IN FLORIDA by THOMAS LYNN

INTERACTIONS BETWEEN Frankliniella bispinosa (Morgan) AND Frankliniella cephalica (D. L. Crawford) IN FLORIDA

THOMAS LYNN SKARLINSKY II

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2016

To my family, Patricia, Solsiree and Patty, whose unconditional support helped me turn a dream into a reality

ACKNOWLEDGMENTS

I extend profound gratitude to my advisor Dr. Joe Funderburk for turning me into

a scientist and committee members Dr. Norman Leppla, Dr. Richard Stouthamer and

Dr. Scott Adkins, for their guidance during the tenure of this study. My sincerest

appreciation to Dr. Paul Rugman-Jones, Associate Research Scientist at the University

of California Riverside, for patiently guiding me through the nuances of PCR and to Mr.

Pedro Millan, South Florida Area Director, for his unwavering support of continued education. I thank my USDA coworkers for their help and encouragement during my

pursuit of higher education. I would also like to thank the following for providing letters of recommendation towards my Graduate school admission: renowned Thysanopterist Dr.

Laurence Mound, State Plant Health Director Mr. Paul Hornby and former Assistant

Director of National Identification Services, Mr. Joe Cavey.

TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

ABSTRACT ...... 9

CHAPTER

1 INTRODUCTION ...... 11

Bidens alba (L.) DC. var. radiata (Sch. Bip.) R.E. Ballard ex Melchert ...... 12 Frankliniella bispinosa (Morgan) 1913:10 Euthrips tritici bispinosus ...... 13 Frankliniella cephalica (D. L. Crawford), 1910: 153 Euthrips cephalicus ...... 14

2 HOST UTILIZATION AND CONGENER ENCOUNTERS ...... 19

Background ...... 19 Materials and Methods...... 20 Bidens alba Samples ...... 20 Bidens alba Samples compared to other Plant Species ...... 21 Molecular Samples ...... 22 Results ...... 24 Bidens alba Samples ...... 24 Bidens alba compared to other Plant Species ...... 26 Molecular Samples ...... 27 Discussion ...... 28

3 A KEY TO SOME Frankliniella LARVAE FOUND IN FLORIDA ...... 42

Background ...... 42 Materials and Methods...... 42 Results ...... 43 Description, Larva II Frankliniella bispinosa (Morgan) ...... 45 Remarks ...... 46 Description, Larva I ...... 46 Material Examined ...... 46 Description, Larva II Frankliniella cephalica (D.L. Crawford) ...... 47 Description, Larva I ...... 47 Material examined ...... 48 Remarks and observations Frankliniella fusca Hinds ...... 48 Material examined ...... 48 Description, Larva II Frankliniella insularis (Franklin) ...... 48 Material examined ...... 49

Description, Larva II Frankliniella kelliae Sakimura ...... 49 Description, Larva I ...... 49 Material examined ...... 50 Remarks and observations Frankliniella occidentalis (Pergande) ...... 50 Material examined ...... 50 Remarks and observations Frankliniella schultzei (Trybom) ...... 51 Material examined ...... 51 Discussion ...... 51

4 CONCLUSIONS ...... 55

LIST OF REFERENCES ...... 56

BIOGRAPHICAL SKETCH ...... 61

LIST OF TABLES

Table page

2-1 Collection data for the specimens sequenced in this study...... 38

2-2 Pearsons correlation coefficients comparison...... 39

2-3 The mean number/flower of thrips from B. alba and other plant species ...... 40

2-4 List of other thrips sampled at the three locations...... 41

LIST OF FIGURES

Figure page

1-1 Bidens alba (L.) DC. var. radiata (Sch. Bip.) R.E. Ballard ex Melchert ...... 16

1-2 Habitus of F. bispinosa and F. cephalica ...... 17

1-3 Antennal III pedicels of F. bispinosa and F.cephalica ...... 18

2-1 Florida B. alba sample locations ...... 35

2-2 Orientation of the ocellar setae pair III of F. bispinosa and F. cephalica ...... 36

2-3 USDA Florida plant hardiness zones ...... 37

3-1 Larva II of F. bispinosa ...... 52

3-2 Larva I of F. kelliae ...... 53

3-3 Abdominal tergites VIII and IX of larvae II ...... 54

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

INTERACTIONS BETWEEN Frankliniella bispinosa (Morgan) AND Frankliniella cephalica (D. L. Crawford) IN FLORIDA

Thomas Lynn Skarlinsky II

May 2016

Chair: Joe Funderburk Major: Entomology and Nematology

Frankliniella bispinosa (Morgan) and Frankliniella cephalica (D.L.Crawford) are morphologically similar species found on the flowers of Romerillo, Bidens alba (L.) DC. in Florida. The actual B. alba/flower thrips distribution in Florida is poorly understood as a result of misidentification of the two similar thrips species and near absence of previous study. To determine the distribution and abundance of the two thrips species on B. alba and if it is a host of the congener species, flowers of B. alba were sampled at

10 mile increments from the southern extreme of the Florida Keys to near the northern border of Georgia. Adults and larvae were identified to species and a morphological instar II larval key was developed. Other plant species were sampled at three distant locations to determine the abundance and distribution of F bispinosa and F. cephalica on B. alba compared to different plant species. Non-destructive DNA template extraction, polymerase chain reaction and sequencing of the cytochrome oxidase c subunit one mitochondrial gene of specimens from the three sample locations were conducted. Three distinct latitudinal regions in Florida were found to have either a single congener species or both congeners on B. alba flowers. Both species utilized B. alba for

9 reproduction and congener encounters on B. alba were more frequent in the central region of Florida. Frankliniella cephalica was competitively superior to F. bispinosa on

B. alba in southern Florida. The molecular sequences revealed intra and interspecific difference among the congeners studied.

CHAPTER 1 INTRODUCTION

The genus Frankliniella (Karny 1910) (Thysanoptera: Thripidae) currently consists of 233 extant species and is the second most species rich genus of the

Thripidae ThripsWiki (2015). The majority of species are found in the Neotropics and to a much lesser extent the Nearctic and Palearctic regions of the world (Mound &

Nakahara 1993). A total of 17 species of are reported to occur in Florida (Diffie et al.

2008). Most members of the genus live in flowers and several are known to inhabit grasses (Mound et al. 2005).

The flower thrips, Frankliniella bispinosa (Morgan) and Frankliniella cephalica

(D.L. Crawford) are common in the ubiquitous wildflower, Bidens alba (L.) DC. var.

radiata (Sch. Bip.) R.E. Ballard ex Melchert, (Asteraceae), (Fig. 1-1). Adult specimens of

both species collected from flowers of B. alba are deposited at the Florida State

Collection of Arthropods, Gainesville (FSCA). Needham (1948) and Nakahara (1992)

reported F. cephalica from flowers of B. alba. Frantz & Mellinger (1990) reported both

species from B. alba in Florida. Adult survey data from central Florida by Childers &

Nakahara (2006) suggested that F. cephalica is more prevalent on B. alba than F.

bispinosa. However, no previous study provided evidence that B. alba is a host plant of

either thrips species. A “host plant” is a plant on which an insect produces offspring

(Mound 2013). The presence of immature life stages provides a quantitative measure of

the plant’s suitability as a reproductive host (Terry 1997).

A significant amount of published research has been dedicated to the description

of the adult life stages of Frankliniella; whereas, few publications describe the immature

stages. Speyer & Parr (1941) published a description of the second instar of

Frankliniella intonsa (Trybom). Miyazaki & Kudo (1986) produced a complete morphological and biometric description of the second instar F. intonsa (Trybom). Kirk

(1987) described the second instar of F. schultzei, and Milne et al. (1997) described the

second instar larvae of F. occidentalis (Pergande). These authors separated only a

single species of Frankliniella from other genera; whereas, Nakahara & Vierbergen

(1998) produced a larval key for the identification of seven species of Frankliniella from

Europe. Mound et al. (2005) described the second instar of F. lantanae, and Masumoto

& Okajima (2013) described the second instar of Frankliniella hemerocallis Crawford. All

of the above larval descriptions and keys were published from Old World locations.

