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IDENTIFYING HOST-STRAIN BEHAVIORAL DIFFERENCES OF FALL
ARMYWORM IN FLORIDA (LEPIDOPTERA: NOCTUIDAE)
CHARLES J. STUHL
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
Charles J. Stuhl
To my family: Without them, I would not be the man I am today
I would like to thank Dr. Robert Meagher for his guidance, support and
encouragement while I pursued this degree. I would also like to thank the members of my
committee, Dr. Heather McAuslane and Dr. James Maruniak for their valuable advice,
expertise and review of this thesis.
I thank Dr. Rodney Nagoshi for his advice, technical support in molecular biology
and insight into the experimental design. I also thank Dr. Paul Mislevy for his donating
the bermudagrass variety utilized in this study.
This research could not have been completed without the assistance of numerous
individuals. I am grateful to Charlie Dillard, whose technical assistance and comic relief
guided me through this project. Special thanks go Peggy Brennan. Her help in setting up
and troubleshooting the olfactometer experiment was immeasurable. I thank Jennifer
Gillett for her tolerance and insight in preparing this thesis. I also thank Gina Posey for
her computing skills and endless support. I would also like to thank Jennifer, Gina and
Delaine Miller for dragging me out of the lab on occasion to enjoy a lunch consisting of
something other than my ubiquitous peanut butter and jelly sandwich. I also thank Debbie
Hall for her assistance in all that was necessary to complete this degree; her knowledge
and guidance was always greeted with a smile.
This would not be complete without mentioning Dr. Everett Mitchell. His passion
in the areas of insect behavior and biological control ignited what has become my passion
as well. I acknowledge the U.S. Department of Agriculture, Agricultural Research
Service for employment, and the opportunity to achieve my goals.
TABLE OF CONTENTS
L IST O F TA B LE S ................ ....... ... .. .... .. ............. ........ .. ..vii
LIST OF FIGURES ..... .......... ......... ........ .... .... .............. viii
ABSTRACT.................................. .............. ix
1 INTRODUCTION ................... ............................ .......... .. .......... 1
2 OVIPOSITIONAL PREFERENCE OF HOST-STRAINS TO CORN AND
Introduction ................. ...........................6.............................
Materials and Methods ................................................8
Strain Isolation and Plant Growth ...........................................8
Oviposition B ioassay ................... .................. ........... ....... .. .... .. .. ...............
Statistical Analysis ...................... ........ ........ .... ...10
Results ...................................... ................................................ 10
D discussion ...................................... ................... .....................10
3 LARVAL PREFERENCE OF HOST-STRAINS TO CORN AND STARGRASS ..18
Introdu action ................ ............... ........................ 18
M materials and M methods .......................... ................ 19
Strain Isolation and Plant Growth .......................................... 19
Choice Test Bioassays ............... ................... ........19
Passing-Over Tests .................... ..................................22
Statistical Analysis ............................................. ..... ... 23
Results.................... .. ......... ..............................23
Choice Tests ................................................23
Passing-Over Tests .................... ..................................24
Discussion ............ .............. .... ..............................24
4 SUM M ARY...................................... ................... .... ......... 43
L IST O F R E FE R E N C E S ............................................................................. 47
BIOGRAPHICAL SKETCH .................................................. ............... 53
LIST OF TABLES
2-1. Number of corn and rice-strain egg masses recovered in the oviposition bioassay.. 13
3-1. Mean ( SEM) number of corn or rice-strain larvae collected from corn or stargrass
sections in the Petri dish choice bioassay......................... ............... 28
3-2. Percent larvae ( SEM) of both strains selecting either corn or stargrass vs. an
empty space in the choice cage bioassay .................................... ......29
3-3. Percent larvae ( SEM) of both strains selecting either corn or stargrass in the
choice cage bioassay ...................... .......... ........ .... 30
3-4. Percent larvae ( SEM) of both strains selecting either corn or stargrass vs. potted
soil in the Y-tube olfactometer bioassay ......................................................... 31
3-5. Percent larvae ( SEM) of both strains selecting either corn or stargrass in the
Y-tube olfactom eter bioassay ............................................................. ............ 32
3-6. Mean ( SEM) number of corn-strain larvae that selected either corn or stargrass in
a Petri dish bioassay ................... ............ .. .... ......... 33
3-7. Mean ( SEM) number of rice-strain larvae that selected either corn or stargrass in a
Petri dish bioassay ...................................... ........................... .. ......... 34
3-8. Percent ( SEM) corn-strain larvae that selected either corn or stargrass in a wind
tunnel bioassay ....................................................35
3-9. Percent ( SEM) rice-strain larvae that selected either corn or stargrass in a wind
tunnel bioassay ....................................................36
LIST OF FIGURES
2-1. Fall arm yw orm collection sites.................................. .................... 14
2-2. Strain isolation bioassay container .................................................................. 15
2-3. Agarose gel showing the rice and corn-strain DNA polymorphism. .........................16
2-4. Dead FAW corn-strain male being used as an oviposition site............... ...............17
3-1. Sm all Petri dish bioassay ........................................................................ 37
3-2. Choice cage bioassay ................... .... ....................................... ........ 38
3-3. Y-tube olfactom eter used in volatile study............................................................ 39
3-4. Petri dish passing-over study ......................................................................... .............40
3-5. W ind tunnel layout ............. ............................ .. ...... 41
3-6. Wind tunnel bioassay................ .. ..................42
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
IDENTIFYING HOST-STRAIN BEHAVIORAL DIFFERENCES OF FALL
ARMYWORM IN FLORIDA (LEPIDOPTERA: NOCTUIDAE)
Charles J. Stuhl
Chair: Robert L. Meagher
Major Department: Entomology and Nematology
Florida is a known overwintering site for the fall armyworm (FAW), Spodoptera
frugiperda (J. E. Smith). Previous research suggests that this insect comprises two
genetically different host-strains: one using large grasses such as corn as a host-plant
(corn-strain), and the other using smaller grasses such as rice and forage grasses
(rice-strain). My study was conducted with insects collected and identified from various
sites throughout Florida. Strain identification was made using the Cytochrome Oxidase
subunit I (CO1) gene as a mitochondrial marker. Once confirmation of strain association
was made, corn-strain larvae were fed a corn (Zea mays L. 'Truckers Favorite') foliage
diet, rice-strain larvae were fed on a type of bermudagrass (stargrass, Cynodon
nlemfuensis Vanderyst var. niemfuensis 'Florona'). The pure strains were established and
colonies reared at the USDA-ARS Gainesville. Successive generations were used in this
study. Female ovipositional site selection and larval host choice between corn and
stargrass plants were behavioral traits measured. Rice-strain females exhibited a strong
ovipositional preference (95%) for stargrass plants. Corn-strain moths oviposited 53% of
egg masses on the test enclosure, rather than on host-plants. Stargrass (30%) and corn
plants (17%) also contained egg masses. The unpredictable behavior of corn-strain
females contradicts previous studies of the two host-strains; suggesting that the
corn-strain is a more generalist feeder than the rice-strain. The conflicting results may be
attributed to my documentation of the enclosure as a non-plant-host variable in the
Larval-choice studies were conducted using multiple bioassays to determine
whether there is a strain preference for corn or stargrass. When given a choice of a
section of each host-plant in a Petri dish bioassay, neonates of both strains chose corn
sections significantly more than stargrass sections. When whole plant material was
presented, corn-strain larvae showed a preference for stargrass; while rice-strain larvae
were evenly distributed between the two plants.
