Assessment of Dicyphus Hesperus (Knight) for the Biological Control of Bemisia Tabaci (Gennadius) in Greenhouse Tomato

ASSESSMENT OF DICYPHUS HESPERUS (KNIGHT) FOR THE BIOLOGICAL CONTROL OF BEMISIA TABACI (GENNADIUS) IN GREENHOUSE TOMATO

PRITIKA PANDEY

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

2018

To my family

ACKNOWLEDGMENTS

I am thankful to all the people who made my master’s study possible and supported me throughout the time. At first, I would like to thank my major advisor, Dr.

Hugh A. Smith for his guidance, support and encouragement throughout my entire graduate school period. Thank you for trusting me and providing me this great opportunity. I would also like to thank my co-advisor Dr. Heather J. McAuslane for her continuous guidance and suggestions. My sincere appreciation to Curtis Nagle, Laurie

Chamber and Justin Carter of the Entomology lab, GCREC for helping me out in my research and arranging all the materials for the research. I would like to thank my parents, Mr. Keshav Dutt Pandey and Mrs. Chhaya Pandey who always encouraged and supported me throughout my studies.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

ABSTRACT ...... 10

CHAPTER

1 INTRODUCTION TO BEMISIA TABACI ...... 12

Introduction ...... 12 Feeding and Damage ...... 14 Life Cycle ...... 15 Management of Bemisia tabaci ...... 16 Chemical Control ...... 16 Cultural Methods ...... 17 Host Plant Resistance ...... 17 Biological Control ...... 18 Dicyphus hesperus ...... 20 Biology ...... 21 Use of D. hesperus for Management of Insect Pests in Greenhouses ...... 22 Challenges to Managing Tomato Pests in Protected and Organic Agriculture ...... 23 Objectives ...... 23

2 EVALUATING THE PREDATORY CAPACITY OF DICYPHUS HESPERUS ON BEMISIA TABACI ...... 25

Introduction ...... 25 Methods and Materials...... 27 Plants and Insects ...... 27 Estimating the Consumption Rate of Bemisia tabaci Eggs by Female Dicyphus hesperus from Two Different Sources ...... 29 Distinguishing Mated and Unmated Females for Predation Study ...... 29 Estimating the Consumption Rate of Bemisia tabaci Eggs by Female Dicyphus hesperus ...... 29 Estimating the Consumption Rate of Bemisia tabaci Nymphs by Female Dicyphus hesperus ...... 31 Predation Behavior of Dicyphus hesperus on Different Life Stages of Bemisia tabaci ...... 32 Statistical Analysis ...... 33 Results ...... 34 Predation of Bemisia tabaci Eggs by Dicyphus hesperus from Two Sources... 34

Predation of Bemisia tabaci Eggs and Nymphs by Mated and Unmated Female Dicyphus hesperus ...... 35 Discussion ...... 36

3 ESTABLISHMENT OF DICYPHUS HESPERUS ON MULLEIN AND TOMATO UNDER CONTROLLED AND GREENHOUSE CONDITIONS ...... 56

Introduction ...... 56 Materials and Methods...... 60 Plants and Insects ...... 60 Life Cycle of Dicyphus hesperus on Tomato and Mullein With and Without Ephestia eggs Under Growth Room ...... 61 Population Development of Dicyphus hesperus on Mullein Plants With Ephestia Eggs Under Greenhouse Conditions ...... 62 Trial at Commercial Greenhouse, Wimauma ...... 62 Trial at GCREC Greenhouse ...... 63 Population Development of Dicyphus hesperus on Mullein With and Without Ephestia eggs and on Tomato With Ephestia Eggs Under Greenhouse Conditions in Gainesville ...... 63 Statistical Analysis ...... 64 Results ...... 64 Life Cycle of Dicyphus hesperus on Tomato and on Mullein With and Without Ephestia eggs Under Controlled Conditions ...... 64 Population Development of D. Hesperus on Mullein Plants With Ephestia Eggs Under Greenhouse Conditions ...... 65 Population Development of Dicyphus Hesperus on Mullein With And Without Ephestia Eggs and on Tomato With Ephestia Eggs Under Greenhouse Conditions ...... 67 Discussion ...... 68

4 SUMMARY ...... 86

LIST OF REFERENCES ...... 88

BIOGRAPHICAL SKETCH ...... 98

LIST OF TABLES

Table page

3-1 Different experiments conducted to evaluate the development of D. hesperus populations under greenhouse conditions...... 72

LIST OF FIGURES

Figure page

2-1 Dicyphus hesperus from Beneficial Insectary company (Redding, CA) ...... 39

2-2 Colony of D. hesperus maintained on mullein plants in growth room...... 40

2-3 Provision of D. hesperus with approximately 0.25 g Ephestia kuehniella ...... 41

2-4 Young female D. hesperus (2-3 d) with green abdomen ...... 42

2-5 Tomato plant with clip cages holding 7-10 pairs of whiteflies...... 43

2-6 Experiment set up in the growth room ...... 44

2-7 Damage to whitefly eggs (A) and nymphs (B) by the predator D. hesperus...... 45

2-8 Survival percentage of female D. hesperus...... 46

2-9 Average number of B. tabaci eggs and nymphs...... 47

2-10 Scatter plot of the number of B. tabaci eggs...... 48

2-11 Number of 24-h periods where consumption of whitefly eggs on tomato...... 49

2-12 Scatter plot of the number of B. tabaci nymphs provided on tomato leaves ...... 50

2-13 Number of 24-h periods where consumption of whitefly nymphs on tomato leaves by D. hesperus females ...... 51

2-14 Survival percentage of mated and unmated female D. hesperus...... 52

2-15 Patterns of damaged...... 53

2-16 Average number of B. tabaci eggs, earlier nymphs and later nymphs ...... 54

2-17 Survival percentage of female D. hesperus ...... 55

3-1 Rearing of D. hesperus on mullein plants ...... 73

3-2 Cage set-up for test of the effect of host plant and supplemental food ...... 74

3-3 First instar D. hesperus was transferred to translucent round plastic box set up development test...... 75

3-4 Sizes of different life stages of D. hesperus...... 76

3-5 Experiment set up with 54 bags of mullien plant...... 77

3-6 Survival of D. hesperus was evaluated on mullein plant...... 78

3-7 Evaluation of best dietary source for the development of D. hesperus...... 79

3-8 Average number of days spent in each instar and total nymphal development time of D. hesperus...... 80

3-9 Adult longevity of D. hesperus...... 81

3-10 Average, minimum and maximum temperature ...... 82

3-11 Average number of different instars and total number of D. hesperus...... 83

3-12 Average number of different instars and total number of D. hesperus...... 84

3-13 Average number of D. hesperus per sample on each treatment ...... 85

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

ASSESSMENT OF DICYPHUS HESPERUS (KNIGHT) FOR THE BIOLOGICAL CONTROL OF BEMISIA TABACI (GENNADIUS) IN GREENHOUSE TOMATO

Pritika Pandey

August 2018

Chair: Hugh A. Smith Cochair: Heather J. McAuslane Major: Entomology and Nematology

Florida produces about 34% of the fresh market tomatoes grown in the United

States. Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is one of the most devastating pests of tomato globally. Dicyphus hesperus Knight (Hemiptera: Miridae) has been evaluated for control of greenhouse whitefly Trialeurodes vaporariorum

Westwood in temperate environments and has potential as a biocontrol agent for B. tabaci. The major objective of this research is to assess the predatory potential of D. hesperus on B. tabaci under Florida’s subtropical condition. An experiment was designed to determine the average number of sweetpotato whitefly eggs or nymphs consumed by female D. hesperus adults per day and during her lifetime on tomato plants. Dicyphus hesperus from a colony and a commercial source, Beneficial Insectary, showed 7% reduction on B. tabaci eggs population. Mated and unmated female D. hesperus did not show any significant difference in their consumption rate when fed on

B. tabaci eggs and nymphs. Average number of eggs consumed by D. hesperus was 10 per day and 83 over their lifetime, with adult survival of 15 days whereas average nymphs consumed was 93 per day and 835 over their lifetime with survival of 25 days.

The average consumption rate of D. hesperus on sweetpotato whitefly eggs, early instar nymphs (1st and 2nd) and later nymphs differed significantly with consumption higher on earlier and later nymphs than on eggs. In addition, we studied the development of D. hesperus on mullein (Verbascum thapsus) and tomato with and without Ephestia kuehniella Zeller eggs as a dietary supplement. The average time for nymphal development on tomato was 24.5 days, on mullein with E. kuehniella was 22.5 days, and on mullein without E. kuehniella was 25.8 days. The adult longevity and survival percentage of D. hesperus was 30.36 d and 91.96% on mullein with Ephestia kuehniella eggs, 22.44 d and 75% on mullein without E. kuehniella eggs and 26.86 d and 87.5% on tomato with Ephestia kuehniella eggs. In greenhouse experiments, the population of

D. hesperus showed a greater increase on tomato provided with E. kuehniella eggs as a supplement prey source than on mullein with supplemental prey.

CHAPTER 1 INTRODUCTION TO BEMISIA TABACI

Introduction

The United States is one of the leading producers of fresh market tomatoes in the world with annual production of about 1.14 million metric tons. Florida contributes about 38% of total tomato production in the United States with recent annual revenue averaging $365,774,000 from 2015-2018 (USDA 2018). In 2017, about 29,000 acres of tomato were planted in Florida with production of about 426739.7 metric tons (USDA

2018). Insect pests are a major problem in Florida. Among them, Bemisia tabaci

Gennadius MEAM1, the sweetpotato whitefly, is the most devastating.

Bemisia tabaci is one of the most serious pests of agriculture globally (Thompson

2011). It causes devastating economic losses to agriculture all over the world, primarily in tropical and sub-tropical regions, but also in protected agriculture in temperate regions (Oliveira et al. 2001). It is recorded as having about 600 different host plant species (Mound and Halsey 1978, Secker et al. 1998). Most host plants belong to the families Fabaceae, Asteraceae, Malvaceae, Solanaceae and Euphorbiaceae (Mound and Halsey 1978). Some vegetable crops produced in greenhouses, including tomato, pepper, beans, eggplant and cucumber are attacked by B. tabaci (Cock et al. 1986). It is now globally distributed on all continents except Antarctica (Martin 1999, Martin et al.

2000).

Bemisia tabaci was first recorded by Gennadius in Greece on tobacco

(Nicotiana sp.) in 1889. Gennadius named the species Aleurodes tabaci, and placed it in the family Aleyrodidae, order Hemiptera, sub-order Sternorrhyncha (Martin 1987).

The first New World Bemisia tabaci was found in the United States on sweetpotato

(Ipomoea batatas Lam) in 1894 and described as Aleyrodes inconspicua Quaintance with the common name, sweetpotato whitefly (Quaintance 1890). In 1914, the genus

Bemisia was first described (Quaintance and Baker 1914) and in 1936, Takashaki replaced the name Aleurodes tabaci with Bemisia tabaci (Takahashi et al. 1936).