Currently, Borbon (2009) is the only publication that described Frankliniella spp. from

the Americas. The author provided a key to the second instar of five Frankliniella

species, with three found only in the Americas (Nakahara 1997).

Bidens alba (L.) DC. var. radiata (Sch. Bip.) R.E. Ballard ex Melchert

Bidens alba (L.) DC. var. radiata (Sch. Bip.) R.E. Ballard ex Melchert is the

accepted name of the synonym Bidens pilosa var. radiata Sch. Bip. that includes the

conspecifics Bidens alba and Bidens alba var. alba (USDA 2015) (Fig 1-1). Common

names include romerillo, shepherds needles and Spanish needles with the latter names

being morphologically indicative of the seeds two barbed points that attach to passing

animals and help to disperse them over a broad range (Daniels & Tekiela 2010).

A member of the Aster family, this is a short lived perennial wildflower plant that

blooms continually and can grow up to five feet tall (Daniels & Tekiela 2010). The flower

head (floret disk) is yellow and consists of 35-50 tightly compacted individual florets with

reproductive components (Huang et al. 2012). Five to eight white petals surround the

upright flower head giving the flower a daisy-like appearance (Daniels & Tekiela 2010). 12

The individual florets go through six floral stages lasting from four to six days, and the pollen is exposed for secondary pollination approximately two days after anthesis initiation (Huang et al. 2012).

This plant was reported as an additional food source by Morton (1962) and for its many folk medicinal attributes (Morton 1962, Silva et al. 2011). The correlated biological activity of B. alba in treating illness has prompted study of its chemical composition resulting in the isolation of 198 chemical compounds (Silva et al. 2011). Furthermore,

Deba et al. (2007) isolated the chemical volatile eugenol from B. alba whilst investigating potential herbicidal and fungicidal activites of the plant. The volatile eugenol was found to be behaviorally more attractive to Frankliniella occidentalis

(Pergande) when given the choice between eugenol and anisaldehyde (Frey et al.

1994). However, Kirk (1985) reported other flower thrips species were more attracted to anisaldehyde than eugenol.

Bidens alba is native to the neotropics (USDA 2015) with the center of diversification of the Bidens pilosa complex in Mexico (Ballard 1986). This plant is found throughout Florida inhabiting disturbed areas and roadsides, and is invasive in perennial and annual crops (Silva et al. 2011, Daniels & Tekiela 2010). Moreover, Bidens is host to the economically important Tomato spotted wilt virus, a species in the genus

Tospovirus (Bunyaviridae) (Cho et al. 1986, Ochoa Martinez et al. 1999).

Frankliniella bispinosa (Morgan) 1913:10 Euthrips tritici bispinosus

Originally described from 4 females collected from blooms of Yucca in Dade City,

Florida, the female of this minute (< 2mm) yellow species usually has darkened medial areas on the abdominal tergites; however, this darkening is not evident in teneral specimens. It is distinguished from most other species found in Florida by the “beer- 13 pint-glass shape” of the basal collar and the swollen pedicel of antennal segment III.

The distal and basal outer margins of the pedicel are produced laterally to acute convergent points similar to the common depiction of a “flying saucer” (Fig.1-3A).

Furthermore the antennal segment II of F. bispinosa has two enlarged distal spine-like setae on the dorsal surface, presumably indicative of the species name, and the posterior abdominal tergite VIII comb is absent medially. The yellow male is smaller than the female, and the male shares all of the aforementioned morphological characters of the female with the exception of the tergite VIII comb. The male has discal oval glandular areas on sternites III-VII (Sakimura & O’Neill 1979). The immature morphology has not been described.

Development and life-table parameters of F. bispinosa fed with pollen of four different plant species were determined by Tsai et al. (1996). The age-fecundity and survival rate of F. bispinosa at different temperatures was reported by Yue (2001).

Childers et al. (2005) reported F. bispinosa is abundant in Florida. Nakahara

(1997) recorded F. bispinosa from Alabama, Georgia, South Carolina, Bahama Islands and Bermuda. Childers & Achor (1991) reported that F. bispinosa damages citrus blossoms. Frantz & Mellinger (1990) reported injury to a wide variety of vegetable crops.

Riley et al. (2011) lists F. bispinosa as a vector of TSWV.

Frankliniella cephalica (D. L. Crawford), 1910: 153 Euthrips cephalicus

Described from numerous females and several males that were collected from

Asteraceae in Guadalajara, Mexico, this small yellow species is morphologically similar

to F. bispinosa (Fig. 1-2). Both possess a distally widened, beer pint glass shaped basal

collar of antennal segment III, two enlarged spine-like distal setae on segment II and a

swollen pedicel on segment III. The basal and distal outer margins of the pedicel, 14 although produced laterally do not converge to a common point as in F. bispinosa, rather the basal outer margins extend slightly more than the distal outer margins (Fig. 1-

3B). The posterior abdominal tergite VIII comb is absent medially. The yellow male is smaller than the female and with the exception of the tergite VIII comb, shares the morphological characters of the female. The male has discal oval glandular areas on sternites III-VII (Sakimura & O’Neill 1979). The immature morphology has not been described.

Widespread in the Caribbean, Central America and Mexico (Mound & Marullo

1996), F.cephalica was reported from the continental United States (TX, FL) (Nakahara

1997), Hawaii (Kumashiro et al. 2001), Japan (Masumoto & Okajima 2004) and Taiwan

(Wang et.el. 2010). It has been recorded from a wide range of plant species (Mound &

Marullo 1996). I found no records of agricultural or horticultural damage, but it is a vector of TSWV (Ohnishi et al. 2006).

Figure 1-1. Bidens alba (L.) DC. var. radiata (Sch. Bip.) R.E. Ballard ex Melchert. (Thomas Lynn Skarlinsky II. Bidens alba. 04/03/2016. Davie, Florida.)

A B

Figure 1-2. Habitus of: F. bispinosa (A); F. cephalica (B). (Thomas Lynn Skarlinsky II. Full Body. 09/11/2015.)

A B

Figure 1-3. Shape of the antennal III pedicel at 600X magnification: F. bispinosa (A); F.cephalica (B). (Thomas Lynn Skarlinsky II. Pedicels. 03/24/2015.)

CHAPTER 2 HOST UTILIZATION AND CONGENER ENCOUNTERS

Background

The evolutionary radiation of the genus Frankliniella (Karny 1910) (Thysanoptera:

Thripidae) is extensive compared to other Thripidae genera. The genus currently consists of 233 extant species and is the second most species rich genus of the

Thripidae ThripsWiki (2015). The majority of species are represented from the

Neotropics and to a much lesser extent the Nearctic and Palearctic regions of the world

(Mound & Nakahara 1993).

The flower thrips, Frankliniella bispinosa (Morgan) and Frankliniella cephalica

(D.L. Crawford) are common in the ubiquitous wildflower, Bidens alba (L.) DC. var.

radiata (Sch. Bip.) R.E. Ballard ex Melchert, (Asteraceae). Adult specimens of both

species collected from flowers of B. alba are deposited in the Florida State Collection of

Arthropods, Gainesville (FSCA). Needham (1948) and Nakahara (1992) reported F.

cephalica from flowers of B. alba. Frantz & Mellinger (1990) reported both species from

B. alba in Florida. Adult survey data from central Florida by Childers & Nakahara (2006) suggested that F. cephalica is more prevalent on B. alba than F. bispinosa. However,

no previous study provided evidence that B. alba is a host plant of either thrips species.

A “host plant” is a plant on which an insect rears its young (Mound 2013). The presence

of immature life stages provides a quantitative measure of the plant’s suitability as a

reproductive host (Terry 1997).

Two species are sympatric when they exist in the same geographic area and

regularly encounter one another. Numerous species of flower thrips in the genus

Frankliniella are sympatric in Florida (Paini et al. 2007). Some are polyphagous and compete for the same flower resources (Northfield et al. 2008). The competitive exclusion principle states that true competitors cannot co-exist sympatrically, leading to extinction or there will be a behavioral shift toward a different ecological niche (Paini et al. 2008).