The ability of the neonate larvae to detect plant volatiles was observed in a Y-tube
olfactometer. Corn-strain larvae showed a strong (yet non-significant) preference for
corn volatiles. Rice-strain larvae were evenly distributed between the two arms of the
A plastic cage/wind-tunnel bioassay was developed to observe movement of
larvae upwind through one host-plant to another. Corn-strain larvae were evenly
distributed between the two plants, regardless of plant position. Rice-strain larvae
showed a strong trend toward whichever host-plant it first encountered. When corn was
the first plant encountered, 70% of the larvae showed a preference for corn; when
stargrass was the first encounter, 67% of the larvae showed a preference for stargrass.
Fall armyworm (FAW), Spodopterafrugiperda (J. E. Smith) is a migratory pest
that makes an annual journey each spring from southern Florida into northern regions of
the United States (Mitchell 1979). The first documented observation ofFAW feeding on
corn, sugarcane, and rice in Florida was by Glover (1856). Quaintance (1897) noted an
outbreak of FAW that occurred during late August at the University of Florida campus,
Lake City. Large numbers of larvae were seen feeding on crab grass (Panicum
sanguinale) and he stated that they were "quite eating up the grass on the southern end of
the college campus." The first reported outbreak in Florida crops was in 1899
Evidence from early in the last century showed that FAW is a native of tropical
and subtropical America (Walton and Luginbill 1916). It has been theorized that
management of FAW during overwintering in southern regions could greatly reduce the
economic impact this pest causes each year during migration. Populations multiply at
overwintering sites in southern Florida and southern Texas, before making their
northward spring migration (Tingle and Mitchell 1977, Sparks 1979). Knipling (1980)
stated that if overwintering populations in Florida were the primary source of the
infestations, a rigid suppression program in the overwintering areas would have a great
impact on the FAW population throughout the southeastern and Atlantic coast regions.
Two closely related populations of one species could diverge, allowing them the
opportunity to establish new niches in the same environment. Sympatric speciation
occurs when one evolutionary lineage splits into two separate species without the
occurrence of geographical isolation (Berlocher and Feder 2002). Most work involving
speciation in insects has been done with Drosophila. Although that work gave valuable
insight into insect speciation, the direct cause still remains unclear (McMillian et al.
1997). Sympatric speciation is likely the outcome of competition for resources. Becerra
and Venable (1999) stated that insects have been shifting among hosts that are
geographically available; and that a shift to a particular plant species is likely if its
geographical range coincides with the geographical distribution of the old host. They
stated that host shifts by phytophagous insects might also be attributed to plant chemical
Previous research into the migration sources of FAW suggests that this insect
comprises of two host-strains: the corn-strain that feeds predominantly on corn (Zea mays
L.), and the rice-strain that feeds on smaller grasses such as rice (Oryza sativa L.) and
bermudagrass (Cynodon dactylon L.) (Pashley et al. 1985, Pashley 1986). Insects
collected throughout Florida overwintering areas are of both strains (Meagher and
Gallo-Meagher 2003, Meagher and Nagoshi 2004, Nagoshi and Meagher 2004).
Molecular data suggest that FAW strains are more likely to be host-associated sibling
species in which the strains appear to be sympatric and tend to use different plant hosts
(Diehl and Bush 1984, Pashley 1986). In addition to speciation being genetically based,
insect behavior plays an important role in the adaptation of FAW to new host-plants.
The FAW larvae will readily feed on at least 60 species of plants, but their
ovipositional preference is on members of the Poaceae rather than other plant species
(Mitchell 1979, Pitre et al. 1983, Whitford et al. 1988). Some favored crops of
agricultural importance that FAW damages include sweet corn, turf grasses, cotton,
peanut, cowpea, potato, and sugarcane. FAW is also the most important pest of
bermudagrass pastures in the southeast (Pencoe and Martin 1982). When populations are
high, FAW can subsist on many types of vegetation it may encounter (Luginbill 1928,
Introduction of monoculture corn crops in North America offered FAW a new
host on a massive scale. Native Americans first introduced this crop to Florida between
1000 and 1500 A.D. (Leonard 2003). With modem agricultural practices, there are now
over 83,000 hectares of corn planted in Florida each year (Nuessly et al. 1999). Florida is
the major source of sweet corn during the winter and early spring, in the United States, as
harvesting is most active from November to June.
The first bermudagrass variety was introduced to Florida in the early 1880s. By
the 1920s, commercial sod was being farmed in the state. After World War II the sod
industry began to develop into the business that it is today. Bermudagrass is now grown
extensively in Florida for pasture and hay, but commercial sod production has risen due
to an increased demand for turf by building contractors and residential homeowners.
Bermudagrass is now being used on golf courses, and this plant covers more than
607,000 hectares of Florida (White and Busey 1987). It has been suggested that FAW
evolved on native grasses or shifted to bermudagrass as a host from corn when the grass
was introduced into the New World (Pashley et al. 1987).
