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SURVEILLANCE AND CONTROL OF Aedes aegypti AND Aedes albopictus WITH A NOVEL LETHAL OVITRAP
By
CASEY NICOLE PARKER
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
2016
© 2016 Casey Parker
To my loving and supportive parents and brothers
4
ACKNOWLEDGMENTS
I would like to thank my committee members for their guidance and patience over
the last few years. This includes Dr. Philip Koehler, Dr. Roberto Pereira, Dr. Rebecca
Baldwin, and Dr. Alexandra Chaskopoulou. Every member of my committee was very
supportive and helped me grow as a scientist. Thank you for your time, expertise, and
wisdom.
I would also like to thank the members of the USDA-ARS laboratory in
Thessaloniki, Greece, Mr. Javid Kashefi and Mr. Emmanuel “Max” Fotakis as well as
the American Farm School. Max spent many hours in the field helping me set mosquito
traps for my research for more than 4 months and the American Farm School provided
a field site for my surveillance research. Also, a very special thanks to „Anda‟ for making
Greece feel like a home.
Dr. James Colee and Dr. Roberto Pereira provided me with guidance on the
statistical analyses involved in my research and I owe both of them thanks.
On a more personal note, I would be remiss if I did not thank my friends and
family. My parents and brothers have consistently encouraged me throughout the
course of my MS program. My friends and lab mates made the time spent in my MS
program fly by and they helped me keep my sanity throughout the whole process. I
would especially like to thank Brittany Campbell, Heather Erskine, Mark Mitola, Joshua
Gibson-Weston, Erin Powell, Kelsey Galicia, and Christopher Crockett for helping me
set up experiments, editing my papers, or simply keeping me motivated while trying to
write my thesis.
To all of these people and the countless others that provided me with their
support and encouragement along the way, thank you.
5
TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
ABSTRACT ................................................................................................................... 10
CHAPTER
1 INTRODUCTION .................................................................................................... 12
2 LITERATURE REVIEW .......................................................................................... 16
Distribution .............................................................................................................. 16 Aedes aegypti ................................................................................................... 16 Aedes albopictus .............................................................................................. 16
Public Health Importance ........................................................................................ 17 Mosquito Feeding Behavior .................................................................................... 18 Oviposition Behavior ............................................................................................... 18 Mosquito Surveillance ............................................................................................. 19 Mosquito Control ..................................................................................................... 20 Lethal Ovitraps ........................................................................................................ 24 Durable Dual-Action Lethal Ovitrap (DDALO) ......................................................... 26
3 SURVEILLANCE OF AEDES ALBOPICTUS POPULATIONS ............................... 28
Materials and Methods............................................................................................ 29 Insects and Field Site ....................................................................................... 29 Adult Surveillance Methods .............................................................................. 30 Adult Surveillance ............................................................................................. 31 Immature Surveillance ...................................................................................... 32 Statistical Analysis ............................................................................................ 32
Results .................................................................................................................... 33 Discussion .............................................................................................................. 35
4 LABORATORY EVALUATION OF THE NOVEL LETHAL OVITRAP AND ITS COMPONENTS ...................................................................................................... 44
Materials and Methods............................................................................................ 45 Insect Rearing and Handling ............................................................................ 45 Leaf Infusion ..................................................................................................... 46 Durable Dual-Action Lethal Ovitrap (DDALO) Treatment and Formulations..... 46 Formulation Efficacy Assay .............................................................................. 47
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Evaluation of Leaf Infusion in DDALO .............................................................. 48 Laboratory Evaluation of DDALO Efficacy and Effects of Aging ....................... 49 Oviposition Preference Assay .......................................................................... 50 Multi-generational Cage Assay ......................................................................... 51 Statistical Analysis ............................................................................................ 52
Results .................................................................................................................... 53 Formulation Efficacy Assay .............................................................................. 53 Evaluation of Leaf Infusion in DDALO .............................................................. 54 Laboratory Evaluation of DDALO Efficacy and Effects of Aging ....................... 55 Oviposition Preference Assay .......................................................................... 55 Multi-generational Cage Assay ......................................................................... 56
Discussion .............................................................................................................. 57
5 CONCLUSION ........................................................................................................ 68
APPENDIX: ZIKA VECTOR CONTROL FOR THE URBAN PEST MANAGEMENT INDUSTRY ............................................................................................................. 71
Zika Virus ................................................................................................................ 71 Incidence and Distribution ................................................................................ 71 Transmission and Symptoms ........................................................................... 72 Zika Virus and Infant Microcephaly .................................................................. 73
Biology and Identification of the Mosquito Vectors ................................................. 73 Integrated Vector Management for Residential Control .......................................... 75
Inspection ......................................................................................................... 75 Resident Cooperation ....................................................................................... 75 Larviciding ........................................................................................................ 76 Adulticiding ....................................................................................................... 77
Adulticiding- Residual Sprays .................................................................... 77 Adulticiding- Space Sprays ........................................................................ 78
Insecticide Resistance ...................................................................................... 79 Monitoring ......................................................................................................... 79
Equipment, Personnel, and Personal Protective Equipment (PPE) ........................ 80 Equipment ........................................................................................................ 80 Personnel and PPE .......................................................................................... 81
Regulatory Corner: Mosquito Spraying Regulations ............................................... 81
LIST OF REFERENCES ............................................................................................... 88
BIOGRAPHICAL SKETCH ............................................................................................ 97
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LIST OF TABLES
Table page A-1 Active ingredient and product type for some residual larvicides. ........................ 85
A-2 Active ingredient and chemical type for some residual adulticides. .................... 85
A-3 Active ingredient and chemical type for some space sprays. ............................. 85
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LIST OF FIGURES
Figure page 3-1 Mean number of Ae. albopictus collected from the AFS campus in the
standard ovitrap, BGS trap, and the CDC LT. .................................................... 40
3-2 Temperature (°C) and precipitation (mm) on the AFS campus ........................... 41
3-3 Percentage of Ae. albopictus collected from total trap catch. ............................. 41
3-4 Mean number of habitats containing immature mosquitoes in the residential and agricultural zone of the AFS campus ........................................................... 42
3-5 Container index on the AFS campus. ................................................................. 43
4-1 Durable dual action lethal ovitrap (DDALO). ....................................................... 60
4-2 Oviposition preference experimental setup. ....................................................... 61
4-3 Simulated tree hole. ............................................................................................ 62
4-4 The effects of different formulations on eggs, larvae, and adult mosquitoes. ..... 63
4-5 The number of mosquito eggs and the number of immature mosquitoes that developed in untreated DDALOs either containing tap water or 20% leaf infusion. .............................................................................................................. 64
4-6 The effects of aging treated and untreated DDALOs in indoor and outdoor environments ...................................................................................................... 65
4-7 The percentage of eggs in each container type in cages with either a treated or untreated DDALO. .......................................................................................... 66
4-8 The number of larvae that develop in each container type in cages with either a treated or an untreated DDALO. ...................................................................... 66
4-9 Number of eggs collected from standard ovitraps .............................................. 67
4-10 Number of live adult mosquitoes present after 4-week study period. ................. 67
A-1 Florida Counties that have reported travel-associated Zika cases as of April 18, 2016. ............................................................................................................ 84
A-2 Aedes aegypti and Aedes albopictus .................................................................. 84
A-3 The eggs of Anopheles, Aedes, and Culex mosquitoes ..................................... 85
A-4 Differences between residual sprays and space sprays. .................................... 86
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A-5 Standard ovitrap ................................................................................................. 86
A-6 Tongue depressor from a standard ovitrap with mosquito eggs. ........................ 87
10
Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Master of Science
SURVEILLANCE AND CONTROL OF Aedes aegypti AND Aedes albopictus WITH A NOVEL LETHAL OVITRAP
By
Casey N. Parker
August 2016
Chair: Philip Koehler Major: Entomology and Nematology
Aedes aegypti and Aedes albopictus are known to flourish in a variety of natural
and residential habitats and are competent vectors of at least 22 different arboviruses
including dengue, chikungunya, and zika. Their global distribution, anthropophilic
nature, and vector competency make them species of interest for control. A surveillance
project was completed in the summer of 2014 that monitored the Ae. albopictus
population in a diverse field site utilizing three surveillance methods. The container
preference of Ae. albopictus within this site was also evaluated. BG-Sentinel and
standard ovitraps were both effective in monitoring the population, but the BG-Sentinel
trap was the first to detect Ae. albopictus early in the season. Monitoring of immature
development sites showed mosquito preference for different containers in the residential
and agricultural areas of the study site. In the residential area, Ae. albopictus primarily
developed in flower pots and water drainage systems, but in the agricultural area,
mosquitoes primarily developed in tires and water drainage systems. Additionally, a
novel durable dual-action lethal ovitrap (DDALO) with combined larviciding and
adulticiding effects, as well as a slow-release polymer, was used to targeting these
11
container mosquito species. Use of the DDALO resulted in high adult mosquito mortality
(~95-100%) in no-choice laboratory cage studies targeting gravid females and
successfully prevented all deposited eggs from hatching. Aging of the trap caused some
loss in activity over time, but traps still caused adult mortality (~50%) and continued to
prevent the successful hatching of eggs for 6 months. Oviposition preference studies
resulted in a significant preference for DDALOs by female mosquitoes in comparison to
other containers. Small cage multi-generational studies resulted in significantly lower
populations of adult mosquitoes in cages containing treated DDALOs after 4 weeks.
Based on successful lab studies, the DDALO could be used as an effective tool for
controlling wild vector populations of Aedes aegypti and Aedes albopictus in
combination with other mosquito control practices.
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CHAPTER 1 INTRODUCTION
Mosquitoes (Culicidae) have been the focus of a growing body of research
relating to an understanding of their biology and control. They are competent vectors of
numerous disease-causing pathogens to both humans and animals, which affect
millions of people every year. Two vector-competent species that have received
increasing attention in recent years are Aedes aegypti, the yellow fever mosquito, and
Aedes albopictus, the Asian tiger mosquito. These mosquitoes are highly invasive and
are capable of vectoring over 22 different arboviruses including the pathogens that
cause zika, dengue fever, chikungunya, and yellow fever. They are also capable of
transmitting dog heartworm (Gratz 2004).
Aedes aegypti and Ae. albopictus originate from Africa and East Asia,
respectively (Gratz 1993, Bonizzoni et al. 2013). These species have spread to all
continents of the globe, excluding Antarctica, due to human-mediated activities. Both
species are present in the U.S. Since their introduction into the U.S., the range of these
mosquito species has continued to grow. These species have a significant preference
for feeding on humans over other animals and prefer to live in urbanized areas (Brown
1966). These characteristics make them particularly dangerous as disease vectors.
Many surveillance methods are used for monitoring the mosquito population in
an area such as the CDC light trap, gravid traps, New Jersey light traps, BG-Sentinel
trap, and standard ovitraps. Due to the differing biology and behavior of various
mosquito species, certain surveillance methods are more effective than others at
trapping adult mosquitoes. For example, CDC light traps are commonly used for
surveillance of Culex species seeking a bloodmeal and gravid traps are used to target
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ovipositing females (Andreadis et al. 2001). However, these commonly used traps do
not collect Ae. aegypti and Ae. albopictus as effectively as other trapping tools such as
the BG-Sentinel trap. Host-seeking Ae. aegypti and Ae. albopictus detect chemical as
and visual cues and the BG-Sentinel trap exploits these behaviors to trap adult
mosquitoes (Farajollahi et al. 2009). Standard ovitraps are also commonly used in
surveillance of Ae. aegypti and Ae. albopictus, but instead, target the gravid females
seeking a container for oviposition by providing an oviposition surface where eggs can
be counted.
Aedes aegypti and Ae. albopictus exhibit ecological plasticity for oviposition sites.
They will oviposit along the sides of small natural and artificial containers such as flower
pots, tires, rain gutters, tree holes and bromeliad plants (Medlock et al. 2006, Hawley
1988). Due to the diverse number of sites that immature mosquitoes can develop in,
containers in rural, suburban, and urban sites are utilized for development (Yiji et al.
2014). The number of potential oviposition sites in an area is often numerous due to the
small amount of water required for larvae to develop and the cryptic nature of these
larval habitats. For this reason, it can be difficult to locate and remove or treat all
potential larval habitats for these mosquito species.
Aedes aegypti and Ae. albopictus are daytime biters, which differs from most
mosquito species. Their daytime biting behavior and preference for small containers as
oviposition sites make these mosquitoes difficult to control with conventional mosquito
control practices. For larviciding to be effective, treatment must reach a majority of larval
development sites, but after heavy rainfall, treatment is usually lost due to overflow of
containers. According to the Florida Mosquito Control Association and the Florida
14
Department of Agriculture and Consumer Services (2012), adulticides are primarily
applied when the target mosquito species are host-seeking, and this is often in the
evening when a majority of mosquitoes are active. Therefore, the daytime-biting
mosquitoes may not be effectively controlled by evening adulticide treatments.
