CONTROL OF AEDES AEGYPTI USING MOSQUITOCIDAL CHIPS ...

CONTROL OF AEDES AEGYPTI USING MOSQUITOCIDAL CHIPS CONTAINING THE INSECT GROWTH REGULATOR PYRIPROXYFEN

By

KRISTEN COLEEN STEVENS

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

2017

© 2017 Kristen Coleen Stevens

To my wonderful Husband. Thank you for being there with me from beginning to end.

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ACKNOWLEDGMENTS

I would like to acknowledge my husband, family and friends for all the support and help

provided in running my research. A special thanks to Lettie Cronin, for all her willingness to

learn and be there for me with rearing and research. I want to thank the rest of the Urban

Entomology Lab for all their help, especially for our wonderful lab technicians. Without the help

of my co-workers in the Urban Lab none of this research would be possible. Finally, I want to

acknowledge my professors Dr. Philip Koehler, Dr. Roberto Pereira, and Dr. Roxanne Connelly,

for all their guidance and editorial work done to perfect my thesis, without them I would not be

the student I am today.

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TABLE OF CONTENTS page

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

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

LIST OF FIGURES .........................................................................................................................8

ABSTRACT .....................................................................................................................................9

CHAPTER

1 LITERATURE REVIEW .......................................................................................................10

Introduction .............................................................................................................................10 Aedes aegypti Biology and Importance ..................................................................................10 Public Health Importance .......................................................................................................11 Larval Control of Aedes aegypti .............................................................................................13

Source Reduction .............................................................................................................13 Biological Control ...........................................................................................................14 Microbial Larvicides .......................................................................................................14 Monomolecular Films .....................................................................................................15

2 EFFECTS OF VARIOUS DOSES OF PYRIPROXYFEN ON LARVAL STAGES OF AEDES AEGYPTI ...................................................................................................................21

Introduction .............................................................................................................................21 Materials and Methods ...........................................................................................................22 Results.....................................................................................................................................25 Discussion ...............................................................................................................................26

3 EVALUATING THE EFFECTIVENESS OF PYRIPROXYFEN TREATED CHIPS WHEN EXPOSED TO DIFFERENT WATER VOLUMES, CONTAINERS AND PERCENTAGES OF OAK LEAF INFUSION ......................................................................31

Introduction .............................................................................................................................31 Materials and Methods ...........................................................................................................32 Results.....................................................................................................................................37 Discussion ...............................................................................................................................38

4 EVALUATING THE LONGEVITY OF CHIPS AND THE EFFECTS THEY WILL HAVE ON AEDES AEGYPTI POPULATIONS AND OVIPOSITION PREFERENCES ....45

Introduction .............................................................................................................................45 Materials and Methods ...........................................................................................................46 Results.....................................................................................................................................49

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Discussion ...............................................................................................................................50

5 CONCLUSIONS ....................................................................................................................56

LIST OF REFERENCES ...............................................................................................................58

BIOGRAPHICAL SKETCH .........................................................................................................65

7

LIST OF TABLES

Table page 4-1 Oviposition in pyriproxyfen-treated and untreated water sources .....................................55

8

LIST OF FIGURES

Figure page 2-1 Percent mortality r SEM of pupae and adultAe aegypti grown in water with different

doses of pyriproxyfen. Striped portion of the bars represent the adult mortality. .............29

2-2 Morphological defects of Ae. aegypti pupae and adults due to exposure to pyriproxyfen. ......................................................................................................................30

3-2 Effects of mosquitocidal chips on percent mortality of Ae. aegypti when used in different types of containers. Controls not shown, due to no mortality. Error bars represent r SEM. ...............................................................................................................43

3-3 Effects of mosquitocidal chips on mortality of Ae. aegypti in the presence of oak leaf infusion. Error bars represent r SEM ................................................................................44

4-1 Percent mortality of Ae. aegypti using an 8.4 µg chip and an 840 µg chip over 16 wks. ....................................................................................................................................53

4-2 Total number of Ae. aegypti eggs laid and adults emerged using five different treatments. ..........................................................................................................................54

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Masters of Science

CONTROL OF AEDES AEGYPTI USING MOSQUITOCIDAL CHIPS CONTAINING THE INSECT GROWTH REGULATOR PYRIPROXYFEN

By

Kristen Coleen Stevens

May 2017

Chair: Philip G. Koehler Major: Entomology and Nematology

Aedes aegypti were exposed to water treated with mosquitocidal chips containing the

insecticide pyriproxyfen in a polymer coating. Chips were tested under different conditions;

different water volumes, in containers made of different material, and in water with different

levels of organic matter. Treated chips caused 100% mortality of Ae. aegypti during their pupal

stage independent of conditions chips were exposed to in water. When tested for longevity, the

chips containing 840 µg of pyriproxyfen killed 100% of Ae. aegypti for 4 sequential months of

the chips being reused in water. Chips containing 8.4 µg of pyriproxyfen ceased to work after the

first week of treatment. When mosquitocidal chips were used in > 25% of the oviposition

containers within their cages, there was a significant control of the mosquito populations.

Mosquitocidal chips work in multiple different environments, last for extended periods of time,

cause significant mosquito population decreases, and are effective in causing mortality in Ae.

aegypti.

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CHAPTER 1 LITERATURE REVIEW

Introduction

Mosquitoes belong to the order Diptera, family Culicidae, and are considered one of the

world’s biggest health threats. Although a major health concern, mosquitoes are consumed by

other predacious organisms. An incredibly versatile organism, they have the potential to lay eggs

in multiple types of water sources from man-made containers, to crab holes, tree holes and even

within mines. Mosquitoes are distributed worldwide with approximately 3,200 known species.

Mosquitoes have become an increasing problem because of their ability to disperse. An

important species of mosquito that is a vector of many pathogens is Aedes aegypti (Linnaeus).

Controlling this mosquito is vital because of its medical importance.

Aedes aegypti Biology and Importance

Aedes aegypti is a mosquito that has moved with humans and is found across the globe.

Also known as the “yellow fever mosquito”, Ae. aegypti was brought over from Africa on

European ships during the colonization of the United States (Brown et al. 2013). Aedes aegypti

has been reported in 23 states and is prominent in South Florida, Texas, and Louisiana (Darsie

and Ward 2005). Aedes aegypti is a small to medium sized mosquito that is brown-black in color.

Notably, the markings on the dorsal surface of its thorax resemble a lyre (Christophers 1960).

Aedes mosquitoesbite during the daytime (Lyerla et al. 2000, Brown et al 2013). The peak biting

times for these diurnal biters are mid-morning and late afternoon. Aedes aegypti develops

through the immature stages in water-holding containers (Christophers 1960, Gubler 1998,

Romero-Vivas et al. 2013). This species can be found in a variety of containers such as clogged

gutters, planters, bird baths, tires, tree holes and many other water-holding containers (Gubler

1998, Lyerla et al. 2000, Richardson et al. 2013). Aedes aegypti’s life cycle, depending on

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conditions develop approximately in 7 d from egg to adult. These mosquitoes have an egg stage,

four larval instars, a pupal stage, and finally, an adult stage (Christophers 1960). Its diurnal

biting, medical importance, and ability to survive in many types of water holding containers

makes Ae. aegypti an important target for control.

Public Health Importance

Aedes aegypti is a species of great interest to public health because it is considered a

major vector of viruses that cause diseases in humans. Aedes aegypti, as its common name

suggests, carries not only yellow fever virus, but also other arboviruses such as dengue,

chikungunya and Zika viruses (Gubler 2002, Morrison et al. 2008, Leroy et al. 2009).

Transmission of yellow fever, first discovered by Walter Reed in 1900, still remains a public

health threat to many countries (Agwu et al. 2016, Vasconcelos and Monath 2016). Yellow fever

is found in subtropical and tropical countries. It is endemic in Africa; however, it poses a threat

in other places such as South America, Asia, Europe, Oceania, and North America (CDC 2015b,

Vasconcelos and Monath 2016). People who are infected with yellow fever may experience

fever, chills, headaches, nausea, vomiting and weakness (CDC 2015b). Despite the existence of a

yellow fever vaccine, yellow fever is considered one of the most important mosquito-borne

diseases in Africa where there are 51,000-380,000 cases annually (Agwu et al. 2016).

Dengue is often referred to as “break bone fever”, and there are two different

manifestations: dengue fever (DF), which is not fatal but is marked by intense pain, and dengue

hemorrhagic fever (DHF), which is fatal in many cases (Slosek 1986, Lyerla et al. 2000, Gubler

2002). Worldwide there are approximately 50-100 million cases per year and there are over 2.5

billion people who are at risk of being infected (Harrington et al. 2005). The first official cases of

dengue were recorded between 1779-1780 in Asia, Africa and North America, but it was not

until 1940 that dengue symptoms became associated with the virus (Gubler and Clark 1995).

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The first documented epidemic of dengue was in the Philippines in 1953-1954 (Gubler 2002). It

was not until the 1970s that dengue became a major public health concern and became the

leading cause of hospitalization and death in children in many countries (Gubler and Clark

1995). Dengue has had some of its greatest impacts in the Americas and was considered the most

important arbovirus affecting humans at the turn of the 21st century (Gubler and Clark 1995,

Gubler 2002).

First discovered in Tanzania, chikungunya is a debilitating and long lasting disease that

affects the joints in humans (Powers and Logue 2007, Becker 2010). Chikungunya outbreaks

have occurred in Africa, Asia (Enserink 2007), and Europe (Becker 2010). Chikungunya has

commonly been referred to as “the third world disease;” in African and Asian countries,

however, that is no longer the case (Enserink, 2007, Becker 2010, CDC 2015a). In December

2013, the first locally transmitted case of chikungunya was reported in the Americas (CDC

2015a). Since then, chikungunya has been seen in the western hemisphere, specifically the

Americas and the Caribbean (Powers and Logue 2007, Becker 2010, CDC 2015a). Recent

research on this disease has shown that chikungunya is being widely distributed by mosquitoes

and has the potential to pass from mother to child during gestation (Enserink 2007).

