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CHARACTERIZING PHENOTYPIC AND GENETIC VARIATIONS IN THE INVASIVE CHILLI THRIPS, SCIRTOTHRIPS DORSALIS HOOD (THYSANOPTERA: THRIPIDAE)

By

VIVEK KUMAR

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2012

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© 2012 Vivek Kumar

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To my family I would have not succeeded if I had not failed

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ACKNOWLEDGMENTS

The list is countless, but I would like to begin by thanking my major advisor Dr.

Dakshina R. Seal who gave me the opportunity to work on this project. His constant

support, encouragement and kindle faith in me always boosted me and kept me focused

towards the goal. My sincere thanks to Vegetable IPM Lab members of TREC, Cathie

Sabines, Carlos, Charles Carter, and Jacinto who played important role in my projects

while being behind the scene. Special thanks to graduate committee member Dr. Cindy

L. McKenzie for her unwavering support and allowing me to use USDA-ARS facility,

without which completion of this project would have been difficult. I would like to extend

my thanks to other committee members Dr. David Schuster, Dr. Lance Osborne, Dr.

James Maruniak and Dr. Shouan Zhang for their valuable suggestions, constructive

criticism and guidance during the doctoral program. I am also grateful to Dr. Ale

Maruniak, John Prokop, and Michael Cartwright for helping me with insect molecular

techniques and Dr. Robert Shatters, Dr. Aaron M. Dickey for scientific advice to

understand the output. I owe sincere thanks to Dr. Wayne Hunter, Lyle Buss and

Thomas Skarlisnky who trained me in various photographic techniques and helped me

identify different thrips species. I thank Dr. Donald Hall, Dr. Heather McAuslane, Debbie

Hall and Maria Bernal for making me aware of the rules and departmental requirements

and updating me with the deadlines.

I also thank to my colleague and friends at University of Florida Deepak

Golasangimath, Megha Kalsi, Amit Gupta, Phalgun Nelaturu, Sudhamshu Acharya,

Ameya and Mithila Gondhalekar, Tamrat Wuletaw, Nichole Dobbs, Xiaodan Mo, Germo

Tatto for making life happy and easier. Great deal of thanks to Dr. Jeet and Seemanti

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Sengupta for encouragements, advice and friendly nature. I will always cherish those

moments.

I am indebted to my parents late Ram Lochan Jha and Kali Jha, and brothers-

Pramod Jha, Ashish Jha, Santosh Jha and other family members who showed

confidence and continuously encouraged me to complete my doctoral program. The

achievements and awards earned during this journey are dedicated to them. Special

thanks to my father-in-law Kuldeep Singh Kakkar, whose never ending challenges

motivated me to go for higher studies in this great nation. Words fail to express thanks

to my wife Garima Kakkar Jha, for her endless, unconditional love and support without

whom I could not have accomplished this task. Finally, I would like to thank almighty

God for giving me eternal life, his blessings and the path shown during my journey.

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TABLE OF CONTENTS page

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

LIST OF TABLES ............................................................................................................ 8

LIST OF FIGURES .......................................................................................................... 9

ABSTRACT ................................................................................................................... 11

CHAPTER

1 LITERATURE REVIEW .......................................................................................... 13

Introduction ............................................................................................................. 13

Background Information .......................................................................................... 14 Economic Host Plants ............................................................................................. 16

Geographical Distribution ........................................................................................ 17 Worldwide distribution ...................................................................................... 17

U. S. invasion ................................................................................................... 17 Host Damage .......................................................................................................... 19

Summary of Damage Symptoms ...................................................................... 20 Identification ............................................................................................................ 20

Life Cycle ................................................................................................................ 21 Management of S. dorsalis ..................................................................................... 22

Cultural Practices ............................................................................................. 22 Chemical Control .............................................................................................. 23

Biological Control ............................................................................................. 25

2 SCIRTOTHRIPS DORSALIS (THYSANOPTERA: THRIPIDAE): SCANNING ELECTRON MICROGRAPHS OF KEY TAXONOMIC TRAITS AND A PRELIMINARY MORPHOMETRIC ANALYSIS OF THE GENERAL MORPHOLOGY OF POPULATIONS OF DIFFERENT CONTINENTS .................. 36

Introduction ............................................................................................................. 36

Materials and Methods............................................................................................ 39 Identification of Specimens ............................................................................... 40

Scanning Electron Microscopy ......................................................................... 40 Morphometric Measurements of Major Body Traits .......................................... 41

Statistical Analysis ............................................................................................ 41 Results .................................................................................................................... 42

Identification of Specimens ............................................................................... 42 Morphometric Measurements of Major Morphological Features ....................... 43

Discussion .............................................................................................................. 45

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3 COUPLING SCANNING ELECTRON MICROSCOPY WITH DNA BAR CODING FOR MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF THRIPS ................................................................................................................... 68

Introduction ............................................................................................................. 68

Materials and Methods............................................................................................ 71 Morphological Identification .............................................................................. 71

Molecular Identification ..................................................................................... 72 PCR protocol and sequencing ................................................................... 72

Results and Discussion........................................................................................... 73

4 INTRAGENOMIC VARIATION IN mtCO1 AND rDNA ITS2 OF THREE MAJOR THRIPS SPECIES, SCIRTOTHRIPS DORSALIS, THRIPS PALMI AND FRANKILINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE) .................. 79

Introduction ............................................................................................................. 79 Materials and Methods............................................................................................ 84

Taxon Sampling ............................................................................................... 84 Morphological Identification of Thrips ............................................................... 84

DNA Processing ............................................................................................... 85 Sequence Alignment and Genetic Distance Matrix .......................................... 86

Results .................................................................................................................... 87 Inter- and Intragenomic Variation ..................................................................... 87

Parsimony Analysis of ITS2 .............................................................................. 90 Discussion .............................................................................................................. 90

Mitochondrial Cytochrome Oxidase I ................................................................ 90 Internal Transcribed Spacer 2 Variation ........................................................... 92

5 SUMMARY ........................................................................................................... 110

LIST OF REFERENCES ............................................................................................. 113

BIOGRAPHICAL SKETCH .......................................................................................... 129

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LIST OF TABLES

Table page 1-1 Plants infested with S. dorsalis as reported in global pest and disease

database. ............................................................................................................ 27

1-2 Confirmed plant hosts of Scirtothrips dorsalis in Florida. .................................... 33

1-3 Choices of insecticides for rotational use against S. dorsalis populations. ......... 35

2-1 Scirtothrips dorsalis populations by year collected, geographical location, host plant, preservative and specimen source.................................................... 50

2-2 Measurements of fourteen morphological characters from five different populations of Scirtothrips dorsalis. .................................................................... 51

2-3 Number of traits in which significant quantitative differences occurred between the various geographic populations of Scirtothrips dorsalis ................. 52

3-1 PCR amplification conditions for two genes........................................................ 77

4-1 Collection date, localities and hosts for specimens used in cloning of rDNA and mtCO1 genes of thrips species of three genera. ......................................... 96

4-2 PCR amplification conditions for two genes........................................................ 97

4-3 Number of clones sequenced and recovered haplotypes for the four individuals of each thrips species. ...................................................................... 98

4-4 The rDNA ITS2 sequences that differ among Scirtothrips dorsalis individuals. .. 99

4-5 The mtCO1 sequences that differ among Scirtothrips dorsalis individuals. ...... 101

4-6 The rDNA ITS2 sequences that differ among Thrips palmi individuals. ............ 102

4-7 The mtCO1 sequences that differ among Thrips palmi individuals. .................. 104

4-8 The rDNA ITS2 sequences that differ among Frankliniella occidentalis individuals. ........................................................................................................ 105

4-9 The mtCO1 sequences that differ among Frankliniella occidentalis individuals. ........................................................................................................ 106

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LIST OF FIGURES

Figure page 2-1 Slide mount of S. dorsalis female showing dark brown antecostal ridge (AR)

on tergites. .......................................................................................................... 53

2-2 Eight segmented antennae with third and fourth segments each possessing forked sensorium. ............................................................................................... 54

2-3 Dorsal view of S. dorsalis head with ocellar triangle, interocellar setae (IOS), hind ocelli (HO) and postocular setae (POS). ..................................................... 55

2-4 Pronotum of S. dorsalis exhibiting horizontal closely spaced sculpture lines...... 56

2-5 Posterior half of the metanotum presents longitudinal striations; medially located metanotal setae arise behind anterior margin. ....................................... 57

2-6 Shaded forewing of S. dorsalis is distally light in color with first and second vein possessing three and two widely spaced setae, respectively. .................... 58

2-7 Abdominal tergites III to VI of S. dorsalis possess small setae medially situated close to each other. ............................................................................... 59

2-8 The posteromarginal comb (row of microtrichia) on segment VIII is complete.... 60

2-9 Discal setae absent on sternites, sternites covered with rows of microtrichia with the exception of the antero-medial region. .................................................. 61

2-10 Simple D1 and funnel-shapped D2 setae on the head of a S. dorsalis larva. ..... 62

2-11 Funnel shaped setae on abdominal terga IX and X of a S. dorsalis larva. ......... 63

2-12 Reticulated pronotum of a S. dorsalis larva illustrating the presence of 6-7 pairs of pronotal setae. ....................................................................................... 64

2-13 Abdominal segments IV-VII of a S. dorsalis larva illustrating the presence of 8-12 setae each. ................................................................................................. 65

2-14 Forefemora of a S. dorsalis larva illustrating the presence of four funnel shaped setae on the distal two-thirds portion. .................................................... 66

2-15 Body of a S. dorsalis larva indicating the presence of granular plaques. ............ 67

3-1 Agarose gel showing PCR results using the ITS2 primers and mtCO1 primer set for the detection of S. dorsalis. .................................................................... 78

4-1 An unrooted semi strict MP tree generated from rDNA ITS2 sequence obtained from 2 female and 2 male individuals of S. dorsalis.. ......................... 107

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4-2 An unrooted semi strict MP tree generated from rDNA ITS2 sequence obtained from 2 female and 2 male individuals of T. palmi. .............................. 108

4-3 An unrooted semi strict MP tree generated from rDNA ITS2 sequence obtained from 2 female and 2 male individuals of F. occidentalis. .................... 109

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Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

CHARACTERIZING PHENOTYPIC AND GENETIC VARIATIONS IN THE INVASIVE

CHILLI THRIPS, SCIRTOTHRIPS DORSALIS HOOD (THYSANOPTERA: THRIPIDAE)

By

Vivek Kumar

May 2012

Chair: Dakshina R. Seal Major: Entomology and Nematology

Chilli thrips, Scirtothrips dorsalis Hood, has been a serious problem across the

globe. Detection of S. dorsalis larvae and adults on host plants is difficult due to the

thigmotactic behavior and tiny stature (< 2 mm in length) of the thrips. In this study, 14

different morphological characters of five different populations of S. dorsalis, collected

from two different continents were compared. The objective of the study was to

determine if certain morphological characters of S. dorsalis adults differ significantly in

size among populations from different geographic regions of the world. These

populations of S. dorsalis did not differ significantly for nine morphological characters,

but statistical differences were observed among five populations for 2-5 characters.

In order to make the identification of thrips simple and more accurate, a non-

destructive taxonomic characterization technique for thrips was developed for larvae

and adults. The method involves the integration of high resolution and magnification

capabilities of Scanning electron microscopy with the DNA bar coding technique. In this

protocol, a single specimen can be identified using both morphological and molecular

identification techniques. The thrips specimen is first identified using morphological

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characters is then further processed for DNA extraction and PCR based assays to

confirm the identification.

The genetic characterization of three economically important thrips species, S.

dorsalis, Frankliniella occidentalis (Pergande) and Thrips palmi Karny was conducted

using one nuclear encoded ribosomal DNA sequence (rDNA ITS2) and one

mitochondria encoded gene (mitochondrial cytochrome oxidase 1). High degree of

intragenomic variations were reported in the ITS2 sequences of the three species

suggesting that ITS2 copies in these thrips do not evolve in concert. Thus, this marker

region is not phylogenetically informative for population studies, and species-specific

PCR identification of the thrips species understudy. However, less intragenomic and

intraspecific variations were observed in conserved mtCO1 region of the three pest

species, indicating that this gene might be more useful than ITS2, in phylogenetic

characterization of the three thrips species. Results from different studies confirmed the

existence of morphological and genetic variation in population of S. dorsalis that

suggests the possibility of this species being a cryptic species complex.

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CHAPTER 1 LITERATURE REVIEW

Introduction

Of more than 5500 species of thrips described in the literature, 1% are considered

serious pests (Morse and Hoddle, 2006). Thrips are polyphagous insects that inhabit

tropical, subtropical and temperate regions of the world (Ananthakrishnan, 1993). The

small size and cryptic nature of thrips pests enable them to occupy microhabitats in the

field and within plants, often making monitoring and identification difficult. Thrips can

reduce crop yield or value indirectly by vectoring plant diseases and directly by using

agriculture products for food and reproduction. They can also act as irritants to the

public or field workers and can negatively affect global trade due to the quarantine risks

(Morse and Hoddle, 2006).

The scientific literature available on the economics of thrips mainly deals with four

thrips species: Thrips tabaci Lindeman, Frankliniella occidentalis (Pergande),

Scirtothrips dorsalis Hood and T. palmi Karny (Morse and Hoddle, 2006). Of these four

thrips species, less information is available on S. dorsalis in the USA owing to its recent

invasion. Since its establishment in Florida in 2005, S. dorsalis has emerged as an

economically important pest of ornamental plants and is considered a potential threat to

vegetable and fruit crops (Seal et al. 2010). In a recent study, Kumar et al. (2012)

reported this pest was economically damaging 12 different species of fruit plants at a

nursery in Miami Dade county of Florida. Nine of these species had not been previously

reported as hosts of this pest in the literature, suggesting that the host range of this pest

is increasing in this region.

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Scirtothrips dorsalis, commonly known as the chilli thrips, is a serious pest of

various vegetable, ornamental and fruit crops in southern and eastern Asia, Africa, and

Oceania (Ananthakrishnan 1993, EPPO 1997). The pest also vectors seven recorded

viruses including Chilli Leaf Curl (CLC) Virus, Peanut Necrosis Virus (PBNV), Tobacco

streak virus (TSV) Watermelon silver mottle virus (WsMoV), Capsicum chlorosis virus

(CaCV) and Melon yellow spot virus (MYSV) (Amin et al. 1981, Mound and Palmer

1981, Ananthakrishnan 1993, Rao et al. 2003, Chiemsombat et al. 2008). According to

the Florida Nurserymen and Growers Association, S. dorsalis is one of the thirteen most

dangerous, exotic pests threatening the industry (FNGLA 2003).

In 2010, the Florida horticultural greenhouse/nursery industry was valued at ca.

$1.74 billion (ERS-USDA 2011). In addition, potential host crops in Florida including

strawberries, peppers, peanuts, cucumbers, cotton, and blueberries were valued at an

additional 1 billion. Even a 10% loss of these products can cause significant impact on

Florida’s economy and may open the market for foreign trade (Derksen 2009).

Background Information

The great reproductive potential, invasion ability and easy adaptation to new areas

are a few of the qualities, which make Scirtothrips species major concerns of agriculture

in many countries (Hoddle et al. 2008). From the beginning, S. dorsalis has been

reported as an opportunistic generalist species that is able to feed on a variety of hosts,

depending upon availability in the region of incidence. From the beginning, S. dorsalis

has been reported as an opportunistic generalist species that is able to feed on a variety

of hosts, depending upon availability in the region of incidence. The first reference to S.

dorsalis was in 1890, when an unidentified thrips species was reported damaging the

tea crop in the Ceylon area of Assam state in India. Watt later supported this

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observation in 1898, but he was not sure that damage was being caused by one or two

species of thrips (Dev 1964). Further investigation confirmed that S. dorsalis was

responsible for damaging the tea crops in all of the major tea growing regions of eastern

India including Darjeeling, Cachar, the Assam Valley, Terai and the Dooars (Dev 1964).

In 1916, this pest was reported infesting castor in the Coimbatore district of the southern

part of India and later was found infesting other hosts in the region including chilli,

groundnuts, mango, beans, cotton, brinjal (Eggplant) and Casia fistula (Ramakrishna

Ayyar 1932, Ramakrishna Ayyar and Subbiah 1935). Young leaves, buds, and tender

stems of the host plants were severely damaged. Thrips repeated puncturing of tender

leaf tissues with their stylet produces ‘sandy paper lines’ on the epidermis of the leaves

and eventual crinkling of leaves. In India, the characteristic leaf curl damage caused by

this pest is known as “Murda” (Hindi meaning- dead body) disease, because

infestations resulted in the death of plants (Kulkarni 1922). Many different scientific

names have been assigned to S. dorsalis since it was first described in 1919 mainly

because of the lack of sufficient scientific literature regarding morphological differences

and variations in host range from the different geographical regions. During the last 100

years, the host range and the bio-geographical range of S. dorsalis have broadened.

The thrips is established in all of the habitable continents except Europe, where

repeated introductions have been intercepted and eliminated. Studying the history of S.

dorsalis aids in the understanding of behavioral and morphological diversity exhibited by

this species as a result of biological and ecological variations that have occurred duing

its migratory period.

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Economic Host Plants

Prior to the introduction of S. dorsalis into the New World, the host range of the

pest included more than 100 plant taxa among 40 families (Mound and Palmer 1981).

Subsequent to the introduction of S. dorsalis into the New World, the pest was found to

attack additional taxa of plants (Venette and Davis 2004). The main wild host-plants

belong to the family Fabaceae, which includes Acacia, Brownea, Mimosa and Saraca.

In its native range of the Indian subcontinent, chilli crops are reported to be attacked by

25 different pests, among which S. dorsalis is considered as one of the most serious

threats (Butani 1976). The pest is responsible for yield losses ranging from 61 to 74%

(Patel et al. 2009). Scirtothrips dorsalis is also abundant on Arachis in India (Amin

1980), sacred lotus in Thailand (Mound and Palmer 1981), and tea and citrus in Japan

(Kodomari 1978). Among the potential economic hosts of this pest listed by Venette

and Davis (2004) are banana, bean, cashew, castor, corn, citrus, cotton, cocoa, cotton,

eggplant, grapes, kiwi, litchi, longan, mango, melon, peanut, pepper, poplar, rose,

strawberry, sweet potato, tea, tobacco, tomato, and wild yams (Dioscorea spp.).

Interestingly, S. dorsalis is not reported reproducing on all of the hosts mentioned in the

literature. Most of the plant hosts were assigned to this pest based on the presence of

adults and their damaging potential. Thus, it is worthwhile to describe the host plant

infested with S. dorsalis as those used for feeding and/or reproduction. Based on

information obtained from the Global Pest and Disease Database (GPDD-2011), S.

dorsalis was reported to feed on (not necessarily reproduce on) more than 225 plant

taxa worldwide in 72 different families and 32 orders of plants (Table 1-1). In Florida, S.

dorsalis has been reported from 61 different plants to date (Table 1-2). Disparities in

host selection in different geographical regions are documented in the literature. For

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example, S. dorsalis is reported on mango in Puerto Rico but not in adjacent Caribbean

islands where it was reported earlier. Scirtothrips dorsalis is a significant pest of citrus in

Japan (Tatara and Furuhashi 1992) and Taiwan (Chang 1995), but not in India or the

United States. Many factors could be attributed to the differences in plant hosts of S.

dorsalis reported from different geographical regions. These various factors could

include variation in competition with other pests, availability of predators in the region of

invasion, availability of hosts, environmental conditions, etc. (Derksen 2009), but could

also be the result of differential biological activity of different S. dorsalis biotypes/cryptic

species, none of which have yet been reported.

Geographical Distribution

Worldwide distribution

Scirtothrips dorsalis is widely distributed along its native range in Asia including

Bangladesh, Brunei Darussalam, China, Hong Kong, India, Indonesia, Japan, Republic

of Korea, Malaysia, Myanmar, Pakistan, Philippines, Sri Lanka, Taiwan, and Thailand.

Further south S. dorsalis occurs in northern Australia and the Soloman Islands

(Macleod and Collins 2006). On the African continent, the pest is reported from South

Africa and the Ivory Coast, with plant health quarantine interceptions suggesting a wider

distribution across West Africa and East Africa (Kenya). Scirtothrips dorsalis is in Israel

as well as in the Caribbean including Jamaica, St. Vincent, St. Lucia, Barbados and

Trinidad. In western Venezuela, S. dorsalis has been found causing serious damage to

grapevine (Macleod and Collins 2006).

U. S. invasion

Changing climatic conditions and globalization have resulted in the increasing

importance of invasive species as recurrent problems around the globe. Pimentel et al.

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(2000, 2005), inferred that approximately more than 50,000 non indigenous species

have already been introduced in the United States, causing an estimated annual

damage of more than $120 billion in forestry, agriculture and other sectors of society.

The rich vegetation and neotropical climate of Florida make the state suitable for the

invasion and establishment of exotic flora and fauna (Ferriter et al. 2006). Scirtothrips

dorsalis is a newly introduced insect pest in Florida believed to have originated in

Southeast Asia. Since 1984, USDA-APHIS inspectors at various ports-of-entry have

intercepted S. dorsalis 89 times on imported plant materials belonging to 48 taxa, most

commonly on cut flowers, fruits and vegetables (USDA 2003). With the exception of

Hawaii, the presence of this tropical south Asian pest was not confirmed in the Western

Hemisphere until 2003. In Florida, S. dorsalis was reported from Okeechobee County in

1991 and from Highland County in 1994 but failed to establish a durable population

(Silagyi and Dixon 2006). In 2003, Tom Skarlinsky (USDA-APHIS-PPQ) reported live

larvae and pupae under the calyx of treated peppers in a shipment of Capsicum spp.

traced back to hot pepper production areas in St. Vincent and the Grenadines, West

Indies (Holtz 2006). Later, with the collaborative efforts of the USDA (APHIS) and IFAS

(University of Florida), S. dorsalis was found established in different agricultural districts

of St. Lucia and St. Vincent (Ciomperlik and Seal 2004), Barbados, Suriname, Trinidad

and Tobago, and Venezuela (Holtz 2006). In 2005, S. dorsalis was found on pepper and

‘Knockout’ Rose plants in retail garden centers in Florida and Texas. Subsequently, S.

dorsalis has been reported many times on different ornamental plants in commercial

nurseries throughout Florida (Hodges et al. 2005). In a collaborative survey over a two-

month period (Oct-Nov 2005), the Florida Department of Agricultural and Consumer

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Services (FDACS) and the University of Florida found infestations 77 times in 16

counties (Holtz 2006). Of the 77 positive observations, 66 were found on roses, 10 on

Capsicum and one on Illicium.

