Evaluation of Frankliniella Bispinosa As a Vector of Tomato Spotted Wilt Virus in Pepper

EVALUATION OF FRANKLINIELLA BISPINOSA AS A VECTOR OF TOMATO SPOTTED WILT VIRUS IN PEPPER

YOLANDA AVILA

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

2004

Yolanda Avila

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my major professor, Dr. Joseph

Funderburk, for his constant support and advice during my program. I would also like to

thank the members of my supervisory committee: Dr. Stuart Reitz for statistical analyses

and manuscript help, Dr. Timur Momol for laboratory space and advisement in my plant

pathology program, and Dr. Heather McAuslane for laboratory space and manuscript help.

I would also like to express gratitude towards those who provided technical support for this research. I give special thanks to Julianne Stavisky and Sara Jane Hague for the wonderful help in collecting data and maintenance of colonies and plants while dividing my time between Gainesville and the Quincy research station. I would like to thank

Hank Dankers for the use of laboratory and ELISA training and Steve Olsen for field plot maintenance.

Finally, I would like to thank my parents, Susan and Francisco Avila, my brother,

Dave, and Oakley Davis for their support and encouragement.

iii

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... iii

LIST OF FIGURES ...... vi

ABSTRACT...... vii

CHAPTER

1 REVIEW OF LITERATURE...... 1

Introduction...... 1 Biology of the Thysanoptera ...... 2 Natural Enemies...... 5 Biology of Orius...... 6 Predator-Prey Interactions ...... 9 Tomato Spotted Wilt Virus...... 12 Thrips As Vectors of the Tomato Spotted Wilt Virus ...... 14 Research Objectives...... 20

2 ACQUISITION OF TOMATO SPOTTED WILT VIRUS AND REPRODUCTION ON PEPPER BY FRANKLINIELLA BISPINOSA, AS COMPARED TO THE KEY VECTOR, FRANKLINIELLA OCCIDENTALIS ...... 22

Introduction...... 22 Materials and Methods ...... 25 Results...... 27 Discussion...... 28

3 TRANSMISSION OF TOMATO SPOTTED WILT VIRUS BY FRANKLINIELLA BISPINOSA VERSUS FRANKLINIELLA OCCIDENTALIS IN PEPPER, AS INFLUENCED BY PREDATION OF ORIUS INSIDIOSUS ...... 33

Introduction...... 33 Materials and Methods ...... 35 Results...... 37 Discussion...... 38

iv 4 POPULATION ABUNDANCE OF FRANKLINIELLA BISPINOSA AND FRANKLINIELLA OCCIDENTALIS IN FIELD PEPPER...... 43

Introduction...... 43 Materials and Methods ...... 44 Results...... 45 Discussion...... 46

5 CONCLUSIONS ...... 48

LIST OF REFERENCES...... 49

BIOGRAPHICAL SKETCH ...... 63

LIST OF FIGURES

Figure page

2-1 Mean number (+ SEM) of F. bispinosa versus F. occidentalis larvae collected per infected pepper plant...... 27

2-2 Mean (+ SEM) percent acquisition of TSWV by F. occidentalis and F. bispinosa on pepper...... 28

4-1 Mean (± SEM) number of F. bispinosa and F. occidentalis per pepper plant collected from field pepper in North Florida from 20 May 2002 through 10 June 2002...... 45

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

EVALUATION OF FRANKLINIELLA BISPINOSA AS A VECTOR OF THE TOMATO SPOTTED WILT VIRUS IN PEPPER

Yolanda Avila

December 2004

Chair: Joseph Funderburk Major Department: Entomology and Nematology

Certain species of thrips are vectors of viral pathogens that infect plants. Of

particular concern to the agricultural community are the potential vectors of the Tomato

spotted wilt virus (TSWV), a tospovirus that has caused extensive economic crop loss.

Determining the role of vectors in the epidemiology of TSWV is crucial in evaluating and

managing the spread of the disease in pepper. The research reported herein focused on

comparing the acquisition of TSWV in pepper by Frankliniella bispinosa to that by the

key vector Frankliniella occidentalis. The reproductive suitability of pepper as a host plant was also evaluated for both species. Pepper was a better reproductive host for F. occidentalis than for F. bispinosa. Frankliniella bispinosa acquired the disease in pepper, but acquisition by F. occidentalis was more efficient.

The TSWV transmission trials were designed to assess the capacity of O. insidiosus to reduce primary spread of TSWV, as well as to compare rates of TSWV transmission by F. occidentalis versus F. bispinosa in the absence of the predator. The results

vii obtained from the study were insufficient to draw conclusions on the ability of O. insidiosus to reduce primary spread of TSWV by predation upon F. occidentalis or F. bispinosa. The results demonstrate the ability of F. occidentalis and F. bispinosa to acquire TSWV from tomato and transmit the virus to pepper. Field studies show the dominant species of thrips in pepper during mid to late summer is F. bispinosa.

viii CHAPTER 1 REVIEW OF LITERATURE

Introduction

Since the early 1900s, an increasing amount of attention has been devoted to studying the insects of the order Thysanoptera. These insects are of great economic concern in the agricultural community due to the amount of crop damage they are capable of inflicting. This newfound interest in thrips as crop pests has resulted in the naming of

5,000 species worldwide (Lewis 1997).

The 5,000 known species of Thysanoptera are classified into two suborders,

Terebrantia and Tubulifera, consisting of nine families. An internal ovipositor characterizes the females of the suborder Tubulifera, whereas the Terebrantians are distinguished by having an external, saw-like ovipositor (Mound et al. 1980).

The Thripidae and Phlaeothripidae families comprise 93% of the species of

Thysanoptera. It is the species of these two families that are typically found on crops.

The Thrips and Frankliniella genera are of most importance to agricultural crops (Mound

1997). The genus Thrips is the largest genus of Thysanoptera, consisting of 275 species worldwide. There are about 180 species in the genus Frankliniella, 90% of which are found only in the neotropics (Mound and Kibby 1998).

Thrips occur worldwide with most of the species originating from the tropics.

Several species are endemic to temperate regions, and a few are found in arctic regions

(Lewis 1997). Of the thrips that are considered serious crop pests, some have limited

distributions, while others are worldwide or have an increasingly expanding range, such

1 2

as the western flower thrips, Frankliniella occidentalis, which has recently expanded its

territory into South America (Mound and Marullo 1996).

Biology of the Thysanoptera

Thrips are minuscule insects, with slender, elongated bodies. Most species are

between 1 to 2 mm in length, with the females generally larger and darker than the males

(Ananthakrishnan 1984). The minute body size of thrips facilitates their ability to cause

excessive amounts of damage to agricultural crops (Moritz 1997).

Thrips oviposit into the plant tissue beneath the epidermis, often resulting in

aesthetic damage to crop plants (Yokoyama 1977, Salguero Navas et al. 1991). F. occidentalis prefers to oviposit along the main vein of leaves or among leaf hairs of cucumber (Kiers et al. 2000). The larvae usually emerge 2-3 days after oviposition,

depending on temperature, relative humidity, and host plant. Larval survival of F.

occidentalis significantly depends on high relative humidity (Shipp and Gillespie 1993).

The life cycle of thrips consists of the egg stage, two feeding larval stages (1st and

2nd instars), two non-feeding pupal stages (pupa and propupa), and the adult stage. Once

development is complete the larvae usually pupate in the soil. The adults are ready to

mate after 2-3 days. The life cycle takes about 20-30 days from egg to adult depending

on temperature (Lewis 1973, Ananthakrishnan 1984).

Thrips tend to overwinter in non-tropical regions. In temperate regions, thrips are

active during spring and summer, and the larvae or adult females diapause in the soil

during the winter (Lewis 1973, Ananthakrishnan 1984). Populations of overwintering

adult thrips have been found in hedge bottoms, grass litter, and bark (Lewis and Navas

1961).

The mouthparts of thrips enable them to feed on a variety of food sources. Thrips

can feed on exposed liquids or liquid contents enclosed in a thin wall of both plant and

animal sources. They feed on the liquid contents of both plants and animals. Regular

food sources include leaf, petal, fruit and seed cell contents, as well as arthropod eggs and

pollen (Kirk 1997b). F. occidentalis is an omnivorous thrips, and has been observed feeding on mite eggs, as well as leaf tissue, nectar, and pollen (Trichilo and Leigh 1986).

Damage to crops occurs when thrips feed on plant tissues, such as leaves, petals,

and fruit. The feeding apparatus of thrips consists of piercing-sucking mouthparts

(Childers 1997). A single, left mandible is used to pierce a hole in the plant surface.

Paired maxillary stylets are linked together to form a single channel for feeding. This

feeding channel is inserted into the plant tissue in order to suck out the contents of the

plant (Kirk 1997b). Further crop damage occurs indirectly through this feeding process

by certain thrips species that vector a pathogen called the Tomato Spotted Wilt Virus

(TSWV). The virus is transmitted through the saliva of the thrips during feeding

(Sakimura 1962b, Hunter and Ullman 1992, Ullman et al. 1997).

Thrips are haplo-diploid, with the males developing from unfertilized eggs and having half the number of chromosomes of females (Mound 1996). Females can be produced sexually or through parthenogenesis, whereas males are produced only through parthenogenesis. F. occidentalis, F. shultzei, and F. fusca are arrhenotokous, producing

males from unfertilized eggs, while some populations of Thrips tabaci are thelytokous,

producing females from unfertilized eggs (Lewis 1973, Ananthakrishnan 1984).

Strong aggregating behavior is characteristic of thrips populations (Lewis 1973).