Zika virus is another major arbovirus transmitted by Ae. aegypti that has recently sparked

public health concerns. Originally isolated from a Rhesus monkey in the Zika Forest of Uganda

in 1947, this virus has since spread (Hennessey et al. 2016). Before 2007, there were only

sporadic cases reported in Africa and Asia, but recently large outbreaks occurred in 2013-2014 in

Yap Island in Micronesia. In 2015, Zika reached an epidemic level in Brazil with estimates of

440,000 to 1,300,000 suspected cases (Hennessey et al. 2016). Zika has mild symptoms such as

fever, rash, nausea, vomiting and joint pain (Hennessey et al. 2016, CDC 2016). Although Zika’s

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symptoms are mild, if pregnant mothers acquire Zika virus during pregnancy, their newborn is at

risk of microcephaly (Rasmussen et al. 2016). Incidences of Zika have been found in the United

States, and as of August 2016 locally-acquired cases have been reported in the US (CDC 2016).

Imported cases have been found in all states except Alaska, and, as of December 2016, 210 local

cases were found in Florida (CDC 2016). This emphasizes the need to control Ae. aegypti.

Larval Control of Aedes aegypti

Because Ae. aegypti larvae are contained, isolated and accessible, larval control is an

effective way of lowering populations of this mosquito. Unlike the adults, the larvae cannot fly

and are more concentrated in one spot, making them easier targets for control efforts. Source

reduction is a primary control method of Ae. aegypti; eliminating the larval habitats can lower

adult populations of this mosquito. While the elimination of Ae. aegypti habitats is important,

there are various biological, microbial, monomolecular films, and chemical control methods that

can be used for larval control.

Source Reduction

Habitat removal or reduction plays a central role in mosquito control, particularly for Ae.

aegypti mosquitos. Adult mosquito oviposition sites and resting places can be made unsuitable

by altering them (Romero-Vivas et al. 2013, Richardson et al. 2013). For Ae. aegypti mosquitoes,

eliminating all water-holding containers helps to decrease the population. These water containers

include abandoned swimming pools, tires, water holding trash, clogged gutters and many more

(Espinoza-Gomez et al. 2002, Gubler 2002, Hales and Panhais 2005). In order to effectively

eliminate these harborages, education programs have been implemented to raise public

awareness of these conducive conditions and to teach people how they can alter the environment

around their home to reduce mosquito habitats (Gubler and Clark 1996, Morrison et al. 2008,

Richardson et al. 2013).

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Biological Control

Biological control is particularly useful for larval control of Ae. aegypti. It has the

advantage of controlling mosquito populations through environmentally safe methods (Becker

2010). Biological control includes the use of mosquito predators which help decrease larval

mosquito populations in places where water sources cannot be removed. These predators include

aquatic animals such as dragonfly larvae, tadpoles, fish, copepods and other aquatic insects

(Rose 2001). These animals eat the aquatic mosquito larvae, thus lowering the population density

(Rose 2001, Martinez-Ibarra et al. 2002, Becker 2010). The larval stages of Toxorhynchites will

feed on other mosquitoes, lowering populations of larval mosquitoes (Aditya et al. 2006). Some

species of larvivorous copepods that have proven to be effective at controlling Ae. aegypti larvae

are Macrocyclops albidus, Mesocyclops longisetus and Mesocyclops aspericorni (Marten et al.

1993). The copepods are effective because of their broad diet, consisting of algae, rotifers,

protozoa and most aquatic organisms that are close to the same body size (Marten et al. 1994,

Nam et al. 1998). Because of their broad diet, copepods are able to reproduce in large numbers,

equal to that of Ae. aegypti, in the containers where Ae. aegypti are found (Marten et al. 1994).

When copepods were introduced into Ae. aegypti larval habitats, they reduced the number of

newly hatched mosquito larvae by 99% (Marten et al. 1994).

Microbial Larvicides

Microbial larvicides are also commonly used to control Ae. aegypti larvae. Microbial

larvicides are typically derived from a microorganism such as bacteria, that are toxic to target

organisms. Two common microbial larvicides that can come in the form of pellets or granules

are Bacillus thuringiensis israelensis (Bti) or Bacillus sphaericus. These bacteria are also

formulated into round donut-like tabs that release Bti into a mosquito larval habitat (Rose 2001,

Ritchie et al. 2010, Bravo et al. 2011). Bti is highly effective in killing mosquitoes, providing

15

control for two wk and is considered environmentally safe. Bti is a bacterium that produces

crystals that, when ingested, act as a stomach toxin to larval mosquitoes. It is the combination of

the toxins that are released from the crystalline bacterial by-product and the alkaline environment

of the mosquito’s midgut that causes the insect’s gut to rupture (Bravo et al. 2007, Bravo et al.

2011). Bti granules remain effective for 30-78 d (Armengol et al. 2006), and wettable powder or

liquid forms of Bti have been effective for two to three weeks at a time. Bti is an attractive

pesticide because it is non-residual, safe for the environment, and non-toxic to humans (Melo-

Santos et al. 2001, Rose 2001, Ritchie 2010).

Spinosad is another microbial larvicide that is used for Ae. aegypti control (Marina et al.

2010, Kovendan et al. 2012). Spinosad is typically sold as a liquid formulation and was shown to

be effective against mosquito larvae (Bond et al. 2004). Spinosad successfully inhibited 91% of

Ae. aegypti larvae from emerging (Bond et al. 2004). Spinosad is not only attractive because of

its toxic effects to mosquitoes, but also, it is considered as a reduced risk pesticide by the US

Environmental Protection Agency because of its low toxicity to mammals (Bond et al. 2004,

Kovendan et al. 2012). Spinosad is naturally derived by the fermentation of a soil actinomycete.

It is a mixture of spinosads A and D which are macrolide lactone molecules (Marina et al. 2010,

Kovendan et al. 2012). Spinosad has a novel mode of action. When ingested, spinosad affects the

central nervous system of the mosquito larvae leading to paralysis and death (Salgado et al.

1998, Marina et al. 2010, Kovendan et al. 2012). Also spinosad has long lasting effects,

providing up to eight wk of inhibition of Ae. aegypti larvae (Marina et al. 2010).

Monomolecular Films

Natural oils and monomolecular films can also be used as larvicidal treatments. In order

for Ae. aegypti larvae to breathe, they use a breathing siphon to penetrate the water’s surface and

thereby obtain air. When oils or surface films are applied at rates between 0.33 to 0.56 mL/m2 to

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larval habitats, the siphon is able to penetrate through the surface of the water; however, the

larvae suffocate due to the film of oil (Nayar and Ali 2003, Becker 2010). There are also some

natural oils, such as essential plant oils, that have shown negative effects leading to mortality of

Ae. aegypti larvae. Some of these plant oils are derived from Lippia sidoides and

Chrysanthemum cinerariaefolium. These plant-based oil larvicides have proven to be effective

against mosquitoes in the laboratory. In the study done by Silva et al. (2008), after 24 h of

exposure at a rate of 100 ppm, plant-based oils caused 100% mortality of Ae. aegypti larvae.

These oils are also non-residual and biodegradable, so they do not remain in the environment

(Carvalho et al. 2003, Cavalcanti et al. 2004, Silva et al. 2008).

Chemical Controls

For chemical control, larvicides come in a variety of forms including oil-based, granule,

pellet, and liquid products. Organophosphates and insect growth regulators are two chemical

classes of pesticides that are used for mosquito larval control. Organophosphates belong to a

class of chemicals that interrupts the central and peripheral nervous systems of insects. They

specifically inhibit the enzyme acetylcholinesterase (AChE) (Pope 2010, Ware and Whitacre

2004). By interrupting the AChE, organophosphates operate in the nerve synapse binding with

AChE causing a buildup of acetylcholine, which leads to the rapid twitching of muscles and

paralysis (Ware and Whitacre 2004). An organophosphate that is used for Ae. aegypti larval

control is temephos (Abate£, BASF). Temephos can be found in a variety of products and is

typically an emulsifiable concentrate or granular formulation; an example of one of these

granular formulations is Abate£ (BASF). Temephos has been proven to be an effective way of

controlling mosquito larvae. When temephos was added to larval habitats at a rate of 1200

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International Toxic Units, there was 98.4% mortality of Ae. aegypti larvae after 3 h (Andrade and

Modolo 1991).

Insect growth regulators (IGRs) are another class of pesticides used for larvicidal

treatment. Often formulated as liquids, IGRs are compounds that disrupt insect growth and

reproduction by interfering with the insect’s development (Belinato 2009, Dhadialla 2012). IGRs

are used to control a variety of insect pests (Graf 1993, Dhadialla 2012). IGRs are uniquely

stage-specific and useful if they are applied against the correct stage. They are particularly useful

in controlling the larval and pupal stages of mosquitoes by interrupting their metamorphosis. As

a result, they are not necessarily fast-acting and can take time to work if an insect is not at the

appropriate stage (Mulla 1991, Graf 1993, Bennet and Reid 1995). IGRs are effective against

insects, but they have little to no effect on mammals and pose no threat to birds and fish (Mulla

1991, Graf 1993, Bennet and Reid 1995, Dhadialla 2012). IGRs, both chitin synthesis inhibitors

(CSI) and juvenile hormone analogues (JHA) are frequently used in mosquito control and can

provide control for up to 15 d (Mulla 1991). When put in a slow release formula, IGRs have been

shown to provide control for up to 3 mo (Graf 1993). IGRs can be very effective and should be

applied only against the aquatic stages of the Ae. aegypti life cycle (Mulla 1991; Belinato 2009,

2013).

The two types of IGRs used in pest control are CSIs and JHAs. CSIs are growth

regulators that inhibit insect development by causing disruptions in the cuticle. CSIs are derived

from the family of benzoylhenyl urea chemical compounds (Graf 1993, Belinato et al. 2009).

These compounds disrupt the chitin synthesis in the cuticle by damaging the polymer in the

insect’s cuticle (Belinato et al. 2009). With a damaged cuticle the insect is unable to molt which

will lead to death (Estrada and Mulla 1986, Mulla 1991).

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Juvenile hormones (JH) or juvenile hormone analogues (JHA) are commonly used IGRs.