Bemisia tabaci has been described as a complex species that constitutes a large number of genetically variable populations, which are referred to as biotypes (Brown et al. 1995). Bemisia tabaci is currently considered a species complex composed of 28 morphologically similar species that can be differentiated by mitochondrial cytochrome oxidase (mtCOI) (Barro et al. 2011). The two most devastating biotypes are B (Costa and Brown 1991) and Q (Guirao et al. 1997, Rosell et al. 1997). Biotype B was determined to have originated from the Middle East-Asia Minor region (Costa and

Brown 1991), and has become one of the most damaging pests in tropical and subtropical regions (Oliveira et al. 2001, De Barro et al. 2011 ). The Q biotype was determined to have originated from the Mediterranean region (Rosell et al. 1997). The B biotype of Bemisia tabaci became established in Florida in the late 1980s and by the early 1990s had caused devastating damage to agricultural crops in warm agricultural regions of the United States from California to Florida. Perring and co-workers defined this species as Bemisia argentifolii (Perring et al. 1992), but this designation has not been widely accepted. After molecular and genetic studies, DeBarro and co-workers described it as B. tabaci Middle East Asia Minor (MEAM1). Bemisia tabaci biotype B and B. argentifolii are now formally named as Bemisia tabaci Middle East Asia Minor

(MEAM1), and the Q biotype is formally named Bemisia tabaci MED (Tay et al. 2012).

Feeding and Damage

Bemisia tabaci has opisthognathus piercing-sucking mouthparts (Gerling et al.

1990). Bemisia tabaci feeds on phloem sap and excretes honeydew, a sugar-rich excreta that provides habitat for saprophytic fungi (sooty mold) and reduces harvest quality (Gerling et al. 1990). It transmits over 150 different plant viruses from seven virus groups including Geminiviridae, Closteroviridae, Carlaviridae, Potyviridae,

Nepoviridae, Luteoviruses and DNA-containing rod-shaped viruses. Of these, the

Geminiviridae and Closteroviridae are the most devastating (Duffus 1987, Duffus et al.

1996). Among the Geminiviridae, B. tabaci transmits Tomato yellow leaf curl virus

(TYLCV) and Tomato yellow mottle virus to tomato (Polston and Anderson 1997).

Bemisia tabaci MEAM1 also induces irregular ripening in tomatoes, leaf silvering in squash, and a white streak in cole crops (Cohen et al. 1992).

Bemisia tabaci adults select suitable parts for feeding that vary according to host species. Bemisia tabaci is apparently attracted to UV light when predisposed to migrate and attracted to the yellow spectrum of light associated with most vegetation when predisposed to colonize plants (Mound 1962). Bemisia tabaci can identify suitable host plants only after landing on them and receiving gustatory information through probing

(Janssen et al. 1989). They typically feed on the underside of the leaf. Younger leaves are more commonly exploited for feeding as well as for oviposition, presumably because younger leaves have higher nutritional quality. In addition, younger leaves may be easier to penetrate than older leaves (Lenteren and Noldus 1990).

Life Cycle

Bemisia tabaci has six life stages: egg, crawler (1st nymphal instar), three sessile instars (2nd, 3rd and 4th nymphal instars) and the adult. Reproduction in B. tabaci is usually sexual, but females can also reproduce by arrhenotokous parthenogenesis

(Dobreanu and Manolache 1969). In this form of parthenogenesis, unmated females produce only male offspring and mated females produce both male and female offspring. Bemisia tabaci oviposit on younger leaves of tomato, eggplant and cotton (Arx et al. 1984) and lay eggs on the underside of leaves. They mostly prefer plant species with smooth or glabrous leaves (Mound 1965). The egg to adult cycle of B. tabaci takes

27.3 d on tomatoes at 250C and 65% RH (Salas and Mendoza 1995). Under these conditions, lifetime fecundity was 194 ± 59.1 eggs per female on tomato, with an egg viability of 86.5%. On the basis of research conducted in Egypt on cotton plants, the number of eggs that B. tabaci can produce varies from 48-394, depending on temperature (Azab et al. 1972). During the course of their lifetime, females oviposit about 250 eggs at 28.5°C, 204 eggs at 22.70C, and 61 eggs at 14.30C (Azab et al.

1972).

Eggs are ovoid and initially cream colored but darken with age to a deep brown.

Eggs are attached to the plant by a pedicel and are 0.21 mm in length and 0.096 mm in width, attached to a stomatal opening or slit produced by the female (Byrne and Bellows

1991). The pedicel facilitates the intake of water from leaves into the eggs. The incubation period of the egg is about 7-8 d on tomato and cotton at 250C and 75% RH.

The first instar emerges from the egg apex. Nymphs are classified into four nymphal instars and the 4th nymphal instar is sometimes called a red-eyed nymph. Whitefly nymphs produce wax that surrounds them. The 1st instar is active for the first few hours

after hatching (named the “crawler” stage) before it settles for the remainder of its nymphal development. The crawler has three-segmented legs and two-segmented antennae. These become one-segmented in 2nd and 3rd instars, which are immobile

(Byrne and Bellows 1991). At 250C and 75% RH the nymphal stages of 2nd and 3rd instar last for 6-7 d each, and the 4th instar lasts for 6-8 d on tomato (Salas and

Mendoza 1995). Adults emerge from nymphal exuvia leaving a T-shaped exit hole in the integument. Generally, the size of B. tabaci varies from 0.85-1.2 mm. Females average

0.96 mm and males are a bit smaller at 0.82 mm (Byrne and Bellows 1991). Female adult longevity is about 19 d while male longevity is about 19.5 d on tomato plants

(Salas and Mendoza 1995). The whitefly has a unique dorsal anus structure known as the vasiform orifice. It is present on the abdominal segment on males and eighth and ninth segments on females (Mound 1983).

Management of Bemisia tabaci

Chemical Control

Historically insecticides used to manage B. tabaci included carbamates (for example: aldicarb, carbaryl, carbofuran), organophosphates (for example: bromophos, demeton, methyl parathion, mevinphos) and pyrethroid insecticides (for example: cypermethrin, fenpropathrin, fenvalerate). Among the most common modes of action used for chemical control of whitefly are sodium channel modulators (pyrethroid insecticides) and nicotinic acetylcholine receptor (NACHR) competitive modulators

(neonicotinoids) (Freeman et al. 2016). Whitefly resistance to insecticides was first recorded in 1985 before the description of biotypes (Barro et al. 2011). Bemisia tabaci has been characterized by a high degree of resistance to many chemical insecticides including organophosphates, carbamates, pyrethroids, insect growth regulators and

chlorinated hydrocarbons (Cahill et al. 1996). Resistance is also detected on α‐ cypermethrin, bifenthrin, pirimiphos‐methyl, endosulfan, imidacloprid, thiamethoxam and alpha‐cypermethrin (Roditakis et al. 2005, Wang et al. 2010).

. Cultural Methods

Cultural methods of pest control rely on the manipulation of the crop environment to reduce damage by harmful insects. Cultural control can play a significant role in the management of B. tabaci. Cultural practices used to manage B. tabaci include repellent metallized plastic mulch, manipulation of planting time, employing a crop-free period, crop rotation, crop sanitation, and trap crops (Zitter and Simons 1980, Cock et al. 1986).

Metallized plastic reflective mulch reflects UV light, which interferes with host location and orientation of whiteflies (Csizinszky et al. 1999). Buckwheat and clover are living mulches that have been evaluated to manage B. tabaci. These species provide habitat for predators that attack whiteflies (Frank and Liburd 2005). Living mulches such as the legumes Arachis pintoi (perennial peanuts) and Styzolobium deeringianum (mucuna)

(Fabaceae), Drymaria cordata (“cinquillo”) (Caryophyllaceae), and Coriandrum sativum

(coriander) (Umbelliferae) reduced numbers of whitefly and incidence of Tomato mottle virus (Hilje and Stansly 2008).

Host Plant Resistance

Reduction in pest population growth through the impact of host plant shape, size, chemistry, nutritional characteristics, or other characters, is known as host plant resistance. Breeding is used to incorporate resistance traits often found in wild species into commercially acceptable varieties (Berlinger 1986). Some varieties of Lycopersicon peruvianum (L) Mill, L. hirsutum and L. hirsutum F. glabratum are resistant to B. tabaci

because of their glandular trichomes (Channarayappa et al. 1992). The toxins secreted by glandular hairs of tomatoes can confer resistance to B. tabaci (Kennedy and

Yamamoto 1979). Densities of leaf trichomes, the existence of acyl sugars in the exudate of glandular trichomes and type of trichomes can alter the relationship between the whitefly and tomato (Simmons 1994). Several wild species of tomato show resistance against B. tabaci. Lycopersicon pennellii (Corr.) D’Arcy contains sugar esters in its glandular exudates (Goffreda et al. 1990). Plant hairs can provide a physical barrier against whiteflies (Duffey 1986). Bemisia tabaci prefers not to oviposit on very hairy leaves (Mound 1965). More whiteflies were found in pubescent and hirsute soybean than glabrous soybean (McAuslane et al. 1995). Bemisia tabaci prefers lightly pubescent varieties to densely pubescent varieties as the hairy varieties may cause a physical barrier and less hairy leaves provide more suitable microclimate with high relative humidity for the pest (Mound 1965).

Biological Control

Biological control uses natural enemies to reduce damage by pests. Natural enemies are predators, parasitoids and pathogens that reduce pest abundance, leading to reduced economic damage. There have been some successes in the biological control of whiteflies (Arnó et al. 2009). There are about 92 species of predatory insects and mites and more than 52 parasitoid species that are known to attack B. tabaci

(Gerling et al. 2001).

Whitefly parasitoids: Parasitoids are host specific and live within or attached to the host’s body. They lay eggs on or in the host and can also kill the host by feeding on it (Emden and Service 2004). Primary parasitoids that are being employed against whiteflies include Encarsia spp. and Eretmocerus spp. (Hymenoptera: Aphelinidae)

(Gerling et al. 1980, De Barro and Coombs 2009), Amitus spp. (Hymenoptera:

Platygasteridae) (Joyce et al. 1999), and Metaphycus spp. (Hymenoptera: Encyrtidae)

(Polaszek et al.1999). About 23 species of Encarsia and 10 species of Eretmocerus have been studied as parasitoids of whitefly (Gerling et al. 2001). Parasitoids Encarsia formosa, Eretmocerus mundus, and Eretmocerus queenslandensis are among the most effective natural enemies that are commercially available (Gerling et al. 2001).

Whitefly predators: Predators of B. tabaci include beetles (Coccinellidae), true bugs (Miridae, Anthocoridae), lacewings (Chrysopidae, Coniopterygidae), mites

(Phytoseiidae) and spiders (Araneae) (Nordlund and Legaspi 1996, Li et al. 2011).

Some coleopteran predators of whitefly are Serangium parcesetosum (Abboud and

Ahmad 1998) found in India, Clitostethus arcuatus (Booth et al. 1996), and Delphastus catalinae (Hoelmer et al. 1993). Some Hemiptera such as Macrolophus caliginosus and

Dicyphus tamaninii are effective predators of greenhouse whitefly (Barnadas et al.

1998, Lucas and Alomar 2002). The mirid, Nesidiocoris tenuis Reuter, is a omnivorous predator that has been evaluated for biological control of whiteflies in greenhouse- grown tomatoes (Calvo et al. 2012). Serangium parcesetosum larvae can consume about 89.2 nymphs of B. tabaci per day and 1119.1 nymphs during the course of larval development at 300C on cotton plants (Sengonca et al. 2005). Nephaspis oculata is another predatory lady beetle introduced for whitefly control; females consume approximately 86 whitefly eggs per day, males consume approximately 79 eggs per day and a pair can consume about 184 eggs per day (Liu et al. 1997). Some effective predators that are commercially available for the control of B. tabaci are Coccinella

septempunctata, Neoseiulus californicus, Neoseiulus cucumeris, D. tamaninii, M. caliginosus, Dicyphus hesperus and Chrysoperla carnea (Gerling et al. 2001).