For these reasons, lethal ovitraps have been explored as novel control methods
for Ae. aegypti and Ae. albopictus (Zeichner and Perich 1999). Lethal ovitraps
specifically target the behavior of these mosquitoes to oviposit in containers. Small
containers impregnated with a pesticide or coated with a sticky surface are used to
attract gravid females. Lethal ovitraps are placed in shaded areas and can be effective
for months and are, therefore, low maintenance (Perich et al. 2003). Lethal ovitraps also
have the potential to affect multiple life stages of the mosquito, depending on the
pesticides used in the trap. In conjunction with an integrated mosquito management
plan, the use of lethal ovitraps could aid in the reduction of Ae. aegypti and Ae.
albopictus populations.
Control of these mosquito species is of high importance due to the threat of
dengue, chikungunya, and zika and the broad range of these species in the U.S. A
novel lethal ovitrap was developed at the University of Florida to help address the
problem of this growing threat. Understanding the distribution, behavior, and biology of
these mosquitoes is crucial for effective control. The goals of this research were to 1)
evaluate different surveillance methods for Ae. albopictus, 2) determine container
preference of field populations of Ae. albopictus within a diverse field site, and 3)
evaluate the efficacy of a novel lethal ovitrap. The hypotheses to be tested were 1)
more than one surveillance method would be effective in monitoring Ae. albopictus
15
populations, 2) mosquitoes would prefer different container types for oviposition, and 3)
the lethal ovitrap would be effective and attractive to Ae. aegypti in laboratory studies.
Surveillance of field populations of Ae. albopictus and their container preference
was evaluated in Thessaloniki, Greece (Chapter 3). Surveillance was completed
through the use of three surveillance methods. Oviposition preference,
multigenerational studies, and efficacy bioassays were completed to evaluate the novel
lethal ovitrap (Chapter 4).
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CHAPTER 2 LITERATURE REVIEW
Distribution
Aedes aegypti
Aedes aegypti originated in Africa (Gratz 1993) and has spread to every
continent excluding Antarctica. Spread is believed to be due to human-mediated
activities such as through shipping (Womack 1993). Ae. aegypti was first described in
the United States in 1828 in Savannah, GA as Culex taeniatus (Christophers 1960).
However, it has likely been present in the United States since the 1640s as evidenced
by intermittent outbreaks of yellow fever and dengue (Eisen and Moore 2013). A study
done by Eisen and Moore in 2013 found Ae. aegypti in at least 27 states in the United
States, but updated maps from the Centers for Disease Control and Prevention (CDC)
show populations of Ae. aegypti in as many as 30 states in the U.S. (CDC 2016).
However, the usual range includes Florida, South Carolina, Georgia, Alabama,
Mississippi, Louisiana, southeastern Texas, and southeastern Arkansas (Eisen and
Moore 2013).
Aedes albopictus
Aedes albopictus originated in East Asia and islands of the western Pacific and
Indian Ocean (Bonizzoni et al. 2013). This mosquito has also spread to every continent,
excluding Antarctica, probably due to human-mediated activities such as commerce in
tires and plants (Bonizzoni et al. 2013). Ae. albopictus was first introduced to the U.S.
(Hawaii) in the 18th century (Karamjit 1991) and to the continental U.S. in 1985 in Texas
(Sprenger and Wuithiranyagool 1986). The CDC estimates that Ae. albopictus is
present in 39 states throughout the U.S. (CDC 2016). Ae. albopictus and Ae. aegypti
17
are present in the same areas but can sometimes develop in different water sources.
Ae. albopictus predominates in rural areas and Ae. aegypti predominates in highly
urbanized environments (Braks et al. 2003).
Public Health Importance
Aedes aegypti and Ae. albopictus are competent vectors of a variety of
arboviruses including dengue fever (DF), dengue hemorrhagic fever (DHF), dengue
shock syndrome (DSS), yellow fever (YF), lacrosse virus (LACV), Potosi virus (POTV),
chikungunya virus (CHIKV), and Zika virus (ZIKV). Aedes aegypti and Ae. albopictus
are also capable of vectoring dog heartworm, a nematode (Gratz 2004). Ae. aegypti and
Ae. albopictus are both primary vectors for chikungunya virus. Ae. aegypti is a primary
vector of the dengue virus while Ae. albopictus is a secondary vector. Ae. aegypti is the
primary vector of yellow fever (Gratz 2004).
It is estimated that 50-100 million people are infected with DF and hundreds of
thousands are infected with DHF each year. In 2007, there were 900,782 cases of DF
and 26,413 cases of DHF reported in the Americas in 11 countries (CDC 2012). In
2015, there was an outbreak of locally acquired dengue in Hawaii. There were 107
laboratory-confirmed cases and most were among residents of the island (Johnston et
al. 2016). According to the Pan American Health Organization (PAHO) there were 748
laboratory confirmed cases of dengue in the U.S in 2015 (PAHO 2015), but as of April
29, 2016, there were no locally acquired cases of dengue in North America for 2016
(PAHO 2016).
Yellow fever is a serious threat in Central and South America as well as areas in
Africa and is the cause of approximately 30,000 deaths per year (Tabachnick 2004). In
addition, mosquitoes are a nuisance to humans and animals. Their bites can cause
18
localized swelling and inflammation. Outdoor activities can also be made impossible by
high numbers of mosquitoes.
Zika virus is transmitted by Ae. aegypti and Ae. albopictus. Sporadic outbreaks of
this disease have occurred since its discovery in 1947, but in 2015, Brazil experienced a
large outbreak of Zika (Campos et al. 2015). ZIKV has been shown to be linked to
microcephaly (Rasmussen et al. 2016) and possibly other neurological conditions such
as Guillan-Barre syndrome. As of April 2016, 44 countries had reported local
transmission of ZIKV (Cao-Lormeau et al. 2016).
Mosquito Feeding Behavior
On average, female Ae. aegypti and Ae. albopictus become receptive to mating
approximately 48 to 72 hours after emergence (Gwadz and Craig 1968). After mating,
females will seek their first bloodmeal (Hien 1976). Aedes aegypti and Ae. albopictus
females are aggressive daytime biters and typically bite outdoors, but can make their
way into structures and bite indoors. They primarily bite in the morning hours between
06:00 and 10:00 (6 AM-10 AM) and in the evening between 16:00 and 22:00 (4 PM-10
PM). Although biting does not cease at night, it does decrease (Estrada-Franco and
Craig 1995). Ae. aegypti and Ae. albopictus are known to be anthropophilic mosquitoes,
but will bite other vertebrate hosts when they are present (Brown 1966).
Oviposition Behavior
The oviposition behaviors of Ae. aegypti and Ae. albopictus are very similar. Both
are mosquitoes that develop in containers and have adapted to laying eggs in artificial
containers such as tires (Medlock et al. 2006). Eggs are laid in artificial as well as
natural containers, with eggs placed just above the flood line of the water so that eggs
will be submerged when the area floods. Flooding of the eggs is necessary for the eggs
19
to hatch and multiple floodings may be necessary (Hawley 1988). Aedes aegypti and
Ae. albopictus lay anywhere from 100-200 eggs per batch and oviposit in areas that
contain favorable media, such as organic matter, for larval development (Nelson 1986).
The number of eggs oviposited is highly dependent on a variety of factors including size
of the female mosquito, size of bloodmeal, and the type of host that was fed on
(Blackmore and Lord 2000, Xue et al. 2008). After the eggs are deposited, they take
between 2-6 days to hatch depending on the temperature (Hawley 1988).
Mosquito Surveillance
Successful control of mosquitoes must begin with surveillance. Understanding
the temporal and spatial distribution as well as the densities of mosquitoes is necessary
when attempting to implement a control program. For adult mosquitoes, two kinds of
surveillance exist: human landing rate and trapping methods. Human landing rates are
recorded by a person who individually aspirates mosquitoes that land on their exposed
legs during a given time period (Krockel et al. 2006).
There are a variety of trapping methods that are used for adult surveillance. The
CDC light trap (CDC LT) is the most commonly used trap for general mosquito
surveillance and is usually baited with dry ice and hung 1.6-1.8 m above the ground.
The trap contains a fan that creates a suction that captures and holds the adult
mosquitoes in the collection container. There is also a light that aids in the attraction of
the trap to mosquitoes (Sholdt 1986). BG-Sentinel (BGS) traps are another method of
adult mosquito surveillance. This trap is black and white and these contrasting colors
are appealing to mosquitoes (Lacroix et al. 2009). The trap contains a fan that creates a
suction, which captures and holds the adult mosquitoes in the collection bag. There is
also a slow release pack of synthetic attractant that is designed to mimic the odors of
20
human skin. The same suction that captures the mosquitoes in the collection bag sends
the odors from the packet out into the environment. Both the CDC LT and the BGS trap
target host-seeking female mosquitoes.
The standard ovitrap is a method of surveillance that does not capture the adult
mosquitoes. Instead, it is used to monitor the population based on the number of eggs
oviposited in the trap. The standard ovitrap is a dark plastic cup and holds
approximately 470 mL of water with two tongue depressors attached on the inside of the
cup. Holes are made on either side of the cup slightly below the middle of the cup.
Water is added to the level of the holes. The standard ovitrap targets gravid mosquitoes
seeking an oviposition site. It is particularly attractive to container-mosquitoes (Fay and
Perry 1965) such as Ae. aegypti and Ae. albopictus.
Larval breeding sites can also be recorded and monitored. This aids in effective
immature mosquito control (O‟Malley 1989). Identifying, monitoring, and quantifying the
percentage of active immature mosquito habitats can be useful information for mosquito
control.
Mosquito Control
Control of mosquitoes can occur in 2 ways: immature control and adult control.
Ae. aegypti and Ae. albopictus are closely related species with almost identical life
cycles and behaviors. The literature on Ae. aegypti is more abundant, therefore Ae.
aegypti control is the focus of the following discussion.
A variety of forms for immature mosquito control exist. These include source
reduction or habitat manipulation, sonic or ultrasonic devices, biological controls, and
larviciding in the form of surface films, bacteria and insect growth regulators.
21
Source reduction is considered to be an essential part of mosquito control. When
doing larval surveillance, the Breteau index is commonly used as an indicator especially
in vector surveillance. The World Health Organization (WHO) defines it as the number
of positive containers per 100 houses inspected. Containers holding water should be
emptied, gutters should be drained if clogged, and discarded tires should be removed.
Mosquito control districts and community members should be involved in source
reduction. Studies done by Hoedojo and Suroso (1990) and Nagpal et al. (2004)
demonstrated the importance of source reduction in controlling dengue vectors
including Ae. aegypti and Ae. albopictus. Hoedojo and Suroso (1990) showed that
premise, container and Breteau indices were reduced from anywhere between 27.1%
and 80% after the implementation of a community-wide source reduction campaign. The
premise index provides a quantitative score based on the conditions of the yard, house
and the degree of shade. The container index is the percentage of water-holding
containers that are infested with immature mosquitoes. The decrease in the different
indices was achieved by implementing community-wide participation in source
reduction. Nagpal et al. (2004) points out that source reduction has to cover key and
amplification breeding sites.
There are a few biological methods that are used in mosquito control. A few
specifically targeted at control of Ae. aegypti are the use of copepods, larvivorous fish
and predatory mosquito larvae. Many studies (Manrique-Saide et al. 1998, Marti et al.
2004, Suarez 1992, Lardeux et al. 1989, Torres-Estrada et al. 2001) have utilized
different species of copepods in the genus Mesocyclops for the control of Ae. aegypti in
laboratory and field settings. These species include Mesocyclops longisetus (Manrique-
22
Saide et al. 1998), Mesocyclops annulatus (Marti et al. 2004), Mesocyclops aspercornis
(Suarez 1992, Lardeux et al. 1989) and other native species. These studies
demonstrated high levels of control of larvae in containers with copepods. Manrique-
Saide et al. (1998) showed a six-fold decrease in survivorship of Ae. aegypti by using
copepods. Additionally, studies done by Torres-Estrada et al. (2001) showed that Ae.
aegypti were significantly more attracted to ovitraps that either contained copepods or
had contained copepods.
Fish have also been used successfully to control mosquitoes in small bodies of
water (Fletcher et al. 1992, Rees et al. 1969). Trichogaster trichopterus, the mosquito
fish, was tested as a control for Ae. aegypti in Bahan Township, Yangon and was found
to decrease entomological indices such as the Breteau and container index. The
Breteau index decreased from 103 to 0 and the container index decreased from 41 to 0
after introduction of the mosquito fish into larvae-infested containers (Htay-Aung et al.
1991). A field study completed in southern Mexico (Martinez-Ibarra 2001) tested the
efficacy of 5 different indigenous mosquito fish species for control of Ae. aegypti. All 5 of
these species were able to successfully decrease the container index from 83-91 to 0 in
all cases.
Bacillus thuringiensis israelenis (B.t.i.) and Bacillus sphaericus (B.sph.) are two
types of naturally occurring soil bacteria that produce toxins that are lethal to mosquito
larvae when ingested. These toxins are produced during sporulation. Timing is crucial
because early 1st instar and late 4th instar larvae do not feed. B.t.i. is the most effective
on Aedes mosquitoes (Mallis 2004). B.t.i. was 100% effective against Ae. aegypti larvae
in a field setting for 2-4 weeks (Batra et al. 2000).
23
Predatory mosquito larvae have also been used to control Ae. aegypti. Gerberg
and Visser (1978) evaluated Toxorhynchites brevipalpis as a means of control. Field
studies indicate that total elimination of Ae. aegypti can be achieved through container
treatments with Tx. brevipalpis. However, after approximately 8 weeks, Ae. aegypti
began to reinfest containers because the predatory larvae had pupated.