Like CSIs, JHAs adversely affect insect development (Graf 1993, Dhadialla 2012). They either

stop the mosquito from reaching the adult stage or are known to cause sterility. JHAs interfere

with normal metamorphosis when they are present in an insect at a time that juvenile hormones

do not normally occur (Graf 1993). As a result, the insect is unable to molt properly into the

adult phase and ultimately dies (Mulla 1991, Dhadialla 2012). Some examples of these IGRs

include methoprene, hydroprene, and pyriproxyfen (Mulla 1991; Graf 1993; Belinato 2009,

2013; Dhadialla 2012).

When used for mosquito larvae control, methoprene is typically sold in various forms

including granular, briquets and liquid. For example, a granular form of methoprene is found in

products such as Altosid£ (Zoecon) (Gordon and Burford 1984). Methoprene has been approved

for use for in potable water by the World Health Organization (WHO), making it an ideal

method of mosquito control (Braga et al. 2005a). WHO recommends that mosquito larvae be

exposed for at least 6 h for methoprene to be effective. The effects of different doses of

methoprene were tested on mosquito larvae and pupae; mortality typically occurred in the pupal

stage and morphological defects were seen in Ae. aegypti larvae (Braga et al. 2005b). Another

study testing the effects of methoprene on Ae. aegypti larvae found that most mortality occurs

during their pupal stage and that it is likely due to protein depletion in the hemolymph (Gordon

and Burford 1984).

Pyriproxyfen is a JHA that has been approved by the U.S Environmental Protection

Agency for use in small containers to kill Ae. aegypti, one reason is because of its relatively low

toxicity to non-target organisms (Ware and Whitacre 2004, Suman et al. 2013). The World

Health Organization (WHO), has also approved pyriproxyfen at 0.01 mg/L, to be put in drinking

19

water (WHO 2008, Invest and Lucas 2008, Seccacini 2014). Pyriproxyfen has been shown to be

an effective control against mosquito larvae (Invest and Lucas 2008). Pyriproxyfen can be found

in products such as NyGuard£ (MGK) (Suman et al. 2013). According to the label, pyriproxyfen

can be applied for mosquito control at the rate of 3 mL per 275 m2, which would equal 1 Pg/L

pyriproxyfen in water (Vythilingam et al. 2005, Juan et al. 2013, Seccacini et al. 2014).

Pyriproxyfen, when used for control of Ae. aegypti, is applied to small water holding

containers. Since Ae. aegypti lay their eggs in a variety of containers, when pyriproxyfen is

applied to their individual containers, there is a risk of pyriproxyfen being absorbed into the

container material (Vythilingam et al. 2005). However, research has shown that pyriproxyfen is

effective in multiple types of containers. Seven different types of container materials were tested

to determine pyriproxyfen’s efficacy in these individual containers (Suman et al. 2013). Results

showed that certain containers more likely to have pyriproxyfen adhere to them than others,

concrete being the least adherent and almost 100% of pupae died. Similar results were seen in a

study done by Vythilingam et al. (2005), to determine the residual effect of pyriproxyfen. They

determined that plastic tubs have a longer residual than earthen jars, suggesting that pyriproxyfen

could have been absorbed by the jars.

Pyriproxyfen not only works effectively to kill mosquito larvae in different containers but

also has a long residual effect, providing control for extended periods of time (Ritchie et al.

2013, Seccacini et al. 2014). Research has shown that at 0.01mg/L pyriproxyfen can have a

residual lasting from 4 months providing 100% control, and up to 6 months with 50% control

(Vythilingam et al 2005, Ritchie et al. 2013). With its ability to cause mortality and its long

residual, pyriproxyfen is a good method of chemical control for Ae. aegypti larvae. Because of

the long residual effect, it can also be used in slow release formulations controlling mosquitoes

20

over an extended period of time. Slow release pyriproxyfen was put into beeswax candles, these

candles were then hollowed out and used as larval rearing containers, which provided 100%

mortality of Ae. aegypti larvae (Juan et al. 2013).

Chemical treatments, although widely used, are often labeled for large bodies of water.

For example, Altosid which contains the IGR methoprene is typically labeled for bodies of water

as small as 50 gallons of water and as large as 9600 gallons of water according to the

manufacturer Zoecon, and the Altosid specimen label. This presents a problem when using it to

control Ae. aegypti, because this mosquito is not found in these large bodies of water, but in

small water containers. Also, typically in Ae. aegypti habitats water is not permanent and can

evaporate, so containers will need to be retreated. There is a need for a small, easy to use,

residual treatment for control of Ae. aegypti in small containers.

The aim of this study is to evaluate a novel form of treatment for Ae. aegypti. This study

evaluates the efficacy of small chips treated with pyriproxyfen in a slow release polymer that can

be distributed in Ae. aegypti habitats. The objectives of this study are:

1. To determine pyriproxyfen’s ability to remain for the duration of Ae. aegypti’s life cycle when applied at varying doses and different larval ages.

2. Evaluate the effectiveness of pyriproxyfen treated chips against Ae. aegypti when exposed to different water volumes, container types and percentages of organic matter.

3. To evaluate the longevity of chips and the effects they will have on Ae. aegypti populations and oviposition preferences.

21

CHAPTER 2 EFFECTS OF VARIOUS DOSES OF PYRIPROXYFEN ON LARVAL STAGES OF AEDES

AEGYPTI

Introduction

Aedes aegypti (L.), the yellow fever mosquito, is considered one of the world’s most

significant health threats, because it is the vector of multiple arboviruses. Aedes aegypti not only

carries yellow fever, but also dengue, chikungunya and Zika viruses (Gubler 2002, Morrison et

al. 2008, Leroy et al. 2009). There are several reasons why Ae. aegypti is a good disease vector.

First, larval stages of Ae. aegypti develop in water containers, as large as swimming pools or as

small as a bottle cap (Espinoza-Gomez et al. 2002, Gubler 2002, Hales and Panhais 2005).

These containers are typically found in urban environments in close proximity to humans, who

are easy targets for arbovirus transmission (Gubler and Clark 1996, Richardson et al. 2013).

Also, Ae. aegypti is a day time feeder, with its peak feeding times occurring between mid-

morning and late afternoon, when humans are involved in outdoor activities (Christophers 1960,

Guber 1998).

Larval control is an important method of limiting Ae. aegypti populations because larvae

develop in small containers where treatments can be applied directly. Some chemicals that are

commonly used to control Ae. aegypti are insect growth regulators (IGRs), which affect the

insect growth and reproduction (Belinato 2009, Dhadialla 2012). IGRs are very effective when

applied during the late larval stages of Ae. aegypti (Mulla 1991, Graf 1993, Bennet and Reid

1995). Another reason IGRs are desirable mosquito control methods of the fact that they pose

little to no threat to mammals, fish and birds (Mulla 1991, Bennett and Reid 1995, Dhadialla

2012). Among IGRs, chitin synthesis inhibitors (CSIs) disrupt the cuticle formation and prevent

the larval mosquitoes from molting, resulting in death during the larval stage. Juvenile hormone

22

analogs (JHAs) stop the mosquito form reaching the adult stage or are known to cause sterility

(Mulla 1991, Graf 1993, Belinato et al. 2009).

Pyriproxyfen is a JHA approved by the U.S. Environmental Protection Agency for

control of Ae. aegypti in small containers (Ware and Whitacre 2004, Suman et al. 2013) and

approved by the World Health Organization (WHO) for use at a rate of 10 PPB in potable water

(WHO 2008, Invest and Lucas 2008). Pyriproxyfen is effective in controlling Ae. aegypti at

concentrations d1 PPB pyriproxyfen in water (Sihuincha et al. 2005, Invest and Lucas 2008), and

primarily resulted in death during the mosquito’s pupal stage. These authors observed very

minimal mortality during the Ae. aegypti larval stage. Bridges et al. (1977), tested the effects of

varying doses of pyriproxyfen (0-150 PPB) on different larval stages. These authors

demonstrated pyriproxyfen’s ability cause larval, pupal and incomplete adult emergence when

applied at different larval stages (Bridges et al. 1977).

The objective of this study was to determine the effect of concentration and timing of

pyriproxyfen treatment in relation to Ae. aegypti larval development and disruption of

metamorphosis. We hypothesized that increasing doses of pyriproxyfen would have more severe

effects on Ae. aegypti larvae of varying ages, as well as pyriproxyfen will be more effective if

applied during Ae. aegypti’s third or fourth instar.

Materials and Methods

Test insects and colony maintenance: Aedes aegypti colonies were acquired from the

Center of Medical, Agricultural and Veterinary Entomology (CMAVE) and the United States

Department of Agriculture, Agricultural Research Service (USDA-ARS), Gainesville, FL. The

Ae. aegypti colony has no known resistance to insecticides. Adults were maintained in 30 cm x

30 cm x 30 cm cages (BioQuip® Lumite Screen Collapsible Cages), provided with 10% sugar

23

solution and tap water in a rearing room (Urban Entomology Lab, at the University of Florida,

Gainesville, FL) maintained at 28° ± 2° C with a relative humidity of 36% ± 5% and a 12:12

(L:D) photoperiod. Newly emerged mosquitoes were allowed one week for mating before being

blood fed, and the colony was blood fed using restrained live domestic chickens (IACUC

Protocol #20163836_01). For oviposition, polypropylene cups (450 mL) were filled with 300 mL

of clean unchlorinated water and placed in the mosquito cage for 72 h after blood feeding. Cups

were lined with filter paper (Fisherbrand® Filter Paper, Fisher Scientific, Pittsburgh, PA) that

served as egg laying sheets for mosquitoes. The cups were removed two to three d after

placement in the cages. The egg sheets were removed from the cups and allowed time to dry.

Once dried, the egg sheets were stored in a small hydration chamber, consisting of a 450-mL

cylindrical container (Tupperware®) containing a 60-mL cup of water and the lid put loosely

over the top. The hydration chamber created moist conditions within the container to help

prevent eggs from desiccating.