Fungal pathogens: Fungal pathogens for the management of B. tabaci include

Beauveria bassiana, Paecilomyces amoenoroseus, P. fumosoroseus, Verticillium lecanii and Metarhizium anisopliae. These entomopathogenic fungi are commercially available and can contribute to the control of B. tabaci (Faria and Wraight 2001). Natural epizootics of entomopathogenic fungi can also suppress B. tabaci populations through infection. The entomopathogenic fungus Lecanicillium muscarium is used as biocontrol agent to control B. tabaci (Cuthbertson et al. 2005).

Paecilomyces fumosoroseus has been used to reduce whitefly populations in the field and greenhouse (Lacey and Kirk 1993, Castineiras 1995). Some fungi are highly effective for nymphal stages of whitefly such as B. bassiana (Vicentini et al. 2001,

Quesada-Moraga et al. 2006) and P. amoenoroseus (Candido 1999). Isaria fumosoroseus (Paecilomyces fumosoroseus designated) is one of the most important fungal pathogens of whiteflies (Huang et al. 2010).

Dicyphus hesperus

Dicyphus hesperus (Hemiptera: Miridae; Subfamily: Bryocorinae; Tribe:

Dicyphini) is an omnivorous predator native to North America (Henry et al. 1988).

Dicyphus hesperus was first described on whiteflower leaf cup, Polymnia canadensis L.

(Compositae) by Knight in 1941. It has since been described from many varieties of plants. Most of the insects of the Bryocorinae subfamily have a wide host range. They are well adapted to plants with sticky glandular hairs (Wheeler and Krimmel 2015).

Dicyphus hesperus is mostly associated with the Solanaceae, Scrophulariaceae,

Rosaceae, Lamiaceae, Ericaceae, and Hydrophyllaceae families (Cassis 1986).

Dicyphus hesperus is a zoophytophagous species that feeds on both plant tissue and prey. It consumes water from plants, and engages in extra-oral digestion of prey

(Gillespie and McGregor 2000, Sinia et al. 2004). Consumption of prey is necessary for the completion of nymphal development on tomato plants. Dicyphus hesperus feeds by inserting its sucking mouthparts into prey and sucking out the inner contents. After consuming whitefly eggs or nymphs, only the chorion or integument remains, with a tiny hole where the mouthparts were inserted (McGregor et al. 1999).

Biology

Dicyphus hesperus requires 42 d to complete its life cycle from egg to adult at

200C on tomato plants (Gillespie et al. 2004), passing through four nymphal stages.

Wing development is initiated during the 3rd nymphal instar. Both males and females are about 3.5 mm long and lightly covered with pale-brown, erect long scales. Adult females insert eggs into plant stem tissue, veins and stalks with a sword-like ovipositor (Cohen

1995, 1998). When Ephestia kuehniella eggs are provided as a dietary supplement, the lifetime fecundity of D. hesperus is about 63.2 ± 10.2 eggs on tomato and 49.6 ± 7.8 eggs on mullein (Verbascum thapsus L., Scrophulariaciae) (Sanchez et al. 2004).

Greenhouses with mullein as well as tomato had higher numbers of D. hesperus than greenhouses with tomato alone (Sanchez et al. 2003). Dicyphus hesperus fed with E. kuehniella eggs produces eggs that hatch in 18 d on tomato at 200C and L16:D8.

The 1st instar lasts for 6-7 d then molts to 2nd instar, which lasts 4-5 d. The 3rd instar lasts 5-6 d and the final (4th) instar lasts for 9-10 d at 20° C. Total nymphal development takes about 25-27 d on tomato plants at 200C (Sanchez et al. 2004). Adult D. hesperus are good fliers and will move throughout large areas to find prey. Nymphal stages of D. hesperus cannot fly but are fast walkers and will move up and down and between plants

which are in contact with each other. Dicyphus hesperus seems more active at nighttime than in daylight (VanLaerhoven et al. 2006). Dicyphus hesperus can complete its life cycle on tomato if prey such as whitefly or mites is available (McGregor et al.

1999). Dicyphus hesperus can complete its life cycle when provided only with plant material on some plant species like mullein, sweet pepper (Capsicum annuum L.) and catnip. It cannot complete its lifecycle without prey on tomato, tobacco (Nicotiana tabacum L.), broadbean (Vicia sativa L.) and corn (Zea mays L.) (Sanchez et al. 2004).

Lack of prey also decreases adult longevity and fecundity (Sanchez et al. 2004).

Use of D. hesperus for Management of Insect Pests in Greenhouses

In British Columbia, Canada, D. hesperus was first examined for its potential to control pests of greenhouse tomato plants, including greenhouse whitefly, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae) and two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae) with effective results (McGregor et al. 1999). Dicyphine mirids are most often described as predators of whiteflies

(Gabarra et al. 1995, Alomar and Albajes 1996, Barnadas et al. 1998), and are considered effective for control of thrips (Thysanoptera: Thripidae), aphids (Hemiptera:

Aphididae), leafhoppers (Hemiptera: Delphacidae), and other small insects (Salim and

Heinrichs 1986, Chua and Mikil 1989, Braman and Beshear 1994, Gabarra et al. 1995).

Banker plants are non-crop plants that directly or indirectly provide habitat, food, and/or prey to the natural enemies that are released within a crop to control a pest

(Frank 2010). The purpose of banker plants is to boost the population of natural enemies in an agricultural production system and sustain the population for long-term pest suppression (Huang et al. 2011). The mullein plant has been evaluated as a banker plant for D. hesperus (Sanchez et al. 2004). Studies have demonstrated that

mullein can be used to establish and increase populations of D. hesperus (Sanchez et al. 2004). Sanchez et al. (2003) demonstrated that mullein can serve as a reservoir for

D. hesperus in commercial greenhouse production of tomato and help suppress populations of T. vaporariorum.

Challenges to Managing Tomato Pests in Protected and Organic Agriculture

Protected agriculture, consisting of greenhouses, screen houses and shade houses, is a small but growing sector of Florida’s agricultural economy (Hochmuth and

Toro 2014). The use of high tunnels for vegetable production increased from zero in

2001 to 186.41 acres in 2013. Hochmuth and Toro (2014) estimate that intensive production of greenhouse tomatoes in Florida could yield 90.71 metric ton per acre with a gross income of $250,000 per acre.

Bemisia tabaci is one of the primary pests of tomato and other horticultural crops.

Chemical pesticides can provide effective control of B. tabaci under certain circumstances, but frequent use of chemicals elevates production costs and can lead to the development of insecticide resistance. Insecticides may also have negative effects on natural enemies as well as agricultural workers and the environment. In addition, many insecticides are not registered for use in protected agriculture and are not compatible with the use of commercial pollinators. As the adoption of protected agriculture is increasing, the use of biological control methods may contribute to effective management of B. tabaci and other pests in protected structures. Dicyphus hesperus has been shown to be an effective predator of greenhouse whitefly, thrips and mites, with potential application for control of B. tabaci.

Objectives

1. Evaluate predation of B. tabaci by D. hesperus

a) Estimate the consumption rate of B. tabaci eggs by female D. hesperus

directly from the insectary and from an established colony b) Estimate the consumption rate of B. tabaci eggs and nymphs by mated vs.

unmated female D. hesperus c) Determine the consumption rate of D. hesperus on different life stages

(eggs, earlier nymphs, later nymphs)

2. Determine the parameters for establishing a population of D. hesperus on

banker plants in the greenhouse. a) Evaluate the life cycle of D. hesperus on tomato and on mullein with and

without E. kuhniella eggs under controlled conditions of relatively constant

temperature b) Determine the survival of D. hesperus on mullein plants with E. kuhniella

eggs under actual greenhouse conditions c) Evaluate the population development of D. hesperus on mullein with and

without E. kuhniella eggs and on tomato with E. kuhniella eggs in the

greenhouse

CHAPTER 2 EVALUATING THE PREDATORY CAPACITY OF DICYPHUS HESPERUS ON BEMISIA TABACI

Introduction

Tomato, Solanum lycopersicum L., is an economically important crop in Florida

(USDA 2016). Approximately 29,000 acres of tomato was produced in Florida in 2017, yielding 426,739.7 metric tons. This comprised about 38% of the total tomato production in the United States and generated $365,774,000 from 2015-2018 (USDA

2018). In recent years, vegetable production in greenhouses has increased from zero in

2001 to 186.41 acres in 2013. Hochmuth and Toro (2014) estimated that intensive production of greenhouse tomatoes in Florida could yield 200,000 lb per acre with a gross income of $250,000 per acre.

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is a severe pest of tomato widely distributed throughout the world. Bemisia tabaci causes direct damage to plants by phloem sap removal and indirect damage by transmitting viruses and producing honeydew that serves as substrate for sooty mold on plants, thereby reducing photosynthesis (Oliveira et al. 2001). Although chemical pesticides provide effective control of B. tabaci under certain circumstances, they may have deleterious effect on natural enemies as well as agricultural workers. The frequent use of chemicals elevates production costs and B. tabaci has also demonstrated the tendency to develop resistance to many classes of insecticides, including organophosphates pesticides (e.g., α‐ cypermethrin, bifenthrin, pirimiphos‐ methyl, endosulfan, pymetrozine, pyrethroids, cyclodienes, and alpha‐ cypermethrin) and neonicotinoid pesticides (e.g., thiamethoxam, imidacloprid) and insect growth regulators

(Cahill et al. 1995, 1996; Roditakis et al. 2005 and Wang et al. 2010).

In addition, many insecticides are not registered for use in protected agriculture and are not compatible with the use of commercial pollinators. As the adoption of protected agriculture is increasing, the use of biological control methods may contribute to effective management of B. tabaci and other pests in protected structures. As a result, an integrated approach with the use of biological control agent should be employed.

Many predators have been reported for the biocontrol of greenhouse whitefly

Trialeurodes vaporariorum (Westwood) and sweetpotato whitefly B. tabaci (Nordlund and Legaspi 1996). Some mirid bugs native to the Mediterranean region, Macrolophus caliginosus Wagner and Dicyphus tamaninii Wagner, are commercially used as natural enemies for whitefly and thrips on tomato and cucumber plants (Barnadas et al. 1998,

Albajes and Alomar 1999). Both the adult and nymph of M. caliginosus and D. tamaninii prey upon all the stages of whitefly and can significantly reduce greenhouse whitefly populations (Lucas and Alomar 2002). The predator Nesidiocoris tenuis Reuter has demonstrated effective control of adult B. tabaci on greenhouse tomato (Calvo et al.

2009).

The Dicyphine mirids are zoophytophagous generalist predators. They have been traditionally neglected for use for biological control of pests because they are facultative plant feeders. They feed on plants to acquire water, and this may produce economic damage (Castañé et al. 2011). However, the potential for economic loss due to feeding by the predator depends on the predator: prey ratio and the actual plant parts upon which they feed (Castañé et al. 2011). Dicyphus hesperus Knight (Hemiptera:

Miridae) is a widely distributed zoophytophagous true bug native to North America

(Henry and Wheeler 1988). The economic damage caused by D. hesperus is minor as they prefer plant leaves over fruits (McGregor et al. 2000). This predator has been evaluated for management of pests on greenhouse-grown tomato plants (Gillespie et al.

2007). Female adult D. hesperus effectively controlled greenhouse whitefly, T. vaporariorum Westwood and two-spotted spider mites, Tetranychus urticae Koch, on tomato plants, consuming on average 24 early instar nymphs and 43 older nymphs of whitefly per day and consuming 28 mites per day (McGregor et al. 1999).

