Culicidae. The family Culicidae consists of approximately 3,200 recognized species. There are three subfamilies: Anophelinae, Culicinae, and Toxorhynchitinae. Most of th e differences between these subfamilies are morphologically apparent in the larval stage. In the larval stage, Anophelinae do not have a siphon on the eighth segment. This adaptation allows the larvae to be submerged under the water but still obtain air. Anophelinae lay eggs which float on the surface of th e water. The other two subfamilies, Culicinae and Toxorhynchitinae, have siphons on the eighth segment during the larval stage and the adult females have palps that are significantly shorter than the proboscis. Toxorhynchitinae species separate themselves from the Culicinae easily because of their predaceous larvae and larger sized adults. In a ddition, they have a uniquely curved proboscis which has been adapted for feeding only on the n ectar of plants (Woodbridge and Walker, 2002). The three subfamilies separate into thirty-eight genera of mosquitoes worldwide. Thirteen of these encompass 77 species in Florida alone: Aedes (Meigen), Anopheles (Meigen), Coquillettidia (Dyar), Culiseta (Felt), Culex (Linnaeus), Deinocerites (Theobald), Mansonia (Blanchard), Ochlerotatus (Lynch Arribalzaga), Orthopodomyia (Theobald), Psorophora (Robineau-Desvoidy), Toxorhynchites (Theobald), Uranotaenia (Lynch Arribalzaga), and Wyeomyia (Theobald) (Public Health Entomology Re search and Education Center, 2002).

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16 Life Cycle and Morphology The mosquito goes through four distinct life st ages, similar to other holometabolus insects. This allows for ample development and separatio n from the adult female and the young. The four stages begin with egg, larva, pupa, and then adult. Most mosquitoes have eggs that are found in various shapes, including ovoid, spherical, rhomboid, elongate, and spindle. Adult female Aedes Anopheles Haemagogus Ochlerotatus Psorophora Toxorhynchites and Wyeomyia lay their eggs individually. Other genera, including Culex Coquillettidia and Mansonia species, lay their eggs together in a single clump, forming an egg ra ft or a submerged cluster which will float on or near the surface of the water (Darsie, 2006). An av erage of 75 eggs per ovary develops two to three days after an adult female mosquito has taken a blood meal. Aedes and Ochlerotatus species lay their eggs on damp soil that will be flooded by water, which is required for hatching. On the other hand, Culex Culiseta and Anopheles lay their eggs on the surface of the water. Most eggs will hatch within 48 hours; others have adapted to with stand subzero winter temperatures before hatching (Harwood and James, 1979). The next stage in mosquito development is the larval stage, which is an active stage that requires an aquatic environment for sustaining the larvae. Mosquito larvae have three distinct body regions: the head, the thorax and the abdomen. Depending on th e species, the head is broad and usually round, with lateral antenna. Toxorhynchites larvae are predaceous and will grasp their prey; however, most other species have mouthparts that consist of brushes and grinding structures that filter bacteria a nd microscopic plants. The next main region is the thorax, which is broader than the head and flattene d. Identification of larval mosquito species is possible with the help of the number, location, and structure of tiny hair-like projections kn own as setae that are found on the different segments of the thorax. The last main distinct body region is known as the abdomen, which consists of nine segments. The abdomen is elongated and cylindrical, with the

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17 first seven segments similar to each other. Th e last two segments are modified for specific functions. The eighth segment, in most species except Anopheles is the respiratory apparatus, known as the siphon. The shape, width, and length of this segment are useful tools in identifying species as well. The last segment is the anal se gment. As the mosquitoes pass through the four different stages of larval development, known as instars, they complete each one by molting and increasing in size. Depending on environmental factors, including temperature, the average mosquito species requires 7 days to complete la rval development. Larvae can survive in water temperatures between 17C and 35C in Florida for a period of one to four days. However, temperatures above or below these will cause an increase in mortality in the population (Nayar, 1968). Following the fourth and last instar, th e larva will molt again and become the third developmental stage, known as th e pupal stage (Darsie, 2006). The third stage in mosquito development is known as the pupal stage, where the juvenile does not feed and completes development. Pupa e are mobile and are often called tumblers because of their jerky movements when they are disturbed. They tumble towards protection, usually deeper into thei r temporary home and then they floa t back up to the surface. The pupa is comma-shaped and has an outer shell of protection known as chitin. The pupa floats on the water because of its low density and receives oxyge n through two breathing tubes called trumpets (Darsie, 2006). The sex can be determined by ex amining the overall size of the pupa along with the ninth segment of the pupas abdomen. The male mosquito has a more prominent ninth segment during this stage of development while the female is larger then the male. The maturation process into an adult mosquito is comp leted in the pupal case. Wh en it is finished, the adult mosquito will split the pupal case and emerge to the surface of the water where it will rest until its body hardens and dries (Floore, 2003).

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18 The last stage of mosquito de velopment is the adult stage. The body of the adult mosquito is slender, with three distinct body regions: the head, thorax, a nd abdomen. Like other insects, they have six legs, which are thin and narrow. They have two wings, which are long and thin, with scales. The surface of the body is covered wi th setae and scales that allow for distinct markings and colorations, providing characteri stics for identification. Females have long, filamentous antennae that are situated between th e eyes on the head, whereas the male has larger, more hairy antennae which allow for distinction fr om the female. The proboscis is prominent and usually projects anteriorly at least two-thirds the length of the abdomen (Woodbridge and Walker, 2002). Adult mosquitoes of both sexes of most species feed regularly on plant sugars throughout their lives. Only female mosquitoes feed on hosts for a blood meal, which is essential for obtaining protein required for egg producti on. Females feed on cold and warm blooded animals and birds. Male mosquito es do not bite, but feed on nectar of flowers or other suitable sugar sources. Females will also feed on nectar for flight energy. Females of some mosquito genera, such as Toxorhynchites feed entirely on plant sugars a nd do not require a blood meal for egg development (Woodbridge and Walker, 2002). A combination of different stimuli influence biting and blood feeding such as carbon dioxide, temperature, moisture, smell, color, and movement (Floore, 2003). During the summer, adu lt mosquitoes have a short life span, usually lasting only a few weeks. However, it has been found that some species can survive through the winter as adults, therefore increasing their ability to have a longer life span of several months (Nasci et al., 2001). Flight Behavior and Ecology Once the mosquito has emerged from the pupal cas e, the adult will seek shelter for a rest period and to allow for hardening of the body. Norma lly a mosquito will take flight during one or two periods per day. This flight period depends on whether the sp ecific species is characterized

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19 as being diurnal, nocturnal, and crepuscular. During these periods, both males and females will take flight without external cues (Woodbridge and Walker, 2002). Generally, mosquitoes will not travel greater than two kilometers. Yet some mosquitoes require long distance flights in order to complete their egg-laying mission. The salt-marsh mosquito, Ochlerotatus taeniorhynchus (Wiedemann) requires long r ound trips to locate hosts for blood meals since they emerge in secluded areas where hosts are not readily available. They have been known to travel long distances with the help of the wind and may be carried hundreds of kilometers from where they first emerged. Ev entually, they make it back to their original breeding sites for oviposition (W oodbridge and Walker, 2002). It is possible to categorize mosquito flight s into three main categories (Bidlingmayer, 1985). A one-way flight with no re turn, which usually lacks an objective and does not meet any need, is known as a migratory flight. Newly em erged mosquitoes will take these flights and rarely respond to stimuli. The destination is accidental as the mosquito relies on wind conditions at the time of departure. Direction of migrati on and the limits of the mosquitos energy bank reserves control the duration of the flight, as well as meteorologi cal conditions during the flight (Bidlingmayer, 1985). When the mosquito undergoe s a physical stimulus, it will usually respond by taking an appetentia l flight. The resting mosquito will begi n a flight to satisfy a physical need, such as taking a blood meal, finding an oviposition site, or moving to a be tter resting place. The appropriate sensory organs will be alert during the appetential flight and will be seeking cues that indicate the presence of the target and the flight will conclude when the objective is located or until the mosquitos energy reserves are depleted (Bidlingmayer, 1985). The final flight category is th e consumatory flight, the subseq uent flight after the female has located her goal (Haskell, 1966). The consumatory flight is usually direct and brief, since

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20 visual and biochemical cues are lost over long distances. If the cue encountered was olfactory a direct upwind flight is conducte d until other cues, visual perc eption, movement or thermal, enable the female to locate her goal more pr ecisely (Gillies and W ilkes, 1972). Consumatory flights do not always have to follow appetential f lights. An example would be the biting of a host that enters the daytime resting site of the female mosquito (Bidlingmayer, 1985). Other factors affecting the flight of mosqu itoes usually involve the weather. The most influential meteorological factor s are light, temperature, humid ity, and wind (Day and Curtis, 1989). Nightly variations in wind, rainfall, and re lative humidity influence mosquito patterns and maybe even feeding success. Daily rainfall patterns can potentially determine whether the mosquito population will continue to build, remain steady, or declin e as it relates to feeding and oviposition behavior, mainly during the rainy season of late summer and early fall in Florida. Research indicates that most mosquito specie s possess a bimodal flight activity pattern during the night, with the larger peak occurring soon after sunset and the smaller peak just prior to dawn (Schmidt, 2003). Mosquito activity can be forecasted using th e four meteorological factors mentioned earlier. The Weather Channel has teamed up w ith the maker of the Mosquito Magnet Pro (MMPro), American Biophysics, to launch the fi rst ever Mosquito Activity Forecast on the website, www.weather.com The website link is managed by a team of meteorologists from the Weather Channel who provide hourly predictions of mosquito activity nationwide. People who want to participate in outdoor activities in areas inhabited by vector species may be able to better plan their activities using this valuable tool (Dilling, 2004). Host Location Behavior The female mosquito will look for a host from which she can obtain a blood meal one to three days following emergence. One study indica ted host seeking was inhibited for a period of

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21 40 hours following a blood meal for the mosquito An. gambiae (Takken et al., 2001). For over half the twentieth century, research has been conducted to determine why mosquitoes are attracted to certain hosts and wh at odors are responsible for the mo squitos attractive behavior to a specific host. The principle difference in the orientation of blood-suck ing insects, compared with other insects, lies in the differences in behavioral re sponses to cues that may be distinct between long and short range. In 1942 it was dem onstrated that unwashed naked children were preferred by Anopheles spp. over freshly washed naked chil dren. This same group showed that the presence of dirty human clot hing attracted more mosquitoes th an an empty hut as the control (Haddow, 1942). The ability of humans to attract anthropophi lic mosquitoes differs among individuals (Khan et al., 1965). Th e results of their studies dem onstrated that one person was more attractive to Ae. aegypti than three other people (Khan et al., 1965). Based on these studies and others, host selection is based on host pref erence and availability, with a combination of visual, olfactory, and physical stimuli to help locate th e host (Takken, 1991). Although many mosquito species display visual re sponse to distinct obje cts at a distance of up to 19 m (Bidlingmayer and Hem, 1980), olfactor y stimuli from host odors are considered to be the strongest cues for location of hosts fo r blood meals (Allan et al., 1987 and Bowen, 1991). Olfactory cues which aid in host-seeking by orienting mosquitoes to a host are known as kairomones (Howse et al., 1998). Extensive work ha s been done to determine the mechanism of mosquito attraction to its host. There is ampl e evidence that host-seeking by mosquitoes is mediated by semiochemicals, chemicals which deliver a message, emanating from the host (Bernier et al., 2003 and Geier et al., 1999). Olfactory cues are detected through an intricate pathway, beginning with the sens illa located on the antennae a nd palpi which detect carbon dioxide (CO2). Age and the physiological state of the mosquito determine whether the detection

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22 of olfactory cues results in a behavioral response (Takken, 1996). Volatile chemicals such as CO2, octenol, and acetone and less volatile substan ces such as lactic acid and fatty acids are present on skin as a result of vertebra te metabolism (Sastry et al., 1980). CO2 is universally attractive to mosquitoes and is probably the be st understood of the volat ile host cues (Gillies, 1980, Gibson and Torr, 1999). Many researchers believe that volatil e compounds act as attractants that help orient the mosquito towards its host and that CO2 can combine with other host odors to elicit a synergistic response (Smith et al., 1970, Gillies 1980, Bernier et al., 2003). Gillies (1980) also found that CO2 and whole-body odors have an orienting effect when presented singly and an enhanced effect wh en presented together Kline and Mann (1998) showed that traps baited with CO2 capture 8-30 times more mosqu itoes than traps without CO2. Around the attractant plume any ins ect with a flight speed less th an approximately 3.5 m/sec will be captured (Kline, 1999). High and low release rates (200 and 500 cc/min) of CO2 are utilized during catches (Kline, 1994). It has been shown that CO2 does not help mosquitoes discriminate different hosts (Mboera and Ta kken, 1997), but may actually functi on as a primer to activate mosquitoes and make them more receptive to other host odors. (Dekker et al., 2005). In 1984, Hall et al., through studying the attracti veness of oxen to Tsetse flies in Africa, identified octenol from the ox. Field tests have de monstrated that octenol serves as a powerful attractant for certain species of mosquitoes and flies (Kline, 1994). Thus, 1-octen-3-ol (octenol) is another olfactory attractant documented as an effective mosquito attractant (Dilling, 2004). This volatile compound has been isolated from ma ny natural sources, mainly plants and fungi (Dijkstra and Wiken, 1976). Chem ically speaking, octenol is an 8-carbon mono-unsaturated alcohol that has an asymmetric center and ther efore two optical isomer s with a terminal double bond (Kline, 1994). This allows for different effects by the different isomers. At the present time,