Mosquito eggs were hatched by placing strips of dried egg sheets into 55 cm x 45 cm x 8

cm into plastic trays (Del-Tec/Panel Control Plastic Trays, Greenville, SC) filled with 8 liters of

clean unchlorinated water. Trays were placed into the rearing room mentioned above. Egg sheets

were left in trays for 24 hours and then removed and allowed to dry on paper towels. After eggs

were hatched, larvae were initially fed 50 mg of ground fish flakes (TetraFin® Goldfish Flakes).

Larvae were monitored daily and given food as needed. Once larvae reach pupation, they were

placed into polypropylene (450 mL) cups and put into adult rearing cages for emergence.

Mosquito rearing for bioassays. To prepare larvae for use in experiments 24 hr after

hatching, 20 first instar larvae were moved from the rearing tray and placed into 120-mL holding

cups filled with 100 mL of clean unchlorinated water. Enough cups were prepared for all

24

treatment and control replications (number of replications dependent on experiment). Fish food

was diluted by adding 100 mg of ground fish flakes (TetraFin® Goldfish Flakes) to 100 mL of

water in a 120 mL cup. The slurry was mixed and 200 µl was pipetted into each cup. Cups with

larvae were kept in an incubator for 4 d and maintained at ca. 31qC and 15% RH.

Chemical source and dilution process. Pyriproxyfen stock solution (60,000 PPB) was

made by combining 60 Pg of technical grade pyriproxyfen (Nylar£ Technical, MGK£ Insect

Control Solutions, Minneapolis, MN) with 1 mL of methanol (Fisherbrand® Filter Paper, Fisher

Scientific, Pittsburgh, PA). Serial dilutions were used to obtain three different concentrations of

pyriproxyfen (1, 3, and 10 PPB). To create the 10 PPB solution, 58 PL of pyriproxyfen stock

solution was added to and mixed with 350 mL of clean unchlorinated water. To create the 3 PPB

solution, 75 mL of the 10 PPB solution was added to and mixed with 175 mL of clean

unchlorinated water. Finally, to create the 1 PPB solution, 25 mL of the 10 PPB dose was added

and mixed with 225 mL of clean unchlorinated water.

Pyriproxyfen direct water treatment. For each treatment, 50 mL of the appropriate

solution (1, 3, and 10 PPB) was added to 120 mL polypropylene cups (WNA¥, Chattanooga,

TN). For controls, 50 mL of clean unchlorinated water with no pyriproxyfen was used. All

treatment and control cups were placed separately inside empty 450 mL polypropylene lidded

cups to prevent cross contamination. Larvae were transferred from holding cups into either

treatment or control cups at 24, 48, 72 and 96 h after emerging from eggs. Cups were placed into

an incubator maintained at ca. 31qC and 15% RH. The larvae were fed every other day according

to mosquito rearing methods. Number of dead and live larvae, pupae and adults were recorded

every 12 hr after larvae were placed into cups. Experiments were run until all specimens either

25

died or emerged as adults for ca. 5-10 d. There was a total of five replicates of each treatment

and control. Replications were done on separate days, over a 1 mo period.

Statistical analysis. Percent mortality of pupae and adults in the experiment was

calculated and then ArcSin transformed. JMP software (SAS Inc, NC, USA) was used to run a

two-way analysis of variance (ANOVA) to determine the effect that time (24, 48, 72, and 96 h)

of treatment and concentration of pyriproxyfen (0,1,3,10 PPB) had on the mortality of the

mosquito larvae. Means were compared using a Student’s-t test.

Results

Effects of stage specific treatment. No larval mortality was recorded throughout the

duration of the experiment. There was no significant difference for the number of mosquitoes

dying as either adults (F = 0.0315, df = 3, P = 0.9924) or pupae (F = 0.1416, df = 3, P = 0.9345),

for larvae that were treated at different ages. Additionally, there was no significant interaction

between dose and age treated for either adults (F = 0.0315, df = 9, P = 1.000) or pupae (F

0.5914, df = 9, P = 0.7976). There were significant differences among the pyriproxyfen doses

both for the numbers of mosquitoes dying as adult (F = 37.004, df = 3, P = <0.0001) and as

pupae (F = 203.02, df = 3, P = <0.0001). The 1 PPB dose did not work as efficiently as the 3 and

10 PPB doses. Mosquitoes exposed to the 1 PPB dose died both as adults and pupae, and this

dose inhibited only 90-95% emergence. In contrast the 3 and 10 PPB doses killed 100% of

mosquitoes as pupae (Fig. 2-1).

Mosquitoes that were subject to the 3 and 10 PPB doses showed different degrees of

malformations. Malformations included insects that were unable to fully emerge from pupal

exuviae (Fig. 2-2A, 2-2B) or died right before emerging (Fig. 2-2C). Pupae categorized as

“albino” (as described by Bridges et al. 1977) were and unable to fully darken and harden their

cuticle, dying as an entirely white pupa (Fig. 2-2D). Adults in the 1 PPB dose were unable to

26

fully emerge from pupal exuviae, often getting stuck around their thorax (Fig. 2-2E), or typically,

their legs were unable to escape the pupal exuviae (Fig. 2-2F).

Discussion

Insect growth regulators such as pyriproxyfen have been shown to be effective against

mosquitoes at low doses, if applied at the correct stage of life (Darriet and Corbel 2006,

Sihuincha et al. 2005, Estrada and Mulla 1986). Our experiments showed that when pyriproxyfen

was applied at three different small doses, significantly different effects were observed as

described previously. Estrada and Mulla (1986) found that, at doses as low as 1 PPB, 95%

mortality occurred in Ae. aegypti. Similarly, Sihuincha et al. (2005), Darriet and Corbel (2006),

and Kamal and Khater (2010) found that, at doses of 1 PPB or lower, 75% or more Ae. aegypti

mortality occurred. The variation between these different studies could be due to different

populations of mosquitoes, experimental set up and conditions, and formulations used in these

studies. However, our studies confirmed that pyriproxyfen can be used at 1 PPB doses to kill

~95% of Ae. aegypti, or at 3 and 10 PBB to kill 100% of Ae. aegypti during the pupal stage.

Pyriproxyfen causes little larval mortality. In our studies, we found that there was no

larval mortality, and other studies (Estrada and Mulla 1986, Darriet and Corbel 2006 and Kamal

and Khater 2010) also showed similar results. Larval mortality levels from 6-14% were observed

by Kamal and Khater (2010), but part of this mortality may be due to mishandling, lack of proper

feeding, or temperature conditions. It is not likely that pyriproxyfen would result in larval

mortality because its mode of action is a juvenile hormone analogue, which results in mortality

or defects in the later stages of the insect’s development (Mulla 1991, Dhadialla 2012).

However, unlike Estrada and Mulla (1986) who saw the highest pupal mortality when

pyriproxyfen was applied at 2 PPB to 4th instar stage of Ae. aegypti, our results showed no

difference in percent mortality when pyriproxyfen was applied to different instars of Ae. aegypti.

27

This was also in contrast to other studies done by Sihuincha (2005) and Darriet and Corbel

(2006) who found that pyriproxyfen had a higher percent mortality when applied during the late

larval stages of Ae. aegypti. Differences in larval mortality could be due to different strains of

mosquitoes or formulations used, and be related to harmful effects from the other components in

the formulation. In our studies, technical grade pyriproxyfen was dissolved in methanol which

may have differed its effect on the mosquitoes. In other studies (Estrada and Mulla 1986),

technical grade pyriproxyfen was used but were not mixed with methanol. Similarly, Darriet and

Corbel (1996) and Kamal and Khater (2010) used a technical grade of pyriproxyfen not mixed in

a solvent and Sihuincha et al. (2005) used a granular form of pyriproxyfen, which could have

provided for the differences seen. Further investigation will be necessary to determine if the

diluent affects mosquito mortality.

Our studies confirmed that pyriproxyfen has negative effects on Ae. aegypti pupae.

Pyriproxyfen mainly prevented Ae. aegypti’s ability to molt from pupae to adult (Bridges et al.

1977, Kamal and Khater 2010). These authors determined that pyriproxyfen caused pupae to

retain some of their larval skin, and mosquito adults either to lose legs attached to the pupal

exuviae or emerge incompletely. We did not observe any pupae that died retaining larval skin;

however, as shown previously by Bridges et al. (1997), some of the larvae in our study

successfully became pupae but died as “albino” pupae, dying without a fully darkened and

hardened cuticle, and adults unable to fully emerge from pupal exuviae. Although, researchers

are not fully aware of why pyriproxyfen has these characteristics on mosquitoes, some recent

research has speculated as to why (Nasr et al. 2010, Bensebaa et al. 2015). Both Nasr et al.

(2010) and Bensebaa et al. (2015), demonstrate how pyriproxyfen’s disruption of chitinases

influences the physical and biochemical process in insects. Bensebaa et al. (2015) demonstrated

28

how pyriproxyfen not only inhibits 20-hydroxyecdysone (20H), an important molting hormone,

but also causes thickening of the pupal cuticle. This thickening of the pupal cuticle is thought to

result in adults that are unable to fully emerge.

Pyriproxyfen’s ability to work at low doses and have little to no effects on mammals

makes it a good chemical for control of Ae. aegypti, with few potential environmental effects.

Also, because it is not stage-specific in Ae. aegypti larvae, pyriproxyfen is a good product for

treatment that homeowners can use immediately in standing water containers around their home.

29

Figure 2-1. Percent mortality r SEM of pupae and adultsAe. aegypti grown in water with

different doses of pyriproxyfen. Striped portion of the bars represent the adult mortality.

30

Figure 2-2. Morphological defects of Ae. aegypti pupae and adults due to exposure to

pyriproxyfen. A) and B) An adult unable to fully emerge from the pupal exuviae, shows part of the exposed undeveloped thorax. C) Adult before emerging, legs are visible in cephalothorax. D) “Albino” pupae, lacking any darkening and hardening of cuticle. E) and F) Deformed Ae. aegypti adults, trapped in pupal exuviae.