Mound and Teulon (1995) explain that aggregation results in full exploitation of a

resource. Thrips populations, in particular Frankliniella spp., tend to aggregate on flowers (Pickett et al. 1988, Yudin et al. 1986, Yudin et al. 1988, Shipp and Zariffa

1991). Gerin et al. (1999) demonstrated that in the absence of flowers, F. occidentalis populations showed no growth, simply maintenance.

Response to color and odor of flowers most likely play an important role in the location of flowers by thrips for aggregation sites. Numerous studies have demonstrated color preference in F. occidentalis, as well as an attraction to various chemical compounds, such as anisaldehyde, emitted from flowers (Teulon et al. 1993, Hollister et al.1995, Teulon et al.1999, Koschier et al. 2000).

Nutritional resources in flowers, such as pollen, are another possible reason for attraction to and aggregation in flowers. The beneficial results of a pollen diet for thrips include increased developmental rate and egg production of F. occidentalis (Trichilo and

Leigh 1988) and F. bispinosa (Tsai et al. 1996).

Flowers also serve as a mating site for thrips. Rosenheim et al. (1990) reported sex ratios of F. occidentalis in cucumber flowers that were biased towards males, suggesting that the species mates in flowers. Additionally, Terry (1995) proposed that fighting behavior in males of F. occidentalis, which usually takes place in flowers, might result in a more advantageous position in the flower for sighting and mating with females.

Attributes of thrips, such as polyphagy and high vagility, aid in their ability to

move successfully between and vector viruses to different cropping systems. High

fecundity and short generation times allow for rapid development of large populations and production of large quantities of offspring, all of which can potentially vector the

virus. Many crop systems are ecologically isolated habitats that provide a new

environment for rapid colonization of opportunistic species, thus furthering the ability of

some species of Thysanoptera to spread viruses (Mound and Teulon 1995).

These characteristics may be inherited from an ancestral group that consisted of

detritovores. It has been suggested that ancestral thrips specialized in obtaining liquid

food from decaying tissues and fungal hyphae. Fungal and microbial decay processes

created microhabitats with ephemeral optimum conditions, giving rise to the r-selected

population characteristics of the Thysanoptera that allow for rapid population growth in

these temporary conditions (Mound 1997).

Natural Enemies

Rapid proliferation is a characteristic of thrips populations, which can arrive at a

crop and breed rapidly, especially at high temperatures (Kirk 1997a). Biological control

programs incorporating natural enemies might prove effective in controlling populations

of thrips that serve as TSWV vectors and reservoir hosts. Insecticide resistance in F.

occidentalis has hastened the need for management alternatives (Immaraju et al. 1992,

Brodsgaard 1994).

Until recently, it was thought that there were no predators, parasites or diseases that

could regulate populations of thrips species in crops. The r-selected population attributes

of thrips were thought to outstrip the capacity of natural enemies to control Frankliniella

spp. Classical biological control by itself was thought to be an unsuccessful tool for

controlling thrips pests (Mound and Teulon 1995). Parrella and Lewis (1997) reviewed

literature on biological control and integrated pest management, and concluded that natural enemies contribute little to the regulation of thrips populations in field crops.

Sabelis and Van Rijn (1997) reviewed literature on arthropod predators of thrips, including mantids, wasps, coccinellids, predatory thrips, mites, and heteropteran

6 predators. Heteropteran insects that feed upon thrips include species within the

Pentatomidae, Reduviidae, Nabidae, Lygaeidae, and Miridae. The most persuasive evidence of predation on thrips, though, comes from the minute pirate bugs in the family

Anthocoridae (Lewis 1973).

Sabelis and Van Rijn (1997) used a simple one-predator-one-prey model in order to estimate the intrinsic capability of predatory arthropods to suppress populations of thrips.

The model assumed a small spatial scale with a coherent local population of prey, that prey density is adequate to stimulate the predators and their offspring to stay until all of the prey are eaten, that predators do not disperse until after the prey are extinct from the local population, rate of predation reaches a continuous plateau, and per capita population growth rates of predator and prey are constant.

The model predicted that suppression of thrips populations occurs for anthocorid:thrips ratios greater than 1:217 and extinction of thrips populations within one week at ratios greater than 1:50. Recent field studies have corroborated this prediction, showing that Orius insidiosus is a successful predators of thrips, causing near extinction of thrips populations with predator:prey ratios of 1:40 (Funderburk et al. 2000).

Biology of Orius

The Anthocoridae family is a group of predatory insects ranging within the new world from southern Canada, throughout the entire United States, and south to the West

Indies, Brazil, and Argentina (Blatchley 1926). These insects mostly feed on aphids, mites, thrips, scales, other arthropods, the eggs and larvae of Lepidoptera, and occasionally pollen and other plant material (Kelton 1978).

Predation upon thrips by Orius spp. has been documented worldwide. Orius tristicolor has been recorded to feed upon Thrips tabaci and F. occidentalis in Canada

(Gilkeson et al. 1990). O. sauteri has been shown to control populations of T. palmi in eggplant in Japan (Nagai 1990). O. armatus is capable of suppressing populations of F. occidentalis in field-grown carnations in Australia (Cook et al. 1996). Additionally, O. insidiosus has been shown to control populations of F. occidentalis in greenhouse and field sweet peppers (van den Meiracker and Ramakers 1991, Funderburk et al. 2000).

There are twenty-one described species of Orius (Herring 1966), with four species occurring in North America (Kelton 1963). Orius insidiosus and Orius tristicolor are the most widespread species of the genus Orius in North America (Kelton 1963).

Orius spp. have an oval shape and a small (1.5-5.0 mm), flattened body and pointed head (Henry 1988). The length of males ranges from 1.75-1.96 mm with width ranging from 0.7-0.84 mm, while the length of the females ranges from 1.82-2.17 mm with width ranging from 0.77-0.98 mm. The females tend to be more robust with darker legs

(Kelton 1963).

During oviposition, the eggs are embedded in the plant tissue at an angle almost perpendicular to the surface. Eggs of O. tristicolor are usually laid singly rather than in clusters (Askari and Stern 1972). O. insidiosus prefers to oviposit in tender stem tissue, leaf petioles and main veins of the plant (van den Meiracker and Sabelis 1993). The 1st,

2nd, and 3rd nymphal stages are yellowish with an orange dorsal scent gland. The 4th and 5th nymphal stages are tan to dark brown and the dorsal scent gland is not as prominent. All nymphal stages have bright red compound eyes (Isenhour and Yeargan

1981a).

Orius spp. search for thrips prey by a probing action, which is accompanied by a side-to-side movement of the head, as observed on soybean leaflets (Isenhour and

Yeargan 1981b). Upon detection, the forelegs are used to subdue the prey (Isenhour and

Yeargan 1981b). Adult O. insidiosus have been observed attacking and killing thrips past

satiation when high densities of thrips were available in small arenas (Isenhour and

Yeargan 1981c).

Orius spp. will feed on plant tissue in the absence of prey, however they cannot

develop on a plant diet. Salas-Aguilar and Ehler (1977) found that O. tristicolor nymphs

fed a diet of green bean had a higher mortality than those fed on a diet of thrips. Coll

(1996) found that plant species differed in their suitability for development of O. insidiosus, which performed better on bean and corn plants than on pepper.

Plant characteristics, such as leaf surface texture, play a significant role in the searching efficiency of the predator. Beekman et al. (1991) hypothesized that plant area, leaf structure, hairiness, plant odor and/or stage of flowering are factors that might explain why the searching behavior in O. insidiosus is more successful on chrysanthemum than on rose.

Coll et al. (1997) found that different plant species altered several aspects of the searching behavior of O. insidiosus, which influenced its searching efficiency. The searching efficiency of O. insidiosus was higher on bean and corn than on tomato, most likely due to the presence of glandular trichomes on the leaf surface of tomato (Coll et al.

1997). Coll and Ridgeway (1995) found that the food plants of the thrips prey play an

important role in determining the functional and numerical responses of O. insidiosus to

its prey. Different plant species influence the ability of the predator to respond to

changes in prey density.

Sabelis and Van Den Meiracker (1999) conducted a case study of O. insidiosus

with F. occidentalis to determine whether the functional response of the predator reaches

a plateau or continues to increase with prey density. It was thought that a plateau might

occur with high prey densities due to foraging time constraints or satiation. Holling

(1959) described a Type II predation, in which the functional response curve would reach an upper limit. Sabelis and Van Den Meiracker (1999) predicted that the functional response of O. insidiosus would not reach a plateau within a realistic range of thrips prey densities, due to satiation, which has many important implications for biological control programs. This characteristic of Orius plays a role in its ability to suppress populations of thrips in short periods of time.

Predator-Prey Interactions

There are many components involved in how the predator, Orius insidiosus, interacts with its thrips prey. Surface structures on the host plant, olfactory cues given off by the host plant and thrips prey, and the availability of other prey species may affect how the predator interacts with its prey in a field setting.

Predator-prey behavior studies include hypothesis testing of odor avoidance by thrips. Venzon et al. (2000) proposed that F. occidentalis could discriminate between odors associated with a predator, O. laevigatus, that had fed on a diet of thrips prey versus a diet of non-thrips prey. Refuge use by F. occidentalis was tested in response to encounters with O. laevigatus that were fed a diet of thrips prey or lepidoptera eggs. The study indicated that spider mite webbing could serve as a refuge for thrips against predators and showed that thrips larvae moved to webbed areas when odors from predators that had fed on other thrips were present. The searching efficiency of O. laevigatus was found to be lower in the areas with spider mite webbing than the areas

without the webbing. Predation and encounter rates were reduced in the areas containing the spider mite webbing.