Venette and Davis (2004) projected the potential geographic distribution of S.

dorsalis in North America to extend from southern Florida to the Canadian border, as

well as to Puerto Rico and the entire Caribbean region. This suggests that this pest

could also become widely established in South America and Central America. The

small size (< 2 mm in length) and thigmotactic behavior of S. dorsalis make it difficult to

detect the pest in fresh vegetation, thus, increasing the likelihood of the transportation of

the pest through international trade of botanicals.

Host Damage

Scirtothrips dorsalis feeding on the meristems, terminals and other tender plant

parts of the host plant above the soil surface results in undesirable feeding scars,

distortion of leaves, and discoloration of buds, flowers and young fruits. The pest is not

reported to feed on mature host tissues. It possesses piercing and sucking mouthparts

and causes damage by extracting the contents of individual epidermal cells, leading to

the necrosis of tissue. The color of damaged tissue changes from silvery to brown or

black. The appearance of discolored or disfigured plant parts suggests the presence of

S. dorsalis. Larvae feed on the lower surfaces of young leaves, causing the leaves to

curl upward. According to Sanap et al. (1987), adults and larvae of S. dorsalis suck the

cell sap of the leaves, causing the leaves to curl upward. Severe infestations of S.

dorsalis cause the tender leaves and buds to become brittle, resulting in complete

defoliation and yield loss. For example, heavy infestations of pepper plants by S.

dorsalis cause changes in the appearance of plants termed “chilli leaf curl”. The

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appearance of discolored or disfigured plant parts suggests the presence of S. dorsalis.

Larvae feed on the lower surfaces of young leaves, causing the leaves to curl upward.

On many hosts, the thrips may feed on the upper surfaces of leaves when infestations

are high. Infested fruits develop corky tissues (Seal et al. 2006a). Sometimes, plants

infested by S. dorsalis appear similar to plant damaged by the feeding of broad mites

Summary of Damage Symptoms

Silvering of the leaf surface

Linear thickening of the leaf lamina

Brown frass markings on the leaves and fruits

Grey to black markings on fruits, often forming a distinct ring of scarred tissue

around the apex

Fruit distortion and premature senescence and abscission of leaves

Identification

Larvae of S. dorsalis are creamish white to pale in color. The sizes of the first

instars, the second instars, and the pupae range between 0.37-0.39, 0.68-0.71 and

0.78-0.80 mm, respectively (Seal et al. 2010). Adults are less than 1.2 mm in length with

dark wings. Dark spots form incomplete stripes seen dorsally on the abdomen (Seal et

al. 2010). There are numerous microtrichia and dark transverse antecostal ridges on the

abdominal tergites and sternites. Three discal setae are located on the lateral

microtrichial fields of the abdominal tergites.and the posteromarginal comb on VIII

segment is complete. The shaded forewings are distally lighter in color with

posteromarginal straight cilia on the distal half and the first and second veins bear three

and two widely spaced setae, respectively (Skarlinsky 2004, Hoddle et al. 2009).

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Detailed information about the morphological identification of S. dorsalis adults and

larvae are in Chapters 2 and 3.

Life Cycle

Thysanopterans have always been recorded as opportunistic species, as their

life history strategies are preadapted from the detriophagous ancestral group developed

in a habitat where optimal conditions of survival are brief (Funderburk 2001). Mating

does not result in fertilization of all the eggs and unfertilized eggs produce males while

fertilized eggs produce females. Sex ratio is in favor of female progeny (Dev 1964). The

stages of the life cycle of S. dorsalis include the egg, first and second instar larva,

prepupa, pupa and adult. Gravid females lay eggs inside the plant tissue (above the soil

surface) and eggs hatch between 5-8 days depending upon environmental conditions

(Dev 1964, Seal et al. 2010). Larvae and adults tend to gather near the mid-vein or

borders of the damaged portion of leaf tissues. Pupae are found in the leaf litter, on the

axils of the leaves, and in curled leaves or under the calyx of flower and fruits. Larval

stages complete in 8-10 days, and it takes 2.6-3.3 days to complete the pupal stage.

The life span of S. dorsalis is considerably influenced by the type of host they are

feeding. For example, it takes 11.0 days to become an adult from first instar larva on

pepper plants and 13.3 days on squash at 28°C. S. dorsalis adults can survive for 15.8

days on eggplant but 13.6 days on tomato plants (Seal et al. 2010). They can grow at

minimal temperature as low as 9.7°C and maximum temperature 33.0°C. Their thermal

requirement from egg to egg is 281-degree days and egg to adult is 265-degree days

(Holtz 2006). Population is multivoltine (having more than one generation per year) in

temperate regions of up to eight generations per year and 18 in warm subtropical and

tropical areas (Nietschke et al. 2008). They start egg laying in late March or early April

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when temperature is favorable for development (Shibao et al.1991) and first generation

adults can be seen from early May (Masui 2007a). However, S. dorsalis cannot

overwinter in regions where temperature remains below -4°C for five or more days

(Nietschke et al. 2008). Prolonged rainy seasons do not affect populations much, but

the population remains more abundant during prolonged dry conditions than in moist

rainy periods.

Management of S. dorsalis

Incursions of S. dorsalis are difficult to manage, and successful eradication is

possible only with early detection and implementation of management practices. Host

crops, which develop from, seeds such as bean, corn or cotton, must be carefully

monitored during the seedling stage of growth because this stage is extremely

susceptible to attack by S. dorsalis (Seal et al. 2010). Symptoms of infestations of S.

dorsalis must be monitored on their susceptible host plants like roses, pepper, cotton,

etc. twice per week and if symptom appears then samples should be sent to a reputable

laboratory for confirmation.

Cultural Practices

Development of effective management practices for S. dorsalis is still in its

infancy. The World Vegetable Center has several recommendations, which could serve

as basic management practices for the control of this pest. It involves crop rotation,

removal of weeds (which may serve as hosts), insecticide rotation and supporting the

maximum use of natural enemies including predators and parasites. In some of the

plant cultivars resistance to S. dorsalis feeding appears to exist. Presence of gallic acid

plays a crucial role in resistance to S. dorsalis in some varieties of the chilli plant (Holtz

2006). In Japan, synthetic reflective (vinyl) film has been used to protect citrus crops

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from S. dorsalis infestations (Tsuchiya et al.1995a). In another study, Tsuchiya et al.

(1995b) reported the use of white aqueous solution, i.e. 4% CaCO3 on mandarin orange

trees along with reflective-sheet mulching to provide better suppression in S. dorsalis.

Use of different colored sticky traps in monitoring and for thrips control has also been

evaluated. Tsuchiya et al. (1995c) found that yellowish-green, green and yellow sticky

board provided effective suppression in pest population on host plant. However,

yellowish-green was most effective in attracting S. dorsalis adults. In a recent study,

Chu et al. (2006) evaluated three different sticky cards (blue, yellow and white) for

sampling of S. dorsalis, and suggested yellow sticky cards could be used efficiently for

population detection and monitoring purposes of this pest.

Chemical Control

Chemical control is a primary mode of management of S. dorsalis and a wide

range of insecticides belonging to different chemical groups is currently used worldwide

to control this pest. In south-central Asia, chemical control is conducted using

quinalphos, dimethoate, phosphamidon and carbaryl. However, monocrotophos and

permethrin gave better suppression of this pest in India and Japan (Sanap et al. 1987,

Shibao 1997). Asaf-Ali et al. (1973) reported that malathion was effective against S.

dorsalis on grapevine. Since their introduction in the Greater Caribbean, there was a

paucity of information for effective management of this insect using modern insecticides.

Seal et al. (2006a) and Seal and Kumar (2010) showed the effectiveness of various

novel chemistries against S. dorsalis. Ten chemical insecticides belonging to seven

different mode of action classes (Table 1-3) provided effective suppression of the pest.

Bethke et al. (2010) suggested the rotational use of three or more insecticides from

different action classes could provide prolonged suppression of the pest population.

24

Pyrethroids have not been reported to provide effective control against S. dorsalis in the

New World. Although it causes an instant reduction in pest population, it also kills

natural controlling agents, ultimately leading to resurgence of pest populations. Various

formulations of imidacloprid, used as either soil drenches or foliar applications provides

control against S. dorsalis without harming any natural controlling agents. Its application

results in suppression of S. dorsalis populations for several days (Table 1-3) after

application of treatments.

Management practices from an ecological point-of-view must be environmental

friendly but from a growers’ viewpoint must be economical, fast acting as well as long

lasting. Different chemical insecticides that could satisfy all concerns, like spinetoram

and various neonicotinoid insecticides do cause significant reduction in S. dorsalis on

pepper crops (Seal et al. 2006b). However, due to the frequent use, insect pests are

under intense selection pressure to developed resistance against these insecticides.

There are many reports where excessive reliance on insecticide has resulted in

resistance development in this pest (Reddy et al. 1992). In India, Reddy et al. (1992)

reported resistance in S. dorsalis populations to a range of organochlorine (DDT, BHC

and endosulfan), organophosphate (acephate, dimethoate, phosalone, methyl-o-

demeton and triazophos) and carbamate insecticides (carbaryl). Recently, S. dorsalis

was reported to develop resistance against monocrotophos, acephate, dimethoate,

phosalone, carbaryl and triazophos (Vanisree et al. 2011). Thus, in order to forestall or

delay development of resistance or minimize the progressive assembly of genes for

resistance through selection in the pest against a particular chemistry, it is necessary to

rotate insecticides from diverse chemical groups, and explore alternative methods of

25

pest control. Inclusion of effective biorational and biological products in a best

management program for S. dorsalis can lead to reduced applications of synthetic

insecticides. Use of biorational and biocontrol products early in the season will delay the

buildup of damaging pest populations on host plants. Furthermore, reduction in the use

of harmful insecticides will increase the population of natural biocontrol agents.

Biological Control

Various biological controlling agents like minute pirate bugs, Orius spp.

(Hemiptera: Anthocoridae) and the phytoseiid mites Neoseiulus cucumeris and

Amblyseius swirskii have been reported to provide effective control of S. dorsalis on

pepper (Dogramaci et al. 2011, Arthurs et al 2009). Adults of Orius insidiosus feed on all

the life stages of thrips, and since it also feeds on aphids, mites, moth eggs and pollen,

its population does not decline when there are periodic drops in the thrips population.

Arthurs et al. (2009) evaluated two phytoseiid mites Neoseiulus cucumeris and

Amblyseius swirskii as potential biological control agents of S. dorsalis and reported A.

swirskii can be a promising tool in managing its population on pepper. Shibao et al.

(2004) showed the effect of predatory phytoseiid mites Euseius sojaensis in regulating

S. dorsalis population, on grapes in Japan. Chow et al. (2008) suggested the use of two

or more natural enemies as a strategy to improve biological control of greenhouse

pests. Predators that warrant further study as potential natural enemies of S. dorsalis

include lacewings (Chrysoperla spp.), several mird bugs, ladybird beetles, and a

number of predatory thrips including Franklinothrips vespiformis, the black hunter thrips,

the six spotted thrips (Leptothrips mali), Scolothrips sexmaculatus, the banded wing

thrips (Aeolothrips spp.), and predatory phytoseiid mites, Euseius hibisci and Euseius

tularensis.

26

Role of entomopathogens like Beauveria bassiana, Metarhizium anisopliae and

Isaria fumosorosea in managing field population of S. dorsalis are still under study.

Beauveria bassiana used with some adjuvants has been reported to control larval

populations of S. dorsalis for the first few days after application but soon the population

of S. dorsalis increases and becomes equivalent to the control plants (Seal and Kumar.

2010). Mikunthan and Manjunatha (2008) showed a significant reduction in S. dorsalis

populations using entomopathogens Fusarium semitectum in pepper fields. However,

commercialization and success of this biorational product in different biogeographical

regions is still in need of evaluation. Therefore, there is an immense need for

developing new strategies to employ best management practices for this serious pest

utilizing cultural, chemical and biological control methods.

27

Table 1-1. Plants infested with S. dorsalis as reported in global pest and disease database.

Plant order Plant family Scientific name Common or trade Name

Alismatales Araceae Colocasia esculenta (L.) Schott Taro Apiales Apiaceae Daucus carota L. Carrot Apiales Araliaceae Schefflera arboricola (Hayata) Merr. Dwarf schefflera, dwarf

umbrella tree Apiales Araliaceae Schefflera spp. J.R. and G. Forst. Schefflera Apiales Araliaceae Hedera helix L. English Ivy Apiales Pittosporaceae Pittosporum spp. Banks ex Gaertn. Cheesewood Apiales Pittosporaceae Pittosporum tobira (Thunb.) W. T.

Aiton Chinese pittosporum

Aquifoliales Aquifoliaceae Ilex crenata Thunb. Japanese holly Aquifoliales Aquifoliaceae Ilex integra Thunb. Mochitree Aquifoliales Aquifoliaceae Ilex spp. L. Holly Asperagales Alliaceae Allium cepa L. Onion Asperagales Alliaceae Allium sativum L. Garlic Aspergales Asparagaceae Asparagus officinalis L. Asparagus Asterales Asteraceae Brachyscome spp. Cass. Chrysanthemum Asterales Asteraceae Chrysanthemum morifolium Ramat. Chrysanthemum Asterales Asteraceae Coreopsis spp. L. Tickseed Asterales Asteraceae Dahlia pinnata Cav. Pinnate dahlia Asterales Asteraceae Dahlia spp. Cav. Garden dahlia Asterales Asteraceae Dimorphotheca aurantiaca DC., non

Horton African daisy, Cape marigold

Asterales Asteraceae Echinops echinatus Roxb. Indian globe thistle Asterales Asteraceae Helianthus annuus L. Sunflower Asterales Asteraceae Sonchus asper (L.) Hill Spiny sowthistle Asterales Asteraceae Tagetes erecta L. African marigold Asterales Asteraceae Tagetes patula L. Indian marigold, French

marigold Asterales Asteraceae Zinnia elegans L. Zinnia Brassicales Brassicaceae Brassica rapa L. var. silvestris

(Lam.) Briggs Colza

Brassicales Moringaceae Moringa oleifera Lam. Drumsticktree Caryophyllales Amaranthaceae Alternanthera sessilis (L.) R. Br. ex

DC. Sessile joyweed

Caryophyllales Amaranthaceae Amaranthus blitum L. Purple amaranth Caryophyllales Amaranthaceae Amaranthus lividus L. Amaranth Caryophyllales Amaranthaceae Amaranthus spp. L. Grain amaranth, Pigweed Caryophyllales Amaranthaceae Celosia argentea L. var. cristata (L.)

Kuntze Celosia

Caryophyllales Amaranthaceae Celosia spp. L. Cock’s comb Caryophyllales Chenopodiaceae Beta vulgaris L. Beetroot Caryophyllales Plumbaginaceae Limonium spp. Mill., nom. cons. Limonium Caryophyllales Plumbaginaceae Plumbago auriculata Lam. Cape leadwort Caryophyllales Polygonaceae Coccoloba uvifera (L.) L. Seaside grape Caryophyllales Polygonaceae Fagopyrum esculentum Moench Buckwheat Caryophyllales Polygonaceae Polygonum esculentum Knotweed Caryophyllaes Portulacaceae Portulaca oleracea L. Pigweed, Little hogweed,

purslane

28

Table 1-1. Continued

Plant order Plant family Scientific name Common or trade Name

Celastrales Celastraceae Euonymus japonica Thunb. Japanese spindletree Celastrales Celastraceae Euonymus spp. L. Euonymus Commelinales Commelinaceae Tradescantia zebrina hort. ex Bosse Wandering jew Cornales Hydrangeaceae Hydrangea spp. L. Hydrangea Cucurbitales Cucurbitaceae Citrullus lanatus (Thunb.) Matsum. &

Nakai Watermelon

Cucurbitales Cucurbitaceae Cucumis melo L. Cantaloupe Cucurbitales Cucurbitaceae Cucumis sativus L. Cucumber Cucurbitales Cucurbitaceae Cucurbita moschata (Duchesne ex

Lam.) Duchesne ex Poir. Pumpkin

Cucurbitales Cucurbitaceae Cucurbita pepo L. Pumpkin, Zucchini Cucurbitales Cucurbitaceae Cucurbita spp. L. Squash Cucurbitales Cucurbitaceae Momordica charantia L. Bitter gourd, bitter melon,

balsam apple Dioscoreales Dioscoreaceae Dioscorea spp. L. Yam Dipsacales Caprifoliaceae Viburnum awabuki K. Koch Viburnum Dipsacales Caprifoliaceae Viburnum odoratissimum Ker Gawl. China Laurestine Dipsacales Caprifoliaceae Viburnum plicatum Thunb. Japanese snowball Dipsacales Caprifoliaceae Viburnum suspensum Lindl. Viburnum Ericales Actinidiaceae Actinidia chinensis Planch. Chinese gooseberry Ericales Actinidiaceae Actinidia deliciosa [A. Chev.] C.F.

Liang et A.R. Ferguson

Kiwifruit

Ericales Balsaminaceae Impatiens spp. L. Impatiens Ericales Balsaminaceae Impatiens walleriana Hook. f. Super Elfin White Ericales Ebenaceae Diospyros kaki Thunb. Persimmon Ericales Ericaceae Pieris japonica (Thunb.) D. Don ex

G. Don Japanese-andromeda

Ericales Ericaceae Rhododendron spp. L. Azalea Ericales Ericaceae Vaccinium corymbosum L. Highbush blueberry Ericales Ericaceae Vaccinium spp. L Blueberry Ericales Sapotaceae Mimusops hexandra Roxb. Palu, rayan Ericales Theaceae Camellia japonica L. Japanese camelia Ericales Theaceae Camellia sasanqua Thunb. Sasanqua camellia Ericales Theaceae Camellia sinensis (L.) Kuntze Tea Ericales Theaceae Eurya japonica Thunb. Eurya Fabales Fabaceae Acacia arabica (Lam.) Willd. Acacia, babul Fabales Fabaceae Acacia auriculiformis A. Cunn. ex

Benth. Darwin black wattle

Fabales Fabaceae Acacia brownii (Poir.) Steud. Heath wattle Fabales Fabaceae Acacia nilotica (L.) Delile Acacia Fabales Fabaceae Acacia spp. Mill. Acacia Fabales Fabaceae Albizia odoratissima (L. f.) Benth. Ceylon rosewood Fabales Fabaceae Arachis hypogaea L. Peanut, groundnut Fabales Fabaceae Brownea spp. Jacq., nom. cons. Brownea Fabales Fabaceae Caesalpinia pulcherrima (L.) Sw. Pride-of-Barbados Fabales Fabaceae Dolichos biflorus L. Horse gram Fabales Fabaceae Dolichos lablab L. Hyacinth bean, labalab

bean Fabales Fabaceae Glycine max (L.) Merr. Soyabean

29

Table 1-1. Continued

Plant order Plant family Scientific name Common or trade Name

Fabales Fabaceae Melilotus indica (L.) All. Hubam clover Fabales Fabaceae Mimosa pudica L. Action plant, sensitive

plant Fabales Fabaceae Phaseolus vulgaris L. Common Bean, green

bean, snap bean Fabales Fabaceae Prosopis cineraria (L.) Druce Jand Fabales Fabaceae Prosopis spicigera L. Khejri Fabales Fabaceae Saraca indica L. Asoka tree Fabales Fabaceae Saraca minor Miq Kembang dedes Fabales Fabaceae Saraca spp. L. Saraca Fabales Fabaceae Tamarindus indica L. Indian tamarind Fabales Fabaceae Tamarindus spp. L. Tamarind Fabales Fabaceae Vicia angustifolia L. Narrow leaf vetch Fabales Fabaceae Vigna radiata (L.) R. Wilczek Mung bean Fabales Fabaceae Melanoxylon spp. Schott Melanoxylon Fabales Fabaceae Melanoxylum spp. Schott Brauna Fagales Fagaceae Castanea crenata Sieb. and Zucc. Japanese chestnut Fagales Fagaceae Quercus glauca Thunb. Ring cup oak Fagales Fagaceae Quercus virginiana Mill. Live oak Geraniales Geraniaceae Pelargonium ×hortorum L. H. Bailey Florist’s geranium Gentianales Apocynaceae Calotropis gigantea (L.) W. T. Aiton Calotropis Gentianales Gentianaceae Eustoma grandiflorum (Raf.)

Shinners Lisianthus

Gentianales Rubiaceae Gardenia jasminoides J. Ellis Jasmine Gentianales Rubiaceae Pentas lanceolata (Forsk.) Deflers Egyptian starcluster Gentianales Rubiaceae Pentas spp. Benth. Pentas Gentianales Rubiaceae Richardia brasiliensis Gomes Tropical Mexican clover Geraniales Geraniaceae Geranium spp. L. Geranium Ginkoales Ginkoaceae Ginkgo biloba L. Ginko Illiales Illiaceae Illicium floridanum J. Ellis Florida anisetree Lamiales Acanthaceae Odontonema strictum (Nees) Kuntze Firespike Lamiales Acanthaceae Strobilanthes dyerianus Mast. Persian shield Lamiales Oleaceae Jasminum multiflorum (Burm. f.)

Andrews Star jasmine

Lamiales Oleaceae Jasminum sambac (L.) Ait. Pikake Lamiales Oleaceae Ligustrum japonicum Thunb. Japanese privet Lamiales Plantaginacae Antirrhinum majus L. Snapdragon Lamiales Lamiaceae Coleus spp. Lour. Coleus Lamiales Lamiaceae Lamium barbatum Sieb. & Zucc. Dead nettle Lamiales Lamiaceae Ocimum basilicum L. Sweet basil Lamiales Lamiaceae Osmanthus heterophyllus (G. Don)

P. S. Green Holly-olive

Lamiales Lamiaceae Plectranthus scutellarioides (L.) R. Br.

Painted nettle

Lamiales Lamiaceae Salvia farinacea Benth. Mealycup sage Lamiales Verbenaceae Duranta erecta L. Golden dewdrops Lamiales Verbenaceae Duranta spp. L. Duranta Lamiales Verbenaceae Verbena spp. L. verbana

30

Table 1-1. Continued

Plant order Plant family Scientific name Common or trade Name

Lamiales Verbenaceae Glandularia ×hybrida (hort. ex Groenl. & Rümpler) G. L. Nesom & Pruski

Florist’s verbana

Laurales Lauraceae Laurus nobilis L. Sweet bay, laurel bay Magnoliales Annonaceae Annona squamosa L. Sugar apple Malphigiales Euphorbiaceae Breynia nivosa (W. Bull) Small Snowflower Malphigiales Euphorbiaceae Euphorbia pulcherrima Willd. ex

Klotzsch Poinsettia

Malphigiales Euphorbiaceae Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg.