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23 manufacturers of mosquito c ontrol equipment such as Mo squito Magnet Pro, (American Biophysics Corporation) recommend octenol as supplementary bait to mosquito traps. Kline (1994) suggested that even though octenol was effective at attrac ting some species, it is not correct to say that the compound will successfully attract all mos quito species (Kline, 1994). The natural release rate of octenol by oxen is 0.043 mg /h (Hall et al., 1984). In studies conducted in 1994, the release rate used was 4 or 40 mg/h (K line, 1994). Octenol is now available in slow release packets, which have a releas e rate of 0.5 mg/hour (Kline, 1999). Another volatile chemical believed to play a ro le in host location is lactic acid, which is a by-product of anaerobic metabolism, common to all mammals. The acidity of freshly secreted sweat is due to the production a nd secretion of lactic acid by the eccrine sw eat glands (Thurmon and Ottenstein, 1952). In one study, both fresh and incubated human sweat was used to catch the malaria mosquito, Anopheles gambiae. They found incubated sweat was more effective than fresh sweat in catching this particular species. Th ey noted a decrease in lactic acid concentration associated with the two day old sweat, and conclu ded that lactic acid ma y not play a role in attracting An. gambiae (Braks and Takken, 1998). An. gambiae seem to be attracted to volatiles of Limburger cheese, which to a human, resemb le human foot odor (De Jong and Knols, 1995). Skin emanations are important because odors fr om live hosts have been shown to be more attractive than any combination of these ch emicals provided in a warm, humid airstream (Woodbridge and Walker, 2002). Some researcher s believe continuous bacterial action in secretions on the human skin re sults in volatiles that functi on as kairomones for mosquitoes (Braks and Takken, 1998). American Biophysics Corporation has been working on additional compounds that have proven useful in trapping mosquitoes. Lurex an d Lurex lactic acid based compounds, when

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24 combined with CO2, are thought to be effective in increasing trap catches of Aedes albopictus (Skuse) (McKenzie et al., 2004). Visual attraction is also a key component in host location. Both male and female adult mosquitoes have two compound eyes and two oc elli. Compound eyes are suited for navigation and sensing movement, patterns, contrast, and colo r, while ocelli are believed to sense light levels, and possibly polarized lig ht (Allan et al., 1987) The compound eyes have relatively poor resolution but overall high light sensitivity (Muir et al., 1992). It has been reported that diurnal species respond to visual charact eristics of hosts such as co lor, brightness, pattern, and movement (Allan et al., 1987). It may be conclu ded that in a human dwelling, when given the choice, the host seeking Mansonia mosquitoes are more attracted to the blue and red spectra than the white, yellow, green, and black (Bhuyan and Da s, 1985). Movement may also play a role in host location by mosquitoes and a consistently small but positive attraction to movement has been affirmed (Wood and Wright, 1968). Within one meter of the host, convective heat and humidity become the main attrac tants rather than chemical or visual stimuli (Woodbridge and Walker, 2002). Physical stimuli such as temperature and humid ity are also attractive to mosquitoes. With the help of a clothed human robot, Brown (1951) wa s able to show that mosquitoes landed three times as often on the clothed robot when the s kin temperature was body temperature (98F) than when the surface temperature was lower, around 50-65F (Brown, 1951). In addition, Brown noticed that moisture coming off of the robots clothing increa sed the landing numbers two to four times, but only at temperatures above 60F. Despite extensive work completed thus far, it has yet to be determined what causes mosqu itoes to locate and feed on a host. It has been

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25 concluded on several occasions that a combinati on of olfactory, visual and physical stimuli are attractive factors. Host Preference As with host-seeking behavior, host preferen ce varies widely among different genera of mosquitoes and this preference may change w ithin genera depending on a geographic location. Some species may feed almost entirely on one host while others who are more opportunitistic may feed on two or three diffe rent vertebrate classes. Culex genus prefers to feed on avian species, but if the population of birds is insuffi cient or unavailable, they will happily feed on mammals (Braverman et al., 1991). Species in Florida such as Aedes Anopheles, Coquillettidia Mansonia and Psorophora prefer to feed on mammals Mosquitoes can be found in high numbers most of the year in Florida on livestock facilities. Unfortunately, some mosquito species are competent disease vectors which threat en livestock industr ies year round. A number of mosquito species are known to feed on horses. Aedes and Ochlerotatus species have been found to be readily attracted to horses (Loftin et al., 1997) as well as Psorophora columbiae (Dyar and Knab) (Kuntz et al., 1982). Anopheles spp., Coquillettidia spp., Culex spp., Culiseta spp., and Mansonia spp. are also equine f eeders (Constantini et al., 1998, Kuntz et al., 1982, and Loftin et al., 1997). Culex nigripalpus Theobald and Culiseta melanura (Coquillett) have been shown to be vector s of WNV and Eastern Equine Encephalitis, respectively (Darsie, 2006). Concerned horse owne rs seek methods to minimize the exposure of animals to mosquitoes. Humans as Attractants Researchers are examining the attraction of the natural host as a way to draw the mosquitoes to traps and away from the host. Ento mologists have used hostbaited traps since the early 1900s for collecting Anopheline mosquitoes during malaria investigations (Mitchell et al.,

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26 1985). In the mid-1960s, researchers were able to determine that people had varying levels of attractiveness by using Ae. aegypti (Linnaeus) and measuri ng bloodfeeding and probing responses (Khan et al., 1965). Human bait catches have been re ported as the standard and most useful method for collecting host-seeking anth ropophilic mosquitoes (Service, 1993). Kline (1994) stated there is a need for new, safe and ef fective ways to kill and control pest and vector mosquito species and to deter blood seeking mo squitoes from feeding on animals and humans. The use of chemical insecticides and topical repellents faces increased restrictions due to environmental concerns a nd mosquito resistance. Several researchers have stated that a di fference exists betwee n individuals (Haddow, 1942, Khan et al., 1965, Schreck et al., 1990, Ca nyon et al., 1998) Therefore, it has been established that human skin odor contains volati le chemical substances that increase mosquito attraction in the laboratory (Schreck et al., 1981, Eiras and Jepson 1991, 1994) and in the field (Gillies and Wilkes, 1972). Once it was determin ed that differences among people existed, attempts were made to identify those differe nces through biological and chemical methods. Samples collected from people in various locations were typica lly analyzed by mass spectrometric detection, e.g. GC/MS, whether the emphasis is on skin emanations, breath, urine, blood, oral cavity, or the total composite of eman ations from an entire person (Bernier et al., 2006). Volatile substances produced by human skin ha ve been shown to act as either attractants or repellents. Many of the volatiles responsible for these actions are found in sweat (Takken, 1991). Smallegange reported that carboxylic acids make up an important part of human sweat (Smallegange et al., 2005). Laboratory studies aimed at elucidating the compounds constituting human-produced odor blends that mosquitoes us e for host location have yielded several active mixtures and individual s ubstances (Smallegange et al., 2005). In their study, An. gambiae rely

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27 on the combination of ammonia, lactic acid, a nd carboxylic acids in its orientation to human hosts, different from the info rmation reported concerning the Ae. aegypti (Smallegange et al., 2005). Humans seem to have uniquely high levels of lactic acid on their skin compared to other animals (Dekker, et al., 2002). Lac tic acid is known to play an im portant role in the host-seeking behavior of another anth ropophilic mosquito species, Ae. aegypti (Acree et al., 1968). Ammonia was also identified as an attractant for Ae. aegypti It is not attractive when tested alone, but it enhances the attractiveness of lactic acid. Fatt y acids of chain length C1-C3, C5-C8, or C-13-C18 had the same effect when mixed with lactic acid. Experiments with An. gambiae females that were done in Y-tube olfactometers showed that the synergistic effect could also be achieved when combining ammonia and lactic acid with on ly one of the short-chain carboxylic acids that was present in their mixture: hexanoic acid (S mallegange et al., 2005). Others reported an attractiveness of an unsaturated carboxylic aci d, 7-octenoic acid, which is a human-specific component, secreted from the aprocrine sweat gla nds in the axillary regions (Smallegange et al., 2005). Braks concluded that the kairomones to which An. gambiae responds were also present in fresh sweat but that the quantit y or quality of the attractive volatiles was e nhanced strongly during incubation. Skin microorganisms are pr esumed to break down sweat-borne compounds into smaller, more volatile co mponents. Furthermore, they no ted that the preference for the incubated sweat decreased after twenty minutes of exposure in the olfactometer. The combined results led them to suggest that the co mponents responsible fo r the preference of An. gambiae for incubated sweat to fresh sweat are highly volatile (Bra ks et al., 2001). Attractants from Other Hosts In addition to human odors, samples from other mammals have been used to attract mosquitoes and have shown high success rates. Researchers found an increase in trap numbers when the attractant used was collected from mice using a closed-air system, without the help of

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28 other attractants (McCall et al ., 1996). Another study in Israel, led by Braverman in 1991, used several different animals as bait in traps to catch Culex pipiens (Linnaeus). Sheep, chickens, calves, and turkeys were the most successful bait s. The calf tended to increase the total trap number when compared to the other three animals. Birds, such as sentinel chickens, are commonl y used for attracting mosquitoes, especially for surveillance techniques and to predict dis ease outbreak (Day and Stark, 1996). Cotton swabs coated with crow uropygial gland secretions cau sed a significant increas e in trap counts when compared to a clean cotton swab, leading research ers to continue work with other avian species (Russell and Hunter, 2005). Previous experiments attempting to use horse odors have been conducted. Several studies were conducted using a vacuum aspirator to v acuum the odors directly from the horses body, which were then fed into a trap in an attempt to increase trap counts. Very little difference was seen when horse odors were collected by this met hod. When a horse is present near a trap, trap counts decrease and the mosquitoes go to the horse (Dilling, 2004). Dekker and fellow researchers measured the amounts of L-Lactic acid in skin-rubbing extracts from humans compared with twelve other mammals and chic kens, including horses. A greater amount was found on humans compared to horses (Dekker, 20 02). Further research needs to be completed with the horse to determine if odors exist that have the capability to attr act mosquitoes. Traps baited with horse odor could concei vably protect the horses nearby.

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29 CHAPTER 2 SEASONALITY OF MOSQUITOES AT THE UNIVERSITY OF FLORIDA HORSE TEACHING UNIT IN NORTH CENTRAL FL ORIDA USING TRAPS BAITED WITH THREE DIFFERENT LURES. Introduction Floridas equine industry is affected by mosquitoes whic h have a direct impact on the management of horse farms. Although nui sance biting, disease transmission and their economic effects have not been studied in hor ses, mosquitoes have been found to have a negative correlation in other livestock species, specifically dairy cattle. Reduced milk yield, lower weight gain, and a compromised i mmune system leading to an increase in disease from stress caused by insects have been noted (Steelman, 1979 and Byford et al., 1992). Since the arrival of the West Nile Virus (WNV) in the United States in 1999, Floridas equine industry has lost millions of dollars through disease prevention, health care, and overall morbidity of inf ected horses (Porter et al., 2003). Mosquito trapping is an effective survei llance tool used to study local mosquito populations. Trapping allows for the determin ation of potential di sease vectors, the prediction of disease transmission, and the study of behavior and other patterns. It is helpful to know the population seasonality trends, which results in more efficient mosquito control, subsequently paving the way for more accurate protective measures which could be implemented on horse farms. Because trapping mosquitoes has become so important to their control, extensive re search has been performed to improve the ability of the traps to work more effectively (Dilling, 2004). Unfortunately, traps may be less succe ssful in many instances on horse farms because the horse becomes a competitor agains t the trap. In this case, the mosquito prefers the horse over the trap, causing a decrea se in mosquitoes trapped and an increase

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30 of mosquitoes that are present on the horse s (Dilling, 2004). Dilling determined that the traps commonly used to study populations of mo squitoes, namely the Center for Disease Control (CDC) trap model 1012 (John W. Ho ck Company, Gainesville, FL) and the Mosquito Magnet Pro (MMPro) (American Biophysics Corp., North Kingston, RI), shown in Figure 2-1, were effective at trappi ng mosquitoes as long as a natural host was not placed in a competitive situation. Many traps use stimuli to mimic host-preference qualities that attract mosquito es, such as heat, carbon dioxide, kairomones, and moisture (Kline and Mann, 1998). It has been thought that adding odors that mimic the scent of the natural host directly to th e trap would improve the efficacy of the trap. Octenol, discovered in 1984 by Hall et al., has been success ful in the past at increasing numbers of certain species captured. Other odors have been isolated as attractants and are also used in mosquito traps. Lurex, a human-based lac tic acid compound and Lurex, a lactic acid + ammonia compound designed for use in the MMPro, are two such products that were used in this study as attractive baits that may potentially enable mo squito traps to outcompete the natural host. There is a definite need fo r volatile baits (odors) that can make mosquito traps more competitive with or out-compete the natu ral host. Therefore, the main objectives of this study were 1) to conduct competitive tra pping studies using three different lures, octenol, Lurex and Lurex3; and 2) to evaluate the to tal mosquito population profiles caught when traps are baited wi th volatile odors (lures). Materials and Methods The lure trapping studies we re conducted at the University of Florida Horse Teaching Unit (HTU) in Gainesville, Florida. The 60-acre facility houses approximately 45 quarter horses used for breeding, teaching, an d training, from weanlings to retired age.