F. occidentalis reacts more readily to odors linked with predators that had fed on

thrips than predators that had fed on other prey. The chemical identity of the volatiles

that elicit refuge-seeking behaviors in thrips is still unknown, although it has been

proposed that it is a combination of thrips alarm pheromone with other metabolic by-

products (Venzon et al. 2000).

Similar aggregation patterns have been found between O. insidiosus and thrips

species. Hansen et al. (2003) found that populations of O. insidiosus were aggregated

with their thrips prey, F. occidentalis, F. bispinosa, and F. tritici, on flowers, rather than

leaves or buds of field peppers. Similar configurations of O. insidiosus and Frankliniella

spp. on flowers have been reported in field and greenhouse peppers (Shipp et al. 1992,

Tavella et al. 1996).

Conversely, Coll and Izraylevich (1997) found that the presence of a predator

might affect where thrips aggregate on plants, as well as affecting their refuge-seeking

behavior. The within plant distribution of thrips differed significantly when O. insidiosus

was present on bean and pepper plants. Higher proportions of thrips were found on

shoots and apices than on leaves and stems when the predator was present. Overall,

thrips were found to change their within-plant distribution when O. insidiosus foraged on

the plant. The study, however, only investigated leaves, stems, and shoot apices, not

flowers.

The similar aggregation patterns of O. insidiosus and thrips prey suggests there are

cues that alert O. insidiosus to the presence of thrips. Venzon et al. (1999) showed that

O. laevigatus had a preference for spider mite and thrips infested plants when tested against clean plants. Olfactometer experiments performed in their study showed that O. laevigatus uses odors to locate plants with spider mites. The searching behavior of O. insidiosus has been reported to rely partially on olfactory perception of volatiles emitted from corn silk when corn is the prey's host plant (Reid and Lampman 1989).

Drukker et al. (1995) found higher numbers of anthocorid predators, primarily consisting of Orius spp., around pear trees that were infested with Psylla spp. prey than around the control trees. The researchers hypothesized that plant synomones played a major role in attracting the anthocorid predators to infested pear trees.

The texture of leaf surfaces also influences predator-prey interactions. Leaf domatia provide refuge for predatory arthropods. Geocoris spp. and Orius spp. are more abundant on domatia bearing plants. Eggs and nymphs of these species are found almost exclusively within domatia. F. occidentalis, which also preys upon mites was found to be more abundant on plants with domatia (Agrawal and Karban 1997).

Leaf domatia and spider mite webbing influence predator abundance by reducing the chance that prey will escape from the leaf surface, by increasing pollen or fungal spore capture, which can serve as an additional food source for the predator, by controlling the micro-environment conditions, such as humidity, or by providing the predator with protection from intraguild predators. It has been found that spider mite webbing provides protection for predatory phtyoseiid mite eggs from the omnivorous F. occidentalis (Roda et al. 2000).

Natural-occurring predators play an extremely important role in regulating pest populations in agricultural crops (Symondson et al. 2002). Little is known, though, about

the predator's ability to control the crop damage caused by primary and secondary spread

of thrips-vectored tospoviruses.

Tomato Spotted Wilt Virus

The Tomato Spotted Wilt virus was first observed in 1906 and described in

Australia (Brittlebank 1919). The disease was soon afterwards identified as having a

worldwide distribution (reviewed in German et al. 1992, Jones and Baker 1991, Sakimura

1962b, Williams et al. 2001, Yeh et al. 1992). Until recently, the virus was considered to

be the only member of the tomato spotted wilt virus group (Matthews 1982, Milne and

Francki 1984). TSWV is now considered a member of the family Bunyaviridae, in which

it is the type speciesof the genus Tospovirus (Francki et al. 1991).

The Tospovirus genus is the only genus in the Bunyaviridae family to include plant-infecting viruses (Francki et al. 1991). The genus is characterized by spherical shape (80-110 nm diameter) and lipid envelope of virions. A Tospovirus virion contains three types of single stranded RNA molecules of differing sizes (S RNA, M RNA, and L

RNA) (de Haan et al. 1990, de Haan et al. 1991, Kormelink 1992, Pappu et al. 2000).

The four structural proteins in a viral particle are the N protein, forming the nucleocapsid, the L protein, forming the interior of the virion, and the glycosides, G1 and G2

(Mohammed 1973). A nonstructural protein (NSs) is found within fibrous paracrystalline

inclusions of infected plant and insect cells, but cannot be detected in healthy plants

(Kormelink et al.1991, Ullman et al. 1992, Ullman et al. 1993).

There are 1050 species of plants susceptible to Tospoviruses, 926 of which are

susceptible to TSWV. These 1050 species belong to 92 different families of plants, most

belonging to the Solanaceae and Compositae families (Peters 1998).

TSWV is capable of causing great amounts of economic loss in agricultural crops and is extremely difficult to control due to its broad host range. Several crops, such as tomato, pepper, lettuce, papaya, eggplant, green beans, artichokes, broad beans, celery, and ornamental plants have experienced significant losses due to the virus (Rosello et al.

1996).

Management techniques for TSWV include growing seedlings under cover, avoiding sequential plantings, weed management, controlling thrips populations, crop rotation with non-susceptible crops, the use of metalized mulch, and the use of resistant varieties (Kucharek et al. 1990, Rosello et al. 1996, Cho et al. 1998, Momol et al. 2004).

Unfortunately, very few successful TSWV resistant hybrids are available. Most of the resistant commercial hybrids are only resistant to certain strains of TSWV and are susceptible to many others (reviewed in Rosello et al. 1996).

Research has demonstrated that high temperatures and a younger growth stage of the plant during time of inoculation may lead to systemic infections in TSWV resistant

Capsicum chinense (Moury et al. 1998, Soler et al. 1998). Many resistant tomato lines have proven to be ineffective; however, lines developed from Lycopersicon peruvianum have demonstrated high levels of resistance (Finlay 1953, Krishna Kumar 1995, Cho et al. 1998).

The symptoms of TSWV are widely varied. Common symptoms are chlorotic and necrotic ring spots on the upper leaf surface, bronzing or yellowing of leaves, purpling of veins on the lower leaf surface, stunting of the main shoot, curling of leaflets, one-sided growth, wilting leaves, dark and light colored fruit spots, and fruit deformation (Ie 1970,

Kucharek et al. 1990, Zitter 1991, Rosello et al. 1996). Symptoms differ greatly between

time of year, plant age, species, cultivar, and virus strain (Jones and Baker 1991). This

considerable variation in symptomology might also be attributed to mixing of virus

strains in the plant (Ie 1970)

The ability to recognize symptoms in order to detect TSWV in the field is a

valuable tool, but serological analyses are critical in diagnosing the virus. A widely used

technique for detecting Tospoviruses is the enzyme linked immunosorbant assay

(ELISA). The direct, or double antibody sandwich (DAS), ELISA uses a polyclonal

antiserum, containing antibodies to the virus, which is created from the blood fluid of an animal (Gonsalves and Trujillo 1986). The antibodies react in a series of steps with the virus antigen, after which an enzyme is added to attach to the antibodies. The final step involves the breakdown of the enzyme with a substrate that produces a color change, indicating presence of the virus (Clark and Adams 1977). ELISA methods have also been developed to test individual thrips for the presence of the TSWV virus (Cho et al.

1988, Bandla et al.1994). Recently, a reverse transcriptase polymerase chain reaction

(RT-PCR) method for TSWV detection has been developed. This protocol involves the sequencing of cDNA from the TSWV viral RNA template, followed by amplification of the cDNA by PCR (Cortez et al. 2001).

Thrips As Vectors of the Tomato Spotted Wilt Virus

Three components are necessary for the occurrence of insect-transmitted plant pathogen epidemics: the vector, the pathogen host plant, and the pathogen. Thrips are excellent vectors of plant pathogens because of their large population sizes and their occurrence over a wide range of host plants and climates (Ullman et al. 1997).

Ten of the 5,000 species of thrips, all belonging to the family Thripidae, are recognized as vectors of plant viruses. These thrips species transmit viruses belonging to

at least four virus groups, including bunyaviruses, ilaviruses, sobemoviruses, and

carmoviruses (Ullman et al. 1997).

Seven thrips species have been identified as Tospovirus vectors (reviewed in

German et al. 1992), six species of which are capable of transmitting TSWV. The TSWV

vectors include Frankliniella occidentalis (Pergande), Frankliniella schultzei (Trybom),

Frankliniella fusca (Hinds), Frankliniella intonsa (Trybom), Thrips tabaci (Lindeman),

and Thrips setosus (Moulton) (Ullman et al. 1997, reviewed in Sherwood et al. 2001).

Frankliniella bispinosa has been suspected of being able to vector TSWV (Tsai et al.

1996, Webb et al. 1998), but has not yet been confirmed as a vector.

Until recently, T. tabaci was considered the main vector of TSWV because of its

worldwide distribution. F. occidentalis is now considered the primary vector of TSWV

due to its increasingly global distribution (Wijkamp et al. 1995a). When compared with

F. shultzei, T. palmi, and T. tabaci, F. occidentalis was found to be the most efficient in

acquiring laboratory isolates of TSWV. Some populations of T. tabaci were unable to

transmit common strains of TSWV (Mau et al. 1991). A recent unpublished laboratory

study comparing the TSWV transmission efficiencies between F. bispinosa and F. occidentalis on jimsonweed showed that F. bispinosa had a higher rate of TSWV acquisition than F. bispinosa (Webb et al. 1997).