Dicyphus hesperus released at a rate of one predator per plant reduced sweetpotato whitefly populations by 88.8% and potato psyllid populations by 90.1% on tomato plants in the greenhouse under a wide range of climatic conditions (Calvo et al.

2016). A predator: prey ratio of 0.5-1:10 is required for the adequate control of western flower thrips, Frankliniella occidentalis Pergande, populations in tomatoes (Shipp and

Wang 2006).

The main objective of this research was to evaluate the effectiveness of D. hesperus for the biological control of B. tabaci (MEAM 1) and to develop baseline information about the number of predators needed to control this pest. To meet this goal, we determined the daily and lifetime consumption rate of D. hesperus on different stages of B. tabaci.

Methods and Materials

Plants and Insects

Mullein seedlings (Verbascum thapsus) were purchased from Gloeckner and

Company (550 Mamaroneck Avenue, Harrison, NY) and were transplanted into 10-cm- diameter pots in a greenhouse at the Gulf Coast Research and Education Center

(GCREC), Balm, Florida. They were provided with 20:20:20 All Purpose nutrient mix

(Hummert International Company, 4500 Earth City Expressway, MO 63045) (10 g for 3 liters), at a rate of 125 ml water per plant every 15 d. Six-week-old mullein plants were placed in a Bug Dorm (60 × 60 × 60 cm, MegaView Science Co. Ltd., Taiwan), which were used to rear D. hesperus. A colony of D. hesperus was established from individuals originally purchased from Beneficial Insectary (Redding, CA) (Figure 2-1), and reared on mullein plants in a growth room (temperature 24-28 0C; RH 48-55 %; L: D

12:12) (Figure 2-2). Ephestia kuehniella Zeller eggs, purchased from Beneficial

Insectary, were provided, attached to a 2-cm adhesive section of a yellow Post-It note

(3M Corporation, St Paul, MN) (Figure 2-3). Approximately 0.25 g (0.1 g Ephestia/100

Dicyphus hesperus) was provided to each Bug Dorm every week as a protein source.

This method for providing E. kuehniella eggs to D. hesperus was developed by Chris

Daye of Beneficial Insectary. HOBO data loggers (Onset Technology Company,

Pocasset, MA) were set up to record temperature and relative humidity.

A colony of B. tabaci MEAM1 established in the early 1990s from field populations in the Bradenton, Florida area and maintained on cotton (Gossypium hirsutum L.) plants was used as a prey source. Cotton plants (DPO935B2RF variety) were grown from seed and transplanted to 20-cm-diameter pots with Fafard (BWI-

Apopka, Plymouth, FL 32768) soil potting mix in a greenhouse. Plants were watered as required and fertilized with a 20:20:20 nutrient mix every week. Whiteflies were reared in the growth room (temperature 24-280C; RH 48-55%; L: D 12:12) with four to six cotton plants per rearing cage. Tomato plants (variety ’Lanai‘) supplied by the tomato breeding program at GCREC were grown from seed in 20-cm-diameter pots with Fafard

potting mix in a greenhouse. Plants were watered as required and fertilized with a

20:20:20 nutrient mix every week.

Estimating the Consumption Rate of Bemisia tabaci Eggs by Female Dicyphus hesperus from Two Different Sources

Dicyphus hesperus used in predation studies were from two sources, Beneficial

Insectary (Redding, CA) and the D. hesperus colony that was established from D. hesperus sent from Beneficial Insectary and maintained in a growth room at GCREC.

Dicyphus hesperus females were randomly selected (mating status unknown) from

Beneficial Insectary and GCREC colony. Females from Beneficial Insectary were used the day after arrival and females from the colony were chosen according to their wing and abdomen color to confirm that they were not older than 72 h (Figure 2-4). The color of wings remains light green for 1 d and the color of abdomen remains bright green for 3 d and then started change to darker green up to 7 d.

Distinguishing Mated and Unmated Females for Predation Study

Individual 4th instar D. hesperus nymphs were placed in small round clear plastic containers (diameter 15 cm) with one mullein leaf and E. kuehniella eggs. When these individuals became adults, females were either kept in isolation to prevent mating, or were confined with males for 3 d to allow mating. Mated females and unmated females were used for egg predation and nymph predation experiments.

Estimating the Consumption Rate of Bemisia tabaci Eggs by Female Dicyphus hesperus

Lanai tomato plants, 6-7 weeks old, were used as a substrate for sweetpotato whitefly oviposition for predation studies. Sweetpotato whitefly adults were aspirated in groups of seven to 10 mating pairs into a glass eye dropper and tapped into a clip cage, which was then attached to a leaflet on a mid-stratum leaf, where they were allowed 24

h to lay eggs on leaves (Figure 2-5). After 24 h, leaves were removed from the tomato plant with a razor blade and all the sweetpotato whitefly eggs on the leaves were counted under the microscope. This was the “pre-count”. The stem of the leaf was then placed in a plastic vial (9 cm long with 3 cm diameter) filled with 60 ml water to maintain leaf turgidity. Parafilm was placed over the top of the vial around the base of the leaf to prevent spillage. The vial and tomato leaf were then placed in a clear Plexiglass cylinder

(30 cm tall with 13 cm diameter) with an organdy top (Figure 2-6). Individual D. hesperus females were then placed in each cylinder, which served as the arena for the predation studies. Ten arenas, labeled 1-10, were set up for each trial of the experiment. Dicyphus hesperus females were confined in arenas 1-5. The remaining arenas, 6-10, were used as an untreated control to test for the disappearance of B. tabaci eggs due to non-predator related factors, such as experimental handling.

Leaves were examined under a microscope after 24 h. The total number of eggs present and eggs damaged due to D. hesperus feeding was recorded from the leaves that were confined with female D. hesperus. Eggs that had been fed upon by D. hesperus consisted of an empty, damaged chorion and were clearly identifiable under the microscope (Figure 2-7a). The total number of eggs present and missing in the controls were also counted to compare with the leaves confined with D. hesperus. The untreated control was added because preliminary studies revealed that leaves confined with D. hesperus sometimes had fewer post-count eggs even after damaged ones were taken into account. Dicyphus hesperus is not thought to consume whole eggs as they have piercing-sucking mouthparts, so it is possible that they dislodged the egg from the leaf in the process of feeding or locomotion. Dicyphus hesperus apparently engages in

extra-oral digestion (Cohen 1995). If this results in partial dissolution of the egg chorion, it could also help explain the disappearance of eggs. The untreated control was added to determine if there were fewer post-count eggs because eggs were being dislodged by D. hesperus or were falling off the plant for reasons unrelated to the activity of the predator, such as experimenter handling.

Each female was provided with a new leaf containing 24-h-old sweetpotato whitefly eggs each day until her death. The experiment was carried out using 34 females randomly selected (mating status unknown) from Beneficial Insectary from

June 21 to August 4, 2016 and 22 females from the GCREC colony from August 17 to

September 21, 2016. Comparing egg predation by mated and unmated female D. hesperus was carried out using 21 mated and 21 unmated female D. hesperus from the

GCREC colony from October 2 to November 19, 2016. In each trial, the number of damaged eggs and missing eggs was recorded for each 24-h period of the female’s life.

Thus, the egg consumption study was carried out using females that had been handled in three different ways: 1) females that had arrived the day previously from

Beneficial Insectary; 2) females that had been reared in a colony at GCREC and that were 72 h old or younger; and 3) females from the GCREC colony that were 72 h old or younger, and that were divided into mated or unmated groups.

Estimating the Consumption Rate of Bemisia tabaci Nymphs by Female Dicyphus hesperus

The predation rate of D. hesperus on B. tabaci nymphs was evaluated following the same methods as used for the egg predation experiment in which mated and unmated D. hesperus females were used. Clip cages were set up on leaves and whiteflies were allowed to oviposit for 24 h. After 24 h the clip cages were removed and

the adults were removed from the plants. Those plants with sweetpotato whitefly eggs were then kept in a Bug Dorm of dimensions 60 × 60 × 60 cm for 9-10 d to allow the eggs to hatch into nymphs. The numbers of 1st and 2nd instar nymphs on leaves were counted and those leaves were then confined with female D. hesperus for 24 h using the same methodology as described for eggs. After 24 h the leaves were removed and the post-count was carried out. Damaged nymphs fed upon by D. hesperus consisted of an empty, damaged integument and were clearly identifiable under the microscope

(Figure 2-7b). Each female D. hesperus was given a new leaf with nymphs until she died. This experiment was performed with 20 mated and 20 unmated females from

November 23 to December 28, 2016.

Predation Behavior of Dicyphus hesperus on Different Life Stages of Bemisia tabaci

The predation behavior of D. hesperus on different sweetpotato whitefly nymphal stages was evaluated. The study was conducted in the growth room at 270C. We placed clip cages on 12 tomato leaflets on mid-stratum leaves from five tomato plants and five to seven pairs of whiteflies were confined on those leaflets for oviposition. Whiteflies were removed from those leaflets after 24 h. The eggs that had been oviposited were allowed to develop to 3rd and 4th instar for 14 d. Five days after the first whiteflies were placed, we confined whiteflies for 24 h on different leaflets and allowed them to grow for

9 d in order to produce 1st and 2nd instar nymphs. To produce eggs for the study, whitefly adults were confined on the leaf for 1 d, then removed, and the plants were used immediately for the experiment. Thus, 2 weeks after the first infestation, eggs and early and late instar B. tabaci nymphs were available. The leaves were then placed in a plastic vial (9 cm long with 3 cm diameter) filled with 60 ml water to maintain leaf

turgidity. The top of the vial was covered with Parafilm wrapped around the base of the leaf to prevent spillage. The vial and tomato leaf were placed in a clear Plexiglass cylinder (30 cm tall with 13 cm diameter) with an organdy top and a single mated 3-4 d- old female D. hesperus from the GCREC colony was confined on a leaf. Thus the experiment evaluated predation of whiteflies of different ages in a no-choice situation.

After 24 h, we counted the number of live and damaged eggs and nymphs. There were four replications (females) per trial of the experiment and the trial was repeated four times for a total of 16 replications (females) of each of the three whitefly-age treatments.

Comparison of predation among B. tabaci eggs, earlier nymphs and later nymphs was done on the basis of the number of damaged individuals of different life stages of B. tabaci.

Statistical Analysis

Predation behavior of D. hesperus was evaluated using two sources: D. hesperus directly brought from Beneficial Insectary and D. hesperus from the colony.

Average number of B. tabaci eggs consumed per day over the entire adult lifetime was calculated for the 34 females directly from Beneficial Insectary and for the 21 females

(less than 72 h old) from the GCREC colony. Statistical comparisons could not be made between the females from the two sources because the experiments were done at different times.

A t-test run in R (R-studio 2013) was applied to compare 21 mated and 21 unmated female D. hesperus for their average daily consumption and lifetime consumption on both B. tabaci eggs and nymphs. A regression analysis was run to determine the correlation between the number of eggs/nymphs provided and the number of eggs/nymphs consumed by D. hesperus.

Predation of female D. hesperus on B. tabaci eggs, earlier nymphs and later nymphs was evaluated. The number of damaged life stages (eggs, 1st / 2nd and 3rd /4th instars) of B. tabaci was recorded separately. Predation of different life stages of B. tabaci was compared using PROC GLM in SAS (SAS institute 2001).