A B

C D

E F

31

CHAPTER 3 EVALUATING THE EFFECTIVENESS OF PYRIPROXYFEN TREATED CHIPS WHEN

EXPOSED TO DIFFERENT WATER VOLUMES, CONTAINERS AND PERCENTAGES OF OAK LEAF INFUSION

Introduction

Aedes aegypti, the yellow fever mosquito, is considered one of the world’s largest health

threats. The most common method of controlling Ae. aegypti is source reduction. One class of

chemicals commonly used to control Ae. aegypti larvae are insect growth regulators (IGRs)

(Belinato 2009, Dhadialla 2012). IGRs are a class of chemicals that disrupt insect growth and

reproduction by interfering with insect development (Graf 1993, Belinato 2009, Dhadialla 2012).

There are two types of IGRs: chitin synthesis inhibitors (CSIs), which disrupt cuticle formation

and thereby interfere with insect development (Graf 1993, Belinato et al. 2009), and juvenile

hormone analogs (JHAs), which also disrupt insect development. Instead of disrupting cuticle

formation, JHAs prevent insects from reaching the adult stage (Graf 1993, Dhadialla 2012).

JHAs cause this response in mosquitoes because they provide juvenile hormones at a time in the

life of the insect when they do not normally occur (Graf 1993).

Pyriproxyfen is a JHA that has been accepted by the U.S. Environmental Protection

Agency for use in small containers to control Ae. aegypti because of its relatively low toxicity to

non-target organisms (Sullivan 2000, Ware and Whitacre 2004, Sumen et al. 2013). The World

Health Organization (WHO) has also approved pyriproxyfen at a rate of 10 PPB for use in

potable water (Invest and Lucas 2008, WHO 2008, Seccacini 2014). That pyriproxyfen is labeled

for mosquito control and relatively non-toxic to non-target organisms renders it useful for control

of Ae. aegypti.

Aedes aegypti develop in a variety of containers. These containers may be as large as

abandoned swimming pools or as small as a bottle cap (Espinoza-Gomez et al. 2002, Gubler et

32

al. 2002). For control of Ae. aegypti, pyriproxyfen is applied to a variety of containers made of

various materials. Pyriproxyfen is known to be an adherent chemical and may potentially adhere

to certain materials that are used in manufacturing of containers (Sullivan 2000, Vythilingam et

al. 2005).

Pyriproxyfen is not only able to be absorbed into container material, but also organic

matter often found in containers where Ae. aegypti occur (Sullivan 2000, Sullivan and Goh

2008). These authors determined that pyriproxyfen, in the presence of suspended organic matter,

such as floating leaves or soil, was readily absorbed. When pyriproxyfen was applied at 18

g/acre it was readily absorbed into organic matter and remained active for two months after

application (Sullivan 2000).

Although effective for mosquito control, pyriproxyfen, like many other chemical

treatments, is labeled for treating large bodies of water. For example, NyGuard£ is labeled for

treatment at 3 mL per 275 m2. This presents a problem for Ae. aegypti control because the

mosquitoes are developing in small containers of standing water, not larger bodies of water.

There is a need for a small, easy-to-use treatment for Ae. aegypti. The objective of this study was

to test the efficacy of mosquitocidal chips treated with pyriproxyfen. The slow release

pyriproxyfen formulation was tested on chips in different volumes of water, container materials

and percentages of organic matter, to determine its ability to work under variable conditions. We

hypothesized that water volume, container material and organic matter would have an effect on

the efficacy of mosquitocidal chips.

Materials and Methods

Test insects and colony maintenance. See Chapter 2, materials and methods on p. 23.

33

Mosquito rearing for bioassays. Twenty-four h after hatching, 20 first instar larvae

were moved from a rearing tray into small 120-mL cups and fed 200 PL of diluted fish food

every other day for three days. One hundred mg of ground fish flakes (TetraFin® Goldfish

Flakes) was diluted in 100 mL of water in a 120-mL cup. Food was mixed well in the water and

pipetted into each cup. Cups were put into an incubator maintained at ca. 31qC and 15% RH.

Mosquitocidal chip formulations. Two chip formulations, containing either 0.01% or

1% pyriproxyfen, were prepared using fumed silica, polymer (Butyl-methacrylate), acetone and

pyriproxyfen. The 0.01% pyriproxyfen formulation was used to prepare the 8.4 Pg chip, it

contained 1% pyriproxyfen, 1% silica, 5% polymer and 93% acetone. To prepare this

formulation, 0.084 g pyriproxyfen and 41.76 g of polymer were dissolved into 785 g of acetone,

and 8.35 g of fumed silica was then added and mixed well using a stir plate with magnetic stirrer.

The 1% pyriproxyfen formulation was used to prepare the 840 Pg chip and contained 1%

pyriproxyfen, 1% silica, 5% polymer and 93% acetone. To prepare this formulation, 8.44 g

pyriproxyfen and 42.20 g polymer were dissolved into 785 g of acetone, and 8.44 g fumed silica

was then added and mixed well using a stir plate with magnetic stirrer. The control formulation

combined the same ingredients and amounts; however, no pyriproxyfen was added to control

solutions. Once prepared, solutions were stored in a 1-liter glass bottle (Pyrex¥, Reusable Media

Storage Bottle, Fisher Scientific).

Chip treatment. Chips (American Olean Satinglo Hex White with Black Dot

Honeycomb Mosaic Ceramic Floor and Wall Tile, Birmingham, AL) were treated using a

micropipette. Before chips were treated with pyriproxyfen formulations, they were cleaned in an

enamel metal tray (30 cm x 16 cm x 5cm, Fisherbrand® Enamel metal utility tray, Pittsburg, PA)

with dish soap (Dawn® Dish liquid) and warm water. Chips were made of ceramic material and

34

contained both a glazed and a non-glazed side. Chips were removed from glue backing, were

placed into an enamel tray with soapy water and then were set on a hot plate. Chips were allowed

to boil for 1 h or until all glue backing dissolved off of chips. Chips were strained using a sieve

and dried on paper towels. All chips were treated using stock solutions. Using a pipette, 100 PL

pyriproxyfen formulation was pipetted onto chip to create the 8.4 Pg or 840 Pg pyriproxyfen

chip. Using a pipette, 100 PL control formulation was pipetted onto chip to create the control

chip. Formulations were pipetted onto the non-glazed side of each chip. Non-glazed sides of each

chip were treated to insure treatments adhered to the tile and remained on chips as long as

possible. Chips were allowed to dry for 24 hr in a fumigation hood to prevent contamination

before being placed in bioassay containers.

Bioassay containers. Bioassay containers were 450 mL polypropylene cups (WNA¥,

Chattanooga, TN). Bioassay containers were filled with 350 mL of clean unchlorinated water.

Containers were treated with pyriproxyfen-treated or control chips, and 10 late 3rd to 4th instar

Ae. aegypti larvae were added to each bioassay container.

Water volume experiment. The purpose of this experiment was to determine whether

mosquitocidal chips are affected by varying water volumes containing Ae. aegypti larvae.

Treatment included 250, 500, 750 and 1000 mL of clean unchlorinated water and 8.4 Pg

pyriproxyfen chips. Controls contained the same volumes of water; however, they contained

untreated chips.

Glass cylindrical vases (1000 mL, Libbey£ Cylinder Vase) were used for each replicate.

Vases were filled with the appropriate volume of water. Using large forceps, either a 8.4 Pg

pyriproxyfen chip or a control chip was carefully grasped on its edges (to prevent disturbing

treatment) and placed at the bottom of the vase. Then, 10 late 3rd/early 4th instar mosquitoes were

35

pipetted into each vase from their rearing cups. There were four replicates of each treatment and

control. Larvae were fed 200 PL of ground fish food every other day. Vases were then put into

an incubator maintained at ca. 31qC and 15% RH. Vases were inspected every 24 h for dead or

live larvae, pupae and adults, all of which were counted. Experiments were run for 4 d or until all

mosquitoes had either died or emerged as adults.

Statistical analysis. Percent mortality of dead insects in experiments was calculated and

then transformed using an ArcSin transformation. JMP software (SAS Inc, NC, USA) was used

to run statistical analysis. Using transformed percent mortality data, a repeated measures

ANOVA, with days after application as the repeated measure, was used to determine significant

difference of the percent mortality. Means were compared using a Tukey’s HSD pairwise

comparison.

Effects of various container materials. The purpose of this experiment was to

determine whether mosquitocidal chips are affected by the different container materials. The

design of this experiment was intended to simulate habitats where Ae. aegypti larvae would

typically be found. The materials used were wood (Artminds£ wooden box, Southfield, MI),

metal (Ashland£ Galvanized Metal Bucket, Ashland, OR), clay (Indigo spice, studio décor,

Irving, TX), ceramic (Indigo spice, studio décor, Irving, TX), plastic 450-mL polypropylene cups

(WNA¥, Chattanooga, TN) and glass (Kimble£ Wide Mouth Jars). Two hundred mL of clean

unchlorinated water were placed into each container with either a 8.4 Pg pyriproxyfen chip for

treatments or an untreated chip for controls. Wood containers were tightly wrapped with a layer

of parafilm in order to prevent leakage for the duration of the experiment.

Chips were placed in the bottom of each container using forceps to avoid disturbing the

treatments on the tile. From rearing cups, 10 late 3rd/early 4th instar mosquitoes were placed in

36

each cup. There were four replicates of each container type with pyriproxyfen treated chips.

There were two replicates of each container type with control chips. Larvae were fed according

to bioassay feeding methods. Containers were put in an incubator maintained at ca. 31qC and

15% RH. Containers were checked every 24 h, and numbers of dead or live larvae, pupae and

adults were recorded. The experiment ran for 4 d until all mosquitoes were either dead or

emerged adults.

Statistical analysis. Percent mortality of insects in experiments was calculated and then

transformed using an ArcSin transformation. JMP software (SAS Inc, NC, USA) was used to run

statistical analysis. Using transformed percent mortality data, a repeated measures ANOVA, with

days after treatment as the repeated measure, was used to determine significant difference of the

percent mortality. Means were compared using a Tukey’s HSD pairwise comparison.