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31 The MMPro trap was used for the study. It is a self-powered mosquito trap that uses propane as an energy source. Upon com bustion, the propane catalytically converts to produce CO2, heat and moisture, which act as attr actants, and electricity to power the fans. MMPros may be used with or without an additional lure. When a mosquito nears the base of the outflow of CO2 and heat, a fan which creat es a counter flow current vacuums the mosquitoes into a collection ne t where they die of dehydration. The MMPro, with its patented technology, is made of st ainless steel with a PVC outer covering and stands about 40 inches tall (Figure 2-1). During the study, a weather station was u tilized to measure the minimum and maximum air temperatures (C) and total rainfall (cm). The station is located just north of the large pond in the west portion of the HTU. The wooden post used to hold the weather station stands 1.5 meters tall, with a permanent rain gauge mounted on the top. Located just below the rain gauge is a waterproof thermometer. Experimental Design The study began September 2, 2005, and was completed September 26, 2006. MMPro traps were placed in 4 predetermine d locations at the HTU (Fig. 2-2). Traps remained at their designated locations for the entire project. Treatments in combination with carbon dioxide (CO2) included three different baits: Octenol (Treatment #1), Lurex (Treatment #2) and Lurex3 (Treatment #3), plus a control treatment of CO2 alone (Treatment # 4). Using a predetermined sche dule, treatments were rotated through the four traps in a 4x4 Latin square design and replicated 9 times (Table 2-1). Each Latin square was completed in 8 weeks. Treatment s were rotated and the lures were replaced every 14 days. The MMPros 9-kg propane ta nks were changed approximately every 19 days. The nylon collection nets were changed two times a week, on Tuesday and Friday

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32 afternoons around 4 pm. The minimum and maximu m air temperatures were recorded, as well as the total rainfall for the past 3or 4-day trapping period. Mosquitoes were stored in a freezer at -25C until counting and identi fication were completed. The species data was combined for the total study to analy ze the seasonality trends at the HTU. Data were analyzed by General Linear M odel (GLM) after tran sformation by log (n + 1) and the means were separated by D uncans Multiple Range Test (SAS 2006). The significance interval was set at P < 0.05. Standard error was calculated from the means using SSPS. Results Traps baited with treatment #1 (Octenol + CO2) captured significantly more mosquitoes than traps baited with the othe r three treatments (Table 2-2). Mean numbers of mosquitoes captured by traps baited with treatments #2 (Lurex3 + CO2) and #3 (Lurex + CO2) were not significantly different from each other, but were significantly lower than those captured by control traps CO2 alone. Thus the addition of the two Lurex baits to CO2 actually reduced the numbers of mosquito es captured (Table 2-2). The mean number of mosquitoes captured by the tr ap at location 1 (Fig. 2-2) was significantly higher than that captured by the trap at location 4, no ma tter which attractant combination was used. Numbers of mosquitoes captured were greatest at location 1 and decr eased from locations 2 through 4 (Table 2-3). There was no signi ficant difference between the mean numbers of mosquitoes captured daily during the two collec tion intervals (nets were collected on Tuesdays and Fridays). Significantly higher nu mbers of mosquitoes were trapped during the month of September (2005) than during the other 12 months of the study (Table 2-4, range of means SE). The mean numbers of mosquitoes captured in October and November were significantly higher than th e remaining ten months of the study. There

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33 was no significant difference in the mean numbers of mosqu itoes captured between the remaining months (Table 2-4). The mean di fferences between the individual mosquito species to the four attractant combinati ons are shown in Table 2-5 and the mean differences of the individual mosquito species trapped in each of the four locations are shown in Table 2-6. Figure 2-3 compares the monthly total num bers of mosquitoes trapped with each attractant combination. Octenol was the most at tractive for the majority of the 13 months, except for February, April, and August, where the control was more effective. A total of 71, 850 mosquitoes were trapped dur ing this study combined (Table 2-7). Data were plotted to show the seasonality diffe rences and mosquito population fluctuations that occurred at the HTU (Figure 2-4). The highest numbers of mosquitoes were caught during the months of October, Novemb er, and December of 2005, but populations decreased during the months of February, Marc h, and June (Fig. 2-4). An increase in mosquito population numbers was seen approximately 2-3 weeks following large rainfalls in October and November (Fig. 2-5) But the same occurrence failed to happen during December of 2005. Mosquito populations were increasing duri ng rain events in June and July, but populations did not peak until late July or early August (Figure 2-5). Average rainfall measured during the study was lo wer than the previous three years at the HTU (Figure 2-6). Minimum and maximum temperatures recorded at the HTU throughout the study (Figure 2-7) further help to explain the trends of mosquito populations. Figure 2-8 illustrates the total mosqu ito species composition for the study. The most prominent species caught was Mansonia spp females (50.6 %) Ma. titillans

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34 (Walker); Ma dyari (Belkin, Heinemann and Page), with Mansonia spp. males (4.7%) combined for a total Mansonia spp. trapped at 55.1%, followed by Anopheles crucians (Wiedemann) (19.6 %) and Coquillettidia perturbans (Walker) (14.5%). To a lesser extent, Culex erraticus (Dyar and Knab) (3.4%), Cx. nigripalpus (Theobald) (2.9 %), Cx. salinarius (2.2 %), Anopheles quadrimaculatus (Say) (1.7 %) were trapped during the study. Psorophora columbiae (Dyar and Knab), Uranotaenia sapphirina (Osten Sacken), and Ochlerotatus infirmatus (Dyar and Knab) combined fo r just over 1% of the total collection. Ma spp. females, An. crucians, Cq. perturbans, Cx. erraticus, Cx. nigripalpus, Cx. salinarius and An quadrimaculatus were trapped in numbers >1,000 over the course of the 13 months and individu al seasonality trends are shown in Figures 2-9 through 2-15. Discussion During this 3-Lure study, octenol combined with CO2 was found to be significantly more effective than Lurex, Lurex3, and CO2 alone at increasing mos quito trap counts on a north central Florida horse farm. In past studies, octenol has proven to be a more effective bait at increasing mosquito trap numbers in the northern states, with Lurex3 being more efficient at trapping Aedes albopictus (Skuse) in the southern states (American Biophysics Corp., 2004). Researcher s from American Biophysics found that Ae. albopictus which is very difficult to catch (J ensen, et al., 1994), was more attracted to MMPro traps when they were baited with Lurex3 instead of octenol.1 The reverse was found in this study with octenol out-trapping Mansonia spp. females, An. crucians Cq. perturbans and Cx. nigripalpus when compared to the other lures, including Lurex3. No 1 McKenzie KE, Bedard SD. 2004. Article retrieved November 2006.

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35 Aedes spp. were captured in this study, which may be a reason for the lower total catch for traps baited with Lurex3. The human-skin-based lures, Lurex and Lurex3, should be recommended for use in environments where Aedes spp., such as Ae albopictus are abundant. Kline et al. (1990) found that there was a highly significant positive response to CO2 by all species except Cx. erraticus and An. quadrimaculatus while working in the phosphate mine pits in central Florida. Both Cq. perturbans and Ma. spp. showed a significant synergistic e nhancement in catch with octenolsupplemented CO2 when compared with CO2 alone. This disagreed with findings by Kline and Mann in 1998, where Cq. perturbans was equally attracted to octenol + CO2 and CO2 alone. Kline (1994) found that the addition of octenol to CO2-baited traps caused a decrease in Culex species trapped, which disagrees with my study. At the HTU, traps baited with the combination of octenol and CO2 captured significantly more Cx. nigripalpus than traps baited with other lures. However, with Cx erraticus there were no significant differences between the numbers captured by traps baited with any of the 4 attractant combinations (Table 2-5). With Cx. salinarius, there was no difference between traps baited with octenol or with CO2 alone. An. quadrimaculatus was more attracted to Lurex and Octenol when combined with CO2 compared to CO2 alone and Lurex3, where there was no difference (Table 2-5). Both of the aforemen tioned studies by Kline et al. made use of traps different than the MMPros, which may have a direct correlation to the differences in performance with the different lures. For some reason, the Mansonia spp. males were attracted to and captured in the MMPro traps. There was no significant diffe rence between octenol baited traps, CO2

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36 alone, or Lurex3 baited traps. Perhaps Ma males related the other 3 lures with areas frequented by Ma spp. females. Instances of Mansonia males frequenting traps and being captured in the MMPros have not be en reported in the literature. Octenol combined with CO2 was found to be the most effective at trapping several key species, including Mansonia spp., An. crucians, Cq. perturbans and Cx. nigripalpus. Cq. perturbans and Cx. nigripalpus are known vectors for equi ne diseases (Woodbridge and Walker, 2002), while Mansonia has not yet been ruled out as a competent vector for WNV (Darsie, 2006). Under conditions simila r to those at the HTU, octenol + CO2 could be used in MMPro traps to increase trap counts of these species. A significant difference was noted in the f our different trap lo cations for the study (Table 2-3). Trap #1 trapped significantly mo re mosquitoes than the other three traps used in the study. Trap #1 was located on the southern most part of the farm, near the covered riding arena and a wet, marshy area that always contained water and downed trees. Trap #2, just south of the large sw amp, caught the most following trap #1, as the swamp provided a consistent breeding gr ound throughout the study. Trap #3 was just north of the swamp, however, was most near the small paddocks that always contained horses, thus presenting a constant natural hos t for the trap to compete against throughout the study. Therefore, trap #3 location trapped the third most mosquitoes. Finally, trap #4 was at the northern most part of the farm, near the feeding barn. This presented natural competitors as well as a constant supply of dust, which caused the trap to clog and malfunction several times. Subsequently, tr ap #4 was down more than any other trap during the study. A 4 x 4 Latin square was in stituted in order to control for location differences. This step allowed the four attr actant combinations to be rotated randomly

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37 throughout the farm, eliminating a constant lo cation for a single lure. Despite the trap randomization, trap #1 was closest to the marsh, where Ma spp. and Cq perturbans both tree hole breeders. This would allow a consta nt supply of mosquito es, especially since these species were trapped the most abundantly during the study. Trap location differences during my study disagreed from what Dilling (2004) discovered, whos traps were in similar locations. She found that tr ap #2 and trap #3 caught significantly more mosquitoes than did trap #1 and trap #4. Campbell (2003) stated that mosquitoes are found in north central Florida 12 months a year, but they are present in much more signifi cant numbers during the warm, wet seasons of summer and fall; this is si milar to the results in my study. Mosquito populations differed throughout the entire study, but were at their highe st during the rainy fall months of September, October, and November, 2005, and again in the late summer months of July, August, and September of 2006 (Figure 2-4). During these peak periods of production, the temperature was also idea l for larval development, rarely dropping below 55F (Figure 2-7). Lower populations we re seen during the c ool, dry months of February and March, as well as the dry, late spring months of May and June. The latter half of 2005 experienced norma l rainfall, resulting in peak mosquito population numbers 2-3 weeks following a major rain event, obs erved in October and November. According to a 30 year study conducted by the University of Utahs Department of Meteorology, Gainesvilles normal yearly rainfall is 51.81 inches, which is close to the rainfall measured during my study. However, the time of year when the rainfall should have been collected (the summer months of June, Jul y, and August) resulted in decreased numbers for the second half of the study. This agrees with several other studies conducted at the

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38 UF HTU, including those of Campbell (2003 ) and Dilling (2004). Both stated that temperature and rainfall appear to be major factors affecting mosqu ito seasonality trends. This trend continued until the nights stayed fairly cool, below 55F, around midDecember. As a result of low temperatures and lower rainfall, the mosquito population numbers did not reach a peak for the remainder of the study. A major rain event occurred in late July, but the mosquito numbers neve r again reached the previous numbers from earlier in the study. The temperature in Gainesvi lle, Florida, does fluctuate in the winter months, and becomes steady during the summ er months, as noted by other research conducted in Florida by Campbell (2003) and D illing (2004). Because of the differences in temperature during the cool season months, the mosquito populations are never able to rise until the spring time due to constant ch ange during the night time, with sudden drops below 55F. During my study, Gainesville experi enced lower rainfall than in previous years (Figure 2-6), resulting in decrease d mosquito populations. In addition, the decreased rainfall caused certain mosquito sp ecies breeding sites to go dry, causing a shift in mosquito species co mplex. Dilling (2004) found the Culex species to be in greater numbers through the majority of her study, because numerous hurricanes made landfall and caused flooded conditions. Culex spp. were not as prevalent in my study (Figure 212, 2-13, 2-14) because of less rainfall (Figure 2-5). Similar population trends were found throughout this study, closely following Dilling (2004) despite differences in trap and baits. During the seasonality study An. crucians and Cx. erraticus were trapped all 13 months. An. crucians and Cx. erraticus peaked during the cool months and remained steady for the remainder of the trial. This closely followed previous work by Dilling (2004). Mansonia spp. was present the entire

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39 study except for February. Cq. perturbans was not present until mid-March, where it began to rise steadily and remained in high numbers for the rest of the study. The Mansonia species and Cq. perturbans have the ability to pierce and attach to the roots of aquatic plants because of the presence of their attenuated siphon which allows them to withstand longer periods of dr ought and develop in pools with less water (Darsie, 2006). This adaptation allowed both species to ma intain higher population numbers through the drought experienced during my study. One species, Ps. columbiae was not noticed in large numbers until June, along with An. quadrimaculatus which disagrees with Dilling (2004), who found Ps. columbiae in greater numbers during October and An quadrimaculatus during November and December. Probably because of climatic differences such as lower rainfall compared to previous years, several species including Cx. nigripalpus, Ps. columbiae, and An. quadrimaculatus were not collected in all 12 months or in such high numb ers as with Dilling in 2004. In the future, attempts would be made to keep the MMPro traps in an operational state by tending to them with regular maintenance, and keeping them clean and running properly. Several times the traps, especia lly the trap at position #4, stopped working because of dust. Furthermore, I would recommend adjusting the CO2 output of the MMPro trap to a level that more closely matches that of the horse, its main competition at the HTU. Horses expire approximately 2000 cc/min, (Pelletier and Leith, 1995) or > 4 times the amount that the MMPro trap releases. This fact could increa se the chances that the trap could be beneficial on a horse farm if in fact olfactor y cues are the main attractant for mosquitoes.