Rubber

Malphigiales Euphorbiaceae Hevea spp. Aubl. Hevea Malphigiales Euphorbiaceae Manihot esculenta Crantz Cassava Malphigiales Euphorbiaceae Poinsettia pulcherrima (Willd. ex

Klotzsch) Graham Christmas flower

Malphigiales Euphorbiaceae Ricinus communis L. Castor bean Malphigiales Euphorbiaceae Ricinus spp. L. Ricinus Malphigiales Euphorbiaceae Sauropus androgynus (L.) Merr. Star-gooseberry Malphigiales Passifloraceae Passiflora edulis Sims Purple granadilla,

passion fruit Malphigiales Salicaceae Populus deltoides Bartr. ex Marsh. Eastern cottonwood Malvales Malvaceae Abelmoschus esculentus (L.)

Moench Okra

Malvales Malvaceae Gossypium herbaceum L. Arabian cotton, Levant cotton

Malvales Malvaceae Gossypium hirsutum Bourbon cotton Malvales Malvaceae Gossypium spp. L. Cotton Malvales Malvaceae Theobroma cacao L. Cocoa Myrtales Combritaceae Calycopteris floribunda Lam. Getonia Myrtales Combritaceae Laguncularia racemosa (L.) C. F.

Gaertn. White buttonwood

Myrtales Combritaceae Concocarpus erectus L. Myrtales Lythraceae Cuphea hyssopifolia Kunth Mexican heather Myrtales Lythraceae Cuphea spp. P. Browne Cuphea Myrtales Lythraceae Lagerstroemia indica L. Crapemyrtle Myrtales Lythraceae Punica granatum L. Pomegranate Myrtales Myrtaceae Syzygium malaccense (L.) Merr. and

L. M. Perry Malay-apple

Myrtales Myrtaceae Syzygium samarangense (Blume) Merrill & Perry

Water apple, wax apple

Myrtales Onagraceae Gaura lindheimeri Engelm. & A. Gray Lindheimer's beeblossom

Nymphaeales Nymphaeaceae Nymphaea pubescens Willd. Red water lily Nymphaeales Nymphaeaceae Nymphaea spp. L. Waterlily Pinales Podocarpaceae Podocarpus macrophyllus (Thunb.)

Sweet Yew plum pine

Pinales Podocarpaceae Podocarpus spp. L'Hir. ex Pers. Plum pine Poales Poaceae Zea mays L. subsp. Mays Corn Proteales Nelumbonaceae Nelumbo lutea L.Willd. Lotus Proteales Nelumbonaceae Nelumbo nucifera Gaertn. Sacred lotus Proteales Nelumbonaceae Nelumbo spp. Adans Lotus

31

Table 1-1. Continued

Plant order Plant family Scientific name Common or trade Name

Proteales Nelumbonaceae Nelumbo nucifera Gaertn. Sacred lotus Ranunculales Berberidaceae Mahonia bealei (Fortune) Carrière Leatherleaf mahonia, Kuo

Ye Shi Da Gong Lao Rosales Moraceae Ficus carica L. Common fig Rosales Moraceae Ficus elastica Roxb. ex Hornem. Rubberplant Rosales Moraceae Ficus spp. L. Ficus Rosales Moraceae Morus spp. L. Mulberry Rosales Rhamnaceae Zizyphus mauritiana Lam. Badari Rosales Rosaceae Amygdalus persica L. Peach Rosales Rosaceae Fragaria ananassa Duchesne ex

Rozier Strawberry

Rosales Rosaceae Fragaria chiloensis (L.) Mill. Strawberry Rosales Rosaceae Fragaria chiloensis (L.) P. Mill. Beach strawberry Rosales Rosaceae Fragaria chiloensis (L.) Mill. var.

ananassa (Duchesne ex Rozier) Ser. Strawberry

Rosales Rosaceae Fragaria virginiana Duchesne Strawberry Rosales Rosaceae Fragaria ×ananassa Duch. Garden strawberry Rosales Rosaceae Photinia glabra (Thunb.) Maxim. Japanese photonia Rosales Rosaceae Prunus avium (L.) L. Sweet cherry Rosales Rosaceae Prunus mume Siebold & Zucc. Japanese apricot Rosales Rosaceae Prunus persica (L.) Batsch Peach Rosales Rosaceae Prunus salicina Lindl. Japanese plum Rosales Rosaceae Prunus spp. L. Cherry, Stone fruit Rosales Rosaceae Pyracantha angustifolia (Franch.)

Schneid. Firethorn

Rosales Rosaceae Pyracantha spp. M. Roemer Firethorn Rosales Rosaceae Pyrus spp. L. Pear Rosales Rosaceae Raphiolepis indica (L.) Lindl. Indian hawthorn Rosales Rosaceae Raphiolepis umbellata Thunb. Yeddo Hawthorne Rosales Rosaceae Rhaphiolepis indica (L.) Lindl. ex Ker

Gawl. Shi Ban Mu

Rosales Rosaceae Rhaphiolepis umbellata (Thunb.) Makino

Japanese-hawthorn

Rosales Rosaceae Rosa chinensis Jacq. Chinese rose Rosales Rosaceae Rosa spp. L. Rose Rosales Rosaceae Rosa ×hybrida L. Knockout Radrazz rose Rosales Rosaceae Rubus spp. L. Blackberry, raspberry Sapindales Aceraceae Acer spp. L. Maple Sapindales Anacardiaceae Anacardium occidentale L. Cashew nut Sapindales Anacardiaceae Mangifera indica L. Mango Sapindales Anacardiaceae Mangifera spp. L. Mango Sapindales Rutaceae Citrus aurantiifolia (Christm.) Swingle Lime Sapindales Rutaceae Citrus limon (L.) Burm. f. Lemon Sapindales Rutaceae Citrus maxima (Burm.) Merr. Pummelo Sapindales Rutaceae Citrus paradisi Macfad. Grapefruit Sapindales Rutaceae Citrus reticulata Blanco var. unshiu

(Marco.) H. H. Hu Satsuma mandarin

Sapindales Rutaceae Citrus sinensis (L.) Osbeck Orange Sapindales Rutaceae Citrus unshiu Marc. Satsuma mandarin

32

Table 1-1. Continued

Plant order Plant family Scientific name Common or trade Name

Sapindales Rutaceae ×Citrofortunella microcarpa (Bunge) Wignands

Calomondin

Sapindales Rutaceae ×Citrofortunella spp. J. W. Ingram & H. E. Moore

Orangequat, citrangequat

Sapindales Rutaceae Fortunella spp. Swingle Kumaquat Sapindales Rutaceae Murraya paniculata (L.) Jack Orange-jasmine Sapindales Rutaceae Poncirus trifoliata (L.) Raf. Trifoliate orange Sapindales Rutaceae Zanthoxylum piperitum (L.) DC. Japanese-pepper Sapindales Sapindaceae Dimocarpus longan Lour. Longan Sapindales Sapindaceae Litchi chinensis Sonn. Litchi, lychee Sapindales Sapindaceae Nephelium lappaceum L Rambutan Sapindales Sapindaceae Nephelium litchi Cambess. Litchi Sapindales Sapindaceae Nephelium longana (Lam.)

Cambess. Longan

Saxifragales Hamamelidaceae Distylium racemosum Siebold & Zucc.

Isu-no-ki

Solanales Convolvulaceae Ipomoea batatas (L.) Lam. Sweet potato Solanales Solanaceae Capsicum annuum L. Chilli, pepper, Jalapeno

pepper, black pearl Solanales Solanaceae Capsicum annuum L. var. anuum Bell pepper Solanales Solanaceae Capsicum chinense Jacq. Bonnet pepper Solanales Solanaceae Capsicum frutescens L. Chilli Solanales Solanaceae Capsicum minimum Blanco Blanco pepper Solanales Solanaceae Datura fastuosa L. Devil's Trumpet Solanales Solanaceae Lycopersicon esculentum Mill. Tomato Solanales Solanaceae Lycopersicon spp. Mill. Tomato Solanales Solanaceae Nicotiana spp. L. Tobacco Solanales Solanaceae Nicotiana tabacum L. Tobacco Solanales Solanaceae Petunia spp. Juss., nom. cons. Petunia Solanales Solanaceae Petunia ×hybrida hort. ex E. Vilm. Petunia Solanales Solanaceae Solanum lycopersicum L. Tomato Solanales Solanaceae Solanum melongena L. Eggplant, aubergine Solanales Solanaceae Solanum nigrum L. Black nightshade Vioales Begoniacae Begonia spp.L. Begonia Vioales Violaceae Viola wittrockiana Gams Field pansy Vioales Violaceae Viola ×wittrockiana Gams Garden pancy Vitales Vitaceae Ampelopsis brevipedunculata

(Maxim.) Trautv. Porcelain berry

Vitales Vitaceae Ampelopsis spp. Michx. Peppervine Vitales Vitaceae Cayratia japonica (Thunb.) Gagnep. Yapu garashi Vitales Vitaceae Vitis pteroclada (Hayata) Hayata Vitis pteroclada Vitales Vitaceae Vitis vinifera L. Grape, grapevine Zingiberales Musaceae Musa spp. L. Banana

33

Table 1-21. Confirmed plant hosts of Scirtothrips dorsalis in Florida.

Scientific name Common or trade name

Antirrhinum majus L. Liberty Classic White Snapdragon

Arachis hypogaea L Peanut or groundnut

Begonia sp. Begonia

Breynia nivosa (W. Bull) Small Snow bush, snow-on-the-mountain

Camellia sinensis (L.) Kuntze Tea

Capsicum annuum L. Jalapeno pepper, Bonnet pepper

Capsicum frutescens L. Chilli pepper

Capsicum spp.

Celosia argentea L. Celosia – red fox

Citrus spp.

Concocarpus erectus

Coreopsis sp. Tickseed

Cuphea sp. Waxweed, tarweed

Duranta erecta L. golden dewdrop, pigeonberry, skyflower

Euphorbia pulcherrima Willd. Poinsettia

Eustoma grandiflorum (Raf.) Shinn. Florida Blue Lisianthus

Ficus elástica Roxb. Ex Hornem. Burgundy Rubber Tree

Gardenia jasminoides J. Ellis Jasmine

Gaura lindheimeri Engelm. & Gray Lindheimer’s beeblossom

Gerbera jamesonii H. Bolus ex Hook. F. Gerber daisy

Glandularia x hybrida (Grönland & Rümpler)

Neson & Pruski

Verbena

Gossypium hirsutum L. Cotton

Hedera helix L. English Ivy

Illicium floridanum Ellis Florida anisetree

Impatiens walleriana Hook. F. Super Elfin White

Jasminum sambac (L.) Ait. Pikake

Lagerstroemia indica L. Crape myrtle

Laguncularia recemosa (L.) Gaertn. f. White buttonwood

Ligustrum japonicum Thunb. Japanese privet

Litchi chinensis Sonn. Litchi

Mahonia bealei (Fortune) Carrière Leatherleaf mahonia

Manilkara zapota(L.) D. Royen Sapodilla

Mangifera indica L. Mango

Murraya paniculata (L.) Jack Orange-jasmine

Ocimum basilicum L. Sweet Basil

Pelargonium x hortorum Bailey Geranium

Pentas lanceolata (Forssk.) Deflers Graffiti White

Persea americana Mill. Avocado

1 Reprinted with permission of Seal and Kumar 2010.

34

Table 1-2. Continued

Scientific name Common or trade name

Petunia x hybrida Petunia Easy Wave Red

Pittosporum tobira (Thunb.) Ait. f. Variegated Pittosporum

Plectranthus scutellarioides (L.) R. Br. Coleus

Pouteria campechiana (Kunth) Baehni Canistel

Rhaphiolepsis indica(L.) Lindl. ex Ker Gawl. Shi Ban Mu

Ricinus communis L. Castor Bean

Rhaphiolepis umbellate (Thunb.) Mak. Yeddo Hawthorn

Richardia brasiliensis Gomes Brazil Pusley

Rhododendron spp. Azalea

Rosa X ‘Radrazz’ ‘Knockout®’ rose

Salvia farinacea Benth. Victoria blue

Schefflera arbicola (Hayata) Merr. Dwarf umbrella tree

Strobilanthes dyerianus Mast. Persian shield

Synsepalum dulcificum (Schumach. & Thonn.) Daniell Miracle fruit

Tagetes patula L. Marigold

Tradescatia zebrina hort. ex Bosse Wandering jew

Vaccinium corymbosum L. Highbush blueberry

Viburnum odoratissimum var. awabuki (K. Koch) Zabel Sweet viburnum

Viburnum suspensum Lindl. Viburnum

Viola x wittrockiana Gams Wittrock's violet

Vitis vinifera L. Grapevine

Zinnia elegans Jacq. Zinnia Profusion White

Sources: Silgayi and Dixon 2006, Klassen et al. 2008, Osborne 2009, Kumar et al. 2012

35

Table 1-32. Choices of insecticides for rotational use against S. dorsalis populations.

Common Name Trade Name IRAC Class Residual Control (days)

Foliar Soil

Adult Larva Adult Larva

Abamectin

Agrimek®, Avid

® 6 2

a 2

a

Acephate

Orthene® 1B 7

b 7

b 7

b 7

b

Chlorfenapyr Pylon® 13 7

a,b 7

a,b - -

Dinotefuran

Venom®, Safari® SG 4A 10 15 0 0

imidacloprid Marathon®, Provado

®,

Admire®

4A 15 15 15 15

Novaluron

Pedestal®, Ramon

® 15 7-14

a, b 7-14

a, b - -

Spinosad

Conserve®, SpinTor

® 5 15 15 _ _

Spinetoram

Radiant® 5 15 15 15 15

Thiamethoxam

Actara®, Platinum

® 4A 10 15 10 15

Borax + orange

oil + detergents

TriCon® 8D 10 10 - -

Beauveria

bassiana

Botanigard® Not applicable 3-7

b, c 3-7

b, c - -

Metarhizium

anisopliae

Met52® Not applicable 7

c 7

c - -

a Seal et al. 2006b.

b Bethke et al. 2010.

c Seal et al. 2007.

2 Reprinted with permission of Seal and Kumar 2010.

36

CHAPTER 2 SCIRTOTHRIPS DORSALIS (THYSANOPTERA: THRIPIDAE): SCANNING

ELECTRON MICROGRAPHS OF KEY TAXONOMIC TRAITS AND A PRELIMINARY MORPHOMETRIC ANALYSIS OF THE GENERAL MORPHOLOGY OF

POPULATIONS OF DIFFERENT CONTINENTS

Introduction

Scirtothrips dorsalis Hood commonly known as the Assam thrips, castor thrips,

chilli thrips, berry thrips or yellow tea thrips (Dev 1964, Asaf- Ali et al. 1973, Seal and

Klassen 2005, Masui 2007b) is a highly polyphagous adventive pest species that

originated in south Asia. With liberalization of trade in agricultural products and the

historic growth in tourism during the past three decades, this tropical and subtropical

pest has spread to all habitable continents except Europe. Scirtothrips dorsalis has

been intercepted numerous times on various flower, fruit and vegetable consignments

imported into Europe, but failed to establish a durable population on that continent

(Vierbergen and Gagg 2009). Recently an incursion was also noted in a glasshouse of a

botanical garden in the United Kingdom, but eradication measures were taken and the

pest was controlled successfully (A. Roy 2011; V. Kumar, personal communication). In

the Americas, S. dorsalis gained its first foothold in Venezuela, where it has been

causing damage to grapevine, Vitis vinifera L. (Vitaceae) since 2000(MacLeod and

Collins 2006, Seal et al. 2010). A few years later Skarlinsy (2003) found S. dorsalis

established in St. Vincent and subsequently this species became widely distributed in

the Lesser Antilles and Puerto Rico (Ciomperlik and Seal 2004) and Surinam

(Ciomperlik et al. 2005). In 2005, S. dorsalis became established in Palm Beach County

Florida on ‘Knockout’® rose (Rosa X ‘Radrazz’) (Coolidge 2005), and in three counties

in Texas. Currently, S. dorsalis has established in 30 counties in Florida and eight

counties in Texas with confirmations in Alabama and Louisiana in 2009 and New York

37

in 2010. In Florida, the pest rapidly distributed throughout the state via the retail trade

industry of nursery plants. Osborne (2009) found this pest reproducing on more than 50

plant species in Florida.

Detection of S. dorsalis larvae and adults in fresh vegetation is difficult due to their

thigmotactic behavior and tiny stature (larvae < 1 mm; adults < 2 mm). Eggs are

deposited within plant tissues and may take a week for the larvae to emerge.

Consequently, chances of transportation of S. dorsalis through state, regional, and

international trade of plant materials for all life stages is high (Seal and Kumar 2010). S.

dorsalis life stages occur on meristems and other tender tissues of all above ground

parts of the host plant. The feeding by this pest causes darkened scarring of extensive

areas on various plant parts, stunted growth of young leaves, reduced yield and

unmarketable fruit. Kuriyama et al. (1991) reported S. dorsalis to be a weak flier and

that the most important route of invasion into the greenhouse was by introduction of

infested pots but not aerial immigration. According to Meissner et al. (2005), the major

pathways of spread of this pest are air passengers, crewmembers and their baggage,

mail including mail delivered by express carriers, smuggled plant parts and windborne

dispersal.

The genus Scirtothrips comprises more than 100 species of thrips. S. dorsalis is

one of the most studied pests in the genus due to its economic importance and global

distribution. Because of the small size of this thrips and morphological similarities in the

genus, the identification to species is a challenge to non-experts. The morphological

traits of taxonomic importance for identification of S. dorsalis are well defined in the

literature. With slide mount images, Hoddle and Mound (2003) illustrated a taxonomic

38

identification key of S. dorsalis along with 20 other Scirtothrips species in Australia.

They noted that, of the 21 species of Scirtothrips, only S. aurantii and S. dorsalis have

microtrichial fields extending fully across the sternites. In S. aurantii, the microtrichia

almost cover the entire surface of the sternites, whereas in S. dorsalis they are

restricted to a complete band across the posterior half of each sternite. Thus, a clear

and accurate taxonomic characterization is required to distinguish between such

species of Scirtothrips. The taxonomic traits of S. dorsalis by Skarlinsky (2004) and

Hoddle et al. (2009) illustrated with thrips slide mount images are very helpful.

Nevertheless, photographs taken at higher magnifications and resolutions using

advanced techniques like Scanning Electron Microscopy (SEM) would be especially

helpful to research, regulatory and extension personnel and, also, for teaching. The

accurate and rapid identification of this invasive and potentially devastating pest is

essential to implement effective plant quarantine and integrated control strategies.

A significant pathway of invasive pests into the Caribbean from south Asia were

travelers whose ancestors had arrived from India as indentured servants following the

abolition of slavery (Klassen et al. 2002). Many of these families traveled back and forth

to visit relatives in India. Thus, the south Florida strain may have originated from the

populations in India. However, when measurements of morphological features of the

Florida 2009 strain were compared to measurements of a population in India reported

by Raizada (1976), five characters (body length, antennal length, prothorax length,

forewing length and hind wing length) out of nine characters studied by Raizada were

larger in the Florida (2009) strain. Conversely, three morphological characters (head

length, abdomen width and ovipositor length) of the Florida population of S. dorsalis

39

were smaller compared with the 1976 Indian population. Thus, there was merit in

making a preliminary comparison of the measurements of the morphological traits

selected by Raizada with corresponding measurements of traits of populations from

different continents and other widely separated locations.

The objectives of this study were a) to produce high-resolution images of

identifying characters of S. dorsalis adults and larvae using SEM, and b) to determine if

certain morphological characters of S. dorsalis adults differ significantly in size among

populations from different geographic regions of the world.

Materials and Methods

The year of sample collection, geographical location (longitude and latitude), host

plant, preservative, and sample source are reported in Table 2-1. Samples of S.

dorsalis were obtained from five geographic regions: New Delhi, India (2008); Shizouka,

Japan (2009); St. Vincent Island, West Indies (2006); Negev, Israel (2009); and Florida,

USA (2009). Florida population of S. dorsalis was collected from an established colony

in the greenhouse; New Delhi and St. Vincent populations were collected directly from

the field. However, no site collection information was provided about Negev and

Shizouka populations. The specimens from New Delhi, India reported by Raizada in

1976 were sampled from cotton (Gossypium hirsutum L.), castor (Ricinus communis L.),

pepper (Capsicum spp.) and other crops. Actual sample techniques depended on the

individual sampler, but, once adults were collected, they were immediately placed in 70-

95% ethanol and eventually mailed to the Tropical Research and Education Center,

UF/IFAS, Homestead, Florida where they were maintained at -20°C until processed for

slide mounting and morphometric analysis.

.

40

Identification of Specimens

Adult female thrips specimens were transferred to vials containing 75% alcohol for

10 min and then for 5 min to a 10% KOH (potassium hydroxide) solution prepared in

50% ethanol. While placed in KOH solution, the insect was gently pounded in the

abdominal region using a fine insect pin to aid the in the removal of its abdominal

contents. For gradual dehydration, the specimens were passed through a series of

alcohol concentrations starting from 65%, followed by 75%, 85%, 90% and 95% for 5-8

min in each concentration. Each specimen was placed ventrally on a slide with a small

drop of Hoyer’s mounting media and covered with a glass cover slip. The specimens

were identified and their morphological traits were compared using taxonomic

characteristics described by Hoddle and Mound (2003), Skarlinsky (2004) and Hoddle

et al. (2009) using a dissecting microscope at a minimum of 10X magnification.

Furthermore, S. dorsalis samples (adults and larvae) collected from Florida were

morphologically characterized using scanning electron microscopy to produce high

quality pictures, displaying the features used for identification. Larvae were identified

using the taxonomic keys of Vierbergen et al. (2010).

Scanning Electron Microscopy

Adult S. dorsalis females and larvae from Florida were collected in 30% ethanol

and dehydrated in a graded series of 50%, 70%, 95%, and twice in 100% ethanol for 30

min in each gradation. Samples were kept in 100% ethanol overnight. On the next d,

samples were dried in 50% and 100% of hexamethyldisilizane (HMDS:ethanol) for 30

min each. Dehydrated samples were placed on stubs consisting of black sticker using

fine forceps. Thereafter, samples were sputter coated with gold/palladium using the

41

Hummer sputtering system (Anatech, USA) and subsequently examined under a Hitachi

S-4800 SEM operated at 10-12 kV.

Morphometric Measurements of Major Body Traits

Fourteen morphological characters were studied in the five populations of S.

dorsalis and the results were compared with the measurements previously reported by

Raizada (1976). Raizada (1976) subjected nine characters of S. dorsalis adults to

morphometric analysis and in the present study; measurements of the pro, meso, and

meta thorax were added for comparisons.