Two categories of insect growth regulators (IGRs) exist for mosquito immature
control: chitin synthesis inhibitors and juvenile hormone analogs. There are a number of
chitin synthesis inhibitors used for larval control of Ae. aegypti. They interfere with the
molting process and production of cuticle of the larva, which prevents normal
development (Becker et al. 2010). Triflumuron is a chitin synthesis inhibitor that has
been evaluated against 6 insecticide-resistant field strains of Ae. aegypti. Under field
conditions, 100% control was achieved in all 6 strains (Belinato et al. 2013). There are
also a number of juvenile hormone analogs (JHAs). Methoprene and pyriproxyfen are
two JHAs that interfere with the mosquito life cycle and result in an incomplete
metamorphosis (Becker et al. 2010). In studies done by Phanthumachinda and
Wattanachai (1978) in semi-field conditions, 0% adult emergence of Ae. aegypti was
achieved at 0.5, 1.0, and 1.5 parts per million of methoprene (Altosid®). Pyriproxyfen
has also been tested in semi-field conditions against Ae. aegypti. When using 100 mg/
liter, 95% control of adult emergence can be achieved for a period of 40 weeks (Ritchie
et al. 2013).
Organophosphates are another class of chemicals used for mosquito control that
affects the central nervous system and cause neuroexcitation, muscle twitching, and
eventually paralysis (Becker et al. 2010). Field studies using temphos show that, even
24
in areas where there is high availability of breeding sites, an eight-fold decrease in larval
infested sites and three-fold decrease in oviposition activity can be achieved (Vezzani et
al. 2004). However, this study was completed in a region where there is no detected
resistance to organophosphates. Resistance of Ae. aegypti to organophosphates has
been detected in a number of countries, especially those in Latin America, including
Cuba, Venezuela, Costa Rica and Jamaica (Magdalena et al. 2000).
Monomolecular films (MMF) are another form of immature mosquito control.
These MMFs disrupt the surface tension of the water and causes larvae and pupae to
die from exhaustion or suffocation. Adults also cannot rest on the surface due to the
disrupted surface tension (Mallis 2004). Monomolecular films are not commonly used
for control of container-mosquitoes like Ae. aegypti because they are more effective in
large standing bodies of water like ponds and brackish water.
Adulticiding, or the chemical control of adult mosquitoes, is an important method
of control especially during times of disease transmission and outbreaks. Space sprays
are most commonly used and are either thermal fogs or Ultra-low volume (ULV) sprays.
Thermal foggers create an insecticidal fog that affects the mosquito on contact (Pan
American Health Organization 1994). ULV sprays utilize small droplets of concentrated
insecticide (~4.5 liters/ ha). ULV sprays can be applied via ground application or aerial
application. For ground application, the optimum droplet size is 5-10 microns, and 10-25
microns for aerial applications (Mount 1970). These applications are primarily done
when mosquitoes are flying and host seeking.
Lethal Ovitraps
Another control method that has received attention over recent years is the lethal
ovitrap. The first lethal ovitrap testing was done by Zeichner and Perich (1999). An
25
insecticide-impregnated oviposition strip was added to the standard ovitrap. The lab
studies showed approximately 98% adult mosquito mortality, but after significant rainfall,
adult mortality dropped to approximately 50% (Zeichner and Perich 1999). This
demonstrated the need for modifications to the lethal ovitrap.
Since 1999, modifications have been made to the lethal ovitrap in attempts to
increase its efficacy and longevity. This has proven difficult, due to the low residual
activities of the pesticide in the lethal ovitrap. After the pesticide is no longer present in
the trap, it becomes a breeding site for mosquitoes. Control with lethal ovitraps can also
be difficult due to the competition of other breeding sites (Ritchie et al. 2008).
In attempts to improve the lethal ovitrap, recent experiments have tested different
colors and patterns on the trap, pesticide combinations and water infusions inside the
trap, and even biodegradable lethal ovitraps (Hoel et al. 2011, Remmers 2001, Santos
et al. 2003).
Different grass and leaf infusions have been evaluated in multiple studies to
determine the oviposition response of different Aedes species (Reiter et al. 1991,
Sant‟ana et al. 2006, Trexler et al. 1998). Studies done by Reiter et al. (1991) showed
that hay infusion significantly more eggs than the cups with only water. Data collected in
other studies agree with the data. Trexler et al. (1998) found that Ae. albopictus
deposited significantly more eggs in cups with oak leaf infusion at various
concentrations than in cups that contained only well water. Studies also evaluated the
attractive effects of different grass infusions made with four species of grass. All grass
infusions collected twice as many mosquito eggs as the control, which contained only
well water (Sant‟ana et al. 2006).
26
There have been varying levels of success with lethal ovitraps treated with
bifenthrin (Williams et al. 2007) and deltamethrin (Perich et al. 2003). Bifenthrin-treated
ovitraps were successful in achieving 92% mortality in laboratory studies and showed
that there was no loss in toxicity of the bifenthrin strip. However, when placed in the
field, untreated ovitraps were more attractive to ovipositing Aedes than the lethal ovitrap
(Williams et al. 2007). In order to optimize the efficacy of the bifenthrin-treated trap,
alternative oviposition sites would need to be removed from the area (Williams et al.
2007). Lethal ovitraps treated with deltamethrin were evaluated in field studies in two
municipalities in Brazil. A decrease in the number of positive containers and a decrease
in the number of pupae and adults in houses that were treated with a lethal ovitrap was
observed (Perich et al. 2003).
One study using lethal ovitraps for control of Ae. aegypti and Ae. albopictus
showed 90-98% efficacy of the trap in a large cage setting. When tested in the field,
treated areas had significantly fewer gravid or parous females when compared to areas
with no treatment (Wesson et al. 2012). Based on similar data, modified lethal ovitraps
may be an effective tool when integrated into a mosquito management program.
Durable Dual-Action Lethal Ovitrap (DDALO)
Researchers at the University of Florida developed a modified lethal ovitrap
based on the idea that an effective lethal ovitrap must have 2 characteristics: 1) it must
outcompete other breeding sites in the area and 2) it must have a long-lasting residual
so that the ovitrap does not become an immature mosquito habitat.
In experiments done by Hoel et al. (2011), black was shown to be the most
attractive color to Ae. aegypti and Ae. albopictus. The inside of the DDALO was also
modified to have numerous ridges and rough surfaces, which mosquitoes prefer for
27
oviposition over smooth surfaces (Yap et al. 1995). The ridges inside the trap also
maximize the surface area on the interior leaving a lot of space for oviposition. The trap
also contains a narrow entrance so that it will not rapidly fill with water and will provide a
stable environment for mosquitoes to oviposit.
The DDALO incorporates a slow-release polymer that is intended to increase the
longevity of the larvicide (pyriproxyfen) and adulticide (permethrin) inside the trap. If
effective, incorporating a larvicide and adulticide allows for two-way control and,
therefore, greater population reductions. The DDALO is intended to work by first
attracting a gravid female to oviposit on the interior of the trap. The attractance is due to
the color of the trap (black with contrasting red lid) as well as being an attractive
oviposition site (ridged interior, calm/protected environment, maximized surface area,
etc.). Once the female lands on the interior of the trap, she would become contaminated
with adulticide. If the female mosquito is still able to oviposit eggs, the larvicide would
prevent adult emergence.
The objectives of this research included a surveillance study of Ae. albopictus for
a full season to determine timing of peak mosquito populations, evaluation of various
surveillance methods for monitoring Ae. albopictus, and identifying preferable immature
development sites. Laboratory studies included evaluating the attractiveness of the leaf
infusion inside the DDALO, the DDALO efficacy and longevity, the oviposition
preference of the mosquitoes and the effects of this DDALO on a small population of
mosquitos.
28
CHAPTER 3 SURVEILLANCE OF AEDES ALBOPICTUS POPULATIONS
From their Asian origins, Ae. albopictus has spread to every continent of the
globe excluding Antarctica (Bonizonni et al. 2013). Many European countries, including
Spain, Italy, France, Greece, Albania, Croatia and the Netherlands, have reported the
presence of Ae. albopictus and many more countries are at high risk of invasion from
this species (Caminade et al. 2012).
Aedes albopictus was first reported in Greece in 2005 (Samindou-Voyadjoglou et
al. 2005) and has been found in different areas of the country including the largest city,
Athens (Giatropoulos et al. 2012). Historically, Greece has suffered from extreme biting
pressures and multiple mosquito related disease epidemics (Spielman and D‟Antonio
2001, Theiler et al. 1960, European Centre for Disease Prevention and Control 2010).
Additionally, Ae. albopictus is an anthropophilic mosquito (Brown 1966) and their
establishment and proliferation can lead to intense nuisance and biting pressure.
Aedes albopictus is found in both residential and agricultural areas developing in
a variety of containers (Medlock et al. 2006). In Greece, there is close proximity
between residential and agricultural areas. However, the containers utilized by the
larvae are likely different between residential and agricultural areas despite the fact that
both areas occur within a small geographical area.
The objectives of this study were to: 1) determine the effectiveness of three
surveillance methods for Ae. albopictus, 2) examine the changes in the Ae. albopictus
populations throughout the summer season, and 3) identify, categorize, and quantify
active immature habitats in the residential and agricultural areas throughout the summer
season. The study was conducted on the American Farm School campus in
29
Thessaloniki, Greece and used standard ovitraps, BGS traps and CDC LT for adult
surveillance.
Materials and Methods
Insects and Field Site
The American Farm School (AFS) is an educational institution that occupies a
45-hectare campus with distinct residential and agricultural zones. Students and faculty
members live in dormitories and houses on the residential side of campus. The
agricultural side of campus includes a full dairy operation, a poultry house, and, at
certain times of the year, a turkey-rearing facility. This diverse field site is representative
of many Greek cities where residential sites and agricultural areas co-exist.
Aedes albopictus adults and active immature habitats have been found on this
campus in the past. There are no mosquito control practices in this area targeting Ae.
albopictus populations. Therefore, the surveillance of the adult population and immature
development sites can be representative of natural fluctuations where control measures
are not present.
Adult and immature development data were collected on wild populations of Ae.
albopictus present at the AFS campus. Competing container-inhabiting mosquito
species were not detected on the campus. However, Ae. cretinus has been found in
parts of Greece (Patsoula et al. 2006) and Culex quinquefasciatus is present in the
neighboring country, Turkey (Gunay et al. 2015). Temperature and precipitation data
were collected throughout the study period using a Davis Vantage Pro 2 weather station
(Hayward, CA) in the center of the campus.
30
Adult Surveillance Methods
Distribution of Ae. albopictus mosquito populations on the AFS campus were
determined through the use of three mosquito trapping/surveillance methods: standard
ovitrap, BGS trap, and CDC LT.
Standard ovitraps were composed of a 480 ml black plastic cup with two circular
holes cut into either side approximately 2.5 cm from the bottom of the cup. These holes
prevent the cup from completely filling with water and allowed for approximately 200 ml
of water to be held in the base of the cup. Two oak leaves were placed in the cup before
the addition of water. Two tongue depressors were secured on the interior of the cup as
a substrate for mosquito oviposition.
The BG-Sentinel (BGS) (Biogents, Resenburg, Germany) trap is a white
collapsible trap with a mesh covering. At the center of the mesh covering is a black
funnel that empties into a catch bag. On the interior of the BGS trap, there is a pouch
where the BG lure is secured. The BG lure is composed of chemicals that are found on
the human skin such as ammonia, lactic acid, and caproic acid. The trap also
incorporates a fan that pulls mosquitoes down the funnel and into the catch bag. This
same current is then pushed out through the mesh covering which disseminates the
odor of the BG lure. Mosquitoes are attracted by these odors and are pulled down into
the removable catch bag.
The CDC light trap (CDC LT) (John W. Hock Company Gainesville, FL) uses a
light to attract mosquitoes that are then pulled into a collection jar by a fan. CDC LTs in
this study were baited with dry ice.
31
Adult Surveillance
Adult surveillance was conducted for 14 consecutive weeks from June 5th to
September 5th 2014. Weeks in the study were numbered 23-36 according to Julian
week numbers. Six sampling stations were used with three sampling locations on the
residential zone and three on the agricultural zone. Each sampling station contained a
CDC LT, a BGS trap, and two standard ovitraps. CDC LTs and BGS traps were used to
collect female mosquitoes seeking a bloodmeal, whereas standard ovitraps were used
to target gravid female mosquitoes.
CDC LTs and BGS traps were run simultaneously every 7 days with
approximately 50 m and a physical barrier (i.e., bush, wall) separating them. CDC LTs
were hung 1 m above the ground, and allowed to run for approximately 15 hours (17:00-
8:00). BGS traps were allowed to run for approximately 24 hours. Standard ovitraps
were placed in two different locations at each sampling site. Each cup was filled with
water and placed on the ground in areas protected from the sun and near vegetation.
Water levels in ovitraps were checked every two days and refilled to the maximum 200
ml as necessary.
Tongue depressors were collected every 7 days and replaced with new ones.
Eggs on the tongue depressor were counted with the aid of a dissecting microscope.