Due to a lack of systematic relationships among the different vector species, it has been hypothesized that each thrips species has arisen independently as a tospovirus vector and that a coevolutionary relationship between thrips and tospoviruses is unlikely

(Mound 2002). The common attribute of the thrips vectors species is that they are the most highly phytophagous species, breeding on and feeding on a large range of hosts.

This greatly increases their probability of finding an infected plant. Mound (1996) proposed that the thrips-tospovirus relationship arose from a either a plant pathogen that optimized its dispersal by acquiring a vector, a thrips pathogen that adapted to infect plants, or a vertebrate pathogen whose ability to infect plants was mediated by thrips.

Transmission of TSWV by thrips is persistent and propagative (Paliwal 1974).

Persistent, or circulative, viruses accumulate in the internal tissues of the insect after acquisition and are transmitted to another plant by feeding. Propagative viruses are persistent viruses that must replicate in its vector before being transmitted (Agrios 1997).

Only first instars may acquire the virus by feeding on infected plant tissue (Sakimura

1963, van de Wetering et al. 1996a, van de Wetering et al. 1996b), but second instars and adults may transmit the virus (Wijkamp and Peters 1993). When acquired by first instars, the TSWV virus persists through the molts to the adult stage (Mau et al. 1991).

The virus must replicate in its thrips vector during a latent period before it can be transmitted by adults and older instars (Ullman et al. 1992, Ullman et al. 1993, Wijkamp et al. 1993). The latent period in viruliferous thrips occurs between acquisition and transmission, during which time the thrips is not infectious. Latent periods of T. tabaci,

F. fusca, and F. occidentalis have been documented to range from 4-18 days, 4-12 days, and 4-7 days, respectively (Sakimura 1963, Wijkamp and Peters 1993), and it has been revealed that this period is highly dependent on temperature. Consequently, at low temperatures, the ability to transmit the virus by second instars of F. occidentalis

(Wijkamp and Peters 1993, Wijkamp et al 1995b) and T. tabaci (Chatzivassiliou et al.

2002) increases significantly.

Receptor-mediated endocytosis is thought to govern penetration of tospoviruses into thrips cells (Ullman et al. 1995, Ullman et al 1997). Bandla et al. (1998) proposed that a 50 k-Da protein found in F. occidentalis acts as a receptor for TSWV particles in vivo. The 50 k-Da protein is only found in thrips vectors and is absent in non-vectors.

Medieros et al. (2000) showed that there is a direct interaction between the G1 glycoprotein that is exposed on the surface of a TSWV virion and the 50 k Da protein found in the midgut cells of F. occidentalis. The study demonstrated the 50 k Da protein acts as a receptor for viral particles.

It is in the salivary glands where the virus replicates. The saliva is injected into the plant tissue while the thrips feeds and subsequently transmits the virus (Ullman et al.

1991, Hunter and Ullman 1992, Ullman et al. 1993, Wijkamp et al. 1993). Movement of viral particles from the digestive system to the salivary glands was originally thought to occur through the hemocoel (Ullman et al. 1997). The hemocoel circulatory current was thought to transport the viral particles to the different organs, including the salivary glands. Nagata et al. (2002) proposed that the virus particle pathway from the midgut to the salivary glands might be through the thread-like ligaments connecting the anterior midgut with the salivary glands, rather than through the hemocoel. Recently, Moritz et al. (2004) theorized that the visceral midgut muscles are fused with salivary glands during the larval stages.

Adult thrips cannot transmit the virus if it was not acquired during the larval stages, even if allowed extensive feeding time (Pittman 1927, Sakimura 1963). Studies of

TSWV acquisition with F. occidentalis show that the midgut epithelial cells obstruct the passage of the viral particles to the hemocoel in adult thrips that have not acquired the

virus during the larval stage. The viral particles are retained and degraded in the midgut of the adult (Ullman et al. 1992). More recently, however, it has been proposed that it is

the apical plasmalemma in the midgut that acts as a barrier, rather than the epithelial cell

layer (Ullman 1997).

The two different methods by which thrips feed, a shallow type, which penetrates

the epidermis, and a deeper type, which penetrates the mesophyll, both result in

innoculation. The minimal amount of time needed for a first instars to acquire the disease while feeding has been documented at 15 minutes (Sakimura 1962b).

TSWV has not been shown to have adverse effects on thrips that have acquired the

virus through short inoculation access periods (6h). Experimental results have shown no

significant differences in developmental time, the number of offspring produced, and

survival rates between virus-infected and healthy F. occidentalis. Wijkamp et al. (1996)

also concluded that the virus is not transovarially passed from adult to offspring.

Efficiency in transmitting the virus has been shown to differ between different

populations of the same species. Van de Wetering et al. (1999b) investigated the

differing efficiencies of F. occidentalis populations originating from various locations

throughout the world to transmit the TSWV virus. Results of the experiment indicated

that transmission efficiency differed within populations, as well as between populations

from differing geographic regions. Chatzivassiliou et al. (2002) further revealed that

differing efficiencies of infectivity in T. tabaci populations are most likely due to

divergent host plant preferences for tobacco versus leek, as well as whether

parthenogenesis was thelytokous or arrhenotokous in nature. Early studies of the vector

efficiencies of pale and dark forms of F. occidentalis have shown that there is no

difference in the ability to transmit the disease between the two color forms (Sakimura

1962a).

Efficiency of TSWV transmission not only varies within thrips species, but also

varies with sex of the thrips species. Experiments have established that F. occidentalis

males transmit TSWV with a higher efficiency due to the different feeding methods of

males and females (van de Wetering et al. 1998, van de Wetering et al. 1999a). The sex

ratio of a thrips population is an important factor in evaluating the spread of TSWV in

crops.

Virus transmission by thrips is thought to be greatly assisted by the presence of weeds and natural vegetation surrounding susceptible crop systems. These weeds serve as reservoirs for the vectors and TSWV, as is the case for numerous other crop viruses

(Duffus 1971, Bos 1981). In a study of weed reservoir sources for TSWV in Hawaii, 44 species of natural vegetation, representing 16 families, were found to have a high incidence of TSWV. This is of particular importance because most of these plants are commonly found growing within vegetable farmlands (Cho et al. 1986). Researchers in

Canada found that 113 of the native plant species in southern Ontario were susceptible to

TSWV, and of those plant species, 86% were ovipository hosts for F. occidentalis

(Stobbs et al. 1992). Similar studies conducted in Australia have demonstrated

comparable results in correlating TSWV incidence with reservoir weeds and thrips

vectors (Latham and Jones 1997, Wilson 1998).

These virus reservoirs are of great significance for thrips vectors that overwinter in

sources of natural vegetation near agricultural areas. Perennial wild plants allow the virus

to persist between plantings of short-lived crops, as well as serve as refuge for

overwintering vector populations that may perpetuate the virus between agricultural

plantings (Bos 1981).

Populations of TSWV vectors, F. fusca (Chamberlin et al. 1992, Johnson et al.

1995, Groves et al. 2001) and F. occidentalis (Chamberlin et al. 1992, Toapanta et al.

1996) have been recorded on wild plant species during winter and spring. Fewer

numbers of thrips have been found diapausing in the soil than overwintering on wild

plants, suggesting that active populations overwintering and reproducing on winter host

plants might play a more significant role in TSWV spread than do diapausing thrips (Cho

et al. 1995, Groves et al. 2001).

Several aspects influence the relationship between the thrips vector and the TSWV

virus. Dispersal and transmission abilities of thrips populations, sources of virus

reservoirs, the association of the thrips vectors with its host plant, and the replicative

nature of the virus are all factors that contribute to the success of certain thrips species to

vector TSWV.

Research Objectives

Thrips pose a serious threat to agricultural crops worldwide due to the direct

feeding damage caused by them as well as their ability to transmit the Tomato Spotted

Wilt Virus. Identifying the species of thrips that serve as vectors of TSWV in a particular

crop, as well as which vector species are of primary concern in that crop, is critical for

understanding the epidemiology of the virus. Epidemics of TSWV occur in field pepper

in Florida, but the role of F. bispinosa in TSWV spread in pepper is not understood.

Insecticide resistance is widespread among thrips species and many insecticides are

known to be detrimental to beneficial insects. Consequently, pest management strategies

that focus on biological control employing natural enemies, rather than insecticide

treatments are essential for regulation of thrips species and as a result, regulation of the

TSWV virus.

One objective of this research is to confirm F. bispinosa as a vector of TSWV. In

particular, the vector competence of F. bispinosa will be evaluated against that of F.

occidentalis. TSWV acquisition will be evaluated for both species and pepper will be

evaluated as a true breeding host for F. bispinosa to further evaluate the specie’s ability to serve as a TSWV vector in pepper. Correspondingly, TSWV transmission by F. bispinosa will be compared with that of F. occidentalis in pepper. TSWV transmission will also be evaluated in the presence and absence of O. insidiosus to determine whether the predator has the ability to reduce primary spread of TSWV in pepper by predation upon thrips.

CHAPTER 2 ACQUISITION OF TOMATO SPOTTED WILT VIRUS AND REPRODUCTION ON PEPPER BY FRANKLINIELLA BISPINOSA, AS COMPARED TO THE KEY VECTOR, FRANKLINIELLA OCCIDENTALIS

Introduction

Thrips are of great economic concern in the agricultural community due to the

amount of crop damage they are capable of inflicting. Damage to crops results when the

adults and larvae of thrips feed on plant structures, such as leaves, petals, and fruit with their piercing-sucking mouthparts (reviewed in Childers 1997). The primary concern, however, lies with the ability of thrips to vector Tomato Spotted Wilt Virus (TSWV), a tospovirus transmitted through the saliva of the thrips during feeding (Hunter and Ullman

1992, Ullman et al. 1997). Tomato spotted wilt was first observed in 1915 in Australia and described as the “spotted wilt” of tomatoes (Brittlebank 1919) and was associated with transmission by thrips (Pittman 1927). The viral etiology was later reported by

Samuel et al. (1930).