Collection records and literature suggests the sympatric species, F. bispinosa and F. cephalica are likely competing for the same B. alba floral resource; however, it is unknown where congener encounters may occur in Florida and how either species is utilizing the flowers. The objectives were to: 1) determine the abundance and distribution of F. bispinosa and F. cephalica on B. alba, 2) to determine areas of geographic sympatry, 3) to evaluate genetic differences between populations, 4) to determine density-dependent relationships and, 5) to compare host plant utilization of

the two congeners.

Materials and Methods

Bidens alba Samples

Samples were taken to determine the abundance and distribution of thrips on B.

alba from January to June 2013. Ten flowers of B. alba were randomly sampled at each

of 49 locations along U.S. Highway 1 and U.S. 27 from the Florida Keys to near the

Georgia border. These data were compiled into a matrix using Microsoft Excel and

imported into ArcGIS 10 (2011) and linked to the corresponding sample locations (Fig.

2-1). Samples were placed in 50 ml plastic vials with 70% isopropyl alcohol. All samples were returned to the laboratory, and the thrips were extracted under 50 to 100X stereoscopic magnification.

Three to five females and three to five males (voucher specimens) from each sample were permanently slide mounted in a Canada balsam media using modified methods from Mound & Marullo (1996). The remaining specimens were slide mounted in Hoyer’s media (Mound & Marullo 1996). The Hoyer’s slides were cured in an oven at approximately 40° C. After curing, slide cover slips were sealed with clear nail polish.

The total number of adult males, adult females, and larvae of each species in each sample was determined. All specimens examined in this study are housed at the USDA,

APHIS, PPQ, Plant Inspection Station, Miami, Florida. Since the larval morphology of F. bispinosa and F. cephalica was unknown a larval key with descriptions was developed.

Initial abundance and distribution data from the B. alba samples indicated the potential of congener encounters on B. alba to be markedly different for three latitudinal regions in Florida. Sample sites north of 30° latitude were categorized as northern; 28° -

30° as central and 24° - 28° as southern (Fig. 2-3). The density of larval and adult F. bispinosa and F. cephalica were compared between regions using analysis of variance procedures (PROC GLM, SAS Institute 2008). Density-dependence was evaluated by correlating the numbers of F. bispinosa and F. cephalica from the B. alba samples

(n=49) (PROC CORR, SAS Institute 2008). The effect of plant host on the densities of adult and larval F. cephalica and F. bispinosa at each location was evaluated using analysis of variance procedures and subsequent orthogonal contrasts (PROC GLM,

SAS Institute 2008).

Bidens alba Samples compared to other Plant Species

Flower samples from B. alba and other plant species were taken from the central and southern Florida regions at three locations to determine the abundance and distribution of F bispinosa and F. cephalica on B. alba compared to other plant species. 21

The sites from the central and southern regions were in Miami Dade County (25.6425,

80.292388), St. Lucie County (27.426389, 80.407222), Alachua County (29.640804,

82.360489) (Fig.2-1). Three samples of 10 flowers of B.alba and other nearby flowering plant species were randomly collected at each location. Samples in Miami Dade County were collected in April 2015, St. Lucie County in June 2015, Alachua County in April

2013 and Gadsden County in June 2013. Samples were processed and curated as previously described.

Molecular Samples

Non-destructive DNA template extraction, polymerase chain reaction (PCR) and sequencing of the cytochrome oxidase c subunit one (COI) mitochondrial gene of specimens (n=36) from the three sample locations followed published procedures

(Rugman-Jones et al. 2010) (Table2-1). Subsequent to extraction, the exoskeletons of

F.bispinosa (n=16), F. cephalica (n=17), Frankliniella. kelliae Sakimura (n=3) were

morphologically identified. Their respective sequences were compared: 1) to confirm

adult/larval morphological observations; 2) to evaluate possible intraspecific and

interspecific genetic differences. In addition, using the same protocol, sequences from

F. cephalica (n=3), Frankliniella. borinquen Hood (n=3) from Puerto Rico and F.

cephalica (n=3) from Costa Rica (Table), were compared to the Florida populations.

Frankliniella borinquen was included because of its morphological similarity to the

Florida congeners (Mound & Marullo 1996) and reported association with flowers of

Bidens sp. (Hood 1941).

Whole genomic DNA was extracted from individual specimens (n=45) using an

EDNA HiSp-ExTM tissue kit (Fisher Biotec, Wembly, WA, Australia) (Rugman-Jones et

22 al. 2009). In a microcentrifuge tube, a single specimen was placed in a 60 µl mix of the proprietary solutions 1A (48µl) and 1B (12µl) and incubated at 95° C for 30 minutes.

Subsequent to incubation 15µl of proprietary solution 2 was added. The tube was gently vortexed and 60µl of the DNA template was transferred to a new microcentrifuge tube, taking care to avoid touching the specimen, and stored at -10°C. Isopropyl alcohol

(70%) was added to the original tube containing the specimen exoskeleton, in preparation for slide-mounting and morphological identification.

PCR was used to amplify 434 bp of the COI gene with the degenerate primer pair mtD-7.2F (5′- ATT AGG AGC HCC HGA YAT AGC ATT-3′) and mtD-9.2R (5′- CAG

GCA AGA TTA AAA TAT AAA CTT -3′) (Brunner et al. 2002) in 25µl reactions in a

GeneAmp® PCR System 9700 thermocycler (Applied Biosystems, Foster City,

California). Each PCR reaction consisted of: 2µl DNA template; 15.2µl of molecular

Biology grade USP sterile purified water (Mediatech Inc, Manassas,VA); 2.5µl of 10x

ThermoPol PCR buffer (New England BioLabs, Ipswich, MA); 1µl of 25 mM MgCl2 (New

England BioLabs); 0.5µl of 20mg/ml bovine serum albumin (BSA), (New England

BioLabs); 0.5µl of each 10 μM primer (synthesized by Integrated DNA Technologies,

Coralville, Iowa); 1.5 U Taq polymerase (New England BioLabs); and 2.5µl of

dNTP/dUTP mix (Thermofisher Scientific,Waltham, MA; product # R0251).

Thermocycling was: initial denaturing 94°C for 3 min; 38 cycles of 94°C for 30 s, 47°C

for 1 min, 68°C for 1 min 30 s and final extension at 68°C for 3 min.

Amplification of a PCR product was confirmed by gel electrophoresis. A single

gel consisted of 100 ml of 1x TBE buffer, 1.5 g Agarose powder and 7µl of SYBR® Safe

DNA gel stain (Invitrogen, Carlsbad, CA). 5 µl of PCR product were mixed with an

23 equal volume of loading dye and loaded into individual wells. A DNA ladder was added to a single well and the electrophoresis was run for 1 hour at 90 Volts (Bio Rad,

Hercules, CA). Products were visualized under UV light and results were recorded with the U Genius3® imaging system (Syngene, Frederick, MD). The amplified DNA was cleaned by combining 10µl of PCR product with 2µl of ExoSAP-IT® (Affymetrix,

Cleveland, OH) and incubating at 37°C for 30 min, and then 80°C for 15 min. The cleaned PCR product was direct sequenced in both directions at the Institute for

Integrative Genome Biology, Core Instrumentation Facility, University of California,

Riverside.

Forward and reverse reads were aligned, and primer sequences were removed, using Sequencher® 4.9 (Gene Codes Corporation, Ann Arbor, MI). All sequences produced in this study were submitted to Gen Bank. Existing F. bispinosa sequences retrieved from GenBank (accessions AB277216, JQ182146), and F. cephalica

(accessions AB277217, AB277219) sequences were compared to the 45 sequences from this study using Bioedit.

Results

Bidens alba Samples

From the 49 B. alba samples, 5,062 individuals of both species were extracted

and identified to determine abundance and distribution. Frankliniella cephalica (adults

58.6%, larvae 17.7%) and F. bispinosa (adults 21.9%, larvae 1.7%) were collected in

the B. alba samples from the Florida Keys to the Georgia border. Other Thysanoptera

species (n=351), extracted and identified from the B. alba flowers included:

Microcephalothrips abdominalis Crawford (n=233), 220 adults, 13 larvae; Haplothrips

24 gowdeyi Franklin (n=75), 48 adults, 27 larvae; Frankliniella tritici Fitch (n=34), 34 adults;

Frankliniella schultzei (Trybom) (n=3), 3 adults; Frankliniella kelliae Sakimura (n=2), 2

adults; Frankliniella insularis (Franklin) (n=2) 2 adults and Leptothrips sp. (n=2), 1 adult,

1 larvae.