Ten female specimens were selected from each of the five populations for

morphometric analysis. Specimens of S. dorsalis were placed individually on

microscope slides with a drop of water: ethanol (50:50) to protect the specimen from

dehydrating. The specimen was spread before measurements were taken. Fourteen

morphological traits were quantified by measuring the lengths of the body, antennae,

head, prothorax, mesothorax, metathorax, ovipositor, forewing and hind wing; and the

widths of the head, abdomen, prothorax, mesothorax, and metathorax (Table 2-2) using

an automontage advanced photography software program (Auto-Montage Pro software,

version 5.02, Syncroscopy, Frederick, MD) and a Leica MZ 12.5 stereomicroscope.

Statistical Analysis

Data on the measurement of various body parts of thrips pertaining to different

geographical regions were subjected to the square root (x + 0.25) transformation to

stabilize variance. Transformed data were analyzed using one-way analysis of variance

(ANOVA, SAS Institute Inc. 2003). The differences among means of length and width

of body segments were separated using Tukey’s HSD procedure (P < 0.05).

Untransformed means and standard errors are reported in Table 2-2.

42

Results

Identification of Specimens

The results of the scanning electron microscopy investigation are depicted in

Figures 2-2 to 2-9 (adult) and 2-10 to 2-15 (larva). Selected characters were used for

positive identification and morphological comparison of S. dorsalis populations prior to

morphometric analysis. The body of adult S. dorsalis is pale yellow in color and bear

dark brown antecostal ridges (AR) on tergites and sternites (Figure 2-1). The head is

wider than long, bearing closely spaced lineations and a pair of eight segmented

antennae with a forked sensorium on each of the third and fourth segments (Figure 2-

2). Of the three pairs of ocellar setae, the third pair, also known as the interocellar setae

(IOS), arises between the two hind ocelli (HO) (Figure 2-3) and is nearly the same size

as the two pairs of post ocellar setae (POS) on the head. The pronotum presents

closely spaced horizontal lineation (Figure 2-4). The pronotal setae (anteroangular,

anteromarginal and discal setae) are short and approximately equal in length. The

posteromarginal seta-II is broader and 1.5 times longer than the posteromarginal setae-I

and III. The posterior half of the metanotum presents longitudinal striations; medially

located metanotal setae arise behind the anterior margin and campaniform sensilla are

absent (Figure 2- 5). The forewings are distally light in color with posteromarginal

straight cilia on the distal half and the first and second veins bear three and two widely

spaced setae, respectively (Figure 2-6). Abdominal tergites III to VI, each present a pair

of small medially located setae (Figure 2-7). The posteromarginal comb on segment VIII

is complete and tergite IX exhibits medially located discal microtrichia (Figure 2-8).

Discal setae are absent on sternites and sternites are covered with rows of microtrichia,

excluding the antero-medial region (Figure 2-9).

43

Several morphological characters can be used for identification of S. dorsalis

larvae. D1 and D2 setae present on the head and abdominal terga IX of larvae are

simple and funnel-shaped, respectively (Figure 2-10 and 2-11), while the D1 setae on

terga X are funnel shaped. The larval pronotum is reticulated and has 6-7 pairs of

pronotal setae present (Figure 2-12). Abdominal segments IV-VII of larvae have a total

of 8-12 setae each (Figure 2-13). The distal two thirds of the forefemora of larvae

possess four funnel shaped setae (Figure 2-14) and the body of larvae possess

granular plaques (Figure 2-15).

Morphometric Measurements of Major Morphological Features

No significant differences were detected among the five S. dorsalis populations for

nine of the morphological characters measured in this study, i.e., body length, antennal

length, length of head, width of head, length of prothorax, width of prothorax, length of

ovipositor and lengths of the forewings and the hindwings (Table 2-2). However,

statistically significant differences were detected among the five populations for

mesothorax (length and width), metathorax (length and width) and width of abdomen

characters.

New Delhi, India (2008) Population. The population from New Delhi did not differ

significantly from the Florida, St. Vincent or Negev populations for any of the

morphological characters under consideration. Significant differences were detected

between this population and the population from Shizouka in that both the metathorax

length and abdomen width were smaller in the New Delhi population (Table 2-3).

The mean lengths of the antennae of the New Delhi (1976) population were

numerically very similar to those of the Negev (2009) population, but shorter than those

of all the other populations in this study. However, the differences in the measurements

44

of all the remaining traits between the New Delhi (1976) and the other populations in

this study were numerically small.

Florida, USA (2009) Population. The Florida population was not significantly

different from the populations of New Delhi or Negev for any of the 14 morphological

characters that were measured (Table 2-3). The Florida population was characterized

by a significantly smaller metathorax than the St. Vincent population, but there were no

significant differences between these two populations for the other 13 characters that

were measured. Five morphological characters of the Florida population were

significantly smaller than the Shizouka population. These differences were most

significant with respect to the lengths and widths of the mesothorax, metathorax and

width of abdomen (Table 2-3).

St. Vincent Island, West Indies (2006) Population. The St. Vincent population

had a significantly longer metathorax than the populations from Florida and Negev and

a significantly narrower abdomen compared with Negev and Shizouka populations.

Shizouka, Japan (2009) Population. The S. dorsalis population from Shizouka

differed significantly from the other four populations for 2 or 5 morphological characters,

depending on the population, suggesting that the Japan population is more robust (i.e.,

mesothorax and metathorax are longer and wider, and abdomen is wider) (Table 2-3).

The metathorax was longer and the abdomen was wider than the New Delhi population.

Further, the Shizouka population had a wider mesothrorax, metathrorax and abdomen,

and longer mesothrorax and metathorax than the Florida population. The metathorax

and abdomen of Shizouka were also wider than the St. Vincent population.

45

Furthermore, the Shizouka population mesothorax was wider and metathorax longer

compared with the Negev population.

Negev, Israel (2009) Population. The population from Negev did not differ

statistically from the New Delhi (2008) and Florida (2009) populations in any of the

morphological traits under study, but differed significantly in two features with St.

Vincent (metathorax length and abdominal width) and Shizouka populations

(mesothorax width and metathorax length).

Discussion

Since its development in the early 1950’s, SEM is considered as an efficient

morphological identification tool with numerous advantages over traditional microscopy;

i) large depth of field, ii) much higher resolution than light microscopy and iii) because

SEM uses electromagnetism instead of lenses, specimens can be magnified to much

higher levels. Thus, the observer has better control of the degree of magnification of the

specimen understudy (Schweitzer 2010). Nevertheless, the use of SEM in taxonomic

characterization of Thysanopteran insects is limited. High-resolution pictures of S.

dorsalis using the SEM techniques will help research, regulatory and extension

personnel to identify this pest with great ease without extensively using complex

taxonomic keys. SEM produced figures provide information about all the major

identification characters of this pest. Two species of the genus Scirtothrips, S. aurantii

and S. dorsalis are unusual in having sternites covered with microtrichia (Hoddle and

Mound 2003, Hoddle et al. 2009). SEM figure 2-9 clearly shows the band of

microtrichia continuous only across the posterior half of the S. dorsalis sternite, a

feature that differentiates this pest not only from S. aurantii, but, insofar as is known,

from all other species of Scirtothrips.

46

No major differences were observed in the body lengths of S. dorsalis adults

recently collected from the five regions. Mean body lengths ranged from 0.85 mm

(Negev population) to 0.98 mm (Florida population). The mean length of adults

collected from New Delhi, India in 2008 was greater than the length previously reported

by Raizada (1976) (0.91 and 0.76 mm, respectively). The mean body length of the

Florida (2009) population is 0.223 mm longer than that of the New Delhi (1976)

population. Likewise, the mean antennal length of the Florida (2009) population of S.

dorsalis is 0.016 mm longer than that of the New Delhi (1976) population. These

differences may be attributed to a possible role of the feeding and reproductive hosts in

regulating body size of the pest.

Insects regulate their body size in response to the temperature surrounding them,

which is often associated with elevation and latitude (Blanckenhorn and Demont 2004,

Tantowijoyoa and Hoffmann 2011). Some studies suggest that there can be positive

correlations between elevation and latitude and body size (Smith et al. 2000). Other

studies suggest there is no correlation, or even a negative correlation between sizes of

body traits and elevation and latitude (Kubota et al. 2007, Hawkins and DeVries 1996).

Variation in size can affect fitness traits like development and reproduction (Berger et al.

2008, Tammaru et al. 2002), somatic and sexual growth (Blanckenhorn 2006, Fischer et

al. 2003), thermoregulation (Bishop and Armbruster 1999) and dispersal ability

(Gutierrez and Menendez 1997). At high temperatures, some populations of thrips

species acquire a smaller and paler form, and at low temperatures, they tend to be large

and dark in color (Murai and Toda 2001, Hoddle et al. 2009). However, results from the

present study did not suggest any significant impact of temperature on body size of S.

47

dorsalis collected from the five regions, but no detailed information on temperatures

during the times of collection were provided.

According to Björkman et al. (2009) body size within certain taxonomic groups

tends to increase with latitude (Bergmann clines), while in certain other groups insect

body size decreases (converse Bergmann clines), and in yet others tends to stay

relatively constant with latitude. In this study, the populations were from the following

latitudes: St. Vincent, West Indies: 13.35 N; Homestead, Florida: 25.48 N; Delhi, India:

28.38 N; Negev, Israel: 34.67 N; and Shizouka, Japan: 36.00 N. On comparing the

measurements of individuals from five regions, the body lengths of S. dorsalis in these

samples did not vary either directly or inversely with latitude (Table 2-2).

All of the specimens recently obtained from New Delhi, Florida, St. Vincent and

Negev were collected from pepper, but the Shizouka thrips population were collected

from tea. Since tea was not a host plant of the other four populations sampled in this

study, it could not be determined if host plants directly affected morphology.

Nevertheless, the Shizouka population differs significantly from the other populations by

having a longer and wider mesothorax and metathorax and an abdomen that is wider,

which is an essential morphological character of females that allows them to produce

more eggs and have higher fecundity and greater fitness (Benitez et al. 2010). In a

concurrent experiment conducted to determine the effect of host plant on the growth of

S. dorsalis, the pest was reared under identical conditions at Homestead, Florida on six

different hosts, i.e., ‘Jalapeño’ pepper (Capsicum annuum L.), ‘Pod Squad’ bean

(Phaseolus vulgaris L.), ‘Black Beauty’ eggplant (Solanum melongena L.), ‘Butternut’

squash (Cucurbita moschata [ex Duchesne Lam.] Duchesne ex Poir.), ‘Solar Set’

48

tomato (Solanum lycopersicum L.) and ‘Knockout’ rose (Rosa chinensis Jacq.) (Seal et

al. 2010). Body lengths and widths of different development stages (10 individuals each)

were measured, and no significant differences in body size of this pest were observed

when S. dorsalis was reared on these six different hosts, indicating that these host plant

species did not differentially induce size alterations in S. dorsalis.

The results from the present study suggest that the Japan population may have

diverged the most from the ancestral population in south Asia. The Japan population

does not appear to be ancestral to the populations in the Americas or Israel based on

morphometric differences. The population in India may be ancestral to the populations

in the New World and Israel and could have been facilitated by the extensive movement

of people between India and Israel, and India and the Caribbean. In addition, Israel has

commercial horticultural ventures in the Greater Antilles. Thus, movement of S. dorsalis

from India to Israel and subsequently to the Caribbean cannot be ruled out. It would be

interesting to determine whether there are substantial genetic or behavioral differences,

and even barriers to reproduction, between the Shizouka and New Delhi populations as

well as between the Shizouka and Florida populations.

Using the automontage system did not appear to introduce inordinately large

errors caused by failure to have each specimen mounted perfectly within the horizontal

plane. Each measurement was repeated on 10 different individuals, and it is well known

that small experimental errors tend to cancel each other. Morphometric analysis is an

efficient tool that is being utilized for identification, determination of larval instar, and

discrimination of cryptic species of several insect species including leaf miners, bees,

beetles, and aphids, (Daly 1985, Ellis and Ellis 2008, Favret 2009, Shibao 2004,

49

Tantowijoyoa and Hoffmann 2011), but has rarely been utilized for thrips. This is

because thrips body size and color are known to be phenotypically plastic in response

to changing environments, which can occur across both small and large spatial scales

(Sakimura 1969, Murai and Toda 2001, Mound 2005). It may be important to collect live

populations from different regions and rear them under identical environmental

conditions in order to ascertain if the apparent stability of morphological traits has a

genetic basis. Future research will concentrate on direct correlation of morphometric

analyses with molecular analyses of the same individual to validate the hypothesis that

the Japan population is not ancestral to populations in the four other regions.

50

Table 2-1. Scirtothrips dorsalis populations by year collected, geographical location, host plant, preservative and specimen source.

aPopulation previously reported by Raizada (1976)

bMexican marigold (Tajetes erecta L.), pepper (C. annum), cotton (Gossypium spp.), castor (Ricinus communis L.), and many others

Year

collected

Geographical location

(latitude and longitude) Host Preservative Source of specimens

1976a India- New Delhi; 28.38 N, 77. 12 E Various

b 70% alcohol Dr. Usha Raizada, University of Delhi, India

2008 India- New Delhi; 28.38 N, 77. 12 E Pepper 95% ethanol Dr. D. R. Seal, University of Florida,

Homestead, Florida, USA

2006 St. Vincent Island, West Indies; 13.15 N,

61.12 W Pepper 70% ethanol

Mr. M. L. Richards, Ministry of Agriculture

and Fisheries, St. Vincent and the

Grenadines, West Indies

2009 United States- Homestead, Florida;

25.28 N, 80.28W Pepper 70% ethanol

Mr. V. Kumar and Dr. D. R. Seal, University

of Florida, Homestead, Florida, USA

2009 Israel- Negev; 30.50 N, 34.91 E Pepper 95% ethanol

Dr. Phyllis Weintraub, Entomology Unit,

Gilat Research Center, D. N. Negev 85280,

Israel

2009 Japan- Shizouka; 34.55 N, 138.19 E Tea 95% ethanol

Dr. Masui Shinichi, Shizuoka Prefectural

Research Institute of Agriculture and

Forestry Tea Research Center, Japan

51

Table 2-2. Measurements of fourteen morphological characters from five different populations of Scirtothrips dorsalis.

All data were analyzed using analysis of variance (ANOVA) procedures with the exception of the India (1976) population previously reported by

Raizada (1976).

Means were separated using Tukey test at the 0.05 level of significance. Means followed by same letter within a row are not significantly different

(P ≤ 0.05).

Morphological

Character

Morphological Character Mean (mm) ± SEM by Population (year collected)

India (1976) New Delhi India

(2008)

Florida (2009) St. Vincent Island

(2006)

Shizouka

Japan (2009)

Negev Israel

(2009)

Body L 0.757 ± 0.021 0.912 ± 0.025a 0.980 ± 0.30a 0.871 ± 0.015a 0.873 ± 0.015a 0.856 ± 0.029a

Antennal L 0.189 ± 0.001 0.198 ± 0.002a 0.206 ± 0.005a 0.196 ± 0.004a 0.218 ± 0.005a 0.182 ± 0.004a

Head L 0.066 ± 0.002 0.052 ± 0.004a 0.054 ± 0.002a 0.062 ± 0.002a 0.060 ± 0.002a 0.054 ± 0.002a

Head W 0.119 ± 0.002 0.120 ± 0.003a 0.111 ± 0.001a 0.120 ± 0.002a 0.120 ± 0.002a 0.122 ± 0.006a

Prothorax L 0.086 ± 0.002 0.086 ± 0.005a 0.098 ± 0.003a 0.088 ± 0.004a 0.099 ± 0.003a 0.098 ± 0.003a

Prothorax W ----- 0.142 ± 0.003a 0.141 ± 0.003a 0.146 ± 0.003a 0.147 ± 0.004a 0.145 ± 0.003a

Mesothorax L ----- 0.055 ± 0.004ab 0.043 ± 0.001b 0.048 ± 0.004ab 0.056 ± 0.003a 0.048 ± 0.002ab

Mesothorax W ----- 0.164 ± 0.004ab 0.149 ± 0.004b 0.166 ± 0.008ab 0.179 ± 0.006a 0.153 ± 0.006b

Metathorax L ----- 0.098 ± 0.003bc 0.097 ± 0.002c 0.108 ± 0.003ab 0.112 ± 0.006a 0.097 ± 0.004c

Metathorax W ----- 0.166 ± 0.004ab 0.160 ± 0.002b 0.162 ± 0.004b 0.181 ± 0.005a 0.176 ± 0.006ab

Abdomen W 0.200 ± 0.004 0.192 ± 0.003bc 0.193 ± 0.005bc 0.182 ± 0.005c 0.213 ± 0.005a 0.207 ± 0.005ab

Ovipositor L 0.156 ± 0.003 0.146 ± 0.003a 0.140 ± 0.004a 0.146 ± 0.004a 0.139 ± 0.004a 0.134 ± 0.005a

Forewing L 0.511 ± 0.016 0.514 ± 0.011a 0.523 ± 0.012a 0.512 ± 0.010a 0.509 ± 0.012a 0.513 ± 0.017a

Hind wing L 0.441 ± 0.012 0.458 ± 0.011a 0.467 ± 0.011a 0.460 ± 0.012a 0.459 ± 0.013a 0.461 ± 0.017a

52

Table 2-3. Number of traits in which significant quantitative differences occurred between the various geographic populations of Scirtothrips dorsalis when compared two at one time.

India 2008 Florida 2009

St. Vincent 2006

Israel 2009 Japan 2009

India 2008 X 0 0 0 2 Metathorax L Abdomen W

Florida 2009 0 X 1 0 5 Metathorax L Mesothorax L

Mesothorax W Metathorax L Metathorax W Abdomen W

St. Vincent

2006 0 1 X 2 2 Metathorax L Metathorax L

Abdomen W Metathorax W Abdomen W

Israel 2009 0 0 2 X 2

Metathorax L Abdomen W

Mesothorax W Metathorax L

Japan 2009 2 5 2 2 X

MetathoraxL Abdomen W

Mesothorax L Mesothorax W Metathorax L Metathorax W Abdomen W

Mesothorax W Abdomen W

Mesothorax W Metathorax L

53

Figure 2-1. Slide mount of S. dorsalis female showing dark brown antecostal ridge (AR) on tergites.

54

Figure 2-2. Eight segmented antennae with third and fourth segments each possessing forked sensorium.

55

Figure 2-3. Dorsal view of S. dorsalis head with ocellar triangle, interocellar setae (IOS), hind ocelli (HO) and postocular setae (POS).

56

Figure 2-4. Pronotum of S. dorsalis exhibiting horizontal closely spaced sculpture lines.

57

Figure 2-5. Posterior half of the metanotum presents longitudinal striations; medially located metanotal setae arise behind anterior margin, campaniform sensilla are absent.

58

Figure 2-6. Shaded forewing of S. dorsalis is distally light in color with first and second vein possessing three and two widely spaced setae, respectively.

59

Figure 2-7. Abdominal tergites III to VI of S. dorsalis possess small setae medially situated close to each other.

60

Figure 2-8. The posteromarginal comb (row of microtrichia) on segment VIII is complete.

61

Figure 2-9. Discal setae absent on sternites, sternites covered with rows of microtrichia with the exception of the antero-medial region.

62

Figure 2-10. Simple D1 and funnel-shapped D2 setae on the head of a S. dorsalis larva.

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Figure 2-11. Funnel shaped setae on abdominal terga IX and X of a S. dorsalis larva.

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Figure 2-12. Reticulated pronotum of a S. dorsalis larva illustrating the presence of 6-7 pairs of pronotal setae.

65

Figure 2-13. Abdominal segments IV-VII of a S. dorsalis larva illustrating the presence of 8-12 setae each.

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Figure 2-14. Forefemora of a S. dorsalis larva illustrating the presence of four funnel shaped setae on the distal two-thirds portion.

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Figure 2-15. Body of a S. dorsalis larva indicating the presence of granular plaques.

68

CHAPTER 3 COUPLING SCANNING ELECTRON MICROSCOPY WITH DNA BAR CODING FOR

MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF THRIPS

Introduction

Changing climatic conditions and globalization has resulted in increasing invasive

species as a recurrent problem around the globe (Masters and Lindsay 2010). During

international trade of plants and animals, importers pay a price to the exporter for their

costs of production and transport, but neither party pays costs associated with invasion

risk (Perrings et al. 2005). A recent study by Pimentel et al. (2005) concludes that more

than 50,000 non indigenous species have already been introduced in the United States

accounting for annual damage of more than $120 billion. Several states in the USA,

including Florida and Hawaii, have always been prone to invasion by non-indigenous

species where more than 25% of animal groups are non-native (Simberloff 1996).

These invasive species, facing no challenge by their natural enemies, thrive in the new

environment (Chenje and Mohamed-Katerere 2006). In addition to the disturbance they

cause to the biodiversity of agro-ecosystems, they pose a significant detrimental impact

on the economic value of crops (Pimentel et al. 2000, Reitz and Trumble 2002). Correct

identification and determination of the possible pathway of introduction of such pests

are a basic requirement in the development of any effective quarantine and pest

management strategy.

In the United States, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) is a

newly introduced pest species of various tropical and subtropical crops that poses a

significant economic threat to U.S. agriculture and trade (Farris et al. 2010). Since the

introduction of S. dorsalis into Florida in 2005, the pest dispersed rapidly across the

state and is causing significant economic damage to horticultural and nursery

69

production (Seal et al. 2010). In 2007, the top two counties in agricultural sales in

Florida were Palm Beach and Miami Dade, contributing around 931 and 661 million

dollars, respectively (ERS-USDA 2008). These two counties were also among the 15

counties in which S. dorsalis was reported to have been established in 2005 (Silagyi

and Dixon 2006). Successful establishment of S. dorsalis on its preferred hosts in these

counties could have a significant impact on agriculture production in the state.

According to an economic analysis, even a loss of 5% due to this pest could result in a

$3 billion loss to the US economy (Garrett 2004). Thus, it is essential to take necessary

measures in order to limit the economic impact of this pest.

The small size (< 2 mm in length) and thigmotactic behavior of S. dorsalis, makes

monitoring and detection of this pest difficult in fresh vegetation. Various life stages of S.

dorsalis can be found on the meristems and other tender tissues of all above ground

parts of their host plants. Because eggs are deposited inside the plant tissues and may

take 6-8 days to hatch (Seal et al. 2010), the probability of dissemination of S. dorsalis

through state, regional and international trade of plant materials is high for all life

stages. Within five years of the introduction of S. dorsalis into the U. S., establishment

has been confirmed in 30 counties of Florida and eight counties of Texas, with

additional positive reports of the interception of this pest in Georgia, New York,

Alabama, Louisiana and California (Kumar et al. 2011). Recently, the pest was reported

to be damaging 12 different crops in a fruit nursery in south Florida, including crops not

previously reported as hosts, demonstrating that this pest is increasing its host range

(Kumar et al. 2012).