Eggs were then hatched by submerging in well water and emerging larvae were fed a
liquid diet of 3% bovine liver powder and 2% brewer‟s yeast in water. After reaching the
pupal stage, mosquitoes were transferred to a 30 x 30 x 30 cm rearing cage (Bioquip®,
Rancho Dominguez, CA, USA) and allowed to emerge. Adult mosquitoes were
aspirated using a mechanical aspirator (Clarke Environmental®, St. Charles, IL, USA)
32
and placed in a -20°C freezer until dead. All adult mosquitoes were identified to genus
and Aedes were identified to species using a key in Becker et al. 2010.
Immature Surveillance
Immature mosquito habitat surveillance was also conducted on the AFS campus
on a bi-weekly basis from June 5th to September 9th of 2014. The campus was divided
into residential and agricultural zones and was further divided into stratified quadrants
for sampling. Each zone was split into four quadrants and surveyed in a „zig-zag‟
pattern. The same two operators moved systematically through the stratified quadrants
in each zone and counted and categorized the water-holding sites and the number of
sites with immature mosquitoes in one of seven ways. The habitats were categorized as
a water drainage system, stagnant water, barrel/bucket, tire, flower pot, tractor, or
fountain. Only outdoor water-holding sites were quantified, but rain gutters were not
included because they were not consistently accessible to the samplers throughout the
surveillance site. A container index was calculated by dividing the number of sites
containing immature mosquitoes by the total number of sites and multiplying that
number by 100.
Statistical Analysis
For the adult surveillance, three separate two-way ANOVAs were performed for
the eggs, larvae, and adults. The number of eggs (ovitrap) or adults (BGS and CDC)
was square root-transformed before analysis. The number of eggs or adults was
analyzed using a two-way ANOVA with zone and week number as independent
variables. A T-test was used for mean separation with α = 0.05. The percent of Ae.
albopictus collected from each trap was calculated and analyzed using a one-way
33
ANOVA with trap type as the independent variable. A T-test was used for mean
separation with α = 0.05.
For the immature surveillance, two separate one-way ANOVAs were performed
for the residential and agricultural zone. The number of sites containing immature
mosquitoes was square root transformed before analysis and habitat type was the
independent variable. A T-test was used for mean separation with α = 0.05. The
container index was analyzed using a chi-squared analysis with the zone and week
number as the independent variables and the container index as the dependent
variable.
Results
The first surveillance method to detect Ae. albopictus was the BGS trap in the
first week of surveillance (Fig. 3-1A). The standard ovitraps detected Ae. albopictus in
the third week of surveillance (Fig. 3-1B) and CDC LT detected Ae. albopictus in the
second week of surveillance (Fig. 3-1C). In the residential zone, Ae. albopictus was first
detected by the BGS trap in the first week of surveillance. In the agricultural zone, both
the standard ovitrap and the BGS trap detected Ae. albopictus in the third week of
surveillance.
For the adult surveillance, zone did not have a significant effect, but week
number significantly affected the number of Ae. albopictus collected for the ovitraps (F =
8.64; df = 13, 167; p < 0.0001), the BGS traps (F = 4.07; df = 13, 83; p < 0.0001), and
the CDC LTs (F = 2.85; df = 13, 83; p = 0.0033). The BGS trap had significantly more
Ae. albopictus in weeks 31 and 32 than in weeks 23-30 and week 35 (Fig. 3-1A). For
the standard ovitraps, number of eggs collected in week 32 was significantly higher than
the eggs collected in weeks 23-30 and 34 and 36 (Fig. 3-1B). The number of eggs
34
collected in weeks 31 and 33 was significantly higher than the number of eggs collected
in weeks 23-30 and 36. The CDC LT collected significantly more Ae. albopictus in week
31 than weeks 23-27, 29, 30, and 36. Weeks 33-35 had significantly more than weeks
23-26 (Fig. 3-1C).
The temperature on the AFS campus ranged between 19°C and 28°C and
temperatures were at their highest in week 33 (Fig. 3-2). Precipitation varied a great
deal throughout the study period ranging from 1.6 mm to 53.4 mm of precipitation. The
largest precipitation event occurred in week 29 (53.4 mm).
The percent of Ae. albopictus collected in the BGS trap, the standard ovitrap, and
the CDC LT was 13 ± 2.4, 100, and 2 ± 0.5, respectively. The percentage of Ae.
albopictus collected in the standard ovitrap was significantly higher than the percentage
collected in the BGS and the CDC LT (F = 1245; df = 2, 37; p<0.0001) (Fig. 3-3). The
percentage of Ae. albopictus collected in the BGS was significantly higher than the CDC
LT.
For the immature surveillance, the habitats types with larvae were significantly
different in both the residential (F = 19.1314; df = 6, 49; p < 0.0001) and agricultural
zone (F = 2.5402; df = 6, 49; p < 0.0320). The primary habitat type utilized by Ae.
albopictus varied between the residential and agricultural zone. Water drainage systems
had significantly more active sites (37 ± 0.56) than all other habitat types in the
residential zone (Fig. 3-4A). The number of active barrels/ buckets (17 ± 0.44) and
flower pots (20 ± 0.76) was significantly higher than all other containers on the
residential side excluding the water drainage systems. In the agricultural zone, the
number of active tires (55 ± 3.44) was significantly higher than all other container types
35
except water drainage systems (28 ± 1.22) (Fig. 3-4B). Tractors were also only present
on the agricultural side of the campus while fountains were only found on the residential
side of the campus. Additionally, tires were primarily present on the agricultural side of
the campus.
The container index exhibited a trend similar to that observed in the adult
surveillance in both the residential and agricultural zone. During the first 7 weeks (23-
29) of surveillance, the container index was low. After week 29, the container index
significantly increased. The container index was higher in the residential zone than the
agricultural zone. Zone and week number had a significant effect on the container
index, X2 (1, N = 223) = 31.6, p < 0.0001 and X2 (7, N = 223) = 63.8, p < 0.0001. The
average container index in the residential zone (44 ± 8) was significantly higher than the
agricultural zone (32 ± 9). The container index was the highest in both the residential
(86) and agricultural zone (79) in week 35.
Discussion
Adult surveillance on the AFS campus using three different surveillance methods
showed trends in the Ae. albopictus population for a full summer season. Because BGS
traps are specifically designed to target container-mosquitoes such as Ae. aegypti and
Ae. albopictus (Lacroix et al. 2009) through the use of the BG lure and the contrasting
black and white coloration of the trap (Kawada et al. 2007), it was not surprising that it
collected more Ae. albopictus than CDC LTs. CDC LTs are most commonly used for
collection of other mosquito genera such as Culex, Anopheles, and Coquilettidia, and
have not been shown to be highly attractive to container-mosquitoes such as Ae.
aegypti and Ae. albopictus (Fay and Eliason 1966, Chan 1985). The primary attractant
for the CDC LT is the light and the CO2 (if the trap is baited with dry ice). Aedes
36
mosquitoes have not been shown to be particularly attracted to light sources (Thurman
and Thurman 1955).
A study done by Farajollahi et al. (2009) showed similar results when comparing
the BGS trap and the CDC LT for collection of Ae. albopictus. This BGS trap were more
effective in collecting Ae. albopictus than both the CDC LT and gravid trap. In contrast,
the current study only used the standard BG lure with the BGS trap as an attractant
instead both the BG lure and CO2. Although traps in the current study only used BG
lures to bait the BGS traps, the BGS still collected more adult Ae. albopictus.
In the current study, changes in the population over time were more clearly seen
in the BGS trap and standard ovitrap based on the statistical connecting letters between
the weeks (Fig. 3-1). Although the efficacy of the standard ovitrap and the BGS trap and
CDC LT cannot be directly compared, a study done by Wright et al. (2015) suggests
that standard ovitraps may be more effective at monitoring populations of Ae. albopictus
than the BGS trap when both Ae. aegypti and Ae. albopictus are present. In the current
study, standard ovitraps showed the same peaks in the population as the BGS trap, but
are more sensitive to the presence of Ae. albopictus due to the fact that only one egg is
required to confirm the presence of this mosquito.
Trap specificity can also play a role when identifying the most appropriate
surveillance method for an area. In the current study, there were no other competing
container-mosquitoes in the area. Therefore, the presence of eggs in the standard
ovitrap confirmed the presence of Ae. albopictus in the area. BGS and CDC LTs
collected Ae. albopictus along with other mosquito species that needed to be identified.
Standard ovitraps are highly targeted towards container-mosquitoes. In areas where
37
there is only one species that will oviposit in containers, standard ovitraps can be a
useful and non-labor intensive method of detecting the presence of a specific species.
When choosing a surveillance method for Ae. albopictus, cost and time spent in
labor and maintenance of a trap may need to be considered. BGS traps are currently
approximately 2 times more expensive than CDC LTs and 200 times more expensive
than the average cost of making a standard ovitrap. However, standard ovitraps require
significantly more labor in refilling cups with water and rearing of eggs collected from the
traps. BGS traps and CDC LTs require labor only in setting up and taking the trap down
and identifying the adult mosquitoes inside the traps.
Considering these factors, if there are no financial constraints to surveillance,
BGS traps would be the preferred surveillance method for Ae. albopictus because they
do not require the level of labor that standard ovitraps do and they collect significantly
more Ae. albopictus than the CDC LT. They also were the first trap to detect the
presence of Ae. albopictus. If there are financial constraints, standard ovitraps would be
the preferred surveillance method because they are cost effective and show similar
peaks to those seen in the BGS trap surveillance.
Changes in the Ae. albopictus population throughout the summer season as
determined by the standard ovitrap, BGS trap, and the CDC LT showed similar trends in
the population. The peak in the Ae. albopictus population can possibly be explained by
the meteorological data collected during the study period (Fig. 3-2). The optimum
development temperature for Ae. albopictus is approximately 30°C (Delatte et al. 2009).
These mosquitoes can develop at lower temperatures, but the developmental time of
the immatures lengthens as the temperature decreases. Additionally, containers where
38
mosquitoes have laid their eggs must flood in order for eggs to hatch. Due to the lower
precipitation and cooler temperatures, mosquitoes were likely not developing in high
numbers during the first 3 weeks of the study. After heavy precipitation, eggs were
flooded and mosquitoes were able to hatch and develop in higher numbers. This may
explain why the highest numbers of Ae. albopictus were collected in weeks 31 and 32.
A study done in Athens, Greece (Giatropoulos et al. 2012) demonstrated similar
trends to the current study. Data collected in Athens resulted in similar peaks in the Ae.
albopictus population in weeks 31 and 32, and another in weeks 39 and 40. The current
study stopped surveillance after week 36, but had surveillance continued, a similar peak
may have been observed in the current study.
For the immature surveillance, habitat preference of Ae. albopictus further
demonstrated their ecological plasticity. Variation in the presence of different immature
habitats in the residential and agricultural zones and the utilization of these containers
based on presence and abundance shows the adaptability of the females when
choosing an oviposition site. Aedes albopictus exploits a wide variety of water-holding
containers and do not usually fly more than 400 m (Marini et al. 2010, Maciel-De-Freitas
et al. 2007). Therefore, they must oviposit in nearby water-holding containers and this
may vary depending on where the mosquito is located.
This is supported by a study done by Yiji et al. (2012). Differences in the types of
habitats utilized by Ae. albopictus was observed between a rural and suburban area.
Based on this study and the current study, tires seem to be a preferred site when they
are available, but when they are absent, other containers that are present and possibly
numerous in the environment will also be utilized. In other areas, like India, the
39
dominant habitat shifts to items like discarded plastic containers or coconut shells
because they are present and available in relatively high numbers (Rao 2010 and
Vijayakumar et al. 2014).
An increase in the container index was likely the cause of a spike in the adult
population. The container index was higher in the residential zone compared to the
agricultural zone in contrast to observation by Vijayakumar et al. (2014). In the current
study, flower pots in the residential zone often contained plants that were actively
watered by residents, therefore increasing the container index. In contrast, the container
index did not increase on the agricultural side until after the heavy precipitation.
Mosquito habitats such as the water drainage systems on the agricultural side stay
flooded due to the regular usage of water for the animal and crop maintenance.
Through these studies, BGS traps and standard ovitraps successfully detected
Ae. albopictus populations throughout a season. Additionally, immature surveillance
revealed that the locations where mosquitoes develop are likely different between
diverse sites based on the presence and abundance of habitats, but this variation did
not result in a change in the adult population. Knowledge of effective surveillance
methods and container preference of this species can be used when designing source
reduction and treatment protocols.
40
Figure 3-1. Mean number of Ae. albopictus collected from the AFS campus in the
standard ovitrap (A), BGS trap (B), and the CDC LT (C). Values sharing a letter within each surveillance method are not significantly different.
41
Figure 3-2. Temperature (°C) and precipitation (mm) during the 15-week surveillance
period.
Figure 3-3. Percentage of Ae. albopictus collected from total trap catch using three
different surveillance methods. Values not sharing a letter are significantly different.
42
Figure 3-4. Mean number of active habitats (containing immature mosquitoes) in the
residential (A) and agricultural zone (B) of the AFS campus. Values sharing a letter within each zone are not significantly different.
43
Figure 3-5. Container index (# of positive sites/ total # of sites multiplied by 100) on the
AFS campus by week.