Frankliniella cephalica was the predominant species identified (adults + larvae =

3,561) from the 27 southern Florida sample sites with F. bispinosa (adults + larvae = 43)

being less numerous. More F. bispinosa (adults + larvae = 1,006) than F. cephalica

(adults + larvae = 307) were collected from the central Florida sites. More F. bispinosa

(adults + larvae =144), than F. cephalica (adults = 1) from the Northern Florida sites, north of the 30° latitude.

There were significant differences (P < 0.05), between regions of Florida in the

number of F. cephalica and F. bispinosa for both life stages (F = 10.5 and 13.5,

respectively; df = 2, 46; P < 0.0002 and 0.0001, respectively). The mean

number/10flowers (SEM) of F.cephalica in southern, central, and northern Florida was

134.4 (22.7), 21.4 (8.3), and 0.2 (0.2), respectively. The mean number/10flowers (SEM)

of F. bispinosa in southern, central, and northern Florida was 1.7 (0.9), 65.3 (16.8), and

25.9 (12.1), respectively. In addition significant differences were found for the number of

adults from the different regions (F = 8.3 and 10.4, respectively; df = 2, 46; P < 0.0009

and 0.0002, respectively). The mean number/10flowers (SEM) of F.cephalica in

southern, central, and northern Florida was 103 (20.4), 12.1 (6), and 0.2 (0.2),

respectively. The mean number/10flowers (SEM) of F. bispinosa in southern, central,

and northern Florida was 1.56 (0.89), 58 (16.6), and 23.3 (11.3), respectively.

Low reproductive utilization by F. bispinosa (larvae = 1) on B. alba was found at the 27 southern locations. In contrast, host utilization for reproduction by F.cephalica

(larvae = 785) was high. Reproductive utilization was higher for F. cephalica (larvae =

114), than F. bispinosa, (larvae = 79) at the 16 central Florida sites. In the 6 northern sites reproductive utilization was low for both species: F. bispinosa (larvae = 4) and F. cephalica (larvae = 0). Significant differences were found for the number of larvae from the different regions (F = 5.7 and 5.9, respectively; df = 2, 46; P < 0.0063 and 0.0052, respectively). The mean number/10flowers (SEM) of F. cephalica in southern, central, and northern Florida was 31.6 (6.5), 9.3 (6), and 0.0 (0.0), respectively. The mean number/10flowers (SEM) of F. bispinosa in southern, central, and northern Florida was

0.19 (0.11), 12.1 (4.9), and 1.67 (0.9), respectively.

To evaluate density-dependent relationships between F. cephalica and F.

bispinosa Log10 (n+1) transformed species data were analyzed using the Pearson

correlation coefficients. Correlations from the total sample sites, (n = 49), between the

adults, the larvae, and the total (larvae + adults) of the two species were assessed

using SAS (SAS Institute 2008). In each comparison (P < 0.05), whether adults (r = -

0.48), larvae (r = -0.53), or total (r = -0.52), there were strong negative correlations

(Table 2-2).

Bidens alba compared to other Plant Species

Thrips specimens were extracted and identified from B. alba and other plant

species to determine abundance and distribution (Table 2-3). From the Alachua County

site, 3736 individuals of both species Frankliniella bispinosa (n=3710) was the most

abundant congener with 89% adults and 11% larvae collected from the flowers of all the

plant species sampled. The adult mean number/flower was between 2.56 for Vicia 26 sativa to 56.6 for Trifolium sp. and larvae 0.1 for B.alba to 2.73 for Trifolium sp. (Table

2-3). Frankliniella cephalica (n=26) was the least abundant with 89% adults and 11%

larvae collected from B. alba and Trifolium sp..

From the St Lucie County site F. cephalica (n=351) was the most abundant with

39% adults and 61% larvae collected. The adult and larval mean number/flower of F.

cephalica was the highest for B. alba compared to all other respective plant species

sampled. The congener F. bispinosa (n=10) was the least abundant with 30% adults

and 70 % larvae collected from Lagerstroemia indica, B. alba and Sphagneticola

trilobata.

From the Miami Dade County site F. cephalica (n=355) was the most abundant

with 72% adults and 28% larvae. The mean number/flower for adults (6.63) and larvae

(3.06) was the highest for B. alba compared to the other plant species sampled whereas

F. bispinosa (n=11) with 64% adults, 36% larvae collected was the least abundant.

Nine additional thrips species were extracted and identified from the three sites.

The most abundant of the other species from the sites were M. abdominalis and Thrips hawaiiensis (Morgan) (Table). Twelve specimens from Miami Dade County were not recognized and were recorded as Phlaeothripinae and Thripidae. Also, in Miami Dade

County the African native species Ceratothripoides brunneus (Bagnall) (Mound & Nickle

2009) was collected for the first time in the continental U.S.

Molecular Samples

Comparison of sequences of the COI mitochondrial gene of F. cephalica (n=23)

with the GenBank accessions (AB277217, AB277219) revealed three distinct

haplotypes: FC1 (n=10); a single substitution at nucleotide 181, FC2 (n=11); three

substitutions at nucleotides 181,199 and 281; FC3 (n=2), with substitutions at 27 nucleotides 181 and 217. For F.bispinosa (n=16) two distinct haplotypes were found

(Fig. 2-2): FB1 (n=15) identical to Genbank accessions (AB277216, JQ182146) and

FB2 (n=1) with a single substitution at nucleotide 79. Congeners with the FC1 haplotype were from Miami Dade County (n=6), St. Lucie County (n=3) and Costa Rica (n=1); FC2 from St. Lucie County (n=3), Alachua County (n=5) and Puerto Rico (n=3); FC3 from

Costa Rica (n=2). Those with the FB1 haplotype were from Miami Dade County (n=3),

St. Lucie County (n=6), Alachua County (n=6) and FB2 from Alachua County (n=1)

Intraspecific sequence divergence of F.cephalica (n=23) was 0.3% - 0.5%; F.

bispinosa (n=16) was 0.3% and interspecific divergence was 13.4% - 13.6%. Sequence

divergence among the four species, F. bispinosa, F. cephalica, F. kelliae and F.

borinquen was 13.4% - 14.6%.

Sequences of the morphologically identified larvae of F. bispinosa (n=7), F.

cephalica (n=4) and F. kelliae (n=1) matched the sequences of the conspecific

confirming larval morphological observations. Furthermore, the sequences confirmed an

undescribed adult morphological character state difference between the congeners. The ocellar setae pair III of F. cephalica are positioned near the anterior margins of the ocellar triangle, whereas in F. bispinosa the ocellar setae pair III are removed posteriorly of the anterior margins of the ocellar triangle and positioned near the anterior edge tangent of the hind ocelli (Fig. 2-2).

Discussion

Congener encounters on B. alba were most frequent in the central region of

Florida. The adults and larvae of F. cephalica were predominate in B. alba flowers in southern Florida. The adults and larvae of both species were common in B. alba flowers in central Florida. The adults of F. bispinosa were predominate in the flowers of B. alba 28 in northern Florida, but there was no indication of reproduction. Tyler-Julian (2013) previously reported that B. alba was not a host for Frankliniella species thrips in northern Florida, although the adults of F. bispinosa reached high densities in the flowers.

The flowers from plant species other than B. alba were sampled for thrips at several locations in this study. The adults and larvae of F. bispinosa were common in the flowers of several plant species in Alachua County. Paini et al. (2007) previously found F. bispinosa larvae in numerous plant species in a survey in northern Florida. In this study, F. bispinosa was collected, but not abundantly, in samples of flowering plants in St. Lucie and Miami-Dade Counties. Childers and Nakahara (2006) reported that F. bispinosa is the predominant flower thrips species in flowering plants in central Florida.