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40 It is important to incorporate mosquito su rveillance with effec tive trapping methods in order to achieve maximum control of disease. It is crucial to combine the technique of surveillance with effective lures, such as octenol combined with CO2 and to monitor the environmental and meteorological factors whic h could potentially in fluence the mosquito populations. Conclusion This study further support ed the fact that CO2 is an effective lure for increasing trap counts. In addition, when combined with octenol, the trap counts can be increased even further. Lurex and Lurex3 significantly suppressed trap counts when combined with CO2. Under similar metrological conditions, octenol + CO2 are the most effective attractant combination for trapping certain species of mosquitoes in the MMPro, especially the Mansonia spp. If this species becomes a competent WNV vector, octenol + CO2 could be used to trap this species. This attracta nt combination could be useful on livestock facilities to lower the total mosquito populat ion that would have access to the animals, especially the Mansonia spp. and Cq. perturbans Under different meteorological conditions, such as a cooler climate, a differe nt mosquito species complex trend might be found and a different lure might be require d if trapping is to be effective. During this study conducted in Gainesvi lle, Florida, 71, 850 mosquitoes were trapped, comprising a total of 10 different species. The most prominent genus trapped was Mansonia, followed by An. crucians and Cq. perturbans The Mansonia genus has not yet been ruled out as a competent WNV in Florida. Possibly due to the lower rainfall, Cx. nigripalpus was in lower than expected numbers, indicating that th e threat of the WNV virus at the HTU was low during the study. Because of the lack of rainfall in the warm summer months, smaller population peaks were seen in July and August, contrary

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41 to previous studies conducted at the HTU. In stead, the large peaks were seen in the late fall month of November, with relatively stable trap counts in December. Mosquitoes were trapped all thirteen months, with the lowest numbers occurring in the cold winter months of January and February. Data from the study suggests that rainfall has a huge impact on mosquito populations by pausing larval development and preventing them from continuing into the pupal stage. Furthermore, low nighttime temperatures also had an adverse effect on population numbers, inhibi ting larval development. Generally, 2-3 weeks following a heavy rainfall, mosqu ito populations increased. In my study temperature and rainfall appear to play major roles in the production of mosquito populations.

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42 Figure 2-1. Mosquito Magnet Pro (American Biophysics Corporation, East Greenwich, RI) mosquito trap.

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43 Figure 2-2. Aerial Photograph of the UF HTU showing the lo cation of the 4 MMPro traps used in the 3-Lure Seasonalit y study conducted from September 2005 September 2006.

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44 0 1000 2000 3000 4000 5000 6000 9101112123456789 MonthTotal # of mosquitoes trapped Lurex Lurex3 Control Octeno l Figure 2-3. Comparison of all four lure co mbinations and total mosquito composition trapped during the 3-lure seasonality study conducted from September 2005 until September 2006. Note: Month number equals corresponding calendar date (i.e. 1 = January).

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45 0 1000 2000 3000 4000 5000 6000 7000 80009 10 1 1 12 1 2 3 4 5 6 7 8 9MonthTotal # of mosquitoes trapped Figure 2-4. Total Mosquito counts as related to months during the 3-Lure seasonality study conducte d at the UF HTU from Septemb er 2005 September 2006. Note: Month number equals corresponding calendar date (i.e. 1 = January).

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46 0 2 4 6 8 10 129 1 0 1 1 1 2 1 2 3 4 5 6 7 8 9MonthRain (cm)0 1000 2000 3000 4000 5000 6000 7000 8000Total Mosquito counts Rainfall (cm) # of mosquitoes Figure 2-5. Total mosquito count from the MMP ro traps related to rainfall in centime ters in the 3-Lure seasonality study conduc ted at the UF HTU from September 2005 through September 2006. Note: Month number equals corres ponding calendar date (i.e. 1 = January).

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47 0 2 4 6 8 10 12 14 169 10 1 1 12 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8MonthAverage monthly rainfall (cm) Figure 2-6. Total rainfall in centimeters meas ured before the 3-Lure seasonality stud y at the UF HTU from September 2003 throug h August 2005. Note: Month number equals corre sponding calendar date (i.e. 1 = January).

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48 -20.00 -10.00 0.00 10.00 20.00 30.00 40.00 50.009 9 9 10 10 10 11 11 11 12 12 12 1 1 2 2 2 3 3 3 4 4 5 5 5 6 6 6 7 7 8 8 9 9 9MonthTemperature (C) Max Temp (C) Min Temp (C) Figure 2-7. Minimum and Maximum temperatur es recorded using the meteorological st ation during the 3-lu re seasonality study conducted at the UF HTU from September 2005 through September 2006. Note: Month number equals corresponding calendar date (i.e. 1 = January).

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49 Cx. salinarius 2.2% An. quadrimaculatus 1.7% Ps. columbiae 0.26% Oc. Infirmatus 0.05% Ur. sapphirina 0.014% Cx. erraticus 3.4% Cx. nigripalpus 2.9% Cq. perturbans 14.5% An. crucians 19.6% Ma. spp. females 50.6% Ma. spp males 4.7% Figure 2-8. Total Mosquito Specie s composition of the 3-Lure seasonality study conducted at the UF HTU conducted from September 2005 through September 2006.

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50 0 1000 2000 3000 4000 5000 6000 70009 10 11 12 1 2 3 4 5 6 7 8 9MonthMansonia spp. females trapped Figure 2-9. Total numbers of Mansonia spp females trapped by the MMPro traps during th e 3-Lure seasonality study conducted at the UF HTU through September 2005 through September 2006. Note: M onth number equals corresponding calendar date (i.e. 1 = January).

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51 0 200 400 600 800 1000 1200 1400 1600 18009 10 1 1 12 1 2 3 4 5 6 7 8 9MonthAnopheles crucians trapped Figure 2-10. Total numbers of Anopheles crucians trapped by the MMPro trap s during the 3-Lure seasona lity study conducted at the UF HTU through September 2005 through September 2006. Note: M onth number equals corresponding calendar date (i.e. 1 = January).

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52 0 200 400 600 800 1000 1200 14009 1 0 1 1 1 2 1 2 3 4 5 6 7 8 9MonthCoquilletidia perturbans trapped Figure 2-11. Total numbers of Coquillettidia perturbans trapped by the MMPro traps during the 3-Lure seasonality study conducted at the UF HTU through September 2005 through September 2006. No te: Month number equals corresponding calendar date (i.e. 1 = January).

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53 0 50 100 150 200 250 300 350 400 4509 1 0 1 1 12 1 2 3 4 5 6 7 8 9MonthCulex erraticus trapped Figure 2-12. Total numbers of Culex erraticus trapped by the MMPro traps dur ing the 3-Lure seasonality study conducted at the UF HTU through September 2005 through September 2006. Note: Mont h number equals correspondin g calendar date (i.e. 1 = January).

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54 0 50 100 150 200 250 300 350 4009 1 0 1 1 1 2 1 2 3 4 5 6 7 8 9MonthCulex nigripalpus trapped Figure 2-13. Total numbers of Culex nigripalpus trapped by the MMPro traps during the 3-Lure seasonality study conducted at the UF HTU through September 2005 through September 2006. Note: Mont h number equals correspondin g calendar date (i.e. 1 = January).

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55 0 50 100 150 200 250 300 350 400 4509 1 0 1 1 1 2 1 2 3 4 5 6 7 8 9MonthCulex salinarius trapped Figure 2-14. Total numbers of Culex salinarius trapped by the MMPro traps during the 3-Lure seasonality study conducted at the UF HTU through September 2005 through September 2006. Note: Mont h number equals correspondin g calendar date (i.e. 1 = January).

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56 0 50 100 150 200 250 300 350 400 4509 10 11 12 1 2 3 4 5 6 7 8 9MonthAnopheles quadrimaculatus trapped Figure 2-15. Total numbers of Anopheles quadrimaculatus trapped by the MMPro traps during the 3-Lure seasonality study conducted at the UF HTU through September 2005 through September 2006. Note: Month number equa ls corresponding calendar date (i.e. 1 = January).

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57 Table 2-1. Four treatment rota tion schedule for the MMPro Traps during the 3-Lure seasonality study at the UF HTU. Date Trap #1 Trap #2 Trap #3 Trap #4 09/02/05 Lurex CO2 alone Octenol Lurex3 09/09/05 Lurex3 Lurex CO2 alone Octenol 09/16/05 Lurex Octenol Lurex3 CO2 alone 09/23/05 CO2 alone Lurex Octenol Lurex3 09/30/05 Lurex3 CO2 alone Lurex Octenol 10/07/05 Octenol Lurex3 CO2 alone Lurex 10/14/05 Lurex Octenol Lurex3 CO2 alone 10/21/05 CO2 alone Lurex Octenol Lurex3 10/28/05 Lurex3 CO2 alone Lurex Octenol 11/04/05 Octenol Lurex3 CO2 alone Lurex 11/11/05 Lurex CO2 alone Octenol Lurex3 11/18/05 Lurex3 Lurex CO2 alone Octenol 11/25/05 Octenol Lurex3 Lurex CO2 alone 12/02/05 CO2 alone Octenol Lurex3 Lurex 12/16/05 Lurex CO2 alone Octenol Lurex3 12/30/05 Lurex3 Lurex CO2 alone Octenol 01/13/06 Octenol Lurex3 Lurex CO2 alone 01/27/06 CO2 alone Octenol Lurex3 Lurex 02/10/06 CO2 alone Lurex Octenol Lurex3 02/24/06 Lurex3 CO2 alone Lurex Octenol 03/10/06 Octenol Lurex3 CO2 alone Lurex 03/28/06 Lurex Octenol Lurex3 CO2 alone 04/11/06 CO2 alone Lurex Octenol Lurex3 04/25/06 Lurex3 CO2 alone Lurex Octenol 05/09/06 Octenol Lurex3 CO2 alone Lurex 05/23/06 Lurex Octenol Lurex3 CO2 alone 06/06/06 CO2 alone Lurex Octenol Lurex3 06/20/06 Lurex3 CO2 alone Lurex Octenol 07/04/06 Octenol Lurex3 CO2 alone Lurex 07/18/06 Lurex Octenol Lurex3 CO2 alone 08/01/06 CO2 alone Lurex Octenol Lurex3 08/15/06 Lurex3 CO2 alone Lurex Octenol 08/29/06 Octenol Lurex3 CO2 alone Lurex 09/12/06 Lurex Octenol Lurex3 CO2 alone

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58 Table 2-2. Mean numbers of total mosqu itoes trapped using each attractant + CO2 combination in the MMPro trapping study conducted from September 2005 through September 2006. Attractant Mean difference trapped (Std. err) n Total trapped Octenol 330.42 (.098)a 99 32,373 CO2 alone (control) 210.81 (.674)b 100 21,167 Lurex3 160.83 (.161)bc 113 11,865 Lurex 131.86 (.103)c 90 15,435 Note: Means for attractant combination which are followed by the same number are not significantly different ( P < 0.05) and n = number of observations. Table 2-3. Mean numbers of total mosquitoes trapped in each trap location in the MMPro trapping study conducted from Se ptember 2005 through September 2006. Trap location Mean difference trapped (Std. err) n Total trapped 1 259.41 (.817)a 104 27, 260 2 216.08 (.005)ab 105 22, 688 3 194.66 (.787)ab 104 20, 243 4 156.43 (.4599)b 89 10, 289 Note: Means for each trap location which are follo wed by the same number are not significantly different ( P < 0.05) and n = number of observations.

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59 Table 2-4. Mean numbers of total mosquitoes trapped for each month during the MMPro trapping study from September 2005 through September 2006. Month Mean difference trapped ( Std. err) n Total trapped September 2005 795. 13 (.2)a 31 24, 649 October 2005 437.81 (68.74)b 27 11, 821 November 2005 430.89 (.88)b 35 15, 081 December 2005 105.37 (.57)c 35 3, 688 January 2006 121.19 (.75)c 32 4, 318 February 2006 35.59 (.758)c 32 1, 139 March 2006 71.92 (.85)c 26 1, 870 April 2006 95.53 (.63)c 32 3, 057 May 2006 118.19 (.98)c 32 3, 782 June 2006 66.64 (.30)c 33 2, 470 July 2006 164.88 (.70)c 25 4, 122 August 2006 136.10 (.02)c 31 4, 219 September 2006 139.65 (.10)c 31 4, 329 Note: Means for each month which are followed by the same number are not significantly different ( P < 0.05) and n = number of observations.

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60 Table 2-5. Mean numbers (standard error) of mo squito species captured for each attractant + CO2 combination for the total MMPro trapping study conducted September 2005 through September 2006. Species Caught Lurex Mean (Std. err) N = 97 Lurex3 Mean (Std. err) N = 105 Control Mean (Std. err) N = 104 Octenol Mean (Std. err) N = 87 Mansonia spp. (females) 52.68 (13.06)b 60.67(10.89)b 91.99(18.91)b 154.46(29.22)a An. crucians 18.20(2.40)c 26.15(60)bc 34.64(4.86)b 60.73(9.62)a Cq. perturbans 8.41(1.50)c 9.50(1.58)c 27.94(4.77)b 57.56(8.99)a Mansonia spp. (males) 4.55(1.22)b 9.31(1.88)a 9.94(2.23)a 12.08(2.35)a Cx. erraticus 4.64(0.96)a 4.29(0.96)a 5.86(1.07)a 9.49(3.62)a Cx. nigripalpus 4.57(1.06)bc 2.90(0.65)c 5.54(1.14)b 7.48(1.59)a Cx. salinarius 1.81(0.38)b 3.96(0.63)ab 4.54(0.84)a 5.03(0.94)a An. quadrimaculatus 3.29(0.66)ab 2.73(0.60)b 1.91(0.45)b 4.65(0.76)a Note: Means for each species which are followe d by the same number are not significantly different ( P < 0.05) and n = number of observations. Species with total trapped numbers > 1,000 are included in table.