Effects of organic matter. Because Ae. aegypti larval habitats often contain significant

amounts of organic matter, this experiment was designed to determine if different percentages of

organic matter in water would affect chip efficacy. Treatments included water containing either

0%, 10%, 30%, 50%, 70% and 90% leaf infusion and the 8.4 Pg pyriproxyfen chip. Controls had

either 0%, 10%, 30%, 50%, 70% and 90% leaf infusion and the control chip. Oak leaf infusion

was prepared using methods demonstrated by Reiter et al. (1991). Fallen oak leaves were

collected from oak trees (Quercus sp.) around the University of Florida campus. Oak leaves (8.3

g) were mixed into one liter of clean unchlorinated water in a glass jar. The infusion was allowed

to ferment for 7 d at 28° ± 2° C and a relative humidity of 36% ± 5%. After 7 d, oak leaves were

removed and the oak leaf infusion was diluted to produce the following percentages in water:

0%, 10%, 30%, 50%, 70% and 90%.

37

Oak leaf infusion (350 mL) was added to bioassay cups. Either a 8.4 Pg pyriproxyfen

chip or a control chip was placed in each cup. Ten late 3rd/early 4th instar mosquitoes were placed

in each cup. There were four replicates of each treatment and control. Larvae were fed 200 PL of

ground fish food every other day. Cups were placed into an incubator maintained at ca. 31qC

and 15% RH. The experiment ran for 5-10 d until all mosquitoes were either dead or emerged

adults. Every 24 h containers were checked for dead or live larvae, pupae and adults.

Statistical analysis. Recorded data of dead mosquitoes was summed and percent

mortality was calculated and transformed using an ArcSin transformation. JMP software (SAS

Inc, NC, USA) was used for statistical analysis. Using transformed percent mortality data, a

repeated measures ANOVA, with days after application as the repeated measure, was used to

determine significant difference of the percent mortality. Means were compared using a Tukey

HSD pairwise comparison.

Results

Varying water volumes. There was a significant difference in times to mortality (F =

261.2, df = 3, P = <0.0001, Fig 3-1) and in mosquito mortality (F = 96.74, df = 4, P = <0.0001,

Fig 3-1) when mosquitocidal chips were used in different water volumes. Additionally, there was

a significant interaction between water volume and time (F = 17.05, df = 12, P = <0.0001).

Pairwise comparisons among water volumes showed that mosquito larvae exposed to chips in

250 mL of water died at significantly faster rates than mosquito larvae exposed to the chips in all

other water volumes (500 mL: p = <0.0028, 750 mL p = 0.0002, 1000 mL p = 0.0174, Fig 3-1).

However, 100% mortality of mosquito larvae was observed in all 4 containers by the fourth day

of treatment. The larvae in 250 mL treatment reached 100% mortality 24 hours prior to larvae

exposed to at all other volumes (Fig 3-1).

38

Effects of various container materials. When mosquitocidal chips were used in

different container materials, container effects over time indicated that there was a significant

difference in mosquito mortality among container materials (F = 16.95, df = 5, P = <0.0001, Fig

3-2) and time (F = 609.35, df = 3, P = <0.0001, Fig 3-2). Also, a significant interaction was

observed in the relationship between container material and time (F = 7.12, df = 15, P =

<0.0001). Tukey’s test demonstrated that no significant difference in mosquito mortality was

observed for mosquitoes in ceramic and clay containers (t = 1.35, df = 35, P = 0.755, Fig 3-2).

However, mosquitoes in ceramic containers died at a significantly faster rate than glass (t = 4.51,

df= 35, P= 0.0009); metal (t= 7.96, df = 35, P =<0.0001); plastic (t=5.59, df = 35, P= <0.0001);

and wood (t = 4.95, df = 35, P = 0.0003, Fig 3-2). Additionally, clay had a significantly faster

mosquito mortality rate than glass (t = 3.16, df = 35, P =0.035); metal (t = 6.61, df = 35, P =

<0.0001); plastic (t = 4.24, df = 35, P = 0.002); and wood (t = 3.60, df = 35, P = 0.012, Fig 3-2).

All other treatments showed no significant difference in mosquito mortality rate, except for glass

and metal (t = 3.45, df = 35, P = 0.0170, Fig 3-2).

Effects of organic matter. When mosquitocidal chips were exposed to various

percentages of organic matter over time, there was no significant difference in mosquito

mortality with different percentages of leaf infusion (F = 0.422, df = 5, P = <0.829, Fig 3-3).

Although not significantly different, in 0% and 10% leaf infusion treatments more mosquitoes

were killed at faster rate than all other percentages of organic matter; however, all treatments

came to 100% mortality on the fourth day of treatment (Fig 3-3). This demonstrates that the

treated chips were able to work regardless of the presence of organic matter.

Discussion

Aedes aegypti are present in different types of containers that vary in water volume

(Espinoza-Gomez et al. 2002, Gubler et al. 2002). Results of our first experiment showed that

39

these mosquitocidal chips were effective in water volumes as low as 250 mL and as much as

1000 mL. Similar to results from chapter 2 (p. 26-27), these varying water volumes result in

different doses of pyriproxyfen within the water. Chapter 2 tested different doses of direct

treatment of pyriproxyfen (1, 3 and 10 PPB). Results from chapter 2 demonstrated that 3 and 10

PPB killed 100% of Ae. aegypti during the pupal stage. Chips are prepared to aim to release

enough pyriproxyfen to have concentrations of 10 PPB in 1000 mL of water. However, in this

experiment, chips that were exposed to different volumes of water that could release amounts of

pyriproxyfen to create doses in the water between 10- 40 PPB. Results were similar to those seen

in chapter 2 because chips could produce similar target doses (10 PPB) and killed 100% of

mosquitoes during their pupal stage. Mosquitoes in the 250 mL treatment were killed at a faster

rate because the amount of pyriproxyfen in the water was approximately 2 to 4 times greater than

in other treatments. It could also be due to having less water to reside in therefore they were able

to quickly acquire food and air from water’s surface.

Results from Estrada and Mulla (1986) as well as our results from chapter 2 (p 26-27),

demonstrated that doses as low as 1 PPB of pyriproxyfen result in 95% mortality of Ae. aegypti.

Similarly, Darriet and Corbel (1996), Sihuincha et al. (2005) and Kamal and Khater (2010)

found that 75% or greater Ae. aegypti mortality occurred at doses as low as 1 PPB. Because of

pyriproxyfen’s efficacy at such small doses, future experiments should include testing these

chips in larger water volumes to achieve lower doses of pyriproxyfen within the water. This will

give a better idea of the mosquitocidal chip’s effectiveness against Ae. aegypti occurring in

larger volumes of water such as clogged gutters or bird baths.

Aedes aegypti development containers not only vary in the amount of water they can hold

but the type of material from which they are made. Our results showed that these mosquitocidal

40

chips can be used in a variety of containers to achieve 100% mortality of Ae. aegypti. However,

some differences in rate of mortality was observed between the six different containers. Notably,

ceramic and clay had the fastest rates of mortality. This contrasted with studies done by

Vythilingam et al. (2005), who found that earthen jars reduced long-term efficacy of

pyriproxyfen. These authors found that pyriproxyfen used at a rate of 10 PPB in earthen jars

seemed to be absorbed and this lessened its efficacy in killing Ae. aegypti after 10 wks. In the

same experiment, pyriproxyfen in plastic tubs had a significantly longer residual and killed 100%

of mosquitoes for >15 weeks. Differences between our results and those found by Vythilingam et

al. (2005) could be attributed to the pyriproxyfen formulation. Vythilingam et al. (2005) used a

granular formulation of pyriproxyfen, whereas we used technical grade pyriproxyfen within a

slow release polymer. It is possible that the technical grade pyriproxyfen when diluted could

have made pyriproxyfen more readily available than granular formulations which may retain

some of the active ingredient. Also, their test purpose was to show long-term residuals, so

containers with chips would have to be tested for extended periods of time to ensure that the

efficacy of the clay container was comparable to earthen jars used in Vythilingam et al. (2005).

Similarly, Suman et al. (2013) tested the absorbency of pyriproxyfen in seven different

types of containers: soft wood, fire clay, glass, rubber, cement and three different plastics. These

authors found that concrete was the least adherent of the materials tested and resulted in the

highest pupal mortality (Suman et al. 2013). They found that all other materials greatly absorbed

pyriproxyfen and reduced its efficacy. Their findings conflict with our results because our clay

seemed to be the least absorbent material besides ceramic, but differences could be due to

strength of concentrations used. Suman et al. (2013) used serial dilutions of NyGuard£ to

achieve their desired concentrations of 0.01 PPB, which was ten times lower than our

41

concentration. Such differences in the formulation of pyriproxyfen also could have contributed to

the different results.

Pyriproxyfen is not only thought to adhere to the containers in which Ae. aegypti are

developing, but also thought to tightly adhere onto organic matter (Schaffer 1988, Sullivan

2000). Schaffer et al. (1990), additionally found that at rates lower than 0.04 PPB, pyriproxyfen

readily absorbed into the organic matter, causing the concentration of pyriproxyfen to decline in

water. Our experiments contrasted those of Schaffer et al. (1990) and Sullivan (2000),

demonstrating that regardless of the presence of organic matter, no significant difference in rate

of mortality was recorded. This indicates that organic matter did not affect the efficacy of

pyriproxyfen in the water. Mosquitocidal chips worked at target rates of 10 PPB in water, killed

100% of Ae. aegypti during the pupal stage.

Differences in our results from those of Schaffer could have resulted from different types

of organic matter. Our experiments used oak leaf infusion, which contains mostly leaf chemicals,

bacteria and minimal debris, but experiments done by Schaffer et al. (1988) involved suspended

organic matter, including leaves and soil, which could have more readily absorbed the

pyriproxyfen. Their studies were done at field sites in ponds containing large amounts of

suspended organic debris. The authors dried out the debris and measured the pyriproxyfen within

that organic matter and found that pyriproxyfen had readily adhered to the organic matter.

Another factor that could have led to the differences in results is the rate of pyriproxyfen used;

Schaffer et al. (1990) used rates smaller than ours. The rate used in our experiment was 10 PPB,

whereas Schaffer et al. (1990) used rates ranging from 0.5 PPB -10 PPB. Future investigation of

chips in a variety of organic matter materials will be necessary to determine the effects that

organic matter has on the efficacy of mosquitocidal chips.