The adults and larvae of F. cephalica were rarely collected in the samples of flowers from other plant species in Alachua, St. Lucie, and Miami-Dade Counties, even though

B. alba was a host at each location.

Gauses’s competitive exclusion principle states that two species cannot coexist sympatrically if they occupy the same ecological niche, leading to extinction of one of the species or there will be a behavioral shift toward a different ecological niche

(Speight et al. 2008). There was no evidence in this study of niche overlap between F. bispinosa and F. cephalica on plant species other than B. alba. Both species utilized B. alba as a host in central Florida in this study, but there was no evidence of reproduction by F. bispinosa in northern or southern Florida. Childers and Nakahara (2006) reported that F. cephalica was monophagous on B. alba and that F. bispinosa was polyphagous in central and southern Florida. The abundance of F. bispinosa was negatively

29 correlated on B. alba with the abundance of F. cephalica, thereby suggesting interspecific competition between the two species. This study did not address the mechanisms involved in this competition.

Paini et al. (2008) previously reported competition between species of

Frankliniella thrips in Florida. Paini et al. (2008) ruled out exploitative competition for the

ability of F. tritici (Fitch) to out-compete F. occidentalis (Pergande) as neither plant

tissue nor pollen was limiting in their study. More likely, interference competition directly

between individuals was the mechanism. Northfield et al. (2011) speculated that

apparent competition most likely explained the ability of F. bispinosa to out-compete F.

occidentalis under field conditions in Florida (i.e., a complex interaction between F.

bispinosa, F. occidentalis, the key predator, and host plants). Funderburk et al. (2015)

after a review of the scientific literature concluded that interactions with native species

clearly limit the abundance of F. occidentalis in Florida, but populations are abundant in

fertilized crop fields where application of insecticides excludes predators and competitor

species.

Florida is divided into several plant hardiness zones (Fig. 2-3). Differences in

abiotic conditions between northern, central, and southern Florida were factors affecting

the abundance and distribution of F. bispinosa and F. cephalica. Local climatic

conditions mediate competitive interactions between congener species (Reitz &

Trumble 2002). In the central and northern regions colder temperatures limited the

availability of B. alba floral resources. Temperatures above 15° C and below 45° C are

necessary for growth and reproduction of B. alba, and freezing temperatures result in

mortality (GISD 2015). Both F. bispinosa and F. cephalica are tropical/subtropical

30 species (Mound and Marullo 1996), and they are not adapted to colder conditions.

Populations of F. bispinosa occur in northern Florida (e.g., Paini et al. 2007), but they are not reported in collections from plant hosts in southern Georgia (e. g., Riley et al.

Yue (2001) reported that adult mortality of F. bispinosa was very high during the first few days at 15 °C, with surviving adults not laying eggs during the first 10 days at this temperature. An additional 44% of the individuals reared at this temperature died at the second instar and pupal stage. The thrips used in the Yue (2001) study were collected from B. alba in South Florida. The data from this study shows F. cephalica was the only species reproducing on B. alba in South Florida. This suggests that Yue (2001) may have misidentified the thrips used in her experiments, but vouchers from her study are not available in the Florida State Collection of Arthropods.

The Alachua samples from this study were collected April 2013 in Gainesville.

The Gainesville average temperature for April was 69°F and 21 of 30 days during April the low temperatures were below 59° F (COAPS 2015). Therefore, based on Yues data, one would expect cooler temperatures to increase adult mortality and increase developmental time of F. bispinosa likely resulting in low abundance levels. However I found F. bispinosa was the most abundant congener sampled in Gainesville, with numbers as high as 56 adults/flower and the abundance of F. cephalica was very low, more closely mirroring predictions from Yues data for F. bispinosa. Assuming the data was erroneously garnered from F. cephalica then temperature may be a mediating factor that is reducing the survivorship of F. cephalica thus limiting its ability to compete

with F. bispinosa for the B. alba floral resource in central and north Florida. Further

temperature survivorship studies are needed for both congeners.

The abundance of F. bispinosa was low from the Miami-Dade samples. However the diversity of other thrips species collected was higher than the other two locations. In general a trend of increased biodiversity is found along latitudinal gradients from the

Polar Regions to the equator (Willig 2003). This latitudinal trend was observed in litter dwelling thrips in China (Wang et al. 2014). It is unclear why the abundance of F. bispinosa was low from the Miami Dade samples. However it may be possible dietary deficiency or some other physical response to environmental conditions in South Florida may be contributing to reduced abundance. The specimens of F. bispinosa were nearly

translucent and appeared malnourished compared to their robust deep yellow

conspecifics in Alachua County. Further sampling and study is needed to validate this

observation.

This study demonstrates the geographical area of congener sympatry is in the

central Florida region and F. cephalica is monophagous in Florida. Records suggest F.

cephalica is polyphagous in other areas of the neotropics. If this is accurate we

speculate this resultant behavioral shift from polyphagy to monophagy in Florida by F. cephalica is evidence of ecological character displacement. The term, “character displacement” was coined by Brown & Wilson (1956) to explain the evolutionary divergence of sympatric and allopatric species. The concept asserts sympatric species are more divergent and readily displace characters amongst the sympatric congeners.

These characters can be morphological, physiological, behavioral or ecological (Brown

& Wilson 1956). More recently Pfennig & Pfennig (2009) reported, based on consensus of previous study that character displacement acts to reduce competitive and reproductive interactions between species. Character displacement: favors the evolution

32 of novel resource-use or reproductive traits; drives divergence between sympatric and allopatric conspecific populations; and both initiates and finalizes the process of speciation (Pfennig & Pfennig 2009).

The morphology and sequence divergence of less than 2-3% (Hebert 2003) suggests the specimens sequenced are the same species. The molecular data revealed intraspecific F. cephalica sequence divergence of the COI gene as evidenced by the three haplotypes. Therefore, it would be reasonable to expect these differences to be greater between populations that are geographically farther apart e.g. the Florida and

Puerto Rico or Costa Rica populations. This was the case between the Florida and

Costa Rica populations, the F3 haplotype was unique to Costa Rica; however the FC2 haplotype found in Florida was shared with the Puerto Rico allopatric population. The incongruence of the Caribbean and Central American populations compared to the

Florida populations highlights the complexity of species divergence and the need for additional study.

The geographical distribution of the two congeners may offer insight toward the overall fitness of the two species. The successful adaptive radiation of F. cephalica throughout a wide range in contrast to the limited distribution of F. bispinosa, primarily in

Florida, suggests F. cephalica may be superior in exploiting novel resources. Moreover with increased agricultural trade, air travel and, general movement of humans throughout the planet it would seem F. bispinosa would have ample opportunity to expand its geographic range. However absence of published reports of its introduction into new areas may suggest it lacks fitness to exploit novel resources in different locations in the world.

Phytotoxic differences in the northern B. alba populations and inability of F. cephalica to produce enzymes necessary to neutralize any toxic effects may explain the absence of F. cephalica in north Florida. Bio-extracts of B. alba at 5% concentration were reported to cause 62% mortality of red spider mites after 96 hours of exposure

(Radhakrishnan & Prabhakaran 2014). Currently plants germinated from seed collected in north Florida, Gadsden County are being grown to maturity in South Florida to verify if north Florida B. alba is an unacceptable host for F. cephalica.

Alachua County

St. Lucie County

Miami Dade County

Figure 2-1. The diamonds indicate the sample locations to determine the abundance and distribution of F. bispinosa and F. cephalica on B. alba. The arrows indicate the sample sites to compare congener abundance on B. alba compared to other plant genera. (ESRI 2011)

A B

Figure 2-2. The orientation of the ocellar setae, pair III in relation to the outer tangents of the ocellar triangle of F. cephalica (A) and F. bispinosa (B). The arrows indicate the anterior position (A) and posterior position (B). (Thomas Lynn Skarlinsky II. Ocellar triangles. 03/24/2015.)