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61 Table 2-6. Mean numbers (standard error) of mo squito species captured for each trap location for the total MMPro trapping study c onducted September 2005 through September 2006. Species Caught Trap #1 Mean (Std. err) N = 107 Trap #2 Mean (Std. err) N = 105 Trap#3 Mean (Std. err) N = 104 Trap#4 Mean (Std. err) N = 87 Mansonia spp. (females) 95.11(22.74)ab 77.89(13.49)ab 116.43(24.14)a 66.15(13.45)b An. crucians 60.15(9.79)a 42.33(4.56)b 20.83(2.46)c 11.94(1.67)c Cq. perturbans 28.50(5.04)b 41.06(7.87)a 13.67(2.76)c 18.46(4.39)bc Mansonia spp. (males) 5.82(1.53)ab 9.20(1.80)b 11.43(2.34)a 9.80(2.25)ab Cx. erraticus 11.13(3.22)a 4.35(0.99)b 5.79(1.53)b 2.20(0.72)b Cx. nigripalpus 6.75) 1.20)a 5.88(1.37)ab 3.66(0.91)b 3.85(1.03)b Cx. salinarius 5.14(0.88)a 5.39(0.89)a 2.72(0.46)b 1.78(0.46)b An. quadrimaculatus 3.91(0.69)a 3.34(0.63)a 2.30(0.54)a 2.94(0.62)a Note: Means for each species which are followe d by the same number are not significantly different ( P < 0.05) and n= number of observations. Sp ecies with total trapped numbers over 1,000 are included in table.

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62 Table 2-7. Total Mosquito Species Count and percen t of total count of mosquito species trapped by MMPro traps in the 3-Lure seasonal ity study at the UF HTU conducted from September 2005 through September 2006. Mosquito Species Total Count Percent of Total Mansonia spp. Anopheles crucians Coquillettidia perturbans Mansonia spp. males Culex erraticus Culex nigripalpus Culex salininarius Anopheles quadrimaculatus Psorophora columbiae Ochlerotatus infirmatus Uranotaenia sapphirina 36217 14071 10392 3361 2433 2055 1554 1264 184 39 10 71580 50.6 19.6 14.5 4.7 3.4 2.9 2.2 1.7 0.26 0.05 0.014

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63 CHAPTER 3 STUDIES USING HORSE ODORS TO AUGMEN T MOSQUITO TRAP COLLECTIONS AT THE UNIVERSITY OF FLORIDA HORSE TE ACHING UNIT, GAINESVILLE, FLORIDA Introduction Mosquito traps may be capable of reducing th e numbers of mosquitoes near a trap, but their primary usage is for surveillance. Trap s are used to catalog species composition and estimate the density of mosquitoes in an area. Va rious trap styles have been developed, spanning a variety of species specificity and trapping efficiencies of each species (Kline, 1999). Some of these traps are designed to mi mic the natural host, usually th rough the release of host-seeking cues that may consist of carbon dioxide (CO2), heat, moisture, and odors (Kline and Mann, 1998). An obstacle that helps render these traps as less effective tool s for mosquito control is that they do not compete well against a natural host wh en the host is near the trap(s). Presumably, this is because the odor profile of the host e licits greater attraction in mosquitoes than CO2 alone or any attractant that is co mprised of simple blends. In the past, it was found that significant di fferences occur between species composition and total numbers of mosquitoes coll ected from mosquito traps compared to those vacuumed from a horse (Campbell, 2003). Th is line of research has been c ontinued in this chapter through experiments that were designe d and conducted to compare simultaneously the mosquitoes captured in traps with those captured near horses. Furthermore, the addition of horse odors to the trap was examined to determine if this bait c ould produce the collection of the same numbers and species composition of mosquitoes which w ould normally be attracted to the horse. Dilling (2004) performed several studies eval uating the use of horse odors to augment mosquito trap collections. In one study, a horse was placed in a feeding slip with a Mosquito Magnet Pro (MMPro) trap directly next to the stall. When the horse was nearby, the numbers of trapped mosquitoes declined due to a preference of the mosquitoes for the live horse. In another

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64 study, the entire body of a horse wa s vacuumed using a modified hand-held vacuum. Exhausted horse volatiles from the vacuum were passe d through a PVC pipe and fed into the CO2 flow of a nearby CDC 1012 trap. Trap catch numbers were not significantly increase d by inclusion of the odors. It is possible that critic al volatiles may have adhered to the inside of the PVC pipe, the flow through the pipe was insufficient to deliver a minimum threshold level of attractants to impart an effect, or volatiles other than CO2 from breath are missing as are non-chemical cues such as heat, moisture, and visual ones. Alternative sampling methods to identify hor se odors may need to be developed and explored as was done to identi fy human odors that attract mos quitoes (Bernier et al., 2000). Some of this development involves collecting sa mples, analyzing and testing the biological efficacy of the samples to attract mosquitoes, followed by chemical sampling to identify the attractants that were involved. The objectives of the studies in this chapter were to 1) Collect hair and dander samples from different locations on a horse and identify the chemical composition of compounds present in these samples using gas chromatography and ma ss spectrometry; 2) determine if a correlation is present between the ability of two different horses to attr act insects and the chemical differences from the skin of these two different horses; 3) determine the ability of horse odor samples to attract and collect mosquitoes in a MMPro; and 4) determine if mosquito species composition and relative numbers of collected mo squitoes in the vacuum aspiration of two different horses compared to the composition and numbers of mosqu itoes caught in traps augmented with collected horse odors. Materials and Methods Three studies were conducted at the University of Florida Horse Teaching Unit (HTU) in Gainesville, Florida. The teaching unit houses ap proximately 40 quarter horses varying in age

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65 and sex on 60 acres of land. Two horses used for these experiments were an 8-year old quarter horse gelding named Steiner (F igure 3-1) and a 4-y ear old quarter horse mare named Lodi (Figure 3-2). Two MMPro traps a nd two portable vacuum aspirators were used to perform these studies. Experimental Design Horse Odor Collection Study Sample odors were collected from two hor ses over the period of May 20 June 30, 2006. Steiner, the eight-year old Quarter Horse gelding, was chosen to represent the animals that would be found on horse farms throughout Florida and Lodi, the four-y ear old black Quarter Horse mare, was chosen because of her hypersensitivi ty to insects such as stable flies and Culicoides spp. Bites from these insects resulted in loss of ha ir on her chest, ears, a nd tail (Figure 3-3). Horses were observed at dusk to determine ar eas where mosquitoes landed. The sites that resulted in the greatest mosquito landings were used as the loca tions where samples were to be taken and analyzed. Horse odors were collected fro m the face, barrel/dorsal side of the abdomen, and the legs using a cotton ball of a 2 diameter The cotton ball was rubbed in the preferred area ~ (5 in2) for 5 min to ensure ample sample extracti on (Figure 3-4). Hair was also removed from the chest of the horse with a sh arp sterilized knife, using a so ft, downward motion to cut hair close to the skin to scrape the dander into a cl ean glass vial. Mane samp les were collected using scissors, with small samples taken from the unders ide of the mane. Sample s were transported to the laboratory after collection and extracted immediately upon return to the laboratory to minimize loss of volatile compounds. Hair samples from various locations on Steine r and Lodi were extracted with 1mL hexane and the dander samples from these horses were ex tracted in 250 L. Extracts were analyzed by gas chromatography/mass spectrometry (GC/MS) on a ThermoFinnigan Trace Single

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66 Quadrupole GC/MS system (Thermo quest Finnigan, San Jose, CA). This system consists of a GC oven, a split/splitless injection port equippe d with a Programmable Temperature Vaporizer (PTV) injector. Injections were performed manually, using 1 L of sample with the GC injection port set at 35oC prior to injection. Upon inje ction, the sample was loaded onto the GC column while the temperature of the injection port was ramped balistically at 14.5 oC/s to 240 oC and held there for 1.0 min. Following this loading phase, the inject ion port is set to clean by a second ballistic ramp at 14.5 oC/s to 240 oC, and held at that temperature for 3 min. The GC oven was held at 35oC for 6 min after injection and then ramped to 250oC at 10 oC/min, and held at that temperature for 25 min. The injector split was 12 :1 (mL/min flow) and the carrier gas was high purity helium set to maintain a constant flow of 1.20 mL/min. Samples were injected onto a 30 mm x 0.25 mm i.d. DB-5ms column with a stati onary phase film thickness of 0.25m. The transfer line into the mass spectrometer was held at 260oC throughout the analysis. The mass spectrometer was tuned and calibrated with pe rfluorotributylamine (PFTBA) prior to the acquisition of data. Additionally, control hexane samples were injected prior to analysis of extracted samples, and a standard hydrocarbon mixture was injected once per day to obtain retention index data to assi st in compound identification. The mass spectrometer was operated in electron ionization (EI) mode, with an average of 70 eV electrons. The ion source was set at 200 oC, the emission current was 350 A, and the detector was set at 350 V; the detector dynode wa s held in the off position until 3.0 min into the analysis. A scan rate of 0.5 s per full s can was used cover the scan range of m/z 35-565. Horse Odor Trapping Study The horse odor trapping study was conduc ted from May 23 September 30, 2006. Two MMPro traps were operated 4 m apart on the south si de of the main body of water, just north of

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67 the large covered arena at the HTU (Figure 3-5) The MMPro is a selfpowered mosquito trap that burns propane catal ytically to produce CO2, heat, and moisture. The output of combustion results in enhanced attraction of some insect species. Combustion of propane also produces electricity to power the fan. MMPros may be operated with additiona l lures or by simply releasing the combustion products without added lure. The principle by which the trap collects insects is patented as counter flow geometry where a mosquito may near the base of the outflow while one fan vacuums the insect into a 2 qt. collection net attached to the bottom of the fan inside the trap (US Patent : 7074830, Durand et al., 2006). The mo squitoes are stored in the net after the fan pulls them into the inside of the trap. A window on the front of the trap provides a view of the collection net within the trap. The trap is construc ted with stainless steel with a PVC outer covering and st ands about 40 tall. Two treatments were tested : horse collected odor + CO2 and CO2 only. Each treatment employing horse odor was collected every 24 h to minimize volatilizat ion of the compounds from the sample. Skin extracts of the horses we re collected by rubbing a cotton ball 2 diameter in a localized area (5 in2) for 5 minutes. Cotton balls were ro tated to ensure that all surfaces were covered with the scent and the length of tim e confirmed the presence of the odors and oils from the skin on the cotton ball. A latex gl oved hand held the cotton balls to minimize contamination from human skin (Dekker et al., 2002). The cotton ball was placed in a modified plastic cartridge, designed by Amer ican Biophysics to hold the Lurex3 lure. The cartridge was placed at the bottom of the trap, near the exit The control MMPro trap was operated with the normal emission of CO2, heat, and water vapor only. The samples were collected from the same physiological area on both horses every 24 h and tested in the traps daily for four consecu tive days. The cotton ba lls containing collected

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68 odorants were changed after each collection was made, around 11 am EST. With each change of cotton, the nylon collection nets were also chan ged. The propane tanks were changed every 18 d to ensure an uninterrupted suppl y of propane during the experiments. Trapped mosquitoes were stored in a freezer at -25C until counting and identification could be conducted. Data were analyzed by GLM and means separated by Duncan s Multiple Range Te st using SAS 2006 after transformation by log (n + 1). Standard erro r of the means was calculated using SAS 2006. Horse Vacuuming Study Species composition and the number of mosquitoes that attempted to blood feed from two different horses were examined on the consecutive nights of September 20 and 21, 2006. Collection began at 7:30 p.m. (around dusk) both days and concl uded one hour later at 8:30 p.m. Each horse was tied at the south end of the cove red arena at the HTU, 61 m apart, one on the far east and the other on the far west side of the arena. Approximately 150 m from the east side of the south side of the arena was a retention pond and near the west side (20 m) was a swampy, wooded area with thick natural vegeta tion, down trees, and standing water. Two individuals vacuumed the horses using por table vacuum aspirators simultaneously for 30 min (Figure 3-6). After the firs t 30 min, the two individuals sw itched horses and used portable vacuum aspirators to vacuum the other horse on the opposite side of the arena for the next 30 min. The same two individuals vacuumed each night. Mosquitoes were vacuumed from all body surfaces of the horses. On night one, individual one vacuumed horse one (Lodi) and individual two vacuumed horse two (Steiner) for the first 30 min. For the next 30 min, horse one (Lodi) was vacuumed by individual two and horse 2 (Ste iner) was vacuumed by individual one. On night two, this same collection scheme was repeated; however, the locations of horses were rotated from left to right side of the arena compared to the previous nights location. Automobile batteries supplied the power, and the two vehicl es were both white to eliminate bias due to