42

Our initial research on mosquitocidal chips has proven that they are effective at

eliminating Ae. aegypti mosquitoes in varying water volumes, container types, and levels of oak

leaf infusion. These chips can serve as an easy-to-use treatment method for Ae. aegypti that are

labeled for their small containers.

43

Figure 3-1. Effects of mosquitocidal chips on percent mortality of Ae. aegypti in varying water volumes. Controls not displayed, due to no mortality. Error bars represent r SEM.

Figure 3-2. Effects of mosquitocidal chips on percent mortality of Ae. aegypti when used in

different types of containers. Controls not shown, due to no mortality. Error bars represent r SEM.

44

Figure 3-3. Effects of mosquitocidal chips on percent mortality of Ae. aegypti in the presence of oak leaf infusion. Error bars represent r SEM

45

CHAPTER 4 EVALUATING THE LONGEVITY OF CHIPS AND THE EFFECTS THEY WILL HAVE ON

AEDES AEGYPTI POPULATIONS AND OVIPOSITION PREFERENCES

Introduction

Pyriproxyfen is an insect growth regulator (IGR), specifically a juvenile hormone

analogue. It is a relatively stable chemical that affects the hormonal system of insects (Bridges et

al. 1977, Sullivan 2000). Pyriproxyfen is responsible for abnormal amounts of juvenile hormones

in the life cycle of an insect at a time they do not normally occur resulting in insects being unable

to molt to the adult stage (Sullivan 2000, Graf 1993).

Pyriproxyfen is known to have a long residual and reside in environments for extended

periods of time (Vythilingam et al. 2005, Seng et al. 2008, Ritchie et al. 2013). Research has

shown that at rates of 10 PPB, pyriproxyfen can have a residual lasting for up to 4 months

providing 100% control and up to 6 months with 50% control (Vythilingam et al. 2005, Ritchie

et al. 2013). Pyriproxyfen has also previously been used in slow release applications and found

to also have a lasting residual. When pyriproxyfen was formed into beeswax candles with the

centers hollowed out and used as larval rearing containers it provided 100% mortality of Ae.

aegypti for 360 d (Juan et al. 2013). Similarly, Seng et al. (2008) tested resin strips treated with

concentrations of 30 or 40 PPB pyriproxyfen in water, and found they would provide 80 – 100%

control of Ae. aegypti for up to 34 wk.

Pyriproxyfen not only has a strong residual but also can significantly decrease

populations of Ae. aegypti. Aedes aegypti exhibit a number of different behaviors that make

controlling them difficult. These behaviors include skip oviposition, where one female will lay

her eggs in numerous containers; they are daytime feeders; and they have the ability to develop

in a wide variety of water holding containers (Harrington and Edman 2001, Gubler 2002, Hales

46

and Panhais 2005). Regardless of these various behaviors, pyriproxyfen has proven to be

effective at reducing populations of Ae. aegypti (Sihuincha et al. 2005, Ohba et al. 2013).

Sihuincha et al. (2005), when treating populations of Ae. aegypti in Peru, found that, over the

course of a 5 mo period, there was 88-96% mortality in their mosquito populations. Sihuincha et

al. (2005) also found that Ae. aegypti showed no oviposition preference, and females were not

deterred by treated containers and consistently laid eggs amongst containers, indicating that

pyriproxyfen does not deter females from oviposition.

Our experiments aimed to test the longevity of mosquitocidal chips, as well as their

ability to decrease Ae. aegypti populations. We tested mosquitocidal chips effect on population

reduction and oviposition preference. We hypothesized that mosquitocidal chips would remain

effective for 4-6 mo killing 100% of Ae. aegypti pupae, and that they would decrease overall

populations of Ae. aegypti. Finally, we hypothesized that Ae. aegypti would show no preference

in selecting containers for oviposition in the presence of mosquitocidal chips.

Materials and Methods

Test insects and colony maintenance. See Chapter 2, materials and methods on p 23.

Mosquitocidal Chip formulations. See Chapter 3, materials and methods on p 34.

Chip treatment. See Chapter 3, materials and methods on p 35.

Chip longevity. Treatments were 0.01% pyriproxyfen chips (8.4 µg) and 1%

pyriproxyfen chips (840 µg). For controls, untreated control chips were used. Treated and control

chips were put into bioassay cups with 350 mL of water, without larvae. Chips were left in water

for 7 d in an incubator maintained at ca. 31qC and 15% RH. After 7 d, chips were carefully

removed so not to disturb chip treatment and placed on a chicken wire rack for 48 h to allow

chips to dry. Chicken wire racks were made using a cookie sheet (Wilton£ Cookie sheet) with

47

chicken wire (50 cm x 40 cm) on top to allow for both sides of the chips to dry evenly. After

chips were removed, ten late third/early fourth instar mosquitoes were added to each of the cups

and were fed 200 µl of ground fish food every other day. There were four replicates of both

treatments and controls. Cups were placed back into the incubator at ca. 31qC and 15% RH. This

procedure was repeated for four sequential weeks. Following the fourth week, chips were

allowed to dry for 1 mo. Chips were then placed in water again for 7 d and experimental

procedures remained the same as above. Mortality was checked for 4-5 d or until all mosquitoes

either died or emerged as adults. Every 24 h dead or live larvae, pupae and adult data was

recorded. Monthly tests were conducted for 4 mo or until there was 50% or less mortality in

bioassays.

Statistical analysis. Mortality data was summed and percent mortality was calculated.

Percent mortality data was transformed using an ArcSin transformation. JMP software (SAS Inc,

NC, USA) was used to run statistical analysis. Using transformed percent mortality data, a two-

way ANOVA was run, using day of treatment as a repeated measure. Then means were

compared using a Student’s-t test.

Population and oviposition effects of chips. This experiment was conducted to

determine if chips have an effect on female oviposition preference and on the overall reduction

of populations of Ae. aegypti. In each cage (60 cm x 60 cm x 60 cm BugDorm Insect Tents

BugDorm-2120F Insect Tent, MegaView Science Co., Ltd., Taichung, Taiwan), there were 4

bioassay cups containing 350 mL of clean unchlorinated water with an oak leaf sachet. Oak leaf

sachets were made using fillable tea bags (Disposable, self-seal tea bags, Otter and Trout Trading

Co, Gainesville, FL), filled with 0.5 g of ground field collected oak leaves. There were 4

treatments. The first treatment contained 3 untreated cups and 1 treated cup with an 8.4 µg

48

pyriproxyfen chip. The second treatment had 2 untreated cups and 2 treated cups. The third

treatment had 1 untreated cup and 3 treated cups. The final treatment contained 4 treated cups.

For the controls, all 4 cups were untreated. There were 4 replicates of each of the 4 treatments

and a control, and repeated twice over a 2-mo period.

The four cups for each treatment were put into 60 cm x 60 cm x 60 cm BugDorm Insect

Tents (BugDorm-2120F Insect Tent, MegaView Science Co., Ltd., Taichung, Taiwan). Forty-

eight h after blood feeding, ten gravid female Ae. aegypti were put into each cage. Mosquitoes

were allowed to oviposit on filter paper for 72 h before egg sheets and adult mosquitoes were

removed from cage. Egg sheets were allowed to dry for 24 hr and eggs were counted. After eggs

were counted chips were temporarily removed, from the cups and sachets were taken out. Eggs

were brushed into cups and lids were placed on containers. Containers were hand shaken for 1

min, to stimulate egg hatching. After shaking, the lid was removed and chips were placed back

into original containers. Larvae that emerged from eggs were fed 200 µl of ground fish food

every other day. A 120-mL cup with 10% sugar water was placed in each cage for emerging

adult mosquitoes to feed on. After 10 d, emerged adults were counted to determine chip effects

on Ae. aegypti populations. Experiments were kept in a greenhouse at ca. 35qC ± 5° C and 25%

± 5° C RH with a photoperiod between 12:12 (L:D) and 14:12 (L:D).

Statistical analysis JMP software (SAS Inc, NC, USA) was used to run statistical

analysis. Percent emergence data was calculated by using the number of eggs laid and number of

adults emerged. Percent emergence data was transformed using an ArcSin transformation.

Percent emergence transformed data and number of eggs laid and adults emerged were analyzed

using a one-way ANOVA and means for percent emergence data were compared using a

Student’s-t-test.

49

Results

Chip longevity. Results demonstrated a significant interaction between duration of chip

use and treatment (F = 4.44, df = 12, P = <0.0001). The 8.4 µg chip mortality was confined to

the first 3 wks after treatment, with >50% mosquito mortality (Fig 4-1a), whereas the 840 µg

chip resulted in 100% mosquito mortality when reused throughout the 16 wk period of the

experiment (Fig 4-1b). Mosquitoes took on average 4-5 d to reach mortality, in the 840 µg chip

treatment and there was very little variability in rate to mortality (Fig 4-1b). The 8.4 µg chip

which aims to achieve 10 PPB concentrations of pyriproxyfen in water, killed less than 50%

during the first week of treatment and then less than 20% for the following two weeks. After 3

wks, it ceased to kill Ae. aegypti larvae. Regardless of treatment, there was 100% mortality

during the pupal stage, when 840 µg chips were used.

Population and oviposition effects of chips. Results showed a significant difference

between the different treatments in the number of live adults that resulted from continuous

population growth for 2 wks (F = 51.87, df = 4, P = <0.0001). There was also a significant

difference in the percent emergence (F = 21.33, df = 4, P = <0.0001). There was no significant

difference in the number of eggs laid in each treatment (F = 0.328, df = 4, P =0.855). These

treatments showed a linear pattern, indicating that with increased treatment there was lower

emergence of adult mosquitoes (Fig 4-2). Female oviposition showed no preference for laying in

either treated or control containers. Number of eggs laid in either treated or untreated containers

corresponded with the percent of treated or untreated cups in cage. Cages containing 75% treated

and 25% untreated containers had approximately 75% of eggs laid in treated containers and 25%

of them laid in untreated containers. This pattern remained for all 5 treatments (Table 4-1).

