1 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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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
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
Materials and Methods............................................................................................ 39 Identification of Specimens ............................................................................... 40
Scanning Electron Microscopy ......................................................................... 40 Morphometric Measurements of Major Body Traits .......................................... 41
Identification of Specimens ............................................................................... 42 Morphometric Measurements of Major Morphological Features ....................... 43
3 COUPLING SCANNING ELECTRON MICROSCOPY WITH DNA BAR CODING FOR MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF THRIPS ................................................................................................................... 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
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
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.
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
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
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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.
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
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
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
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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.
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Figure 2-10. Simple D1 and funnel-shapped D2 setae on the head of a S. dorsalis larva.
63
Figure 2-11. Funnel shaped setae on abdominal terga IX and X of a S. dorsalis larva.
64
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.
66
Figure 2-14. Forefemora of a S. dorsalis larva illustrating the presence of four funnel shaped setae on the distal two-thirds portion.
67
Figure 2-15. Body of a S. dorsalis larva indicating the presence of granular plaques.
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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
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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
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.
74
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.
75
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
76
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.
77
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
78
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).
79
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
80
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-
81
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
82
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),
*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.
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
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.
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.
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.
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.
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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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.