44
CHAPTER 4 LABORATORY EVALUATION OF THE NOVEL LETHAL OVITRAP AND ITS
COMPONENTS
Aedes aegypti and Ae. albopictus are invasive mosquito species that have
expanded their range in recent decades from their African and Asian origins,
respectively (Gratz 2004, Bonizzoni et al. 2013). Their global expansion has likely been
due to human-mediated activities such as the commerce of tires and lucky bamboo
plants (Womack 1993, Bonizzoni et al. 2013) and they are now present on every
continent of the globe excluding Antarctica.
Both species are competent vectors of a variety of disease-causing arboviruses
including dengue, chikungunya, and zika (Gratz 2004, Campos et al. 2015). These
diseases affect millions of people every year and many more people are at risk. These
mosquito species also have a significant preference for feeding on humans, a fact which
has an impact on the risk of disease transmission (Ponlawat and Harrington 2005,
Sivan et al. 2015).
Control of Ae. aegypti and Ae. albopictus is challenging due to their biology and
behavior. Bloodfeeding by these species usually occurs during the day (Estrada-Franco
and Craig 1995) while a majority of mosquitoes feed after sunset. For this reason,
traditional mosquito adulticiding misses the peak activity of Ae. aegypti and Ae.
albopictus. Additionally, Ae. aegypti and Ae. albopictus primarily oviposit their eggs in a
wide variety of natural or artificial containers (Medlock et al. 2006). Due to the wide
variety of containers that these mosquitoes can develop in, treating all potential habitats
can be nearly impossible. Applications of larvicides to containers can offer little to no
residual activity and is very time consuming. Source reduction can be effective (Hoedojo
and Suroso 1990), but is also difficult because habitats are cryptic and numerous.
45
Lethal ovitraps are a form of control for container-mosquitoes, such as Ae.
aegypti and Ae. albopictus, that has been explored in recent years. Early studies using
lethal ovitraps used an insecticide-impregnated oviposition strip inside of a standard
ovitrap. These studies were effective in causing adult mosquito mortality, but decreased
in efficacy after heavy rainfall (Zeichner and Perich 1999). Many studies have modified
lethal ovitraps in attempts to increase attractiveness and efficacy, but this has proven
difficult due to the low residual activities of the pesticides within the lethal ovitrap (Hoel
et al. 2011, Remmers 2001, Santos et al. 2003). Lethal ovitraps are also difficult to
implement because of the competition of other development sites (Ritchie et al. 2008).
A novel lethal ovitrap developed at the University of Florida incorporated a slow-
release polymer, adulticide, and a larvicide. The objectives of the current study were to
evaluate the efficacy of the pesticide formulation and the attractance of the leaf infusion
in the novel lethal ovitrap. Additionally, the efficacy and attractiveness of the novel lethal
ovitrap was evaluated.
Materials and Methods
Insect Rearing and Handling
Insecticide-susceptible Aedes aegypti from the Center for Medical, Agricultural,
and Veterinary Entomology (CMAVE, USDA-ARS) USDA strain were maintained at the
University of Florida Urban Entomology Laboratory in Gainesville, FL. Adult rearing
rooms were maintained at 26±1°C, 55% RH, and a photoperiod of 12:12 (L:D) h. Adults
were given access to a 10% sucrose solution and bloodfed weekly. Mosquitoes were
bloodfed by placing the legs of a chicken inside the fabric sleeve of the rearing cage
and allowing mosquitoes to feed until a majority of females had taken a full bloodmeal.
Moist filter paper inside 16 oz cups (WNA, Covington, KY) was provided for oviposition
46
and eggs. Eggs were stored at 26±1°C in Ziploc® (SC Johnson, Racine, WI) twist top
containers with a small 60 ml cup of water inside which maintained approximately 80%
RH. Eggs were hatched by submerging in well water and resulting larvae were provided
larval diet of ground goldfish flakes (Tetra Fin®, Blacksburg, VA) as needed and
maintained at 30±1°C in an Isotemp incubator (Fisher Scientific, Waltham, WA).
Mosquito pupae were transferred into small containers and allowed to emerge into a 30
x 30 x 30 cm rearing cage (Bioquip®, Rancho Dominguez, CA, USA). For laboratory
assays, adult mosquitoes were aspirated using a mechanical aspirator (Clarke
Environmental®, St. Charles, IL, USA), chilled in -20°C environment until immobile, and
placed on a chilled petri dish for sexing and counting.
Leaf Infusion
Fallen leaves collected from oak trees in Gainesville, FL were mixed with water in
a glass jar at a rate of 8.3 g of oak leaves per 1 L of water. The mixture was covered
with a glass lid and allowed to ferment for a period of 7 days at 26±1°C. After 7 days,
oak leaves were removed from the mixture so only the liquid infusion remained (Reiter
et al. 1991). Infusion not used in experiments was stored in a freezer. In laboratory
assays, 20% leaf infusion mixture was made by mixing leaf infusion with well water.
Durable Dual-Action Lethal Ovitrap (DDALO) Treatment and Formulations
The DDALO (Fig. 4-1) was a black trap approximately 22 cm tall. It had ridges on
either side of the device, a water drainage hole on the front of the trap, and an opening
at the top of the trap. DDALOs hold approximately 1.2 L of water. Traps were 3D-printed
using an RTV process, made out of urethane, and manufactured by Artemis Plastics
(Ocala, FL).
47
Traps were treated with 5 ml of formulation using a single action airbrush
(Paasche®, Chicago, IL, USA) at 40 PSI. Adulticide + larvicide formulation consisted of
0.01% pyriproxyfen, 0.7% permethrin, 1% fumed silica, 5% iso-buthyl-methacrylate
polymer, and 93.29% acetone by weight. Adulticide-only treatment consisted of 0.7%
permethrin, 1% fumed silica, 5% iso-buthyl-methacrylate polymer, and 93.3% acetone.
Larvicide-only treatment consisted of 0.01% pyriproxyfen, 1% fumed silica, 5% iso-
buthyl-methacrylate polymer, and 93.99% acetone. Untreated formulation consisted of
1% fumed silica, 5% iso-buthyl-methacrylate polymer, and 94% acetone.
Formulation Efficacy Assay
To assess the efficacy of different formulations, 160-ml cups (Dart®, Mason, MI)
were treated with 0.80 ml of one of the following treatments using a single action
airbrush (Paasche®, Chicago, IL, USA) at 40 PSI: 1) water only, 2) untreated
formulation, 3) larvicide formulation, 4) adulticide formulation, or 5) adulticide + larvicide
formulation. Formulation was applied to the cups using a single action airbrush
(Paasche®, Chicago, IL, USA) at 40 PSI. A total of 150 cups were treated with 30 cups
for each treatment.
Three separate bioassays were done to evaluate the effects of each formulation
type on the eggs, larvae, and adults of Ae. aegypti. For the egg bioassay, ten eggs were
placed in 50 of the treated cups (10 for each of the 5 treatments). Eighty ml of well
water was added to the cups to induce egg hatch. Egg hatch was recorded after 1, 2, 3,
4, 8, 12, 24, 48, and 72 hrs. Ten replicates were performed for each treatment type.
For the larval bioassay, ten 3rd to 4th instar larvae were pipetted into 50 of the
treated cups with 80 ml of well water. Mortality was recorded at 1, 5, and 20 min, and 1,
2, 3, 4, 8, 12, 24, 48, and 72 hr. Percent mortality of the larvae was also recorded.
48
Mortality was characterized by the lack of movement by the mosquito larvae after the
water was agitated. Ten replicates were performed for each treatment type.
For the adult bioassay, ten bloodfed females Ae. aegypti mosquitoes were
placed in 50 of the treated cups. Bloodfeeding and sorting was as described above. Lids
were placed on top of the cup to prevent mosquitoes from escaping the container. A
small hole was cut in the lid and a sugar-water soaked cotton wick was pulled through
the hole. Mortality was recorded at 1, 5, and 20 min, and 1, 2, 3, 4, 8, 12, 24, 48, and 72
hr. Mortality was characterized by the inability of the mosquito to right itself. Ten
replicates were performed for each treatment type.
Evaluation of Leaf Infusion in DDALO
To assess the attractiveness of the leaf infusion in the DDALO, one untreated
DDALO containing 700 ml of 20% leaf infusion/80% well water and one untreated
DDALO containing 700 ml of well water were placed inside of a cage (61 cm x 61 cm x
61 cm). A 60-ml cup with was filled with 10% sucrose solution. A cotton wick was
inserted into a small hole in the lid of the cup and the lid was placed on the cup. A small
artificial plant was placed inside the cage as a resting site. Recently bloodfed, female
Aedes aegypti were chilled, counted into groups of 20 as described above, placed in the
cage with the DDALOs, and allowed to oviposit for a period of 5 days. On the fifth day,
DDALOs were removed from the cage and eggs in each DDALO were dislodged by
vigorously agitating the liquid inside the trap and pouring it over a fine mesh. Eggs
retained on the mesh were counted and then replaced into their original trap. DDALOs
were then filled completely to the water drainage hole with well water (~1.2L) to induce
egg hatch. Larval diet of ground goldfish flakes was provided as needed for a period of
7 days and the number of larvae in each DDALO was recorded on the seventh day. A
49
replicate consisted of the pairing of a DDALO with water and a DDALO with leaf infusion
inside a cage. A total of 12 replicates were used on two different days.
Laboratory Evaluation of DDALO Efficacy and Effects of Aging
A treated or untreated DDALO containing 700 ml of 20% oak leaf infusion in well
water was placed in a BugDorm dome cage (Taqichung, Taiwan) with dimensions 60 x
60 x 60 cm covered with 150 x 150 mesh screen. Recently bloodfed, female Ae. aegypti
were chilled, counted into groups of 20 and placed in the cage with the DDALOs. A 60-
ml cup with was filled with 10% sucrose solution. A cotton wick was inserted into a small
hole in the lid of the cup and the lid was placed on the cup. Mosquitoes were allowed to
oviposit for a period of 5 days and adult mosquito mortality was recorded on the fifth
day. DDALOs were removed from the BugDorm cages and filled with well water (~1.2 L
total) to induce egg hatch. Larval diet of ground goldfish flakes was provided as needed
for a period of 7 days and the number of larvae in each DDALO was recorded on the
seventh day. After larvae were removed from the DDALOs, treated and untreated traps
were either filled with 700 ml of well water and aged outdoors or water was emptied and
the trap was aged inside of the laboratory. These procedures were repeated monthly for
6 months with traps aged in the outdoor and indoor environment. Traps were aged from
July of 2015 to January of 2016. Outdoor traps were exposed to temperatures ranging
from 9°C-35°C. Traps were placed directly next to a building in a location that received
shade during parts of the day and direct sunlight during parts of the day (~6 hours of
sunlight). Traps that were aged indoors were kept under a fume hood in an air-
conditioned space (approximately 23°C). A replicate consisted of the evaluation of a
treated DDALO and an untreated DDALO. A total of 5 replicates per month were
completed for traps aged indoors and outdoors.
50
Oviposition Preference Assay
To assess the oviposition preference of Ae. aegypti, the following containers with
20% leaf infusion were placed inside of a cage (61 cm x 61 cm x 61 cm): a small plant
saucer (35 ml), a mason jar (550 ml), a simulated tree hole (375 ml), and either a
treated or untreated DDALO (700 ml) (Fig. 4-2). The simulated tree hole (Fig. 4-3) was
constructed by placing a 480 ml black inverted cup on a 480 ml upright cup and
connecting them with hot glue. A small entrance, approximately 2.5 x 2.5 cm, was cut
where the two cups were glued together using a rotary tool (Dremel®, Racine, WI). The
cage also contained a 60-ml cup filled with 10% sucrose solution. A small artificial plant
was placed inside the cage as a resting site, and all other containers were placed
equidistant from each other in a circular pattern around the artificial plant. Location of
containers was alternated between replicates to adjust for any positioning effect. Aedes
aegypti females were bloodfed and sorted into groups of 20, placed in the cages, and
allowed to oviposit for a period of 5 days. On the fifth day, all containers were removed
from the cage and eggs in each container were counted by vigorously agitating the
liquid inside the container and pouring it over a fine mesh. Eggs retained on the mesh
were counted and replaced into their original container. All containers were then filled
with well water (~1.2 L total) to induce egg hatch. Larval diet of ground goldfish flakes
was provided as needed for a period of 7 days and the number of larvae in each
container was recorded on the seventh day. The proportion of hatched eggs was not
expected to be different between containers. Therefore, the minimum expected egg
count for each container was assumed to be equal to the number of larvae. Treated
DDALOs had no larval development and the minimum expected egg count was
estimated using a washing factor. The washing factor was calculated as the total
51
number of larvae divided by the number of eggs that were washed from the untreated
DDALO. An average washing factor was calculated from the individual washing factors
from each of the replicates. The minimum expected egg count for treated DDALOs was
calculated by multiplying the number of eggs washed from the treated DDALO by the
average washing factor. A replicate consisted of the evaluation of oviposition preference
in a treated and an untreated cage. A total of 12 replicates were prepared on four
different days.