Results

Predation of Bemisia tabaci Eggs by Dicyphus hesperus from Two Sources

For both predator sources, the number of intact B. tabaci eggs on tomato leaves after 24 h was lower in the treatment provided with female D. hesperus than in the control without the predator (Beneficial Insectary: t = 7.2; df = 344; P < 0.0001 and

GCREC colony: t = 5.45; df = 322; P < 0.0001). However, the total reduction in B. tabaci egg population on treatment (Insectary) was 13.03%, including 6.91% with visible damage and 6.13% which were missing compared to the control with 5.6% missing.

The reduction in B. tabaci egg population by the colony D. hesperus was 13.43%, including 7.14% with visible damage and 6.29% missing compared to the control treatment with 6.97% missing. The average consumption was 2.7 ±0.2 eggs per day and 14.1 ±1.7 over the lifetime by D. hesperus from Beneficial Insectary and 3.3 ± 0.9 per day and 25.5 ±7.1 over the lifetime by D. hesperus from GCREC colony. Female D. hesperus from Beneficial Insectary survived on average 5 ±0.4 d (Figure 2-8 a) whereas the average survival for colony D. hesperus was 7.4 ±0.4 d (Figure 2-8 b). Dicyphus hesperus apparently engages in extra-oral digestion (Cohen 1995). If this results in partial dissolution of the egg chorion, it could also help explain the disappearance of eggs. The untreated control was added to determine if there were fewer post-count eggs because eggs were being dislodged by D. hesperus or were falling off the plant for

reasons unrelated to the activity of the predator, such as experimenter handling. The missing data from treatment and control were compared and found to be very similar.

This suggests that the missing data were due to handling or egg hatching not by predator activity. So, the control was not used for further experiments.

Predation of Bemisia tabaci Eggs and Nymphs by Mated and Unmated Female Dicyphus hesperus

The daily consumption rate of mated and unmated female D. hesperus on B. tabaci eggs did not differ significantly (F = 1.29; df = 1,40; P = 0.2054) but lifetime consumption rate of mated D. hesperus females on eggs was significantly higher than that of unmated D. hesperus (t = 5.37; df = 40; P < 0.0001) (Figure 2-9a). Daily nymph consumption (t = 0.43; df = 40; P = 0.6684) as well as lifetime nymph consumption (t =

0.57; df = 1,40; P = 0.5708) by mated and unmated female D. hesperus did not differ significantly (Figure 2-9b). On average, female D. hesperus consumed eight times more nymphs than eggs of B. tabaci in a 24-h period and 10 times more nymphs than eggs in their lifetime.

The number of eggs damaged increased as the number of eggs provided to female D. hesperus increased up to certain level where the damage averaged 20 eggs per day, and then the regression line leveled off (R2 = 0.168) (Figure 2-10). During most

24-h exposure periods, female D. hesperus damaged only up to 20 eggs, however, on a few days, a few predators consumed up to 100-120 eggs per day (Figure 2-11). The consumption of nymphs increased as the number of nymphs provided to D. hesperus increased (R2= 0.44) (Figure 2-12). On most days, female D. hesperus fed on 50 to 100 nymphs, however, on several days female predators fed on up to 300 nymphs per day

(Figure 2-13). Predation rate increases as the number of available prey increases, and

then levels off (Figure 2-9; Figure 2-11). The predation over lifetime was also analyzed and there was no pattern seen in the damaged eggs and nymphs with the age of female

D. hesperus although, the consumption was low at the near the end of the lifespan

(Figure 2-15). The survival of mated and unmated D. hesperus fed on whitefly eggs and nymphs was not significantly different (t = 1.017; df = 40; P = 0.32 and t = 1.04; df = 40;

P = 0.31) respectively. Predators had 100% survival up to 6th day when fed on eggs and all were dead by day 15. All D. hesperus provided with whitefly nymphs survived for at least 9 days and all predators were dead by day 25 (Figure 2-14).

The daily consumption and lifetime consumption of D. hesperus of B. tabaci eggs, earlier nymphs and later nymphs were significantly different (F = 209.89; df =

2,769; P < 0.01) and (F = 142.06; df = 2,774; P < 0.001) respectively (Figure 2-16). The consumption rate on earlier and later whitefly nymphs was not significantly different.

The survival % of predators fed on whitefly early and later nymphs was greater than on eggs (Figure 2-17). One hundred percent of D. hesperus fed on older nymphs survived for 10 d. Thirty percent of D. hesperus fed on older nymphs survived for 31 d. One hundred percent survived for 9 d and 10% survived for 26 d when fed on earlier instar nymphs. One hundred percent survived for 4 d and 10% survived for 27 d when provided with whitefly eggs only (Figure 2-17).

Discussion

The female D. hesperus from the GCREC colony ate more during the course of their lifetime as they lived longer. The lifetime consumption of Insectary D. hesperus was low as they were short- lived (Figure 2-8). This could be because of the age of predator when used in the predation study. The age of females from the GCREC colony were known as we choose young females, based on their abdomen color, so they likely

had a longer remaining lifespan than the females of unknown age from Beneficial

Insectary.

The average consumption rate of both mated and unmated female D. hesperus on B. tabaci eggs was about 10 eggs per day when an average of 94 eggs (ranging from 0-120 eggs) (Figure 2-11) were provided and the average consumption of B. tabaci nymphs was about 92 nymphs per day when an average of 246 nymphs (ranging from

10- 300 nymphs) were provided to daily them (Figure 2-13). This rate of feeding is substantially higher than that shown by other hemipteran predators, such as M. caliginosus and D. tamaninii, which fed on only five and 13 B. tabaci nymphs respectively when 40 B. tabaci nymphs were provided (Barnadas et al. 1998). The reason for higher consumption rate of D. hesperus in our study could be because higher number of prey nymphs were provided to the predator in comparison to the studies using M. caliginosus and D. tamaninii. Dicyphus hesperus are known as potential predators of greenhouse whitefly and have been reported consuming 23.7 ± 6.5 younger nymphs per day and 43.2 ± 15.8 older nymphs per day. However, the number of nymphs provided to them was not recorded (McGregor et al. 1999). Our research supports that D. hesperus can be a potential predator for B. tabaci control. The predators live longer when fed on whitefly nymphs than on eggs. This could be because of size differences between eggs and nymphs; nymphs are bigger and likely easier to locate.

The major concern of using omnivorous mirid predator for biological control is their ability to damage plants, as the predator needs the plant for water sources and also to survive in the case of prey scarcity (Naranjo and Gibson 1996). Some

zoophytophagous predators can show direct damage to plants but in the case of D. hesperus the feeding damage on tomato fruit is related to prey availability under greenhouse conditions; if there is sufficient prey available for the predator, plant damage will be minimal (Shipp and Wang 2006). Other research also supports the use of D. hesperus as a predator and did not record any negative impact on tomato fruit

(Gillespie et al. 2007). Overall, all the results suggest that the release of D. hesperus would be more effective if the pest population is high on tomato crops. It has considerable potential to improve the biological control of B. tabaci in the greenhouse.

These studies provide baseline data to support the use of D. hesperus for sweetpotato whitefly management in greenhouse tomatoes in Florida. However, the potential of D. hesperus needs to be evaluated in commercial greenhouse before it can be recommended as a biological control agent for B. tabaci.

Figure 2-1. Dicyphus hesperus from Beneficial Insectary company (Redding, CA) used for predation study and also for establishing the GCREC colony. Photo courtesy of author.

Figure 2-2. Colony of D. hesperus maintained on mullein plants in growth room (temperature 25-280C, RH 48-55% and L:D 12:12) Photo courtesy of author.

Figure 2-3. Provision of D. hesperus with approximately 0.25 g Ephestia kuehniella Zeller eggs (0.1g Ephestia/100 D. hesperus) as a supplemental prey source Photo courtesy of author.

Figure 2-4. Young female D. hesperus (2-3 d) with green abdomen used for the experiment to determine daily consumption and lifetime consumption of B. tabaci eggs and nymphs. Photo courtesy of author.

Figure 2-5. Tomato plant with clip cages holding 7-10 pairs of whiteflies confined for 24 h to lay eggs for use in predation study in the growth room. Photo courtesy of author.

Figure 2-6. Experiment set up in the growth room where tomato leaves with B. tabaci eggs/nymphs were confined with a female D. hesperus for 24 h with continuous re-provision until her death. Photo courtesy of author.

A B

Figure 2-7. Damage to whitefly eggs (A) and nymphs (B) by the predator D. hesperus. Photo courtesy of author.

Figure 2-8. Survival percentage of female D. hesperus from Beneficial Insectary (A) and from GCREC colony (B) when fed on B. tabaci eggs on tomato leaves.

Figure 2-9. Average number of B. tabaci eggs and nymphs consumed by mated and unmated female D. hesperus (21 replication in each treatment) daily and over their lifetimes on tomato plants in an experiment conducted in the growth room of GCREC under controlled conditions. Errors bars indicate 1 SEM. The letters (Uppercase letter for daily consumption and lowercase for lifetime consumption) on the top of bars indicate significant difference. Bars within a variable topped by the same letters do not differ significantly (t-test; α < 0.05).

120

100 y = -0.0006x2 + 0.2527x - 6.2448 R² = 0.1683 80

40 No. of damaged eggs ofNo.damaged

0 0 50 100 150 200 250 300 350 400 No. of eggs provided

Figure 2-10. Scatter plot of the number of B. tabaci eggs provided on tomato leaves to female D. hesperus (pre-count) and number damaged in a 24-h period. Dicyphus hesperus female studied = 42, Predation days (number of days all 42 females were provided with eggs) (n) = 343.

180

160

140

120

100

80 Predation days Predation 60

0 0-20 21-40 41-60 61-80 81-100 101-120 More Frequency of damaged eggs

Figure 2-11. Number of 24-h periods where consumption of whitefly eggs on tomato leaves by D. hesperus females fell into damage frequencies shown on the x axis. The experiment was conducted in the growth room of GCREC.

350 y = -0.0004x2 + 0.5846x - 20.795 R² = 0.4404 300

250

200

150 No. of damaged nymphs ofNo.damaged 100

0 0 100 200 300 400 500 600 700 Pre-count

Figure 2-12. Scatter plot of the number of B. tabaci nymphs provided on tomato leaves to female D. hesperus (pre-count) and number damaged in a 24-h period. Dicyphus hesperus females studied = 42, Predation days (number of days all 42 females were provided with nymphs) (n) = 480.

200

180

160

140

120

100

Predation days Predation 80

Frequency of damaged nymphs

Figure 2-13. Number of 24-h periods where consumption of whitefly nymphs on tomato leaves by D. hesperus females fell into damage frequencies shown on the x axis. The experiment was conducted in the growth room of GCREC.

Figure 2-14. Survival percentage of mated and unmated female D. hesperus (A) when fed on B. tabaci eggs and (B) when fed on B. tabaci nymphs on tomato leaves.

160 A Pre-count 140 Damage

120 eggs 100

60 Bemisia tabaci Bemisia

40 No. ofNo. 20

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

400 Pre-count B 350 Damage

300 nymphs 250

200

150 Bemisia tabaci Bemisia

100 No. ofNo. 50

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Days

Figure 2-15. Patterns of damaged (A) eggs and (B) nymphs by D. hesperus throughout their lifetime.

b Daily consumption 1100 1050 Lifetime consumption 1000 950 b 900 850 800

B. tabaciB. 750 700 650 600 550 500 450 400 350 300 250 a 200 No. of different stages ofstages ofNo.different 150 B B 100 A 50 0 Eggs Earlier nymphs Later nymphs

Figure 2-16. Average number of B. tabaci eggs, earlier nymphs and later nymphs consumed by female D. hesperus daily and over their lifetimes on tomato plants in an experiment conducted in the growth room of Gainesville under controlled conditions. Data shown are means ± SE and the letters A and B indicate significant differences within treatments for daily consumption and a and b for lifetime consumption of D. hesperus (Tukeys, α < 0.05). The experiment was performed in Gainesville growth room. N =16 number of D. hesperus females

100 Later nymphs 90 Earlier nymphs 80 Eggs 70

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Figure 2-17. Survival percentage of female D. hesperus when fed on B. tabaci eggs, earlier nymphs and later nymphs on tomato leaves. The experiment was performed in Gainesville growth room. N =16 number of D. hesperus females.