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Development of effective management practices of S. dorsalis populations will

depend upon clarifying the taxonomy, biology and ecology of this species. The biology,

host preference, distribution and chemical control of this pest have been reported

previously (Seal et al. 2006a, Seal et al. 2010, Seal and Kumar 2010). Correct

identification of thrips, including S. dorsalis, has always been difficult due to their small

size and cryptic nature (Farris et al. 2010). Using traditional taxonomic keys, adult thrips

are identified to genus, but due to the intraspecific morphological variations in many

species, identifying them to species requires substantial expertise (Rugman-Jones et al.

2006). For many taxa of thrips it is impossible to assign an immature to a particular

species in the absence of adults (Brunner et al. 2002). Therefore, an accurate standard

method is desirable to validate the species designations of thrips larvae as well.

Taxonomists involved with identification of Thysanoptera, mount specimens on

slides for morphological identification under a light microscope. Mounting specimens on

slides is often time consuming and labor intensive, and requires expertise and

knowledge of distinct characters visible through microscopy (Bisevac 1997). The

method also involves the risk of specimen collapse and the disintegration of specimens

can have a devastating impact on projects involving the global collection and

identification of pest species. The use of genetic markers offers an additional tool to

supplement the phenotypic identification of thrips specimens. The integration of

morphological and genetic marker techniques for identification of thrips has certain

limitations. First, a sufficiently large number of specimens are required in order to

confirm identifications using both techniques. Second, when a mixed population of

thrips specimens labeled as one species (which is very common) is received, then

71

morphological identification data do not corroborate with molecular identification. Third,

sometimes only larvae of any thrips population are available for identification.

Since the development of the scanning electron microscope (SEM) in the early

1950’s, the technique has been used for morphological identification. SEM provides

many advantages over traditional microscopy including a larger depth of field, a higher

resolution and a higher level of magnification (Schweitzer 2010). These characteristics

of SEM can be coupled with genetic marker techniques to develop a simple, reliable,

robust tool for accurate identification of larval and adult thrips by closely studying their

morphological characters with confirmation of species diagnosis with DNA bar coding

using the same speciman. Because individual specimens can be used for both

morphological identification using SEM and for molecular identification using

polymerase chain reaction (PCR), the aforementioned limitations associated with using

traditional morphological identification integrated with molecular identification are

reduced. Thus, the specific objective of this study was to develop methodology for S.

dorsalis identification that allowed comparing the morphological characters using SEM

and the molecular PCR based assay utilizing the same individual (larva or adult) so that

results could be directly correlated.

Materials and Methods

Morphological Identification

Larvae and adults samples of S. dorsalis subjected to morphological

characterization under scanning electron microscope in the previous study (chapter 2),

were used in this study. High quality pictures, displaying features used for S. dorsalis

identification were obtained for photo-documentation of the specimens. Larvae were

identified using the keys of Vierbergen et al. (2010), and adult female thrips were

72

identified using taxonomic characteristics described by Skarlinsky (2004) and Hoddle et

al. (2009).

Molecular Identification

After morphological identification of S. dorsalis adults and larvae, gold/palladium

sputter coated specimens were removed from each stub using a fine forceps and were

placed in 95% ethanol for 15 min before proceeding to their DNA extraction. Sputter

coated adult females and larvae were subjected to DNA extraction by placing

specimens individually into 1.5-ml labeled Eppendorf tubes, adding 50 µl of DNA lysis

buffer (De Barro and Driver 1997, McKenzie et al. 2009), and grinding the specimen

with a plastic pestle. Tubes were placed in a metal boiling rack and boiled at 95°C for 5

min and were then placed directly on ice for 5 min. Tubes were centrifuged at 8,000 × g

for 30 s, and the supernatant (crude DNA lysate) was transferred to another labeled

tube and stored at -80°C until further analysis. Aliquots from the same individual thrips

DNA extract were used for molecular identification to confirm morphological

identification data.

PCR protocol and sequencing

PCR amplifications for the mtCO1 gene and ITS2 rDNA were performed

separately using universal mtCO1 (Folmer et al. 1994) and Thrips-ITS2 (Campbell et

al.1993, Toda and Komazaki 2002, Rugman-Jones et al. 2006). The 25 µl PCR

reactions for CO1 and ITS2 gene consisted of 12.5 µl of go Taq PCR mastermix

(Promega Corporation, Madison, USA) and 2 µl of DNA template and 10 pmol of each

primer.The PCR reactions were run using the conditions described in Table 3-1, in a

PTC-200 Peltier thermal cycler (MJ Research, Watertown, MA). The amplified products

were cleaned using ExoSAP-IT PCR Clean-up Kit (GE Healthcare Limited, Amersham,

73

UK) following the recommended protocol. Samples were sequenced after dilution of the

cleaned sample with 15 µl of water. The process was repeated twice to get a

concordant reliable result.

Fifty nanograms of total thrips genomic DNA was used in BigDye sequencing

reactions. All sequencing was performed bidirectionally with the amplification primers

and BigDye Terminator cycle sequencing kits (Applied Biosystems, Foster City, CA) at

the Genomics Core Instrumentation Facility of USHRL-USDA, Fort Pierce, FL.

Sequence reactions were analyzed on an Applied Biosystems 3730XL DNA sequence

analyzer, and compared and edited using Sequencher™5.0- Build 7081 (Gene Codes

Corporation, Ann Arbor, MI). Thrips species determination was based on direct

sequence comparisons using the web-based National Center for Biotechnology

Information BLAST sequence comparison application

(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Results and Discussion

The results of sequencing both mtCO1 and ITS2 rDNA of individual larvae and

adult thrips concurred 100% with the positive controls (known S. dorsalis specimens)

(Figure 3-1) and with the morphological identification using SEM. PCR reactions

repeated twice also confirmed the concordant results (Figure 3-1), suggesting that

coupling SEM morphological and molecular identification techniques can be accurately

and efficiently used for detecting larvae and adults of S. dorsalis. The methodology

would likely be useful in identifying different thrips genera, even to the species level.

Sequences obtained were deposited in Genbank with successive accession numbers

JN578861 and JN578862.

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Misidentification of thrips specimens using molecular identification based on

genetic information available in databases such as Genbank and EMBL is very common

(Porco et al. 2010) until a voucher specimen or photo-documentation is available to

confirm the identity. The current study was undertaken to improve the identification of S.

dorsalis larvae and adults by coupling both morphological and molecular identification

techniques using the same specimen. The figures 2-1 to 2-15 along with figure captions

explains the keys features important for taxonomic identification of S. dorsalis adult and

immature stages. The high-resolution SEM pictures of S. dorsalis produced in the

previous study can be effectively used by research, regulatory and extension personnel

to identify this pest with greater ease.

Although SEM has numerous advantages over light microscopy, its use in

taxonomic identification of thrips has not been widely explored (Chandra and Verma

2010). Lack of sufficient phenotypic variation among closely related thrips species or the

limitation of light microscopy to characterize these variations (Brunner et al. 2002) can

often lead to misidentification of thrips specimens. In such cases, the qualities of SEM

may be useful to distinguish between two species. The correct identification of a pest

species is essential to assure that appropriate management strategies are employed.

Because different thrips species might differ in susceptibility to different insecticides,

failure to correctly identify the problematic thrips and to correctly select the most

efficacious insecticide might result in decreased yields and exports of harvested crops

(Timm et al. 2008). Thus, utilization of SEM in taxonomic identification of these minute

insects can supplant or enhance traditional taxonomic identification techniques.

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The use of genetic markers is becoming more fully integrated with classical

taxonomic techniques for identification of species of interest. Integration of these

techniques has enhanced the quality of diagnostic tools, which has resulted in the

discovery of new species and in the understanding of inter- and intra-species variability

among the species (Carew et al. 2011).

Accurate identification of a pest is necessary to access previously reported

biological information concerning the organism and becomes extremely important when

the study organism is a part of cryptic species complex (Rugman-Jones et al. 2010). In

a recent study, Hoddle et al. (2008) reported that S. dorsalis collected from three

different regions of the world were morphologically identical but were genetically

distinct, and the genetic diversity in the species was extensive. Often taxonomic

identification is conducted using a compound microscope at maximum magnification of

650 to 1,000 times, which may be a limitation in identifying additional information

needed to differentiate two morphologically similar thrips species. SEM can magnify an

entire specimen or a particular body area of a specimen up to 500,000 times, which can

be crucial in searching for new morphological characters to differentiate among cryptic

species or a species complex within a thrips population.

Compared to other available integrated methods of insect identification, such as

sonication of specimens for DNA extraction (Hunter et al. 2008) or the automated high-

throughput DNA protocol (Porco et al. 2010), the current novel technique is simple and

quick, utilizes fewer specimens for identification, provides high yield of DNA and can be

easily mastered by non-experts. Another integrated technique available for thrips

identification involves piercing the abdominal region of the specimen using a minute pin

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and processing the extracted gut content for molecular identification prior to the slide

mount (Rugman-Jones et al. 2006). This method requires great skill to keep the

specimen intact and save the specimen for slide preparation. Because thrips are soft-

bodied, minute insects, specimens can be damaged while puncturing the abdomens or

during slide preparation. In the method reported here, an unknown thrips specimen

(larva/adult) can be identified at higher magnification using SEM and then the same

gold/palladium sputter-coated specimen can be used directly for DNA extraction. In

addition, the high magnification of SEM can be efficiently used for taxonomic

identification of thrips larvae in the absence of adults, which can be further confirmed

using the genetic marker tool. Thus, the method can conserve specimens and avoid

problems concerning mixed sample populations. Future research will concentrate on

making the method more economical and more efficient in order to increase wider

adoption of the method.

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Table 3-1. PCR amplification conditions for two genes of Scirtothrips dorsalis.

PCR Primer Set PCR amplification conditions (25-µl reactions)

mtCO1 primers

LCO1490:5'-GGTCAACAAATCATAAAGATATTGG-3'

HCO2198: 5'-TAAACTTCAGGGTGACCAAAA AATCA-3'

94°C 2 min

35 cycles of

94°C 30 s

54°C 1 min

72°C 1 min

72°C 10 min

ITS2 primers

ITSF: 5'-TGTGAACTGCAGGACACATG-3'

ITSR- 5'AATGCTTAAATTTAGGGGGTA-3'

94°C 2 min

35 cycles of

94°C 30 s

48°C 1 min

72°C 1 min

72°C 10 min

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Figure 3-1. Agarose gel showing PCR results using the ITS2 primers and mtCO1 primer set for the detection of S. dorsalis. The marker fragment was successfully amplified from S. dorsalis DNA (lanes 2- 5 and 9-12). Lanes 1 and 8 are the 1Kb DNA ladders. Lanes 2, 3, 9, and 10 are S. dorsalis adults. Lanes 4, 5, 11, and 12 are S. dorsalis larvae. Lanes 6 and 13 are negative controls and lanes 7 and 14 are positive controls (known specimens of S. dorsalis).

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CHAPTER 4 INTRAGENOMIC VARIATION IN mtCO1 AND rDNA ITS2 OF THREE MAJOR THRIPS

SPECIES, SCIRTOTHRIPS DORSALIS, THRIPS PALMI AND FRANKILINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE)

Introduction

Correct identification is a fundamental step in the development of sound

management practices against a pest. Identification helps in attaining previously

reported information against the subject species (Rugman-Jones et al. 2010) that

supports in planning and implementation of an appropriate biological research strategy.

The morphological identification of various species in the order Thysanoptera can be

difficult because of the high degree of polymorphism within and among species (Murai

and Toda 2001, Hoddle et al. 2009, Kakkar et al. 2011), the similarity in developmental

stages of different species (Brunner et al. 2002), and the lack of taxonomic experts to

differentiate thrips specimens to the species level (Asokan et al. 2007). The presence

of cryptic species makes identification more difficult because the delimiting boundary

between two species is unknown (Hoddle et al. 2008, Rugman-Jones et al. 2010).

However, molecular identification is not limited by these factors (Asokan et al. 2007); it

is cost effective, rapid, and can be accomplished by non-taxonomic experts (Rubinoff et

al. 2006). Various molecular markers have been developed for use in species

determination. These include several nuclear genes (i.e, 16S rRNA, 18S rRNA, 28S

RNA ) (Barr et al. 2005) and internal transcribed spacers (rDNA ITS) (Rugman-Jones et

al. 2006), as well as the mitochondrial cytochrome c oxidase 1 (mtCO1) gene (Rugman-

Jones et al. 2010).

A portion of the mtCO1 gene is widely used as a DNA barcode for taxon

characterization of animals i.e., taxon identification, species delimitation and

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phylogenetic placement (Rubinoff et al. 2006). This gene is putatively conserved

among members of a species and diverged by 3% or more among different species

(Hebert et al. 2003, Song et al. 2008), making it well suited for this purpose (Brunner et

al. 2002). However, this has proven not to be the case for several arthropod groups

(Gellissen and Michaelis 1987, Zhang and Hewitt 1996, Parfait et al. 1998, Bensasson

et al 2001, Campbell and Barker 1999). In some of these cases, the presence of

substantial intra- and intergenomic variation has confounded the traditional 3%

divergence cut-off between species. These two variations have been attributed to i)

duplication of the CO1 fragment (Campbell and Barker 1999), (ii) nuclear heteroplasmy,

where multiple copies of mtDNA undergo coamplification (Petri et al. 1996, Thomas et

al, 1998), (iii) amplification of mtDNA haplotypes of maternally inherited symbionts

(Parfait et al. 1998), and (iv) nuclear integration of mitochondrial sequences producing

pseudogenes (numts) (Song et al. 2008). The first three events are rare phenomena

and have been reported in few organisms. However, numts (nonfunctional copies of

mtDNA) have been reported in more than 82% of eukaryotes (Bensasson et al. 2001).

These can be co-amplified with target mtDNA and can interfere in PCR-based

identification and phylogenetic study by producing within individual sequence

divergence. Numts coamplified with conserved orthologous mtDNA can be identified by

the presence of indels, point mutations and in-frame stop codon (Song et al. 2008).

Due to some of these problems faced when delimiting species based on a single

gene, some researchers use mtCO1 along with the second internal transcribed spacer

in the nuclear ribosomal DNA (ITS2) for taxon characterization (Navajas et al. 1994,

1998, Ruiz et al. 2010). The internal transcribed spacer of the nuclear ribosomal 5.8S-

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28S gene exists in multiple copies within the nuclear genome. This region, like that of

mtCO1, is believed to have low intraspecific and high interspecific variability (Fairley et

al. 2005) making it useful for delimiting cryptic species (Li and Wilkerson 2007). The

fixed intra- and interspecific differences in the non-coding ITS2 region are ensured by

concerted evolution (Li and Wilkerson 2007). Concerted evolution is a universal

biological phenomenon in which members of a multicopy gene family do not evolve

independently and rapid spread of mutation is observed in all the members of the gene

family (Liao 1999, Wörheide et al. 2004). In nuclear rDNA, homogenization of mutations

acts as quality control to maintain intra- and intergenomic uniformity (Fairley et al.

2005). Molecular processes governing concerted evolution involves a variety of DNA

recombination and repair and replication mechanisms in the form of unequal crossing

over, gene conversion and gene amplification (Zimmer et al. 1980, Liao 1999).

Nevertheless, several authors have reported considerable intragenomic and/or

intergenomic variation in the ITS region of arthropods (Vogler and Desalle 1994, Tang

et al. 1996, Benevolenskaya et al. 1997, Rich et al. 1997, Navajas et al. 1998, Leo and

Barker 2002, Fairley et al. 2005, Li and Wilkerson 2007, Vesgueiro et al. 2011) raising

questions regarding the suitability of this marker region for taxonomic characterization.

These variations can be attributed to i) faster rate of mutation among copies than the

speed of homogenization (Fritz et al. 1994, Fairley et al. 2005), ii) duplication of DNA

sequence produces new genes, which can either evolve independently to acquire new

biochemical function or can remain non-functional as pseudogenes in the genome

(Murti et al. 1992); coamplification of such pseudogenes with the target ITS gene can

bring ambiguity in taxon characterization (Mayol and Rossello 2001), iii) slow

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homogenization of duplicated genes has been reported to produce divergent sequences

that evolve independently (Brunner et al. 1986), and iv) physical location of duplicated

genes on the chromosome influencing its participation in recombination which decides

its fate for concerted or divergent evolution (Murti et al. 1992, Liao 1999). Given that

variations may exist in the mtCO1 and ITS2 genes of an individual, the ability of PCR to

amplify sequences of these genes may lead to inaccurate identification and

phylogenetic placement of the individual. Thus, determining the magnitude of intra- and

intergenomic variations in the two genes is of paramount importance for any given

species.

Worldwide, a large part of the literature dealing with economic thrips is focused on

four major species i.e., Frankliniella occidentalis Pergande (western flower thrips),

Scirtothrips dorsalis Hood (chilli thrips), Thrips tabaci Lindeman (onion thrips), and

Thrips palmi Karny (melon thrips) (Morse and Hoddle 2006). Of these four species, F.

occidentalis, S. dorsalis, and T. palmi are well known for their significant economic

impact on agriculture and horticulture industries in Florida. These species are highly

polyphagous and cause direct feeding damage to fruits, leaves or flowers of their hosts.

Apart from causing direct or cosmetic damage to the host, the species are well known

for their ability to transmit important and damaging plant viruses. Twelve out of 14

species of virus within the genus Tospovirus are vectored by these three species of

thrips (Whitfield et al. 2005). Frankliniella occidentalis transmits the Tomato spotted wilt

tospovirus (TSWV), one of the most important members of the genus which has 1,090

host species in 85 different families of plants. Financial losses attributed to TSWV were

estimated at more than $1 billion/year in the early 1990’s (Goldbach and Peters 1994).

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Other economically important tospoviruses vectored by S. dorsalis and T. palmi

includes Capsicum chlorosis virus, Watermelon silver mottle virus, and Melon yellow

spot virus (Chiemsombat et al. 2008). With the continued global expansion of these

thrips vectors, the agriculture sectors in developing countries with limited resource input

can suffer maximum damage. In addition, greater economic losses from plant diseases

are expected with the emergence of new varieties and species of tospoviruses, which

could be acquired and transmitted by the thrips (Pappu et al. 2009).

High level of variation in the basic biology, life history, host selection, pest status,

vector efficiency and resistance to insecticides exist in different thrips species.

Misidentification of thrips species can lead to the misapplication of management

practices, resulting in wasted money, resources and time (Rosen 1986). Considering

the importance of correct identification, the objective of this study was to determine the

suitability of two widely used marker genes i.e. mtCO1 and ITS2, for the identification of

F. occidentalis, S. dorsalis and T. palmi. Frey and Frey (2004) reported intragenomic

variation in the mtCO1 gene of T. tabaci; however, no published information is available

regarding such variation in the ITS2 gene of thrips species. Thus, in the current study

the intragenomic and intraspecific variation in the mtCO1 and ITS2 gene of three thrips

species was studied using the clone approach. Since thrips are known to exhibit

haplodiploidy (Ananthakrishnan 1993), male and female thrips specimens were selected

within each species to investigate any gender-based differences in ITS2 genes.

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Materials and Methods

Taxon Sampling

The adult male and female thrips specimens that were used for mtCO1 and ITS

sequence determination during the study were obtained from either wild populations (T.

palmi) or laboratory colonies (S. dorsalis and F. occidentalis). The date of sample

collection, geographical location (longitude and latitude), host plant, and collector are

reported in Table 4-1. The actual sample technique depended upon the individual

collector, but once adults were collected, they were immediately placed in 70-95%

ethanol. The specimens were shipped at room temperature to the US Horticultural

Research Lab in Fort Pierce, FL, where all samples subsequently were stored in 90%

ethanol at -80°C.

Morphological Identification of Thrips

Thrips specimens were placed individually into vials containing 75% alcohol for 10

min and then into vials containing a 10% KOH (Potassium hydroxide) solution prepared

in 50% ethanol for 5 min. While in the KOH solution, each specimen was macerated

gently in the abdominal region using a fine insect pin to aid in the removal of abdominal

contents. For gradual dehydration, each specimen was passed through a series of

ethanol concentrations starting from 65%, followed by 75%, 85%, 90% and 95% for 5-8

min at each concentration. Each specimen was placed ventrally on a slide with a small

drop of Hoyer’s mounting media and covered with a glass cover slip. The adult thrips

specimens were identified using morphological characteristics described by Hoddle et

al. (2009) using a dissecting microscope at a minimum of 10X magnification. The

identity of thrips specimens were reconfirmed by USDA-APHIS entomologist, Thomas

Skarlinsky (Thysanoptera specialist- eastern region- Miami, FL).

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DNA Processing

Using cohorts from the sample that were morphologically identified, DNA was

isolated from individual thrips by placing a single thrips specimen in a labeled 1.5-ml

Eppendorf tube, adding 25 µl of DNA lysis buffer (De Barro and Driver 1997, McKenzie

et al. 2009), and grinding the specimen with a plastic pestle. The pestle was rinsed with

an additional 25 µl of DNA lysis buffer and collected in the same tube. Tubes were

placed in a metal boiling rack and boiled at 95°C for 5 min and then placed directly in

ice for 5 min. The tubes were then centrifuged at 8,000 × g for 30 s, and the supernatant

(crude DNA lysate) was transferred to another tube and stored at -80°C for future

processing. Aliquots from the same individual thrips DNA extract were used for both

mtCO1 and ITS2 marker analysis so that thrips identity using both genes could be

directly compared.

Polymerase chain reaction (PCR) amplifications of the mtCO1 gene for S. dorsalis

and T. palmi were performed using universal CO1 primers LCO1490 and HCO2198

designed by Folmer et al. (1994). Mitochondrial CO1 gene amplification for F.

occidentalis was conducted using mt D-7.2 and mt D-9.2 primers designed by Brunner

et al. (2002). Amplification of rDNA ITS2 gene for all the three species was conducted

using Thrips-ITS2 primer (Campbell et al.1993, Toda and Komazaki 2002, Rugman-

Jones et al. 2006). The complete primer sequence along with the PCR conditions are

described in Table 4-2. The 25 µl PCR reactions for mtCO1 and ITS2 consisted of 2 uL

of the DNA sample, 12.5 µl of go Taq PCR mastermix (Promega Corporation, Madison,

USA) and 10 pmol of each primer. PCR reactions were performed in a PTC-200 Peltier

thermal cycler (MJ Research, Watertown, MA). Amplification of the correct PCR

products was verified by electrophoresis in a 1.5% agarose gel, stained with ethidium

86

bromide. Before sequencing, the amplified products excised from the gel were cleaned

using nucleospin®Extract II, PCR clean up, Gel extraction kit (Macherey-Nagel, Inc.