FAW is active year-round in southern Florida, and this area serves as a reservoir
for the yearly migration throughout the northeastern United States. It is believed that
FAW uses bermudagrass as its primary host, thus increasing the population, which in turn
migrates to other food and forage crops (Fuxa 1989, Pitman et al. 2002). However, this
observation may be disputed because those observations were made before the existence
of two host-strains was discovered. Sweet corn production is also at its peak during the
winter months in southern Florida. This may account for the ability of both populations
to increase in numbers during winter.
Previous studies on host sensory behavior in moths showed that moths rely on
multiple sensory inputs for host location (Ramaswamy 1988). Selection of a suitable
oviposition site by the female is initiated by chemical and tactile cues (Rojas et al.).
Singer (1984) proposed for Lepidoptera, the host-plant is selected by the adult female.
Environmental pressures may account for females selecting a host that is not optimal for
larval development. Their limited mobility makes neonate larvae dependent on the adult
female to select the most nutritious host, although neonate larvae do use chemoreception
in host-plant location (Showler 2001). Many lepidopterous larvae are highly mobile at
older instars, and have the ability to seek out a suitable food source (Berdegue et al.
1998). Larvae use olfaction and gustation to provide information for food-plant
discrimination (Hanson and Dethier 1973, de Boer and Hanson 1987). Insect feeding
behavior is influenced by chemical components of the host-plants that assist in food
finding and acceptance (Thorsteinson 1960). Olfaction can induce orientational
responses to plant hosts in larvae with prior feeding experiences, although a polyphagous
species may not be equipped with the inherent response to host-plants (Carlsson et al.
Research objectives. These studies were performed to identify behavioral
differences between the two FAW host-strains from a Florida perspective. Areas of
concentration were ovipositional preference and larval host selection to corn and a type
of bermudagrass known as stargrass.
OVIPOSITIONAL PREFERENCE OF HOST-STRAINS TO CORN AND
Fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) is a generalist insect pest
that can develop on many host-plant species. Although an economic pest of numerous
crops, it has a preference for plants in the family Poaceae (Luginbill 1928). Cotton and
soybean can also be injured by FAW feeding, but are usually only attacked when
populations are extremely high, or when preferred host-plants are scarce (Pitre et al.
FAW is thought to be a native of tropical and subtropical America (Walton and
Luginbill 1916). Florida and southern Texas are known overwintering sites for FAW
from where populations expands into the eastern and central United States during the
course of the spring and summer (Mitchell 1979, Sparks 1979). Suppressing
overwintering populations in southern Florida before migration has been offered as a
possible management strategy to reduce the impact of this pest (Knipling 1980). It is
difficult to develop successful management programs due to the fact that this insect is
able to sustain life upon a wide variety of food-plants (Pencoe and Martin 1981).
Host selection by a generalist insect may be accomplished by visual, chemical and
tactile cues. Adult females use all three senses to find suitable ovipositional sites
(Zacharuk and Shields 1991). In response to plant cues, an orientational movement may
initiate the behavioral process leading to host location and acceptance for oviposition and
feeding (Jallow et al. 1999).
Host-plant selection in Lepidoptera for larvae is assumed to be the choice of the
ovipositioning female (Singer 1984). Adult FAW females can be indiscriminate in their
selection of oviposition sites. Pitre et al. (1983) observed that females will oviposit on
non-plant material despite the presence of a host-plant nearby, and Thomson and All
(1984) found eggs laid on objects such as survey flags. FAW females usually place their
eggs on the underside of leaves of the food-plant, but eggs have been found on leaves
upon which the larvae are not known to feed (Quaintance 1897). Physical stimuli may
have a greater impact than close-range chemical cues on ovipositional selection with
FAW (Rojas et al. 2003). In order to enhance larval development and survival by
providing a suitable diet, many insects prefer to oviposit on certain plant species
(Showler 2001). The limited mobility of neonate larvae makes them highly dependent on
the female parent's ability to select the most nutritious host (Smits et al. 1987).
In a search for the geographical sources of Louisiana migrants, Pashley et al.
(1985) collected specimens in the Caribbean, Florida, Louisiana, Texas, and Mexico and
discovered that there were two genetically differentiated host-strains. One host-strain
feeds predominantly on corn (Zea mays L.) (corn-strain), while the other feeds
predominantly on small grasses such as bermudagrass (Cynodon dactylon L.) and rice
(Oryza sativa L.) (rice-strain) (Pashley 1986). Although ovipositional preference is
potentially one mechanism that maintains strain fidelity, only one limited study has been
completed (Whitford et al. 1988). My study was designed to determine the ovipositional
preference of the two FAW host-strains to corn or to a forage grass.
Materials and Methods
Strain Isolation and Plant Growth
FAW egg masses and larvae of various instars were collected in during 2003 from
multiple sites throughout Florida. FAW were collected from the University of Florida
Dairy Research Unit, Hague; University of Florida Range Cattle Research and Education
Center (REC), Ona; University of Florida, Everglades REC, Belle Glade; and sweet corn
fields in Miami-Dade County (Fig. 2-1). Eggs and larvae collected at these locations
were reared to pupation on a pinto bean diet (Berger 1963). A single adult male-female
pair was placed in an oviposition cage (Fig. 2-2). This cage consisted of a cylindrical
inverted 473 mL plastic food container (Solo Cup Co., #Mkl6) lined with a 7 cm x 7.6
cm coffee filter (Bunn, BCF). Holes of-5.0 mm were placed in the top positioon to allow
for airflow. A hole -1.5 cm was placed in the inverted lid (Solo, ML8) in which a
braided cotton roll (Richmond Dental, #200205) cut to a length of 5 cm was inserted.
This allowed for absorption of liquid for adult nourishment. The cage was placed over a
177 mL container (Ft. Howard, S306), which held a plastic souffle cup (Solo, P100) with
a 10% honey/sugar solution. Females were allowed to freely deposit eggs on the inner
surface of the coffee filter.
Upon death, male and female moths were analyzed separately for strain
identification utilizing a PCR technique which amplified the Cytochrome Oxidase
subunit I (COI) gene that was used as a mitochondrial marker (Levy et al. 2002, Nagoshi
and Meagher 2003a, Nagoshi and Meagher 2003b). Eggs were collected daily, and
labeled according to pair mating. Newly emerged larvae were reared on pinto bean diet
until strain identification was verified. Once confirmation of strain association was
made, F2 larvae were placed on either a corn or stargrass (Cynodon nlemfuensis
Vanderyst var. nlemfuensis 'Florona') foliage diet, according to their strain host
preference. Adults of the corn-strain (Hague, F3-Fo1) and rice-strain (Ona and Miami
colonies, which were combined, F3-F10) were used; however, generations of both strains
were used concurrently.