TSWV is capable of causing extensive economic loss in agricultural crops. The virus has a broad host range, infecting 926 plant species, and causing an estimated crop

loss of one billion US dollars per year throughout its host range (reviewed in Prins and

Goldbach 1998). Tomato, tobacco, lettuce, pepper, papaya, eggplant, green beans,

artichokes, broad beans, celery and ornamental plants have experienced significant losses

due to the virus (reviewed in Rosello et al. 1996).

Ullman et al. (1997) described the virus-vector relationship and reviewed relevent

scientific literature. The larvae and the adults are the only stages involved in acquisition

22 23

and transmission. Both the first and second instars acquire TSWV. The prepupae and

pupae do not feed and neither acquire nor transmit the virus. The virus survives

moulting, pupation, and the replacement of tissues during the prepupal and pupal stages of thrips development. The adults that successfully acquire the virus as larvae are responsible for its transmission and spread.

Outbreaks of tomato spotted wilt are difficult to manage and only a few

preventative measures exist. Growing seedlings under cover, avoiding sequential plantings, weed management, controlling thrips populations and crop rotation with non- susceptible crops, and the use of metalized mulch are a few of the management techniques that can be employed (Kucharek et al. 1990, Rosello et al. 1996, Cho et al.

1998, Momol et al. 2004). Unfortunately, host plant resistance is not a viable management strategy as very few TSWV resistant hybrids have been developed; most of them are only resistant to certain strains of TSWV, while being susceptible to many others. Strains of TSWV have developed that can overcome resistance (reviewed in

Rosello et al. 1996). Regulation of viruliferous thrips has not been successful due to increasing resistance to broad-spectrum insecticides (Immajaru et al. 1992, Brodsgaard

1994). Moreover, primary spread of TSWV cannot be prevented by insecticide use because the adults successfully transmit the virus before death (Nagata et al. 1999).

The thrips species known to transmit TSWV are Thrips tabaci (Lindeman), Thrips

setosus (Moulton), Frankliniella occidentalis (Pergande), Frankliniella schultzei

(Trybom), Frankliniella fusca (Hinds), and Frankliniella intonsa (Trybom) (reviewed in

Sherwood et al. 2001). F. occidentalis is now considered the primary vector of TSWV

due to its increasingly global distribution and its ability to transmit new strains of TSWV

(Wijkamp et al. 1995a). Frankliniella bispinosa (Morgan) has been suspected as a vector of TSWV (Tsai et al. 1996, Webb et al. 1998), but has not yet been confirmed as a vector.

The species is distributed in parts of the southeastern US, Bermuda, and the Bahamas

(Nakahara 1997).

A common attribute shared by these thrips vectors is a strong propensity towards aggregating behavior (Lewis 1973). Mound and Teulon (1995) explain that aggregation results in full exploitation of a resource. Frankliniella spp., in particular, tend to aggregate in flowers (Yudin et al. 1986, Pickett et al. 1988, Yudin et al. 1988, Shipp and

Zariffa 1991), and both F. occidentalis and F. bispinosa have been observed in pepper flowers. This tendency to aggregate in flowers, as well as other population attributes such as polyphagy and high vagility aid in the ability of Frankliniella spp. to move successfully between cropping systems. High fecundity and short generation times allow for rapid development of large populations and production of large numbers of offspring, all of which can potentially acquire the virus in the larval stage. Many crop systems are ecologically isolated habitats that provide a new environment for rapid colonization of opportunistic species, thus furthering their ability to spread the TSWV.

The identification of thrips species that can vector TSWV provides insight into the epidemiology of the disease. Reproduction on a particular host and acquisition of virus from that host are two key factors that determine vector competence. Adults of many thrips species are found in large numbers in flowers of plants that are not reproductive hosts (Mound 1997). Adult F. bispinosa have been observed in pepper flowers along with the adults of other species (Reitz et al. 2003, Hansen et al. 2003), but it remains unclear whether this species is capable of completing its entire life cycle on pepper. The

“host plants” for thrips species cited in the literature are often only “finding places” and only inform us that a certain species can be found in abundance on that particular plant, but nothing else about the life cycle or biology of the thrips (Mound and Teulon 1999).

An experiment was conducted to assess pepper as a suitable reproductive host for F. bispinosa, as well as to determine the ability of F. bispinosa to acquire the virus on pepper compared to the key TSWV vector, F. occidentalis.

Materials and Methods

‘Camelot’ sweet pepper plants were transplanted into either 16 ×16 cm or 13.5 ×

10.5 cm pots containing 3B soil mixture (Fafard , Agawam, MA) and maintained under greenhouse conditions. Greenhouse plants were fertilized with Peat-Lite  special 15-

16-17 fertilizer (Scotts-Sierra Horticultural Products Company, Marysville, OH) and

Miracle-Gro  Bloom Booster 10-52-10 (Miracle-Gro, Marysville, OH). Virus acquisition experiments were conducted in growth rooms that maintained the temperature between 23◦ and 25◦ C and a 14-hr photoperiod.

Between 6 and 8 weeks of age, pepper plants were mechanically inoculated with a

TSWV source obtained from leaves of field peppers at the Quincy North Florida

Research and Education Center. The pepper plants showed symptoms of TSWV and were

later confirmed by ELISA. Peppers used in the virus acquisition study were inoculated

by grinding infected leaf material in sodium sulfite solution (0.26g Na2SO3/50 ml

deionized water) and lightly rubbing extracts on 3 to 4 leaves of each plant using

diatomaceous earth and cheesecloth.

After 7 to 10 days, TSWV infected plants were confirmed by the double antibody

sandwich (DAS) ELISA (Clark and Adams 1977). The ELISA assays were performed as

described by Gonsalves and Trujillo (1986), with the exception of using glass tubes and

6.6 mm polystyrene balls (Precision Plastic Ball Co., Chicago, IL) in place of a

microplate. The polystyrene balls were coated with purified antisera obtained from a

lettuce isolate of TSWV (Agdia Inc., Elkhart, IN). TSWV infected samples were

determined qualitatively by visual inspection of color change.

Individual TSWV infected pepper plants in 16 × 16 cm pots were enclosed in 35 ×

15 cm polyethylene tubing (n=23 and 25 for F. bispinosa and F. occidentalis respectively). The tubes were covered at the top with fine mesh to prevent thrips escape, and there were two 2.5 × 2.5 cm side openings covered with mesh. Ten adult females of

F. occidentalis or F. bispinosa were introduced per cage, and they were allowed to lay eggs for 6 days, after which time the adults were removed. The larvae were collected from the plant at days 6, 8 and 10 by visual inspection of the plants. The larvae were transferred to green bean pods. After developing to the adult stage, each was tested for presence of the virus using an indirect ELISA method that detects the presence of the nonstructural (NSs) protein encoded by TSWV small RNA (Bandla et al. 1994). The

NSs protein is present in thrips cells as a result of TSWV replication, demonstrating that the thrips have acquired the virus. This assay distinguishes between adult thrips that have acquired TSWV as larvae, in which the virus is replicating and transmissible to plants, and adult thrips that have fed on TSWV-infected tissue, but cannot transmit the virus.

The mean number of larvae of F. occidentalis and F. bispinosa recovered from each acquisition trial plant, as well as percent acquisition of TSWV by F. occidentalis and F. bispinosa, were compared using two-sample t-tests. A significance level of 0.05 was used for the survival and acquisition data.

Results

Significantly more F. occidentalis larvae were collected per infected plant than F.

bispinosa (t = -4.08, df = 58, P = 0.0001) (Fig 2-1). Pepper appears to be a more suitable

reproductive host for F. occidentalis than for F. bispinosa. Both species acquired the virus from infected pepper (Fig. 2). A significantly higher percentage of F. occidentalis acquired the virus than did F. bispinosa (t = -2.07, df = 53, P = 0.04).

Number of 30 larvae recovered per plant 20

F. bispinosa F. occidentalis

Figure 2-1. Mean number (+ SEM) of F. bispinosa versus F. occidentalis larvae collected per infected pepper plant.

Percent TSWV Acquisition 15 .

0 F. bispinosa F. occidentalis

Figure 2-2. Mean (+ SEM) percent acquisition of TSWV by F. occidentalis and F. bispinosa on pepper.

Discussion

Reproduction on a host plant, as well as virus acquisition and transmission of a virus on a host plant, determine vector competence. This study focused on two of these factors, reproduction and virus acquisition. The research confirms that F. bispinosa can successfully reproduce on pepper as well as acquire TSWV from pepper.

Previous research showed that pepper is a host for the adults of F. occidentalis and

F. bispinosa under field conditions in Florida. Thrips larvae were present in peppers in these studies, but the species of the larvae was not determined (Funderburk et al. 2000,

Ramachadran et al. 2001, Hansen et al. 2003, Reitz et al. 2003). The research presented here demonstrates the suitability of pepper as a reproductive host for F. bispinosa, but the number of larvae of F. occidentalis recovered, the key vector of TSWV, was 3.7-fold greater.

Reproduction on a host is a major factor governing vector competence in a particular crop. Greater offspring production results in a greater number of larvae that will acquire the virus as first instars and potentially develop into viruliferous adults.