Bethlehem, PA). Ligation and transformation of amplified DNA was done using

the TOPO® TA Cloning® Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s

instructions. Transformed cultures were cultivated in 1.5 ml of Luria-Bertani medium

overnight containing 50 ug/ml kanamycin. Plasmids were extracted using

Wizard® Plus SV Minipreps DNA Purification System (Promega Corporation, Madison,

WI), dissolved in 0.1X TE and sent for sequencing. All sequencing was performed bi-

directionally with the amplification primers and ABI Prism® BigDye® Terminator v3.1

Cycle Sequencing Kits (Applied Biosystems, Foster City, CA) at the Genomics Core

Instrumentation Facility of USHRL-USDA. Sequence reactions were analyzed on

an Applied Biosystems 3730XL DNA Analyzer (Applied Biosystems).

Sequence Alignment and Genetic Distance Matrix

Sequence base-calling was verified using Sequencher™5.0- Build 7081 (Gene

Codes Corporation, Ann Arbor, MI) and then aligned suing ClustalW 2.1 (Larkin et al.

2007) in mesquite (Maddison and Maddison 2011). Thrips species determination was

based on direct sequence comparisons using the web-based National Center for

Biotechnology Information (NCBI) BLAST sequence comparison application

(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Mean uncorrected “p” pairwise distances were

calculated by PAUP version 4.0 (Swofford 1998) and it was used to assess sequence

differentiation within and between individuals of same species. The aligned mtCO1 and

ITS2 sequences were analyzed by maximum parsimony (MP) as implemented in PAUP

(Li and Wilkerson 2007). Each indel was considered as a single character, and

regardless of size, indels were coded as 0 or 1 (Simmons and Ochoterena 2000). In

87

order to minimize possibility of Taq random error, single unique mutations were

disregarded. Rate of Taq polymerase error was determined by recloning a cloned and

sequenced fragment of a fifth S. dorsalis individual. Following the protocol of Li and

Wilkerson (2007), parsimony analysis was conducted using the heuristic search option

with tree-bisection-reconnection branch swapping algorithm. Parsimony bootstrapping

was conducted using 1,000 pseudoreplicates with 10 random taxon addition replicates

for each pseudoreplicate.

Results

Inter- and Intragenomic Variation

Scirtothrips dorsalis. Evidence for intragenomic variation in the ITS2 of S.

dorsalis was observed when individual clones of PCR products were analyzed. To

assess the frequency of mutation in rDNA ITS2, 137 clones were sequenced from two

females (SD-1, SD-2) and two males (SD-3, SD-4) as individual specimens, ranging

from 23-46 clones/individual thrips (Table 4-3). Pairwise alignment of sequences from

all the clones produced a consensus sequence of about 507 bp with GC content of

57%. When compared together, 137 clones of S. dorsalis produced 71 paralogous

haplotypes (Table 4-4) of variable divergences. These haplotypes differed in 87

mutation points (nucleotides) out of 507 bp, amounting to 17% variable sites in

haplotypes of S. dorsalis. Out of 71 variant haplotypes, 61 haplotypes consisted of a

single clone from an individual specimen, and only four shared haplotypes (clones from

two or more specimens) were observed. The frequency of the most common haplotype

was 10.9% (Table 4-3). Maximum sequence divergence of 1.9% was observed between

haplotypes. At the majority of nucleotide sites, haplotypes differed due to single base

indels or substitutions, but two base substitutions/indels were observed at nucleotide

88

positions 100, 107, 144, 338 and 462. Indels were observed at position 99, 100, 101,

107, 112, 113, 142, 144, 416 and 417.

Alignment of 132 mtCO1 clones from four S. dorsalis individuals produced 23

paralogous haplotypes. Haplotypes differed in 42-nucleotide positions in a total of 655

bp (AT- 72%), suggesting a site variation of about 6.4%. The most common haplotype

*SD (Table 4-5) consisted of 110 clones (83.3%) from four individuals (Table 4-3). At all

nucleotide positions haplotypes differed in single base indels or substitutions, except at

position 475 where haplotype SD-2.5 exhibited C and SD-2.11 had an A while all others

had a T (Table 4-5). Maximum sequence divergence of 0.4% was observed between

haplotypes. The uncorrected “p” distance matrix showed variation among clones of S.

dorsalis, where the intragenomic base differences ranged from 0.1% to 3.4% for the

ITS2 gene and from 0.0% to 0.9% for the mtCO1 gene (Table 4-3). The intergenomic

variation in this specis was similar to the intragenomic deviation, ranging from 0.1% to

3.8% and 0.0 to 0.9% for the ITS2 and mtCO1 genes, respectively.

Thrips palmi. Variations were observed within and between clones of the ITS2

gene of the four specimens of T. palmi (Table 4-6). Alignment of 149 clones from the

four individuals produced 76 paralogous haplotypes. Haplotypes differed from each

other in 79 mutation points (Table 4-6) in the 564 bp sequence (GC- 56%), amounting

for 14% site variation. Sixty-five haplotypes consisted of a single clone from the same

specimen and six shared haplotypes from two or four different specimens were

observed. The frequency of the most common haplotype was 14.7% (Table 4-3). At

nucleotide position 139, haplotypes differed in two base substitutions, where haplotype

T.P-1.11 and TP-2.6 exhibited A and C, respectively instead of T. Indels were observed

89

at position 56, 249 and 250. Sequence divergence of 1.5% was observed between

haplotypes.

Compared to ITS2, not much sequence variation observed among mitochondrial

CO1 clones of T. palmi. Fifteen paralogous haplotypes were obtained from 120 clones

of four individuals. Haplotypes differed from each other in 25 nucleotides positions in

655 bp (AT- 70%), amounting to site variation of 3.8%. Haplotype TP-1.1 comprised of

34 clones from individual TP-1 and shared haplotype TP* consisted of 73 clones from

the other three individuals (Table 4-7). Maximum sequence divergence among

haplotypes was about 0.6%. Based on distance matrix, intragenomic variation of 0.0 to

2.6% and 0.0% to 0.7% and intergenomic variation of 0.0% to 2.8% and 0.0% to 1.0%

was observed for ITS2 and mtCO1 clones of T. palmi, respectively (Table 4-3).

Frankliniella occidentalis. Compared to the other two thrips species, less site

variation (5.7%) was observed among haplotypes of ITS2 in F. occidentalis. Out of 105

ITS2 clones, 14 different haplotypes were obtained (Table 4-8). These haplotypes

differed in 23 nucleotide positions in 456 bp (GC- 54%). The frequency of the most

common haplotype was 76%. Maximum sequence divergence of 1.5% was observed

between haplotypes.

In the case of mtCO1 clones, a site variation of 5.2% was observed among 150

clones in 434 bp (AT- 68%) (Table 4-8). Frequency of the most common haplotype was

88%. Maximum sequence divergence among haplotypes was about 0.6%. Distance

matrix analysis showed 0.4% to 1.1% and 0.2% to 1.1% intragenomic variation among

clones of ITS2 and mtCO1 and the value of intergenomic variation between 0.4% to

1.5% and 0.2% to 1.5%, respectively.

90

In all the three thrips species no sex based haplotype diversity was observed in

male and female clones of the rDNA ITS2 gene (data not shown), which indicates

existence of similar rDNA arrays in both sexes.

Parsimony Analysis of ITS2

Based on the variations observed in the ITS2 regions of the three thrips species,

MP analysis was conducted only for this region. For clear representation of tree

topology of S. dorsalis and T. palmi, polytomys were removed and selective haplotypes

were included to produce the unrooted consensus MP tree (Figure 4-1, 2). Bootstrap

values were provided as branch support. The unrooted MP tree (Figure 4-1) showed a

high degree of intragenomic variations among the clones of same individual specimen

of S. dorsalis. These variations were equivalent to intergenomic variations i.e., variation

between clones of different individuals of a species. Similar variations were seen in the

MP tree generated from rDNA ITS2 sequences obtained from four individuals of T.

palmi (Figure 4-2). In the case of F. occidentalis, less intra- and intergenomic variations

were observed; thus, the single most parsimonious tree was presented without

removing polytomys (Figure 4-3).

Discussion

Mitochondrial Cytochrome Oxidase I

On comparing intra- and intergenomic variability observed in the sequence of the

mtCO1 and ITS2 genes, the relatively low variation in mtCO1 and variant paralogous

haplotype suggests that this gene could be used for the taxonomic characterization of

the three thrips species. Corresponding to our results, Frey and Frey (2004) observed

2.5% nucleotide divergence in mtCO1 clones of T. tabaci and suggested that this was

low enough to allow molecular diagnosis of the species. Nucleotide divergence of the

91

mtCO1 sequence for the thrips observed in this study was much lower (0.4% seen in S.

dorsalis and 0.6% seen in both T. palmi and F. occidentalis).

In the current study, there is the possibility that Taq polymerase induced error

could contribute to variation among mtCO1 clones. Since, Taq polymerase lacks the

ability of 3’-5’ proof reading (Barnes 1994), it may interfere in identifying and quantifying

the actual level of intra- and intergenomic variation in the gene. However, Taq error rate

(mutations per nucleotide per cycle) can vary between 1 x 10-4 to 1x 10-5 (Eckert and

Kunkel 1991, Pray 2008, Vesgueiro et al. 2011) and are proportionally related to the

length of the product. In this study, single unique mutations were disregarded to

minimize the possibility of Taq random error. Based on the results obtained from cloning

a clone product, the Taq error rate was observed about 1 out of 10,000 bases. In the

three thrips species, 65,000 to 86,500 bases were sequenced for CO1 clones, thus,

artifacts due to Taq polymerase account for an insignificant percentage of the clones

and sequence variation can be attributed to nuclear integrated gene fragment copies or

mitochondrial heteroplasmy. The results suggest the presence of a large number of

numts which were co-amplified with target mtDNA using conserved universal primers in

the three thrips species. The presence of numts can be identified by indels, point

mutation and in-frame stop codons (Song et al. 2008). Due to the presence of an

altered genetic code, numts are not expressed correctly and they randomly accumulate

high degrees of mutations resulting in divergence of numts from the orthologous

sequence (Zhang and Hewitt 1996). They induce nonsense mutations that result in

presence of stop codon in the coding DNA, which makes it possible to identify them

(Frey and Frey 2004). However, in the current study, stop codon were not seen in the

92

mtCO1 sequence of all three thrips species, but presence of indels and point mutations

confirmed the presence of nonfunctional numt haplotypes. Presence of numts has

previously been reported in a number of arthropods including aphids, crickets, locusts

and grasshoppers (Zhang and Hewitt 1996, Parfait et al. 1998, Bensasson et al. 2001,

Gellissen and Michaelis 1987). In the case of variant T. palmi haplotype, TP-1.1 that

comprises 34 functional clones (Table 4-7); heteroplasmy may be a possible cause

behind this variation. Heteroplasmy has been reported in bark weevils and

grasshoppers (Boyce et al. 1989, Song et al. 2008) which can be identified by absence

of stop codon in paralogous haplotypes exhibiting multiple clones from the same

individual. However, the results do not provide clear evidence of the possible reason

behind variant haplotypes and it is worth mentioning that the low frequency of variant

mtCO1 haplotypes in the three species do not interfere in their PCR-based

identification. The possible origin of such variations can be traced by using allele-

specific single cell-PCR and cytological methods of using high sensitive gold and silver

staining with which any event of heteroplasmy and nuclear integration can be observed

(Frey and Frey 2004).

Internal Transcribed Spacer 2 Variation

Extensive inter- and intragenomic variations were observed in the ITS2 gene of S.

dorsalis and T. palmi. There were a number of base substitutions and indels accounting

for sequence variabilities. The unrooted MP tree for both species (Figure 4-1 and 4-2)

showed cluster of clones from the same individuals that were as different from each

other as from other individuals. Out of 71 paralogous haplotypes, only four shared

haplotypes were observed where clones from SD-1 align together with clones from SD-

3 and SD-4 (Figure 4-1), but clones from SD-2 did not align with clones from other

93

individuals and formed separate haplotypes. In the case of T. palmi six shared

haplotypes were observed where TP* (Figure 4-2) comprised ITS2 clones from all four

individuals indicating low variability among clones compared to S. dorsalis. ITS2 clones

from F. occidentalis, FO-1 did not align with clones from other individuals and formed a

separate cluster together (Figure 4-3). Alignment of clones is of primary importance to

ensure the concerted evolution among members of the gene family (Vesgueiro et al.

2011). However, intra- and intergenomic variability in ITS2 clones in this study failed to

support this hypothesis and questions the reliability of this gene for taxon

characterization. Due to high within and between individual variability, it may be

possible that during phylogenetic analysis, members from one individual species can

form clades with another and provide ambiguous results.

Ignoring the insignificant percentage of Taq contamination in the ITS2 clones, high

variability within this gene can be attributed to the evolutionary process causing failure

of concerted evolution. Genetic exchange between homologous or non-homologous

chromosomes by unequal crossing over or gene conversion (Zimmer et al. 1980, Liao

1999) is important in maintaining sequence homogeneity in a multigene family such as

rDNA. In this process, mutation generated in one region is rapidly transferred to all the

members of multigene families, even if repeats are located on different chromosomes

(Tautz et al. 1988). Due to this homogenization of mutation in noncoding regions like

ITS2, no intragenomic and fixed rate of intergenomic variation can be expected (Li and

Wilkerson 2007). However in the current study, within individual variations in ITS2 was

similar among individual differences, which clearly suggests the rate of mutation within

this gene is higher than homogenization of mutation. This may serve as a possible

94

reason behind high intragenomic variation in ITS2 within the thrips species. In a similar

study, distant populations of black fly had been reported to exhibit multiple copies of

ITS1 region (Tang et al. 1996). Although concerted evolution occurred in the ITS region

in each of the black fly isolated populations, interbreeding between these populations

was interpreted as a source of multiple copies of the ITS1 in this pest. Gasser et al.

(1998) reported that interbreeding populations of a parasitic nematode (red stomach

worm, Haemonchus contortus) was the source of intragenomic variation in the ITS2

region of the pest (Leo and Barker 2002). Considering the global distribution of S.

dorsalis, T. palmi and F. occidentalis, the intragenomic variations in the ITS2 gene of

these pests due to interbreeding between geographically isolated populations cannot be

ruled out. Because the thrips populations used in the present study were only from

Florida, the possible role of interbreeding in genetic divergence could not been

determined. Future studies focused on assessing such variations in the global

populations of individual thrips species could explain interbreeding in divergent

evolution of the ITS2 gene.

Pseudogenes can also produce variant haplotypes in the ITS2 gene, resulting in

the high degree of intra- and intergenomic variation observed in this study. Due to the

heterogeneous nature of rDNA, infrequent existence of nonfunctional operons or

pseudogenes can be noted within a genome (Harpke and Peterson 2006). Presence of

pseudogenes can be identified by a high degree of indels in a nucleotide sequence

(Scholin et al. 1993, Santos et al. 2003). In this study, a considerable amount of indels

were found in haplotypes of S. dorsalis suggesting involvement of nonfunctional

pseudogenes in generating intragenomic variation. A better understanding of the

95

presence of pseudogenes could have been provided by analyzing the secondary

structure of the ITS2 gene (Bailey et al. 2003). Secondary structural stability is important

for the proper functioning of rRNA and even a minute modification in the structure of the

ITS region can cease the production of mature rRNA’s (Musters et al. 1990, Henry et al.

1994, Thornhill et al. 2007). Structural variation can affect cleavage efficiency of the

precursor RNA and may lead to formation of multiple pseudogenes of variable

divergence (Li and Wilkerson 2007). Intragenomic variation due to pseudogenes has

been reported in Drosophila melanogaster, Anopheles marajoara (Benevolenskaya et

al. 1997, Li and Wilkerson 2007) and other organisms (Brownell et al. 1983,

Razafimandimbison et al. 2004).

Although the possible reason for ITS2 variation of these thrips species could not

be traced, it can be concluded that the ITS2 copies in these thrips do not evolve in

concert based on the degree of intra- and intergenomic variability. Thus, this marker

region is not phylogenetically informative for population studies, and should not be used

for species-specific PCR identification of the three major economic thips species

studied. Ambiguity in correct identification, due to intra- and intergenomic variability

within a gene can be avoided by incorporating other marker regions and alternate

molecular tools as well as searching for additional biological and ecological information

segregating thrips populations.

96

Table 4-1. Collection date, localities and hosts for specimens used in cloning of rDNA and mtCO1 genes of thrips species of three genera.

Scientific name (M=male,

F= female) Specimen no.

Individual code

Date collected Host Locality Coordinates Collector

Scirtothrips dorsalis (F)

CLM9.13 SD-1 Aug. 7- 2007 Indian

Hawthorne USA, Florida-

Apopka 28.63 N, 81.55W

Dr. Lance Osborne

Scirtothrips dorsalis (F)

CLM9.14 SD-2 Aug. 7- 2007 Indian

Hawthorne USA, Florida-

Apopka 28.63 N, 81.55W

Dr. Lance Osborne

Scirtothrips dorsalis (M)

CLM9.15 SD-3 Aug. 7- 2007 Indian

Hawthorne USA, Florida-

Apopka 28.63 N, 81.55W

Dr. Lance Osborne

Scirtothrips dorsalis (M)

CLM9.16 SD-4 Aug. 7- 2007 Indian

Hawthorne USA, Florida-

Apopka 28.63 N, 81.55W

Dr. Lance Osborne

Thrips palmi (F)

CLM85.5 TP-1 Mar. 11- 2010 Vlaspek

cucumber USA, Florida- Homestead

25.50 N, 80.49W Vivek Kumar

Thrips palmi (F)

CLM85.6 TP-2 Mar. 11- 2010 Vlaspek

cucumber USA, Florida- Homestead

25.50 N, 80.49W Vivek Kumar

Thrips palmi (M)

CLM85.9 TP-3 Mar. 11- 2010 Vlaspek

cucumber USA, Florida- Homestead

25.50 N, 80.49W Vivek Kumar

Thrips palmi (M)

CLM85.10 TP-4 Mar. 11- 2010 Vlaspek

cucumber USA, Florida- Homestead

25.50 N, 80.49W Vivek Kumar

Frankliniella occidentalis (F)

CLM87.20 FO-1 Apr. 16- 2011 Green beans USA, Florida- Tallahassee

30.48N, 84. 17W Dr. Stuart

Reitz Frankliniella

occidentalis (F) CLM87.22 FO-2 Apr. 16- 2011 Green beans

USA, Florida- Tallahassee

30.48N, 84. 17W Dr. Stuart

Reitz Frankliniella

occidentalis (M) CLM87.25 FO-3 Apr. 16- 2011 Green beans

USA, Florida- Tallahassee

30.48N, 84. 17W Dr. Stuart

Reitz Frankliniella

occidentalis (M) CLM87.30 FO-4 Apr. 16- 2011 Green beans

USA, Florida- Tallahassee

30.48N, 84. 17W Dr. Stuart

Reitz

97

Table 4-2. PCR amplification conditions for two genes of Scirtothrips dorsalis, Thrips palmi and Frankliniella occidentalis.

*Primers used for mtCO1 amplification of Frankliniella occidentalis with annealing temperature of 52°C

Annealing temperature for CO1 and ITS2 amplification of Thrips palmi was 40°C and 48°C, respectively.

PCR Primer Set PCR amplification conditions

(25 µl reactions)

mtCO1 primers

LCO1490:5'-GGTCAACAAATCATAAAGATATTGG-3’

HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'

*mt D-7.2F: 5'-ATTAGGAGCHCCHGAYATAGCATT-3'

*mt D9.2R: 5'-CAGGCAAGATTAAAATATAAACTTCTG-3'

94°C 2 min

35 cycles of

94°C 30 s

54°C 30 s

72°C 1 min

72°C 10 min

ITS2 primers

ITSF: 5'-TGTGAACTGCAGGACACATG-3'

ITSR: 5'-AATGCTTAAATTTAGGGGGTA-3'

94°C 2 min

35 cycles of

94°C 30 s

52°C 1 min

72°C 1 min

72°C 10 min

98

Table 4-3. Number of clones sequenced and recovered haplotypes for the four individuals of each thrips species.

Internal transcribed spacer 2 Cytochrome oxidase 1

Individual No. of clones

sequenced

No. of different

haplotypes

Frequency of most

common haplotypes

(%)

Uncorrected “p” distance

matrix of clones

No. of clones

sequenced

No. of different

haplotypes

Frequency of most

common haplotypes

(%)

Uncorrected “p” distance matrix

of clones

SD-1 23 17 21.7 0.001-0.022 33 4 90.9 0.003-0.006 SD-2 26 19 23.0 0.003-0.038 44 13 72.7 0.001-0.009 SD-3 42 21 33.3 0.001-0.023 24 4 87.5 0.001-0.006 SD-4 46 19 26.0 0.001-0.034 31 5 87.0 0.0-0.007

All Scirtothrips dorsalis clones

137 71 10.9 0.001-0.038 132 23 83.3 0.0-0.009

TP-1 41 23 24.3 0.0-0.023 42 9 80.9 0.0-0.007 TP-2 38 18 18.4 0.001-0.023 24 3 91.6 0.001-0.004 TP-3 31 16 22.5 0.001-0.026 24 1 100 * TP-4 39 28 15.3 0.001-0.026 30 4 90 0.003-0.006

All Thrips palmi clones 149 76 14.7 0.0-0.028 120 15 60.8 0.0-0.010

FO-1 20 4 17 0.004-0.008 42 6 90 0.002-0.009 FO-2 17 2 16 0.004 31 4 87.0 0.002-0.011 FO-3 36 7 30 0.004-0.011 46 5 86.9 0.002-0.011 FO-4 32 3 30 0.004-0.008 31 2 96.7 0.004

All Frankliniella occidentalis clones

105 14 76 0.0-0.015 150 14 88.6 0.002-0.013

*Because of only one haplotype, the distance matrix could not be calculated

Uncorrected “p” distance matrices between clones from same individual represents intragenomic variation, and uncorrected “p” distance matrices between clones of all individuals of the same species represents intergenomic variation.

99

Table 4-4. The rDNA ITS2 sequences that differ among Scirtothrips dorsalis individuals.