Plants were grown in 550 mL pots, in a greenhouse at ambient temperature
(-30C) and were fertilized once weekly with Miracle-Gro 15-30-15 plant food; no
pesticides of fungicides were applied. Plant age during experimentation was
approximately three weeks for both field corn ('Truckers Favorite') and 'Florona'
stargrass. 'Florona' stargrass is a long-lived, persistent perennial grass similar to
bermudagrass types that was observed growing at the Range Cattle REC in Ona in 1973
(Mislevy et al. 1989). Previous research showed that it was an excellent host for FAW
(Meagher, unpublished data).
Eight pairs of adults from one strain -48 h old were released in a screen enclosure
placed inside a Conviron plant growth chamber. Each strain was tested separately. The
enclosure measuring 178 (L) x 76 (W) x 120 (H) cm was constructed of 1.9 cm PVC pipe
and nylon window screen. Five corn and five stargrass plants were placed haphazardly
within the enclosure. The chamber's environment was set at 23.9 + 2C, 80% RH with
a 14/10 day/night cycle. Two plastic souffle cups (Solo, P100) with a saturated cotton
ball containing a 10% honey/sugar solution were placed inside the enclosure for moth
nourishment. Females were allowed to freely oviposit within the enclosure. The
numbers of egg masses were counted on each host-plant after a period of 72 h. The inner
surface of the enclosure was also inspected as a possible surface for oviposition. Six
replicates were performed for each strain.
Analysis of variance (PROC MIXED, Contrasts, Littell et al. 1996) was used to
examine variation among oviposition substrates.
PCR analysis of insects collected in the four sites indicated the presence of both
corn and rice-strain populations in Florida (Fig. 2-3). This information supports previous
findings of populations collected and analyzed in Florida (Meagher and Gallo-Meagher
2003, Meagher and Nagoshi 2004, Nagoshi and Meagher 2004). Insects collected from
Ona and Miami were determined to be rice-strain, and those collected from Hague were
corn-strain. Insects collected from corn in Belle Glade were a mixture of the two strains,
and not utilized in this study.
FAW females oviposited on both host-plants as well as on the top and sides of the
enclosure. The two host-strains showed a significant difference in their placement of egg
masses (Table 2-1). The greatest amount of egg masses oviposited by rice-strain females
was found on stargrass plants (95.4%), as opposed to corn (2%) or the enclosure (2%).
Alternately, corn-strain females did not discriminate between host-plants in placement of
their eggs. Both host-plants had fewer egg masses than the interior walls of the enclosure
on which more than 50% of eggs were laid. Corn-strain females even oviposited on the
remains of a dead adult (Fig. 2-4).
The results of this study clearly identify a strain behavioral distinction between
the two host-strains. The initial scope of this experiment was to identify the ovipositional
preference on two known host-plants. Egg masses rather than eggs per mass were
recorded because egg masses deposited provides a better indication of the development of
a FAW infestation than eggs per mass due to high neonate mortality (Pitre et al. 1983).
Rice-strain females clearly showed a preference for stargrass plants as an ovipositional
substrate. However, 53% of the egg masses deposited by corn-strain females were on the
enclosure surrounding the host-plants. Previous studies make reference to FAW being
indiscriminate in its selection of oviposition sites, depositing eggs on objects as well as
plants (Thomson and All 1984). Showler (2001) indicated that Spodoptera exigua
(Htibner) deposited twice as many egg masses on chamber walls and plant pots than on
host-plants. He also indicated that the limited mobility of S. exigua neonates made them
highly dependent on the female's ability to select the most nutritious host. Prior to the
identification of host-strains, FAW was considered a single host-strain polyphagous
insect. The indiscriminate ovipositional behavior previously noted may have been that of
Previous research concerning the ovipositional preference of FAW strains has
shown that differences do exist between the two strains. Whitford et al. (1988) presented
each strain with corn, sorghum, bermudagrass and centipedegrass. Rice-strain females
showed preference for grasses and corn-strain females for corn and sorghum. However,
it was not stated in their study whether eggs masses were found in locations other than on
plants. Also, they used colonies that were reared on an artificial pinto bean diet whereas
insects in my study were reared on host-plant material. Test results may be
unintentionally altered if insects are reared or collected from various food-plants or
artificial diets (Pencoe and Martin 1981). Pashley et al. (1995) stated that the
ovipositional preference of the corn-strain is more specialized, and this strain rarely
occurs in pastures. The unpredictable oviposition of corn-strain females in my study
contradicts her results. My results suggest that corn-strain females display more
generalist ovipositional behavior than rice-strain females.
The indiscriminate behavior of corn-strain females indicates that chemical and
tactile cues are of a lesser importance to this strain. The rough surface of the screened
enclosure was the most desired ovipositional site. Rojas et al. (2003) stated that host
location in FAW is not influenced by plant volatiles but that surface texture alone affects
ovipositional behavior. In their study, grooved and pitted surfaces were preferred
ovipositional sites rather than smooth surfaces. Unfortunately, it was not stated which
strain was used in the experiments, although the colonies used were collected from a corn
habitat. The ovipositional preferences that they observed indicate that corn-strain
females were probably used in their tests.
An herbivore whose preferred host-plant varies in abundance will utilize a lesser
host when the ideal host is not available. Competition or natural enemies at other trophic
levels may result in poor performance on a particular host (Price et al. 1980, Thompson
1988). Although FAW are reported to prefer plants in the grass family, it has been shown
in many studies that they will readily oviposit and feed on plants of other families.
Therefore, it may be chemical stimulants within members of the grass family that
influence a female's ovipositional preference (Pitre et al. 1983).
Table 2-1. Number of corn and rice-strain egg masses recovered in the oviposition
Substrate Mean number of egg masses'
Corn 1.8 0.6 b 0.2 0.3 b
Stargrass 3.3 0.9 ab 8.3 1.5 a
Enclosure 5.8 1.0 a 0.2 0.14 b
1 Means SEM followed by the same letter within strains were not significantly different
(P < 0.05). ANOVA statistics: n = 6 reps; F = 5.5; df = 2, 10; P = 0.0247 and F = 24.1;
df = 2, 10; P < 0.0001 for corn and rice-strain, respectively.