Figure 2-3. USDA Florida plant hardiness zones. The upper horizontal black lines indicates the 30° latitude boundary and the lower line indicates the 28° latitude boundary separating the north, central and southern latitudinal regions. (USDA 2012)

Table 2-1. Collection data for the specimens, adults (AD) and larvae (LV) sequenced in this study. Thrips Specimen number Location Plant species species (lifestage) Date Miami Dade Bidens alba F. cephalica 001-004 (AD), 005-006 (LV) 29-IV-2015 (25.6425, 80.2923) Cassia javanica F. kelliae 025 (AD), 026 (LV) Handroanthus F. kelliae 027 (AD) impetigonosus F. bispinosa 028 & 030 (AD), 029 (LV) St. Lucie Bidens alba F. cephalica 007-010 (AD), 011-012 (LV) 16-VI-2015 (27.4263, 80.4072) Lagerstroemia F. bispinosa 031, 035, 036 (AD), indica 032-034 (LV) Alachua Bidens alba F. cephalica 014, 016, 017,022, 024 (AD) 09-IV-2013 (29.6408, 82.3604) F. bispinosa 013, 015, 018, 019, 023 (AD) 020,021 (LV) Costa Rica Sphagneticola F. cephalica 037-039 (AD) 27-VII-2011 (9.9422, 84.1463) trilobata Puerto Rico Bidens alba F. cephalica 040, 043, 044 (AD) 17-VI-2015 (18.0886, 67.105) F. borinquen 041,042, 045 (AD)

Table 2-2. Pearsons correlation coefficients comparison of the larvae, adults and total larvae + adults of F. cephalica and F. bispinosa from the B. alba samples. F. cephalica F. cephalica F. cephalica adults larvae total F. bispinosa r = -0.48 r = -0.48 r = -0.52 adults p = 0.0004 p = 0.0004 p = 0.0001 F. bispinosa r = -0.53 r = -0.53 r = -0.46 larvae p = 0.0001 p = 0.01 p = 0.0008 F. bispinosa r = -0.50 r = -0.48 r = -0.52 total p = 0.0002 p = 0.0005 p = 0.0001

Table 2-3. The mean number/flower of F. cephalica adults (FCAD), larvae (FCLV) and F. bispinosa adults (FBAD), larvae (FBLV) sampled from B. alba and other plant species. Location Plant FCAD FCLV FBAD FBLV Miami Dade Bidens alba 6.63 3.06 0 0 Sphagneticola trilobata 0.33 0.03 0 0.1 Cassia javanica 0 0 0 0 Handroanthus impetigonosus 0 0 0.03 0.03 Asystasia gangetica 1.73 0.03 0 0 Cassia fistula 0 0 0 0 Delonix regia 0 0 0.1 0.1 St. Lucie Bidens alba 4.46 6.76 0 0.03 Sphagneticola trilobata 0.13 0.33 0 0.1 Polygala rugellii 0 0 0 0 Arachissp. 0 0 0.1 0.1 Alachua Bidens alba 0.7 0.03 3.3 0.1 Trifoliumsp. 0.07 0.03 56.6 2.73 Vicia sativa 0 0 2.56 0.33 Cestrum aurantiacum 0 0 21.6 1.56 Raphiolepis indica 0 0 27.6 7.2

Table 2-4. List of other thrips adults (AD) and larvae (LV) sampled at the three locations. Location Plant Thrips species (number, lifestage)

Miami Bidens alba Microcephalothrips abdominalis (13 AD, 1 LV) Dade Sphagneticola trilobata Microcephalothrips abdominalis (115 AD, 10 LV) Frankliniella insularis (1 AD) Haplothrips gowdeyi (1 AD) Thripidae sp. (1 LV) Cassia javanica Frankliniella kelliae (1 AD, 1 LV) Phlaeothripinae sp. (4 AD, 3LV) Handroanthus impetigonosus Frankliniella insularis (4 LV) Frankliniella kelliae (25 AD, 5 LV) Phlaeothripinae sp. (1AD, 2 LV) Thripidae sp. (2 LV) Asystasia gangetica Ceratothripoides brunneus (32 AD) Frankliniella schultzei (6 LV) Frankliniella kelliae (1 AD) Cassia fistula Frankliniella kelliae ( 1LV) Delonix regia Frankliniella kelliae (57 AD, 87 LV) Frankliniella insularis (27 AD, 2 LV) Frankliniella occidentalis (1 LV) Frankliniella schultzei (1 LV) Franklinothrips vespiformis (1 AD) St. Lucie Bidens alba Frankliniella kelliae (1 AD) Microcephalothrips abdominalis (13 AD) Haplothrips gowdeyi (1 AD, 1 LV) Sphagneticola trilobata Microcephalothrips abdominalis (74 AD, 34 LV) Polygala rugellii Haplothrips gowdeyi (7 AD, 1 LV) Arachissp. Frankliniella fusca (50 AD, 3 LV) Haplothrips gowdeyi (1 AD) Alachua Bidens alba Haplothrips gowdeyi (2 AD, 2 LV) Trifoliumsp. Haplothrips gowdeyi (7 LV) Vicia sativa Frankliniella fusca (1 AD) Raphiolepis indica Thrips hawaiiensis (7AD, 1LV)

CHAPTER 3 A KEY TO SOME Frankliniella LARVAE FOUND IN FLORIDA

Background

The paucity of larval morphological identification is a continuing problem in all studies on Thysanoptera (Mound 2013). For example, studies conducted in Florida

(Reitz 2002; Northfield et al. 2008; Frantz & Mellinger 2009; Baez et al. 2011) only reported total thrips larvae of all species because no larval identification key was available. Furthermore, the lack of larval species identification can result in the misinterpretation of a host plant. A host plant is a plant on which an insect rears its young (Mound 2013), and the presence of immature life stages provides a quantitative measure of a plant’s suitability as a reproductive host (Terry 1997). Some species of

Frankliniella are competent vectors of species of plant viruses in the genus Tospovirus

(Bunyaviridae), while other species are not vectors (Webster et al. 2015). Only the larvae acquire tospoviruses; therefore, the ability to identify the larvae is important in research and management programs involving tospoviruses. My objective was to create a larval morphological identification tool to separate Frankliniella species encountered in the study of host utilization.

Materials and Methods

Larval character states and chaetotaxy used in this key are adapted from

Vierbergen et al. (2010) and Speyer & Parr (1941). Descriptions of taxa indicated by an asterisk (*) in the key are based on Nakahara and Vierbergen (1998) and the author’s further examination of larval specimens. Character states of F. insularis are from a S.

Nakahara unpublished description (personal communication), from Vierbergen et al.

(2010), and from the examination of larval specimens collected with adults. The species

Frankliniella cephalica (D.L Crawford), Frankliniella bispinosa (Morgan) and Frankliniella kelliae Sakimura are based on examination of larvae with adults. In addition, non- destructive DNA template extraction, PCR and sequencing of the COI mitochondrial gene of F. cephalica, F. bispinosa and F. kelliae were conducted (Rugman-Jones et al.

2010), to confirm larval morphological observations. The molecular data is presented in chapter two. All specimens examined in this study are housed at the USDA, APHIS,

PPQ, Plant Inspection Station, Miami, Florida.

All specimens were examined with a Leica DM LB2 compound microscope under phase contrast at 100X, 200X and 400X magnification. Measurements used in the key and descriptions are in microns at 400X magnification. Images were taken with a

Helicon Focus 6.1.0 system and adjusted for visual clarity with Adobe® Photoshop®

Elements 10. All images are at 400X magnification. The larval specimens were mounted in Hoyer’s medium, stored in an oven at 38° C for a minimum of two weeks and sealed. The adults were mounted in Canada balsam or Hoyer’s mediums.

The larvae II of Frankliniella are distinguished from other Thripinae larvae by having the campaniform sensillae of abdominal tergite IX separated by more than 1.7X the distance between the D1 setae of the tergite. Also, variably sized sclerotized teeth are usually present on the posterior margin of tergite IX. Nakahara and Vierbergen

(1998) give a complete diagnosis of the Frankliniella larvae II.

Results

1. ̶ ̶̶ ̶ ̶ Pronotum with 6 pairs of setae (Fig.2A); mesonotum with 5 pairs of setae (Fig. 3-

2A) ...... instar I

1′. ̶ ̶ ̶ Pronotum with 7 pairs of setae (Fig. 1B); mesonotum with 8 pairs of setae (Fig.