1 2 3 4 5 6 7 8 9

1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

2

6

2

7

2

8

2

9

3

0

3

1

3

2

3

3

3

4

3

5

3

6

3

7

3

8

3

9

4

0

4

1

4

2

4

3

4

4

4

5

4

6

4

7

4

8

4

9

5

0

5

1

5

2

5

3

5

4

5

5

5

6

5

7

5

8

5

9

6

0

6

1

6

2

6

3

6

4

6

5

6

6

6

7

6

8

6

9

7

0

7

1

7

2

7

3

7

4

7

5

7

6

7

7

7

8

7

9

8

0

8

1

8

2

8

3

8

4

8

5

8

6

8

7

S. no. Haplotype Ratio 1 6

1

2

3

2

4

3

7

9

8

4

9

9

1

0

0

1

0

1

1

0

7

1

0

9

1

1

1

1

1

2

1

1

3

1

2

6

1

3

3

1

4

1

1

4

2

1

4

3

1

4

4

1

4

7

1

8

1

1

8

4

1

9

0

2

0

5

2

0

6

2

1

9

2

2

2

2

2

4

2

3

3

2

3

4

2

3

6

2

3

7

2

4

1

2

4

2

2

5

0

2

5

3

2

5

8

2

6

2

2

6

8

2

7

5

2

8

2

2

9

3

2

9

6

3

0

2

3

0

3

3

1

8

3

2

4

3

3

5

3

3

8

3

4

4

3

4

8

3

5

5

3

6

5

3

7

1

3

7

7

3

8

0

3

8

4

3

8

6

3

9

3

3

9

4

3

9

8

4

0

1

4

0

3

4

0

8

4

1

2

4

1

4

4

1

6

4

1

7

4

2

3

4

3

0

4

3

1

4

3

6

4

3

7

4

3

9

4

4

7

4

5

1

4

5

2

4

5

9

4

6

0

4

6

1

4

6

2

4

7

3

4

7

7

4

8

5

4

9

5

1 SD-1.1 1/137 A C T C T A A G C G C A A − C T T T T T G C G T T C G C T G T T A C G T C A T G T G A G C C G C A C A C T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

2 SD-1.2 1/137 A T T C T A A G C G T A A A C T T T − C − C G C T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

3 SD-1.3 1/137 A T T C T A A G C G C A A − C T T T T T G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

4 SD-1.4 1/137 A C T C T A A G C G T A A A C T T T − C − C G C T C G C T G T T A C G T C G G G T G A G C C G C A C A T T C C A A C C A C G A A T A T T − − A T T T G G C G C T C C C G T T T

5 SD-1.5 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C G T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G T T T

6 SD-1.6 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C G T T C C A A C C G C G A A T A T T − − A T A T G G C G C T C C C G T T T

7 SD-1.7 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C G T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

8 SD-1.8 2/137 A C T C T A A G C G C A A − C T T T T T G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

9 SD-1.9 1/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C G T T C C G A C C G C G A A T A T T − − A T A T G G C G C T C C C G T T T

10 SD-1.10 1/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G C T T

11 SD-1.11 1/137 A C T C T A A G C G T A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G T T T

12 SD-1.12 1/137 A C T C T A A G C G C A A − C T T T T C G C G T T C G C T G T T A C G T C A T G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

13 SD-1.13 1/137 A C T C T A A G C G − A A A C T T T − C C G T T C A C T G T T A C G T C A G G T A A G C C G C A C A T T C C A G C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

14 SD-2.1 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A T C G C G A A T A A C C T A T T T G G C G C T C C C G T T T

15 SD-2.2 6/137 A C T C T A A G C G T A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A T A T C C C A A C C G C G A A T A A C C T A T T T G G C G C T C C A G T T C

16 SD-2.3 1/137 A C T C T A A − − − T C A − − T C T T C G C G T T C G C T G T T A T G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T C G G A G C T C C C G T T T

17 SD-2.4 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G G G C C G C A C A T T C C A A T C G C G A A T A A C C T A T T T G G C G C T C C C G T T T

18 SD-2.5 2/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T T C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − − T T T G G C G C T C C C G T T T

19 SD-2.6 1/137 A C T C T A A G C G T A A A C T T T − C − C G T T C G C T G T T A C A T C A G G T G A G C C G C A T A T C C C A A C C G C G A A T A A C C T A T T T G G C G C T C C A G T T C

20 SD-2.7 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T A C A A C C G C G A A T A T T − − A T T T G G C G C T C C G G T T C

21 SD-2.8 2/137 G C T C T A A G C G T A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A T A T C C C A A C C G C G A A T A A C C T A T T T G G C G C T C C A G T T C

22 SD-2.9 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A T C G C A A A T A A C C T A T T T G G C G C T C C C G T T T

23 SD-2.10 1/137 A C T C T A A G C G T A A A C T T T − C − C G T T T G C T G T T A C G T C A G G T G A G C C G C A T A T C C C A A C C G C G A A T A A C C T A T T T G G C G C T C C A G T T C

24 SD-2.11 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C T G C A C A T T C C A A T C G C G A A T A A C C T A T T T G G C G C T C C C G T T T

25 SD-2.12 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T T T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

26 SD-2.13 1/137 A C T C T A A G C G T A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A T A T C C C A A C C G C G G G T A A C C T A T T T G G C G C T C C A G T C C

27 SD-2.14 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T T A G G T G A G T C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

28 SD-2.15 1/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T A C C G T T T

29 SD-2.16 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C T G T T A C G T C A G G T G A G T C G C A C A T T C C A A C C G C G A A T A A C C − A T T T G G C G C T C C A A T T C

30 SD-2.17 1/137 A C T C T A A G C G T A A − C T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A A T T G G C G C T C C C G T T T

31 SD-2.18 1/137 A C T T T A A − − − T C A − − T C C − C − C G T T C G C T G T T A C G T C A G G T G A G T C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

32 SD-2.19 1/137 A C T C T A A G C G T A A A C T T T − C − C G T T C G C T G C T A C G T C A G G T G A G C C G C A T A T C C C A A C C G C G A A T A A C C T A T T T G G C G C T C C A G T T C

33 SD-3.2 14/137 A C T C T A A G T G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

34 SD-3.3 1/137 A C T C T A A G C G C A A − C T T T T C A C G T T C G C T G T T A C G T C A G G T G A G C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T C

35 SD-3.5 1/137 A C T C T A A G C G − A A A C T T T T C G C G T T C G C T G T T A C G T C A T G T G A G C C G C C A T T C C A A C C G C G A A C A T T − − A T T T G G C G C T C C C G T T T

36 SD-3.6 1/137 G C T C T A A G C G − A A A C T T T T C G C G T T C G C T G T T A C G T C A T G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

37 SD-3.7 1/137 A C T C T A A G T G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C A C T C C C G T T T

38 SD-3.8 1/137 A C T C T A A G T G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G A T G A G C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

Nucleotide position

Mutation number

100

Table 4-4. Continued

SD* -1.20,26 -3.1,7,10,37 SD** -1.27 -3.8,13 SD*** -1.5,18,19,28,39 -4.1,4,8,16,20,23,25,29,38 SD**** -1.15 -3.5,6,25,32,48 -4.4,12,18,24,26,28,34,39 Columns 1, 2 and 3 are unique sequence number (S. no.), haplotype code, and number of clones of haplotype per total no. of clones from each of four individual specimens. For example, SD- 3.2 is S. dorsalis specimen number 3, set of like clones number 2, which was found in 14 of a total of 137 clones. Haplotypes consisting of clones of more than one individual specimen have been marked with asterisks. The coding for these haplotypes consists of bold digits denoting the specimen number, which is followed by the clones it exhibited. For example, SD** -1.27 -3.8,13, indicates that haplotype SD** consisted of one clone (clone no. 27) from specimen number 1 and two clones (8 and 13) from specimen number 3.

1 2 3 4 5 6 7 8 9

1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

2

6

2

7

2

8

2

9

3

0

3

1

3

2

3

3

3

4

3

5

3

6

3

7

3

8

3

9

4

0

4

1

4

2

4

3

4

4

4

5

4

6

4

7

4

8

4

9

5

0

5

1

5

2

5

3

5

4

5

5

5

6

5

7

5

8

5

9

6

0

6

1

6

2

6

3

6

4

6

5

6

6

6

7

6

8

6

9

7

0

7

1

7

2

7

3

7

4

7

5

7

6

7

7

7

8

7

9

8

0

8

1

8

2

8

3

8

4

8

5

8

6

8

7

S. no. Haplotype Ratio 1 6

1

2

3

2

4

3

7

9

8

4

9

9

1

0

0

1

0

1

1

0

7

1

0

9

1

1

1

1

1

2

1

1

3

1

2

6

1

3

3

1

4

1

1

4

2

1

4

3

1

4

4

1

4

7

1

8

1

1

8

4

1

9

0

2

0

5

2

0

6

2

1

9

2

2

2

2

2

4

2

3

3

2

3

4

2

3

6

2

3

7

2

4

1

2

4

2

2

5

0

2

5

3

2

5

8

2

6

2

2

6

8

2

7

5

2

8

2

2

9

3

2

9

6

3

0

2

3

0

3

3

1

8

3

2

4

3

3

5

3

3

8

3

4

4

3

4

8

3

5

5

3

6

5

3

7

1

3

7

7

3

8

0

3

8

4

3

8

6

3

9

3

3

9

4

3

9

8

4

0

1

4

0

3

4

0

8

4

1

2

4

1

4

4

1

6

4

1

7

4

2

3

4

3

0

4

3

1

4

3

6

4

3

7

4

3

9

4

4

7

4

5

1

4

5

2

4

5

9

4

6

0

4

6

1

4

6

2

4

7

3

4

7

7

4

8

5

4

9

5

39 SD-3.9 1/137 A C T C T A A G T G − A A A C T T T − C − C G T C C G C T G T T A C G T C A G G T G A G C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

40 SD-3.10 1/137 A C T C T A A G C G − A A A C T T T T C G T G T T C G C T G T T A C G T C A T G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

41 SD-3.11 1/137 A C T C T A A G C G C A A − C T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G A G A A T A T T − − A T T T G G C G C T C C C G T T T

42 SD-3.12 1/137 A C T C T A A G C G C A A − C T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G T A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

43 SD-3.13 1/137 A C T C T A A G T G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

44 SD-3.14 1/137 A C T C T A A G T G − A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C G C C C C C G T T T

45 SD-3.15 1/137 A C T C T A A G C G − A A A C T T T T C G C G T T C G C T G T T A C G T C A T G T G A G C C G C G C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

46 SD-3.16 1/137 A C T C T A A G C G C A A − C T T T T C G C G T T C G C T A T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

47 SD-3.17 1/137 A C T C T A A G T G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A T C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

48 SD-3.18 1/137 A C T C T A A G C G − A A G C T T T T C G C G T T C G C T G T T A C G T C A T G C G A G C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

49 SD-3.19 1/137 A C T C T A A G C G − A A A C T T T T C G C G T T C G C T G T T A C G T C A T G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C C C C C G T T T

50 SD-3.20 1/137 A C T C T A G G T G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C T T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

51 SD-4.1 1/137 A C T C T A A G C G C A A − C T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A T A T C C T A A C C G C G A A T A A C C T A T T T G G C G C T C C A G T T T

52 SD-4.2 12/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A T A T C C T A A C C G C G A A T A A C C T A T T T G G C G C T C C A G T T C

53 SD-4.3 1/137 A C T C C A A − − − T C G − − T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T A G C G C T C C A G T T C

54 SD-4.4 1/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C T G C G A A T A T T − − A T T T G G C G C T C T C G T T T

55 SD-4.5 1/137 A C T C T A A G C G T A A A C T T T − C − C G T T C G T T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G T T T

56 SD-4.6 1/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G A C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G T T C

57 SD-4.7 1/137 A C T C T A A − − − T C A − − C T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G A C G C T C C C G T T T

58 SD-4.8 1/137 A C T C T A A − − − T C A − − T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A T A T C C T A A C C G C G A A T A A C C T A T T T G G C G C T C C A G T T C

59 SD-4.9 1/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T C T G G C G C T C T C G T T T

60 SD-4.10 1/137 A C T C T G A − − − T C A − − T T T T C G C G T T C G C T G T C A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

61 SD-4.11 1/137 A C T C T A A − − − T C A − − T T T − C − C G T T C G C C G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G T T C T C G T T T

62 SD-4.12 1/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C A C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G T T T

63 SD-4.13 1/137 A C C C T A A G C G C A A − C T T T T T G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G T T T

64 SD-4.14 1/137 A C T C T A A − − − T C A − − T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T C G G C G C T C C C G T T T

65 SD-4.15 1/137 A C T C T A A G C G C A A − C T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T G T T − − A T T T G G C G C T C C C G T T T

66 SD-4.16 1/137 A C T C T A A G C G − A A A C T T T − C − C A T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G T T T

67 SD-4.17 1/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

68 SD* 6/137 A C T C T A A G C G − A A A C T T T T C G C G T T C G C T G T T A C G T C A T G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

69 SD** 3/137 A C T C T A A G C G T A A A C T T T − C − C G C T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

70 SD*** 15/137 A C T C T A A G C G − A A A C T T T − C − C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C T C G T T T

71 SD**** 14/137 A C T C T A A G C G C A A − C T T T T C G C G T T C G C T G T T A C G T C A G G T G A G C C G C A C A T T C C A A C C G C G A A T A T T − − A T T T G G C G C T C C C G T T T

Mutation number

Nucleotide position

101

Table 4-5. The mtCO1 sequences that differ among Scirtothrips dorsalis individuals.

SD*-1.3,4,6,7,9,10,11,12,13,17,18,20,21,22,23,24,26,28,29,31,32,33,35,36,37,40,45,46,47,48-2.5,6,7,8,10,15,16,19,20,21,22,23,24,26,27,28,29, 30,31,32,33,34,36,38,40,41,42,43,44,45,46,48 -3.2,6,7,10,11,12,15,16,17,18,20,21,22,24,25,27,28,29,30,31,34 -4.9,10,17,19,20,22,24,25,26,27, 28,29,30,32,33,34,35,36,38,40,41,42,43,45,46,47,48

1 2 3 4 5 6 7 8 9

1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

2

6

2

7

2

8

2

9

3

0

3

1

3

2

3

3

3

4

3

5

3

6

3

7

3

8

3

9

4

0

4

1

4

2

S. no. Haplotype Ratio

2

2

2

4

3

1

3

4

4

0

6

2

8

1

8

8

1

2

0

1

4

2

1

6

0

1

9

3

1

9

5

2

1

4

2

3

6

2

4

1

2

4

9

2

7

9

2

9

3

3

0

9

3

2

6

3

3

1

3

3

2

3

3

6

3

4

3

3

7

5

4

0

6

4

3

2

4

3

5

4

6

7

4

7

5

4

9

1

4

9

7

5

1

9

5

4

5

5

5

6

5

7

6

5

7

8

6

0

1

6

1

3

6

1

6

6

2

8

1 SD-1.1 1/132 G T T T T A T A G C T A A A T T A C A A A A A T A C T C T A T A T T T A T T T T A T

2 SD-1.2 1/132 G T T T T A T A A T T A A A T T A C A A A A A T A C T C T A T G T T T A T T C T A T

3 SD-1.3 1/132 G T T T T A T G A T T A A A T T A C A A A A A T A C T C T A T A T T T A T T T C A T

4 SD-2.1 1/132 G T T C T G T A A T T A A A T T A C A A A A A T A C T C T A T A T T T A T T T T A T

5 SD-2.12 1/132 G T T T T A T A A T T A G A T T A C A G A A A T A C T C T A T A T T T A T T T T A T

6 SD-2.2 1/132 G T T T C A T A A T T A A A T T A C A A A G A T A C T C T A T A T T T A T T T T A T

7 SD-2.3 1/132 G T T T T A T A A T T A A A C T A C A A A A A T A C T C T A T A T T T A T C T T A T

8 SD-2.4 1/132 G T T T T A T A A T T A A A T T A C A A A A A T G C T C T A T A T T T A T T T T A T

9 SD-2.5 1/132 G T T T T A T A A T T A A A T T A C A A A A A T A C T C T A C A T T T A T T T T A T

10 SD-2.6 1/132 G T T T T A T A A T T A A A T T A C A A A A A T A C T C T A T A T T T A C T T T A T

11 SD-2.7 1/132 G C T T T A T A A T T A A A T T A C A A A A A T A C T C C A T A T T T A T T T T G T

12 SD-2.8 1/132 G T T T T A T A A T T A A A T T A C A A A A A G A T T C T G T A T T T A T T T T A T

13 SD-2.9 1/132 A T T T T A T A A T T A A A T T A C A A A A A T A C T C T A T A C T T A T T T T A T

14 SD-2.10 1/132 G T T T T A T A A T T G A A T C A C A A A A A T A C T T T A T A T T T A T T T T A T

15 SD-2.11 1/132 G T T T T A T A A T T A A A T T A C G A A A A T A C T C T A A A T C T A T T T T A T

16 SD-3.1 1/132 G T C T T A T A A T T A A A T T A C A A A A G T A C T C T A T A T T T A T T T T A T

17 SD-3.2 1/132 G T T T T A T A A T T A A A T T G A A A A A A T A C T C T A T A T T T A T T T T A T

18 SD-3.3 1/132 G T T T T A T A A T T A A G T T A C A A A A A T A C − C T A T A T T T A T T T T A T

19 SD-4.1 1/132 G T T T T A T A A T T A A A T T A C A A A A A T A C T C T A T A T T T G T T T T A T

20 SD-4.2 1/132 G T T T T A C A A T T A A A T T A C A A A A A T A C T C T A T A T T T A T T T T A C

21 SD-4.3 1/132 G T T T T A T A A T − A A A T T A C A A A A A T A C T C T A T A T T T G T T T T A T

22 SD-4.4 1/132 G T T T T A T A A T T A A A C T A C A A G A A T A C T C T A T A T T C A T T T T A T

23 SD* 110/132 G T T T T A T A A T T A A A T T A C A A A A A T A C T C T A T A T T T A T T T T A T

Mutation number

Nucleotide position

102

Table 4-6. The rDNA ITS2 sequences that differ among Thrips palmi individuals.

1 2 3 4 5 6 7 8 9

1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

2

6

2

7

2

8

2

9

3

0

3

1

3

2

3

3

3

4

3

5

3

6

3

7

3

8

3

9

4

0

4

1

4

2

4

3

4

4

4

5

4

6

4

7

4

8

4

9

5

0

5

1

5

2

5

3

5

4

5

5

5

6

5

7

5

8

5

9

6

0

6

1

6

2

6

3

6

4

6

5

6

6

6

7

6

8

6

9

7

0

7

1

7

2

7

3

7

4

7

5

7

6

7

7

7

8

7

9

S. no.Haplotype Ratio 1

1

7

5

1

5

6

6

8

7

9

8

0

9

7

1

0

5

1

0

8

1

1

8

1

2

1

1

2

8

1

3

0

1

3

9

1

4

4

1

5

3

1

7

1

1

7

4

1

9

2

2

0

4

2

0

8

2

1

2

2

2

2

2

3

1

2

4

9

2

5

0

2

5

5

2

6

0

2

6

1

2

6

2

2

6

7

2

7

8

2

8

0

2

8

9

3

0

2

3

0

4

3

0

5

3

1

0

3

1

1

3

2

0

3

2

7

3

3

1

3

3

4

3

5

1

3

5

2

3

5

5

3

5

9

3

7

6

3

8

7

3

9

1

3

9

4

3

9

8

3

9

9

4

0

0

4

0

8

4

1

7

4

2

1

4

2

3

4

3

0

4

3

1

4

3

2

4

3

6

4

5

7

4

6

8

4

8

1

4

8

8

4

9

0

4

9

5

4

9

6

4

9

8

5

0

0

5

0

3

5

1

3

5

1

5

5

2

6

5

3

7

5

5

4

5

6

2

1 TP-1.1 1/149 A A A T G A T T C T T A A T T A A A T G G T T G A T T G A C C C A A T C A T C T G G C T T G G T T T G G A T C T G C A C G T T A G T T T T T T T A C C A C T G

2 TP-1.2 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G C T T G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

3 TP-1.3 1/149 A A A − G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A T T G T T G

4 TP-1.4 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C T A A T C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A T T G C T G

5 TP-1.5 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T C A C C C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C C C T G

6 TP-1.6 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G C T T G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C C C T G

7 TP-1.7 2/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T C A C C C A A C C A T T T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A T T G C T G

8 TP-1.8 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G A T T T A G A T C T G C A T G C C A G T T T T T T T A C C C C T G

9 TP-1.9 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T C A T C C A A C C A T T T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C C C T G

10 TP-1.10 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T C T G G A T C T G C A T G C C A G T T T T T T T A C C C C C G

11 TP-1.11 1/149 A A A T G A T T C T T A A T A A A A T G A T T G A T T G A C C C A A T C A T C T G G C T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A T T G T T G

12 TP-1.12 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C C C T A

13 TP-1.13 1/149 A A A T G T C T C T T A A T T A A A C G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C C C T G

14 TP-1.14 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T C A C C C A A C C A T T T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C C C T G

15 TP-1.15 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T T G G T T T G G A T C T G C A C G T T A G T T T T T T T A C C C C T G

16 TP-1.16 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C C C T G

17 TP-1.17 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A T C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A T T G T T G

18 TP-2.1 1/149 A A G T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A C C A T T T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

19 TP-2.2 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A C C A C T G

20 TP-2.3 1/149 A A A T G T C T C T T A A T T G A A T G A T T G A T T G A C C C A A T C A T C T G G C T T G G T T T G G A T C T G C A T G C C G G T T T T T T T A C C A C T G

21 TP-2.4 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T T A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

22 TP-2.5 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T A G A T C T G C G T G C C A G T T T T T T T A C C A C T G

23 TP-2.6 1/149 A A A T G A T T C T T A A T C A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T A C A T G C C A G T T T T T T T A C C A C T G

24 TP-2.7 1/149 A A A T A A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C A C T G

25 TP-2.8 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A T G T T A G T T T T T T T A C C A C T G

26 TP-2.9 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T A G A T C T G C A T A C C A G T T T T T T T A C C A C T G

27 TP-2.10 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G C T T G G T T C G G A T C T G C A C G T T A G T T T T T T T A C C A C T G

28 TP-2.11 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G C T T G G T T T G A A T C T G C A C G T T A G T T T T T T T A C C A C T G

29 TP-2.12 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A C G T T A G T T T T T T T A C C A C T G

30 TP-2.13 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T C A T T G T T G

31 TP-2.14 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T A G A T C T G C A C G T T A G T T T T T T T A C C A C T G

32 TP-3.1 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C C G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A C C A C T G

33 TP-3.2 7/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C A G C A T G C T A G T T T T T T T A C C A C T G

34 TP-3.3 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G A G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

35 TP-3.4 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C G T C T G G C T T G G T T T G G A T C T G C A T G T C A G T T T C T T T A T T G T T G