University of Fforda Dary Research Unit, Hague
University of Florida Range Cattie REC, Ona
Universty of Florida Everglades REC, Belle Glade
Figure 2-1. Fall armyworm collection sites
Figure 2-2. Strain isolation bioassay container
Figure 2-3. Agarose gel showing the rice and corn-strain DNA polymorphism. The
rice-strain pattern is a 569 bp PCR band, while the corn-strain fragment is cut
by MspI to produce two fragments of 497 and 72 bp
Figure 2-4. Dead FAW corn-strain male being used as an oviposition site
LARVAL PREFERENCE OF HOST-STRAINS TO CORN AND STARGRASS
In order for an immature insect to sustain their growth and development, they
must be voracious feeders. Food location and feeding behavior of larval herbivores are
important attributes of their biology (Zacharuk and Shields 1991). Fall armyworm
(FAW) [Spodopterafrugiperda (J. E. Smith)] is a polyphagous species that damages a
wide range of agricultural crops. This species has two host-strains, one that feeds
predominantly on corn (Zea mays L.) (corn-strain), and another that feeds predominantly
on small grasses such as bermudagrass (Cynodon dactylon L.) and rice (Oryza sativa L.)
(rice-strain) (Pashley et al. 1985, Pashley 1986). As with other moth species, these two
strains exhibit differences in physiological characters that may or may not be affected by
differences in larval or adult behavior (Pashley 1993, Futuyma and Philippi 1987).
Larvae of both strains feed and develop on corn and grasses, although development can
be significantly influenced by plant host (Pashley 1988, Whitford et al. 1992, Pashley et
al. 1995, Veenstra et al. 1995).
It is not known whether FAW adults or neonate larvae select the host-plant on
which development will take place. It has been suggested that female moths in general
select the ovipositional substrate that will best sustain their progeny, utilizing visual,
chemical and tactile cues in their search. Corn-strain females can be indiscriminate in
their selection of oviposition sites, depositing eggs on objects as well as host-plants (Pitre
et al. 1983, Thomson and All 1984, Chapter 2).
Therefore, it can also be suggested that it is the newly emerged larva that is
making the "choice" of host. Gustatory and tactile cues are of primary importance for
food selection to the immature stage (Zacharuk and Shields 1991). Larvae spin threads
and descend downward on or near the desired host, and some may be carried a distance
by the wind. This may be an adaptive behavior allowing individuals to disperse from the
location of the egg mass and thus prevents competition among siblings (Claycomb 1954).
Behavioral analysis of larval Bombyx mori L., Manduca sexta L., and Pieris brassicae L.,
has shown evidence of a high degree of chemosensory specificity at the receptor level
(Ishikawa et al. 1969, Schoonhoven 1969). With the diversity of chemicals in green
plants, their role in insect feeding behavior has created many theories as to their influence
on host selection (Hsiao 1974).
Host selection in immature FAW has received limited study. Pashley et al. (1995)
found that both host-strains exhibited a strong preference for corn over bermudagrass in
Petri dish bioassays. The current study was conducted using multiple experimental
bioassays to determine if the two host-strains demonstrate preference for corn or stargrass
(Cynodon nlemfuensis Vanderyst var. nlemfuensis 'Florona'), a plant closely related to
bermudagrass (Mislevy et al. 1989).
Materials and Methods
Strain Isolation and Plant Growth
Insect culturing and plant growth were conducted using the same colonies and
plant-growing techniques as in Chapter 2.
Choice Test Bioassays
These tests were designed to compare preference of neonate larvae of both
host-strains for either corn or stargrass. Three separate bioassays were performed. The
first experiments were conducted using 9-cm diameter polystyrene Petri dishes (Thomas
Scientific, #3488-B32). New growth leaf sections were taken from each plant type, and
trimmed along the top and sides to achieve a uniform size (- 5 cm x 2 cm). One section
of each plant host was placed haphazardly on filter paper discs (Thomas Scientific,
#4712B25) moistened with 3 mL deionized water. Sections were placed 2 cm from
the center, along the outer edge of the Petri dish (Fig. 3-1). Twenty newly hatched larvae
were placed in the center of each dish, and the lid put into place. Ten replicates were
performed for each strain. Petri dishes were placed in a RevcoTM incubator at 23.9 2C
with a 14/10 day/ night cycle, -80% RH. The number of larvae on or under each leaf
section was counted 24 h after introduction.
The second bioassay used a clear acrylic plastic cage measuring 51 (L) x 25 (W) x
28 (H) cm with a testing area of 51 x 25 x 18 cm. This cage allowed for whole plants to
be tested rather than plant sections. Potted plants were placed in an elongated recess that
was removable, and allowed for the soil/plant interface to be level with the floor surface
(Fig. 3-2). During testing, the cage was placed in an environmentally controlled room at
23.9 2C with a 14/10 day/night photoperiod, 80% RH.
The first experiments tested corn or stargrass plants vs. an empty space. The
position of the host-plant was alternated within the cage for each replicate. Egg masses
containing an unknown number of eggs were placed in the center between the plant and
the empty space. The number of neonate larvae on the plant or in the empty space was
counted 24 h after introduction. There were three replicates each of corn or rice-strain
larvae selecting either corn or stargrass vs. no plant. The second experiment tested corn
vs. stargrass plants. Plant location was alternated for each replicate with a total five
replicates completed. Egg masses were placed in the center between plants and counts
were made 24 h later. Thus, the choice tests conducted in the plastic cage were plant vs.
no plant and corn vs. stargrass.
The third bioassay used was conducted with a Y-tube olfactometer. This unit was
constructed of 2.5 cm O.D. clear Plexiglas tubing. The body of the Y-tube measured
58.0 cm, and the arms measured 15.24 cm (Fig. 3-3). Airflow entered the olfactometer
by passing through a stainless steel column of activated charcoal. Airflow entering each
arm of the Y-tube was set at 0.2 L/min. One-week old plants in 550 mL pots were placed
in clear 3.8 1-glass jars. Jar lids were modified to allow airflow to enter and exit the
container, thus allowing plant volatiles to be carried into the arms of the olfactometer.