CHAPTER 3 ESTABLISHMENT OF DICYPHUS HESPERUS ON MULLEIN AND TOMATO UNDER CONTROLLED AND GREENHOUSE CONDITIONS

Introduction

The tomato, Solanum lycopersicum L., has been cultivated in the United States for decades, both outdoors and under glass, for fresh market consumption and for processing. Florida is a leading producer of tomato and contributes about 34% of the total U.S. production with recent annual revenue averaging $424,118,000 (USDA 2017).

In 2016, about 30,000 acres of tomato were planted in Florida with production of about

815,360,000 lb (USDA 2017). Protected agriculture, consisting of greenhouses, screen houses and shade houses, is a small but growing sector of Florida’s agricultural economy (Hochmuth and Toro 2014). The use of high tunnels for vegetable production increased from zero in 2001 to 186.41 acres in 2013. Hochmuth and Toro (2014), estimated that intensive production of greenhouse tomatoes in Florida could yield

200,000 lb per acre with a gross income of $250,000 per acre. Arthropod pests are a primary constraint on the production of greenhouse tomatoes in Florida. One of the most serious pests limiting tomato production in south Florida is Bemisia tabaci

(Gennadius) MEAM1, the sweetpotato whitefly.

Commercial agricultural yield losses due to whitefly have been reported all over the world, primarily in tropical and sub-tropical regions, but also in protected agriculture in temperate regions (Oliveira et al. 2001). The sweetpotato whitefly transmits Tomato yellow leaf curl virus (TYLCV) and also causes irregular ripening of tomato (Jones

2003). Chemical insecticides can provide effective control of B. tabaci under certain circumstances but frequent use of chemicals elevates production costs. It is also a very challenging pest to control by chemicals due to its high reproduction rate and potential

to develop resistance to insecticides, as well as the side effects on beneficial organisms used in IPM programs. The rapid build-up of insecticide resistance in this pest requires the development of alternative management approaches (Castle et al. 2010).

The adoption of biological control has increased over recent decades, as it is a more sustainable method of pest control, reducing the likelihood that arthropods will develop resistance against chemical insecticides. After the outbreaks of B. tabaci in the southern regions of the United States in 1992, the study of establishment and augmentation of natural enemies against this pest increased. Predators such as

Macrolophus caliginosus Wagner, Dicyphus taminii Wagner and parasitoids such as

Encarsia formosa Gahan and Eretmocerus eremicus Rose and Zolnerowich have been used successfully for whitefly control (Gerling et al. 2001). There are still some challenges for the use of biological control agents such as frequent mass release of natural enemies, crop damage by omnivorous predators, and plant structure hindering the efficiency of natural enemies. Parasitic Hymenoptera that have been used for whitefly management include E. formosa and E. eremicus (Arnó et al. 2011). Repeated releases of whitefly parasitoids are required for effective control, which is not always economically viable (Greenberg et al. 2002). Encarsia formosa has low efficiency for pest control on tomato in the greenhouse because of trichomes and exudates present on tomato leaves. Sticky honeydew produced by whitefly hinders the parasitoid’s movement and lowers pest foraging efficiency (Levin 1973). The predatory mirids

Macrolophus pygmaeus Rambur and Nesidiocoris tenuis Rambur demonstrated efficacy against whitefly but their use has been constrained by impacts on non-target species and the potential risk of crop injury (Albajes et al. 2006, Van Lenteren et al. 2006).

Dicyphus hesperus Knight has been evaluated for the control of whitefly and the presence of the predator decreases the population of B. tabaci by about 88% (Calvo et al. 2016). In a previous study conducted in British Columbia, Canada, D. hesperus completed its development on the greenhouse whitefly, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), and two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae) and also controlled these pests

(McGregor et al. 1999). Predatory mirids such as D. hesperus are well adapted to plants like tomato as they have pretarsal claws (curved, elongated) to remove plant exudates from their antennae, proboscis and body; this behavior allows them to attach to and live on sticky hosts. They have long, slender legs and walk on tiptoe to minimize contact with exudates (Wheeler and Krimmel 2015).

The need to release bio-control agents repeatedly can substantially increase the cost of control. Methods for establishing populations of biocontrol agents in the greenhouse with a limited number of releases can increase the viability of biocontrol as a management option for pests of protected structures (Huang et al. 2011). The banker plant system is an open-rearing system that supports augmentation and conservation of predators within a cropping system. Banker plants are non-crop plants that act as long- lasting rearing units for natural enemies and are infested with herbivores or provisioned with supplement food (Stacey 1977, Hansen 1983). The main purpose of banker plants is to maintain, and hopefully increase, the predator population even if the pest is not present (Bennison 1992). It is important to establish the population of D. hesperus even in the absence of prey for effective biocontrol of pests. The use of an alternative host

plant is sometimes necessary to help to preserve the predator and maintain their population density (Sanchez et al. 2003).

In previous studies, mullein, Verbascum thapsus L., was used as an alternative host to evaluate the growth response of D. hesperus on tomato plants. Whitefly populations were successfully controlled on tomato by D. hesperus in greenhouses with and without mullein plants, however, the abundance of predators was higher on the tomato plants with mullein plants than on tomato plants only (Sanchez et al. 2003).

Dicyphus hesperus built up to higher populations on mullein than on pepper or eggplant when used as alternative host plants with tomato (Nguyen-Dang et al. 2016). In addition, the reproduction rate and longevity of D. hesperus was higher on mullein plants provided with Ephestia kuehniella Zeller eggs as supplemental prey sources than on mullein plants with no supplemental prey (Sanchez et al. 2004). Ephestia kuehniella eggs are produced by commercial insectaries and are frequently used as a dietary supplement to maintain certain predators, such as D. hesperus and M. caliginosus

(Gillespie and Mcgregor 2000, Callebaut et al. 2004).

Based on the previous studies referenced above, I chose to investigate mullein plant as an alternative host plant for the establishment of D. hesperus in tomato crops.

For the practical and effective use of D. hesperus, the predator must remain reproductively active throughout the cropping season. So, the main objectives of my research were to evaluate the establishment capacity of D. hesperus on different supplemental food sources so that it can control whitefly before pest populations reach unmanageable levels. In particular, I evaluated: 1) the survival and development of D. hesperus on mullein as a banker plant and on the main crop, tomato, with and without

E. kuhniella eggs as supplemental prey, under controlled conditions, and 2) the population development of D. hesperus under commercial greenhouse conditions.

Materials and Methods

Plants and Insects

Tomato plants (variety ’Lanai‘), supplied by the tomato breeding program at the

UF/IFAS Gulf Coast Research and Education Center (GCREC), Balm, FL, were grown from seed in 20-cm-diameter pots with Fafard 2 Mix (BWI-Apopka, Plymouth, FL 32768) soil potting mix in a greenhouse. Plants were watered as required and fertilized with

Jacks 20:20:20 All Purpose nutrient mix (Hummert International Company, 4500 Earth

City Expressway, MO 63045) (10 g for 3 liters) in 125 ml water every week. Mullein seedlings (Verbascum thapsus) were purchased from Gloeckner and Company (550

Mamaroneck Avenue, Harrison, NY) and were transplanted into 10-cm-diameter pots in a greenhouse at GCREC. Each mullein plant was provided with 20:20:20 water-soluble nutrient (10 g for 3 liters) in 125 ml water every 15 d.

A colony of D. hesperus, established from individuals originally purchased from

Beneficial Insectary (Redding, CA), was reared in a growth room (temperature 24-280C,

RH 48-55 %; L: D 12:12) on 6-week-old mullein plants contained in a Bug Dorm (60 ×

60 × 60 cm, MegaView Science Co. Ltd., Taiwan) (Figure 3-1). Ephestia kuehniella eggs, purchased from Beneficial Insectary, were provided attached to a 2 cm by 7.5 cm adhesive section of a yellow Post-It note (3M Corporation, St Paul, MN). Approximately

0.25 g of eggs (0.1 g Ephestia/100 D. hesperus) was provided to each Bug Dorm every week as a protein source (method developed by Chris Daye, Beneficial Insectary).

HOBO data loggers (Onset Technology Company, Pocasset, MA) were set up to record temperature and relative humidity.

Life Cycle of Dicyphus hesperus on Tomato and Mullein With and Without Ephestia eggs Under Growth Room

The development time of D. hesperus from 1st instar nymph to adult, survival of nymphs to adulthood, and adult longevity were evaluated on mullein and tomato plants with and without eggs of E. kuehniella as a dietary supplement. The four treatments evaluated were: 1) mullein with E. kuehniella eggs; 2) mullein without E. kuehniella; 3) tomato with E. kuehniella; and 4) tomato without E. kuehniella.

In order to produce individual D. hesperus 1st instar nymphs to track under the four treatments, five plants each of tomato and mullein of about 4 weeks old were kept separately in individual cages (35 x 35 x 60 cm) (Bio-Quip®, Compton, CA) in a growth room (temperature 24-280C; RH 48-55 %; L: D 12:12). Five pairs of male and female D. hesperus that had been adults for 1 week were released into each cage. Each cage was provided with 0.25 g E. kuehniella eggs on 2-cm adhesive strips every week. When a 1st instar nymph appeared on a mullein or tomato plant, it was removed and placed into a translucent round plastic box (5 cm height and 13 cm diameter) containing either a new tomato or mullein leaf, with or without E. kuhniella eggs, depending on the treatment. First instar nymphs produced by adults reared on tomato were used in the tomato treatments, and nymphs produced by adults reared on mullein were used in the mullein treatments. Leaf petioles were covered with wet cotton to maintain turgidity

(Figure 3-2 and 3-3). Eight replicates (1st instar nymphs) were selected for each treatment. The trial was repeated three times for a total of 24 replicates for each of the four treatments. The four rearing substrate treatments were arranged in a randomized complete block design in a growth room under the same conditions as described above.

Nymphs were observed every day and moved to a new leaf every 3-4 d. The date of

molting from one nymphal instar to the next was recorded according to the change in size and the presence of cast skin (Figure 3-4). Dates of molts were recorded until individuals reached the adult stage to calculate the days spent in each instar stage, and days required to complete development from nymph to adult under the four treatments.

In order to determine treatment effects on adult longevity, adults were kept on the same treatment until they died.

Population Development of Dicyphus hesperus on Mullein Plants With Ephestia Eggs Under Greenhouse Conditions

Two experiments were conducted to determine the rate of population development of D. hesperus and the potential for establishment on mullein with an

Ephestia supplement under actual greenhouse conditions. One trial was carried out in a commercial greenhouse in Wimauma, Florida and one trial was carried out in a greenhouse at GCREC (Table 3-1).