50

Discussion

The need for an easy to use, long-lasting treatment for Aedes aegypti, has become

increasingly necessary. Mosquitocidal chips are a potential solution to this problem.

In Chapter 3 (p 38-39), the 8.4 µg chip in all experiments worked to eliminate 100% of

mosquitoes, which contrasted to the experiment reported here. Results were similar to those seen

by Seng et al. (2008), who tested the longevity of pyriproxyfen embedded into resin chips. In our

study, it was the 840 µg chip (target dose in water = 1000 PPB), that killed 100% of Ae. aegypti

for 16 wks. Seng et al. (2008) found that pyriproxyfen at a target dosage of 30 PPB killed 92.6%

after 34 wks. Because our chip is almost thirty times stronger than that of Seng et al. (2008), it is

expected that the 840 µg chip would kill 100% of mosquitoes for a longer period than was used

in this study. This conclusion is also supported by the findings of Sihuincha et al. (2005) that,

when studying pyriproxyfen under field conditions at target concentrations of 50, 67 and 83

PBB, found that pyriproxyfen killed 95% of Ae. aegypti pupae for 20 wks.

Vythilingam et al. (2005), found that at rates of 10 PPB, pyriproxyfen can have a residual

lasting up to 4 mo. This differed from our experiments where the 8.4 µg chip (target dose in

water = 10 PPB), did not work to kill Ae. aegypti for any length of time. Explanations for the

differences between our results and those of Vythilingam et al. (2005) are most likely due to

formulation of pyriproxyfen used and presence of organic matter. Vythilingam et al. (2005), used

granular formulations of pyriproxyfen which allowed for complete dispersal of pyriproxyfen into

water in a short period of time. Whereas, in our experiments, mosquitocidal chips are designed to

slowly release pyriproxyfen over time, so not all pyriproxyfen was released into the water at

once. This difference in formulation may be a reason for why the 8.4 µg chip did not work as

efficiently as direct treatment of the water. Another reason for why the 8.4 µg chip may not work

as well, is explained by Roberts and Hutson (1998), Sullivan (2000) and Sullivan and Goh

51

(2008). These authors tested the chemical properties of pyriproxyfen and found it to be soluble in

water up to 400 PPB. However, pyriproxyfen more readily adheres into organic matter. In the

chip longevity experiment, chips were removed from bioassay cups before larvae were added.

This contributed to the inability of the 8.4 µg chip to work. In Chapter 2 (p. 26-27), chips were

left in containers for the duration of the experiments, and larvae were fed ground fish flakes

during these experiments, allowing for the presence of organic matter for pyriproxyfen to adhere

to and subsequently be ingested by mosquito larvae. According to Sullivan and Goh (2008) and

Schaffer et al. (1988), pyriproxyfen is highly susceptible to photo degradation and has a half-life

of fewer than 10 days, whereas when absorbed into soil it has a much longer half-life of 10-20

wk. When chips and organic matter are both present pyriproxyfen readily adheres to the ground

fish food making it accessible to Ae. aegypti larvae. Perhaps the 8.4 µg chip did not release

enough pyriproxyfen in the absence of organic matter to be absorbed when organic matter (fish

food) was present, preventing its ability to work. Further research will have to be conducted to

confirm these assumptions. Chips will have to be tested in the presence and absence of fish food

or other suspended organic matter to determine if pyriproxyfen is being readily absorbed.

Our laboratory studies have also shown that mosquitocidal chips will reduce mosquito

populations, demonstrating results that are in line with those of Sihuincha et al. (2005). These

authors found using laboratory and field studies on Ae. aegypti in Peru, that, over a 5 mo period,

pyriproxyfen prevented 100% of mosquitoes from hatching. This was similar to our studies

where we found that when populations of mosquitoes were in a situation with 100% treated

containers 98% control of Ae. aegypti population occurred, perhaps 100% mortality was not

reached because chips are designed to slowly release pyriproxyfen, allowing for minimal

emergence of Ae. aegypti because not all mosquitoes had enough exposure to the treatment.

52

Aedes aegypti perform skip oviposition, where they spread their eggs over multiple water

holding containers (Harrington and Edman 2001, Gubler 2002). One of the aims of this

experiment was to determine if pyriproxyfen repelled mosquitoes from laying their eggs. Our

experiments showed that mosquitocidal chips did not repel mosquitoes from laying their eggs.

Female Ae. aegypti were not deterred to oviposit on cups containing treatment as previously

observed by Sihuincha et al. (2005) who found that at direct doses of pyriproxyfen as high as

31,250 PPB, demonstrated no repellency to treatment and Ae. aegypti continued to lay their eggs

in treated and untreated containers. In our experiments there was a slight preference for Ae.

aegypti to lay in the treated cups over the untreated cups in 50:50 treatment. This could be due to

cup placement within the cage, or just a random occurrence, however, other experiments are

needed to confirm the possibility of Ae. aegypti oviposition preference.

The ability of mosquitocidal chips to work for extended periods of time independent of

reuse demonstrates their ability to effectively lower populations of Ae. aegypti. These

mosquitocidal chips have the potential for being an effective treatment of Ae. aegypti in the field.

53

Figure 4-1. Percent mortality of Ae. aegypti using an 8.4 µg chip and an 840 µg chip over 16 wks. A) Percent mortality of Ae. aegypti using an 8.4 µg chip over a period of 16 wks. B) Percent mortality of Ae. aegypti using an 840 µg chip over a period of 16 wks. Error bars represent ± SEM

54

Figure 4-2. Total number of Ae. aegypti eggs laid and adults emerged using five different treatments. Treatments are as follows: (1) 100% treated (2) 75% treated – 25% untreated (3) 50% treated – 50% untreated (4) 75% untreated – 25% treated (5) 100% untreated. Error bars represent ± SEM.

100% TRT 75% TRT 25% UT

50% TRT 50% UT

25% TRT

100% UT

Treatments

No.

of E

ggs a

nd A

dults

A A A

A A

D

A

D

C

B

1.0 ± 0.34 4.8 ± 2.51 18.5 ± 4.20 52.0 ± 12.14 56.2 ± 6.54

Total Eggs Total Adults

% EMERG:

0

55

Table 4-1. Oviposition in pyriproxyfen-treated and untreated water sources

Treatment Treated Untreated No. of eggs % of eggs No. of eggs % of eggs

100% TRT 516 ± 71.9 100% 0 0% 75% TRT 25% UT

355 ± 75.8 74% 122 ± 62.5 26%

50% TRT 50% UT

420 ± 75.7 78% 116 ± 49.6 22%

25% TRT 75% UT

140 ± 40.5 24% 439 ± 83.7 76%

100% UT 0 0% 625 ± 82.9 100%

56

CHAPTER 5

CONCLUSIONS

Aedes aegypti, the yellow fever mosquito, is a threat to public health on a global scale. .

Despite many existing methods for Ae. aegypti control, there is a need for an easy method to

control these mosquitoes, such as the use of mosquitocidal chips as described here, which

represent an easy treatment method that homeowners can use to control Ae. aegypti. In Chapter

2, the potential of pyriproxyfen and its ability to control mosquitoes was examined before its

application to mosquitocidal chips. As previously demonstrated by other authors, pyriproxyfen

killed > 92% of mosquitoes at doses as low as 1 PPB, but results showed that the 10 PPB dose

was more consistent, the dose to obtain this concentration in water was targeted for use with the

8.4 µg mosquitocidal chips.

In Chapter 3, tests with mosquitocidal chips under 3 different conditions with varying

water volumes, different container materials and varying organic matter levels, provided

information on potential field conditions in which these chips are supposed to work. These

experiments demonstrated that organic matter did not have an effect on the chip efficacy, and

mosquito mortality was 100% during their pupal stage regardless of the level of organic matter.

Furthermore, chips with the higher dose of 840 µg of pyriproxyfen provided 4 months of 100%

Ae. aegypti control, and resulted in significant mosquito population decreases.

This research demonstrates that these mosquitocidal chips are a robust mosquito control

method that can work under multiple conditions. Regardless of what type or size containers they

may be used in, the mosquitocidal chips can eliminate 100% of the Ae. aegypti in the containers

to which they are applied. Also, the mosquitocidal chips may work in killing mosquitoes beyond

4 months, eliminating the need for retreatment within the mosquito season. Population control

57

can be obtained even if only part of the mosquito development containers are treated with

mosquitocidal chips. Aedes aegypti occupies multiple containers around people’s homes due to

skip oviposition so nearly all containers should be treated in order to prevent high mosquito

populations, although, mosquitocidal chips can have significant effects on the mosquito

population even when just part of the containers are treated.

Although the mosquitocidal chips work well to eliminate Ae. aegypti, as with other

control methods, they have some drawbacks. The lower dose mosquitocidal chip (8.4 µg chips)

did not perform well in longevity trials, probably due to the low dose of pyriproxyfen being

bound to the organic matter in the water. However, the 840 µg of pyriproxyfen, preformed as

expected for more than 4 months. It is possible that the low dose chips was less efficient due to

the low amount of organic matter in the water which would bind the pesticide and then be

ingested by the mosquito larvae. Further testing on the role of suspended organic matter in the

presence of these chips are needed.

The role of mosquitocidal chips in oviposition preference of Ae. aegypti needs further

research. An unexpected egg laying preference for containers treated with mosquitocidal chips

needs further investigation in tests with different layouts of treated and untreated cups in cages

and in the field.

This research explores a novel technique for controlling Ae. aegypti that may soon be

determined to be an effective method of controlling Ae. aegypti and other mosquitoes that

develop in small bodies of water.

58

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BIOGRAPHICAL SKETCH

Kristen Stevens received her bachelor’s degree from the University of Florida in

Entomology in May of 2015. In the fall of 2015, she began her master’s degree in Entomology at

the University of Florida, studying Urban Entomology, specifically mosquito control. Kristen

worked with Aedes aegypti and novel mosquitocidal chips as a control method. After her

graduation in May of 2017, Kristen is beginning her career as technical director for Clegg’s Pest

Control in Durham, NC.