Airflow exited the Y-tube by providing a vacuum at 0.40 L/min. The first experiment
tested corn or stargrass plants vs. a pot containing moistened soil. The position of the
host-plant was alternated for each replicate. Egg masses containing an unknown number
of eggs were placed at the midpoint in the body of the Y-tube. A black 9-cm filter paper
disk (Thomas Scientific #4740C 10) was placed encircling the area outside of the tube
above the egg mass. This allowed for larvae to emerge in an area that mimics the
underside of a leaf. Larvae were allowed free movement within the olfactometer. The
number of larvae in each arm was counted after 24 h. There were three replicates each of
corn or rice-strain larvae selecting either corn or stargrass vs. the potted soil. The second
experiment tested larval attraction to the volatiles of corn vs. stargrass plants. Egg
masses were placed in a similar fashion, and the number of larvae in each arm was
counted after 24 h. There were seven replicates each of corn or rice-strain larvae selecting
either corn or stargrass plants.
These tests were conducted to determine if larvae would continue to disperse once
they came in contact with a plant source. Two bioassays were conducted. Sections of
corn and stargrass leaf material were placed on filter paper discs (Thomas Scientific,
#0898V87) moistened with ca. 3 mL deionized water and cut to fit the dimensions of a
140 x 15 mm polystyrene Petri dish (Thomas Scientific, catalog #3488C10). The plant
material that was being "passed-over" was cut to dimensions large enough to span the 14
cm diameter of the Petri dish. Another plant section was trimmed to a uniform size (- 5
cm x 2 cm) and placed 30 mm from the center, and 20 mm along the outer edge of
the Petri dish (Fig. 3-4). Twenty neonate larvae were placed in the dish opposite the leaf
section and the lid put into place. Petri dishes were placed in a RevcoTM incubator at 23.9
2C with a 14/10 day/ night cycle, -80% RH. The number of larvae on or under each
leaf section was counted 24 h after introduction. This method was performed for ten
replicates each of corn-strain passing over corn to stargrass and over stargrass to corn,
and rice-strain passing over corn to stargrass and over stargrass to corn.
The second bioassay used a wind tunnel design. The plastic cage used in the choice
tests was modified to have inflow and outflow of air (0.25 m/s). Air exiting the cage was
vented to the outside to prevent plant volatiles from reentering the cage. Plants were
arranged in the cage so that larvae would have to pass through one plant host to reach the
other (Figs. 3-5 and 3-6). Newly emerged larvae from an egg mass were placed in the
downwind position behind the first plant. There were five replicates each of corn-strain
larvae passing through corn to stargrass and through stargrass to corn; and rice-strain
larvae passing through corn to stargrass and through stargrass to corn. The number of
larvae on each plant was counted 24 h after introduction.
Nonparametric statistical analysis was performed using the Kruskal-Wallis test
(Minitab 14, SAS Institute, 8.0). For the Petri dish test, the number of larvae on each
section was compared between host-plants; for the plastic cage tests, the percent larva on
each plant was compared.
Results from the Petri dish bioassay suggested that larvae of both strains showed
strong preferences for corn over stargrass, as over 80% of the larvae were found on corn
sections (Table 3-1).
In the choice cage bioassay, the first test showed that both stains exhibited a
strong preference for whichever host-plant was present as opposed to no plant (Table
3-2). Therefore, no directional bias was observed in this bioassay. The choice test
between plants showed that corn-strain larvae exhibited a trend towards selecting
stargrass compared to corn, while rice-strain larvae were evenly distributed between the
two plants (Table 3-3).
The first experiment with the Y-tube olfactometer demonstrated that larvae would
select the arm that contained plant volatiles rather than air from moistened soil (Table
3-4). The second experiment showed that corn-strain larvae displayed a trend towards
the volatiles emitted from the corn plant. Although not significant, 68% of the
corn-strain larvae collected were present in this arm (Table 3-5). This contrasted with
rice-strain larvae, which did not show a significant preference for either plant (Table 3-5).
Corn-strain larvae demonstrated a preference to select the first plant section they
encountered in the Petri dish bioassay, selecting corn when it was first encountered and
selecting stargrass when it was first encountered (Table 3-6). Almost 89% of the
rice-strain larvae selected stargrass when first exposed to stargrass, however, when first
exposed to corn they distributed themselves evenly between the two host-plants (Table
The wind tunnel bioassay with whole plants provided slightly different results.
Corn-strain larvae were evenly distributed across both plants no matter which host was
first encountered (Tables 3-8). Rice-strain larvae selected corn (69.6%) when it was the
plant first encountered (Table 3-9). There was a trend for rice-strain larvae to select
stargrass (67.4%) when it was first encountered; however the difference was not
significant (Table 3-9). Thus, corn-strain larvae showed a preference for the first plant
encountered in the Petri dish bioassay, but were evenly distributed between plants in the
plastic cage wind tunnel. Rice-strain larvae showed a trend to accept the first plant
encountered in the plastic cage; response in the Petri dish was mixed.
Previous research conducted in Chapter 2 suggested that there are differences in
ovipositional preference between corn-strain and rice-strain moths. However, corn-strain
moths were just as likely to oviposit on non-plant materials as host-plants. Therefore, if
corn-strain females are not selecting quality host-plants then it is possible that neonates
are making the host-plant selection.
Experimental studies that have previously examined the performance of the two
host-strains indicated that rice-strain larvae would readily accept corn as a host
(Pashley et al. 1995). The result from the Petri dish experiment supports these findings,
in that rice and corn-strain larvae accepted corn sections as their feeding site significantly
more often than stargrass sections. When the two strains were presented a choice of
whole plants, corn-strain larvae exhibited a trend towards stargrass, while rice-strain
larvae were evenly distributed between plants. Perhaps the corn plant sections released
large amounts of volatiles in the closed Petri dish that influenced larval behavior in a
different manner compared to whole plants.
The olfactometer study was designed to determine if FAW neonates could detect
plant volatiles that may guide them to a food source. Results of the first experiment
suggested that the neonates have the capacity to detect plant volatiles as part of their
food-finding behavior since larvae selected olfactometer arms that contained plant
volatiles over those that contained volatiles from moistened soil. Observations suggested
that larvae "sampled the air" by raising their heads and swaying back and forth. The
comparison between volatiles of each host-plant suggested that corn-strain larvae
exhibited a trend for preference for corn over stargrass, while rice-strain larvae were
evenly dispersed in both arms of the olfactometer when presented the two plants.