Trial at Commercial Greenhouse, Wimauma

Fifty-four gauze bags with bamboo sticks as support and binder clips to close the top were set up with one mullein plant (8-10 weeks old) in each cage in a 200 ft bay of a commercial greenhouse on June 14, 2016 (Figure 3-5). Cages were arranged in four blocks in the greenhouse. Thirteen cages were placed in the northeast and northwest corners of the bay, and fourteen cages were placed in the southeast and southwest corners of the bay. Five pairs of D. hesperus were placed in each cage provided with

Ephestia eggs that were attached to 2-cm adhesive section of a yellow Post-It note every week. Two weeks after setup, starting on June 14, two cages were sampled randomly from each corner each week for 7 weeks, with the last sample taken August

16. Dicyphus hesperus of all stages were aspirated from each cage and kept in ethanol vials. Later, the number and life stage of each individual from each cage was recorded.

Trial at GCREC Greenhouse

Single 5-6 weeks old mullein plants in 15-cm-diameter pots were maintained in cages (35 x 35 x 60 cm) (Catalog #1466BV, Bio-Quip®, Compton, CA). Ninety cages were set up in the greenhouse on August 17, 2016, arranged on six greenhouse benches in groups of 15, corresponding to six replications of the experiment (Figure 3-

6). Five pairs of male and female D. hesperus were placed on mullein in each cage.

About 0.25 g E. kuehniella eggs were placed in each cage each week. Starting on

September 1, one cage was sampled randomly from each block (bench) each week for

14 weeks, with the last sample taken December 2. Dicyphus hesperus of all stages were aspirated from each cage and kept in ethanol vials. Later, the number and life stage of each individual from each cage was recorded.

Population Development of Dicyphus hesperus on Mullein With and Without Ephestia eggs and on Tomato With Ephestia Eggs Under Greenhouse Conditions in Gainesville

The population development of D. hesperus on three different combinations of plant and supplemental food was studied in the greenhouse in Gainesville, FL (Table 3-

1). On August 14, 2017, 60 mullein plants and 30 tomato plants, 6-8 weeks old, were established individually in cages (35 x 35 x 60 cm) (Bio-Quip®, Compton, CA) for the following treatments: 1) mullein plant with Ephestia eggs, 2) mullein without Ephestia eggs, and 3) tomato plants with Ephestia eggs (Figure 3-7). Each treatment was replicated six times. Five pairs of 1-week-old D. hesperus males and females were introduced into each cage and treatments were arranged in a randomized complete block design with six blocks. After 3 weeks, one plant was randomly selected from each

of the three treatments in each of the six blocks. All nymphs and adults were collected carefully with the help of an aspirator from the cages and the plants and were counted.

Additional plants were selected from each treatment in each block on the sixth, ninth, twelfth and fifteenth week after introduction of the mated adult D. hesperus. A fresh 6- week-old plant was placed in each cage on week 12 without removing the original plant.

Experiment conditions were summarized (Table 3-1).

Statistical Analysis

Life history parameters of D. hesperus reared under four different treatments were compared with PROC GLIMMIX and TUKEY-HSD was used for mean separation

(SAS Institute 2001). Nymphal survival was analyzed by using PROC LOGISTIC (SAS

Institute 2001). Trial was set as a random factor in the analysis.

All the stages (1st, 2nd, 3rd, 4th and adult [male and female]) of D. hesperus were recorded separately for each of the 7 weeks from the commercial greenhouse data and

14 weeks from GCREC greenhouse data. Means of each life stage of D. hesperus were plotted to identify population trends. No statistical analysis was conducted.

The population development of D. hesperus in the Gainesville greenhouse experiment was compared among the three treatments with ANOVA using PROC

MIXED in SAS (SAS Institute 2001). Population trends were plotted over 15 weeks.

Mean separation was done by Tukey-Kramer in SAS.

Results

Life Cycle of Dicyphus hesperus on Tomato and on Mullein With and Without Ephestia eggs Under Controlled Conditions

There were significant differences in total nymphal development time among the treatments (F = 10.44; df = 2, 57; P < 0.0001) (Figure 3-8). The nymphal development

time of D. hesperus reared on mullein with E. kuehniella eggs and tomato with E. kuehniella eggs was shorter than on mullein without E. kuehniella eggs whereas D. hesperus did not reach the 3rd instar on tomato without E. kuehniella eggs (Figure 3-8).

The duration of the 1st instar did not differ with and without E. kuehniella eggs on tomato treatments but was significantly shorter on mullein with E. kuehniella than without (F =

5.20; df = 3,90; P = 0.0023). The duration of the 2nd instars did not differ among treatments (F = 1.92; df = 3,76; P = 0.133). The 3rd instar development time was significantly shorter when reared on mullein with E. kuehniella eggs compared to mullein without E. kuehniella eggs and tomato with E. kuehniella eggs (F = 8.06; df =

2,56; P = 0.0008) and the development of 4th instar differ among treatments (F = 23.50; df = 2,56; P < 0.0001) (Figure 3-8).

Survivorship of nymphs varied among the four treatments. The percentage of D. hesperus surviving on mullein and on tomato with E. kuehniella eggs was not significantly different but was significantly greater by 15% than the percentage surviving on mullein without E. kuehniella eggs (F = 1.31; df = 2,1; P = 0.5262) (Figure 3-9). Adult longevity of D. hesperus on mullein with and without E. kuehniella eggs and tomato with

E. kuehniella eggs differed significantly and was longest on mullein with E. kuehniella eggs than on tomato with E. kuehniella eggs; both were longer than on mullein without

E. kuehniella eggs (F = 39.75; df = 2,57; P < 0.0001) (Figure 3-9).

Population Development of D. Hesperus on Mullein Plants With Ephestia Eggs Under Greenhouse Conditions

In the commercial greenhouse in Wimauma, D. hesperus were reared on mullein with E. kuehniella eggs and population growth was studied for 7 weeks. The temperature recorded by HOBO in the bay throughout the 7-week duration of the

experiment ranged from 23.87 to 43.840C (Figure 3-10 A). High in temperature has a negative effect on nymphal development (Gillespie et al. 2004) and about 37.6% of the time during 7 wk duration of my study, the temperature was above 270C. The average number of 1st instar/2nd instar per sample (combined, as they were difficult to differentiate accurately) increased in the 3rd week and then declined at the end of 7th week whereas the average number of 3rd instar and 4th instar increased until the 4th week and then declined. The average number of adults increased until the 5th week and remained constant until the 7th week (Figure 3-11). Seven weeks after introducing five male and five female D. hesperus per plant, the average number per plant had increased 1.6-fold (Figure 3-11). The population initially increased 2 weeks after establishment when nymphs first began to emerge, and continued to increase up to the

5th week, after which the population decreased (Figure 3-11).

The population development of D. hesperus on mullein was observed in the

GCREC greenhouse for 14 weeks (Figure 3-12). Temperature conditions over the 14- week experiment ranged from 15.37 to 36.130C with 77.2% RH (Figure 3-10 B). About

37.6% of the time over 14 weeks, the temperature was over 270C that could have negative impact on population growth of predator. The average number of 1st /2nd instar

D. hesperus per sample (cage) reached up to 40 per sample during the 2nd and 3rd weeks and then decreased down to 10. Numbers of 3rd and 4th instars increased up to

15 in 4th week and again decreased to 6. The adult population maintained its density of approximately 15 per plant throughout the 14-week experiment. Total number of D. hesperus increased on 3rd to 4th weeks to 70 and then decreased to 35 and remained

constant (Figure 3-12). The number of D. hesperus had increased 3.5-fold per plant by the end of week 14 (Figure 3-12).

Population Development of Dicyphus Hesperus on Mullein With And Without Ephestia Eggs and on Tomato With Ephestia Eggs Under Greenhouse Conditions

The population development of D. hesperus was evaluated in a Gainesville greenhouse under three different treatments: mullein with and without E. kuehniella eggs, and on tomato with E. kuehniella eggs in the greenhouse. In previous trials, D. hesperus completed its life cycle on these three diets but not on tomato without a supplemental protein source (Figure 3-8). Therefore, tomato without E. kuehniella eggs was not evaluated in this trial. HOBO data loggers were set up to record the temperature, which averaged 25.30C, ranging from 16.2 to 34.80C over the 15-week experiment (Figure 3-10 C). About 26% of the time over 15 weeks the temperature was above 270C, which may have had a negative impact on predator nymphal development.

The development of D. hesperus was significantly different on three treatments (F =

8.24; df = 2, 64; P = 0.0007) and the averaged totals of different instars of D. hesperus for 15 weeks were calculated (Figure 3-13). The experiment was started with five male and five female adult D. hesperus per plant. Populations increased 5-fold on tomato with E. kuehniella eggs, and 2.5-fold on mullein with E. kuehniella eggs but did not increase on mullein without Ephestia eggs by the end of 15 weeks (Figure 3-13). The population increased gradually on tomato provided with supplemental food of E. kuehniella eggs. The population remained constant on mullein with E. kuehniella eggs for a while then increased after the 09h week while on mullein without E. kuehniella eggs, the predator maintained its initial population density (Figure 3-13).

Discussion

The availability of alternative prey sources on the host plants in the agroecosystem is beneficial for D. hesperus as they provide additional nutrients that help in their development and survival (Sanchez et al. 2004). The development time of

D. hesperus was shorter on mullein and tomato when provided with E. kuehniella eggs, similar to the results of previous research (Sanchez et al. 2004). We found that D. hesperus could not complete its life cycle on tomato plant without any prey sources which was also noted by Sanchez et al. (2004). Nymphal development duration was longer when the predator was reared on mullein without any supplemental food source.

Longer development time of D. hesperus may be disadvantageous for biological control because whitefly reproduction capacity is high (Jones 2003). Adult longevity was longer on mullein and tomato with a prey source, which clearly indicates that D. hesperus needed supplemental food to enhance reproduction (McGregor et al. 2000). In my laboratory experiment, 92% of D. hesperus were able to complete their life cycle on mullein and 88% of D. hesperus on tomato plant with E. kuehniella eggs but only 75% completed their life cycle on mullein without a prey source (Figure 3-6). The faster growth of D. hesperus on mullein and tomato plant with prey source is likely due to the presence of more nutritious food (i.e., E. kuehniella eggs). Using tomato plant as a banker plant may contribute to the early establishment of D. hesperus in main crops when provided with supplemental food source and also help to maintain predator populations even after prey populations decrease. Tobacco plants have been used as banker plants for early establishment of Macrolophus pygmaeus in tomato greenhouses. The reproduction of predator was highest in the presence of banker plants and tomato plants than just with tomato plants (Bresch et al. 2014).

In the commercial greenhouse study, the population development of D. hesperus on mullein with Ephestia eggs was recorded and population trends plotted. We found that the population did not grow during the 7-week period. The nymphs of D. hesperus emerge 10-14 d after oviposition and complete their nymphal development in 24-32 d

(Sanchez et al. 2003). Seven weeks may not have been sufficient time to allow the predator population to grow significantly.

In the GCREC greenhouse, the experiment was conducted for 14 weeks on mullein plants provided with supplemental food sources. The result showed that the population of the predator increased three-fold. The increase in the population in 4th week might be because of emergence of 1st instars. After the 4th week the population remained steady till the 14th week. Mullein can help maintain the predator population when E. kuehniella eggs are also provided. Although the predator can maintain their population on mullein only it would be more fruitful if a supplemental prey source was provided to increase the reproduction and predator population density. More the number of predators available, the greater the chances of pest population control.