Undoubtedly, a larger proportion of adults capable of transmitting the virus will develop on virus hosts that are most suitable as reproductive hosts for the vector (Peters et al.

1996).

Acquisiton of TSWV by ingestion of infected tissue must occur during the window of opportunity in the larval stage in which the visceral midgut muscles are fused with the salivary glands, allowing access of viral particles to the site of viral replication (Moritz et al. 2004). The presence of the NSs protein in adult thrips demonstrates that TSWV replication in the vector has occurred and viral acquisition is complete Bandla et al.

1994). These results show a significantly higher proportion of F. occidentalis versus F. bispinosa acquired the virus from pepper.

Differences in feeding preferences and host suitability between F. occidentalis and

F. bispinosa may result in varying abilities of each species to acquire TSWV depending on the host plant. Laboratory observations have revealed higher rates of acquisition of

TSWV for F. bispinosa than for F. occidentalis when the larvae fed on Datura stramonium (Webb et al. 1998). Thrips will ingest larger quantities of cytoplasm from plant cells when the plant is recognized as a more suitable host (Broadbent et al. 1987).

Larger amounts of plant tissue ingested by a thrips increases the likelihood of ingesting sufficient quantities of viral particles to allow for successful replication inside the thrips.

Ullman et al. (1992) correlated ingestion of higher titer of TSWV by F. occidentalis larvae with an increased production of viruliferous adults. The suitability of a host plant

is a key factor in determining which species plays a greater role in the spread of TSWV in field pepper.

Pepper supports higher population growth and higher rates of TSWV acquisition by

F. occidentalis than F. bispinosa, indicating that F. occidentalis may be a better vector

than F. bispinosa in pepper. The research herein has demonstrated a higher proportion of

TSWV acquisition by F. occidentalis, but does not account for ecological factors that

influence thrips populations in the field, such as parasitism and predation that may reduce

thrips populations, nor does it account for behavioral differences in the two thrips species

that might affect vector competence and the ability to colonize in the field. The

difference in mobility of the two species may have implications when assessing the

vector competence of these thrips species in a field situation.

Studies on mobility show that F. bispinosa are more vagile than F. occidentalis.

Ramachandran et al. (2001) looked at the mobility of F. bispinosa and F. occidentalis in

untreated peppers in a greenhouse study. Limited movement and lower colonization rates

by F. occidentalis was demonstrated. The males and females of F. bispinosa, however,

were much more mobile. Rates of acquisition were lower for F. bispinosa, but the

species moves between plants much quicker than F. occidentalis, suggesting the

probability of this species to come into contact with more plants to which it can

potentially transmit virus. Although TSWV acquisition by F. occidentalis was

significantly greater, the more vagile F. bispinosa may account for a greater proportion of

TSWV infection in field pepper based upon the interspecific variation in mobility of the

two species.

The greater mobility of F. bispinosa may also facilitate its escape from predation.

Orius insidiosus has been shown to be a successful predator of Frankliniella species,

reducing populations of thrips close to extinction in field pepper (Funderburk et al. 2000,

Ramachandran et al. 2001). Reitz et al. (2004) found O. insidiosus preys more

effectively on F. occidentalis than F. bispinosa in laboratory experiments in which both

species were present. F. bispinosa may be mobile enough to avoid predation by O.

insidiosus, suggesting that this TSWV vector may be more difficult to control under field

settings.

Correspondingly, it is believed that the reason insecticides toxic to F. bispinosa in laboratory assays have little effect under field conditions is the rapid colonization ability of the species (Eger et al. 1998, Funderburk et al. 2000). The more active F. bispinosa

may be more significant as a TSWV vector based upon its ability to avoid predation by

O. insidiosus and recolonize crops after insecticide application faster than F. occidentalis.

Populations of F. bispinosa have been observed to decline long after those of F.

occidentalis in North Florida (Chellemi et al. 1994, Funderburk et al. 2000,

Ramachandran et al. 2001).

Abundance of F. bispinosa and F. occidentalis in certain geographical regions should also be considered when assessing these species as a potential threat to field pepper in Florida. Hansen et al. (2003) found F. bispinosa to be more common than F. occidentalis in central Florida, whereas F. bispinosa was the dominant species in North

Florida. F. bispinosa may be more of a concern as a vector of TSWV than F.

occidentalis in central Florida, whereas F. occidentalis may be more of a threat to agriculture in North Florida.

Evidence has been provided here that confirms preliminary reports of F. bispinosa as a vector of TSWV (Webb et al. 1998). Reproduction and TSWV acquisition in pepper by F. bispinosa have been demonstrated. Lower rates of larval production, as well as lower rates of virus acquisition, by F. bispinosa as compared with the key vector F. occidentalis indicate that F. occidentalis may be the more efficient vector in pepper at the individual level, however species-specific attributes may play a role in the ability of both vectors to vector TSWV under field conditions at the population level. Variation in behavior and mobility in F. bispinosa and F. occidentalis should be considered when assessing the vector competence of both species in field pepper. Rapid colonization by F. bispinosa may play a significant role in the species ability to vector TSWV in field pepper, particularly in central Florida where F. bispinosa is the predominant thrips species. Epidemics of TSWV occur in field pepper in Florida (Gitaitis et al. 1998), but the role of each species in disease epidemiology under field conditions is not understood.

CHAPTER 3 TRANSMISSION OF TOMATO SPOTTED WILT VIRUS BY FRANKLINIELLA BISPINOSA VERSUS FRANKLINIELLA OCCIDENTALIS IN PEPPER, AS INFLUENCED BY PREDATION OF ORIUS INSIDIOSUS

Introduction

Vast numbers of thrips are capable of arriving at a crop and breeding rapidly, especially at high temperatures (Kirk 1997a). The opportunistic strategy of thrips arises from their ancestral beginnings as fungal detritovores who survived in short-lived optimal conditions (Mound 1997). Agricultural crops provide the optimal conditions for rapid estaplishment and breeding of many pest species of thrips.

Thrips populations can grow steadily despite regular applications of toxic insecticides (Nagai 1990, Funderburk et al. 2000). Resistance to broad-spectrum insecticides by F. occidentalis (Immajaru et al. 1992) fosters the need for alternative

control measures.

Recent research has demonstrated the ability of anthocorid predators to successfully

regulate populations of pest thrips (Van den Meiracker and Ramakers 1991, Funderburk

et al. 2000). The anthocorids are predatory insects with a worldwide distribution

(Blatchley 1926). These heteropteran predators mostly feed on aphids, mites, thrips,

scales, other arthropods, the eggs and larvae of lepidoptera, and occasionally pollen and

other plant material (Kelton 1978).

Predation upon thrips by Orius spp. has been documented throughout North

America, Asia, Australia and Europe. (Gilkeson et al. 1990, Nagai 1990, van den

Meiracker and Ramakers 1991, Cook et al. 1996). Hansen et al. (2003) found that

33 34

populations of O. insidiosus were aggregated with their thrips prey, F. occidentalis, F.

bispinosa, and F. tritici, on flowers, rather than on leaves and buds of field peppers.

Similar concentrations of O. insidiosus and Frankliniella spp. on flowers have been reported in field and greenhouse peppers (Shipp et al. 1992, Tavella et al. 1996).

O. insidiosus has the ability to suppress adults and larvae of F. occidentalis in field pepper, leading to a decline of the population towards extinction during the period of highest population abundance in pepper for F. occidentalis. The reproductive vigor of F. occidentalis can overpower the suppressive effects of insecticide applications, but not the ability of O. insidiosus to reduce its populations (Funderburk et al. 2000). A predator:prey ratio of 1:40 in untreated field pepper reduces F. occidentalis populations close to extinction (Funderburk et al. 2000, Ramachandran et al. 2001).

Research has been conducted to elucidate the means by which Orius spp. are such highly effective predators. Similar aggregation patterns of O. insidiosus and its thrips prey suggest there are cues that alert O. insidiosus to the presence of thrips. Teerling et al. (1993a) reported that O. tristicolor uses the alarm pheromone produced by second instars of F. occidentalis to find its thrips prey. Correspondingly, Venzon et al. (1999) showed that O. laevigatus prefers plants infested with spider mites and thrips over uninfested plants. Olfactometer experiments performed in their study show that O. laevigatus use odors to locate plants with spider mites. O. insidiosus had previously been reported to respond to volatiles emitted from corn silk when corn is the prey's host plant

(Reid and Lampman 1989).

Anthocorid predators play an important role in regulating pest thrips populations in agricultural crops (Symondson et al. 2002). O. insidiosus can successfully be used as a

35 beneficial insect to reduce pest populations unaided by insecticides (Funderburk et al.

2000). Llittle is known, though, about the capacity of O. insidiosus to control primary and secondary spread of thrips vectored tospoviruses. The TSWV transmission trials described in this chapter were designed to compare percent transmission between F. occidentalis and F. bispinosa in the absence of the predator, as well as to evaluate the ability of O. insidiosus to reduce spread of TSWV by predation upon thrips.

Materials and Methods

‘Camelot’ sweet pepper plants were transplanted into 16 x 16 cm or 13.5 x 10.5 cm pots containing 3B soil mixture (Fafard ©, Agawam, MA) and maintained under greenhouse conditions. Greenhouse plants were fertilized bi-weekly with Peat-Lite © special 15-16-17 fertilizer (Scotts-Sierra Horticultural Products Company, Marysville,

OH) and Miracle-Gro Bloom Booster © 10-52-10 (Miracle-Gro, Marysville, OH).

TSWV transmission experiments were conducted in growth rooms that maintained a temperature between 23◦ and 25◦ C and the photoperiod at 14:10 [L:D].