Chemical senses have been shown to be important in food choice behavior (Chang 1985,
de Boer and Hanson 1987), although olfaction in larvae is usually limited to short range
orientation (Zacharuk and Shields 1991).
The olfactometer study was also conducted to determine if neonates exhibited a
predisposition to host-plants or was prior feeding required to be attracted to a particular
plant host (induction). Several workers have shown with other lepidopteran larvae that
neonates generally don't show an orientation response to specific host-plants (Saxena and
Schoonhoven 1978, Saxena and Schoonhoven 1982). The same results were found with
Spodoptera littoralis (Boisduval), which showed that neonates did not have a preference
for host-plants but third instar larvae reared on a host exhibited an increase in
orientational response in their attraction to a host-plant (Carlsson et al. 1999). The
sensory requirements and development of the immatures are most likely more limited
than that of older instars (de Boer and Hanson 1987). Induction of feeding is less evident
in a choice test when close relative plant species are used. The more distant relation of
the two plants will bring forth a stronger induction of feeding response (de Boer and
Fall armyworm neonate larvae have the ability to be mobile upon emergence and
disperse from the location of the egg mass (Claycomb 1954). The passing-over study
was designed to assess larval ability to choose one host-plant over another after coming
in contact with one host. In the Petri dish bioassay, corn-strain larvae readily accepted
whichever host-plant section they first encountered. However, when rice-strain larvae
first encountered corn, an equal number would pass over the corn to feed upon the
stargrass. Conversely, when stargrass was first encountered, it was preferred as a suitable
host. The plastic cage wind tunnel was constructed to allow larvae to be mobile over a
longer distance than that of a Petri dish and allowed for live plant material to be used
rather than plant sections. When corn-strain larvae first encountered either corn or
stargrass, they distributed themselves evenly between the two plants. However,
rice-strain larvae selected and accepted corn if it was the first plant that they encountered,
and displayed a trend to accept stargrass when it was first encountered. This behavior
supports the theory that the corn-strain is the more generalist feeder of the two strains
(Whitford et al. 1988).
Significant nutritional and developmental differences have been noted between the
two strains when feeding on corn and bermudagrass. Whitford et al. (1988) noted larvae
and pupae of the corn-strain were heavier than rice-strain immatures when reared on
identical hosts. Also, rice-strain larvae developed faster on bermudagrass than other host
grasses presented. Corn-strain pupae were heavier when fed a diet of corn or
bermudagrass than on an artificial diet. These developmental differences between the
two strains were noted by Pashley (1988), who showed that corn-strain larval
development was similar when fed corn or bermudagrass. The rice-strain female's
preference for grass in the ovipositional study and larval preference in the wind tunnel
indicates strain orientation toward bermudagrass.
Table 3-1. Mean ( SEM) number of corn or rice-strain larvae collected from corn or
stargrass sections in the Petri dish choice bioassay
Corn 14.4 1.21 a 16.5 0.83 a
Stargrass 3.3 1.17b 1.1 + 0.06 b
Means with same letter are not significantly different, corn-strain P < 0.0001,
rice-strain P < 0.0001; n = 10.
Table 3-2. Percent larvae ( SEM) of both strains selecting either corn or stargrass vs. an
empty space in the choice cage bioassay
Host-plant % Larvae P value'
Corn 94.9 5.1
Corn- Empty 5.1 5.1
strain Stargrass 95.9 2.2
Empty 4.1 2.2
Corn 99.6 0.3
Rice- Empty 0.4 0.3
strain Stargrass 85.7 7.2
Empty 14.2 7.2
For each comparison, n = 3, df = 1.
Rice- Empty 0.4 0.3
strain Stargrass 85.7 + 7.2
Empty 14.2 + 7.2
For each comparison, n = 3, df = 1.
Table 3-3. Percent larvae ( SEM) of both strains selecting either corn or stargrass in the
choice cage bioassay
Table 3-4. Percent larvae ( SEM) of both strains selecting either corn or stargrass vs.
potted soil in the Y-tube olfactometer bioassay
Host-plant % Larvae P value'
Corn 68.5 + 5.1
Corn- Soil 31.6 5.1
strain Stargrass 64.2 + 7.1
Soil 34.9 + 7.4
Corn 81.1 6.6
Rice- Soil 18.9 6.6
strain Stargrass 67.8 5.7
Soil 32.1 + 5.7
For each comparison, n = 3, df = 1.
Table 3-5. Percent larvae ( SEM) of both strains selecting either corn or stargrass in the
Y-tube olfactometer bioassay
Host-plant % Larvae P value'
Corn 68.2 + 15.0
Corn-strain r 1 0.1102
Stargrass 31.8 15.0
Corn 53.8 + 9.0
Rice-strain 90 0.5653
Stargrass 46.2 9.0
For each comparison, n = 7, df = 1.
Table 3-6. Mean ( SEM) number of corn-strain larvae that selected either corn or
stargrass after first being directionally exposed to corn or stargrass in a Petri
First exposure Plant section selected Larvae P value'
Corn to Corn 18.5 0.47
Stargrass Stargrass 0.7 + 0.21
Stargrass to Stargrass
SFor each comparison, n = 10, df = 1.
16.6 + 0.79
2.0 + 0.72
Table 3-7. Mean ( SEM) number of rice-strain larvae that selected either corn or
stargrass after first being directionally exposed to corn or stargrass in a Petri
First exposure Plant section selected Larvae P value'
Corn 9.0 + 1.56
Corn to Stargrass 0.7035
Stargrass 8.5 1.61
Stargrass to Corn Srr
For each comparison, n = 10, df = 1.
14.2 + 0.94
1.8 + 0.49
Table 3-8. Percent ( SEM) corn-strain larvae that selected either corn or stargrass after
first being directionally exposed to corn or stargrass in a wind tunnel bioassay
Corn to Stargrass
Stargrass to Corn Srr
For each comparison, n = 5, df = 1.