In one of the previous studies, the development time of D. hesperus from Woody,

California and Summerland, British Columbia decreased with increase in temperature up to 270C. The mortality of D. hesperus nymphs increased in both populations when temperature increased from 27 to 350C (Gillespie et al. 2004). In Florida greenhouses, however, 24-h average temperature sometimes reaches up to 400C. An effect of extreme temperature on the mortality of nymphs and fitness of adult was likely to be a factor for low population growth of D. hesperus. In commercial greenhouse trial and in the GCREC greenhouse trial the temperature was above 270C about 37.5% of the time

and in Gainesville greenhouse trial, the temperature exceeded 270C about 26% of the time.

From the first development experiment conducted under controlled conditions, we found that D. hesperus can survive on mullein and tomato with prey and mullein without prey. So, I studied dynamics of D. hesperus on all three treatments in the greenhouse. Tomato plant with prey source (i.e. E. kuehniella eggs) serves as a best source for establishment of D. hesperus and mullein plant with E. kuehniella eggs also showed population growth but D. hesperus on mullein plant without E. kuehniella eggs could only maintain their population. Just to maintain the predator population, mullein can be good source but to increase the population of predators mullein plant alone will not be an option. This implies that mullein plants alone cannot be a banker plant system; it is the prey source provided to them that plays a vital role to establish predator population.

The lifecycle period of D. hesperus was shorter on mullein with E. kuehniella eggs than on tomato with E. kuehniella eggs but the population growth rate was twice as high on tomato with E. kuehniella eggs than on mullein with E. kuehniella eggs. This indicate that the fecundity could be higher on tomato than on mullein, similar to previous research where fecundity on tomato was 63 and on mullein was 49 (Sanchez et al.

2003). The other reason for increase in population on tomato plants supplemented with

E. kuehniella eggs might be due to high nutrition from prey. From my study I conclude that D. hesperus do not need alternative host plants as long as tomato plants have supplemental prey sources such as E. kuehniella eggs but in the absence of prey they cannot build their population. It would be effective to use supplement source in the

development of predator on tomato crop also on banker plant. Tomato plant can be a good banker plant for earlier establishment of D. hesperus in the presence of pest or alternative prey. Mullein plants can also be used as alternative host plants to conserve predator populations when the main crop is not available. Those plants can also influence the performance of D. hesperus for the biological control of pest. It is important to learn more about the predator establishment on different plant species.

Banker plant system has potential to improve biological control efficiency. Further research is needed to integrate adoption of biological control method with banker plant system.

Table 3-1. Different experiments conducted to evaluate the development of D. hesperus populations under greenhouse conditions.

Location Dates Daily average Replicates Treatments No. of Temp. (0C) avg. min- avg. Sampling max times

Commercial June 14 - Aug 24.56 – 32.43 8 Mullein plant with 7 greenhouse, 16, 2016 E. kuehniella Wimauma eggs GCREC Aug 17- Dec 1, 21.47 – 32.43 6 Mullein plant with 14 greenhouse, 2016 E. kuehniella Balm eggs Research Aug 14 - Oct 22.18 – 31.03 6 Mullein with E. 5 greenhouse, 27, 2017 kuehniella eggs Gainesville Mullein without E. kuehniella eggs Tomato with E. kuehniella eggs

Figure 3-1. Rearing of D. hesperus on mullein plants provided with E. kuehniella eggs in the growth room of GCREC. Photo courtesy of author.

Figure 3-2. Cage set-up for test of the effect of host plant and supplemental food on D. hesperus development. Five pairs of D. hesperus were kept in each cage and each cage represents one treatment (mullein plant with and without E. kuehniella eggs and tomato plants with and without E. kuehniella eggs). Experiment was conducted under controlled conditions in a growth room. Photo courtesy of author.

Figure 3-3. First instar D. hesperus was transferred to translucent round plastic box set up for development test. Each box represents one treatment (mullein plant with or without E. kuehniella eggs or tomato plants with or without E. kuehniella eggs). Mullein and tomato leaves were kept in each box (dependent on treatment) with wet cotton to maintain leaf turgidity. Experiment was conducted in the growth room. Photo courtesy of author.

0.48 cm

0.32 cm

0.16 cm

st th 1 instar 2nd instar 3rd instar 4 instar Adult

Figure 3-4. Sizes of different life stages of D. hesperus. Photo courtesy of author.

Figure 3-5. Experiment set up with 54 bags of mullien plant each with five male and five female of D. hesperus provided with E. kuehniella eggs in the commercial greenhouse, Wimauma. Photo courtesy of Hugh A. Smith.

Figure 3-6. Survival of D. hesperus was evaluated on mullein plant with E. kuehniella eggs. Ninety cages of mullein plant, each with five pairs of D. hesperus, were set up in the greenhouse of GCREC. Photo courtesy of author.

Figure 3-7. Evaluation of best dietary source for the development of D. hesperus was performed in a greenhouse in Gainesville by setting up five pairs of D. hesperus in 90 cages (30 of each of the three treatments: 1) mullein plant with E. kuehniella eggs, 2) mullein without E. kuehniella eggs, and 3) tomato with E. kuehniella eggs). Photo courtesy of author.

30 a Mullein with Ephestia b 25 Mullein without Ephestia b Tomato with Ephestia 20 Tomato without Ephestia

a b Number ofDays Number 10 c b ab a a b a ab a a a a 5

0 1st instar 2nd instar 3rd instar 4th instar Total nymphal development Different stages of D. hesperus

Figure 3-8. Average number of days spent in each instar and total nymphal development time of Dicyphus hesperus in four treatments: mullein plant with Ephestia eggs, mullein plant without Ephestia eggs, tomato plant with Ephestia eggs and tomato plant without Ephestia egg conducted in the growth room. Data shown are means ± SE and the letters a, b and c indicate significant differences within instar and total nymphal development time of D. hesperus (Tukeys, α < 0.05).

100 a a 90 Adult longevity 80 b 70 Survival % 60 50 40 A B 30 C 20

Number of days and Survival % andSurvival ofdays Number 10 0 Mullein with Mullein without Tomato with Tomato without Ephestia Ephestia Ephestia Ephestia

Figure 3-9. Adult longevity of D. hesperus reared from first instar nymphs on four treatments: mullein plant with Ephestia eggs, mullein plant without Ephestia eggs, tomato plant with Ephestia eggs and tomato plant without Ephestia eggs. Experiment conducted in the growth room of GCREC. Data shown are means ± SE and the letters A, B and C indicate significant differences within treatments for adult longevity and a, b and c for survival of D. hesperus (Tukeys, α < 0.05).

Figure 3-10. Average, minimum and maximum temperature of (A) commercial greenhouse (Wimauma) during the 7-wk duration, (B) GCREC greenhouse during the 15-wk duration and (C) Gainesville greenhouse over 15 weeks duration of experiments to determine establishment and population growth of D. hesperus. Four HOBO dataloggers, two in front and two in back side of greenhouse were set up and temperature was recorded over a 24-h period. Data shown are high and low temperatures averaged on a weekly basis.

30 1st & 2nd Instar

3rd Instar 25 4th Instar

Adults 20 per sampleper Total

15 D. hesperus D. 10

Number ofNumber 5

0 1 2 3 4 5 6 7 Weeks

Figure 3-11. Average number of different instars and total number of D. hesperus per sample over 7 weeks in a trial conducted in the commercial greenhouse in Wimauma, starting with five pairs of D. hesperus on each mullein plant provided with Ephestia eggs.

80 1st and 2nd instar

70 3rd instar 4th instar 60 Adults

per sampleper 50 Total no. D. hesperus 40

D. hesperus D. 30

20 Number ofNumber 10

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Weeks

Figure 3-12. Average number of different instars and total number of D. hesperus per sample over 14 weeks in a trial conducted in the GCREC greenhouse, starting with five pairs of D. hesperus on each mullein plant provided with Ephestia eggs.

100 a Tomato with Ephestia 90 Mullein with Ephestia a 80 Mullein without Ephestia

70 a per sampleper 60 a a 50

Dicyphus Dicyphus hesperus 30 a b 20 No. ofNo. b 10 b b

0 0 5 10 15 20 Weeks

Figure 3-13. Average number of D. hesperus per sample on each treatment (Tomato with Ephestia eggs, Mullein with and without Ephestia eggs) over 15 weeks. The experiment was completed in the greenhouse in Gainesville and data shown are means ± SE. The letters a, b and c describe significant differences in population size of D. hesperus on three different treatment each week.

CHAPTER 4 SUMMARY

Sweetpotato whitefly, Bemisia tabaci Gennadius, is a major pest worldwide in subtropical and tropical agriculture and also in greenhouse production. It has a wide host range recorded in more than 600 species and transmits numerous viruses including Tomato leaf curl virus. The effective control measures used are chemical insecticides, cultural methods and host-plant resistance. Bemisia tabaci MEAM 1 has developed resistance against many chemicals that lead to develop a biological control method. Dicyphus hesperus was evaluated as an effective predator against greenhouse whitefly, thrips and mites under the greenhouse condition. The predation potential of D. hesperus on B. tabaci eggs, early nymphs and later nymphs was studied by recording daily consumption and lifetime consumption of female D. hesperus on sweetpotato whitefly eggs and nymphs under controlled conditions. Predation was significantly higher on nymphs than on eggs and the predator survived for longer time when fed on whitefly nymphs than on eggs. The consumption rate of predator depends on the pest population density; if the pest population was high in density the consumption rate was also high and vice versa.

The lifecycle of D. hesperus was studied on mullein with and without Ephestia eggs and on tomato with and without Ephestia eggs under controlled condition. There was a significant difference among the treatments; the nymphal development time was shorter on mullein and tomato with Ephestia eggs and longer on mullein without

Ephestia eggs; the predator could not complete the lifecycle on tomato with Ephestia eggs. The establishment of D. hesperus was studied in the greenhouse on mullein with and without Ephestia eggs and on tomato with Ephestia eggs for 15 weeks. There was

a significant difference in population growth among treatments. Dicyphus hesperus population was more on tomato with Ephestia eggs than on mullein with Ephestia eggs and the predator just maintained their population on mullein without Ephestia eggs. The results indicate that the D. hesperus can establish in main crops when the pest is present or when provided with supplement prey source.

Overall, our results confirmed that D. hesperus can be effective control agent for

B. tabaci in the greenhouse tomato if the pest density is high. The consumption rate was significantly influenced by the different sweetpotato life stages as they preferred nymphs to eggs. This data can be useful for further greenhouse study about the potential of predator to control whitefly such as number of D. hesperus needed to control pest population in the greenhouse. Additionally, the interaction of D. hesperus with other biological agents as well as with other bio-pesticides for the control of pest can be next step. Our results showed that predator establishment was better on tomato than on mullein provided with supplemental prey source, which is useful to understand the predator growth in the presence of actual pest. Therefore, D. hesperus can be potentially considered as an effective predator for the control of B. tabaci in the greenhouse.

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BIOGRAPHICAL SKETCH

Pritika Pandey was born in Nepal. She received her M.S. in entomology and nematology from University of Florida in August of 2018 and B.S. in agriculture from

Institute of Agriculture and Animal Science, Tribhuwan University, Chitwan, Nepal. She showed her interest in entomology during her undergraduate program. After her B.S. graduation she worked as Livelihood officer in OXFAM International Government

Organization in Nepal where she conducted IPM trainings and plant protection work.

She was admitted to the University of Florida in 2016 and started her work under the supervision of Dr. Hugh A. Smith at Gulf Coast Research and Education Center

GCREC- Balm, FL. During her master’s study, she received two travel awards from

Graduate Student Council and GCREC travel grant. She wants to develop her career on researching insects and their ecology.