Multi-generational Cage Assay
To assess the effects of the DDALO on a small population of Ae. aegypti, the
following containers with 20% leaf infusion were placed inside of a cage (61 cm x 61 cm
x 61 cm): a small plant saucer (35 ml), mason jar (550 ml), simulated tree hole (375 ml),
standard ovitrap (200 ml) and either an insecticide-treated or untreated DDALO (700
ml). A small artificial plant was placed inside the cage as a resting site and all other
containers were placed equidistant from each other in a circular pattern around the
artificial plant. Location of containers was alternated between replicates to adjust for any
positioning effect and a 60-ml cup filled with 10% sucrose solution was provided as
described above.
Recently bloodfed, female Ae. aegypti were counted into groups of 20 and
placed in the cages. Mosquitoes were bloodfed 5 days a week (Monday-Friday). To
bloodfeed the mosquitoes during the experiment, a 3.5 by 0.5 cm hole was cut into the
lid of a 3.5 by 1 cm petri dish and a cotton wick was placed in this opening. The bottom
of the petri dish was filled with bovine blood. Placing the lid on the petri dish allowed the
bovine blood to wick up through the cotton wick.
52
Eggs were collected from the standard ovitraps weekly (Tuesday) by removing
the two tongue depressors on the interior of the standard ovitrap. Eggs on the tongue
depressors were counted with the aid of a dissecting microscope and replaced into their
original cage. Tongue depressors were submerged in the water in the mason jar to
induce egg hatch. Water was added to containers weekly to submerge eggs and a
larval diet of goldfish flakes was provided to all containers every 2 days. After 4 weeks,
all live adult mosquitoes present in the cage were aspirated, placed in the freezer for 24
hours, and counted. A replicate consisted of the evaluation of a treated and an
untreated cage. A total of 12 replicates were prepared on four different days with three
replicates prepared on each of the four days.
Statistical Analysis
Formulation efficacy was analyzed with three separate statistical tests. Percent
hatch (egg), percent mortality (larva and adult), time to hatch (egg), and time to death
(larva and adult) were analyzed using a non-parametric Wilcoxson test with treatment
as the independent variable. Steel-dwass test was used for mean separation with α =
0.05.
Leaf infusion efficacy was analyzed using a non-parametric test. The number of
eggs and the number of immatures was analyzed using a Wilcoxson test with treatment
as the independent variable. Steel-dwass test was used for mean separation with α =
0.05.
Laboratory evaluation of the DDALO and the effects of aging was analyzed using
a multivariate analysis of variance (MANOVA) with repeated measurement. The percent
mortality and the number of emerged adults was analyzed using a MANOVA with time
53
(0-6 months) as the repeated measurement variable and location and treatment as the
independent variables.
Oviposition preference was analyzed using a two-way ANOVA. The number of
larvae from each container was square root transformed before analysis. The proportion
of eggs in each container type and the number of larvae was analyzed using an ANOVA
with container type and cage treatment (and interaction) as the independent variables.
A T-test was used for mean separation with α = 0.05.
Data from the multigenerational cage assay was analyzed using non-parametric
tests. The number of eggs collected in each of the four weeks and the number of adults
at the end of the 4-week period was analyzed using a Wilcoxson test with treatment as
the independent variable. Steel-dwass was used for mean separation with α = 0.05. The
number of adults was analyzed using a Wilcoxson test with treatment as the
independent variable.
Results
Formulation Efficacy Assay
Formulations containing the adulticide had no egg hatch while the larvicide
formulation, untreated formulation, and water only had an average of 64-67% of eggs
hatch (Fig. 4-4 A). This was a significant difference in the percentage of successfully
hatched eggs. Average time to egg hatch (h) after flooding was not significantly different
between the untreated formulation (32.8 ± 2.4), water only (34.9 ± 2.4), and larvicide
formulation (30.5 ± 2.0) treatments (Fig. 4-4 A) (2 = 1.65; df = 2; p = 0.4388). However,
the percent of eggs that hatched was significantly different between treatments (2 =
39.2; df = 4; p < 0.0001) (Fig 4-4 B).
54
The average percent mortality of larvae for the untreated formulation and water
only (0%) were significantly lower than the percent mortality for the larvicide formulation,
adulticide formulation, and adulticide + larvicide formulation (100%) (Fig. 4-4 C) (2 =
49; df = 4; p < 0.0001). The time to mortality (h) for the mosquito larvae was significantly
different between the larvicide formulation (65.5 ± 2.7), adulticide formulation (1.5 ±
0.1), and adulticide + larvicide formulation (2.6 ± 0.5) (2 = 21.19; df = 2; p < 0.0001).
Pyriproxyfen formulation had a significantly higher time to mortality then permethrin
formulation and pyriproxyfen and permethrin formulation (Fig. 4-4 D).
The percent mortality for adults was significantly higher for adulticide formulation
and adulticide + larvicide formulation (100%) than it was for larvicide formulation,
untreated formulation, and water only (0%) (Fig. 4-4 E) (2 = 49; df = 4; p < 0.0001). The
time to mortality (h) was not significantly different between the adulticide formulation
(1.2 ± 0.04) and the adulticide + larvicide formulation (1.1 ± 0.03) (Fig. 4-4 F) (2 = 1.25;
df = 1; p = 0.2636).
Evaluation of Leaf Infusion in DDALO
The number of eggs laid in the DDALO traps was significantly different between
the leaf infusion and water treatments (2 = 14.52; df = 1; p < 0.0001). The average
number of eggs laid in the DDALO with the leaf infusion was 137 ± 12.5 while the
average number of eggs in the DDALO with water was 48 ± 8.7 (Fig. 4-5). The number
of larvae that developed in the DDALO traps was also significantly different between the
two treatments (2 = 17.28; df = 1; p < 0.0001). The number of larvae that developed in
the DDALO with the leaf infusion was 478 ± 25.6 and the number of larvae in the
DDALO with water was 47 ± 4.8 (Fig. 4-5).
55
Laboratory Evaluation of DDALO Efficacy and Effects of Aging
Location (indoor or outdoor) did not have a significant effect on the percent adult
mortality (F = 0.94; df = 1, 16; p = 0.3479), but DDALO treatment (treated or untreated)
did have a significant effect on the percent adult mortality (F = 2841.3; df = 1, 16; p <
0.0001) (Fig. 4-6A). Aging had a significant effect on the adult mortality (F = 145.2; df =
6, 11; p < 0.0001) and there was a significant interaction between both time and location
(F = 10.46; df = 6, 11; p < 0.0001) and time and treatment (F = 132.0; df = 6, 11; p <
0.0001). Percent adult mortality from treated traps was approximately 98% at time 0 and
decreased to 50% after 6 months of aging (Fig. 4-6A). Percent mortality did not change
over time for untreated traps, but did change over time for the treated traps.
Location (indoor or outdoor) did not have a significant effect on the number of
larvae that developed in the traps (F = 3.18; df = 1, 16; p = 0.0934), but DDALO
treatment (treated or untreated) did (F = 2733.2; df = 1, 16; p < 0.0001) (Fig. 4-6B).
Aging had a significant effect on the number of larvae that developed (F = 367.1; df = 6,
11; p < 0.0001) and there was a significant interaction between aging and location (F =
25.87; df = 6, 11; p < 0.0001) and aging and treatment (F = 367.1; df = 6, 11; p <
0.0001). The average number of mosquito larvae that developed in untreated DDALOs
was 510 ± 37 while the no larvae developed in treated DDALOs for the duration of the
study.
Oviposition Preference Assay
There was no significant difference in the minimum expected number of eggs
collected from the cages containing a treated DDALO or an untreated DDALO (F =
0.016; df = 1; p = 0.0.9004) (Fig. 4-7). The number of eggs laid was significantly
different between container types (F = 30.15; df = 3; p < 0.0001) and there was an
56
interaction between treatment and the number of eggs laid in the different containers (F
= 4.25; df = 3; p = 0.0074). The number of eggs in the simulated tree hole (222 ± 29.2)
and the DDALO (1103 ± 243.9) was significantly higher than the number of eggs found
in the mason jar (62 ± 11.3) and the plant saucer (11 ± 3.04). DDALOs from untreated
cages and simulated tree holes from treated cages had significantly more eggs than all
other containers except the simulated tree hole from the untreated cage. Simulated tree
holes from untreated cages and DDALOs from treated cages had more eggs than the
mason jars and plant saucers from both treated and untreated cages (Fig. 4-7).
There were significantly more larvae in cages with untreated DDALOs than
cages with treated DDALOs (F = 9.4; df = 1; p<0.0029) and the number of larvae that
developed differed between container types (F = 7.2; df = 3; p = 0.0002) (Fig. 4-8).
There was an interaction between cage type and container type (F = 8.83; df = 3; p <
0.0001). Untreated DDALOs produced significantly more larvae than all other container
types from both treated and untreated cages.
Multi-generational Cage Assay
The number of eggs collected from standard ovitraps was not significantly
different between treated and untreated cages after 1 week (2 = 0.1026; df = 1; p =
0.7488), but there were significantly more eggs in the untreated cages after 2 (2 =
8.3077; df = 1; p = 0.0039), 3 (2 = 7.4363; df = 1; p = 0.0064) and 4 weeks (2 =
6.5641; df = 1; p = 0.0104) (Fig. 4-9). The number of adults present in the in treated
cage (119 ± 11.8) after the 4-week study period was significantly fewer than the
untreated cage (524 ± 26.1) (2 = 8.3077; df = 1; p = 0.0039) (Fig. 4-10).
57
Discussion
Similar studies evaluating the attractiveness of leaf infusion in containers have
showed results similar to those presented (Ponnusamy et al. 2008, Trexler et al. 1998).
Decaying vegetation in water can potentially provide food for immature mosquitoes
(Chua et al. 2004), increasing their likelihood of survival. Therefore, significantly higher
oviposition in DDALOs with oak leaf infusion is expected. The addition of leaf infusion to
the DDALO can increase the attractiveness of the trap, therefore making it more
effective against Ae. aegypti and Ae. albopictus.
Through the evaluation of various pesticide formulation, those containing
permethrin were effective in controlling eggs, larvae and adults. Formulations with only
pyriproxyfen did not prevent eggs from hatching, did not kill adult mosquitoes, but did
cause mortality in larval mosquitoes. Approximately 65% of eggs in formulations not
containing permethrin successfully hatched. This could mean that multiple floodings
were needed to hatch the remaining eggs, or the other eggs were not viable.
Previous studies done with pyriproxyfen also show successful control of Ae.
aegypti and Ae. albopictus larvae in both laboratory (Hatakoshi et al. 1987) and field
studies (Doud et al. 2014). However, pyriproxyfen has not been shown to cause adult
mortality at low concentrations (0.01%). In contrast, permethrin has been widely used in
mosquito control and has been successful in causing mortality in susceptible strains of
Ae. aegypti (Seccacini et al. 2006). In contrast with presented results, a lethal ovitrap
study showed only 47% adult mortality in a susceptible strain of Ae. aegypti (Zeichner
and Perich 1999) while the current study demonstrated 100% mortality from a
susceptible strain of Ae. aegypti. This discrepancy could be a result of varying
experimental methods, strain, or the formulation that was used in the study.
58
Evaluation of multiple pesticide formulations against different life stages of Ae.
aegypti and aging of the DDALO revealed the necessity for both a larvicide and
adulticide contained inside the lethal ovitrap. While the permethrin formulation was
successful at causing adult and larval mortality, aging of the DDALO showed a
decrease in the adult mortality over time. Different slow-release formulations of
pyriproxyfen have demonstrated residual activity (Ritchie et al. 2013). Therefore,
pyriproxyfen can prevent a container from becoming an immature development site
after one pesticide degrades.
Aging of the DDALO for a 6-month period suggests that these traps could be
placed in the field for an entire season without needing to be replaced. Previous studies
done with lethal ovitraps demonstrate mortality approximately 3 months post-treatment
(Perich et al. 2003), but after aging of the DDALO, at least 6 months of control should
be achieved. A longer lasting trap results in decreased of the lethal ovitrap becoming an
immature habitat for container-mosquitoes.
Multiple sensory cues contribute to the attractance of a mosquito to a container
for oviposition. These cues can include the size, shape, color, and contents of the
container (Chua et al. 2004). The most attractive containers in the current study were
DDALOs and the simulate tree holes. This could be due to the dark color of the
containers, which has been shown to attract Ae. aegypti and Ae. albopictus (Chua et al.
2004). Additionally, both containers were accessible to mosquitoes for oviposition, but
did not allow for excessive collection of water in the container. Based on results by
Chua et al. (2004), the size of the entrance to a container has a significant impact on
the preferred oviposition sites of these mosquitoes. Additionally, rough surfaces have
59
been shown to be more attractive than smooth surfaces (Wong et al. 2011), which could
be why more oviposition is seen in containers with rough surfaces (DDALO and
simulated tree hole) when compared to containers with smooth surfaces (mason jar).
Numerous factors play a role in the effectiveness of a lethal ovitrap in controlling
Ae. aegypti and Ae. albopictus. Female mosquitoes detect cues such as the color and
content of the trap. In order to be effective, lethal ovitraps must contain a pesticide
formulation that effectively controls one or more life stages of the mosquito for extended
periods of time and outcompetes other water-holding containers present in the
environment. Through these studies, the DDALO caused significant mortality even after
aging, was a preferred oviposition site in comparison to common immature habitats, and
caused a significant decrease in a small mosquito population over time. Based on this
information, the DDALO could be implemented as a part of an integrated mosquito
management plan to control Ae. aegypti and Ae. albopictus in an area.