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69 vehicle color. A 1995 white Ford Ranger with a 12-V Interstate ba ttery was parked on the east side and a 1995 white Lincoln Continental with a 12-V Auto Zone battery was parked on the west side for both consecutive nights. Mosqu itoes were stored in a freezer at -25C until counting and identification could be comple ted. Data were analyzed by GLM and means separated by Duncans Multiple Range Test usi ng SAS 2006 after transformation by log (n + 1). Standard error was calculated using SAS 2006. Results Horse Odor Collection Study Figure 3-7 is a chromatogram depicting the compound peaks observed from the analysis of hair from the horse, Steiner. One of the mo st abundant compound peaks based on peak area is that at 13.39 min in the chromatogram. The mass spectrum corresponding to this peak could not be matched with any of the library mass spectra. Additionally, this chroma togram had significant peaks at 19.01 (geranylacetone) and at 28.60 (9-octadecenamide). Additional compounds identified in this examination of Steiner are listed in Table 3-1. There are differences in compounds on the hair between the horses examined in this study; these are reported in Table 32. The chromatogram in Figure 3-8 demonstrates visually the remarkable differences in compounds collected from the hair of Steiner an d Lodi. Figure 3-9 shows very similar results from the dander of both horses. It also demonstrates the peak at 13.39 min, an unknown compound that has not previously been detected nor reported in horse odor, nor other host odor samples from humans, chickens, or other mammals th at have been studied pr eviously (Bernier et. al., 2000; Bernier, U.R., pers. communication). Horse Odor Trapping Study There were a total of 6,282 mosquitoes captu red during the five m onth horse odor study. The species composition trapped throughout the st udy on Lodi and Steiner are shown in Figures

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70 3-10 and 3-11, respectively. The three most abundant species collected on both horses was Coquillettidia perturbans the Mansonia spp., and Culex nigripalpus When odor from Lodi was collected and added to CO2, 1,758 mosquitoes were trapped compared to the control trap with just CO2 which caught only 1,523 mosquitoes (Figure 3-12 ). In this same figure, Steiners odor + CO2 used in the trap caught only 1,437 mosqu itoes, compared to 1,564 mosquitoes for CO2 alone. The mean numbers of mosq uitoes trapped using the odors fr om Lodi and Steiner are in Tables 3-3 and 3-4, respectively. The breakdown of species composition trapped for both horses are listed in Tables 3-5 and 3-6, for Lodi and Steiner resp ectively. There was no significant difference ( P < 0.05) between the treatment groups for St einer or Lodi, including comparisons against each other, nor was there a significant difference for catches in different locations. Horse Vacuuming Study A total of 474 mosquitoes were vacuumed fr om Steiner on the east side of the arena, compared to only 411 mosquitoes when he wa s located on the west side. There were 437 mosquitoes aspirated from Lodi when she wa s located on the east side compared to 381 mosquitoes from the west side. The mean numbers for each horse and for both sides of the arena are in Table 3-7. No significant difference ( P < 0.05) was found in the sp ecies composition and the number of mosquitoes aspirated when mosqu itoes were compared between the two horses, nor was a significant difference in collections found for location in th e arena, nor for the individual person who vacuumed the horse. The total number of mosquitoes vacuumed off of both horses for both nights was 1,703 with th e most prominent species trapped being Mansonia spp. comprising 86% of those captured, followed by Cq. perturbans at just 8% and Cx. erraticus in third with just 4% of th e total catch. Nearly the same species profile was found for both horses, with only slight differences in total percen tage of species for each horse as illustrated in

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71 Figure 3-13 and 3-14. Table 3-8 and 3-9 illustrate the total mosqu ito species composition trapped for each horse. Discussion Horse Odor Collection Study Results from GC/MS analysis of horse hair and dander reveal that the compounds present on the horses contain some similarities and diffe rences compared to those present on other mammals, such as humans and bovines. Aldehydes such as nonanal and decanal are common on the skin and other surfac es of just about all animals and co uld possibly play a role in insect attraction to host odors. Anothe r compound class containing member s that were present on the horses was the alcohols and the role of many of these in th e host-seeking process remains unknown, although 1-octen-3-ol is a known mosquito attractant (T akken and Kline, 1989). An interesting aspect regarding horses as hosts fo r mosquito blood meals fr om is that they are appealing to mammal feeders, such as Cq. perturbans as well as avian feeders, such as those of the genus Culex Therefore, examination of horse odors may reveal clues about chemicals involved in the hostseeking process. Of notable interest is that the chromatogram s of both horses dander contained a peak at 13.39 min (Figure 3-7, 3-8, and 3-9). The identity of this compound is still under investigation. Until identification can be made, it will not be possi ble to determine whether or not it is crucial to mosquito location of the horses. Despite this optimism, it should not be discounted that other cues may be vital for host location, su ch as body temperature, respired CO2, production of lactic acid or even other volatile compounds such as oc tenol, methane, or excess nitrogen excretion from bodily fluids such as urination. The similarities in both horses dander may e xplain how the equine species as a whole, combined with the aforementioned cues, has an increased ability to attract insects. Despite

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72 similarities in chemical compos ition of the horse dander, the hair samples taken from both horses revealed a different chemical composition, which could be attributable to the deposition of exogenous compounds on the outside surface (hair) of the horse. Lodi had several compounds on her hair that were terpene-based, similar to those found in nature on plants and trees, as well as in pressure-treated wood, like fence posts. Since Lodi and Steiner were pastur ed in separate fields on different ends of the HTU, this may explai n the differences in the profile of compounds present in the extracts of their hair. Both horse s were restricted from bathing and excessive brushing throughout the study in an attempt to minimize contamination by exogenous chemicals from shampoos and contaminants on the equipment. These precautions were also used during the horse odor trapping study. Despite these efforts, substances hi ghly likely of exogenous origin were found in the analysis, such as compounds found in plastic gloves that were worn when samples were collected to prevent contamina tion of oils and other compounds present on the surface of human skin. Hair contaminants found on Steiner during the study are noted when possible in Table 3-1, Table 3-2 co mpares compounds that may be unique to a horse or that were found from the dander of both horses. All of the samples were similarly for each horse with respect to loca tion on the horse, such as the face, legs, and the neck. However, it wa s difficult to control for environmental conditions. Lodi was kept in a large fiel d during the summer, with greate r exposure to the environment including the sun, dirt, and exogenous chemicals, such as those from vegetation. When samples were collected from Lodi, it was apparent that th ey contained sweat and dirt from her habitat. Steiner was kept in a smaller pa ddock with greater shading, result ing collections from his hair and skin that contained less sweat than Lodi.

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73 However, the differences in composition of th e samples from each horse could not simply be attributed to the sweat of one horse versus the lack of such on the other. Bernier (1995) showed that human sweat was too aqueous, with very low volatile content and contained mostly water and salts. It could be bene ficial to thoroughly analyze the sweat from horses to determine its chemical make up and then compare it to ot her mammals, such as the human. Another useful study that could be valuable to understanding host preference of mosquitoes may be the comparison of lactic acid content in the sweat of horses as related to others. Since lactic acid has been a component in trap studies with Aedes spp. mosquitoes, the combination of this compound with others identified in horse odor could be an important lure for more efficient trapping of mosquitoes. Horse Odor Trapping Study It has been shown that differences exist be tween individual people and their ability to attract mosquitoes (Schreck et al., 1990). Therefor e it is likely to expect variation in attraction between individual horses. In addition to explor ation of differences th at exist between horses with respect to compounds present on the hair and dander, it is important to find out how a mosquito trap would fare with respect to coll ecting mosquitoes in clos e proximity of a horse. When comparing trapping of mosquito spec ies for either horse during this study, the results are essentially identical as seen in Tables 3-5 and 36 and Figures 3-10 and 3-11. The similar species profile of both hor ses could be due to the season of year when the particular treatment was run. Cq. perturbans was the most abundant species trapped; this closely follows the time of year when these mosquitoes show preference for mammals. Mansonia spp. was trapped effectively throughout this study, as seen in the seasonal ity study discussed in previous research by Dilling (2004). Finally, Cx. nigripalpus was the third most abundant species trapped and its appearance did not occur until mid-J une, very similar to the seasonality study.

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74 The GC/MS analysis of collected horse odors dem onstrated that there is little difference in the dander between the two horses in the study and that the only difference between them was on their hair. It is extremely likely that these diffe rences are due to exogenous chemicals from the environment.. The difference in the horses themselv es, though slight, could ha ve played a role in the small discrepancies between the total ca tch numbers for each horse. As previously mentioned, Dilling (2004) found a slight differe nce between an appaloosa mare and a paint gelding and their ability to compete against a trap. Even though there was no significant difference in that study, the appaloosa mare gene rally caught more mosquitoes than the paint gelding. In this study, the mares odors when combined with CO2 caught more mosquitoes compared to CO2 alone and compared to the ge ldings odors combined with CO2 similar to the findings of Dilling (2004). In this study, no sign ificant differences were found in mosquito numbers collected by odors plus CO2 compared to CO2 alone from either horse (Figure 3-12). Although differences were not found in this study, previous studies indicat e that the differences between a mare and a gelding or a stallion and a gelding could have a direct impact on the mosquitos host-seeking behavior (Dilling, 2004). It could be very impor tant to study hormone levels between the female and the male horse; in cluding an intact male hor se (the stallion) in future studies. One factor that may have impacted the study is the time constraint, as the period ran from May 2006 until early October 2006. Accordingly, th e time and month varied for each set of treatments, and this may have affected both the species composition as well as the total numbers of mosquitoes trapped. In addition, this area was under the conditions of a drought during this five month period, so the total number of mosqu itoes present was expected and certainly lower than the previous year. Therefore, the decrea se in total mosquito numbers may confound the

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75 interpretation of the results from this study. An additional factor was that only two MMPro traps were available for comparison at a time. Subsequently, the individual studies for each horse were run separately. This means that the numbers of total feeding mosquitoes on a given night during the study likely differed from one pe riod to another. Furthermore, th e traps were placed near trap #2 of the three-lure study, just south of the large body of water (F igure 3-5). This area contained varying numbers of nearby horses and these horses fluctuated throughout the entire 13 months all around the farm. The location of a horse was usua lly not permanent and the numbers in close proximity to the test site change d constantly. This fluctuation in natural hosts in the vicinity may have affected the trap counts. Future studies involving horse odors combined with CO2 used in traps could be modified by restricting the time of year by increasing the number of traps used in the comparison, as well as testing several different hor ses. In addition, it could be beneficial to increase the CO2 output of the MMPro trap to more closely follow that of the horse, which is around 2000 cc/min at rest (Pelletier and Leith, 1995) comp ared to 200 500 cc/min of CO2 that is emitted from the MMPro trap (Takken and Kline, 1989). Horse Vacuuming Study It has been noted that horses will more proficiently attract mosquitoes when compared to a mosquito trap using commercial bait when both ar e in close proximity. Therefore, it is important to investigate the ability of the factors that re sult in superior attracti on of insects by the horse whether with olfactory cues or a combination of ot her factors that trigger the mosquito and affect host preference. The species composition of v acuumed mosquitoes from both horses closely followed Dillings study (2004), ag ain probably due to the low rainfall and warm nights. There were slight numerical differences of mosquitoes aspirated from the two horses yet there was no statistical difference noted, as seen in Table 3-7. Contrary to the horse odor trapping

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76 experiment using the two horses, more mosquitoes were aspirated from Steiner on the east side of the arena when compared to Lodi. This also he ld true when Steiner st ood on the west side of the arena. The mosquitoes showed similar increases in activity both nights with respect to the time frames of collection. The early time from 7:00 7:30 pm had less mosquitoes feeding with a drastic increase as dusk set in. With this incr ease in landings and biting, the horses became agitated and used their tails to swat off mos quitoes. Both horses also stomped and flinched, which made it more difficult to aspirate mosqu itoes and collect them in the container. The feeding locations were simila r for both horses; the smaller Cx. spp. tended to prefer the legs and near the hooves and came out to feed during the first 30 min peri od. The feeding location of the Cx. spp. was similar to what Dilling (2004) found, near the coronet band of one appaloosa mare. Very little mosquitoes fed on the coronet band in this study; however, it was obvious that the smaller mosquitoes preferred the legs rather th an the other portions of the body. This could be because Cx. spp. are avian feeders and adapted to feeding in areas of the host with less skin and muscle tissue, like the legs of the horses which are more similar to th e physiological makeup of birds than the larger areas on the body. The larger species, such as the Cq. perturbans and Ma. spp were later feeders and preferred the highe r up sections of th e body, including the face, forehead and the top of the hip. A small difference, though not significant, was noted between the two far sides of the arena; regardless of which horse was there, a gr eater number of mosquitoes were vacuumed from the east side when compared to the west. There was a large retention pon d near the east side which still had some standing water. This ma y have attributed to the greater number of mosquitoes nearby, leaving the breeding ground, in search of a blood meal. When the

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77 mosquitoes left their resting site, they may ha ve stopped at the first host they came upon, which in both cases was the hors e on the east side of th e arena at the south end. It was very difficult to draw any conclusions fr om two nights, so it would be beneficial to repeat the aspirating experiment an additional number of repetitions throughout the season to determine if differences really do exist between horses and their ability to attract more mosquitoes than another. Conclusions Mosquitoes affect livestock species and humans all over the world through disease transmission and resultant morbidity and possibl y mortality. When mosqu ito traps are used on various livestock facilities, they become less effe ctive when natural hosts are in close proximity. It is important to find ways to improve trapping so that they are more e ffective in decreasing the numbers of mosquitoes that are able to feed on animals. When various samples were collected and analyzed using GC/MS, it was found that ho rses were very similar to other mammals including humans and that there could be a compound found on the dander of horses that is unique to them. This compound, in combination with other host-seeking cues, could be an important key in the amazing ability of the horse to attract the mosquito. Further analysis will need to be completed to determine the nature of the compound and the role that it may or may not play in mosquito attraction. Once this compound has been di scovered it could be used with other cues in mosquito traps in order to increase their ability to collect mosquitoes and other haematophagous insects. When odor samples were collected from the horse s and used in the mosquito trap, it was found that the trap count did not exhibit a significant difference between the uses of odors from either horse. Additional field research is needed to confirm that horse odor can increase a trap count and that the mosquito is not just feeding oppor tunistically. Furtherm ore, a horse exhales

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78 CO2 at a rate of 2000 cc/min at rest, over 4 time s the amount that the MMPro trap releases (Pelletier and Leith, 1995). It coul d be beneficial to adjust the CO2 output from the MMPro to be more quantitatively similar to that of the horse. To further investigate the differences between two horses, mosquitoes were aspirated off of each horse and the number s were compared, with no statistically significant differences observed in the numbers and species aspirated. There appeared to be a location factor so further research need s to be completed in order to confirm the differences between horses. A better understand ing of horse odors and the role that they play in host-seeking could lead to more efficient traps designed to compete against natural hosts.