To generate the viruliferous thrips used in the TSWV transmission trials, approximately fifty first instars obtained from virus-free colonies of both species reared on green bean pods were moved individually by paintbrush onto TSWV-infected tomato fruit in thrips-proof containers and reared to the adult stage. The techniques used to rear

TSWV-infected thrips produced more F. occidentalis than F. bispinosa, resulting in the completion of fewer TSWV transmission trials with F. bispinosa. The TSWV infected F. occidentalis colonies, as well as the healthy colonies, were much more prolific than the

F. bispinosa colonies. F. bispinosa generally took 2-3 days longer than F. occidentalis to develop into adults.

Adult thrips were sampled for TSWV using an indirect ELISA method that

detected the nonstructural protein encoded by TSWV S RNA (Bandla et al. 1994). Six of

11 F. occidentalis sampled from the TSWV-infected colonies tested positive for TSWV.

Due to the limited number of F. bispinosa produced for the TSWV transmission trials, only three were tested for TSWV. None of the F. bispinosa tested positive for TSWV.

Four uninfected pepper plants in 13.5 cm × 10.5 cm pots were placed peripherally around a center uninfected release plant in a 55 cm × 30 cm × 48 cm polyethylene cage vented with three 15 cm × 15 cm mesh openings. Flowering pepper plants of 6 to 8 weeks of age were used because F. bispinosa and F. occidentalis do not infest field peppers until flowering and then they aggregate in the flowers (Hansen et al. 2003).

Twenty putatively viruliferous thrips of both species were released on the center plants of the test cages. Additional trials were conducted for each thrips species with the addition of one adult O. insidiosus per cage. The predator was released on the center plant one hour after the thrips were released. The length of each TSWV transmission trial was three weeks.

The four peripherally placed pepper plants were tested for TSWV using ELISA once a week for three weeks. The center release plant was not tested for TSWV infection. TSWV infected plants were confirmed by the double antibody sandwich

(DAS) ELISA (Clark and Adams 1977). The ELISA procedures were completed as described by Gonsalves and Trujillo (1986), with the exception of using glass tubes and

6.6 mm polystyrene balls (Precision Plastic Ball Co., Chicago, IL) in place of a microplate. TSWV positive and negative samples were detected qualitatively by visual inspection. A TSWV trial with three consecutive weeks of TSWV testing on the pepper

37 plants by DAS ELISA was considered a successful completion. Nineteen trials introducing putatively TSWV infected F. occidentalis were successfully completed.

Eight of these trials were conducted with one O. insidiosus introduced per cage and the remaining eleven trials were conducted without introduction of O. insidiosus. Eight trials introducing F. bispinosa were completed. In three of these trials one O. insidiosus was introduced and in five trials no O. insidiosus were introduced.

Results

TSWV transmission by F. occidentalis occurred in three trials in which a predator was present. In two of those trials, all four pepper plants were infected with TSWV and the remaining trial produced one TSWV-infected plant. TSWV transmission by F. occidentalis also occurred in three trials without the predator (Table 3-1). Two of those trials produced two TSWV-infected plants, while the remaining trial produced one infected plant. These experiments confirm that F. occidentalis can transmit TSWV from tomato to pepper. There appears to be no difference in TSWV transmission by F. occidentalis between the trials that introduced the predator and those that did not.

TSWV transmission occurred in two of the F. bispinosa trials in which O. insidiosus was present. In both of those trials only one plant was infected with TSWV.

TSWV transmission also occurred in two trials in which O. insidiosus was absent (Table

3-1). One trial produced three TSWV infected plants and the other trial produced four infected plants. These data confirm that F. bispinosa can transmit TSWV. There appears to be no difference in TSWV transmission by F. bispinosa between trials that introduced the predator and those that did not.

Table 3-1. Transmission of TSWV to healthy pepper plants by two species of thrips in the presence and absence of the predator, Orius insidiosus. Presence of TSWV No Thrips Species O. insidiosus Trials completed Transmission Transmission Present 8 3 5 F. occidentalis Absent 11 3 8 Present 3 2 1 F. bispinosa Absent 5 2 3

Discussion

TSWV transmission to a healthy plant occurs during feeding by adult viruliferous

thrips that have acquired the virus as larvae, resulting in a systemic infection of the

inoculated plant (Ullman et al. 1997). There are a multitude of factors that can affect the

ability of viruliferous thrips to transmit virus. Sakimura (1963) originally reported an

irregular retention of the virus by TSWV-infected thrips. Sakimura’s research showed

that the latent period of F. occidentalis can range from 4-18 days and the virus retention

period can range between 2-30 days. Some thrips retained the replicative form of the

virus for life, while others were infective for only a short period of time. Only half of the

thrips tested remained infective until death. It appears that although the virus is

persistent, the ability of a viruliferous thrips to be infective is sometimes sporadic.

Sakimura (1963) speculated that this erratic infectivity might be caused by varying

amounts of virus ingested by larval thrips. More recent laboratory investigations have

shown that many TSWV infected thrips do not efficiently inoculate plants (Cho et al.

1991, Ullman et al. 1992a).

Differing distribution of virions in the host plant has been suggested to have an

effect on ingestion by larvae (Ullman et al. 1992a). The viruliferous colonies used in the

transmission trials were reared on infected tomato fruit rather than infected leaf tissue in order to reduce handling of the larvae. Transferring larvae from infected leaf tissue to

green bean pods often resulted in decreased survival. The infected tomato fruit was able to sustain the larvae until the adult stage. It is unknown whether thrips that have fed on fruit tissue or leaf tissue of pepper are exposed to differing virus titers.

Differing concentrations of virus titer may exist in leaf versus fruit tissue. This might have an effect on acquisition and retention of viral particles by the thrips, thus affecting the ability to transmit TSWV. Moreover, thrips have a very small window of opportunity to become viruliferous (Peters et al. 1996). Not all first instars will ingest the virus. Of the larvae that do ingest the virus, the virus may not replicate in all of them. As demonstrated by the sporadic infectivity of some thrips, even if the virus is replicating in the thrips host, this will not necessarily lead to that thrips being able to transmit the virus

(Sakimura 1963, Cho et al. 1991, Ullman et al. 1992a).

I estimate that approximately 50% of the F. occidentalis used in the TSWV transmission trials were virus-infected. Fifteen to twenty F. occidentalis were released in each trial to ensure that there would be TSWV-infected thrips in every trial, yet virus transmission did not occur in all trials.

The percentage of TSWV transmission was similar whether O. insidiosus was present or not. TSWV transmission occurred in 3 out of 8 trials with the predator present

and 3 out of 11 trials with the predator absent. Because virus transmission by F.

occidentalis in the presence of O. insidiosus was limited and the same amount of virus transmission occurred in the trials in which O. insidiosus was present, no conclusions can be made with respect to the influence of O. insidiosus on virus transmission.

F. occidentalis produces an alarm pheromone, which can be triggered by the presence of an anthocorid predator (Teerling et al 1993a). Teerling et al. (1993b)

40 identified a two-component alarm pheromone consisting of decyl acetate and dodecyl acetate in a molar ratio of 1.5:1. The study found that F. occidentalis did not tend to move far in response to the pheromone. Each developmental stage tested moved approximately 9.5 mm from the treatment secretions. Nymphal dropping and reduced oviposition by adults were also documented responses to contact with the pheromone. It is possible that the presence of O. insidiosus caused alarm pheromone release in F. occidentalis, which might have triggered faster dispersal of thrips to the other plants.

This is a plausible explanation for the virus transmission that occurred in the experiments in which O. insidiosus was present.

Correspondingly, recent research has shown that F. occidentalis exhibits avoidance behavior in response to the odors given off by anthocorid predators. F. occidentalis larvae seek refuge in spider mite webbing, where predation rates by Orius spp. are reduced, after encounters with a predator fed on a diet of thrips prey. F. occidentalis reacts more readily when it encounters O. laevigatus that fed on thrips prey versus non- thrips prey (Venzon et al. 2000). The chemical that elicited refuge seeking behavior in F. occidentalis was thought to be composed of thrips alarm pheromone. Baez et al. (2004) also observed that in the presence of O. insidiosus, F. occidentalis is more likely to disperse from flowers, suggesting that F. occidentalis is able to detect the predator.

It is also possible, yet not as likely, that F. occidentalis did not disperse from the center release plant, resulting in limited virus transmission in many of the peripheral plants in the TSWV transmission experiments. The center release plant was not tested for TSWV infection because one of the objectives of the experiments was to evaluate dispersal by thrips and the corresponding spread of TSWV. The failure of the peripheral

plants in some of the virus transmission trials to become infected might be due to a

failure of the thrips to disperse from the center release plant in those experiments. The

location of the released thrips was not tracked during the TSWV experiments.

In the future, the TSWV transmission experiments should be simplified in order to

demonstrate virus transmission by thrips. Any components not essential for

demonstrating virus transmission, such as dispersal ability of thrips and the predator

aspect, should be taken out. Transmission can be demonstrated on leaf discs rather than a

whole plant. A leaf disc assay using a petunia hybrid has been developed and used

successfully to document transmission of tospoviruses by thrips (Wijkamp and Peters

1993, Wijkamp et al. 1995). Petunia X hybrida ‘Blue Magic’ produces a local lesion

response to tospoviruses consisting of brown or black lesions that develop within two to

three days. A leaf disc assay using a local lesion response rather than a whole plant

producing systemic infection would greatly simplify the experiment. When using the leaf disc assay, it would be easier to keep track of individual thrips and ensure that they were

feeding and not falling to the ground. A single infected thrips could be placed on a leaf

disc of healthy tissue and allowed to feed for 24 h. The appearance of local lesions on a

leaf disc represents virus transmission. Virus transmission efficiency of F. occidentalis

and F. bispinosa could be expressed as the number of adults that transmitted the virus to

healthy leaf discs.