58.4 + 8.37
41.6 + 8.37
46.7 + 8.13
56.9 + 8.08
Table 3-9. Percent ( SEM) rice-strain larvae that selected either corn or stargrass after
first being directionally exposed to corn or stargrass in a wind tunnel bioassay
First exposure Plant Selected % Larvae P value'
Corn 69.6 + 7.14
Corn to Stargrass 0.0163
Stargrass 30.4 + 6.97
Stargrass to Corn
1 For each comparison, n = 5, df = 1.
Figure 3-1. Small Petri dish bioassay
Figure 3-2. Choice cage bioassay
Figure 3-3. Y-tube olfactometer used in volatile study
Figure 3-4. Petri dish passing-over study
Corn Grass Corn Grass
Height = 15 cm
Figure 3-5. Wind tunnel layout
Figure 3-6. Wind tunnel bioassay
Results show two FAW host-strains that exhibit host specificity at larval and adult
stages. FAW eggs and larvae were collected and identified using PCR techniques and
results confirmed that both corn and rice-strain populations reside in Florida corn and
grass habitats (Meagher and Gallo-Meagher 2003, Meagher and Nagoshi 2004, Nagoshi
and Meagher 2004). Behavioral differences between the two host-strains involving
ovipositional site preference and larval host-selection to corn and stargrass were
The rice-strain exhibited a significant preference for utilizing stargrass as its
ovipositional substrate. Whitford et al. (1988) indicated this behavioral difference when
each strain was presented with corn, sorghum, bermudagrass and centipedegrass. Their
study demonstrated the rice-strain's ovipositional preference for grass that indicated a
degree of adaptation to bermudagrass. However, the corn-strain indiscriminately placed
egg masses within and onto the enclosure. When a host-plant was selected, stargrass was
selected over corn. Previous FAW ovipositional studies do not indicate if eggs masses
were located in other areas than on plants. Pashley et al. (1995) concluded that
ovipositional preference in the corn-strain was more specialized, and that this strain
rarely occurs in pastures. The ovipositional selection of corn-strain females in my study
indicated contradictory results since these females oviposited on PVC, screen, stargrass,
and corn leaves. Corn-strain individuals have been found in grass habitats along with
rice-strain (Meagher and Gallo-Meagher 2003). It is not known which strain affects
crops other than corn and grass, but my results of my ovipositional study suggest it may
be the corn-strain.
My study differs from other research in that moths were reared on natural plant
material as opposed to an artificial diet. It is not known what effect that larval feeding on
an artificial diet has on the adult's ability to adequately select a suitable host.
When the two host-strains are presented with a choice for feeding, there appears
to be certain trends inherent to each host-strain in food selection behavior. The bioassays
conducted support previous findings in regards to host-strain behavioral differences. The
Petri dish choice study showed a significant preference for corn by both strains. These
results support the findings of Pashley et al. (1995) that the two host-strains would accept
corn as their host. An even distribution of larvae was noted when the two strains were
presented a choice between potted corn and stargrass plants.
The ability for FAW neonates to detect plant volatiles alone to guide them to a
food source has yet to be determined. Veenstra et al. (1995) suggested that physiological
and biochemical adaptations by the host-strains may account for host-plant use in FAW.
It may be that their dispersal is initiated by visual and chemosensory cues. Corn-strain
larvae exhibited a preference for corn over stargrass volatiles in the olfactometer.
Rice-strain larvae were evenly dispersed in both arms of the olfactometer when presented
the two plant hosts. The strong attraction to stargrass exhibited by the rice-strain when
visual cues were available may explain the results obtained from the host-plant choice
experiments. The olfactometer study also gave an indication that the neonates have
sensilla that assist in their food-finding behavior. Older instars may give a clearer insight
to sensory structures that aide in food-finding behavior. As the immature stage develops
through several molts, cuticular structures of existing sensilla are replaced with new
sensilla (Zacharuk and Shields 1991).
The passing-over experiments were designed to examine the dispersal of both
strains. The Petri dish bioassay indicated that corn-strain larvae would readily feed upon
whichever host-plant section they first encountered. Rice-strain larvae were evenly
distributed upon corn and stargrass when corn was encountered first, but when stargrass
was first encountered many of the neonates selected stargrass as a suitable host. Using
the plastic cage wind tunnel design simulated a more realistic test since whole plants
were used rather than plant sections. Corn-strain larvae evenly dispersed between both
plants no matter which was encountered first, however, rice-strain larvae tended to stop
and feed at the first plant encountered. An improvement of the bioassay in the future
would be to limit the number of larvae used since dispersion to either plant may have
been due to crowding rather than plant attraction.
In conclusion, these experiments confirm that there are behavioral differences
between the two host-strains of FAW. The studies performed suggest that the adult
female determines host selection in the rice-strain by selecting grass plants for
oviposition. Corn-strain moths are much less selective in egg mass placement. Neonates
of both strains do not have prior feeding experiences on a host-plant and appear to be
unbiased in their food-finding behavior. Plant volatiles and visual cues assist the
neonates in dispersal, but their ability to seek out a suitable host when not placed in the
vicinity is negligible. When food sources are consumed, older larvae become mobile in
search of new hosts. The ovipositional preference and food-seeking behavior observed
suggests that the corn-strain is the more generalist feeder.
Strain identification is important for population control and management because
previous research has shown toxicological and host genotypic differences between
strains. Rice-strain larvae were shown to be more susceptible to various insecticides and
more susceptible to transgenic Bacillus i/i#l iligie'll\i Berliner (Bt) cotton than corn-strain
larvae (Pashley et al. 1987, Adamczyk et al. 1997). Laboratory and field studies have
shown distinct differences in feeding of bermudagrass genotypes, with rice-strain larvae
generally able to gain more weight and consume more plant material than corn-strain
larvae (Quisenberry and Whitford 1988.
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Charles J. Stuhl was born on Long Island, New York on January 13, 1965. A few
years after graduation of high school, he enlisted in the US Army. Entering the military in
1986, he spent a three-year tour of duty as a combat medic in Germany. Upon completion
of his military duty, he attended Gupton-Jones College of Mortuary Service in Atlanta,
Georgia, receiving an A.S. degree in mortuary science. He relocated to Florida to work in
the funeral industry. After a few years in his new career, he decided to return to college
and pursue a B.S. degree in entomology at the University of Florida, and was awarded his
degree in 2000. He began working at the US Department of Agriculture, Agriculture
Research Service as an undergraduate, and hopes to make of long career in their employ.
He currently resides in Santa Fe, Florida, and plans on pursuing a PhD. in entomology at
the University of Florida.