60
Figure 4-1. Durable dual action lethal ovitrap (DDALO).
22cm
Trap entrance Water
drainage
Ridges
61
Figure 4-2. Oviposition preference experimental setup. Photograph courtesy of author.
62
Figure 4-3. Simulated tree hole. Photograph courtesy of author.
63
Figure 4-4. The effects of five different formulations on percent egg hatch (A), time to
egg hatch (h) (B), percent larval mortality (C), time to larval mortality (h) (D), percent adult mortality (E), and time to adult mortality (h) (F).
64
Figure 4-5. The number of mosquito eggs and the number of immature mosquitoes that
developed in untreated DDALOs either containing tap water or 20% leaf infusion.
65
Figure 4-6. The effects of aging treated and untreated DDALOs in indoor and outdoor
environments on the percent adult mortality (A) and the number of larvae that develop in the traps (B).
66
Figure 4-7. The percentage of eggs in each container type in cages with either a
treated or untreated DDALO.
Figure 4-8. The number of larvae that develop in each container type in cages with
either a treated or an untreated DDALO.
67
Figure 4-9. Number of eggs collected from standard ovitraps in treated and untreated
cages over the 4-week study period.
Figure 4-10. Number of live adult mosquitoes present in treated and untreated cages
after the 4-week study period.
68
CHAPTER 5 CONCLUSION
Female Ae. aegypti and Ae. albopictus are dependent on a bloodmeal to develop
their eggs. They are aggressive daytime biters and domestic forms of Ae. aegypti
preferentially feed on humans over other hosts (McBride et al. 2014), increasing the risk
of transmission of pathogens. After obtaining a bloodmeal, multiple cues are used to
locate a suitable oviposition site. Based on the research presented (Chapter 4) a novel
lethal ovitrap has promising potential as a control method due to its attractiveness and
lethality against Ae. aegypti and Ae. albopictus.
In order for a lethal ovitrap to be effective against container-mosquitoes it must 1)
be more attractive than other containers in the area for oviposition and 2) it must cause
mortality for extended periods of time. The DDALO was designed with these
characteristics in mind and met both of the above criteria (Chapter 4).
Aedes aegypti and Ae. albopictus do not lay all of their eggs in one location.
Instead, they exhibit skip oviposition (Rey and O‟Connell 2014) where they oviposit their
eggs in multiple containers. Therefore, the DDALO could not be used as a stand-alone
control strategy. However, attracting a majority of eggs to one lethal location would still
cause substantial decline in adult populations. This, in combination with other mosquito
control practices such as source reduction and chemical sprays could have a significant
effect on the adult population.
Surveillance methods used in mosquito control varies between mosquito control
districts. The best surveillance method for a mosquito control program will vary
depending on factors such as habitat type, population density, and financial constraints.
In reference to Ae. aegypti and Ae. albopictus specifically, this study has shown that
69
several surveillance methods are effective in monitoring populations of these container
mosquito species (Chapter 3). The ability to use multiple surveillance methods for
monitoring a mosquito population offers flexibility when doing surveillance under
different circumstances.
Immature habitat surveillance also revealed important characteristics of Ae.
albopictus population dynamics. These mosquitoes are highly adaptable and have the
ability to develop in a wide assortment of containers. Based on the current study, it
seems that Ae. albopictus will exploit many water-holding containers in different areas
depending on what is available. Despite a variation in the type of containers used for
oviposition, the adult density of mosquitoes remains the same, further demonstrating
the ecological plasticity of this species. Knowing this, any water-holding containers
should be considered immature habitats, and tipped and cleaned regularly.
Due to the fact that the types of containers available for Ae. albopictus
development can vary even in a small geographical range, it is crucial to customize
treatment plans to specific locations. In the studied field sites, DDALO treatments would
be best placed near primary immature habitats like tires in the agricultural zone and
water drainage systems in the residential zone. In addition, nearby larval habitats
should be eliminated to reduce competition with the DDALO. The best control will be
achieved when the biology and the behavior of these unique mosquitoes is highly
considered.
Increased impact on the Ae. aegypti and Ae. albopictus populations could also
be achieved through community efforts. If DDALOs are made commercially available,
individuals will have an additional opportunity to contribute to the mosquito control effort
70
in their own backyard. Some studies have demonstrated short-term success in
community-wide source reduction programs (Hoedojo and Suroso 1990, Nagpal et al.
2004). If a community-wide DDALO program was initiated, the impact could be longer
lasting due to the residual activity of the insecticides. Increased involvement from the
public with this novel method in control could likely help reduce populations of these
problematic mosquitoes.
With the imminent threat of Zika, the importance of controlling Ae. aegypti and
Ae. albopictus is at an all-time high. For this reason, novel targeted measures of
mosquito control must be implemented if disease transmission and the proliferation of
these invasive species is to be prevented. Based on the field and laboratory studies
presented here, using the DDALO before heavy populations of mosquitoes are present
and placing traps near competitive oviposition sites could provide highly attractive and
lethal sites for oviposition by Ae. aegypti and Ae. albopictus. This novel technology may
provide a significant contribution in the prevention of vector-borne disease transmission,
such as dengue, chikungunya, and zika virus.
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APPENDIX ZIKA VECTOR CONTROL FOR THE URBAN PEST MANAGEMENT INDUSTRY
Appendix is a fact sheet on Zika virus that was made for the urban pest
management industry. Document was published through the Electronic Data
Information Source (EDIS) of the University of Florida/ Institute of Food and Agricultural
Sciences Extension. Publication #ENY-891.
Authors: Casey Parker, Roxanne Connelly, Dale Dubberly, Roberto Pereira, and
Philip Koehler.
Zika Virus
Incidence and Distribution
Zika is a mosquito-transmitted virus that has recently spread to the Americas.
Zika virus (ZIKV) was discovered in 1947 in Africa where it was isolated from a Rhesus
monkey in the Zika forest of Uganda. Until recently, ZIKV occurred in a very narrow
range in Africa and parts of Asia. In 2007, a disease outbreak occurred on the Yap
Islands in Micronesia, and in 2013, an outbreak occurred in French Polynesia. In 2015,
a large outbreak occurred in Brazil, and ZIKV has since spread through Central and
South America. According to the World Health Organization (WHO), 44 countries have
reported local transmission of ZIKV, and many have reported travel-associated cases of
the virus. According to the Centers for Disease Control and Prevention (CDC), from
January to April 13, 2016, there were 358 travel-associated cases from 40 states in the
United States and 471 locally acquired cases in US territories. There have also been 7
cases of sexual transmission of Zika within the United States. As of April 18, 2016, 15
counties in the state of Florida had reported travel-associated Zika cases (Fig. A-1).
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ZIKV is expected to continue to spread, but the extent of the impact to specific
geographical areas is difficult to predict.
Transmission and Symptoms
The primary mode of transmission for ZIKV is through the bites of female Aedes
species mosquitoes, particularly Aedes aegypti (yellow fever mosquito) and Aedes
albopictus (Asian tiger mosquito) in the Americas. For a female mosquito to become
infected, she must first feed on an infected human or primate host. The virus from the
human blood the female mosquito ingests begins to increase in number and moves
throughout the mosquito‟s body. This process, known as the “extrinsic incubation
period,” takes approximately 10 days. If the virus makes it to the mosquito‟s salivary
glands, she may transmit the virus to future hosts through her bite. It is estimated that
humans are infectious for the first 3–12 days of the illness.
Other modes of transmission include from pregnant mother to child, sexual
transmission, and blood transfusion. For more information on these modes of
transmission, consult your local health department or http://www.cdc.gov/zika/
transmission/index.html.
The illness caused by ZIKV is very similar to dengue
(http://edis.ifas.ufl.edu/in699), but is milder in most cases. Symptoms of ZIKV infection
include fever, rash, joint pain, and red eyes, sometimes accompanied by muscle aches
and headaches. However, approximately 80% of infected individuals are asymptomatic.
Although hospitalizations or fatalities are highly uncommon for this disease, there is a
causative link between Zika and microcephaly and an association between Zika and
increased risk of Guillain-Barré syndrome, a rare disorder where the body‟s immune
system attacks nerves, causing paralysis. Infections of ZIKV can be hard to diagnose
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due to the similarity in symptoms with two other mosquito-borne diseases, dengue and
chikungunya and there are few laboratories that have the appropriate molecular tests
for the virus. As of April 2016, there was no vaccine available to prevent ZIKV infection
in humans, and treatment includes rest, pain relievers, and fever reducers. Aspirin is not
recommended until dengue infection has been ruled out due to the increased risk of
bleeding. Any person who has previously been infected with ZIKV is likely immune to
future infections.
Zika Virus and Infant Microcephaly
Health agencies and multiple scientific journal articles have now confirmed that
infection with Zika virus can cause microcephaly. Microcephaly is a condition wherein
infants‟ heads are much smaller than those of typical babies. This neurological condition
is rare and only occurs in approximately 2–12 of every 10,000 live births in the United
States. However, in 2015, an increased number of microcephaly cases was reported in
Brazil that correlated with a recent outbreak of Zika in May of the same year. Zika virus
can be passed from a mother to her child in the womb, increasing the risk of
microcephaly and other birth defects. The CDC recommends that pregnant women do
not travel to areas with local transmission of ZIKV.
Biology and Identification of the Mosquito Vectors
Outside of Africa, the likely primary vector of Zika is Aedes aegypti. Aedes
albopictus has not been confirmed as a vector, but it has been implicated as the Zika
vector in Gabon. Both Aedes aegypti and Aedes albopictus are established in the
United States and both are considered invasive species that continue to expand their
range. Populations of Aedes aegypti declined after the introduction of Aedes albopictus
in the 1980s. However, populations of Aedes aegypti are now resurging. These species
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most often feed on human hosts and live in close proximity to humans. Adult females
lay their eggs primarily in containers that can hold water. Examples include flower pots,
corrugated pipes, clogged rain gutters, or discarded tires, but natural containers such as
tree holes and bromeliad plants are often utilized.
It is important to be able to identify the adult vectors and their eggs and the
presence of larvae in aquatic habitats. Aedes aegypti and Aedes albopictus (Fig. A-2)
are dark-colored mosquitoes (dark brown or black) with white scaling on different parts
of their body. The pale white scaling on the thorax of Aedes aegypti is lyre-shaped with
two lines in between the sides of the lyre shape. Aedes albopictus has a single white-
scaled line down its thorax. Aedes aegypti and Aedes albopictus both have bands of
white scales on their legs.
The eggs of Aedes aegypti and Aedes albopictus can be identified by where and
how they are deposited (Fig. A-3). The eggs of Aedes aegypti and Aedes albopictus are
laid singly on moist surfaces such as the edges of containers. When these containers
eventually flood, the eggs will hatch. Anopheles eggs are also laid singly, but they have
“floats” on either side, unlike the eggs of Aedes. Culex eggs are different from both
Aedes and Anopheles because the eggs are deposited in rafts on the surface of the
water.
The vectors of ZIKV are day-biting mosquitoes, unlike many of the Florida
mosquito species that bite at night. After bloodfeeding, the females rest in a shaded
area until they are ready to lay their eggs in a container. Their daytime feeding behavior,
fondness for feeding on humans, and exploitation of water-holding containers around a
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home make these mosquitoes efficient disease vectors and very difficult to control.
However, they generally do not fly distances greater than 500 meters.
Integrated Vector Management for Residential Control
Pest control companies can aid in mosquito control by offering treatments to
residential and commercial areas. Below are the components of an Integrated Vector
Management plan for control of Aedes aegypti and Aedes albopictus.
Inspection
Before any treatments are made, operators/technicians should do a thorough
inspection of the property to identify larval habitats and adult resting locations. All water-
holding containers should be identified and noted, including those that are not easily
accessible such as rain gutters or corrugated pipes. When identifying larval habitats, it
is important to note that mosquito larvae can develop in containers as small as a bottle
cap. Any water-holding containers should be emptied or discarded, if possible. Adult
mosquitoes often rest in shaded locations such as overgrown vegetation, the open
space beneath a stilt house, or in crawl spaces. Overgrown vegetation can be trimmed
to reduce the resting locations of the adults.
Resident Cooperation
In addition to any pesticide treatments that are done by pest control companies
or local mosquito control, residents should practice preventative measures to protect
themselves and to aid in the mosquito control process. The CDC recommends wearing
long-sleeved shirts and long pants, staying in air-conditioned or screened places, and
wearing EPA-registered insect repellents. To prevent mosquitoes from developing
around the home, residents should empty any containers holding water at least once
per week, dispose of discarded tires, clean rain gutters, chlorinate pools, and stock
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ornamental ponds with fish. Bird baths and other permanent water-holding containers
should be scrubbed along the inner walls to remove mosquito eggs. To reduce resting
habitats for the adults, residents should trim overgrown vegetation near the residence.