A whole plant TSWV transmission trial could be conducted as well. Viruliferous

thrips could be released onto a caged pepper plant and allowed to feed for 24 h. Adult

thrips could be removed from the plants 24 h after feeding by application of imidacloprid.

The plant would then be tested by DAS ELISA 7-14 d after the inoculation access period

during which the thrips fed. With this experimental design, the number of thrips released

per cage, as well as the inoculation access period, could be varied. A predator can also be released in the whole plant TSWV transmission experiment. A leaf disc assay can provide useful information on rates of TSWV transmission by thrips, whereas a whole plant system in which a predator is introduced can provide a more realistic evaluation of

TSWV transmission by thrips in a field situation.

The TSWV transmission study has demonstrated that both F. occidentalis and F.

bispinosa are capable of transmitting the virus to pepper once acquired in the larval stage.

This is the first time transmission of TSWV to pepper by F. bispinosa has been demonstrated. The effect of O. insidiosus on transmission of TSWV in pepper by F.

bispinosa and F. occidentalis is not understood.

CHAPTER 4 POPULATION ABUNDANCE OF FRANKLINIELLA BISPINOSA AND FRANKLINIELLA OCCIDENTALIS IN FIELD PEPPER

Introduction

Frankliniella spp. are the most abundant species of pest thrips in Florida agricultural crops and surrounding wild host plant communities (Salguero-Navas et al.

1991, Chellemi et al. 1994, Puche et al. 1995, Toapanta et al. 1996). Frankliniella bispinosa has been confirmed as a vector of TSWV. The research presented in this thesis shows that F. bispinosa can reproduce (Chapter 2), as well as, acquire and transmit

TSWV in pepper (Chapter 3). Determining the population abundance of TSWV vectors, in particular, F. bispinosa and F. occidentalis, in field pepper can provide insight into the epidemiology of TSWV in pepper.

In North Florida, where this study was conducted, populations of F. occidentalis and F. bispinosa are low during the winter and increase rapidly during early spring

(Toapanta et al. 1996). The highest abundance of Frankliniella spp. occurs in May.

During this time, many of the wild plant species that can serve as reservoirs for TSWV infection and thrips vectors are flowering. Populations of Frankliniella spp. tend to decline in June in northern Florida (Chellemi et al.1994).

Seasonal population abundance of F. occidentalis and F. bispinosa may influence trends in TSWV incidence in pepper. Toapanta et al. (1996) noted that F. occidentalis is usually the predominant species until April, when F. bispinosa populations increase and become the dominant thrips species. TSWV incidence early in the growing season may

43 44

be attributed to F. occidentalis, whereas F. bispinosa most likely accounts for TSWV

spread during mid to late summer in pepper in North Florida. Seasonal patterns of F.

occidentalis and F. bispinosa do exist, but proportions of Frankliniella spp. in field

pepper have been observed to change from one season to the next (Reitz et al. 2003).

TSWV epidemics occur only through inoculation by primary vectors that colonize

crops. Determining population abundance of important vector species may help predict

epidemics of TSWV in field pepper. A field experiment was conducted at the University

of Florida, North Florida Research and Education Center in Quincy, Florida to determine

population abundance of F. occidentalis and F. bispinosa in pepper during the 2002 growing season to determine the significance each vector might have in the spread of

TSWV in pepper.

Materials and Methods

Four cohorts of ‘Camelot’ sweet peppers (1 Cohort = 40) in 16 cm X 16 cm pots were transplanted into the field upon flowering. The experiment was a random design, with each cohort randomly placed within a plot of 600 peppers of the same age as the cohorts. The plot consisted of seven raised beds 18 m long and 0.9 m apart with 30 cm spacing within the rows. Each bed was covered with black plastic mulch and irrigation water was supplied through a trickle tube placed in the center of each bed.

The first cohort was transplanted into the field on 13 May 2002. After one week, forty flowers were randomly collected and preserved in 70% ethyl alcohol. This procedure was repeated with the remaining cohorts, with the second cohort transplanted on 20 May 2002, the third cohort transplanted 27 May 2002, and the fourth cohort transplanted on 3 June 2002.

The flower samples from the cohorts were sampled for F. bispinosa and F. occidentalis under a stereo microscope at 40X. Identification of adults was based on

setation of the head and prothorax, as well as presence of antennal spines (Mound and

Kibby 1998). Larval thrips were not identified to species because larval thrips have little effect on primary infection. There is also very limited available information on the diagnostics of larval thrips. The mean number of F. bispinosa and F. occidentalis per plant sample was compared using a paired difference t-test.

Results

Individual sampling dates were tested seperately. There were significantly more F.

bispinosa than F. occidentalis on 20 May 2002 (t = 4.72, df = 19, P = 0.0001), 27 May

2002 (t = 3.32, df = 19, P = 0.0036), 3 June 2002 (t = 3.38, df = 19, P = 0.0032), and on

10 June 2002 (t = 3.47, df = 19, P = 0.0026) (Figure 4 -1).

6 5 4 Number of F. bispinosa Thrips 3 F. occidentalis 2 1 0 20 May 2002 27 May 2002 3 June 2002 10 June 2002

time in which the population samples were collected. Reitz et al. (2003) also observed low TSWV incidence in field pepper in North Florida during the 2000 and 2001 growing seasons. F. bispinosa was the dominant thrips vector in field pepper between 20 May

2002 and 10 June 2002. F. bispinosa is often a dominant thrips species after April in

North Florida, when populations of F. occidentalis tend to decline (Toapanta et al. 1996).

The role of each species in the epidemiology of the TSWV remains unknown, but the relative abundance of each species in the field may provide insight as to which species poses a more significant threat to field pepper in North Florida. Laboratory assays conducted as part of this research have demonstrated that F. occidentalis is more efficient in acquiring TSWV, however, F. bispinosa may account for more TSWV spread in the field based on the higher proportion of F. bispinosa found in field pepper in early to mid summer.

Population abundance of thrips vectors may provide some insight into the epidemiology of TSWV in field pepper, but it does not provide information on where those primary thrips populations may have originated. The amount of inoculum present in the alternate cropping system or wild host plants from which the thrips migrated remains unknown. It is possible that the original host plants harbored very little TSWV infection, resulting in low incidence of primary infection when the thrips dispersed to field pepper.

Further insight on populations of TSWV-infected thrips can be provided by conducting serological assays on field-collected thrips. Bandla et al. (1994) developed an

ELISA method for detecting the presence of the nonstructural (NSs) protein in thrips. The

NSs protein is only present in thrips when the virus has replicated in the vector, demonstrating that the thrips is able to transmit the virus to healthy plants. If performed on field-collected thrips, this technique would provide a greater understanding of how thrips vectors spread TSWV.

The population abundance of F. bispinosa and F. occidentalis does provide some insight into the epidemiology of TSWV in field pepper, but identifying proportions of those vector populations that are viruliferous would facilitate the prediction of TSWV epidemics in field pepper.

CHAPTER 5 CONCLUSIONS

The research reported herein focused on the assessment of Frankliniella bispinosa as a vector of the tomato spotted wilt tospovirus in pepper. Vector competence was defined as the innate ability of an insect to acquire and transmit a virus from a particular host plant, as well as reproduce on that host. Pepper was evaluated as a true breeding host for F. bispinosa. F. bispinosa produced fewer larvae per pepper plant than did F. occidentalis. Evidence has been provided that confirms F. bispinosa vectors TSWV.

Laboratory assays demonstrated that F. bispinosa does not acquire the virus from pepper as efficiently as does the key TSWV vector, F. occidentalis, but interspecific variations in movement and dispersal, should be considered when assessing the significance of both of these species to the epidemiology of TSWV in field pepper.

F. occidentalis and F. bispinosa transmit the virus to pepper. No comparisons could be evaluated on the efficiency of TSWV transmission in pepper by the two species.

No conclusions were reached about the ability of O. insidiosus to reduce TSWV spread by suppressing populations of F. occidentalis or F. bispinosa. It is possible that O. insidiosus might trigger faster dispersal by thrips vectors, thus contributing to more rapid spread of the virus in the field. Further research is necessary to investigate this issue.

There were significantly more F. bispinosa than F. occidentalis collected in field pepper during summer 2002. Although F. bispinosa acquires TSWV less efficiently in pepper than does F. occidentalis, F. bispinosa may account for more TSWV spread in pepper during the mid to late summer due to its larger population size and behavior.

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BIOGRAPHICAL SKETCH

Yolanda Avila was born on December 4, 1977, to Francisco and Susan Avila in

Long Island, New York. She received a Bachelor of Science degree in Environmental

Resource Management from the Pennsylvania State University in 2001. As an undergraduate, she worked as a research assistant for an Integrated Pest Management

Program, working with the Colorado potato beetle, European Cornborer and Fall

Armyworm. She also volunteered on an Earthwatch Expedition to the Brazilian Amazon where she assisted with research on dung beetles as seed dispersers in tropical rain forests. She has been working on a concurrent Master of Science program in the

Entomology and Nematology department and the Plant Pathology department under the guidance of Dr. Joseph Funderburk and Dr. Timur Momol. During her plant pathology studies she has been working as a research assistant in a University of Florida Plant

Virology laboratory. She will graduate with both degrees in December 2004.