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79 Figure 3-1. Steiner, sorrel quarter horse gelding used for odor co llections and mosquito trapping studies.

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80 Figure 3-2. Lodi, black quarter hor se mare used in the odor colle ctions and mosquito trapping studies.

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81 Figure 3-3. Illustration of hypersen sitivity found on Lodi, black quarter horse mare used for odor collections and mosquito trapping studies.

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82 Figure 3-4. Method of collecting horse odors fr om different locations on the body using cotton balls to collect horse odor for mosquito trapping studies.

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83 Figure 3-5. Aerial view of the University of Florida HTU sh owing the location of the two MMPro traps used during the horse odor st udy (yellow) and those used during the seasonality study (red).

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84 Figure 3-6. Portable vacuum aspirator (DC In sect Vac. BioQuip, Rancho, Dominguez, CA) and technique of aspirating mosquitoes off of the horses used for horse vacuuming studies.

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85 RT: 3.00 31.00 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 28.92 20.80 3.70 13.44 16.64 4.05 6.33 5.81 15.97 6.70 29.07 4.98 30.40 28.00 27.91 27.41 7.08 26.02 7.30 7.64 18.68 25.22 7.98 24.78 23.54 18.83 15.12 8.32 10.43 12.23 22.08 14.04 19.71 17.46 NL: 2.41E6 TIC F: MS UB-051806B Figure 3-7. Chromatogram from the analysis of ex tracts from collected hair from Steiner, Equus caballus at the Univ ersity of Florida HTU. unknown compound

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86 Figure 3-8. Chromatograms illust rating differences in peaks and abundances of compounds from the chest hair from Steiner (top), to that of Lodi (bottom).

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87 Figure 3-9. Chromatograms comparing the dander from Steiner (top), to that of Lodi (bottom) unknown compound

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88 Cq. perturbans 34.8% Ma. spp 32.8% Ma. spp males 7.5% An. quadrimaculatus 3.6% An. crucians 6.3% Cx. salinarius 3.0% Cx. erraticus 0.5% Ps. columbiae <0.001% Cx. nigripalpus 11% Oc. infirmatus 0.3% Figure 3-10. Total mosquito species compositi on for horse odor trapping study using samples from Lodi in the MMPro traps from May 2006 until October 2006 at the UF HTU.

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89 Cq. perturbans 38.7% An. quadrimaculatus 3.2% Cx. salinarius 7.6% Cx. erraticus 2.8% An. crucians 9.6% Ma. spp males 4.6% Ma. spp 21.2% Cx. nigripalpus 12.2% Ps. columbiae <0.01% Figure 3-11. Total mosquito species compos ition for horse odor study using samples from Steiner in the MMPro traps from May 2006 until October 2006 in trapping study at the UF HTU.

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90 1758 1564 1523 1437 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Lodi (CO2 only) Lodi (odor + CO2) Steiner (CO2 only) Steiner (odor + CO2)a a a a Figure 3-12. Total mosquitoes trapped using the horse odors in the MMPro traps; samples from Lodi and Steiner. Note: Totals were not found to be statistically different (p<0.05), and these are indi cated by the same letter.

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91 A n. crucians 0.28% Cx. erraticus 3.8% Cq. perturbans 19.6% Cq. perturbans males 4.0% Ma. spp 72.3% Figure 3-13. Mosquito species comparison (represented as a percent of the total mosquitoes collected) aspi rated from Lodi during the horse vacuuming study conducted at the UF HTU.

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92 An. crucians 0.37% Cx. erraticus 4.1% Cq. perturbans 9.3% Cq. perturbans males 2.2% Ma. spp 84.0% Figure 3-14. Mosquito species comparison (represented as a percent of the total mosquitoes collected) aspirated from Steiner during horse vacuuming study conducted October 2006.

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93 Table 3-1. Compounds found on Steiner, Equus caballus from samples collected for analysis by gas chromatogr aphy and mass spectrometry. Compound GC (tR) (min) Unknown Nonanal Decanal Decanal Nonanoic acid Undecanal Undecenal Undecanoic acid Dodecenal Geranylacetone Diethyl phthalate, contaminant Unsaturated alcohol, or an aldehyde Farnesol related compound 1-heptdecanol Tetradecanal Dioctyladipate Long Chain Hydrocarbon Diisooctyl phthalate (contaminant) Suspected long chain amide Cholesterol Cholestanol 13.44 14.04 15.12 15.73 15.97 16.64 17.24 18.68 18.82 19.05 20.80 23.35 23.54 26.01 27.91 28.92 30.40 30.83 36.06 49.30 49.78

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94 Table 3-2. Comparison of compounds f ound on the dander of two horses. Com p oun d GC (t R ) ( min ) Horse1 Terpene, alpha-pinene 10.61 L 2-methylnonane 11.36 S Cyclosiloxane 11.73 B Octanal 12.15 B 2-ethyl-1-hexanol 12.64 B Unknown Compound 13.39 B Terpene, 4-Carene 13.69 L 2-hydroxyacetophenone 13.84 B Nonanal 14.00 B Terpene, p-menth-1-en-4-ol 14.59 L Benzyl ester of acetic acid 14.96 L Unsaturated alcohol 15.08 B Decanal 15.64 B Terpene or terpene-like, e.g. geraniol or myrcene 15.91 L Terpene or terpene-like, e.g. geraniol or myrcene 16.28 B Unsaturated aldehyde 16.48 B Undecanal 16.60 B Caryophellene 18.78 L 1-dodecanol 19.34 B Tridecanal 19.81 B 4-methoxy-6-(2-propenyl)-1,3-benzodioxole 19.96 B Terpene-related 20.06 L Aldehyde or unsaturated alcohol 21.05 B Tridecanol 21.78 B Pentadecanal 22.22 B Aldehyde 23.32 B Alcohol 23.98 B Hexadecanoic acid 24.75 B Aldehyde, long chain 25.40 B Alcohol, long chain 25.99 S Diisoctyl maleate or related 26.49 S Dioctyl maleate or related 27.26 B 9-octadecenamide 28.60 B 1Key: Lodi (L), Steiner (S) and Both (B).

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95 Table 3-3. Mean numbers (standard deviation) of mosquitoes captured per trapping interval using the odors from Lodi in the MMPro traps. Interval Mean difference trapped ( SD) n Treatment Control 109.88 ( 18.33)a 95.20 ( 15.62)a 16 16 Note: Means followed by the same numb er are not significantly different ( P < 0.05) and n= number of observations. Table 3-4. Mean numbers (standard deviation) of mosquitoes captured per trapping interval using the odors from Steine r in the MMPro traps. Interval Mean difference trapped ( SD) n Treatment Control 119.75 ( 22.75)a 130.33 ( 26.07)a 12 12 Note: Means followed by the same numb er are not significantly different ( P < 0.05) and n= number of observations

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96 Table 3-5. Total mosquito species and percent of total mosquitoes trapped using the odors from Lodi in the MMPro traps during the horse odor trapping studies. Mosquito Species Total trapped Percent of total (%) Coquillettidia perturbans Mansonia spp. Culex nigripalpus Mansonia males Anopheles crucians Anopheles quadrimaculatus Culex salinarius Culex erraticus Ochlerotatus infirmatus Psorophora columbiae Total 1143 1076 362 246 207 117 100 16 10 4 3281 34.8 32.8 11.03 7.5 6.3 3.6 3.04 0.49 0.31 0.12 100.0% Table 3-6. Total mosquito species count and perc ent of total mosquitoes trapped using the odors from Steiner in the MMPro traps during the horse odor trapping studies. Mosquito Species Total trapped Percent of total (%) Coquillettidia perturbans Mansonia spp. Culex nigripalpus Anopheles crucians Culex salinarius Mansonia males Anopheles quadrimaculatus Culex erraticus Ochlerotatus infirmatus Psorophora columbiae Total 1162 636 365 288 229 137 98 83 10 3 3001 38.7 21.2 12.2 9.6 7.6 4.6 3.3 2.8 0.33 0.1 100.0%

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97 Table 3-7. Mean numbers (standard deviation) of mosquitoes captured per trapping interval for the vacuum aspirator study conducted in October 2006. Interval Mean difference trapped ( SD) n Lodi Steiner West East 204.50 ( 181.41)a 221.25 ( 139.92)a 198.00 ( 152.07)a 227.75 ( 170.14)a 4 4 4 4 Note: Means followed by the same numb er are not significantly different ( P < 0.05) and n= number of observations Table 3-8. Total mosquito species count and pe rcent of total mosquito es trapped using the vacuum aspirator on Lodi in October 2006 at the UF HTU. Mosquito Species Total Count Percent of Total (%) Mansonia spp. 687 84.0 Coquillettidia perturbans 76 9.30 Culex erraticus 34 4.15 Culex nigripalpus 18 2.20 Anopheles crucians 3 0.37 Total 818 100.0% Table 3-9. Total mosquito species count and pe rcent of total mosquito es trapped using the vacuum aspirator on Steiner in October 2006 at the UF HTU. Mosquito species Total count Percent of total (%) Mansonia spp. Coquillettidia perturbans Culex erraticus Culex nigripalpus Anopheles quadrimaculatus Total 774 58 41 7 2 885 87.6 6.60 4.63 0.79 0.23 100.0%

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98 CHAPTER 4 CONCLUSIONS AND IMPLICATIONS Mosquito trapping is an effective tool used to monitor species composition in the area and allows professionals to predict possible di sease outbreaks in a population. Many different commercial traps and lures are available to attrac t and trap the mosquitoes. It is important to study the efficacy of these different traps and lures in different situ ations, some that are effective on North Florida Horse farms. Disease preventio n and a decrease in nuisance biting are crucial for minimal economic loss to equine owners. Severa l lures were tested in comparison to a natural host at the University of Florid a HTU. Octenol, Lurex, and Lurex3 were combined with CO2 and tested against a control trap the Mosqu ito Magnet Pro trap operated with CO2 alone. It was found that throughout the study, octenol proved to be the most effective lure when used in close vicinity of a natural host. There was a significant differen ce between the three lures tested; octenol was more effective, followed by the control trap (CO2 alone), with no difference between Lurex and Lurex3. An additional set of studies examined the odors of a horse as an attractant. Odor samples from two different horses were collected and used in the MMPro traps. The traps were operated for 24 h. No significant difference in mosquitoes trapped was found between the odors of the two horses. An additional study was conducted where the same two horses were vacuumed to determine if either horse had an increased ability to attract mosqu itoes. No significant differences were found between the two horses. The series of research studies conducted at the UF HTU have indicated possible directions for future studies. The horse odor studies answered several questions, yet raised several more. It was found that the hair and dander of the two horses was very similar chemically. When the

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99 analyzed samples were used in the traps, the resu lts were less clear. It would be beneficial to continue studying different horses, both for chemi cal analyses and efficacy in mosquito traps. In addition to increasing the number of horses in these studies, it woul d be beneficial to compare attraction to various locations of the hor se, similar to studies in humans. Alternatively, horse sweat samples could be analyzed and then compared to humans and other mammals. Then the samples could be used in the mosquito traps and compared to the other horse hair and dander samples. Other sample collection methods could be explored as well. Actual pieces of hair and shavings of horse hair coul d be used in the traps. More could be done with the horses themselves. In these series of st udies, a castrated male and a female were used, both with different horm onal profiles. A stallion could be used as well as a gelding and a mare, to compare the different effects of hormones and the ability to attract mosquitoes. More repetitions of the studies c onducted here would suppor t the results and make the horse a more valuable tool for mosquito surveillan ce and control. Mosquito trapping is an effective tool at monitoring local species composition and predicting potential disease outbreaks. However, wh en the natural host is in the area, the traps effectiveness decreases and other methods must be used. If the horse is in fact effective at increasing trap numbers, more experiments are need ed to refine the use of the horse and horse odors as trap lures.

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100 APPENDIX ADDITIONAL INFORMATION ABOUT FLORIDA MOSQUITOES Table A-1. Classification of the family Culicidae Tribe Genera Anopheline Culicinae Toxorhynchitinae Aedeomyiini Aedini Culcini Culisetini Ficalbiini Hodgesiini Mansoniini Orthopodomyiini Sabethini Uranotaeniini Anopheles, Bironella, Chagasia Aedeomyia Aedes, Ochlerotatus, Verrallina, Ayurakitia, Armigeres, Eretmapodites, Haemagogus, Heizmannia, Opifex, Psorophora, Udaya, Zeugnomyia Culex, Deinocerites, Galindomyia Culiseta Ficalbia, Mimomyia Hodgesia Coquillettidia, Mansonia Orthopodomyia Sabethes, Wyeomyia, Phoniomyia, Limatus, Trichoprosopon, Shannoniana, Runchomyia, Johnbelkinia, Isostomyia, Tripteroides, Malaya, Topomyia, Maorigoeldia Uranotaenia Toxorhynchites The classification of all mosquitoes into 3 subf amilies, 10 tribes of Culicinae, and 38 genera is based on Knight and Stone (1977).