Pest control companies can provide their customers with brochures like those
produced by the CDC ( http://www.cdc.gov/zika/fs-posters/index.html ) or the Florida
Department of Health (http://www.floridahealth.gov/diseases-and-conditions/zika-
virus/index.html). These brochures cover a wide array of topics, but “Help Control
Mosquitoes that Spread Dengue, Chikungunya, and Zika Viruses” and “Mosquito Bite
Prevention” are particularly useful to homeowners by informing them of the effective use
of insect repellents, how to mosquito-proof their home, and how to prevent mosquitoes
from developing around their home.
Larviciding
Larvicidal treatments are specifically applied to water where mosquitoes lay their
eggs and larvae are able to develop. Three biologically derived larvicides are Bacillus
thuringiensis israelensis (Bti), Bacillus sphaericus (Bsph), and spinosad. These
larvicides act as stomach or internal toxins once they have been ingested by the
mosquito larvae. Residents should see dead larvae in containers approximately 1–2
days after treatment.
Other larvicides registered for use are known as insect growth regulators (IGRs)
and include methoprene, pyriproxyfen and novaluron. IGRs kill insects by disrupting or
preventing mosquito development. Some products used for immature mosquito control
must be ingested, and others work by contact, but both types are effective. Residents
may notice larvae, but these are not likely to survive until the adult stage.
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A list of some active ingredients used for control of mosquitoes in the aquatic
stage can be found in Table A-1. Reductions in mosquito populations take longer to
occur when larviciding treatments are done (~2 weeks or more) because the current
adult mosquito population is not being controlled, but it will prevent the next generation
of adults from emerging. Product labels should be read thoroughly for specific treatment
instructions before any application is done.
Adulticiding
Aedes aegypti and Aedes albopictus are difficult to control in the adult stage
because they are host-seeking at a different time (during the day) than the majority of
other mosquito species. Their host-seeking behavior occurs when humans are most
active. Therefore, spraying for these mosquitoes when they are host-seeking results in
increased pesticide exposure to humans. Aedes aegypti and Aedes albopictus also rest
in areas that are often protected from pesticide treatments.
It can be hard for mosquito control districts to control these day-biting
mosquitoes. Additionally, mosquito control districts may be constrained financially and
may not be equipped to treat all individual residences thoroughly. Also, some counties
in Florida do not have an organized mosquito control district.
Adulticiding- Residual Sprays
Residual treatments, also known as barrier or surface treatments, are long-term
applications typically lasting several weeks. These treatments are most easily and
thoroughly applied using a mist blower so that the insecticide forms a deposit on
surfaces. Mosquitoes resting on these treated surfaces come in contact with a lethal
dose of pesticide.
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Residual applications should be applied to areas where adult mosquitoes rest
such as the vegetation near a home. There are many label restrictions on many
insecticides, so it is important to read, understand and follow all label language. Areas
over impervious surfaces cannot be treated with pyrethroid insecticides, and residual
sprays should not be applied to the air. A list of some residual adulticide active
ingredients is available in Table A-2.
It is important to note that the equipment required for doing residual treatments
for mosquitoes is different from the equipment used by many pest control operators for
general household pests. Compressed-air sprayers are not appropriate for mosquito
treatment due to poor coverage on vegetation, and power spraying is also not
recommended for mosquito treatments because it is not targeted, puts out too much
pesticide, and could contribute to further insecticide resistance in mosquitoes.
Adulticiding- Space Sprays
Some locations, such as areas with little or no vegetation, are not suitable for
residual sprays, but can be treated with space sprays. These sprays (Table 3) target
mosquitoes that are flying and are therefore sprayed into open air. It is important to
target areas such as the space underneath stilt houses, under crawl spaces, or shaded
regions with no vegetation. Space sprays contribute to immediate knockdown of
mosquito populations but do not provide long-term control and should not be applied to
surfaces. Due to the short-term nature of space sprays, they should be reapplied as
needed, according to the label. Space sprays use equipment such as ultra-low-volume
(ULV) sprayers or foggers. Space spray applications have no residual activity but
provide immediate-knockdown of flying mosquitoes. Applications made during periods
of maximum flying and host-seeking activity are often the most effective.
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Insecticide Resistance
Various counties throughout the state of Florida have reported permethrin and
bifenthrin resistance in these mosquitoes. The extent of resistance in the state is
currently under investigation. To delay and prevent further insecticide resistance, it is
important to practice an integrated approach that includes, in order of priority: source
reduction, larviciding, and adulticiding. Monitoring the mosquito population and
resistance status should be a part of all mosquito control activities. Rotation of
chemicals can also be useful in delaying insecticide resistance. However, pyrethroids
and a pyrethroid/neonicotinoid mixture are the only chemical classes available for
residual sprays, making rotation difficult. For space sprays, both organophosphates and
pyrethroids are available for vector control. Major differences between residual sprays
and space sprays are presented in Figure A-4.
Monitoring
Effectiveness of treatment for mosquitoes that develop in containers can be
monitored through the use of standard ovitraps (Fig. A-5), which consist of dark plastic
cups (~500 ml) with two holes on either side of the cup for water drainage. Two tongue
depressors are secured on the interior with binder clips, and the cup is filled with water.
These monitors should be placed in a shaded area around the home near vegetation.
Cups can be secured with small tent stakes so that they are not knocked over by wind
or animals. The monitors should be checked weekly for the presence of eggs, and new
tongue depressors should be installed. If eggs are present (Fig. 6), a retreatment of the
house should be considered. If possible, count the number of eggs on the tongue
depressors weekly with the aid of a microscope to detect any reductions in the
population over time.
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Many mosquito control programs throughout the state routinely do adult
surveillance of mosquitoes and may be able to provide historical or current data on
Aedes aegypti or Aedes albopictus populations in an area. This data may aid in
treatment of an area and understanding the historical mosquito pressures.
Equipment, Personnel, and Personal Protective Equipment (PPE)
Equipment
Mist blowers are low-volume sprayers used to control both larval and adult
populations of mosquitoes. Mist blowers use high air velocity with relatively low fluid
pressures, and with flow rates of several ounces per minute. Mist blowers dispense
small droplets of pesticide though a nozzle mounted within an open cylinder that can be
aimed and that thereby permits precise treatment of mosquito resting areas. Backpack-
sized units can be used to treat areas up to several acres quickly and efficiently. Mist
blowers are particularly valuable if they are used to administer thorough residual
applications to hard-to-treat areas that likely harbor resting adult mosquitoes. Backpack-
type power mist blowers are highly portable and allow rapid treatment of up to several
acres by individual vector-control technicians. Although mist blowers are best suited for
liquid applications, some manufacturers offer the option of equipping them with hoppers
for use with larvicidal pellets or granules.
Space sprays use equipment such as ultra-low-volume (ULV) sprayers or
foggers that deliver small particle droplets (< ~30 microns) that can impinge on the
mosquito cuticle and deliver a lethal dose of pesticide. These types of applications have
minimal residual activity but provide immediate knockdown of flying mosquitoes. Both
ULV sprayers and foggers can be handheld machines, or they can be mounted on a
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truck. Ultra-low-volume sprayers can also be used in aerial applications for wide-area
control.
Personnel and PPE
Any person conducting insecticide treatments for mosquitoes should wear long
sleeves and long pants in addition to using mosquito repellents. The CDC recommends
DEET, IR3535, oil of lemon eucalyptus (OLE), and picaridin for long-lasting protection
from mosquitoes. DEET is a commonly used repellent and is highly effective. Repellents
should be provided to operators/ technicians doing mosquito work. Repellents should be
applied to exposed skin and clothing but not worn underneath clothing. They should not
be applied over irritated skin such as cuts or wounds. They should also be removed
after completing treatments and returning indoors.
When doing mosquito pesticide applications, operators should wear eye
protection and gloves in addition to long pants and long-sleeved shirts. Face masks,
dust masks, or respirators can be worn as an added precaution. Some insecticide labels
recommend the use of a respirator when products are being applied. Refer to
insecticide label instructions for required PPE for different products.
Regulatory Corner: Mosquito Spraying Regulations
With the threat of a Zika epidemic in Florida, it is important that licensed pest
control companies understand the regulations concerning mosquito control, which are
set by either the Structural Pest Control Act (FS Chapter 482) or the Mosquito Control
Act (FS Chapter 388).
Pest control companies licensed in the categories of General Household Pest
(GHP) or Lawn and Ornamental (L&O) may perform pest control, including mosquito
control in, on or under a structure, lawn, or ornamental (Florida Statutes Section
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482.071). This law refers to spraying residential and commercial properties as a part of
normal business practices. However, if a company is doing community-wide mosquito
control using handheld, truck-mounted, or aerial large-scale methods throughout
neighborhoods, agricultural areas, other public areas, or in a contract agreement with a
local mosquito control program, then the company must have a Public Health (PH)
license or be operating under the direct supervision of an individual holding a Public
Health pest control license. See the following regulations and contact the regulatory
agency shown below if you have any further questions.
The Public Health (PH) license is substantially different from GHP or L&O license
of the Structural Pest Control Act. The rules implemented by the Florida Department of
Agriculture and Consumer Services (FDACS) for the PH license are:
5E-13.021 (21) “Public health pest control” – a category or classification of
licensure that includes private applicators, federal, state, or other governmental
employees using or supervising the use of general or restricted-use pesticides in
public health programs for the management and control of pests having medical
and public health and nuisance importance.
5E-13.039 (2) Applicators licensed in public health pest control may directly
supervise no more than 10 unlicensed employees
5E-13.040 (1) It is a violation of these rules for a person to apply a pesticide
intended to control arthropods on property other than his own individual
residential or agricultural property unless he is licensed to do so or is working
under the direct supervision of a licensed applicator, as allowed under subsection
5E-13.039(2), F.A.C.
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5E-13.021 (28) “Direct supervision” – supervision by licensed applicators, who
are responsible for the pesticide use activities and actions of unlicensed
individuals. The licensed direct supervisor must be in immediate contact, either
directly or by electronic means, including, but not limited to, cell phones, radios
and computers.
Contact FDACS for more information on the licensing and certification
requirements under Chapters 482 or 388, Florida Statute.
Bureau of Licensing and Enforcement
Division of Agricultural Environmental Services
Florida Department of Agriculture and Consumer Services
(850)-617-7997
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Figure A-1. Florida Counties that have reported travel-associated Zika cases as of April
18, 2016 (highlighted in red). Figure courtesy of author and Roberto M. Pereira.
Figure A-2. Aedes aegypti (left) and Aedes albopictus (right). Photograph courtesy of
the Florida Medical Entomology Laboratory, University of Florida.
85
Figure A-3. The eggs of Anopheles (left), Aedes (center), and Culex (right) mosquitoes.
Individual eggs are approximately the size of a grain of pepper. Figure courtesy of the Centers for Disease Control and Prevention Environmental Health Services.
Table A-1. Active ingredient and product type for some residual larvicides.
Active ingredient Product type Bti Microbial Bsph Microbial Spinosad Microbial Methoprene Insect Growth Regulator Pyriproxyfen Insect Growth Regulator Novaluron Insect Growth Regulator Temphos Organophosphate Table A-2. Active ingredient and chemical type for some residual adulticides.
Active ingredient Chemical type Alpha-cypermethrin Pyrethroid Bifenthrin Pyrethroid Lambda-cyhalothrin Pyrethroid Tau-fluvalinate Pyrethroid Deltamethrin Pyrethroid Imidacloprid/beta-cyfluthrin Neonicitinoid/Pyrethroid Table A-3. Active ingredient and chemical type for some space sprays.
Active ingredient Chemical Type Etofenprox Pyrethroid Permethrin Pyrethroid d-Phenothrin (Sumithrin) Pyrethroid Pyrethrins/Pyrethrum Pyrethroid Deltamethrin Pyrethroid Chlorpyrifos Organophosphate Malathion Organophosphate Naled Organophosphate
86
Figure A-4. Differences between residual sprays and space sprays.
Figure A-5. Standard ovitrap used for monitoring Aedes aegypti and Aedes albopictus.
Photograph courtesy of author.
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Figure A-6. Tongue depressor from a standard ovitrap with mosquito eggs.
Photograph courtesy of author.
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BIOGRAPHICAL SKETCH
Casey N. Parker was born and raised in Ocala, FL to Gerri Wynn and Adam
Parker. Casey has three younger brothers, Brandon, Chase, and AJ. She grew up on a
thoroughbred horse farm and has always loved biology and the outdoors. She attended
West Port High School where she graduated Summa Cum Laude in 2010. She then
attended the University of Florida in the fall of 2010 and graduated in the spring of 2014
with a Bachelor of Science degree in entomology and nematology and a minor in
leadership. She immediately started working on her MS program in the summer of 2014
at the University of Florida evaluating a novel lethal ovitrap for the control of container-
breeding Aedes mosquitoes and graduated with her MS degree in summer of 2016.
During her MS program, Casey attended many professional conferences including the
annual meetings of the American Mosquito Control Association, the Florida Mosquito
Control Association, the Society for Vector Ecology, the Arbovirus Surveillance
workshop, the Southeast Pest Management Conference and the National Pest
Management Association. She has presented at many of these conferences and has
also given talks for CEUs and Master Gardeners.
After graduating with her MS degree in Entomology and Nematology, she plans
to earn a Master of Public Health degree as well as a PhD at the Florida Medical
Entomology Laboratory in Vero Beach, FL working with mosquitoes. Casey is
passionate about research and vector control and looks forward to career in this field.