3-1C)……………………………………………………………………………… instar II 43

2. (1) Dark band of abdominal tergite IX extends to and sometimes anterior to the

campaniform sensilla………………………………………………….....F. f usca Hind*

2′. ̶̶̶ ̶ ̶ Dark band extends anterior to the D1 setae by about the distance of the diameter

of the D1 setal socket (Fig. 3-3A-E)…………………………………………...... 3

3. (2) Posteromarginal teeth, abdominal tergite IX, between the D1 setae, longer than

the basal width of the D1 setae (Fig. 3-3 A, B, D, E)………………………………..4

3′. ̶̶̶ ̶ ̶ ̶ Posteromarginal teeth, abdominal tergite IX, between the D1 setae, equal to or

less than the basal width of the D1 setae (Fig. 3-3C, F)..…………………………..7

4. (3) Dorsal setae of abdominal tergites VIII broadly pointed or acute (Fig 3-3B, C,

E) ……………………………………………………………………………………….5

4′. ̶ ̶ ̶ ̶ Dorsal setae of abdominal tergites VIII blunt (Fig 3-3A, D, F)……………..……….6

5. (4) Abdominal tergite VIII discal plaques with poorly developed or rounded

microtrichia (Fig. 3-3B); D1 setae of tergite VIII usually less than

40um……………………………………...... F. cephalica (D.L.Crawford)

5′. ̶ ̶ ̶ ̶ Abdominal tergite VIII discal plaques with well-developed or pointed microtrichia

(Fig. 3-3E); D1 setae of tergite VIII usually greater than

40um..……………………………………………………...F. occidentalis (Pergande)*

6. (4) Abdominal tergite VIII discal plaques with broadly pointed to rounded microtrichia

(Fig. 3-3D); D1 setae of tegite VIII 20-30um long……...... F. kelliae Sakimura

6′. ̶ ̶ ̶ ̶ Tergite VIII discal plaques with fine narrowly pointed microtrichia (Fig. 3-3A); D1

setae of tergite VIII 30-40um long……..………..………….....F. bispinosa (Morgan)

7. ̶ ̶ ̶ ̶ ̶ Abdominal posteromarginal teeth tergite IX minute, less than the basal width of

the D1 setae (Fig. 3-3F); abdominal tergal setae blunt; antennal segment VII 20-

25um long; anterior margin of dark band tergite IX often emarginate between the

D1 setae …………. ……...... F. schultzei (Trybom)*

7′. ̶ ̶ ̶ ̶ Abdominal posteromarginal teeth tergite IX small, most equal to the basal width

of the D1 setae, occasionally some teeth laterad of the D1 setae are longer (Fig.

3-3C); abdominal tergal setae broadly pointed; antennal segment VII 27-33um

long; anterior margin of dark band tergite IX variously

shaped...………………………………………………….……….F. insularis (Franklin)

Description, Larva II Frankliniella bispinosa (Morgan) (Figs. 3-1A, B, C, 3-3A)

Head with the D1-D4 setae apically pointed (Fig 3-1A); D1 setae 10-13um long;

D2 21-25um long; D3 17-20um long and D4 23-30um long. Pronotal setae apically pointed. Meso and metanotal setae with a combination of broadly pointed to apically acute setae. The abdominal tergal setae are apically blunt and appear as if the tips were trimmed by scissors. Abdominal plaques tergites II-VI with some developed microtrichia anteromedially of the D1 setae; most plaques anterolateral of the D1setae with developed microtrichia. Tergite VII plaques anterior of the dorsal setae with well-

developed microtrichia and those posterior of the setae rounded. Nearly all plaques on

tergite VIII with long finely pointed microtrichia. Tergite IX with a dark band in which the

anterior margin extends anteriorly about the diameter of a D1 setal socket. The

posteromarginal teeth tergite IX, are longer than the basal width of the D1 setae;

approximately 12-15 teeth are present between the D2 setae. Ventral posteromarginal

teeth terga IX are minute, about 1/3 the size of the dorsal teeth. Tergite X with a dark

band that extends to the campaniform sensilla of the tergite.

Remarks

Larvae examined from a sample of 24 adult Frankliniella tritici Fitch and 1 adult

F. bispinosa collected with 15 larvae II collected from Lagerstroemia indica, Gadsden

County, Florida were compared to a sample of 14 adult F. tritici with 6 Larvae II collected

from Lagerstroemia indica, Prince Georges County, Maryland. No morphological

differences were detected between the larvae II vouchers of F. bispinosa and the larva II

collected with both samples of F. tritici adults. Therefore, larvae II that key to F.

bispinosa from areas of Florida where populations of F. tritici are encountered should be

reported as F. bispinosa/tritici.

Description, Larva I

Head with the D1-D4 setae apically pointed. Pronotal, meso and metanotal setae

with both apically blunt and broadly pointed setae. Meso and metanotal plaques,

anterior to the posteromarginal row of major setae with rounded microtrichia and those

posterior of the setal row without microtrichia. Abdominal segments I-VIII tergal plaques

with microtrichia. Approximately 12 thin posteromarginal sclerotized teeth between the

D2 setae. Usually, 1-5 small sclerotized teeth lie within the dark band between the D1

setae of the tergite (Fig. 3-2B). Terga IX and X with dark bands similar to the larva II.

Material Examined

12 instar I, 88 instar II larvae. Host: Bidens alba. Collected: 3-I-2013 to 12-VI-

2013. Counties: Glades, Highland, Polk, Osceola, Lake, Sumter, Marion, Alachua,

Lafayette. 2 instar I, 1 instar II. Host: Coreopsis sp.. Collected: 9-IV-2013. County:

Alachua. 36 instar I, 46 instar II. Host: Trifolium sp.. Collected: 9-IV-2013. County:

Alachua. 1 instar I, 8 instar II. Host: Vicia sativa. Collected: 9-IV-2013. County: Alachua.

42 instar I, 5 instar II. Host: Cestrum aurantiacum. County: Alachua. 137 instar I, 79

46 instar II. Host: Raphiolepsis indica. Collected: 9-IV-2013. County: Alachua. 3 instar I.

Host: Sphagneticola trilobata. Collected: 16-VI-2015. County: St. Lucie. 1instar II. Host:

Handroanthus impetigonosus. Collected: 29-IV-2015. County: Miami Dade. 1 instar I, 2 instar II. Host: Delonix regia. Collected: 29-IV-2015. County: Miami Dade. 3 instar II.

Host: Lagerstroemia indica. Collected: 16-VI-2015. County: St. Lucie. 1 instar II. Host:

Asystasia gangética. Collected: 29-IV-2015. County: Miami Dade. 17 instar II. Host:

Lagerstroemia speciosa. Collected: 6-VIII-2010. County: Miami Dade.

Description, Larva II Frankliniella cephalica (D.L. Crawford) (Fig. 3-3B)

Head with the D1-D4 setae apically pointed: D1 setae 10-15um long; D2 12-

18um long; D3 12-15um long and D4 22-25um long. The abdominal setae, tergites II-

VII, are broadly pointed. The D1 setae of abdominal tergite IX are blunt and D2 pointed.

The dorsal abdominal plaques segments II-VI with few poorly developed or rounded microtrichia anterior of the dorsal setae; most plaques anterior of the dorsal setae segments VII and VIII with rounded microtrichia. Occasionally, a few developed or pointed microtrichia present on tergite VIII. Tergite IX with a darkened band in which the anterior margin extends anteriorly about the diameter of a D1 setal socket. The posteromarginal teeth tergite IX, are longer than the basal width of the D1 setae; approximately 12-15 teeth present between the D2 setae. The ventral posteromarginal teeth are minute about 1/3 the size of the dorsal teeth. Tergite X with a transverse dark band that extends to the campaniform sensilla of the tergite.

Description, Larva I

Head with the D1-D4 setae apically pointed. Dorsal thoracic and abdominal setae

I-VIII apically pointed. Tergite IX D1 and D2 setae blunt. Tergite X D1 setae blunt. Meso and metanotal plaques, anterior to the posteromarginal row of major setae with rounded

47 microtrichia and those posterior of the setal row are crescent shaped and lack microtrichia. Tergal plaques abdominal segments I-VIII with microtrichia. Approximately

10-12 thin posteromarginal sclerotized teeth between the D2 setae of tergite IX. No sclerotized teeth lie within the dark band between the D1 setae of the tergite. Terga IX

and X with dark bands similar to the larva II.