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101 Table A-2 List of mosquitoes in Florida Genus species Anopheles Aedes Ochlerotatus Psorophora Culex Deinocerites Culiseta Coquillettidia Mansonia Orthopodomyia Wyeomyia Uranotaenia Toxorhynchites albimanus, atropos, barberi, bradl eyi, crucians, diluvialis, georgianus, grabhamii, inundatus, maverlius, nyssorhynchus, perplexens, punctipennis, quadrimaculatus, smaragdinus, walkeri aegypti, albopictus, cinereus, vexans atlanticus, bahamensis, canade nsis, dupreei, fulvus pallens, hendersoni, infirmatus, mathesoni, mitchellae, scapularis, sollicitians, stiticus, taeniorhynchus thelcter, thibaulti, tormentor, tortilis, triseriatus, ciliata, columbiae, cyanescens, di scolor, ferox, horrida, howardii, johnstonii, mathesoni, pygmaea atratus, bahamensis, biscaynensis, cedecei, erraticus, iolambdis, mulrennani, nigripalpus, peccato r, pilosus, quinquefasciatus, restuans, salinarius, tarsalis, territans cancer inornata, melanura perturbans dyari, titillans alba, signifera mitchellii, smithii, vanduzeei lowii, sapphirina rutilus septentrionalis, rutilis rutilis, The classification of Florida mosquitoes by Ge nus species was taken from Richard Darsie, 2006.

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102 LIST OF REFERENCES Acree F, Turner RB, Gouck HK, Beroza M, Smith, N. 1968. L-Lactic acid: a mosquito isolated from humans. Science 161:1346-1347. Allan SA, Day JF, Edman JD. 1987. Visual Ecology of biting flies. Ann Rev Entomol 32:297316. Bernier UR, Kline DL, Posey KH. 2006. Human Eman ations and related natural compounds that inhibit mosquito host-finding ability. Insect Repellents: Principles, Methods, and Uses Ch.4:77-99. Bernier UR. 2006. Personal Communication. ARS USDA Center for Medial, Veterinarian, Agriculture Entomology Research, Universi ty of Florida, Gainesville, Florida. Bernier UR., Kline DL., Posey KH, Booth MM, Yost RA, Barnard DR. 2003. Synergistic attraction of Aedes aegypti (L.) to binary blends of L-Lactic acid and acetone, dichloromethane, or dimethyl disulfide. J. Med. Entomol 40:653. Bernier UR, Kline DL, Barnard DR, Schreck CE Yost RA. 2000. Analysis of human skin emanations by gas chromatography/mass sp ectrometry. 2. Identification of volatile compounds that are candidate attrac tants for yellow fever mosquito ( Aedes aegypti ). Analytical Chemistry 72 (4):747-756. Bernier UR. 1995. Mass spectrometric investigations of mosquito attrac tion to human skin emanations. Dissertation, University of Florida, Gainesville, Florida. Bhuyan M, Das SC. 1985. Field trails on colour affinity of host seeking Mansonia mosquitoes. Indian J Med Res 82:139-140. Bidlingermayer WL. 1985. The measurement of adult mosquito population changes some considerations. J. Am. Mosq. Control Assoc 1(3):328-348. Bidlingmayer WL, Hem DG. 1980. The range of visu al attraction and the e ffect of comparative visual attractants upon mosquito (Diptera: Culicidae) flight. Bull Entomol Res 70:321-342. Bowen MF. 1991. The sensory physiology of hos t-seeking behavior in mosquitoes. Ann Rev Entomol 36:139-158. Braks MAH, Meijerink J, Takken W. 2001. The response of the malaria mosquito, Anopheles gambiae to two components of human sweat, ammonia and L-lactic acid, in an olfactometer. Physiology Entomology 26:142-148. Braks MA, Takken W. 1998. Incubated human sw eat but not fresh sweat attracts Malaria mosquito Anopheles gambiae Sensu Stricto Journal of Chemical Ecology 25: 663-672.

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104 Dilling SC. 2004. Evaluation of Mosquito Trapping E fficiency and Determination of Seasonality for mosquitoes at the University of Florid a Horse Teaching Unit. Thesis, University of Florida. Gainesville, Florida. Eiras AE, Jepson PC. 1994. Responses of female Aedes aegypti (Diptera: Culicidae) to host odours and convection currents us ing olfactometer bioassay. Bull Entomol Res 84:207-211. Eiras AE, Jepson PC. 1991. Host Location by Aedes aegypti (Diptera: Culicidae): a wind tunnel study of chemical cues. Bull Entomol Res 81:151-160. Floore T. 2003. Mosquito Information. In: Th e American Mosquito Control Association. Retrieved January 16, 2006 from http://www.mosquito.org/info/php Geier M, Bosch OJ, Boeckh J. 1999. Ammonia as an attractive componet of host odour for the Yellow Fever mosquito, Aedes aegypti. Chem. Senses 24:647-653. Gibson G, Torr SJ. 1999. Visual and olfactory responses of haematophagous Diptera to host stimuli. Medical and Veterinary Entomology 13: 2-13. Gillies MT. 1980. The role of Carbon Dioxide in hos t finding by mosquitoes (Diptera: Culcidae): a review. Bull Entomol Res 70, 525-532. Gillies MT and Wilkes TJ. 1972. The range of at traction of animal baits and carbon dioxide for mosquitoes. Studies in a fres hwater area of West Africa. Bull. Entomol. Res 61:389-404. Haddow AJ. 1942. The mosquito fauna and clim ate of native huts at Kisumu, Kenya. Bull. Ent Res 33:91-142. Hall DR, Beevor PS, Cork A, Nesbit BF, Va le GA. 1984. 1-Octen-3-ol: a potent olfactory stimulant and attractant for tset se isolated from cattle odours. Insect Sci Applic 5:335-339. Harwood RF, James MT. 1979. Entomology in Human and Animal Health MacMillian Publishing Co., New York. Haskell PY. 1966. Flight Behavior. In: Insect Behaviour Symp. R. Entomol. Soc London 3:2945. Howlett FM. 1910. The influence of temp erature upon the biting of mosquitoes. Parasitology 3: 479-484. Howse PE, Jones OT, Stevens IDR. 1998. Pheromones and Behavior in Insect Pheromones and their use in Pest management London: Chapman & Hall. pp. 3-6. Jensen T, Willis OR, Fukunda T, Barnard DR 1994. Comparison of Bi-Directional Fay, OmniDirectional, CDC, and Duplex Cone Traps for sampling adult Aedes albopictus and Aedes aegypti on North Florida. J Am. Mosq. Control Assoc 10(1):74-78.

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105 Khan AA, Maibach HI, Strauss WG, Fenley WR. 1965. Screening humans for degrees on attractiveness to mosquitoes. J Econ. Entomol 58:694-697. Kline DL. 1999. Comparison of two American Biophys ics mosquito traps: the professional and a new counterflow geometry trap. J Am. Mosq. Control Assoc 15(3): 276-282. Kline DL, Mann MO. 1998. Evaluation of butanone carbon dioxide, and 1-octen-3-ol as attractants for mosquitoes associated with north central Florida ba y and cypress swamps. J Am Mosq. Control Assoc 14, 289-297. Kline DL. 1994. Olfactory Attractants for mosqu ito surveillance and control: 1-octen-3-ol. J Am Mosq. Control Assoc 10(2):280-287. Kline DL, Wood JR, Morris CD. 1990. Evaluation of 1-octen-3-ol as an attractant for Coquillettidia perturbans, Mansonia spp. and C ulex spp associated with phosphate mining operations. J Am Mosq. Control Assoc Dec;6(4):605-11 Knight KL, Stone A. 1977. A Catalog of the Mosquitoes of the World (Diptera: Culicidae) Entomological Society of America, College Park. Kuntz KJ, Olson JK, Rade BJ. 1982. Role of Do mestic animals as hosts for blood-seeking females of Psorophora columbiae and other mosquito species in Texas USA ricelands. Mosq. News 42:202-210. Loftin KM, Byford RL, Loftin MJ, Craig ME, St einer RL. 1997. Host preferences of mosquitoes in Bernalillo County, New Mexico J Am Mosq. Control Assoc 13:71-75. Mboera LEG, Takken W. 1997. Carbon Dioxide chemotropism in mosquitoes (Diptera: Culicidae) and its potential in vector su rveillance and management programmes. Rev. Med. And Vet. Ent 85: 355-368. McCall PJ, Harding G, Roberts J, Auty B. 1996. Attraction and Trapping of Aedes aegypti (Diptera: Culicidae) with hos t odors in the laboratory. J Med. Entomol 33(1):177-9. Mitchell CJ, Darsie RF, Moath TP, Sabattini MS, Daffner J. 1985. The use of animal-baited net trap for collecting mosquitoes during the We stern Equine Enchepal itis investigation in Argentina J. Am. Mosq. Control Assoc 1(1):43-47. Muir LE, Thorne MJ, Kay BH. 1992. Aedes aegypti (Diptera:Culicidae) vision: spectral sensitivity and other perceptual parameters of the female eye. J Med. Entomol 29(2): 278281. Nasci RS, Savage HM, White DJ, Miller JR, Cropp BC, Godsey MS, Kerst AJ, Bennett P, Gottfried K, Lanciotti RS. 2001. We st Nile virus in overwintering Culex mosquitoes, New York City, 2000. Emerg. Infect. Dis 7(4):742-745. Nayar JK. 1968. Effects of larval and pupal environm ental factors on biological status of adults in emergence in Aedes taeniorhynchus. Bull. Entomol. Res 58(4):811-827.

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106 Norusis MJ. 2005. SPSS 14.0 Statistical Analysis Program. Prentice Hall. Pelletier N, Leith DE. 1995. Ventilation and carbon di oxide exchange in exercising horses: effect of inspired oxygen fraction. Journal of Applied Physiology 78(2):654-662. Public Health Entomology Research and Education Center (PHEREC). 2002. Retrieved September 21, 2006 from http://www.pherec.org/traini ng/mosquitolistflorida.html Porter MB, Long MT, Getman LM, Giguere S, Mackay RJ, Lester GD, Alleman AR, Wamsley HL, Franklin RP, Jacks S, Buergelt CD Detrisac CJ. 2003. West Nile virus encephalomyelitis in horses: 46 cases (2001). J Am. Vet. Med. Assoc 222(9): 1241-1247. Rose RI. 2001. Pesticides and public health: in tegrated methods of mosquito management. Emerg. Infect. Dis 7(1):17-24. Russell CB, Hunter FF. 2005. Attraction of Culex pipiens/restuans (Diptera: Culicidae) mosquitoes to bird uropygial gland odors at two elevations in the Niagara region of Ontario. J Med Entomol 42(3):301-5. Sastry SD, Buck KT, Janak J, Dressler M, Pr eti G. 1980. Volatiles emitted by humans. In: Wiley J, ed. Supplementary Volume to Biochemical Applications of Mass Spectrometry. New York: Interscience. P 1085-1129. Schmidt RF. 2003. Relationship of landing count observations to the time of sunset. Wing Beats of the Florida Mosquito Control Association 14(3):12-19. Schreck CE, Kline DL, Carlson DA. 1990. Mosquito a ttraction to the substanc es from the skin of different humans. J of Am. Mosq. Control Assoc 6:406-410. Schreck CE, Smith N, Carlson DA, Price GD, Ha ile D, Godwin DR. 1981. A material isolated from human hands that attracts female mosquitoes. J Chem Ecol 8:429-438. Service MW. 1993. Mosquito Ecology-Field sampling methods 2nd ed. London: Chapman and Hall. Shorey HH. Interaction of insects w ith their chemical environment, in Chemical Control on insect behavior: Theory and Application H.H. Shorey and J.J. McKelvey (Eds.), New York: Wiley, 1977. pp. 1-5. Silva IM, Eiras AE, Kline DL, Bernier UR. 2005. Laboratory Evaluation of Mosquito traps baited with a synthetic hu man odor blend to capture Aedes Aegypti J Am Mosq Control Assoc 21(2)229-233. Smallegange RC, Qiu Yu Tong, JA van Loon J, Takken W. 2005. Synergism between ammonia, lactic acid, and carboxylic acids as kairomone s in host-seeking behavior of the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae). Chem. Senses 30:145-152.

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108 BIOGRAPHICAL SKETCH Aimee Camille Holton was born in Gainesville, Florida, in 1981 to Quinn and Suzanne Holton. She has lived in Gainesville, Florida her whole life, attended and graduated from Santa Fe High School in Alachua, Florida in 2000. She went to Santa Fe Community College and earned an Associate of Arts degree in 2002, and then she transferred to the University of Florida. She earned a Bachelor of Science degree in anim al science with a major in equine industry. Following graduation in December of 2004, she was accepted into a graduate program under Saundra TenBroeck with a concentration in equi ne science management. Throughout the course of her graduate program, she served as a teach ing assistant for equine reproductive management, sales preparation of thoroughbred yearlings, and psychology and traini ng I and II under the supervision of Mr. Joel McQuagge She graduated with a Master of Science degree in animal science with a minor in veterinary entomol ogy in August of 2007. Aimee plans to pursue a career as a teacher of biological sciences for secondary students.