36 TP-3.5 1/149 G A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A G C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A T T G T T G

37 TP-3.6 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A T G T T A G T T T T T T T A C C A C T G

38 TP-3.7 1/149 A A A T G T C T C T T G A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C A G C A T G C T A G T T T T T T T A C C A C T G

39 TP-3.8 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C A G C A T G C T A G T T T T T C T A C C A C T G

40 TP-3.9 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T C T T T A T T G C T G

Mutation number

Nucleotide position

103

Table 4-6. Continued

TP*-1.1,16,17,36,40,47 -2.1,5,24,26,31,34,46 -3.12,21,45 -4.2,9,14,21,31,37 TP**-1.3,8,12,19,22,24,26,33,38,41 -2.8,10,32,35,39,40,45 TP***-1.30 -4.30 TP****-1.20,23 -2.3,4,19,43 TP*****-1.31-9.23,28,32 -2.7,22,27,36,38,44 TP******-1.9,10,39 -3.16,19,20,25,27,35

1 2 3 4 5 6 7 8 9

1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

2

6

2

7

2

8

2

9

3

0

3

1

3

2

3

3

3

4

3

5

3

6

3

7

3

8

3

9

4

0

4

1

4

2

4

3

4

4

4

5

4

6

4

7

4

8

4

9

5

0

5

1

5

2

5

3

5

4

5

5

5

6

5

7

5

8

5

9

6

0

6

1

6

2

6

3

6

4

6

5

6

6

6

7

6

8

6

9

7

0

7

1

7

2

7

3

7

4

7

5

7

6

7

7

7

8

7

9

S. no. Haplotype Ratio 1

1

7

5

1

5

6

6

8

7

9

8

0

9

7

1

0

5

1

0

8

1

1

8

1

2

1

1

2

8

1

3

0

1

3

9

1

4

4

1

5

3

1

7

1

1

7

4

1

9

2

2

0

4

2

0

8

2

1

2

2

2

2

2

3

1

2

4

9

2

5

0

2

5

5

2

6

0

2

6

1

2

6

2

2

6

7

2

7

8

2

8

0

2

8

9

3

0

2

3

0

4

3

0

5

3

1

0

3

1

1

3

2

0

3

2

7

3

3

1

3

3

4

3

5

1

3

5

2

3

5

5

3

5

9

3

7

6

3

8

7

3

9

1

3

9

4

3

9

8

3

9

9

4

0

0

4

0

8

4

1

7

4

2

1

4

2

3

4

3

0

4

3

1

4

3

2

4

3

6

4

5

7

4

6

8

4

8

1

4

8

8

4

9

0

4

9

5

4

9

6

4

9

8

5

0

0

5

0

3

5

1

3

5

1

5

5

2

6

5

3

7

5

5

4

5

6

2

41 TP-3.10 1/149 G A A T G T C T C T T A A T T A A G T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A C C A C T G

42 TP-3.11 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C G A T C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

43 TP-3.12 1/149 A A A T G T C T C T T A A T T A A A T A A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G G T C T G C A T G C C A G T T T T T T T A C C A C T G

44 TP-3.13 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A T T A C T G

45 TP-4.1 3/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T T G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

46 TP-4.2 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C A C T G

47 TP-4.3 2/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G C T T G G T T T G G A T C T G C A C G T T A G T T T T T T T A C C A T T G

48 TP-4.4 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C T T A A T C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A T T G

49 TP-4.5 4/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C T C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C A C T G

50 TP-4.6 1/149 A A A T G A T T C T T A A T T A G A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A T T G T T G

51 TP-4.7 1/149 A A A T G A T T T T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A C T T G C A T G C T A G T T T T T T T A T T G T T G

52 TP-4.8 1/149 A A A T G T C T C T T A A T T A A A T G A T C G A T T G A C C C A A T C A T C T T G T T G G G T T T G G A C C T G C A T G C C A G C T C T T T T A C C A C T G

53 TP-4.9 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C T C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A T T G T T G

54 TP-4.10 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C T C A A T C A T C T G G T T G G G C T T A G A T C T G C A T G C C A G T A T T T T T A C C A C T G

55 TP-4.11 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C T C A A T C A T C T G G T T G G G T T T A G A T C T G G A T G C C A G T T T T T T T A C C A C T G

56 TP-4.12 1/149 A G A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G C T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

57 TP-4.13 1/149 A A A T G A T T C T T A A T T A A A T G A T T A A T T G A C T C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C A C T G

58 TP-4.14 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C T C A A T C A T C T G A T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C A T T G

59 TP-4.15 1/149 A A A T G T C T C T C A A T T A A A T G A T T G A T T G A C C C A A T C A T C T T G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

60 TP-4.16 1/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T T G T T G G G T T T G G A T C T G C A T G C C A G T T T T C T T A C C A C T G

61 TP-4.17 1/149 A A A T G T C T C T T A G T T A A A T G A T T G A T T G A C T C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C A C T G

62 TP-4.18 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T G T T G T T G

63 TP-4.19 1/149 A A A T G A T T C T T A A T T A A A T G A C T G G T T G A C T C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C A C T G

64 TP-4.20 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G G C C C A A T C A T C T G G T T G G G T T T G G G T C T G C A T G C C A G T T T T T T T A C C A C T G

65 TP-4.21 1/149 A A A T G T C T T T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

66 TP-4.22 1/149 A A A T G T C T C T T A A T T A A A T G A T T A A T T G A C C C A A T C A T C T G G C T T G G T T T G G A T C T G C A C G T T A A T T T T T T T A C C A T T G

67 TP-4.23 1/149 A A A T G T C T C C T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G C T T G G T T T G G A T C T G C A C G T T A G T T T T T T T A C C A T T G

68 TP-4.24 1/149 A A A T G A T C C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A C T T G C A T G C T A G T T T T T T T A T T G T T G

69 TP-4.25 1/149 A A A T G A T T C T T A A C T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A C C A C T G

70 TP-4.26 1/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A C C T G G T C G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

71 TP* 22/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

72 TP** 17/149 A A A T G A T T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T A G A T C T G C A T G C C A G T T T T T T T A C C A C T G

73 TP*** 2/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A C C C C T G

74 TP**** 6/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G C T T G G T T T G G A T C T G C A C G T T A G T T T T T T T A C C A C T G

75 TP***** 10/149 A A A T G T C T C T T A A T T A A A T G A T T G A T T G A C C C A A T C A T C T G G T T G G G T T T G G A T C T G C A T G C C A G T T T T T T T A C C A C T G

76 TP****** 9/149 A A A T G A T T C T T A A T T A A A T G A T T G A − − G A C C C A A C C A T C T G G C T G G G T T T G G A T C T G C A T G T C A G T T T T T T T A T T G T T G

Mutation number

Nucleotide position

104

Table 4-7. The mtCO1 sequences that differ among Thrips palmi individuals.

TP* -2.11,12,13,19,20,24,26,27,28,29,31,32,35,38,39,40,41,42,43,44,46,48 -3.3,5,7,9,11,14,15,16,18,19,22,23,26,27,35,36,40,41,42,43,44,45, 47,48 -4.8,14,15,16,19,20,21,22,23,24,26,27,28,29,31,32,34,36,37,39,40,42,44,45,46,47,48

1 2 3 4 5 6 7 8 9

1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

S. no. Haplotype Ratio

3

0

4

4

7

9

1

2

9

1

6

0

1

8

7

2

3

4

2

6

7

2

7

0

2

8

0

3

0

8

3

1

3

3

2

8

3

3

9

3

6

3

3

8

6

3

9

3

3

9

4

4

0

8

5

7

2

5

7

3

5

7

8

5

9

8

6

0

1

6

4

3

1 TP-1.1 34/120 C T A T T T C T T T A A A A A A T T C A T T T A C

2 TP-1.2 1/120 C T A T T T C T T T A A A A A A T T A A T C T A C

3 TP-1.3 1/120 C T A T T T T T T T A A A A A A T T C A T T T A C

4 TP-1.4 1/120 T T A T T T C T T T A A A A A A T T C A T T T A C

5 TP-1.5 1/120 C T A T T T C T C T G A A A A A T T C A T T T A C

6 TP-1.6 1/120 C T A T T T C T T T A A A A A A T T C A C T T A C

7 TP-1.7 1/120 C T G T T T C T T T A A A A A A T T C A T T T A T

8 TP-1.8 1/120 T T A T T T C T T T A A A A A A T T C G T T T A C

9 TP-1.9 1/120 C T A T T T C T T T A A A A A A − − C A T T T A C

10 TP-2.1 1/120 C C A T − C C T T T A G A A A A T T C A T T C A C

11 TP-2.2 1/120 C C A T T C C C T T A A A A G A T T C A T T C A C

12 TP-4.1 1/120 C C A T T C C T T C A A G A A A T T C A T T C A C

13 TP-4.2 1/120 C C A T T C C T T T A A A G A A T T C A T T C G C

14 TP-4.3 1/120 C C A T T C C T T T A A A A A G T T C A T T C G C

15 TP* 73/120 C C A T T C C T T T A A A A A A T T C A T T C A C

Nucleotide position

Mutation number

105

Table 4-8. The rDNA ITS2 sequences that differ among Frankliniella occidentalis individuals.

FO* 2.8,16,22,23,29,30,34,35,37,38,40,41,43,44,45,48 -3.2,3,4,7,8,9,10,11,12,13,14,15,16,17,18,20,22,23,24,25,27,28,30,31,32,33,38,39,41,48 -4.1,8,9,14,16,18,19,21,22,23,24,25,26,29,30,31,32,33,34,35,36,37,39,40,42,44,45,46,47,48

1 2 3 4 5 6 7 8 9

1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

2

6

S. no. Haplotype Ratio

3

4

3

7

7

0

9

2

1

2

7

1

4

9

1

5

8

1

6

9

1

7

3

1

7

9

2

3

7

2

4

0

2

4

6

2

5

2

2

5

8

2

6

4

2

8

9

2

9

1

2

9

5

2

9

9

3

1

3

3

2

8

3

8

6

3

8

7

4

1

6

4

3

2

1 FO-1.1 17/105 A A G A T T C T T G G A C C A T G T T G A T − − G A

2 FO-1.2 1/105 A G G A T T T T T G G A C C A T G T T G A T − − G A

3 FO-1.3 1/105 A A G G T T C T T G G A C C A T G T T G G T − − G A

4 FO-1.4 1/105 A A G A T T C T T G G A C C A T G T T G G C − − G A

5 FO-2.1 1/105 G A G A T T C T C G G A C C A T G T T G G C T T G A

6 FO-3.1 1/105 A A G A C T C T T G A A C C A T G T T G G C T T G A

7 FO-3.2 1/105 A A G A T T C T T G G A C C G T G T T A G C T T G A

8 FO-3.3 1/105 A A G A T T C C T G G A C T A T G T T G G C T T G A

9 FO-3.4 1/105 A A G A T T C T T A G A T C A T G T T G G C T T G A

10 FO-3.5 1/105 A A G A T C C T T G G A C C A C G T T G G C T T T A

11 FO-3.6 1/105 A A G A T T C T T G G A C C A T G T C G G C T T G G

12 FO-4.1 1/105 A A G A T T C T T G G A C C A T T A T G G C T T G A

13 FO-4.2 1/105 A A A A T T C T T G G G C C A T G T T G G C T T G A

14 FO* 76/105 A A G A T T C T T G G A C C A T G T T G G C T T G A

Mutation number

Nucleotide position

106

Table 4-9. The mtCO1 sequences that differ among Frankliniella occidentalis individuals.

FO* -1.1,2,3,4,5,6,7,11,12,13,14,15,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,41,43,46 -2.1,2,3,4,5,6,7,9,12,13,14,16, 17,18,19,20,22,23,24,25,,28,29,30,33,34,35 -3.1,2,3,4,5,7,8,9,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,32,33,34,35,37,40, 42,43,44,46,47,48 -4.1,7,8,9,10,11,12,14,15,16,18,19,21,22,23,24,25,26,27,28,29,30,31,32,34,35,36,37,38,39

1 2 3 4 5 6 7 8 9

1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

S. no. Haplotype Ratio

3

0

3

1

3

4

4

1

4

8

8

8

1

1

3

1

1

4

1

3

3

1

3

7

1

5

8

1

8

9

1

9

0

2

1

6

2

5

2

2

6

8

2

9

7

3

0

3

3

2

1

3

5

2

3

8

3

3

9

9

4

1

4

1 FO-1.1 1/150 T T T T T A T C A T T T A T A T C T C A G T T

2 FO-1.2 1/150 T T T T C A T T A T T T A T A T T T C G G T T

3 FO-1.3 1/150 T T T T C A T C A T T T G T A T T T C A G C T

4 FO-1.4 2/150 T T T T C G T C A T T T A T A T T T C A G T T

5 FO-1.5 1/150 C T T T C A T C A T T T A T A T T C C A G T T

6 FO-2.1 2/150 T T T T C A C C A T T T A T A T T T C A G T T

7 FO-2.2 1/150 T T T T C A T C A T T T A C A T T T C A G T C

8 FO-2.3 1/150 T T C T C A C C A C T T A T A T T T C A G T T

9 FO-3.1 2/150 T T T T C A T C A T T T A T A T T T T A G T T

10 FO-3.2 2/150 T T T T C A T C G T T T A T A T T T C A G T T

11 FO-3.3 1/150 T C T T C A T C A T T T A T A C T T C A G T T

12 FO-3.4 1/150 T T T T C A T C A T A T A T G T T T C A A T T

13 FO-4.1 1/150 T T T C C A T C A T T C A T A T T T C A G T T

14 FO* 133/150 T T T T C A T C A T T T A T A T T T C A G T T

Nucleotide position

Mutation number

107

SD*-1.20,26 -3.1,7,10,37 SD**-1.27 -3.8,13 SD***-1.5,18,19,28,39 -4.1,4,8,16,20,23,25,29,38 SD****-1.15 - 3.5,6,25,32,48 -4.4,12,18,24,26,28,34,39

Figure 4-1. An unrooted semi strict MP tree generated from rDNA ITS2 sequence obtained from 2 female and 2 male

individuals of S. dorsalis. Clones from different individuals have been coded in different colors. Bootstrap values are on the branches. Haplotypes consisting clones of more than one individual have been marked with asterisks. Coding of these haplotypes consists of bold digits denoting the specimen number, which is followed by the clones it exhibited. For example, SD**-1.27 -3.8,13, means shared haplotype SD** consists of one clone

(27) from specimen number 1 and two clones (8 and 13) from specimen number 3.

Blue: Specimen no.1, Red: Specimen no. 2, Green: Specimen no. 3, Dark blue: Specimen no. 4, Brown: Shared haplotype

108

TP*-1.1,16,17,36,40,47 -2.1,5,24,26,31,34,46 -3.12,21,45 -4.2,9,14,21,31,37 TP**-1.3,8,12,19,22,24,26,33,38,41 -2.8,10,32,35,39,40,45 TP***-1.30 -4.30 TP****-1.20,23 -2.3,4,19,43 TP*****-1.31-9.23,28,32 -2.7,22,27,36,38,44 TP******-1.9,10,39 -3.16,19,20,25,27,35

Figure 4-2. An unrooted semi strict MP tree generated from rDNA ITS2 sequence obtained from 2 female and 2 male individuals of T. palmi. Clones from different individuals have been coded in different colors. Bootstrap values are on the branches.

Blue: Specimen no.1, Red: Specimen no. 2, Green: Specimen no. 3, Dark blue: Specimen no. 4, Brown: Shared haplotypes

109

FO*-2.8,16,22,23,29,30,34,35,37,38,40,41,43,44,45,48 -3.2,3,4,7,8,9,10,11,12,13,14,15,16,17,18,20,22,23,24,25,27,28,30,31,32,33,38,39,41,48 - 4.1,8,9,14,16,18,19,21,22,23,24,25,26,29,30,31,32,33,34,35,36,37,39,40,42,44,45,46,47,48

Figure 4-3. An unrooted semi strict MP tree generated from rDNA ITS2 sequence obtained from 2 female and 2 male individuals of F. occidentalis. Clones from different individuals have been coded in different colors. Bootstrap values are on the branches.

Blue: Specimen no.1, Red: Specimen no. 2, Green: Specimen no. 3, Dark blue: Specimen no. 4, Brown: Shared haplotypes

110

CHAPTER 5 SUMMARY

In the insect order Thysanoptera, the genus Scirtothrips Shull contains more than

100 thrips species, among which 10 species have been reported as serious pests of

agricultural crops (Rugman-Jones et al. 2006). Within this devastating genus,

Scirtothrips dorsalis Hood is an emerging pest of various economically important host

crops in the United States. Scirtothrips dorsalis is a polyphagous pest with more than

100 reported hosts among 40 different families of plants (Mound and Palmar 1981).

However, in the past two decades increased globalization and open agricultural trade

has resulted in the expansion of the geographical distribution and host range of the

pest. In a recent study, Kumar et al. (2012) reported this pest attacking 11 different

hosts at a fruit nursery in Homestead, Florida. Interestingly, they were found to

reproduce on nine plant taxa that had never been reported as hosts in the literature.

The small size and cryptic nature of adults and larvae enables S. dorsalis to inhabit

microhabitats of a plant and in the field, often making monitoring and the identification

difficult. Scirtothrips dorsalis’ life stages may occur on meristems and other tender

tissues of all above ground parts of host plants. Consequently, the opportunity of trans-

boundary transportation of S. dorsalis through the trade of plant materials is high.

Existence of any variation in phenotypic and genetic makeup of such a pest makes

identification much more difficult. Thus, the overall goal of this study was i) to develop a

reliable and accurate technique to identify S. dorsalis from single specimens and ii) to

determine the extent of morphological and genetic variations in populations of S.

dorsalis.

111

Accurate identification of S. dorsalis is a fundamental requirement in development

of effective quarantine and management strategies. Using Scanning Electron

Microscopy (SEM), high resolution images of adults and larvae of S. dorsalis were

produced which will assist growers and extension personnel in identifying the pest with

greater ease. Furthermore, a comparison of morphological traits of S. dorsalis

populations from different geographical regions was conducted which can help in

understanding the phenotypic variation of this pest. Specimens of S. dorsalis were

obtained from five distinct geographical regions: New Delhi, India; Shizouka, Japan;

Negev, Israel; St. Vincent; and Florida, United States. Fourteen morphological

characters of each of 10 adult specimens of S. dorsalis were measured and compared

among the five populations. No significant differences were observed between the body

lengths of the various S. dorsalis populations, which ranged from 0.85 mm (Negev) to

0.98 mm (Florida). When comparing 12 morphological characters, no significant

differences were detected among the New Delhi, St. Vincent, Negev and Florida

populations. However, when S. dorsalis adult specimens from the populations of each

of these four regions were compared with specimens from the Shizouka population,

significant differences were detected for two or five morphological characters,

depending on the population. Thus, speciemens from the Japan population are more

robust (i.e., mesothorax and metathorax is longer and wider, abdomen is wider) than

specimens from the other populations. In addition, the mean lengths of body size

among different populations did not vary directly or inversely with latitude.

Morphological and molecular techniques were coupled to develop a novel, quick,

reliable and simple diagnostic method for identifying individual thrips specimens.

112

Individual specimens (larvae and adults) of S. dorsalis are first subjected to

morphological identification using high-resolution SEM. Then, the gold/palladium sputter

coated thrips specimens are further processed for DNA extraction and PCR assay for

molecular identification. The results of the study indicated that the sequence results of

both mtCO1 and ITS2 rDNA genes of individual larvae and adults of S. dorsalis were in

agreement with the taxonomic identifications conducted using SEM. Our results suggest

that the two techniques together could be used to validate the identification of various

thrips species using single specimens.

The genetic characterization of three economically important thrips species,

Scirtothrips dorsalis, Frankliniella occidentalis (Pergande) and Thrips palmi Karny was

conducted using the mtCO1 and ITS rDNA genes. The high level of inter- and

intragenomic variation of the ITS gene in all the three of the species would most likely

preclude the use of this gene in molecular identification of the species. However, less

intragenomic and intraspecific variation was observed in the conserved mitochondrial

CO1 region of the three pest species, indicating that this gene might be more useful for

their taxonomic characterization. Results from different studies confirmed the existence

of morphological and genetic variation in population of S. dorsalis that suggests the

possibility of this species being a cryptic species complex.

113

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Amin, P. W., D. V. R. Reddy, and A. M. Ghanekar. 1981. Transmission of tomato

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Ananthakrishnan, T. 1993. Bionomics of Thrips. . Ann. Rev. Entomol. 38: 71-92.

Arthurs, S., C. L. McKenzie, J. Chen, M. Dogramaci, M. Brennan, K. Houben, and

L. Osborne. 2009. Evaluation of Neoseiulus cucumeris and Amblyseius swirskii (Acari: Phytoseiidae) as biological control agents of chilli thrips, Scirtothrips dorsalis (Thysanoptera: Thripidae) on pepper. Biol. Control. 49: 91-96.

Asaf-Ali, K., E. Abraham, S. Thirumurthi, and T. Subramaniam. 1973. Control of

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BIOGRAPHICAL SKETCH

Vivek Kumar was born in a small village named Ghoghardiha pertaining to

Madhubani district of Bihar in India. He received his bachelor’s degree in botany

(honors) at Sri Guru Teg Bahadur Khalsa College, University of Delhi, India. In the

same year, he met his wife Garima Kakkar who was also a fellow student at the class

and they got married after a span of 10 years. Vivek earned his master’s degree in

agrochemicals and pest management from University of Delhi in 2005. After receiving

master’s degree he worked as Senior Research Fellow at Department of Entomology,

Indian Agricultural and Research Institute under direction of Dr. A.V. N. Paul. In August

2007, he began a new journey and started his doctoral program at Department of

Entomology and Nematology, University of Florida. His doctoral work under supervision

of Dr. Dakshina R. Seal was focused on studying morphological and genetic variation in

population of an invasive thrips species, chilli thrips Scirtothrips dorsalis Hood. He

received doctoral degree in spring 2012. Besides his Ph. D. project, he was also

involved in several side projects in his lab. During his doctoral program, he received 7

different awards or scholarships for academic achievement and his contribution towards

agricultural research. He also attended several regional, national or international

conferences and presented/coauthored in more than 25 (4 invited) conference papers.

During his doctoral program, he published 9 refereed journal articles and 5 non refereed

articles and meeting proceedings. After completion of doctoral program, he will be

joining Dr. Lance Osborne’s lab at the University of Florida, to work as postdoctoral

research associate. His long-term goal is to pursue a research career in the innovative

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and exciting field of entomology at the cutting edge of nature and technology to improve

existing crop protection strategies and develop novel methods of pest control.