Material examined

534 instar I, 763 instar II larvae. Host: Bidens alba. Collected: 3-I-2013 to 16-VI-

2015. Counties: Monroe, Miami Dade, Broward, Palm Beach, Hendry, St. Lucie,

Glades, Highlands, Polk, Osceola, Lake, Sumter, Marion, Alachua, Suwannee,

Lafayette.

Remarks and observations Frankliniella fusca Hinds

The primary character that separates this species from the others treated in this

key is the longitudinally broad dark band of tergite IX. For a complete description, see

Nakahara & Vierbergen (1998).

Material examined

1 instar I, 1 instar II. Host: Arachis sp.. Collected: 16-VI-2015. County: St. Lucie.

Description, Larva II Frankliniella insularis (Franklin) (Fig. 3-3C)

Head with D1-D4 setae broadly pointed: D1 setae 20-23um long; D2, 25-30um long; D3, 20-30um long and D4, 27-37um long. All the abdominal tergal setae are broadly pointed apically. The abdominal plaques segments II-VII with few poorly developed or rounded microtrichia anterior of the dorsal setae; those on VIII are more abundant. Sometimes a few pointed microtrichia present on tergite VIII. Tergite IX with a darkened band in which the anterior margin extends anteriorly about the diameter of a

D1 setal socket. The posteromarginal teeth tergite IX are about the same length as the

48 basal width of the D1 setae; approximately 18-21 teeth present between the D2 setae.

The ventral posteromarginal teeth are minute about 1/2 the size of the dorsal teeth.

Tergite X with a transverse dark band that extends to the campaniform sensilla of the tergite.

Material examined

96 instar II larvae. Host: Abutilon chitenendii. Collected: 27-III-2010. County:

Miami Dade.

Description, Larva II Frankliniella kelliae Sakimura (Figs. 3-2A, B, 3-3D)

Head with the D1, D2 and D4 setae apically broadly pointed, D3 apically pointed;

D1 setae 10-13 long; D2, 15-18um long; D3, 17-20um long and D4, 22-25um long.

Pronotal setae apically blunt. Meso and metanotal setae with a combination of apically truncate to broadly pointed setae. The abdominal tergal setae are apically blunt and appear as if the tips were trimmed by scissors. Abdominal tergites I-VII plaques with rounded microtrichia. Tergite VIII plaques with a combination of broadly pointed to rounded microtrichia. Tergite IX with a darkened band in which the anterior margin extends anteriorly about the diameter of a D1 setal socket. The posterormarginal teeth tergite IX, are longer than the basal width of the D1 setae; approximately 15-18 teeth are present between the D2 setae. The ventral posteromarginal teeth are minute about a 1/3 the size of the dorsal teeth. Tergite X with a transverse dark band that extends to the campaniform sensilla of the tergite.

Description, Larva I

Head with the D1, D2 and D4 setae apically blunt; D3 pointed. Pronotal, meso and metanotal setae apically blunt. Meso and metanotal plaques with some reduced microtrichia on the anterior rows. The plaques are crescent-shaped giving the

49 integument a sculptured or wrinkled appearance (Fig. 3-2A). Plaques, abdominal tergites I-V, similar in appearance as the thoracic plaques with reduced microtrichia on the anterior and lateral margins of the tergites. Tergal plaques abdominal segments VI-

VIII with microtrichia. Approximately 12 thin posteromarginal sclerotized teeth present

between the D2 setae, tergite IX. Usually 1-5 small sclerotized teeth lie within the dark

band between the D1 setae of the tergite (Fig. 3-2B). Terga IX and X with dark bands

similar to the larva II.

Material examined

1 instar I, 1 instar II. Host: Cassia javanica. Collected: 29-IV-2015. County: Miami

Dade. 1 instar I, 5 instar II. Host: Handroanthus impetigonosus. Collected: 29-IV-2015.

County: Miami Dade. 1 instar II. Host: Cassia fistula. Collected: 29-IV-2015. County:

Miami Dade. 18 instar I, 69 instar II. Collected: 29-IV-2015. County: Miami Dade. 74 instar II. Collected: 29-IV-2015. County: Miami Dade. 20-I-2009.

Remarks and observations Frankliniella occidentalis (Pergande) (Fig. 3-3E)

Dorsal abdominal setae broadly to finely pointed. Nearly all plaques on abdominal tergite VIII with long finely pointed microtrichia. Usually 12-15 posteromarginal teeth are present between the D2 setae and 4-6 teeth between the D1

setae of tergite IX. The length of the posteromarginal teeth exceed the basal width of

the D1 setae and often exceed the diameter of the D1 setal sockets.

Material examined

15 instar II. Host: Gerbera sp.. Collected: 12-III-2009. County: St. Lucie. 16 instar

II. Host: Capsicum annuum. Collected: 13-IV-2009. County: St. Lucie. 2 instar II. Host:

Nasturtium sp. Collected: 13-IV-2009. County: St. Lucie

Remarks and observations Frankliniella schultzei (Trybom) (Fig. 3-3F)

Dorsal abdominal setae apically blunt. Most plaques on abdominal tergite VIII with reduced apically rounded microtrichia.

Material examined

18 instar II. Host: Lycopersicon esculentum. Collected: 14-IX-2015. County: Palm

Beach. 8 instar II. Host: Lycopersicon esculentum. Collected: 7-IX-2009. County:

Hendry.

Discussion

The number of obvious morphological characters available for larval study was less compared to those available for the adults. Furthermore, many of the larval characters and their respective states were not reliable for separating these congener species. Larval characters in this key that we found useful in separating the species may be difficult to discern for the occasional user. Therefore, we recommend using this key to confirm larval identities with vouchered adults when possible. Also, phase contrast microscopy should be used. Subtle differences of the integument and microtrichia were not otherwise visible under bright-field microscopy.

Figure 3-1. The larva II of F. bispinosa: dorsal setae pairs, D1-D4 of head (A); dorsal setae pairs, D1-D7 of pronotum (B); dorsal setae pairs, D1-D8 of mesonotum (C). (Thomas Lynn Skarlinsky II. Larva II. 01/11/2016)

Figure 3-2. The larva I of F. kelliae: dorsal setae pairs, D1-D6 of pronotum and D1-D5 of mesonotum (A), arrow indicates small sclerotized teeth between D1 setae of abdominal tergite IX (B). (Thomas Lynn Skarlinsky II. Larva I. 01/11/2016)

A B

C D

E F

Figure 3-3. Abdominal tergites VIII and IX of larvae II: dorsal setae, D1, D2 of tergites VIII and IX F. bispinosa (A); F. cephalica (B); F. insularis (C); F. kelliae (D); F. occidentalis (E); F. schultzei (F). (Thomas Lynn Skarlinsky II. Tergites VIII and IX. 01/11/2016)

CHAPTER 4 CONCLUSIONS

The Florida sympatric species F. bispinosa and F. cephalica are utilizing B. alba for reproduction and congener encounters on B. alba were more frequent in the central region of Florida. Frankliniella cephalica was the most successful congener utilizing

B.alba flowers for reproduction and food acquisition in southern Florida and both species were proficient using B. alba for both purposes in central Florida. The fitness of both species indicated by different respective adult densities from B. alba suggest F. cephalica is competitively excluding F. bispinosa in the south and F. bispinosa is competitively excluding F. cephalica in central and north Florida. Local climatic conditions may be mediating competitive interactions between the two congeners in central and north Florida. However it is unclear what factors or mechanisms are mediating their interactions in the south.

The molecular data supported morphological observations. In addition, sequences from the 45 specimens’ revealed intra and interspecific difference among the congeners studied. Additional sequencing of nuclear genes would further validate the mitochondrial sequences and possibly detect evidence of hybridization in central Florida where congener encounters are more common on B. alba.

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BIOGRAPHICAL SKETCH

Thomas Lynn Skarlinsky II was born and raised in Ashtabula, Ohio. He received a Bachelor of Science in parasitology and medical entomology from Bowling Green

State University in 1987. He subsequently served as a U.S. Peace Corps volunteer in

Guatemala until 1989. Shortly thereafter, he married his beautiful wife Patricia, who

gave him two wonderful daughters, Solsiree and Patty. Since 1989 he has worked for

the USDA in Miami, Florida. He currently is an entomology identifier at the Miami Plant

Inspection Station and specializes in the study of Neotropical Thripidae.